Patho-Biotechnology

Biotechnology intelligence unit

The chapters in this book, as well as the chapters of all of the five Intelligence Unit series, are available at our website.

Roy Sleator and Colin Hill

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Sleator • Hill

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Patho-Biotechnology

Sleator ISBN 978-1-58706-304-6

9 781587 063046

Patho-Biotechnology

Biotechnology Intelligence Unit

Patho-Biotechnology Roy Sleator, PhD

Alimentary Pharmabiotic Centre University College Cork Cork, Ireland

Colin Hill, PhD

Alimentary Pharmabiotic Centre University College Cork Cork, Ireland

Landes Bioscience Austin, Texas USA

Patho-Biotechnology Biotechnology Intelligence Unit Landes Bioscience Copyright ©2008 Landes Bioscience All rights reserved. No part of this book may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Printed in the USA. Please address all inquiries to the publisher: Landes Bioscience, 1002 West Avenue, Austin, Texas 78701, USA Phone: 512/ 637 6050; Fax: 512/ 637 6079 www.landesbioscience.com ISBN: 978-1-58706-304-6 While the authors, editors and publisher believe that drug selection and dosage and the specifications and usage of equipment and devices, as set forth in this book, are in accord with current recommendations and practice at the time of publication, they make no warranty, expressed or implied, with respect to material described in this book. In view of the ongoing research, equipment development, changes in governmental regulations and the rapid accumulation of information relating to the biomedical sciences, the reader is urged to carefully review and evaluate the information provided herein.

Library of Congress Cataloging-in-Publication Data Patho-biotechnology / [edited by] Roy Sleator, Colin Hill. p. ; cm. -- (Biotechnology intelligence unit) Includes bibliographical references and index. ISBN 978-1-58706-304-6 1. Microbial biotechnology. 2. Pathogenic bacteria--Therapeutic use. I. Sleator, Roy. II. Hill, Colin, PhD. III. Series: Biotechnology intelligence unit (Unnumbered) [DNLM: 1. Genetic Vectors--therapeutic use. 2. Bacteria. 3. Bacterial Toxins--therapeutic use. 4. Gene Therapy--methods. QU 470 P297 2008] TP248.27.M53P38 2008 616.9'041--dc22 2008019688

Dedication Dedicated to the memory of Mrs. Helen Carey

About the Editors...

ROY SLEATOR, PhD, graduated from University College Cork, Ireland in 1997 with a first class honours degree in Microbiology, before going on to complete a PhD in 2001 at the National Food Biotechnology Centre, BioResearch Ireland. Following his PhD studies, Dr Sleator was granted a prestigious EMBARK Post-doctoral Fellowship from the Irish Research Council for Science Engineering and Technology, to investigate the mechanisms of bacterial stress response. This work was recognized in 2004 with the inaugural joint European Society for Clinical Microbiology and Infectious Diseases/Federation of European Microbiology Societies Research Fellowship. This was followed, in 2006, by the highly prestigious Pierce Prize—the highest award bestowed on a scientist by the Society for Applied Microbiology in recognition of an excellent contribution to the field of bacteriology. In 2006 Dr Sleator was awarded a Health Research Board Principal Investigatorship to further develop the area of Patho-biotechnology and in 2008 was appointed as a Principal Investigator at the Alimentary Pharmabiotic Centre, University College Cork.

About the Editors...

COLIN HILL, PhD, has a PhD in molecular microbiology and works in the Microbiology Department of University College Cork, Ireland. He is also a Principal Investigator in the Alimentary Pharmabiotic Centre, a multidisciplinary research centre focusing on the role of gut microbiota in health and disease. His main interests are in infectious disease, particularly in defining the mechanisms of virulence of foodborne pathogens and in developing strategies to prevent and limit the consequences of microbial infections in the gastrointestinal tract. He has published more than 200 peer-reviewed papers and holds 10 patents in this area. He has also served on the Scientific Committee of the Food Safety Authority of Ireland for many years. In 2005, Professor Hill was awarded a DSc by the National University of Ireland in recognition of his contributions to research.

CONTENTS Preface.......................................................................................................xvii 1. Application of Bacterial Pathogens in Replacement Therapy ......................1 John R. Tagg, Jeremy P. Burton and Philip A. Wescombe Directed Modulation of the Indigenous Microbiota— Historical Perspectives ........................................................................................2 Criteria for Selection of Effector Strains for Replacement Therapy ..............3 Bacteriocin-Mediated Interactions between Streptococcal Commensals and Pathogens in the Oral Cavity and the Implications for Replacement Therapy ....................................................................................5 Specific Examples of the Application of Potential Bacterial Pathogens in Replacement Therapy......................................................................................8 Dental Caries Due to S. mutans ............................................................................8 Upper Respiratory Tract Infection .....................................................................10 Prevention of Otitis Media ...................................................................................10 Prevention of Urinary Tract Infection ...............................................................11 Prevention of Vaginal Infection ...........................................................................11 Final Thoughts .........................................................................................................11 2. Improvement of Insect Pathogens as Insecticides through Genetic Engineering.................................................................................................15 Brian A. Federici, Bryony C. Bonning and Raymond J. St Leger Genetic Engineering to Optimize Baculovirus Insecticides .........................16 Biology of Baculoviruses ........................................................................................16 Genetic Engineering to Optimize Speed of Kill ..............................................17 Baculovirus for Bt Toxin Delivery ......................................................................19 Genetic Engineering for Increased Virulence.................................................. 20 Genetic Engineering of Bacillus thuringiensis (Bt) ......................................... 20 Molecular Biology of Bt Insecticidal Proteins ................................................. 23 Bt Endotoxin Mode of Action ............................................................................ 23 Genetic Regulation of Cry and Cyt Protein Synthesis .................................. 24 Recombinant Bacterial Insecticides Based on Bt ............................................ 24 Safety Concerns about Wild Type and Recombinant Bacterial Insecticides ........................................................................................ 27 Basic Biology and Genetic Engineering of Insect-Pathogenic Fungi ..........29 Genetic Engineering Entomopathogenic Fungi ..............................................31 Overview of Engineering Saprophytic Competence or Containment .......31 Engineering Improved Virulence ........................................................................32 3. Phage Therapy: A Trojan Horse Approach to the Control of Intracellular Pathogens...........................................................................41 Lawrence Broxmeyer Waksman’s “Antibiotics” ...................................................................................... 42 Intracellular Quest ................................................................................................. 42 Novel Concept ........................................................................................................ 43 Results ....................................................................................................................... 44

4. Bacterial Ghosts as Vaccine and Drug Delivery Platforms ........................50 Ulrike Beate Mayr, Verena Juliana Koller, Petra Lubitz and Werner Lubitz Characterization of BG..........................................................................................51 BG as Vaccines .........................................................................................................53 BG with Extended Properties...............................................................................53 BG as Carrier Vehicles for DNA and/or Cytostatic Drugs in Tumour Therapy ........................................................................................... 56 5. In Vivo Remote Control of Bacterial Vectors for Prophylaxis and Therapy ................................................................................................60 Holger Loessner and Siegfried Weiss Remote Control of Bacterial Vectors by Physical Means ...............................61 Drug-Mediated Remote Control of Bacterial Vectors....................................63 6. Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles ........71 Pablo Daniel Becker, Miriam Noerder and Carlos Alberto Guzmán Plasmid Design ....................................................................................................... 72 Live Attenuated Bacterial Carriers .................................................................... 77 Conclusions and Outlook .................................................................................... 88 7. Bacteria Mediated Gene Therapy Strategies ..............................................98 Sophie Conchon and Georges Vassaux Mechanism of Bacteria-Mediated Gene Transfer ........................................... 98 Bacteriolysis of Tumors ....................................................................................... 100 Bacterial Vectors for Immunotherapy ............................................................. 103 Other Examples of Bacterial Gene Therapy Strategies ................................ 105 Emerging Technologies ....................................................................................... 106 8. Use of Intracellular Bacteria for the Development of Tools for Tumor Therapy and the Detection of Novel Antibacterial Targets .....................112 Christoph Schoen, Jochen Stritzker, Thilo M. Fuchs, Stephanie Weibel, Ivaylo Gentschev, Aladar A. Szalay and Werner Goebel Secretion of Microbial and Tumor Antigens in Salmonella enterica Serovar Typhi Ty21 by T1SS ......................................................................... 113 Bactofection of Tumor Cells with Bacterial Carriers Introducing DNA and RNA for Antigens and Prodrug-Converting Enzymes ...... 113 Growth of E. coli in Solid Tumors Transplanted into Mice........................115 Metabolic Pathways as Evaluated Targets for Virulence Attenuation and Screening of Antimicrobials ..................................................................117 9. Bacterial Vectors for RNAi Delivery ........................................................121 Thu Nguyen and Johannes H. Fruehauf Transkingdom RNA Interference ..................................................................... 122 Bacteria-Mediated RNA Delivery.................................................................... 124

10. Viral Pathogens as Therapeutic Delivery Vehicles ...................................126 Helen O’Shea Gene Therapy......................................................................................................... 127 Viruses as Vectors ................................................................................................. 127 Making Recombinant Viruses .......................................................................... 131 Retroviral Vectors................................................................................................. 131 Lentiviral Vectors .................................................................................................134 Adenoviral Vectors ............................................................................................... 135 Adeno-Associated Viral Vectors ....................................................................... 136 Herpes Simplex Viral Vectors ............................................................................ 138 Vaccinia Viral Vectors ......................................................................................... 139 RNA Viral Vectors ............................................................................................... 139 Nonviral Methods of DNA Transfer............................................................... 140 Comparison of Vectors ....................................................................................... 140 Strategies for Gene Therapy ............................................................................... 140 Future Development of Viral Vectors for Gene Therapy............................. 141 11. Promiscuous Drugs from Pathogenic Bacteria in the Post-Antibiotics Era .......................................................................145 Arsenio M. Fialho, Tapas K. Das Gupta and Ananda M. Chakrabarty Pathogenic Bacteria as Anticancer Agents ..................................................... 146 Immune Action Alone Does Not Explain Microbial Antitumor Activity ......................................................................................... 147 Bacterial Proteins as Anticancer Agents ......................................................... 147 Azurin Inhibits Cancer Cell Growth by Interfering in Multiple Steps .............................................................................................. 149 Azurin as a Promiscuous Drug...........................................................................152 Antiparasite Activity of Azurin and Laz .........................................................153 Azurin/Laz as Inhibitors of Entry/Growth of the HIV-1 Virus............... 154 Azurin/Laz as a Scaffolding Protein................................................................ 158 12. Attack and Counter-Attack: Targeted Immunomodulation Using Bacterial Virulence Factors ............................................................163 Mohammed Bahey-El-Din, Brendan T. Griffin and Cormac G.M. Gahan Vibrio cholerae Toxin (CT) and Escherichia coli Heat-Labile Enterotoxin (LT) ............................................................................................. 164 Listeria monocytogenes Listeriolysin O (LLO) ............................................... 166 Bordetella pertussis Virulence Factors .............................................................. 167 Clostridium difficile Toxin A ............................................................................. 168 Helicobacter pylori Neutrophil-Activating Protein (HP-NAP) ................. 169

13. Cosmetic and Therapeutic Applications of Botulinum Toxin in the Head and Neck ...............................................................................173 Nwanmegha Young and Andrew Blitzer Pathophysiology .................................................................................................... 173 Clinical Applications............................................................................................174 Cosmetic Applications .........................................................................................174 Uses in the Upper Face.........................................................................................174 Uses in the Lower Face and Neck ......................................................................174 Other Cosmetic Uses .......................................................................................... 177 Facial Synkinesis ................................................................................................... 177 Therapeutic Applications.................................................................................... 177 Hyperfunctioning Muscle Disorders ............................................................... 178 Pain/Nociceptive Disorders............................................................................... 183 Headache ................................................................................................................ 184 Trigeminal Neuralgia .......................................................................................... 184 Discussion .............................................................................................................. 184 14. The Use of Recombinant Phage Lysins for the Control of Bacterial Pathogens ..................................................................................................187 Marianne Horgan, Aidan Coffey, R. Paul Ross, Jim O’Mahony, Gerald F. Fitzgerald and Olivia McAuliffe Endolysin Structure ............................................................................................. 188 Important Properties of Phage Lysins ............................................................. 193 Potential Applications of Phage Endolysins ................................................... 195 15. Engineered Pharmabiotics with Improved Therapeutic Potential...........206 Roy Sleator and Colin Hill Improving Probiotic Stress Tolerance..............................................................206 ‘Designer Probiotics’ ............................................................................................208 Biological Containment ..................................................................................... 210 Index .........................................................................................................213

EDITORS Roy Sleator

Alimentary Pharmabiotic Centre University College Cork Cork, Ireland Chapter 15

Colin Hill

Alimentary Pharmabiotic Centre University College Cork Cork, Ireland Chapter 15

CONTRIBUTORS Note: Email addresses are provided for the corresponding authors of each chapter. Mohammed Bahey-El-Din Department of Microbiology and

School of Pharmacy University College Cork Cork, Ireland Chapter 12

Pablo Daniel Becker Department of Vaccinology Helmholtz Centre for Infection Research Braunschweig, Germany Chapter 6

Andrew Blitzer Head and Neck Surgical Group and

NY Center for Voice and Swallowing Disorders New York, New York, USA Email: [email protected] Chapter 12

Bryony C. Bonning Department of Entomology Iowa State University Ames, Iowa, USA Chapter 2

Jeremy P. Burton BLIS Technologies Ltd Centre for Innovation University of Otago Dunedin, New Zealand Chapter 1

Lawrence Broxmeyer MAR-Research Institute Whitestone, New York, USA Email: [email protected] Chapter 3

Ananda M. Chakrabarty Department of Microbiology and Immunology University of Illinois College of Medicine Chicago, Illinois, USA Email: [email protected] Chapter 11

Aidan Coffey Department of Biological Sciences Cork Institute of Technology Bishopstown, Cork, Ireland Email: [email protected] Chapter 14

Sophie Conchon INSERM CIC04 Biothérapies Hépatiques Université de Nantes Nantes Atlantique Universités Institut des Malades de l’Appareil Digestif Nantes, France Email: [email protected]

Thilo M. Fuchs ZIEL, Abteilung Mikrobiologie Technische Universität München Freising, Germany Chapter 8

Chapter 7

Cormac G.M. Gahan Department of Microbiology Alimentary Pharmabiotic Centre

Tapas K. Das Gupta Department of Surgical Oncology University of Illinois College of Medicine Chicago, Illinois, USA

School of Pharmacy University College Cork Cork, Ireland Email: [email protected]

Chapter 11

and

Chapter 12

Brian A. Federici Department of Entomology University of California, Riverside Riverside, California, USA Email: [email protected]

Ivaylo Gentschev Lehrstuhl für Biochemie Biozentrum

Arsenio M. Fialho Institute for Biotechnology and Bioengineering Center for Biological and Chemical Engineering Institute Superior Tecnico Lisbon, Portugal

and

Chapter 2

Chapter 11

Gerald F. Fitzgerald Department of Microbiology University College and

Alimentary Pharmabiotic Centre Cork, Ireland Chapter 14

Johannes H. Fruehauf Skip Ackerman Center for Molecular Therapeutics GI Cancer Laboratory Beth Israel Deaconess Medical Center Harvard Medical School Boston, Massachusetts, USA Email: [email protected] Chapter 9

and

Institut für Molekulare Infektionsbiologie Universität Würzburg Würzburg, Germany Genelux Corp. San Diego Science Center San Diego, California, USA Chapter 8

Werner Goebel Max-von-Pettenkofer-Institut Ludwig-Maximilians-Universität München München and

Lehrstuhl für Mikrobiologie Biozentrum Universität Würzburg Würzburg, Germany Chapter 8

Brendan T. Griffin School of Pharmacy University College Cork Cork, Ireland Chapter 12

Carlos Alberto Guzmán Department of Vaccinology Helmholtz Centre for Infection Research Braunschweig, Germany Email: [email protected] Chapter 6

Marianne Horgan Moorepark Food Research Centre Teagasc Fermoy and

Department of Microbiology University College Cork Cork, Ireland Chapter 14

Verena Juliana Koller Department of Medicinal Chemistry University of Vienna Vienna and

BIRD-C GmbH&CoKEG Kritzendorf, Austria Chapter 4

Holger Loessner Department of Molecular Immunology Helmholtz Centre for Infection Research Braunschweig, Germany Email: [email protected] Chapter 5

Petra Lubitz Department of Medicinal Chemistry University of Vienna Vienna and

BIRD-C GmbH&CoKEG Kritzendorf, Austria Chapter 4

Werner Lubitz Department of Medicinal Chemistry University of Vienna Vienna and

BIRD-C GmbH&CoKEG Kritzendorf, Austria Email: [email protected] Chapter 4

Ulrike Beate Mayr Department of Medicinal Chemistry University of Vienna Vienna and

BIRD-C GmbH&CoKEG Kritzendorf, Austria Chapter 4

Olivia McAuliffe Moorepark Food Research Centre Teagasc Fermoy Cork, Ireland Chapter 14

Thu Nguyen Skip Ackerman Center for Molecular Therapeutics GI Cancer Laboratory Beth Israel Deaconess Medical Center Harvard Medical School Boston, Massachusetts, USA Chapter 9

Miriam Noerder Department of Vaccinology Helmholtz Centre for Infection Research Braunschweig, Germany Chapter 6

Jim O’Mahony Department of Biological Sciences Cork Institute of Technology Bishopstown, Cork, Ireland Chapter 14

Helen O’Shea Department of Biological Sciences Cork Institute of Technology Bishopstown, Cork, Ireland Email: [email protected] Chapter 10

R. Paul Ross Moorepark Food Research Centre Teagasc Fermoy and

Alimentary Pharmabiotic Centre Cork, Ireland Chapter 14

Christoph Schoen Universität Würzburg Institut für Hygiene und Mikrobiologie Würzburg, Germany Email: [email protected] Chapter 8

Raymond J. St Leger Department of Entomology University of Maryland College Park, Maryland, USA Chapter 2

Jochen Stritzker Lehrstuhl für Biochemie Biozentrum and

Aladar A. Szalay Lehrstuhl für Biochemie Biozentrum and

Institut für Molekulare Infektionsbiologie and

DFG-Forschungszentrum für Experimentelle Biomedizin Universität Würzburg Würzburg, Germany and

Genelux Corp. San Diego Science Center San Diego, California, USA Chapter 8

John R. Tagg Department of Microbiology and Immunology University of Otago Dunedin, New Zealand Email: [email protected] Chapter 1

Georges Vassaux INSERM CIC04 Biothérapies Hépatiques Université de Nantes Nantes Atlantique Universités Institut des Malades de l’Appareil Digestif Nantes, France Chapter 7

Institut für Molekulare Infektionsbiologie Universität Würzburg Würzburg, Germany

Stephanie Weibel Lehrstuhl für Biochemie Biozentrum

Genelux Corp. San Diego Science Center San Diego, California, USA

Institut für Molekulare Infektionsbiologie Universität Würzburg Würzburg, Germany

and

Chapter 8

and

Chapter 8

Siegfried Weiss Molecular Immunology Helmholtz Centre for Infection Research Braunschweig, Germany Chapter 5

Philip A. Wescombe BLIS Technologies Ltd Centre for Innovation University of Otago Dunedin, New Zealand Chapter 1

Nwanmegha Young Department of Surgery Division of Otolaryngology Yale University School of Medicine New Haven, Connecticut, USA Chapter 13

PREFACE “Who is mighty? He who makes an enemy into a friend.” —[Hebrew proverb] In 2006, we coined the term ‘Patho-biotechnology’ to describe the exploitation of pathogens, or pathogen derived factors, for beneficial applications in biotechnology, food and medicine1. This concept encompasses three broad areas: i. The first approach (outlined in Chapters 1-10) involves the use of selected pathogens as effective prophylactic and/or therapeutic agents by replacement technology. The rationale for this “fighting fire with fire” approach being that for most species the strongest niche competitors are often the same or closely related species. ii. The second approach (outlined in Chapters 11-14) involves the isolation and purification of pathogen-specific immunogenic proteins for direct application, thus removing the necessity for potentially harmful bacterial carrier platforms. iii. The third approach (outlined in Chapter 15) provides an alternative to either (i) or (ii) above. This approach involves equipping non-pathogenic bacteria with the genetic elements necessary to survive the many stresses encountered outside the host as well as the myriad of antimicrobial hurdles faced during host transit and/or colonisation. Roy Sleator, PhD 1. Sleator RD, and Hill C. Patho-biotechnology; using bad bugs to do good things. Curr Opin Biotech 2006; 17:211-216.

Acknowledgements Firstly we would like to thank the Alimentary Pharmabiotic Centre (APC) for championing the Patho-biotechnology concept and for its continued support of both editors. We would also like to extend our sincerest thanks to Ron Landes, Megan Klein, Cynthia Conomos, Erin O'Brien and Celeste Carlton for making the book a reality.

The concept of yin and yang is a fundamental principle of traditional Chinese medicine describing two primal opposing but complementary forces creating balance. Patho-biotechnology seeks to attain balance by exploiting pathogens (representing yin; the dark or negative force) for biotechnological and medical applications (representing yang; the light or positive force).

Chapter 1

Application of Bacterial Pathogens in Replacement Therapy John R. Tagg,* Jeremy P. Burton and Philip A. Wescombe

Abstract

A

s our knowledge of the cellular content and intercellular dynamics of the indigenous microbiota increases, so too does our ability to rationally adjust its composition to heighten its specific pathogen-excluding capabilities. In replacement therapy the principal objective is to modulate the composition of the indigenous microbiota, in instances where it is considered potentially vulnerable to pathogen overgrowth. This is achieved by supplementing the existing bacterial population with “effector” strains that are equipped to competitively oust or repel members of targeted bacterial species thought to have disease-causing capability. Often however it proves difficult to lodge new bacterial entities within the existing microflora of the host; finely-tuned molecular mechanisms intrinsic to the established community typically operate to maintain a population status quo. One attribute of bacteria that may assist their establishment and survival within these complex ecosystems appears to be their production of and/or expression of specific immunity to anti-competitor peptide antibiotics called bacteriocins. For most bacteria, the strongest niche competitors are probably other bacteria of the same or closely-related species. Hence, the effector strains chosen for replacement therapy often are either naturally-occurring or engineered low-virulence variants of the targeted pathogenic species. In the future it should increasingly prove possible to judiciously engineer effector strains, not only to reduce their virulence, but also to express traits that will enhance their genetic stability, niche competitiveness and physical resilience in the face of the various challenges that confront them both ex vivo and in situ.

Introduction Optimising Our Body’s Front-Line Microbial Defences—Good Luck or Good Management?

Soon after birth, pioneer microbes colonize all of the accessible surfaces of healthy humans. An orderly succession of site-specific acquisitions and eliminations soon leads to the development of climax microbial communities, each exquisitely adapted to the physical and physiological idiosyncrasies of the various ecosystems that collectively constitute the homeland of our indigenous microbiota—the first cellular line of defence of the human tissues against incursions or uprisings of potentially-pathogenic microbes. This indigenous microbiota (or normal microflora) presents the first effective barrier to infection of our healthy tissues. It typically forms with no purposeful human intervention as a result of the adventitious seeding of the tissues in those first days of life. It is somewhat salutary to reflect upon the fact that although we equally inherit the genetic complement of our tissues from both of our parents, the composition of the (numerically) major cellular component of our body, our indigenous microbiota, is more likely to mirror that of our primary *Corresponding Author: John R. Tagg—Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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care-giver- typically our mother- although it will also include assorted offerings from other close contacts. The first opportunity to orchestrate the composition of our microbial defences is at this stage of pioneer settlement. Fine-tuning of the mother’s indigenous microbiota by the deliberate implantation of potentially-beneficial (pathogen-antagonistic) bacteria in the period just prior to birthing may ultimately provide the most practical and successful means of achieving these objectives. Once fully established the mature (climax community) indigenous microbiota appears remarkably resistant to change unless subjected to major microbiocidal assault, such as exposure to broad-spectrum antibiotics or territorial invasion by ultra-competitive microbes. The term replacement therapy is used to describe the process of attempting to effect compositional changes within the established microflora that are of benefit to the host. The goal is implantation of relatively innocuous “effector” bacteria that are capable of excluding or at least preventing the harmful proliferation of disease-causing microbes, without adversely disturbing the natural balance of the existing microbial ecosystem.1 The reader is referred to the excellent account by Michael Wilson of the “Microbial Inhabitants of Humans” for an in depth discussion of the role of the indigenous microbiota in influencing the delicate equilibrium between health and disease.2

Directed Modulation of the Indigenous Microbiota— Historical Perspectives

The origins of bacterial replacement therapy can (like so many other iconic concepts in microbiology) be traced to Pasteur (Table 1). Pasteur explored the use of “common bacteria”, probably Escherichia coli, to interfere with the growth of Bacillus anthracis in vitro (using urine as a culture medium) and in animals.3 This was the pre-antibiotic era and physicians of that time had no other rational means at their disposal to prevent or limit infection, other than by dosing their patients with putatively-innocuous commensal bacteria or virulence-attenuated pathogenic species.4 Later, a nonpathogenic E. coli strain isolated by Nissle5 and subsequently marketed as Mutaflor, was promoted for its purported ability to suppress the proliferation of undesirable E. coli and enterobacteria. Only recently the inhibitory activity of this strain has been attributed to a mixture of the microcins H47 and M.6 With the spectacular advent of penicillin, closely followed by an entourage of other “miracle drugs”, the practice of replacement therapy was largely put on the “back burner” until the unanticipated failures and foibles of antibiotics encouraged some hardy devotees of the pasteurian principles to once again delve into the dark world of human-directed inter-germ warfare. A major prompt for this resurgence of interest in replacement therapy was the spectacular increase in serious hospital infections of neonates with Staphylococcus aureus (often penicillin-resistant) in the 1950s. The counter strategy was preemptive colonization of the nasal mucosae and umbilicus of babies in newborn nurseries with the low virulence Staph. aureus 502A in order to prevent infection by more virulent strains.7,8 Other early bacterial replacement therapy ventures included the pharyngeal application of alpha hemolytic streptococcus strain 215 in neonates to Table 1. Some milestones in the development of probiotics and replacement therapy Date

Event

Reference(s)

1877

Pasteur’s use of a coliform to inhibit Bacillus anthracis

3

1906

Metchinikoff’s book on health benefits of lactobacilli

10

1925

Development by Nissle of the E. coli component of Mutaflor

5,6

1966

Use of strain 502A to control Staph. aureus infections

7,8

1980

“Viridans” streptococci to reduce upper respiratory tract infections

9

2000

Proposal to use genetically-modified S. mutans for dental caries control

15,24

2004

Modulation of the oral microbiota using S. salivarius K12

13,14

Application of Bacterial Pathogens in Replacement Therapy

3

establish a “normal” microflora that would provide increased protection against upper respiratory tract pathogens.9 Meanwhile, the use of intestinal probiotics, first popularized by the flamboyant Metchnikoff in his book The Prolongation of Life: Optimistic Studies,10 steadily gained popularity and this in turn has no doubt helped to allay public concerns about the practice of deliberately seeding “friendly” microorganisms in non-intestinal body sites in order to confer a health benefit. The conventional intestinal probiotics include the dairy product-associated lactic acid bacteria (LAB), especially Bifidobacterium spp., Lactobacillus spp., Streptococcus thermophilus and Enterococcus faecium. Many of these LAB have been granted “generally regarded as safe” (GRAS) status and indeed no untoward outcomes appear to have followed their ad libitum consumption by healthy humans. Interestingly, the beneficial outcomes from use of intestinal probiotics appear largely to be achieved without substantial or persistent colonization of the host’s microflora and, as such, their application does not properly conform to the criteria of “replacement therapy.” Nevertheless these intestinal probiotics do have the potential to effect health benefits, such as short-term modulation of host immunity as a consequence of their fleeting interactions with host cells during their transit of the gastrointestinal tract. The term bacteriotherapy is probably a more appropriate term than replacement therapy as a descriptor of the conventional applications of the intestinal probiotics.11 According to the currently adopted WHO/FAO definition probiotics are “Live microorganisms which when administered in adequate amounts confer a health benefit on the host”.12 This definition legitimizes the inclusion within the term probiotic of effector microorganisms having their origins and target tissues not limited to the human intestinal tract. Indeed, our own efforts to extend the application of probiotics to the oral cavity has resulted in the development of Streptococcus salivarius replacement therapy,13 with S. salivarius strain K12 currently used either as the sole effector for halitosis treatment and prevention of streptococcal pharyngitis or in combination with conventional intestinal probiotics (Bifidobacterium and Lactobacillus spp.) to replenish the oral and intestinal microbiotas following the use of broad-spectrum antibiotics (www.blis.co.nz).14 Features of replacement therapy that differentiate it from conventional probiotic applications include (a) the colonization of host tissues, (b) the effecting of a persisting modification to the composition of the microflora and (c) the targeted removal or exclusion of specific pathogens. One highly publicized recent development in the field of bacterial replacement therapy is the proposal to implant a genetically-modified, low-virulence (but strongly competitive) S. mutans strain to control dental caries in human subjects.15 Others have proposed that the performance of conventional probiotics might be enhanced by engineering into their genomes certain stress tolerance or virulence-associated loci obtained from successful pathogens.16,17 Stay tuned for further exciting developments in this re-awakening field.

Criteria for Selection of Effector Strains for Replacement Therapy

Most of the bacteria proposed for use in replacement therapy (also sometimes called bacterial interference)18,19 have been selected on the basis that they are strongly competitive with specific target pathogens. Certain desirable characteristics have been delineated for the effector bacteria (Box 1). Ideally these bacteria will be powerful niche-competitors and typically the most effective will be of species either the same or closely-related to the pathogen. As such, the prime strains for use in replacement therapy are unlikely to have GRAS status. Often they are naturally-occurring or laboratory-attenuated low-virulence variants of potentially-pathogenic species. One could reasonably ask—when does a microbe merit classification as a “pathogen”? Any microbe that upon binding to and/or entering our tissue cells elicits a detrimental tissue response could potentially be considered a “pathogen”. Indeed, possibly any member of our indigenous microbiota could suddenly find itself cast in the role of “opportunistic pathogen” following damage to the host’s immune system (as in AIDS) or its normal microflora (e.g., associated with chemotherapy) (Fig. 1). Acute tissue responses to microbes can be beneficial to the host and may heighten the short-term defences against infection, as appears to be the case following consumption of some intestinal probiotics.20 The extent to which the adjuvant-like stimulation of intestinal and systemic immunity

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Box 1. Attributes of an ideal effector bacterium in replacement therapy

• Active against all members of the target species • Effects no collateral damage to other microbes in the indigenous microbiota • Readily establishes as a stable and persisting member of the relevant component of the indigenous microbiota • Genetically stable • Not directly pathogenic to or causing predisposition of the host to other disease conditions • Able to be eliminated if necessary by use of low-risk antibiotics • Easily grown and processed into a stable form for widespread distribution • Reliably detected and quantified within the indigenous microbiota

elicited by such probiotics can achieve persistent (albeit non targeted) protection against potential pathogens in the intestinal tract (or in more remote sites such as the oral cavity) has not yet been clearly established.21 A number of possible contributing mechanisms have been proposed to account for the ability of replacement therapy effector strains to limit proliferation of their target species (Box 2). Successful effector strains, in addition to not themselves causing adverse tissue damage, provide a degree of protection against overgrowth/infection by certain potentially-pathogenic microbes. For many of the “classical” examples of replacement therapy (e.g., Staph aureus 502A, alpha hemolytic streptococcus strain 215 and S. salivarius TOVE-R) the process of selection of the effector

Figure 1. Factors influencing the pathogen or commensal status of microbes.

Application of Bacterial Pathogens in Replacement Therapy

5

Box 2. Mechanisms of interference attributed to replacement therapy effector strains • • • •

Blockage of pathogen attachment site(s) Alteration in the microenvironment (e.g., change in pH) Competition for essential nutrients (substrate or vitamins) Production of inhibitory substances ranging from complex entities (bacteriophages, bacteriocins and bacteriolytic enzymes) to simple molecules (ammonia, lactic acid, free fatty acids and hydrogen peroxide)

strain appears to have been rather empirical and the mechanism(s) operating in situ to confer host protection remain ill-defined. The effector strain attribute that has attracted most contemporary research interest and which appears to offer considerable potential for human intervention is the production of specifically-targeted anti-competitor antibiotics called bacteriocins. Increased knowledge of their molecular and genetic basis and of the modes of killing activity of these molecules has informed the recent choices of replacement therapy strains that produce either multiple bacteriocins (e.g., S. salivarius K1222) or high yields of bacteriocins (e.g., S. mutans BCS3-L123,24) having inhibitory activity directed relatively specifically against particular target pathogens.

Bacteriocin-Mediated Interactions between Streptococcal Commensals and Pathogens in the Oral Cavity and the Implications for Replacement Therapy

The oral cavity is a complex micro-environment having a wide variety of ecological habitats and microbial niches. In order to implement replacement therapy within this ecosystem one option is to introduce a naturally-occurring, low-virulence isolate of a species that will either (i) competitively occupy a particular niche/habitat required by the target pathogen or (ii) seed into the saliva diffusible inhibitory agent(s) capable of controlling the numbers/metabolism of the target pathogen located at some other oral site which is infiltrated by that saliva. As an alternative option, the effector strain may be a genetically-modified, virulence-enfeebled variant of the pathogen species. By necessity such a strain must however have a strong competitive edge over its more virulent cousins in the battle for niche supremacy. We are particularly interested in factors that may influence the population balance between commensal and potentially-pathogenic streptococci in the human oral cavity. The commensal S. salivarius and potential pathogens Streptococcus pyogenes and Streptococcus mutans are found only in the human mouth. S. salivarius is the numerically predominant oral bacterial species and is principally located on the highly-papillated tongue surface.25 It has very low virulence potential in the normal healthy host.26 Carriage of S. pyogenes (in the pharynx) and of S. mutans (in dental plaque) is common, especially in school-aged children. When present in small numbers they cause no pathology, presumably any excessive proliferation being held in check either by other members of the oral microflora or by the host’s immune defences. Failure of these controls can result in development of streptococcal pharyngitis or dental caries. We speculate that due to its strategic location and dominant numbers S. salivarius is well-placed to perform an important population surveillance and management “i.e., sentinel” role within the human oral microbiota, contributing to modulation of the population levels of other streptococcal species (including potential pathogens). We further propose that the most influential instrument of population control associated with S. salivarius is its highly flexible ability to acquire and (when necessary) decommission genetic loci encoding a remarkable variety of bacteriocins and bacteriocin-like inhibitory substances (BLIS). The bacteriocins of gram positive bacteria are anti-competitor antibiotics, classified recently into four clusters27 and having in common their ribosomally-synthesised composition and targeted killing

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of other bacteria, most typically including those that are relatively closely-related to the producer. Reports of bacteriocins exhibiting toxicity for eukaryotic cells are rare, though bacteriocin loci are sometimes closely linked to virulence determinants.28 Several observations (Fig. 2) support an important role for S. salivarius in the control of population levels of other oral microbes:

A. On the Tongue

1. Low populations of S. salivarius on the tongue can correlate with relatively high proliferation of Candida albicans (leading to oral thrush)29 or of odiferous anaerobes (resulting in halitosis).30 By contrast, a healthy tongue microflora appears to result from abundant space occupancy by S. salivarius. Besides, some S. salivarius also appear capable of producing substances inhibitory to gram-negative bacteria or candida. 2. A high proportion of S. salivarius produce BLIS activity against other streptococcal species, especially S. pyogenes and multiple bacteriocins are commonly produced by individual isolates.31 For example, the oral probiotic S. salivarius K12 produces both salivaricin A and salivaricin B, as well as various other (as yet uncharacterized) bacteriocins.22 When a salivaricin A-producing S. salivarius population is present, some other bacterial species on the tongue show increased levels of specific resistance to this bacteriocin.32 3. S. salivarius bacteriocins appear typically to be encoded by loci on very large (mega) plasmids (size range 160-220 kb).33 4. Bacteriocin-encoding mega-plasmid DNA is transmissible in vivo between different S. salivarius strains.33 5. Loci encoding homologs of some bacteriocins (salivaricin A, salivaricin B and SA-FF22) produced by various other oral streptococcal species, including S. mitis, S. pyogenes, S. equisimilis and S. mutans, have also been detected in S. salivarius mega-plasmids.33 These

Figure 2. Sentinel role of S. salivarius in the human oral cavity.

Application of Bacterial Pathogens in Replacement Therapy

7

observations are consistent with S. salivarius mega-plasmids functioning as flexible receptacles for the acquisition and expression of bacteriocin loci putatively gathered from (or donated to) a variety of other oral streptococci. 6. A S. salivarius strain initially expressing both salivaricin A and salivaricin B was found, following implantation within the oral microflora of a subject, to undergo mutations first within the salivaricin B locus and then the salivaricin A locus leading successively to loss of production of both of these bacteriocins (but not of the corresponding bacteriocin immunities).34 The fact that the nonproducing, but immune, variants came to predominate within the subject’s microflora indicated that these bacteria may have had an ecological advantage in that particular habitat. The metabolic burdens imposed upon a bacterium by expression of bacteriocin immunity and even more so by the expression of both bacteriocin immunity and the bacteriocin (killer) peptide itself has been used to account for a pattern of cyclic dominance of either bacteriocin sensitive, bacteriocin immune or bacteriocin producing variants of a strain in a particular habitat. The model, first advanced in relationship to colicin-producing E. coli has been likened to the childhood rock-scissor-paper game, such that bacteriocin producers win (increase in numbers) at the expense of bacteriocin-sensitive bacteria.35,36 In a predominantly bacteriocin-producing population, variants will flourish that have dispensed (by mutation) with the metabolic cost of inhibitor production whilst retaining the protective shield of specific immunity. Similarly, in the colicin example, it was shown that in a population of bacteriocin immune (but nonbacteriocin producing) cells the conditions will favour proliferation of mutants that are defective in immunity expression. And so a changing cycle of predominant variants continues to operate within the population. Further study is required to determine how well this model applies to the population dynamics of bacteriocinogenic S. salivarius and cohabiting streptococcal species within the human oral microflora.

B. In the Pharynx

7. Although principally located on the tongue, S. salivarius is also detected in substantial numbers within the pharyngeal microbiota.25,37 Here, bacteriocin-producing strains may directly interact with and interfere with the proliferation of S. pyogenes. 8. S. salivarius bacteriocins released into the saliva from other oral sites can inhibit target species such as S. pyogenes, providing these bacteriocins accumulate to effective levels within the pharyngeal biofilm. 9. S. pyogenes strains typically harbour at least partial loci for one or more bacteriocins of the lantibiotic class (salivaricin A,38 SA-FF2239 or streptin40—but, apparently not salivaricin B). Although relatively few S. pyogenes produce lantibiotic peptides, most appear to have retained the ability to express immunity (insensitivity) to various of these lantibiotics. The action of salivaricin A against S. pyogenes strains expressing salivaricin A immunity is bacteriostatic rather than bacteriocidal. It is possible that the presence of salivaricin A in pharyngeal biofilms encourages relatively slow proliferation of S. pyogenes, such as when they are present in the carriage state. 10. It has recently been shown that expression by S. pyogenes of salivaricin A immunity can confer protection against the killing action of defensins (cationic molecules similar to lantibiotics produced by phagocytes).41 Hence it is speculated that the expression of lantibiotic immunity determinants may be important for intracellular persistence (carriage) of S. pyogenes within the pharyngeal epithelium. 11. In other studies it has been shown that ingestion of preparations of S. salivarius K12 can be followed by elevation of salivary levels of gamma interferon, potentially providing heightened short term protection to the host against viral infection.42 Teichoic acid-containing wall fragments present in the freeze-dried S. salivarius K12 preparations may have a role in eliciting this response.

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C. On the Tooth Surface (in Dental Plaque)

12. Some S. salivarius produce extracellular dextran, a relatively insoluble polymer similar to that formed by S. mutans, that enables them to adhere to and persist within dental plaque.43-45 Plaque-associated S. salivarius producing anti-S. mutans BLIS could help control the level of S. mutans. 13. S. salivarius may release into the saliva various extracellular products which (following permeation into the plaque matrix) are capable of impacting upon S. mutans numbers or metabolism. These include dextranase (potentially weakening the plaque matrix and increasing access of inhibitory agents to embedded S. mutans),46 urease (releases ammonia from urea and neutralizes acid)47,48 and BLIS inhibitory to S. mutans.49

Specific Examples of the Application of Potential Bacterial Pathogens in Replacement Therapy Staph Aureus Infection and Carriage

Serious Staph. aureus infection of neonates in the 1950s-1960s, combined with increased occurrence of penicillin resistance led to implementation of preemptive colonization of the nasal mucosa and umbilicus of neonates with the putative low virulence strain 502A Staph. aureus to prevent infection by more virulent phage type 80/81 strains.50 Although effective at precluding infection by the epidemic strains of Staph. aureus a variety of infections with the colonizing strain were reported51 and the death of a neonate from meningitis due to strain 502A52 ultimately led to abandonment of the approach. Never was the actual mechanism of the observed interference established. Elimination of Staph. aureus nasal carriage continues to be identified as an important goal to reduce nosocomial infections and bacterial interference remains a consideration for dealing with this condition.53 In one recent study human subjects were inoculated intranasally with a mixture of Staph. aureus (including strain 502A) and subsequently tested for persistence of carriage by either their own or the newly-introduced strains.54 It was concluded that the host-parasite relationship in Staph. aureus carriage has considerable specificity. Not all subjects became carriers and the predominant colonizing strain(s) varied widely from person to person. As an alternative approach, commensal nasal isolates of nonstaphylococcal species have been evaluated for their in situ competitiveness. Seeding the nares with corynebacterium strain Co304 eliminated Staph aureus from ca. 70% of carriers after 15 days treatment.55 No anti-staphylococcal bacteriocin-like inhibitory activity appeared to be produced in vitro by the corynebacterium.

Dental Caries Due to S. mutans

On the basis of epidemiological observations and laboratory-based research the species S. mutans is generally considered the most influential of the purported etiological agents of dental caries in humans. A key cariogenic attribute of S. mutans is its production of large quantities of lactic acid from dietary carbohydrate. Interestingly, S. mutans seems insensitive to most of the bacteriocins produced by other species of oral streptococci when tested in vitro.56 Nevertheless, many S. mutans produce bacteriocins (called mutacins) that have strong activity against other S. mutans.57 Based on the premise that bacteriocin producers are likely to have an advantage over non producers in niche competition, Jeffrey Hillman selected S. mutans JH1001, which displays particularly potent mutacin activity against other tested S. mutans, for further development as a candidate strain for replacement therapy.58 When several mutants of this strain (e.g., JH1005 and JH1140) producing increased mutacin activity were tested in human subjects it was found that colonization efficacy and persistence correlated directly with the level of mutacin activity.23 The strong inhibitory activity of strain JH1140 has been attributed to its production of the epidermin-like lantibiotic mutacin 1140.59,60 It seems important to obtain some further data relating to the occurrence in S. mutans of strains producing either mutacin 1140 or the closely-similar mutacin B-Ny266 61,62 since such strains (being relatively immune to mutacin 1140) may not readily be supplanted by an introduced interfering strain solely producing mutacin 1140. Interestingly

9

Application of Bacterial Pathogens in Replacement Therapy

Table 2. Replacement therapy using bacteria of varying degrees of potential pathogenicity Target Pathogen/Disease

Effector Strain(s)

Reference(s)

Staph. aureus

Staph. aureus 502A Corynebacterium sp. strain Co304

50 55

S. mutans

S. mutans BCS3-L1 S. salivarius TOVE R

15 43,44

Upper respiratory tract pathogens

viridans streptococcus 215 Three S. sanguis and one S. mitis

9 69

S. pyogenes

S. salivarius K12

14

Otitis media

Two S. sanguis, two S. mitis and one S. oralis

73

Urinary tract infections

E. coli 83972

74

Vaginal infections

L. casei ss. rhamnosus GR-1 L. fermentum B54

76 76

however, it appears that strain JH1140 also harbors genetic loci encoding mutacins IV, K8 and Smb—a bacteriocin repertoire unsurpassed to date in this species.63 Having identified a particularly competitive S. mutans, Hillman’s strategy was to then obtain a genetically-crippled variant of this strain having reduced acid-producing capability. The lactate dehydrogenase gene was replaced with the alcohol dehydrogenase (ADH) B gene from Zymomonas mobilis. The resulting effector strain (BCS3-L1) has no lactate dehydrogenase activity and its ADH levels are around 10-fold higher than those of the parent.15 Growth on a variety of sugars and polyols resulted in substantially higher pH values. Studies in rodents indicated the engineered effector strain was genetically stable and that it produced no deleterious side effects due to prolonged colonization.24 It was significantly less cariogenic than wild type S. mutans in gnotobiotic rats64 and the caries levels in conventional rats colonized with this strain were similar to those in S. mutans-free animals. In both groups the caries levels were significantly lower than in animals infected with wild type S. mutans. Clearly it is important that a mutant S. mutans effector strain should be genetically stable prior to application in human clinical trials. Since bacteriophage and conjugation-mediated gene transfer have not been previously reported in S. mutans they were considered unlikely to contribute to in situ re-acquisition of the lactate dehydrogenase gene by strain BCS3-L1. For use in human clinical trials, additional mutations were introduced to enable rapid elimination of the strain in case of adverse side effects and to increase its genetic stability.65 Strain A2JM contains a mutation in the gene (dal) for alanine racemase, which makes the strain dependent on exogenous D-alanine. This is required to be provided as a dietary supplement for participants in colonization trials. Also, a mutation in comE (required for uptake of environmental DNA) renders the strain less capable of being transformed. Interestingly, colonization studies with these mutacin 1140-producing strains appear to have resulted in highly specific exclusion of other mutans streptococci—a finding that seemingly contrasts with the broad activity spectrum of the producer strains when tested in vitro. This indicates that bacteriocin producers do not necessarily eliminate all sensitive co-inhabitants in complex ecosystems such as dental plaque. Presumably the distinct micro-habitats present within biofilms and the differing physiological growth states of the inhabitants enable the individual members of heterogeneous populations to co-exist more readily than could be predicted from the outcome of strain-strain confrontations in more homogeneous environments such as laboratory culture media.

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An alternative candidate explored for potential bacterial interference control of mutans streptococcus populations was S. salivarius strain TOVE-R. This rough colony-forming (dental plaque-localising) strain was reported to successfully compete with S. mutans 10449 and S. sobrinus 6715-13 on the teeth of rats.43,44 No bacteriocin agent that could account for the observed competition could be detected.45

Upper Respiratory Tract Infection

Studies of the normal oropharyngeal microflora of neonates indicated that when alpha haemolytic streptococci were predominant there were fewer generalized infections.66 Implantation of an alpha haemolytic streptococcus (strain 215) in neonates having an “abnormal” microbiota (where alpha streptococci did not comprise >90% total count) protected them from subsequent infections.9 The effector strain was selected by screening the oral microfloras of healthy neonates for a strain able to inhibit the growth of Staph. aureus, enterobacteria and Pseudomonas sp.—potential pathogens commonly found to colonize the pharynx of neonates. The pharynx is the principal habitat of S. pyogenes. Laboratory studies indicated that some viridans streptococci produce compounds that can inhibit or kill S. pyogenes and it has been inferred that they may be important in preventing colonization and growth of S. pyogenes in susceptible hosts. In prospective studies Crowe and associates67 showed that children subsequently acquiring S. pyogenes in their throats had lower proportions of inhibitory viridans streptococci than those who did not become colonized. Twice as many adults harbour inhibitory viridans streptococci,68 perhaps contributing to the relative resistance of adults to streptococcal infection. In a group of patients susceptible to recurrent strep tonsillitis, spraying the throat with inhibitory alpha streptococci for 10 days after antibiotic therapy significantly reduced the risk of recurrent infection. The effector strains had been isolated from the throats of healthy individuals and comprised three S. sanguis and one S. mitis, all of which displayed strong inhibitory activity against S. pyogenes.69 In a larger follow-up study the same four strains were sprayed twice daily for 10 days after antibiotic therapy and 24% of 189 test subjects became reinfected with S. pyogenes compared to 42% of 93 receiving placebo.70 Subsequent studies have shown that children having S. salivarius producing inhibitory activity attributable to the lantibiotic salivaricin A are less likely to acquire S. pyogenes than children harboring nonlantibiotic-producing S. salivarius.71 This led to the hypothesis that the presence of certain bacteriocin-producing S. salivarius in the oral cavity could provide protection against S. pyogenes. S. salivarius strain K12 which produces two anti-S. pyogenes lantibiotics is now distributed as a colonizing strain in the oral probiotic preparation BLIS K12 Throat Guard (www.blis.co.nz).

Prevention of Otitis Media

Acute otitis media (AOM) is perhaps the most common bacterial disease of young children and is caused by certain members of the indigenous nasopharyngeal microbiota, especially Streptococcus pneumoniae, nontypable Haemophilus influenzae and Moraxella catharralis. Infection recurrences are common. Strains of alpha streptococci produce substances that kill or inhibit the growth of these pathogens in vitro and are also thought to play a role in controlling their numbers in situ. Children having recurrent AOM have lower proportions of alpha streptococci in their nasopharyngeal microflora and the strains present have less activity against AOM pathogens.72 A clinical trial was conducted by Roos and associates (2001)73 involving 108 children under the age of 6 given a suspension of alpha streptococci in skimmed milk (or control) twice daily for 10 days after a 10-day antibiotic course. This was repeated after one month. The mixture contained two S. sanguis, two S. mitis and one S. oralis. The proportion of the treated children who experienced AOM recurrences during the next three months was 22%, while the control (untreated) group had a recurrence rate of 42%. Interestingly in a similar study with the same organisms, but no prior use of antibiotics, no differences were observed between test and control in the number of AOM episodes or in the prevalence of AOM pathogens in the nasopharynx. This shows that although the effectors are able to prevent colonization by AOM pathogens they are unable to displace the pathogens once these are established in the nasopharynx.

Application of Bacterial Pathogens in Replacement Therapy

11

Prevention of Urinary Tract Infection

The clinical observation that asymptomatic bacteruria may provide some protection against overt urinary tract infection (UTI) raises the prospect of implantation of avirulent E. coli as a means of prophylaxis against UTI in those predisposed to such infections. E. coli 83972 has been used to colonize the urinary tract of volunteers for several months with no sign of fever or systemic infection.74 There are no reports of deterioration of bladder or kidney function associated with colonization. The authors conclude that the approach has good potential for patients vulnerable to UTI.75

Prevention of Vaginal Infection

The vagina is often infected by faecal bacteria such as E. coli and can act as a reservoir for these bacteria which are subsequently able to initiate bladder infections. Some lactobacilli appear specially capable of protecting the vagina against colonization by uropathogens such as E. coli by (1) preventing their adhesion to the epithelium (2) producing surfactants which displace the uropathogens (3) inhibiting their growth or killing them with hydrogen peroxide, bacteriocins or fatty acids. One such strain is L. rhamonsus GR-1 which has been used in trials to prevent UTI because of its in vitro ability to inhibit the growth of pathogens and to interfere with their attachment. In one study 55 women were given GR-1 and also L. fermentum RC-14 weekly for a year. This reduced their UTI rate from 6 a year to 1.6 during the trial year.76

Final Thoughts

Replacement therapy is theoretically applicable to the control of any bacterial infection of a host surface. It provides the potential for a single application to effect long-term protection with minimal compliance, cost or education for the subject. Since the most effective bacterial niche competitors are typically bacteria closely-related to the target pathogens it is not surprising that most of the bacteria developed for use in replacement therapy strategies will also have at least a modicum of pathogenic potential.

References

1. Bibel DJ. Bacterial interference, bacteriotherapy and bacterioprophylaxis. In: Aly R, Shinefield HR, eds. Bacterial Interference. Boca Raton: CRC Press Inc., 1982:1-12. 2. Wilson M. Microbial Inhabitants of Humans. Cambridge: Cambridge University Press, 2005. 3. Pasteur L, Joubert JF. Charbon et septicémie. C R Soc Biol Paris 1877; 85:101-115. 4. Florey HW. The use of micro-organisms for therapeutic purposes. Yale J Biol Med 1946; 19:101-117. 5. Nissle A. Weiteres uber Grundlagen und Praxis der Mutaflorbehandlung. Dtsch Med Wocenschr 1925(44):1809-1813. 6. Patzer SI, Baquero MR, Bravo D et al. The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN. Microbiology 2003; 149(Pt 9):2557-2570. 7. Shinefield HR, Wilsey JD, Ribble JC et al. Interactions of staphylococcal colonization. Influence of normal nasal flora and antimicrobials on inoculated Staphylococcus aureus strain 502A. Am J Dis Child 1966; 111(1):11-21. 8. Aly R, Maibach HI, Shinefield HR et al. Bacterial interference among strains of Staphylococcus aureus in man. J Infect Dis 1974; 129(6):720-724. 9. Sprunt K, Leidy G, Redman W. Abnormal colonization of neonates in an ICU: Conversion to normal colonization by pharyngeal implantation of alpha hemolytic streptococcus strain 215. Pediatr Res 1980; 14:308-313. 10. Metchnikoff E. The Prolongation of Life: Optimistic Studies. New York: Putnam, 1908. 11. Huovinen P. Bacteriotherapy: The time has come. BMJ 2001; 323(7309):353-354. 12. FAO/WHO. Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria. Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria 2001. 13. Burton J, Chilcott C, Tagg J. The rationale and potential for the reduction of oral malodour using Streptococcus salivarius probiotics. Oral Dis 2005; 11(s1):29-31.

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14. Tagg JR. Prevention of streptococcal pharyngitis by anti-Streptococcus pyogenes bacteriocin-like inhibitory substances (BLIS) produced by Streptococcus salivarius. Indian J Med Res 2004; 119 Suppl:13-16. 15. Hillman JD. Genetically modified Streptococcus mutans for the prevention of dental caries. Antonie Van Leeuwenhoek 2002; 82(1-4):361-366. 16. Sleator RD, Hill C. Patho-biotechnology; Using bad bugs to make good bugs better. Sci Prog 2007; 90(Pt 1):1-14. 17. Sleator RD, Hill C. ‘Bioengineered Bugs’—A patho-biotechnology approach to probiotic research and applications. Med Hypotheses 2008;70(1):167-9. 18. Brook I. Bacterial interference. Crit Rev Microbiol 1999; 25(3):155-172. 19. Tagg JR, Dierksen KP. Bacterial replacement therapy: Adapting ‘germ warfare’ to infection prevention. Trends Biotechnol 2003; 21(5):217-223. 20. Corthesy B, Gaskins HR, Mercenier A. Cross-talk between probiotic bacteria and the host immune system. J Nutr 2007; 137(3 Suppl 2):781S-790S. 21. Hull MW, Chow AW. Indigenous microflora and innate immunity of the head and neck. Infect Dis Clin North Am 2007; 21(2):265-282, v. 22. Hyink O, Wescombe PA, Upton M et al. Salivaricin A2 and the novel lantibiotic salivaricin B are encoded at adjacent loci on a 190-kilobase transmissible megaplasmid in the oral probiotic strain Streptococcus salivarius K12. Appl Environ Microbiol 2007; 73(4):1107-1113. 23. Hillman JD, Dzuback AL, Andrews SW. Colonization of the human oral cavity by a Streptococcus mutans mutant producing increased bacteriocin. J Dent Res 1987; 66(6):1092-1094. 24. Hillman JD, Brooks TA, Michalek SM et al. Construction and characterization of an effector strain of Streptococcus mutans for replacement therapy of dental caries. Infect Immun 2000; 68(2):543-549. 25. Sklavounou A, Germaine GR. Adherence of oral streptococci to keratinized and nonkeratinized human oral epithelial cells. Infect Immun 1980; 27(2):686-689. 26. Burton JP, Wescombe PA, Moore CJ et al. Safety assessment of the oral cavity probiotic Streptococcus salivarius K12. Appl Environ Microbiol 2006; 72(4):3050-3053. 27. Heng NCK, Wescombe PA, Burton JP et al. The diversity of bacteriocins produced by gram-positive bacteria. In: Riley MA, Chavan MA, eds. Bacteriocins—Ecology and Evolution. Berlin, Heidelburg: Springer-verlag, 2007:45-92. 28. Wescombe PA, Heng NCK, Jack RW et al. Bacteriocins associated with cytotoxicity for eukaryotic cells. In: Proft T, ed. Microbial Toxins, A Critical Review. Wymondham: Horizon Press, 2005:399-448. 29. Liljemark WF, Gibbons RJ. Suppression of Candida albicans by human oral streptococci in gnotobiotic mice. Infect Immun 1973; 8(5):846-849. 30. Kazor CE, Mitchell PM, Lee AM et al. Diversity of bacterial populations on the tongue dorsa of patients with halitosis and healthy patients. J Clin Microbiol 2003; 41(2):558-563. 31. Dempster RP, Tagg JR. The production of bacteriocin-like substances by the oral bacterium Streptococcus salivarius. Arch Oral Biol 1982; 27(2):151-157. 32. Tompkins GR, Tagg JR. The ecology of bacteriocin-producing strains of Streptococcus salivarius. Microb Ecol Health Dis 1989; 2:19-28. 33. Wescombe PA, Burton JP, Cadieux PA et al. Megaplasmids encode differing combinations of lantibiotics in Streptococcus salivarius. Antonie Van Leeuwenhoek 2006; 90(3):269-280. 34. Tagg JR, Wescombe PA, Burton JP. Oral streptococcal BLIS: Heterogeneity of the effector molecules and potential role in the prevention of streptococcal infections. International Congress Series 2006; 1289:347-350. 35. Czaran TL, Hoekstra RF, Pagie L. Chemical warfare between microbes promotes biodiversity. Proc Natl Acad Sci USA 2002; 99(2):786-790. 36. Riley MA, Gordon DM. The ecological role of bacteriocins in bacterial competition. Trends Microbiol 1999; 7(3):129-133. 37. Carlsson J, Grahnen H, Jonsson G et al. Early establishment of Streptococcus salivarius in the mouths of infants. J Dent Res 1970; 49:415-418. 38. Simpson WJ, Ragland NL, Ronson CW et al. A lantibiotic gene family widely distributed in Streptococcus salivarius and Streptococcus pyogenes. Dev Biol Stand 1995; 85:639-643. 39. McLaughlin RE, Ferretti JJ, Hynes WL. Nucleotide sequence of the streptococcin A-FF22 lantibiotic regulon: model for production of the lantibiotic SA-FF22 by strains of Streptococcus pyogenes. FEMS Microbiol. Lett 1999; 175(2):171-177. 40. Wescombe PA, Tagg JR. Purification and characterization of streptin, a type A1 lantibiotic produced by Streptococcus pyogenes. Appl Environ Microbiol 2003; 69(5):2737-2747. 41. Phelps HA, Neely MN. SalY of the Streptococcus pyogenes lantibiotic locus is required for full virulence and intracellular survival in macrophages. Infect Immun 2007; 75(9):4541-4551.

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42. Chilcott CN, Crowley L, Kulkarni V et al. Elevated levels of interferon gamma in human saliva following ingestion of Streptococcus salivarius K12. Joint meeting of New Zealand Microbiological Society and New Zealand Biochemistry and Molecular Biology. Dunedin, NZ 2005. 43. Tanzer JM, Kurasz AB, Clive J. Inhibition of ecological emergence of mutans streptococci naturally transmitted between rats and consequent caries inhibition by Streptococcus salivarius TOVE-R infection. Infect Immun 1985; 49(1):76-83. 44. Tanzer JM, Kurasz AB, Clive J. Competitive displacement of mutans streptococci and inhibition of tooth decay by Streptococcus salivarius TOVE-R. Infect Immun 1985; 48(1):44-50. 45. Kurasz AB, Tanzer JM, Bazer L et al. In vitro studies of growth and competition between S. salivarius TOVE-R and mutans streptococci. J Dent Res 1986; 65(9):1149-1153. 46. Ohnishi Y, Kubo S, Ono Y et al. Cloning and sequencing of the gene coding for dextranase from Streptococcus salivarius. Gene 1995; 156(1):93-96. 47. Chen YY, Clancy KA, Burne RA. Streptococcus salivarius urease: Genetic and biochemical characterization and expression in a dental plaque streptococcus. Infect Immun 1996; 64(2):585-592. 48. Burne RA, Marquis RE. Alkali production by oral bacteria and protection against dental caries. FEMS Microbiol Lett 2000; 193(1):1-6. 49. Balakrishnan M, Simmonds RS, Tagg JR. Diverse activity spectra of bacteriocin-like inhibitory substances having activity against mutans streptococci. Caries Res 2001; 35(1):75-80. 50. Light IJ, Walton RL, Sutherland JM et al. Use of bacterial interference to control a staphylococcal nursery outbreak. Deliberate colonization of all infants with the 502A strain of Staphylococcus aureus. Am J Dis Child 1967; 113(3):291-300. 51. Blair EB, Tull AH. Multiple infections among newborns resulting from colonization with Staphylococcus aureus 502A. Am J Clin Pathol 1969; 52(1):42-49. 52. Houck PW, Nelson JD, Kay JL. Fatal septicemia due to Staphylococcus aureus 502A. Report of a case and review of the infectious complications of bacterial interference programs. Am J Dis Child 1972; 123(1):45-48. 53. Perl TM, Golub JE. New approaches to reduce Staphylococcus aureus nosocomial infection rates: treating S. aureus nasal carriage. Ann Pharmacother 1998; 32(1):S7-16. 54. Nouwen J, Boelens H, van Belkum A et al. Human factor in Staphylococcus aureus nasal carriage. Infect Immun 2004; 72(11):6685-6688. 55. Uehara Y, Nakama H, Agematsu K et al. Bacterial interference among nasal inhabitants: eradication of Staphylococcus aureus from nasal cavities by artificial implantation of Corynebacterium sp. J Hosp Infect 2000; 44(2):127-133. 56. James SM, Tagg JR. A search within the genera Streptococcus, Enterococcus and Lactobacillus for organisms inhibitory to mutans streptococci. Microb Ecol Health Dis 1988; 1:153-162. 57. Chikindas ML, Novák J, Caufield PW et al. Microbially-produced peptides having potential application to the prevention of dental caries. Int J Antimicrob Agents 1997(9):95-105. 58. Hillman JD, Johnson KP, Yaphe BI. Isolation of a Streptococcus mutans strain producing a novel bacteriocin. Infect Immun 1984; 44(1):141-144. 59. Hillman JD, Novak J, Sagura E et al. Genetic and biochemical analysis of mutacin 1140, a lantibiotic from Streptococcus mutans. Infect Immun 1998; 66(6):2743-2749. 60. Smith L, Novak J, Rocca J et al. Covalent structure of mutacin 1140 and a novel method for the rapid identification of lantibiotics. Eur J Biochem 2000; 267(23):6810-6816. 61. Mota-Meira M, Lacroix C, LaPointe G et al. Purification and structure of mutacin B-Ny266: A new lantibiotic produced by Streptococcus mutans. FEBS Lett 1997; 410(2-3):275-279. 62. Bekal-Si Ali S, Hurtubise Y, Lavoie MC et al. Diversity of Streptococcus mutans bacteriocins as confirmed by DNA analysis using specific molecular probes. Gene 2002; 283(1-2):125-131. 63. Robson CL, Wescombe PA, Klesse NA et al. Isolation and partial characterization of the Streptococcus mutans type AII lantibiotic mutacin K8. Microbiology 2007; 153(Pt 5):1631-1641. 64. Johnson CP, Gross SM, Hillman JD. Cariogenic potential in vitro in man and in vivo in the rat of lactate dehydrogenase mutants of Streptococcus mutans. Arch Oral Biol 1980; 25(11-12):707-713. 65. Hillman JD, Mo J, McDonell E et al. Modification of an effector strain for replacement therapy of dental caries to enable clinical safety trials. J Appl Microbiol 2007; 102(5):1209-1219. 66. Sprunt K, Leidy G. The use of bacterial interference to prevent infection. Can J Microbiol 1988; 34(3):332-338. 67. Crowe CC, Sanders WE, Longley S. Bacterial interference. II. Role of the normal throat flora in prevention of colonization by group A Streptococcus. J Infect Dis 1973; 128:527-532. 68. Sanders CC, Nelson GE, Sanders WE. Bacterial interference. IV. Epidemiological determinants of the antagonistic activity of the normal throat flora against group A Streptococci. Infect Immun 1977; 16:599-603.

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69. Roos K, Grahn E, Holm SE et al. Interfering alpha-streptococci as a protection against recurrent streptococcal tonsillitis in children. Int J Pediatr Otorhinolaryngol 1993; 25(1-3):141-148. 70. Falck G, Grahn-Hakansson E, Holm SE et al. Tolerance and efficacy of interfering alpha-streptococci in recurrence of streptococcal pharyngotonsillitis: A placebo-controlled study. Acta Otolaryngol 1999; 119(8):944-948. 71. Dierksen KP, Tagg JR. The influence of indigenous bacteriocin producing Streptococcus salivarius on the acquisition of Streptococcus pyogenes by primary school children in Dunedin, New Zealand. In: Martin DR, Tagg JR, eds. Streptococci and Streptococcal Diseases: Entering the New Millenium. Wellington: Securacopy, 2000:345-348. 72. Brook I. The role of bacterial interference in otitis, sinusitis and tonsillitis. Otolaryngol Head Neck Surg 2005; 133(1):139-146. 73. Roos K, Hakansson EG, Holm S. Effect of recolonisation with “interfering” alpha streptococci on recurrences of acute and secretory otitis media in children: Randomised placebo controlled trial. BMJ 2001; 322(7280):210-212. 74. Trautner BW, Hull RA, Darouiche RO. Escherichia coli 83972 inhibits catheter adherence by a broad spectrum of uropathogens. Urology 2003; 61(5):1059-1062. 75. Trautner BW, Hull RA, Darouiche RO. Prevention of catheter-associated urinary tract infection. Curr Opin Infect Dis 2005; 18(1):37-41. 76. Reid G, Charbonneau D, Erb J et al. Oral use of Lactobacillus rhamnosus GR-1 and L. fermentum RC-14 significantly alters vaginal flora: randomized, placebo-controlled trial in 64 healthy women. FEMS Immunol Med Microbiol 2003; 35(2):131-134.

Chapter 2

Improvement of Insect Pathogens as Insecticides through Genetic Engineering Brian A. Federici,* Bryony C. Bonning and Raymond J. St Leger

Abstract

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iruses and microorganisms that cause disease in insects have been under evaluation as insecticides for more than a century. Only Bacillus thuringiensis (Bt) has been a commercial success and even so it still represents less than 1% of insecticide usage worldwide. The development of recombinant DNA techniques, however, has made it possible to significantly improve the insecticidal efficacy of viruses, bacteria and fungi that attack insects. Here we review the recent literature on advances made with model viral, bacterial and fungal pathogens of insects, showing how the efficacy of wild type pathogens can be improved ten-fold or more using genetic engineering techniques. These advances have been achieved by combining new knowledge derived from basic studies of the molecular biology and genomics of these pathogens with technical developments that enable increased gene expression and the use of genes from other organisms that encode highly specific enzymes and insecticidal proteins to improve efficacy. With increasing public concern over the continued use of synthetic chemical insecticides, these new types of biological insecticides offer a range of environmentally compatible options for cost-effective control of insect pests and vectors of disease that continue to plague agriculture and human health.

Introduction

Like all organisms, insects are hosts to many viruses and microorganisms that cause disease and mortality in insect populations. In fact, viruses and fungi often cause disease outbreaks, referred to as epizootics, that reduce insect populations by greater than 90%. These typically occur in insects such as caterpillars that attack field and vegetable crops, forests and even in pests of turf grasses. In other cases, fungi, especially under conditions of high moisture, devastate populations of aphids (known commonly as plant lice), beetle pests of vegetables and fruit crops and even such insects as house flies. Bacteria only rarely cause epizootics, but several types are known to be highly pathogenic to insects, acting initially through insecticidal proteins. Owing to these properties, for well over a century entomologists and microbiologists have had an interest in using these pathogens as biological control agents and insecticides to control pest insect and vector populations, in the latter case meaning populations, for example, of aphids that transmit viral diseases to plants and mosquitoes that vector the causative agents of malaria and filariasis. At present, less than 1% of the insecticides used worldwide for pest and vector control are based on insect pathogens. Those used most widely are different subspecies of the bacterium, Bacillus thuringiensis (Bt), which constitute approximately 80% of the pathogens used as insecticides. Of the remaining 20%, a few baculoviruses have been successful as classical biological control agents, *Corresponding Author: Brian A. Federici—Department of Entomology, University of California, Riverside, California 92521, USA. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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or have achieved moderate success as viral insecticides in several developing countries. Fungi have been less successful and essentially have only been used in niche markets in a few countries, or where control programs are subsidized by governments or international agencies. Examples of the latter include the use of fungi for locust control in the Middle East and Africa and baculoviruses for control of forest pests in the United States and Canada. The advent of recombinant DNA techniques—in essence, genetic engineering—has provided a myriad of opportunities to enhance the efficacy and thus cost-effectiveness of the above pathogens as insect control agents. Insect tolerant crops such as Bt cotton and Bt corn based on the insecticidal proteins of B. thuringiensis, now a multibillion Euro industry, are the best example to date. But molecular biology and the various techniques associated with this field of science have also provided tools that are being used to generate new types of viruses, bacteria and fungi that can be used for pest and vector control. In this chapter, we review some of the most promising developments in this new and exciting field.

Genetic Engineering to Optimize Baculovirus Insecticides

Baculoviruses are large, occluded, double-stranded DNA viruses of insects, isolated primarily from the Lepidoptera1 that have been the subject of study for a variety of applications.2 For pest management purposes, baculoviruses have a number of beneficial attributes such as specificity and environmental safety and can be applied without risk of impacting nontarget organisms, a key detriment to the current use of classical chemical insecticides. One of the most restrictive limitations for the use of baculovirus insecticides in maintaining insect populations below the economic injury threshold however is the time taken for a given virus to kill the host insect. It can take anywhere from a few days to several weeks for an insect to succumb to baculovirus infection, according to the host-virus combination. Over the past two decades, this limitation has been successfully addressed by genetic engineering by using one or more of three different strategies: (i) deletion of a baculovirus gene thereby reducing the time taken by the baculovirus to kill the host insect, (ii) insertion of a gene encoding a toxin, hormone or enzyme such that expression of the transgene by the baculovirus would result in death of the host insect, (iii) insertion of a gut-active toxin into the occlusion body of the baculovirus on the basis that toxin action in the gut would synergize baculovirus infectivity. Some of the recombinant baculovirus insecticides that have been developed are competitive with classical chemical insecticides in suppression of pest insect populations below the economic threshold. In addition to enhancing the speed of action (i.e., reducing the lethal time, LT50) of baculovirus insecticides, genetic engineering has also be used to increase the virulence (i.e., reduce the lethal dose LD50 of the virus) and to modify the host range.3 Although a number of baculovirus genes are known to function as host range determinants, there is insufficient understanding for production of a “designer baculovirus” for use against a particular set of pests in a given cropping system. Here we provide a synopsis of the genetic optimiziation of baculovirus insecticides. The reader is referred to more comprehensive reviews on the biology, use, development and ecology of wild type and genetically enhanced baculovirus insecticides for more detailed information.4-10

Biology of Baculoviruses

Rather unusually, baculoviruses (family Baculoviridae) have two phenotypes. Baculoviruses are found in the field in the form of occlusion bodies (OB; Fig. 1) referred to as polyhedra and granules for the genera Nucleopolyhedrovirus (NPV) and Granulovirus (GV) respectively.9 Within each occlusion body is one or more occlusion-derived viruses (ODV), which are enveloped virions containing one or more rod-shaped nucleocapsids (Fig. 1). On ingestion by a suitable host insect, the polyhedrin or granulin matrix of the OB dissolves in the alkaline conditions of the gut, thereby releasing the ODV. The ODV penetrate the peritrophic membrane that lines the gut and infect the midgut epithelial cells. Initial rounds of replication in an infected cell result in production of the second baculovirus phenotype, budded virus (BV). BVs consist of singly enveloped nucleocapsids that leave the cell to disseminate virus within the host insect (Fig. 1). A switch occurs late in the infection cycle from production of BV, to retention of nucleocapsids within the nucleus and

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Figure 1. Transmission electron micrograph of a midgut cell of a larva of Heliothis virescens infected with AcMNPV. Polyhedra (P), occlusion-derived virus (ODV) and budded virus (BV) between the basement membrane and the plasma membrane are shown. NM, nuclear membrane; pep, polyhedral envelope protein. Scale bar = 1 μm. TEM courtesy of Hailin Tang, Iowa State University.

production of ODV which are subsequently embedded within OB. Eventually the host insect is overwhelmed by the infection and OB are released into the environment following disruption of the fragile cuticle of the host insect.

Genetic Engineering to Optimize Speed of Kill Gene Deletion

The baculovirus gene egt encodes ecdysteroid UDP-glucosyl transferase (EGT), which catalyses the conjugation of sugar molecules to ecdysteroids thereby rendering the ecdysteroids inactive.11,12 When baculovirus expressed EGT accumulates to a critical level, the inactivation of ecdysteroids blocks molting of the host insect, thereby prolonging the actively feeding larval stage and conserving resources for use by the virus. egt has been found in the majority of baculoviruses characterized to date and is classified as an auxiliary gene, i.e., a gene that is not essential for baculovirus replication but provides some competitive advantage.13 Deletion of egt from the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome resulted in more rapid death and an approximately 40% reduction in feeding damage caused by infected larvae of Trichoplusia ni and Spodoptera frugiperda compared to those infected with wild type AcMNPV.14 The same approach also reduced the survival time of larvae infected with Lymantria dispar MNPV and Helicoverpa

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armigera NPV.15,16 The more rapid death of the host larvae was associated with a reduction in the yield of progeny virus. However, the impact of egt deletion on survival time was not consistent between hosts and varied according to the stage used for bioassay.17-20 Deletion of egt is commonly combined with insertion of a gene encoding an insecticidal agent (Fig. 2).

Gene Insertion

The most commonly used approach for genetic optimization of the speed of kill of baculovirus insecticides is insertion of a gene encoding a toxin, hormone or enzyme, into the baculovirus genome. On replication of the virus within the host insect, the toxic agent is produced and the insect succumbs to the effect of that agent. Several recombinant baculoviruses have been constructed for overexpression of the host insect’s own hormones or enzymes such as diuretic hormone,21 eclosion hormone,22 prothoracicotrophic hormone23 and juvenile hormone esterase24. The impact of these transgenes in reducing the survival time of the host ranged from no impact to moderate impact (Fig. 2). For these genes in particular, the lack of effect may result from the insect’s ability to regulate

Figure 2. Improving the speed of kill of baculovirus insecticides by genetic engineering. The genetic modification and percent improvement in speed of kill (or paralysis) relative to the wild type or control virus are shown for each study. Abbreviations and references: WT, wild type; BeIT, insectotoxin-1;49 BT, Bacillus thuringiensis endotoxin;41 PTTH, prothoracicotrophic hormone;23 EH, eclosion hormone;50 JHE, juvenile hormone esterase;24 enhancin, MacoNPV enhancin;51 DTX9.2, spider toxin;52 gelatinase, human gelatinase A;35 ORF603D, deletion of AcMNPV orf603;53 chitinase;54 DH, diuretic hormone;21 c-MYC transcription factor;55 TalTX-1, spider toxin;52 PBAN, pheromone biosynthesis activating neuropeptide;56 JHE-KK, stabilized JHE;57 AaIT, scorpion-derived insect toxin;25,27 egtD+AaIT, insertion of aait at the egt locus;15 egtD, deletion of ecdysteroid UDP-glucosyltransferase gene (egt);14 LqhIT1/LqhIT2, simultaneous expression of scorpion-derived toxins LqhIT1 and LqhIT2;34 LqhIT2, insect toxin 2 derived from Leiurus quinquestriatus;58 URF13, maize pore-forming protein;59 egt+tox34, insertion of tox34 under the early DA26 promoter at the egt locus;19 cv-PDG, glycosylase;48 cathepsin L;35 As II, sea anemone toxin;33 Sh I, sea anemone toxin;33 μ-Aga-IV, spider toxin;33 LqhIT2(ie1), LqhIT2 under the early ie1 promoter;28 AaIT + pyrethroid, co-application of pyrethroid with low dose of AcMNPV expressing AaIT;60 tox34.4, mite toxin;49,61 Pol+BT, expression of authentic polyhedrin with polyhedrin-BT-GFP fusion proteins (see text).43 Reprinted with permission from ref. 4.

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such factors; misexpression of such hormones or enzymes would be detrimental to survival of the insect and hence an efficient system for appropriate regulation is essential for insect survival. A wide range of genes encoding insect-specific toxins isolated from various venomous creatures such as scorpions, spiders, parasitic wasps and sea anemones have been inserted into baculovirus genomes. These toxins act on the major ion channels such as the Na+, K+, Ca2+ and Cl− channels and generally result in rapid paralysis (Fig. 2). The excitatory toxin AaIT derived from the North African scorpion Androctonus australis and the excitatory and depressant toxins from the yellow Israeli scorpions of the genus Leiurus have received particular attention. Field testing of recombinant baculoviruses expressing these toxins having been conducted in US, China and UK and some were competitive with pyrethoid insecticides.5,11,18,25-32 Combinations of toxins have also been tested and in some instances were shown to synergize for improved toxic effect.33,34 Among the fastest of the recombinant baculovirus insecticides is one that expresses a cathepsin-L like protease that targets the basement membrane.35 This virus was constructed to determine whether disruption of the basement membrane barrier to virus dissemination within the host insect, would enhance the speed of kill by the virus. However, rather than facilitating virus dissemination, baculovirus expression of this protease caused sufficient damage to basement membrane and the underlying tissues to result in more rapid death of the host insect.36,37 One of the common factors associated with genetic optimization for increased speed of kill, is that the faster the virus kills the host insect, the fewer OB are produced (Fig. 3). Hence, large scale production of these recombinant baculoviruses in vivo becomes a challenge. An elegant solution has been developed that uses a tetracycline-induced suppressor for suppression of toxin expression when laboratory reared insects are fed on tetracycline-containing artificial diet.38

Baculovirus for Bt Toxin Delivery

Toxins derived from the bacterium Bacillus thuringiensis act on the midgut epithelium of the insect.39 Hence, baculovirus delivery of Bt toxins within the insect does not enhance insecticidal efficacy.40-42 In an alternative approach, the Bt toxin Cry1Ac was produced as a polyhedrin-Cry1Ac-GFP

Figure 3. Baculovirus insecticides that kill quickly yield few polyhedra. Data are shown for the yield of polyhedra at death following infection with wild type virus AcMNPV C6 compared to viruses expressing the scorpion-derived insect-specific neurotoxin AaIT (AcMLF9.AaIT), or the basement membrane-degrading protease, ScathL (AcMLF9.ScathL).

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fusion protein with trypsin sensitive linkers by the recombinant virus ColorBtrus. This virus expressed the fusion protein as well as authentic polyhedrin to produce polyhedra that occluded GFP and Bt toxin, which were released on exposure to trypsin in the insect gut. Bioassays in Plutella xylostella showed that the LD50 of ColorBtrus was 100-fold lower than that of wild type virus and the ST50 was reduced by 60% when compared to the wild type AcMNPV, with no significant difference between ColorBtrus and treatment with Cry1Ac alone.43 In this case, the baculovirus simply served as an alternative delivery system for release of Cry1Ac within the insect gut rather than contributing to the insecticidal impact of the virus. Hence the use of wild type virus for comparative purposes in these bioassays was inappropriate. However, the advantage of this dual mode of action approach lies in the efficacy of ColorBtrus against species such as Spodoptera exigua that are not sensitive to the action of Cry1Ac but are susceptible to infection by AcMNPV and conversely against species that are not susceptible to AcMNPV but are sensitive to Cry1Ac. In effect, by combining the two insecticidal agents, the range of insects that can effectively be targeted has been expanded.

Genetic Engineering for Increased Virulence

There are several examples of baculoviruses that have been genetically engineered to reduce the amount of virus required for a fatal infection of the targeted insect pest. Enhancin is a metalloprotease commonly expressed by baculoviruses that degrades insect intestinal mucin in the peritrophic membrane.44 Insertion of the enhancin gene derived from Trichoplusia ni GV enhanced AcMNPV virulence by 2 to 14-fold in various insect species.45 Conversely, deletion of two enhancin genes from Lymantria dispar MNPV reduced viral potency 12-fold compared to wild type virus.46 Deletion of the gene encoding the polyhedral envelope protein that surrounds the OB of AcMNPV (Fig. 1) resulted in a 6-fold increase in infectivity against first instar Trichoplusia ni compared to that of wild type virus.47 The OBs of this virus that lacked the polyhedral envelope protein dissolved more readily in alkali than OBs of wild type virus. Hence, the reduction in effective dose was attributed to the rapid release and increased availability of ODV in the gut for infection. Curiously, engineering of AcMNPV to express an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, in an effort to reduce the susceptibility of the virus to UV inactivation, significantly increased the virulence of the virus in some species.48 The dose required to kill S. frugiperda larvae was reduced 16-fold by expression of cv-PDG. A possible explanation for this observation is that cv-PDG is toxic to midgut cells, with the toxicity facilitating virus entry.48 Some of the best recombinant baculovirus insecticides developed induce feeding cessation within 24 hours of infection, making them competitive with classical chemical insecticides (Fig. 2).4 This degree of efficacy along with the demonstrated safety of recombinant baculoviruses combines to make them useful tools for crop protection. The actual use of genetically engineered baculoviruses in crop protection, as promising as it is, awaits governmental protocols that facilitate registration at a reasonable cost, and for some, more cost-effective mass production methods for large-scale application.

Genetic Engineering of Bacillus thuringiensis (Bt) Basic Biology of Bt

In this section, we focus our attention on the insecticidal bacterium Bacillus thuringiensis (Bt), as this species has been the most successful commercial microbial insecticide and also has been the subject of the overwhelming majority of genetic engineering studies to improve efficacy. We also introduce information on B. sphaericus (Bs), another insecticidal bacterium, as genes from this species have been used to improve the efficacy of Bt. We first briefly describe the biology of these two species. Bt is actually a complex of bacterial subspecies that occur commonly in such habitats as soil, leaf litter, on the surfaces of leaves, in insect feces and as a part of the flora in the midguts of many insect species.62,63 Bts are characterized by the production of a parasporal body during sporulation that contains one or more protein endotoxins in a crystalline form (Fig. 4). Many of these endotoxins are highly insecticidal to certain insect species. Bt endotoxins are actually protoxins

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activated by proteolytic cleavage in the insect midgut after ingestion. The activated toxins destroy midgut epithelial cells, killing susceptible insects within a day or two of ingestion. In insect species only moderately sensitive to the toxins, such as Spodoptera species (caterpillars commonly known as armyworms), the spore contributes to pathogenesis by germinating and producing vegetative insecticidal proteins (Vips), proteases and phospholipases. Bt also produces other insecticidal compounds including β-exotoxin and Zwittermicin A. Some of these are synergistic and thus their combined actions often result in death of recalcitrant lepidopteran species.

Figure 4. Sporulated cells of Bacillus thuringiensis and parasporal protein crystals. A Phase contrast micrograph of cells from a sporulated culture of B. thuringiensis just prior to lysis. Parasporal protein crystals (arrowheads) lie adjacent to oval spores. B Scanning electron micrograph of typical Cry1 and Cry2 crystals purified from a sporulated culture of B. thuringiensis subsp. kurstaki, isolate HD1. The parasporal body of this isolate consists of a bipyramidal crystal that contains Cry1Aa, Cry1Ab and Cry1Ac, which cocrystallize and a separate “cuboidal” crystal composed of Cry2Aa molecules. C Carbon replica of a typical bipyramidal Cry1 type protein crystal exhibiting the lattice of Cry1A molecules that compose the crystal. The HD73 isolate of B. thuringiensis subsp. kurstaki only expresses a single cry1Ac gene and its parasporal body contains only a single crystal, such as this one, which measures approximately 1 μm from point-to-point along the longitudinal axis. D Transmission electron micrograph through a parasporal body of the HD1 isolate of B. thuringiensis subsp. kurstaki illustrating the embedment of the cuboidal Cry2A crystal (P2) in the bipyramidal crystal (P1). Bar in D = 200 nm. Micrograph in C by C. L. Hannay.

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The most widely used Bt is the HD1 isolate of B. thuringiensis subsp. kurstaki (Btk), an isolate that produces four major endotoxin proteins, Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa packaged into two crystalline parasporal bodies. The three Cry1 proteins cocrystallize forming a bipyramidal crystal, whereas Cry2Aa forms a separate cuboidal crystal (Fig. 4). This isolate is used as the active ingredient in numerous commercially available bacterial insecticides (Dipel, Foray, Thuricide) used to control many lepidopteran pests in field and vegetable crops and forests.64 Another successful Bt is B. thuringiensis subsp. israelensis (Bti), which is highly toxic to the larvae of many mosquito and blackfly species. This isolate produces a parasporal body that contains four major endotoxins, Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa (Fig. 5). The three Cry proteins are related to those of Btk, but have insect spectra limited to mosquitoes, black flies and related dipterans species. Cyt proteins are unrelated to Cry proteins.63,64 Several commercial products based on Bti are available and are used to control both nuisance and vector mosquitoes and black flies. Formulations of Bti (VectoBac, Teknar) proved particularly important in the World Health Organization’s Onchocerciasis Control Program in West Africa, where they were used during the 1980s and 1990s in rotation with the chemical insecticide, temephos, to virtually eliminate larval populations of the black fly vector, Simulium damnosum, of the filarial worm that causes this disease. A third isolate of Bt that has been developed commercially

Figure 5. Sporulating cell of Bacillus thuringiensis subspecies israelensis and parasporal bodies characteristic of this subspecies as revealed by transmission electron microscopy. A Sporulating cell illustrating the developing spore (Sp) and parasporal body. The parasporal body (PB), composed primarily of Cry4A, Cry4B, Cry11A and Cyt1A proteins, is assembled outside the exosporium membrane (E). B Portion of sporulating cell just prior to lysis. The Cry11A crystal (*) lies adjacent to the Cyt1A and Cry4A and Cry4B inclusions. C Purified parasproal body showing the components of the parasporal body. In this subspecies, the individual protein inclusions are enveloped in a multilamellar fibrous matrix (arrowheads) of unknown composition, which also surrounds the crystals holding them together. A typical mature parasporal body of this subspecies measures 500-700 nm in diameter. Bar in A = 100 nm.

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is the DSM2803 isolate of B. thuringiensis subsp. morrisoni (strain tenebrionis). This isolate produces Cry3Aa, which forms a cuboidal parasporal body toxic to many coleopterous insects and is available in some countries as an insecticide. All of the above isolates are essentially used as bacterial insecticides, applied as needed. These are available in a variety of formulations including emulsifiable concentrates, wettable powders and granules for use against a wide range of pests and vectors in different habitats. On a worldwide basis, millions of hectares are treated annually with products based on Bt. Recent estimates indicate the worldwide market is about 60-80 million Euros.

Basic Biology of Bacillus sphaericus (Bs)

Since the mid-1960s it has been known that many isolates of Bs are toxic to mosquito species. Over the past three decades, three isolates have been evaluated for mosquito control, 1593 from Indonesia, 2297 from Sri Lanka and 2362 from Nigeria.65 The 1593 and 2297 isolates were obtained from soil and water samples at mosquito breeding sites, whereas 1593 was isolated from a dead adult black fly. The toxicity of Bs, like Bt, is the result of protein endotoxins that are produced during sporulation and assembled into a parasporal body. Bs is unusual in that the main toxin is a binary toxin, i.e., composed of two protein subunits (BinA and BinB). These are proteolytically activated in the mosquito midgut to release peptides of, respectively, 43 and 39 kDa, that associate to form the binary toxin, with the former protein constituting the binding domain and the latter the toxin domain. The toxins bind to microvilli of the midgut epithelium, causing hypertrophy and lysis of cells, destroying the midgut and killing the mosquito larva. Recently, a commercial product known as VectoLex (Valent BioSciences, Libertyville, Illinois) has come to market for control of Culex mosquito larvae and certain species of Anopheles mosquitoes.

Molecular Biology of Bt Insecticidal Proteins

Parasporal body proteins account for Bt’s activity for most insect pests. These proteins are referred to as δ-endotoxins, the δ referring to their early designation in a series of insecticidal factors and endotoxin referring to their assembly into inclusions within the cell after synthesis.64,66 In the early 1980s, shortly after the development of recombinant DNA techniques, it was discovered that Bt endotoxins were encoded by genes carried on plasmids. This discovery quickly led to a general understanding of endotoxin genetics and molecular biology, including mode of action, through the cloning and sequencing of numerous genes, along with characterization of the toxicity and target spectrum of the protein encoded by each gene. These studies revealed that Bt endotoxins fall into two broad classes, Cry (for crystal) and Cyt (for cytolytic) proteins. Mode of action studies show that each type, after ingestion and proteolytic activation, bind to and cause lysis of midgut epithelial cells, which results in bacteremia, typically caused by Bt, and insect death. Cry proteins require surface glycoproteins on midgut microvilli for initial binding to exert toxicity, whereas Cyt proteins bind directly to the lipid portion of the microvillar membrane.

Bt Endotoxin Mode of Action

As noted above, Cry and Cyt proteins are actually protoxins that must be ingested and processed by midgut enzymes to yield active toxins. Most have evolved to dissolve from the environmentally stable endotoxin crystals and be activated under the alkaline conditions, pH 8-10, that are characteristic of the midgut lumen of caterpillars and mosquito larvae. Once activated, Cry molecules bind to glycoprotein or glycolipid receptors. In general, the former are aminopeptidases, alkaline phosphatases, or cadherins on the midgut epithelial cell microvilli. Toxin molecules oligomerize and insert into the membrane causing cell lysis. Cyt proteins are thought to have a similar mode of action, with the exception of requiring protein receptors for binding. Instead, they bind directly to the microvillar lipid bilayer. The underlying hypothesis for Cry and Cyt protein mode of action is known as colloid-osmotic lysis. Toxin oligomers are thought to form cation-selective pores that cause an influx of cations, especially K+, into the cell. The cell then takes in water, compensating for the cation influx to

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maintain ionic balance and subsequently swells and lyses. The actual cause of larval death is not known, but is thought to be nerve paralysis that results from a rise in blood pH due to the inflow of alkaline midgut juices into the hemolymph followed by bacteremia and death. While this is the current paradigm, there is some evidence that neither toxin type forms cationic pores. If fact, evidence is quite strong that Cyt proteins act as membrane detergents.

Genetic Regulation of Cry and Cyt Protein Synthesis

The principal genetic factors controlling the yield of endotoxin synthesis in Bt are promoters, a 5ʹ mRNA stabilizing sequence and 3ʹ transcriptional termination sequences. The relative stability of each endotoxin is also a factor that affects yield. With respect to promoters, Bt endotoxin synthesis is typically under the control of two strong sporulation-dependent promoters, BtI and BtII. BtI is transcribed by sigma-35 complexed with the RNA polymerase, whereas BtII transcription is regulated by sigma-28. While this is the typical state for Bt promoters, Cry3A synthesis is under the control of a weak promoter active during vegetative growth. Moderate levels of Cry3A synthesis occur in the bacterium due to the presence of a mRNA stabilizing sequence of 9 nucleotides referred to STAB-SD present in the 5ʹ region of the cry3A transcript.67 Endotoxin synthesis can be increased by as much as 10-fold when this sequence is spliced into expression constructs for many proteins and placed under the control of Bt sporulation-dependent promoters.68-70 The 3ʹ terminus noncoding terminus of most Bt genes contains a stem-loop structure that acts as a transcription terminator, but these structures also stabilize the transcript, apparently by retarding 3ʹ exonuclease degradation. This extends transcript half-life, leading to higher endotoxin synthesis than would occur in the absence of these terminators. Several other factors enhance synthesis of Bt endotoxins during or after translation. For example, a 20-kDa protein encoded as the third ORF of the cry11A operon enhances net synthesis of many endotoxins, apparently acting as a chaperone. A 29-kDa protein encoded by the cry2Aa operon facilitates crystallization and therefore yield of Cry2A. Lastly, different endotoxin proteins vary in their stability, some, such as Cry3A are much more stable than others, for example, than Cry4A.68-70 Generally, the more stable a protein, the higher the yield when these are synthesized at high levels using expression vectors.

Recombinant Bacterial Insecticides Based on Bt

The most common strategy for constructing recombinant Bt strains is using a shuttle expression vector that contains replication origins for both B. thuringiensis and E. coli, a multiple cloning site and genes for antibiotic resistance, for example to ampicillin and erythromycin for easy selection of transformants. A shuttle vector such as pHT3101 containing a gene of interest is amplified in E. coli, isolated and then introduced into a candidate Bt strain by electroporation. In many cases, cry and cyt genes of B. thuringiensis inserted into shuttle vectors were expressed under the control of their own promoters, which generally results in a high yield of the encoded protein. In terms of promoter strength, cyt1A promoters are among the strongest known among cry and cyt genes. In addition, as mentioned above, the cry3A upstream 5ʹ mRNA stabilizing sequence (STAB-SD) improves stability of cry3A transcripts and concomitantly the yield of certain Cry proteins. To optimize Cry protein yields in Bt, a recombinant expression vector, pSTAB was developed. This vector was constructed by inserting the 660-bp DNA fragment containing cyt1A promoters combined with the STAB-SD sequence into the multi-cloning site of pHT3101. Using the pcyt1A/STAB expression vector, which combined these different genetic elements, it was possible to significantly increase yields of several Cry proteins. For example, by expressing the cry3A gene using this vector, yields twelve-fold greater than those obtained with the wild type strain of B. thuringiensis subsp. morrisoni (isolate DSM2803) from which this gene was cloned were obtained (Fig. 6). Cry3A yield obtained per unit medium using cyt1A promoters alone, i.e., lacking the STAB-SD sequence, was only about two-fold higher than that of the wild-type DSM280 strain. This demonstrates that most of the enhancement was due to inclusion of the STAB-SD sequence.68

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Figure 6. Enhanced synthesis of Cry3A through use of sporulation dependent promoters and the STAB-SD mRNA stabilizing sequence. A) Size of wild type Cry3A crystals in sporulated cells of B. thuringiensis subsp. morrisoni strain tenebrionis. B) Sporulated Bt cell in which expression of cry3A is controlled by the three cyt1A sporulation-dependent promoters. C and D) respectively, longitudinal and cross-sections through Cry3A crystals in sporulated Bt cells in which expression of cry3A was under the control of cyt1A promoters and the transcript included the STAB-SD sequence for transcript stabilization. The combination of cyt1A promoters and STAB-SD yielded at least ten-fold more protein per cell than the wild type DSM 2803 isolate. Aside from the significant increase in Cry3A yield, these results show that the small size of the crystals in the wild type strain is due primarily to the control of expression by sA, not an inherent property of Cry3A. All micrographs are the same magnification; bar in B = 300 nm.

The significant increase in Cry3A yield obtained using cyt1A promoters combined with the STAB-SD sequence led to tests of this expression vector for enhancing synthesis of other Bt endotoxins. The level of enhancement using this expression system varied depending upon the candidate protein. For example, yields of Cry11B and the Bs binary toxin were increased substantially, as much as eight-fold (Fig. 7), whereas yields of proteins such as Cry11A and Cry2A increased only 1.5 to two-fold. Perhaps the best example of the successful use of cyt1A promoters combined with STAB-SD comes from engineering recombinant Bti strains. This vector was used to produce several different recombinant strains that vary in complexity, ranging from a strain that produces only a single endotoxin to strains that produce as many as five endotoxins. In the simplest case, pcyt1A/STAB was used to synthesize the Bin toxin of Bs 2362. Using this construct, Bin synthesis was eight-fold higher than that obtained with wild type Bs 2362. The toxicity of this strain was 13-fold better than wild type Bs against Culex species.69,70 To improve toxicity while at the same time preventing or delaying the evolution of resistance, several strains were constructed in which toxin complexity was increased and Cyt1A was added for resistance management. One strain constructed using this strategy was a recombinant that synthesized the Bin toxin, Cyt1A and Cry11B. In this recombinant, the mosquitocidal proteins were from three different species; Bin from Bs 2362, Cry11B—a protein more toxic than Cry11A—from Bt subsp. jegathesan and Cyt1A from Bti. This recombinant was constructed using a dual-plasmid expression system with two different plasmids, each with a different antibiotic resistance gene for selection. The resulting recombinant B. thuringiensis produced three distinct crystals and was 3 to 5 times as toxic to Culex species as either Bti IPS-82 or Bs 2362 (Fig. 8). To construct a recombinant with an even greater range of endotoxins for both increased toxicity and resistance management, the IPS-82 strain of Bti, which produces the complement of toxins characteristic of this species, was transformed with pPHSP-1, the pcyt1A/STAB plasmid that produces a high level of Bs Bin

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Figure 7. Cloning of the Bacillus sphaericus binary toxin operon followed by engineering the operon into a shuttle expression vector designed to synthesize the binary toxin under the control of the three wild type tandem cyt1A promoters combined with the STAB-SD sequence. A) Electron micrograph of sporulated cells showing (arrows) crystals of the binary toxin on the internal side of the exosporium membrane. B) Scanning electron micrograph of purified binary toxin crystals synthezied in an acrystalliferous strain of B. thuringiensis subsp. israelensis using the E. coli-B. thuringiensis shuttle vector shown in C) (circular vector map). The top portion of C shows the structure of the B. sphaericus operon (top) and the engineered operon in which expression is driven by cyt1A pomoters. Bars in A and B equal approximately 1 mm. Micrograph in A courtesy of Dr. Jean-Francois Charles of the Institut Pasteur, Paris, France.

toxin (Fig. 9).69,70 This recombinant was more than ten-fold more toxic than either of the parental strains to larvae of Cx. quinquefasciatus and Cx. tarsalis. Aside from high efficacy, as noted above, this new bacterium is much less likely to select for resistance in target populations, as it combines Cyt1A from Bti with Bti Cry toxins and Bs Bin. The resistance management properties of this bacterium are currently under evaluation. The markedly improved efficacy and resistance-delaying properties of this new bacterium make it an excellent candidate for development and use in vector control programs, especially to control Culex vectors of West Nile and other viruses as well as species of this genus that transmit filarial diseases. Moreover, larvae of Anopheles gambiae, the key vector of malaria in many regions of Africa, have recently been show to be as sensitive to this recombinant as Culex mosquitoes (Federici et al unpublished). This is because of the high sensitivity of this species to the Bs Bin toxin. This indicates this Bti/Bs recombinant should also be more cost-effective against other species of anopheline larvae sensitive to Bs Bin than the insecticides based on the wild type parental strains of these bacterial species. The improvements in activity noted above result from the increase in toxicity per unit weight of fermentation medium. These increases significantly reduce production costs for obtaining the same level of pest or vector control. The extent to which these savings are potentially passed along to consumers, as opposed to being used to increase company profits has not been determined, as the recombinant strains discussed have not yet been commercialized.

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Figure 8. Construction de novo of a unique mosquitocidal strain of B. thuringiensis that produces individual endotoxin crystals of the Bs Bin binary toxin, Cyt1A and Cry11B. A) Vector bearing the cyt1A and B. sphaericus bin genes. B) Vector bearing the cry11B gene from B. thuringiensis subsp. jegathesan. These were transformed successively into the 4Q7 acrystalliferous strain of B. thuringiensis subsp. israelensis to yield the final recombinant strain that produces Bs Bin, Cyt1A and Cry11B. C) Phase-contrast micrograph of sporulated recombinant cells of B. thuringiensis 4Q7 containing individual crystals of Cyt1A, Cry11B and the B. sphaericus binary toxin. The spore is on the right in each of these cells. D) Analysis of endotoxin content in wild type and recombinant strains of B. thuringiensis. M, molecular size marker; lane 1, B. thuringiensis subsp. israelensis 4Q7 producing B. sphaericus binary toxin and Cyt1A (4Q7/p45S1); lane 2, B. thuringiensis subsp. israelensis 4Q7 producing Cry11B (4Q7/pPFT11Bs-CRP); lane 3, B. thuringiensis subsp. israelensis 4Q7 producing Cry11B, Cyt1A and B. sphaericus binary toxin (4Q7/p45S1-11B). The numbers at the base of lane 3 indicate the approximate ratio of each toxin produced in the Cry11B, Cyt1A, Bin recombinant in comparison to, respectively, the Cyt1A plus Bin recombinant (lane 1) and the Cry11B recombinant (lane 2). Equal amounts of culture medium were loaded in each well. Bar in C = 1 mm.

Safety Concerns about Wild Type and Recombinant Bacterial Insecticides

In determining what types of tests should be done to evaluate the safety of bacterial insecticides, early tests were based primarily on those used to evaluate chemical insecticides. However, the tests have evolved over the decades and are now designed to evaluate the risks of Bt, specifically the infectivity of the bacteria and toxicological properties of proteins used as active ingredients. The tests are grouped into three tiers, I-III.71 Tier I consists of a series of tests aimed primarily at determining whether an isolate of a Bt subspecies, as the unformulated material, poses a risk if used at

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Figure 9. Synthesis of Bacillus sphaericus 2362 binary toxin in B. thuringiensis subsp. israelensis. A) Transmission electron micrograph of the recombinant strain Bti4Q7/BsB, engineered to synthesize only the B. sphaericus binary toxin. A single large crystal (BsB) is adjacent to the spore. B) Transmission electron micrograph of the recombinant crystalliferous strain, BtiIPS-82/ BsB, engineered to synthesize the B. sphaericus binary toxin in a wild type strain of Bacillus thuringiensis subsp. israelensis. This strain produces the typical IPS-82 parasporal body (Bti) and Bs 2362 binary toxin crystal (BsB). C) Comparative endotoxin yields produced per unit medium by wild type B. sphaericus strain 2362 and wild type and engineered strains of B. thuringiensis subsp. israelensis constructed to synthesize the Bs 2362 binary toxin. Lanes: 1, wild type control B. sphaericus 2362 strain; 2, recombinant Bti4Q7 strain, Bti4Q7/BsB, engineered to produce Bs2362 toxin; 3, recombinant BtiIPS-82 strain, BtiIPS-82/BsB, that produces the Bs2362 toxin and typical Bti parasporal body; 4, wild type control Bti IPS-82 strain.

high levels, typically at least 100 times the amount recommended for field use, to different classes of nontarget organisms. The principal tests include acute oral, acute pulmonary (inhalation) and acute intraperitoneal evaluations of the material against different vertebrate species, with durations from a week to more than a month, the length depending on the organism. In the most critical tests, the mammals are fed, injected with and forced to inhale millions of Bt cells in a vegetative or sporulated form. Against invertebrates, the tests are primarily feeding and contact studies. Representative nontarget vertebrates and invertebrates include mice, rats, rabbits, guinea pigs, various bird species, fish, predatory and parasitic insects, beneficial insects such as the honeybee, aquatic and marine invertebrates and plants. If there is clear infectivity or acute toxicity in any of these tests, then the candidate bacterium would be rejected. If uncertainty exists, then Tier II tests must be conducted. These tests are similar to those of Tier I, but require multiple consecutive exposures, especially to organisms where there was evidence of toxicity or infectivity in the Tier I tests, as well as tests to determine if and when the bacterium was cleared from nontarget tissues. If infectivity, toxicity, mutagenicity, or teratogenicity is detected, then Tier III tests must be undertaken. These consist of tests such as two-year feeding studies and additional testing of teratogenicity and mutagenicity. The tests can be tailored to further evaluate the hazard based on the organisms in which hazards were detected in the Tier I and II tests. The safety protocols also apply to recombinant baculoviruses and fungi. It must be realized that due to the genetic engineering methods used to construct Bt crops, the safety criteria are more strict than those for many registered synthetic chemical insecticides. Nevertheless, unlike Bt crops, which have a narrow spectrum of insecticidal activity, many chemical insecticides are known to be toxic to nontarget invertebrates, as well as to vertebrates such as fish and humans, especially if not used properly.

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To date, not a single registered Bt insecticides based on Cry or Cyt proteins has had to undergo Tier II testing.62,71,72 In other words, no moderate or significant hazards or risks have been detected with any Bt subspecies used commercially or any Bt crop against any of the nontarget organisms studied, including mammals. As a result, all Bt insecticides (and Bt crops) registered in the US are exempted from a tolerance requirement, i.e., a specific level of insecticide residue allowed on a crop just prior to harvest. Moreover, no washing or other requirements to reduce levels consumed by humans are required. In fact, Bt insecticides can be applied to crops such as lettuce, cabbage and tomatoes just prior to harvest and Bt crops have no restrictions for human consumption. It is important to realize that such a statement cannot be made for any chemical insecticide. Aside from the various safety studies required by the US Environmental Protection Agency, programs have been mounted to monitor the health effects of spraying Bt insecticides directly on human populations. Two such recent studies, for example, were conducted during the late 1990’s, one in Victoria, Canada and another in Auckland, New Zealand.73,74 In both cases the Bt spray programs were undertaken to eliminate lepidopteran forest pests that had invaded these countries. To eliminate these pests, suburban residential areas inhabited by thousands of people were spayed periodically for several weeks, until the pests were eradicated. During the spray programs and for months thereafter, the human populations were monitored for the presence of the Bt applied and for symptoms of disease. Bacteria were easily recovered from nasal samples, for example and from monitoring particulates in the air. In Auckland, New Zealand, some discomfort followed the sprays, but “most residents saw their health as unaffected by the spray program and there was no significant increase in visits to general practitioners or alternative health care providers.”73 Similar results were obtained in the populations monitored in Victoria during the Bt spray program—the “human health surveillance program failed to detect any correlation between the aerial application of B. thuringiensis subsp. kurstaki HD1-like bacteria and short-term health effects in the general adult population.”74 This evidence of little or no significant health effects on human populations subjected to Bt sprays is in sharp contrast to the well-known toxic effects many chemical pesticides have on humans.75,76 Despite these and previous studies, there are several putative cases in the literature where it is claimed that certain isolates of B. thuringiensis have caused infections in humans. The evidence in support of these claims is very weak and has been reviewed recently in the context of the high degree of safety that Bt exhibits toward mammals.75 Specific safety studies on the various recombinant bacteria described above have not yet been conducted. The reasons for this include a shortage of funding for such studies and marketing concerns by the companies that produce bacterial insecticides that the public may not accept the use of genetically engineered bacteria. There is, however, no reason to think that recombinant bacteria like those described above will have properties that would adversely affect most nontarget organisms and certainly any effects, including toxicity to these organisms, would be much less than those of synthetic chemical insecticides. Nevertheless, it is likely that it will be years before recombinant bacterial insecticides will join other recombinant organisms, such as Bt crops, as components of biological control and integrated pest management programs for agricultural pests and vectors of human and animal diseases.

Basic Biology and Genetic Engineering of Insect-Pathogenic Fungi

Insect pathogenic fungi are key regulatory factors in insect pest populations. Unlike bacteria and viruses that have to be ingested to cause disease, fungi infect insects by direct penetration of the cuticle (Fig. 10). They therefore allow microbial control of insects which feed by sucking plant or animal juices, as well as for the many coleopteran pests that have no known virulent viral or bacterial diseases. Notwithstanding the potential of many fungi as insect control agents, only a handful have been commercialized and most attention has focused on the ascomycetes Metarhizium anisopliae and Beauveria bassiana. These have been used for insect control in many countries including the USA, China, Brazil, Australia, the Philippines and several African countries.77,78 Several strains of M. anisopliae are produced commercially e.g., by Biocare, Australia; BCP, South Africa; Bayer,

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Figure 10. Typical infection pathway for insect pathogenic fungi. Unlike bacteria and viruses that need to be ingested to cause disease, fungi such as Metarhizium anisopliae kill insects by direct penetration of the cuticle followed by multiplication in the hemocoel. During infection processes the fungus therefore has to adapt to several distinct environments including the hydrophobic wax-rich epicuticle, the protein-chitin procuticle and the cellular hypodermis before reaching the solute rich hemolymph. Genomic studies show that each step requires differential expression of hundreds of genes including those for cuticle degrading enzymes, toxin production, stress responses, immune evasion and cell wall re-organization.

Germany; and several Brazilian companies and released on a large scale without any reports of risks to vertebrates.79 The devastating plagues of locusts in the mid-1980s provided a sense of urgency to insect fungus research in general and use of M. anisopliae in particular and a requirement for standardized products that advanced our knowledge in a range of areas such as formulation, quality control and storage.80,81 Industrial production of M. anisopliae is now highly automated allowing Metarhizium products to be competitively priced compared with established insecticides.80,82 However, the slow speed of kill and inconsistent results of mycoinsecticides in general compared with chemicals has deterred development. An example is the use of M. anisopliae to kill adult mosquitoes inside Tanzanian houses as the current protocol only reduces the number of bites four-fold.83,84 These studies indicate that this level of reduction will not be sufficient to provide adequate protection against disease transmission, however, they suggest that improving infection rates using a more aggressive fungal strain will contribute in a significant and sustainable manner to the control of vector-borne diseases such as malaria, dengue and filariasis.83,84 More virulent mosquitocidal strains of M. anisopliae might be found by screening wild strains. However, intensive searches in the ‘60s and ‘70s failed to identify strains active at low doses. This is consistent with intensive efforts to control other pests; historically fungal pathogens of plant and insect pests have not met expectations because of low virulence (slow kill and high inoculum loads). Gressel et al85 suggested that this was because an evolutionary balance has developed between microorganisms and their hosts, even when the biocontrol agent is used at very high levels. Slow kill by Metarhizium strains is linked with a strategy of optimizing utilization of nutrients by growing within the living

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host.86 Thus, there is a consensus that sufficient virulence for cost effective biocontrol can be achieved only by transferring genes to the microorganism.85,87 Ultimately, various traits of fungal pathogens, including host range, production capacity, stability and virulence and saprophytic competence will be enhanced through genetic manipulations. Currently, M. anisopliae is the most tractable entomopathogenic fungal model for a genetic engineering approach. It is one of the most commonly isolated insect pathogenic fungi and contains strains with wide host ranges and strains specific for certain locusts (within sf. acridum), beetles (e.g., sf. majus), crickets, hemipterans, etc and are unable to infect other insects. M. anisopliae offers EST collections, microarray analyses,88,89 promoters that allow expression of foreign genes90,91 and gene disruption technology.92 Identification of types of genes whose manipulation would have potential in mycoinsecticide development is easier in an organism such as M. anisopliae, for which there are extensive physiological and biochemical data.

Genetic Engineering Entomopathogenic Fungi

Genetic studies of entomopathogenic fungi were traditionally hampered by low transformation frequencies. This has been remedied by adapting a method of Agrobacterium mediated transformation, that is relatively straightforward for both B. bassiana and M. anisopliae.93,94 Agrobacterium-mediated transformation has been successful with very diverse fungi including members of the Ascomycetes, Basidiomycetes, Zygomycetes and Oomycetes, indicative of the potential of this transformation system for introducing biotechnology to insect pathogenic fungi such as Erynia species and Lagenedium species that have so far not been transformed. Agrobacterium may therefore provide a simple standardized method for transformation of essentially any entomopathogenic species that would obviously be novel and useful. Genome-wide functional screening using Agrobacterium insertional mutagenesis are under way in M. anisopliae to identify genes involved in particular phenotypes or morphogenetic process, e.g., sporulation, appressoria formation, attenuation of virulence. A mutant with enhanced virulence has been identified that may provide new ideas on genetically manipulating virulence (Fang, Bidochka and St. Leger, Unpublished).

Overview of Engineering Saprophytic Competence or Containment

Most studies on insect pathogens have focused on their virulence. However, if the pathogen is expected to function as a classical biocontrol agent and persist in the environment, then factors influencing this will include a wide range of climatic (solar radiation, temperature, water availability, precipitation and wind), edaphic (soil types) and biotic (antagonists) conditions.95,96 Genetically based resistance to these parameters could also be a distinct advantage both during infection and product preparation and storage. Considerable variability exists among taxa and strains within species in their thermal characteristics, requirements for relative humidity and susceptibility to irradiation.97-99 This provides evidence for strong selective pressures and the existence of a range of naturally available tools for developing tolerance to environmental constraints. The genetic mechanisms of resistance to environmental parameters are not well understood but are probably governed by polygenic factors that may therefore be too complex to be readily amenable to genetic manipulation. However, progress has been made in understanding susceptibility to damage by the UV-B (290-315 nm) portion of the solar spectrum; a major impediment to the successful commercialization of entomopathogens for field crops. Recent studies have shown that the degree of conidial pigmentation and levels of DNA repair enzymes contribute to tolerance and that there is a relationship between this tolerance and the geographical origin of the insect host.100 To date it has been axiomatic that a recombinant pathogen of any kind would be applied as an inundative insecticide. Environmental persistence is not required and might be regarded as a drawback by a company seeking repeat sales and as a threat to the environment. A science-based regulatory system currently has three requirements for enhanced biocontrol agents: (i) limited off site dispersal, (ii) poor long term persistence and (iii) limited possibility of recombination with other pathogens.101 Based on methods developed with transgenic plants, Gressel101 has suggested it

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may be possible to develop technology that would block the spread of transgenic fungi beyond the natural barriers that already exist. This would involve using a two step procedure to mitigate spread of/and gene flow from transgenically hypervirulent insecticidal organisms: a) by using transgenic mitigating (TM) genes that in the antisense form suppress sporulation or other traits and b) obviating potential recombination to native microbial strains by flanking the hypervirulence gene with the TMs so that they are inherited together and would be detrimental to any recombinant. Thus, linking a hypervirulence gene with one or more antisense genes suppressing sporulation could serve the dual purpose of blocking dispersal and recombination. This assumes that because of gene linkage, the deleterious genes will be nearly inseparable from the primary transgene and will not segregate. Deleterious effects will render the rare recipients of the linked constructs sufficiently unfit to compete with the wild type of that species and with other organisms. This approach has not been applied to a fungus but has been extensively tested as a failsafe to mitigate introgressions of transgenes from crops to weeds.102 The practical need for this may be small in M. anisopliae as it seems to be almost exclusively asexual; recombination is rare and most likely limited to heterokaryon formation between multiple closely-related strains. Using fungus tagged with either green or red fluorescent protein we have observed unstable diploid formation between colonies of the same M. anisopliae strain at field sites, but not yet between different strains (Wang and St. Leger, Unpublished). However, the availability of failsafe procedures will alleviate biosafety concerns and the general concepts may be applicable to other microbial agents.

Engineering Improved Virulence

The most attractive initial candidates for this approach include cuticle-degrading enzymes and toxins that are encoded by single genes as they are highly amenable to manipulation by gene transfer. Many of the cuticle-degrading enzymes that act synergistically to solubilize cuticles are multiple gene products with distinctive activity profiles.90,103-105 The variability of molecules with activity against host substrates increases the range of tools naturally available to develop biotechnological procedures for pest control. Furthermore, these molecules possess pathogenic specializations that distinguish them from similar molecules produced by saprophytes. For example, stronger binding, due to the positively charged surface groups on the subtilisin protease Pr1 contribute to increasing Pr1 activity 33-fold against insoluble cuticle proteins compared to proteinase K from a related saprophyte.106 Pr1 is also resistant to proteinase inhibitors (serpins) in hemolymph and even to being in a rapidly melanizing suspension, mimicking the insect defense response.107 In the first example of a recombinant pathogen with enhanced virulence, additional copies of the gene encoding the regulated Pr1 were inserted into the genome of M. anisopliae under the control of the gpd promoter such that the gene was constitutively overexpressed.90 At the commencement of the experiment, it was expected that if over expression of a cuticle degrading protease was to have an effect on virulence, it would be to speed up penetration. However, the actual effect of overexpression was to cause massive melanization in the body cavity and cessation of feeding 40 hr earlier than controls infected with wild type. In contrast to the wild-type, transgenic strains continued to produce Pr1 in the haemocoel of Manduca sexta caterpillars following penetration of the cuticle. This activated a host trypsin-like enzyme that is involved in a cascade terminating in prophenoloxidase activation. Microarray experiments have since shown that WT M. anisopliae stops expressing protease genes in the hemolymph, presumably to prevent this from happening.108 Engineering studies often do not turn out as expected. Experimenters may know in considerable detail the properties of an individual gene and its protein product. They may know how it interacts with other gene products produced concurrently with it in the native organism. However, transfer it to another organism and the results are unpredictable because they involve unforeseen interactions with proteins that it would not normally interact with. In the particular case of a pathogen it may be harder to predict consequences because there will be interactions between two genomes, as with the pathogen protease and host phenoloxidase pathway. However, a fortunate by-product of this interaction is that insects killed by transgenic strains and extensively melanized were very poor substrates for fungal growth and sporulation. This reduces transmission of the recombinant fungi, which assisted in obtaining permission for a field trial.109

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Fungal pathogenicity is polyphylectic and virulence is certainly multifactorial. The ability to penetrate the host cuticle and tolerance of the host immune system may be common denominators, but pathogenic fungal species might have reached these qualities through different evolutionary paths. The particular factors important for virulence, such as adhesins, pigments, extracellular enzymes, or other secreted products, could therefore be very specific to the pathogen. The availability of these genes raises the possibility of creating novel combinations of insect specificity and virulence by recombining them in other fungi, bacteria or viruses to produce improved pathogens. Thus, the Pr1 gene and an esterase gene from M. anisopliae have each been used to increase virulence of B. bassiana.110 Subtilases similar to Pr1 have improved the biocontrol potential of fungal pathogens of other fungi111 and nematodes.112 Subtilisn Pr1 was cloned and studied based on the assumption that cuticle degrading enzymes would be required for penetrating host barriers. In recent years, hypothesis-driven cloning of genes assumed to be involved in pathogenicity has been replaced by EST and microarray approaches that allow experimenters to look into the intricacies of infection processes and let the pathogen inform on what it is doing during infection processes.81 These techniques allow side effects occurring in constructed strains to be accessed and a greater range of engineering possibilities to be exploited from knowledge of the interrelated regulatory and metabolic processes going on in cells. Although they dramatically accelerate the gain of information, the bottleneck is in translating gene expression profiling onto hypothesis-driven approaches which result in clearly proven function of single genes and new ideas for genetically enhancing virulence. Nevertheless, construction of deletion strains for highly expressed genes has already led to the identification of separate adhesins essential for binding to insect cuticle and plant surfaces,113 a perilipin that regulates lipolysis, osmotic pressure and formation of infection structures114 and an osmosensor that signals to penetrant hyphae that they have reached the haemocoel (Wang and St. Leger, Unpublished). These genes all have as yet un-realized potential to engineer changes in host range; either increasing it or diminishing it and illustrate the power of expression profiling for revealing previously unsuspected stratagems of infection. Most importantly for fungal transgenesis, microarray studies putatively identified M. anisopliae promoters capable of expressing homologous and heterologous genes in a regulated fashion and that vary in levels of expression. Confirmation of regulation was achieved by putting regulatory regions of genes in front of the green fluorescent reporter gene.92 The most highly expressed gene (Mcl1) during growth in hemolymph (5.6% of total transcripts) encodes a cell wall protein with a long collagenous domain. Immunofluorescence demonstrated that the protein coats the fungus during growth in hemolymph and gene knockout confirmed that Mcl1 is required for immune evasion.92 The mutant is rapidly attacked by hemocytes and has reduced virulence to Manduca sexta. RT-PCR confirmed that Mcl1 is expressed during growth in the hemolymph of a diverse array of insect species, consistent with the broad host range of the M. anisopliae strain (ARSEF 2575) that it was acquired from. However, it was not expressed in other media, consistent with it being involved in pathogenesis.92 The highly expressed Mcl1 promoter seems optimal for targeted expression of transgenes encoding toxins to the hemolymph. Aside from the possibility of increasing virulence, regulation of toxin expression to growth in the hemolymph has safety considerations, by precluding casual release of the toxin by the fungus living as a saprophyte. In addition, specificity is usually controlled by infection events at the level of the cuticle,115 so altering postpenetration events should not reduce environmental safety derived from species selectivity. A problem that can arise from using the pathogen own genes and toxins to improve virulence is that the hosts have had millions of years to evolve resistance so it could be advantageous to take toxin genes from heterologous organisms. The Mcl1 promoter has been used to drive expression in M. anisopliae of the insect-selective 70 aa AaIT neurotoxin from the scorpion Androctonus australis. Having M. anisopliae specifically express AaIT in the hemolymph increased virulence 20-fold against the model lepidopteran Manduca sexta and nine-fold against adult Aedes aegypti in terms of insecticidal activity per number of spores (3 to 6 transgenic spores to kill a mosquito as cf. ∼35 wild type spores).91 In comparison to wild type M. anisopliae, expression of AaIT in this fungus also produced useful

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prelethal effects including reduced mobility and feeding, as well as muscle contractions in both adults mosquitoes and larval blowflies (Figs. 11 and 12). AaIT has already provided the some of most promising recombinant baculoviruses,31 with improved performance against lepidopteran larvae in several field trials.116 AaIT acts on the neuronal sodium channel causing presynaptic excitatory effects. Interestingly, lepidopterans are relatively tolerant to this toxin compared to locusts, beetles and crickets.31 Baculoviruses are primarily pathogens of lepidopterans. However, many insects not susceptible to baculoviruses are targeted by M. anisopliae. These studies are providing an opportunity: (1) to diversify the deployment of AaIT, a very well studied toxin, which like M. anisopliae has already passed many regulatory hurdles and (2) to directly compare the efficacy of fungal toxins with the most frequently studied arthropod toxin. The best studied M. anisopliae toxins are the destruxins.117 Unfortunately for the purposes of genetic engineering, destruxins are secondary metabolites and encoded by genes that are too large (ca 20 Kb) for convenient molecular manipulations. However, a strain of M. anisopliae has been identified that produces an acute protein toxin active at 0.7 μg/100 mg and other toxins from M. anisopliae and B. bassiana are being isolated.118 The toxins from arthropod venoms, M. anisopliae and B. bassiana affect different aspects of insect biology and therefore could be targeted synergistically to produce a large magnitude of hypervirulence and reduce the probability of resistance evolving to a single transgene product. A strain of B. bassiana transformed with AaIT and an esterase from M. anisopliae kills caterpillars faster than strains transformed with either gene separately, suggesting at least additive effects (Pava-Ripoll and St. Leger, Unpublished). The approximately 1 million arachnid toxins include specific toxins for many different organisms, including microorganisms and they will likely remain a major resource for genetically modified plants and biopesticidal delivery systems. In fact, their potential appears virtually limitless as long as they are provided with suitable delivery systems.119,120 However, comparing arthropod and fungal toxins could usefully increase interest in fungi as a resource of genes for biotechnology. Fungi have been under-exploited to date. This is particularly true for the insect pathogens, even though they are exceptionally rich sources of novel biologically active substances.121,123,124

Figure 11. Mosquitoes infected with wild type fungus Metarhizum anisopliae or M. anisopliae synthesizing AaIT. Those infected with the transgenic strain (right) demonstrated prelethal effects including spasmic leg and wing movements several hours before death. They invariably died with extended wings, indicative of muscle contraction, which thus provides a useful marker for mortality due to AaIT. The green color is due to conidia produced by the fungus on the surface of the infected mosquitoes. A color version is available at www.eurekah.com.

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Scientists today are not limited to the immense diversity of peptide sequences that already occur in nature but can derive synthetic multifunctional genes that are hybrids of different activities. Chitinases characterized in B. bassiana, lack chitin-binding domains. Fang et al122 characterized several B. bassiana hybrid chitinases where the chitinase was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain from the silkworm Bombyx mori chitinase fused to the B. bassiana chitinase showed the greatest ability to bind to chitin compared to other hybrid chitinases. Constitutive expression of this hybrid chitinase gene by B. bassiana reduced time to death by 23% compared to the wild-type fungus showing that genetic components of the host insect incorporated into the fungal pathogen can increase virulence. The same authors have produced a strain of B. bassiana expressing a multifunctional fusion of protease and chitinase that simultaneously targets the major structural components of cuticle and rapidly digests it (Unpublished). As expression systems, Metarhizium and Beauveria are as easy to use as the commercially available yeasts but with the added advantage of also providing a delivery system into the insect. This contrasts sharply with insect transgenesis for example, that requires training and expertise well beyond the casual. Evidently, insect pathogenic fungi could provide a tractable model system for screening novel effectors or fusion products produced by gene shuffling, or that are hybrids of different vectors. After screening, the most potent effectors could be delivered by the fungus, another microbe and/or in a transgenic insect or plant.

Conclusions

The knowledge and variety of techniques generated by molecular biology and genomics over the past twenty years have been put to use to generate recombinant insect-pathogenic viruses, bacteria and fungi that are much more efficacious than the wild type strains from which they were derived. Techniques for mass production of recombinant bacteria and fungi have been developed that have reduced the costs of these down to those of many chemical insecticides, but production of recombinant bacteria typically are much more environmentally compatible. Mass production of recombinant viruses remains costly, but studies are underway to bring these costs down. While

Figure 12. Effects of Metarhizium anisopliae engineered to produce the scorpion toxin AaIT on blowfly larvae. Blow fly larvae were injected with aliquotes of hemolymph from larvae of the moth, Manduca sexta, infected with wild type M. anisopliae or M. anisopliae expressing the scorpion toxin AaIT. The scorpion toxin causes immediate contraction and paralysis of the blowfly.

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acceptance by the public remains an issue, the extensive plantings of Bt crops in countries like the US, Australia, Argentina and China indicates recombinant microbial insecticides will eventually will be accepted and will play an important role in controlling insect pests and vectors of disease.

Acknowledgements

The research reported here on is based in part on financial support from various governmental agencies, grants to (1) B. Bonning from the USDA (NRI 2003-35302-13558) as well as Hatch Act and State of Iowa funds; to (2) B. Federici from the U.S. National Institutes of Health (NIH AI045817 and AI0547780 and to (3) R. St Leger from the U.S. National Science Foundation (MCB-0542904) and U.S. Department of Agriculture (Grant 2006-03692).

References

1. Adams JR, Bonami JR. Atlas of Invertebrate Viruses. Boca Raton: CRC Press, 1991. 2. Bonning BC. Insect viruses: Biotechnological applications. Maramorosch K, Shatkin AJ, eds. Advances in Virus Research, vol 68. San Diego: Elsevier, 2006. 3. Miller LK, Lu A. The molecular basis of baculovirus host range. In: Miller LK, ed. The Baculoviruses. New York: Plenum Press, 1997:217-235. 4. Kamita SG, Kang K-D, Hammock BD et al. Genetically modified baculoviruses for pest insect control. In: Gilbert LI, Iatrou K, Gill SS, eds. Comprehensive Molecular Insect Science. 6th ed. Oxford: Elsevier, 2005:271-322. 5. Black BC, Brennan LA, Dierks PM et al. Commercialization of baculoviral insecticides. In: Miller LK, ed. The Baculoviruses. New York: Plenum Press, 1997:341-388. 6. Inceoglu AB, Kamita SG, Hammock BD. Genetically modified baculoviruses: A historical overview and future outlook. Adv Virus Res 2006; 68:323-360. 7. Cory JS, Myers JH. The ecology and evolution of insect baculoviruses. Annu Rev Ecol Evol Syst. 2003; 34:239-272. 8. Moscardi F. Assessment of the application of baculoviruses for control of Lepidoptera. Annu Rev Entomol 1999; 44:257-289. 9. Bonning BC. Baculoviruses: Biology, biochemistry and molecular biology. In: Gilbert LI, Iatrou K, Gill SS, eds. Comprehensive Molecular Insect Science. 6th ed. Oxford: Elsevier Pergamon, 2005:233-270. 10. Bonning BC, Boughton AJ, Jin H et al. Genetic enhancement of baculovirus insecticides. In: Upadhyay RK, ed. Advances in Microbial Control of Insect Pests. London: Kluwar Academic/Plenum Publishers, 2002:109-125. 11. O’Reilly D. Baculovirus-encoded Ecdysteroid UDP-glucosyltransferases. Insect Biochem Mol Biol 1995; 25(5):541-550. 12. OReilly DR, Miller LK. A baculovirus blocks insect molting by producing ecdysteroid UDP-glucosyl transferase. Science 1989; 245:1110-1112. 13. O’Reilly D. Auxiliary genes. In: Miller LK, ed. The Baculoviruses. New York: Plenum Press, 1997:267-300. 14. O’Reilly DR, Miller LK. Improvement of a baculovirus pesticide by deletion of the EGT gene. Bio/ Technology 1991; 9:1086-1089. 15. Chen X, Sun X, Hu Z et al. Genetic engineering of Helicoverpa armigera single-nucloepolyhedrovirus as an improved pesticide. J Invertebr Pathol 2000; 76:140-146. 16. Slavicek JM, Popham HJR, Riegel CI. Deletion of the Lymantria dispar multicapsid nucleopolyhedrovirus ecdysteroid UDP-glycosyl transferase gene enhances viral killing speed in the last instar of the gypsy moth. Biol Control 1999; 16(1):91-103. 17. Bianchi FJJA, Joosten NN, Vlak JM et al. Greenhouse evaluation of dose- and time-mortality relationships of two nucleopolyhedroviruses for the control of beet armyworm, Spodoptera exigua, on chrysanthemum. Biol Control 2000; 19:252-258. 18. Sun X, Wang H, Sun X et al. Biological activity and field efficacy of a genetically modified Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus expressing an insect-selective toxin from a chimeric promoter. Biol Control 2004; 29:124-137. 19. Popham HJR, Li Y, Miller LK. Genetic Improvement of Helicoverpa zea Nuclear Polyhedrosis Virus as a Biopesticide. Biol Control 1997; 10(2):83-91. 20. Treacy MF, All JF, Ghidiu GM. Effect of ecdysteroid UDP-glucosyltransferase gene deletion on efficacy of a baculovirus against Heliothis virescens and Trichoplusia ni (Lepidoptera: Noctuidae). J Econ Entomol 1997; 90:1207-1215. 21. Maeda S. Increased insecticidal effect by a recombinant baculovirus carrying a synthetic diuretic hormone gene. Biochem Biophys Res Commun 1989; 165:1177-1183.

Improvement of Insect Pathogens as Insecticides through Genetic Engineering

37

22. Eldridge R, Horodyski FM, Morton DB et al. Expression of an eclosion hormone gene in insect cells using baculovirus vectors. Insect Biochem 1991; 21:341-351. 23. O’Reilly DR, Kelly TJ, Masler EP et al. Overexpression of Bombyx mori prothoracicotropic hormone using baculovirus vectors. Insect Biochem Mol Biol 1995; 25(4):475-485. 24. Hammock BD, Bonning B, Possee RD et al. Expression and effects of the juvenile hormone esterase in a baculovirus vector. Nature 1990; 344:458-461. 25. Stewart LMD, Hirst M, Ferber ML et al. Construction of an improved baculovirus insecticide containing an insect- specific toxin gene. Nature 1991; 352:85-88. 26. Maeda S, Volrath SL, Hanzlik TN et al. Insecticidal effects of an insect-specific neurotoxin expressed by a recombinant baculovirus. Virology 1991; 184:777-780. 27. McCutchen BF, Choudary PV, Crenshaw R et al. Development of a recombinant baculovirus expressing an insect-selective neurotoxin: Potential for pest control. Biotechnology 1991; 9:848-852. 28. Harrison RL, Bonning BC. Use of scorpion neurotoxins to improve the insecticidal activity of Rachiplusia ou multicapsid nucleopolyhedrovirus. Biol Control 2000; 17:191-201. 29. Treacy MF, Rensner PE, All JN. Comparative insecticidal properties of two nucleopolyhedrovirus vectors encoding a similar toxin gene chimer. J Econ Entomol 2000; 93:1096-1104. 30. Sun X, Chen X, Zhang Z et al. Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton. J Invertebr Pathol 2002; 81:63-69. 31. Zlotkin E, Fishman Y, Elazar M. AaIT: from neurotoxin to insecticide. Biochimie 2000; 82(9-10):869-881. 32. Zlotkin E, Moskowitz H, Herrmann R et al. Insect sodium channel as the target for insect-selective neurotoxins from scorpion venom. ACS Symp Ser 1995; 591:56-85. 33. Prikhodko GG, Popham HJR, Felcetto TJ et al. Effects of simultaneous expression of two sodium channel toxin genes on the properties of baculoviruses as biopesticides. Biol Control 1998; 12:66-78. 34. Regev A, Rivkin H, Inceoglu B et al. Further enhancement of baculovirus insecticidal efficacy with scorpion toxins that interact cooperatively. FEBS Lett 2003; 537(1-3):106-110. 35. Harrison RL, Bonning BC. Use of proteases to improve the insecticidal activity of baculoviruses. Biol Control 2001; 20:199-209. 36. Li H, Tang H, Harrison RL et al. Impact of a basement membrane-degrading protease on dissemination and secondary infection of Autographa californica multiple nucleopolyhedrovirus in Heliothis virescens (Fabricus). J Gen Virol 2007; 88(Pt 4):1109-1119. 37. Tang H, Li H, Lei SM et al. Tissue specificity of a baculovirus-expressed, basement membrane-degrading protease in larvae of Heliothis virescens. Tissue Cell 2007; 39:431-443. 38. McCutchen BF, McCutchen BF, McCutchen BFs. Production of recombinant baculoviruses. US patent 6,322,781 B1, 2001. 39. Soberon M, Fernandez LE, Perez C et al. Mode of action of mosquitocidal Bacillus thuringiensis toxins. Toxicon 2007; 49(5):597-600. 40. Martens JWM, Knoester M, Weijts F et al. Characterization of baculovirus insecticides expressing tailored Bacillus thuringiensis Cry1A(b) crystal proteins. J Invertebr Pathol 1995; 66:249-257. 41. Merryweather AT, Weyer U, Harris MPG et al. Construction of genetically engineered baculovirus insecticides containing the Bacillus thuringiensis subsp. kurstaki HD-73 delta endotoxin. J Gen Virol 1990; 71:1535-1544. 42. Ribeiro BM, Crook NE. Expression of full-length and truncated forms of crystal protein genes from Bacillus thuringiensis subsp. kurstaki in a baculovirus and pathogenicity of the recombinant viruses. J Invertebr Pathol 1993; 62(2):121-130. 43. Chang JH, Choi JY, Jin BR et al. An improved baculovirus insecticide producing occlusion bodies that contain Bacillus thuringiensis insect toxin. J Invertebr Pathol 2003; 84:30-37. 44. Wang P, Granados RR. An intestinal mucin is the target substrate for a baculovirus enhancin. Proc Natl Acad Sci USA 1997; 94:6977-6982. 45. Wang P, Hammer DA, Granados RR. Interaction of Trichoplusia ni granulosis virus-encoded enhancin with the midgut epithelium and peritrophic membrane of four lepidopteran insects. J Gen Virol 1994; 75:1961-1967. 46. Popham HJR, Bischoff DS, Slavicek JM. Both Lymantria dispar nucleopolyhedrovirus enhancin genes contribute to viral potency. J Virol 2001; 75(18):8639-8648. 47. Ignoffo CM, Garcia C, Zuidema D et al. Relative in vivo activity and simulated sunlight-uv stablility of inclusion bodies of a wild-type and an engineered polyhedral envelope-negative isolate of the nucleopolyhedrosis virus of Autographa californica. J Invertebr Pathol 1995; 66:212-213. 48. Petrik DT, Iseli A, Montelone BA et al. Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme. J Invertebr Pathol 2003; 82:50-56. 49. Carbonell LF, Hodge MR, Tomalski MD et al. Synthesis of a gene coding for an insect-specific scorpion neurotoxin and attempts to express it using baculovirus vectors. Gene 1988; 73:409-418.

38

Patho-Biotechnology

50. Eldridge R, O’Reilly DR, Miller LK. Efficacy of a baculovirus pesticide expressing an eclosion hormone gene. Biol Control 1992; 2:104-110. 51. Li Q , Li L, Moore K et al. Characterization of Mamestra configurata nucleopolyhedrovirus enhancin and its functional analysis via expression in an Autographa californica M nucleopolyhedrovirus recombinant. J Gen Virol 2003; 84(Pt 1):123-132. 52. Hughes PR, Wood HA, Breen JP et al. Enhanced bioactivity of recombinant baculoviruses expressing insect-specific spider toxins in lepidopteran crop pests. J Invertebr Pathol 1997; 69:112-118. 53. Popham HJR, Pellock BJ, Robson M et al. Characterization of a variant of Autographa californica nuclear polyhedrosis virus with a nonfunctional ORF 603. Biol Control 1998; 12:223-230. 54. Gopalakrishnan B, Muthukrishnan S, Kramer KJ. Baculovirus-mediated expression of a Manduca sexta chitinase gene: Properties of the recombinant protein. Insect Biochem Mol Biol 1995; 25(2):255-265. 55. Lee SY, Q u X, Krause M. Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment. J Gen Virol 1997; 78:1. 56. Ma PWK, Davis TR, Wood HA et al. Baculovirus expression of an insect gene that encodes multiple neuropeptides. Insect Biochem Mol Biol 1998; 28:239-249. 57. Bonning BC, Ward VK, Meer Mv et al. Disruption of lysosomal targeting is associated with insecticidal potency of juvenile hormone esterase. Proc Natl Acad Sci USA 1997; 94(12):6007-6012. 58. Froy O, Zilberberg N, Chejanovsky N et al. Scorpion neurotoxins: Structure/function relationships and application in agriculture. Pest Manag Sci 2000; 56:472-474. 59. Korth KL, Levings CS. Baculovirus expression of the maize mitochondrial protein URF13 confers insecticidal activity in cell cultures and larvae. Proc Natl Acad Sci USA 1993; 90:3388-3392. 60. McCutchen BF, Betana MD, Hoover K et al. Interactions of recombinant and wild-type baculoviruses with classical insecticides and pyrethroid-resistant tobacco budworm (Lepidoptera: Noctuidae). J Econ Entomol 1997; 90:1170-1180. 61. Burden JP, Hails RS, Windass JD et al. Infectivity, speed of kill and productivity of a baculovirus expressing the itch mite toxin Txp-1 in second and fourth instar larvae of Trichoplusia ni. J Invertebr Pathol 2000; 75:226-236. 62. Glare TR, O’Callaghan M. Bacillus thuringiensis: Biology, Ecology and Safety. Chichester: J Wiley & Sons, 2000. 63. Federici BA. In: Bellows TS, Fisher TW, eds. Handbook of Biological Control. San Diego: Academic Press, 1999. 64. Schnepf E, Crickmore N, Van Rie J et al. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 1998; 62(3):775-806. 65. Charles JF, Nielson-LeRoux C, Delecluse A. Bacillus sphaericus toxins: Molecular biology and mode of action. Annu Rev Entomol 1996; 41:451-472. 66. de Maagd RA, Bravo A, Berry C et al. Structure, diversity and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu Rev Genet 2003; 37:409-433. 67. Agaisse H, Lereclus D. STAB-SD: A Shine-Dalgarno sequence in the 5ʹ untranslated region is a determinant of mRNA stability. Mol Microbiol 1996; 20(3):633-643. 68. Park HW, Ge BX, Bauer L et al. Optimization of Cry3A yields in Bacillus thuringiensis by use of sporulation-dependent promoters in combination with the STAB-SD mRNA sequence. Appl Environ Microbiol 1998; 64(10):3932-3938. 69. Federici BA, Park HW, Bideshi DK et al. Recombinant bacteria for mosquito control. J Exp Biol 2003; 206(Pt 21):3877-3885. 70. Park HW, Bideshi DK, Wirth MC et al. Recombinant larvicidal bacteria with markedly improved efficacy against culex vectors of west nile virus. Am J Trop Med Hyg 2005; 72(6):732-738. 71. Betz FS, Forsyth SF, Stewart WE. In: Laird M, Lacey LA, Davidson EW, eds. Safety of Microbial Insecticides. Boca Raton: CRC Press Inc., 1990:3-10. 72. Federici BA. Effects of Bt on nontarget organisms. J New Seeds 2003; 5:11-30. 73. Petrie K, Thomas M, Broadbent E. Symptom complaints following aerial spraying with biological insecticide Foray 48B. N Z Med J 2003; 116(1170):U354. 74. Valadares De Amorim G, Whittome B, Shore B et al. Identification of Bacillus thuringiensis subsp. kurstaki strain HD1-Like bacteria from environmental and human samples after aerial spraying of Victoria, British Columbia, Canada, with Foray 48B. Appl Environ Microbiol 2001; 67(3):1035-1043. 75. Siegel JP. The mammalian safety of Bacillus thuringiensis-based insecticides. J Invertebr Pathol 2001; 77(1):13-21. 76. Betz FS, Hammond BG, Fuchs RL. Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regul Toxicol Pharmacol 2000; 32(2):156-173. 77. Milner R, Samson P, Bullard G. FI-1045: A profile of a commercially useful isolate of Metarhizium anisopliae var. anisopliae. Biocontrol Sci Technol 2002; 12:43-58.

Improvement of Insect Pathogens as Insecticides through Genetic Engineering

39

78. Driver F, Milner R, Trueman J. A taxonomic revision of Metarhizium based on a phylogenetic analysis of rDNA sequence data. Mycol Res 2000; 104:134-150. 79. Khetan S. Microbial Pest Control. 1st ed. New York: Marcel Dekker Publishers, 2001. 80. Thomas M, Read A. Can fungal biopesticides control malaria? Nature Rev Microbiol 2007; 5:377-383. 81. St. Leger R. Metarhizium anisopliae as a model for studying bioinsecticidal host pathogen interactions. In: Vurro M, Gressel. J, eds. Novel Biotechnologies for Biocontrol Agent Enhancement and Management. Netherlands: Springer, 2007:179-204. 82. Langewald J, Kooyman C. Green muscle, a fungal biopesticide for control of grasshoppers and locusts in Africa. In: Vincent C, Goettel M, Lazarovitis G, eds. Biological Control, a Global Perspective. London: CABI International, 2007:311-318. 83. Scholte EJ, Knols BG, Samson RA et al. Entomopathogenic fungi for mosquito control: A review. J Insect Sci 2004; 4:19. 84. Scholte EJ, Ng’habi K, Kihonda J et al. An entomopathogenic fungus for control of adult African malaria mosquitoes. Science 2005; 308(5728):1641-1642. 85. Gressel J, Meir S, Herschkovitz Y et al. Approaches to and successes in developing transgenically enhanced mycoherbicides. In: Vurro M, Gressel J, eds. Novel Biotechnologies for biocontrol agent enhancement and management. Netherlands: Springer, 2007:277-296. 86. Charnley A. Fungal pathogens of insects: Cuticle degrading enzymes and toxins. Adv Bot Res 2003; 40:241-321. 87. Wraight S, Jackson M, Kock Sd. Production, stabilization and formulation of fungal biocontrol agents. In: Butt T, Jackson C, Morgan N, eds. Biocontrol Agents: Progress, Problems and Potential. London: CAB International, 2001:253-287. 88. Wang C, St Leger RJ. Developmental and transcriptional responses to host and nonhost cuticles by the specific locust pathogen Metarhizium anisopliae var. acridum. Eukaryot Cell 2005; 4(5):937-947. 89. Freimoser FM, Hu G, St Leger RJ. Variation in gene expression patterns as the insect pathogen Metarhizium anisopliae adapts to different host cuticles or nutrient deprivation in vitro. Microbiology 2005; 151(Pt 2):361-371. 90. St Leger R, Joshi L, Bidochka MJ et al. Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc Natl Acad Sci USA 1996; 93(13):6349-6354. 91. Wang C, St Leger RJ. A scorpion neurotoxin increases the potency of a fungal insecticide. Nat Biotechnol 2007; 25:1455-1456. 92. Wang C, St Leger RJ. A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proc Natl Acad Sci USA 2006; 103(17):6647-6652. 93. Fang W, Pei Y, Bidochka MJ. Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens. Can J Microbiol 2006; 52(7):623-626. 94. Leclerque A, Wan H, Abschutz A et al. Agrobacterium-mediated insertional mutagenesis (AIM) of the entomopathogenic fungus Beauveria bassiana. Curr Genet 2004; 45(2):111-119. 95. Inglis G, Goettel M, Butt T et al. Use of hyphomycetous fungi for managing insect pests. In: Butt T, Jackson C, Magan N, eds. Fungi as Biocontrol Agents. London: CAB International, 2001:253-287. 96. Roberts DW, St Leger RJ. Metarhizium spp., cosmopolitan insect-pathogenic fungi: Mycological aspects. Adv Appl Microbiol 2004; 54:1-70. 97. Drummond J, Heale J, Gillespie A. Germination and effect of reduced humidity on expression of pathogenicity in Verticillium lecani. Ann Appl Biol 1987; 111:193-201. 98. Bidochka MJ, Kamp AM, Lavender TM et al. Habitat association in two genetic groups of the insect-pathogenic fungus metarhizium anisopliae: Uncovering cryptic species? Appl Environ Microbiol 2001; 67(3):1335-1342. 99. Rangel DE, Braga GU, Anderson AJ et al. Influence of growth environment on tolerance to UV-B radiation, germination speed and morphology of Metarhizium anisopliae var. acridum conidia. J Invertebr Pathol 2005; 90(1):55-58. 100. Braga GU, Rangel DE, Flint SD et al. Conidial pigmentation is important to tolerance against solar-simulated radiation in the entomopathogenic fungus Metarhizium anisopliae. Photochem Photobiol 2006; 82(2):418-422. 101. Gressel J. Failsafe mechanisms for preventing gene flow and organism dispersal of enhanced microbial biocontrol agents. In: Vurro M, Gressel J, eds. Novel Biotechnologies for Biocontrol Agent Enhancement and Management. Netherlands: Springer, 2007:353-362. 102. Al-Ahmad H, Galili S, Gressel J. Tandem constructs to mitigate transgene persistence: Tobacco as a model. Mol Ecol 2004; 13(3):697-710. 103. St Leger RJ, Joshi L, Bidochka MJ et al. Characterization and ultrastructural localization of chitinases from Metarhizium anisopliae, M. flavoviride and Beauveria bassiana during fungal invasion of host (Manduca sexta) cuticle. Appl Environ Microbiol 1996; 62(3):907-912.

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104. St Leger RJ, Joshi L, Bidochka MJ et al. Biochemical characterization and ultrastructural localization of two extracellular trypsins produced by Metarhizium anisopliae in infected insect cuticles. Appl Environ Microbiol 1996; 62(4):1257-1264. 105. Bagga S, Hu G, Screen SE et al. Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene 2004; 324:159-169. 106. St Leger RJ, Frank DC, Roberts DW et al. Molecular cloning and regulatory analysis of the cuticle-degrading-protease structural gene from the entomopathogenic fungus Metarhizium anisopliae. Eur J Biochem 1992; 204(3):991-1001. 107. St Leger RJ, Cooper RM, Charnley AK. The effect of melanization of Manduca sexta cuticle on growth and infection by Metarhizium anisopliae. J lnvertebr Pathol 1988; 52:459-470. 108. Wang C, Hu G, St Leger RJ. Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation. Fungal Genet Biol 2005; 42(8):704-718. 109. Hu G, St Leger RJ. Field studies using a recombinant mycoinsecticide (Metarhizium anisopliae) reveal that it is rhizosphere competent. Appl Environ Microbiol 2002; 68(12):6383-6387. 110. Gongora C. Transformacion de Beauveria bassiana cepa Bb9112 con les genes de la proteina verde fluorescente y la protease pr1A de M. anisopliae. Rev Colom Entomol 2004; 30:1-6. 111. Flores A, Chet I, Herrera-Estrella A. Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1. Curr Genet 1997; 31(1):30-37. 112. Ahman J, Johansson T, Olsson M et al. Improving the pathogenicity of a nematode-trapping fungus by genetic engineering of a subtilisin with nematotoxic activity. Appl Environ Microbiol 2002; 68(7):3408-3415. 113. Wang C, St Leger RJ. The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects and the MAD2 adhesin enables attachment to plants. Eukaryot Cell 2007; 6(5):808-816. 114. Wang C, St Leger RJ. The Metarhizium anisopliae Perilipin Homolog MPL1 Regulates Lipid Metabolism, Appressorial Turgor Pressure and Virulence. J Biol Chem 2007; 282(29):21110-21115. 115. St. Leger R, Screen S. Prospects for strain improvement of fungal pathogens of insects and weeds. In: Butt T, Jackson C, Morgan N, eds. Fungal biocontrol agents: Progress, problems and potential. London: CAB International, 2001:219-238. 116. Sun X, Chen X, Zhang Z et al. Bollworm responses to release of genetically modified Helicoverpa armigera nucleopolyhedroviruses in cotton. J Invertebr Pathol 2002; 81(2):63-69. 117. Pal S, St Leger RJ, Wu LP. Fungal peptide Destruxin A plays a specific role in suppressing the innate immune response in Drosophila melanogaster. J Biol Chem 2007; 282(12):8969-8977. 118. Q uesada-Moraga E, CarrascoDíaz J, Santiago-Álvarez C. Insecticidal and antifeedant activities of proteins secreted by entomopathogenic fungi against Spodoptera littoralis (Lepidoptera, Noctuidae). J Appl Entomol 2006; 130:442-452. 119. Whetstone PA, Hammock BD. Delivery methods for peptide and protein toxins in insect control. Toxicon 2007; 49(4):576-596. 120. Edwards M, Gatehouse A. Biotechnology in crop protection: Towards sustainable insect control. In: Vurro M, Gressel J, eds. Novel Biotechnologies for Biocontrol Agent Enhancement and Management. Netherlands: Springer, 2007:1-24. 121. Isaka M, Kittakoop P, Kirtikara K et al. Bioactive substances from insect pathogenic fungi. Acc Chem Res 2005; 38(10):813-823. 122. Fan Y, Fang W, Guo S et al. Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase. Appl Environ Microbiol 2007; 73(1):295-302. 123. French-Constant RH, Dowling A, Waterfield NR. Insecticisal toxins from Photorhabdus bacteria and their potential use in agriculture. Toxicon 2007; 49:436-451. 124. Liu S, Li H, Sivakumar S et al. Virus-derived genes for insect resistant transgenic plants. In: Bonning BC, ed. Insect Viruses: Biotechnological Applications. San Diego: Academic Press, 2006:428-457.

Chapter 3

Phage Therapy:

A Trojan Horse Approach to the Control of Intracellular Pathogens Lawrence Broxmeyer*

Abstract

I

f biotechnology describes the exploitation of living systems or their products towards biomedical processes, then few entities have a greater biotechnological potential than the use of bacteriophages to cure human illness. Many researchers studying infectious diseases have little doubt that phage therapy has a contribution to make and will achieve mainstream medical relevance in the 21st century. This would come not a moment too soon. Today multi-drug-resistant and “extremely” drug-resistant forms of pathogens are reaching epidemic proportions and causing death on a global scale. But until recently, there seemed neither promise nor assurance that the use of such phage therapy would indeed kill pathogens intracellularly, in white blood cells, where they live and propagate; a necessary condition for cure. For this very reason many a National Institute of Health (NIH) grant application regarding phagotherapy had to be turned down. By late 2002, the question as to whether a relatively nonvirulent pathogen, armed with the right bacteriophage, could kill, intracellularly, a related yet virulent bacteria was addressed. Such a Trojan horse approach, would use threatening nonvirulent microbes to run search and destroy missions in the body’s macrophages against life threading, antibiotic-resistant pathogen. This study culminated in results of intracellular killing of two extremely virulent mycobacteria on a surprising scale.

Introduction

Although ever since Sinclair Lewis’s Arrowsmith popularized Felix D’Herelle’s epic laboratory saga of bacteriophage viruses, which ate and could destroy bacteria,1 the concept of injecting bacterial phage viruses into the body to kill serious bacterial disease has fascinated man. But the mechanics of such a systematic attack has had a checkered history. Yet long before penicillin was even known, in the early 1930s, drug giants Eli Lilly, ER Squibb and Abbott all manufactured “phage” preparations in the form of potions and injections. That was before a pivotal, poorly designed, 1934 US JAMA study by Eaton and Bayne-Jones would prove fatal, finding systemic phage therapy to have mixed results, concluding that body fluids strongly inhibit and destroy most bacteriophages before they could reach target tissue.2 Almost as if in answer, Dubos, discoverer of gramacidin, published one of the best early animal studies of phage treatment, supported by the US military.3 Dubos, answering virtually all of the scientific concerns raised in the flawed Eaton and Bayne—Jones JAMA report, used intraperitoneal phage at high concentrations to fight bacteria injected into the brain at lethal levels and showed that there was no problem with the phages reaching and destroying them, even in that privileged site. The phages multiplied rapidly there and remained at substantial levels in the blood as well, as long as there were sensitive bacteria in which *Lawrence Broxmeyer—MAR-Research Institute, 148-14A 11th Avenue, Whitestone, New York 11357, USA. Email: [email protected]

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they could reproduce. But despite this impressive study, antibiotics soon thereafter became seen as a much greater “magic bullet”, easily produced and applied, while phage workers in the West became diverted to molecular biology.

Waksman’s “Antibiotics”

Just how close the genesis of what Selman Waksman coined as “antibiotics” and phages were was no more clearly seen than in the discovery of Streptomycin. Both Dubos and Waksman were soil microbiologists, the same soil that bacteriophages are isolated from. Waksman had, since college, been almost fixated on one group of soil organisms in particular … the streptomycin family and called streptomyces. For some time it had been known that something in the soil was killing tuberculosis. Like Dubos before him, Waksman had discovered that soil microbes were the creators of certain chemicals which Waksman now called “antibiotics”. Chester Rhines experiment, of which Waksman was a part, had proven that tuberculous germs, when added to certain soil, died.4 Waksman thought he knew just what caused this … either a streptomyces or its chemical products. Eventually Waksman decided solely upon its chemical products or “antibiotics” as being responsible. But streptomyces, as an actinobacteria exactly like tuberculosis and of the genus mycobacteria, also was in a position to initiate a great deal of phage transfer between TB and itself, some of it deadly. The actinobacteria include some of the most common soil life. And the general gene sequence and chromosomal organization of other soil-born Mycobacteria besides TB, such as Mycobacterium smegmatis, was very similar to that of the closely related streptomyces, from which Waksman and Schatz, his codiscoverer, worked to isolate tuberculosis-killing streptomycin in well-manured soil.5 But both Schatz and Waksman ignored the possibility that bacteriophages, such as the mycobacteriophage discovered by Froman at Olive View in 1952 from closely related organisms, could also be behind the death that TB faced from certain soils. Today, of course, in this day of multi-drug-resistant (MDR) Staphycoloccus aureus (MRSA) and tuberculosis (MDR and “Extreme” TB), we realize that antibiotics are no longer infallible. Epidemics of drug resistant tuberculosis are currently raging in Europe and Asia6 and M. avium (fowl tuberculosis), for which there never was a satisfactory treatment, is described with increasing frequency in non-AIDS populations.7 On the other hand, phages adapt naturally and very quickly, as they have done for millions of years, so a sustained resistance by bacteria susceptible to a particular phage is unlikely. A limitation of most antimicrobial agents is that their modes of action require having the microbial target in active replication. Pathogens such as M. avium and M. tuberculosis, on the other hand, can have a long latent or dormant phase of infection in humans.8 The Russians took phages, the natural predators of bacteria, far more seriously than the West, soon producing phages 80% effective against serious enterococcal infections. But even in the States, a 1996 combined University of Texas-Austin and Emory study by Levin and Bull showed that mice infected with fatal doses of Escherichia coli had a 92% survival rate with phage therapy as opposed to a 33% survival rate with the antibiotic streptomycin.9

Intracellular Quest

But the gold standard, the acid test of bacteriophage therapy, had yet to be met. Such a study would show in no uncertain terms phages ability to kill intracellular bacteria, not ‘from without’, but rather to kill them in their intracellular macrophagal and monocyte habitats, the lack of proof of which brought constant criticism towards older studies. Thus, when Sula, in a 1981 Czech study, used parenterally injected mycobacteriophage DS-6A to destroy M. tuberculosis in guinea pigs with the approximate effectiveness of Isoniazid (INH), a first line TB drug,10 phage experts still questioned whether the killing was occurring intracellularly or extracellularly, just by virtue of the high phage concentrations he used. Nevertheless, Sula showed good clearing of all tuberculous lesions, including miliary nodules and granulomas in autopsied specimens. In focusing his attention towards tuberculosis, not only was Sula after a virulent disease which presently infects 1.8 billion people or one-third of the planet, but the leading cause of infectious death, with at least

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1.8 million lives snuffed-out annually and an estimated track record of 1 billion deaths between the years 1850 and 1950 alone.11 By the mid-1980s, tuberculosis, like a comeback plague, returned with a vengeance, both in the US and abroad. Menacing new strains resistant to known antibiotics emerged. Yet in the last 40 years, no new classes of anti-TB drugs had been developed.12 In his publication on phage therapy, Sula emphasized the subsequent need for a study on the phagotherapy of antibiotic resistant M. avium-intracellulare (MAI) or fowl tuberculosis, for which definitive antibiotic cure is still not possible.13 Since Sula though, not only have new phages been uncovered that attack MAI and TB, but so too has the development of ‘shuttle phasmid’ delivery systems. By 1982 Brenner used the term “shuttle phasmid” to denote phages that in the laboratory replicated in nonpathogenic, nondisease causing forms of microorganisms (certain E. coli and M. smegmatis) as plasmids (extrachromosomal, circular, genetic materials), while at the same time replicating in pathogenic bacteria as potentially lethal agents.14 Such a scenario opened up the theoretical possibility that bacteria, nonpathogenic to man and with the capacity to generate phages known to kill bacterial pathogens already in the body, could be parenterally injected to cure disease—all the while protecting, nurturing and delivering these viral phages. This phenomenon in nature is referred to as “lysogeny”. Lysogeny is how one colony of bacteria kills another by means of its phage weaponry without itself being harmed.15

Novel Concept

By late 2002, a phage treatment study appeared in The Journal of Infectious Diseases16 designed to prove, once and for all, that the intracellular killing of pathogens by phage was both possible and demonstrable. The study confirmed that at least inside the macrophage, bacteriophages, when properly introduced into a relatively nonvirulent microbe, had an intracellular killing rate for virulent AIDS human and fowl tuberculosis in excess of modern day antibiotics. The vehicle used was M. smegmatis, a moderately benign relative of tuberculosis17 found in smegma from human genitalia as well as soil, dust and water. A common environmental mycobacteria, first identified in 1884, Rich (18), described it as the smegma bacillus, citing it as a nonpathogenic acid-fast bacillus repeatedly found not only in man but other animals. Rich mentions that when injected into the body, smegmatis incites mononublear-like reactions, giant and epitheloid cells, but doesn’t survive very long in the tissues of warm-blooded animals, gradually disappearing.18 The thought behind our 2002 study16 was that perhaps a less virulent pathogen in the same family, armed with an appropriate phage, would perform systemic search and destroy missions against tuberculosis. Supporting this possibility was the fact that once systemic, M. smegmatis shares antigenic determinants (epitopes) with TB and the other mycobacteria, aiding in its approach to the target.19 In addition, there is evidence that phage activated smegmatis biosynthesizes a fat-splitting lipase,20 which could easily penetrate TB’s first line of defense; it’s heavily lipid laden shell.21 M. smegmatis was used to generate TM4 mycobacteriophage, a phage which were it compared to antibiotics, would be called “broad-spectrum” in that it infects both fast (M. smegmatis) and slow (M. tuberculosis) growing mycobacteria. TM4 was already known to have lytic action against MAI and TB and has been used extensively to deliver reporter genes to other mycobacteria.21 M. smegmatis was signaled out as the study’s veritable Trojan horse to attack M. tuberculosis and M. avium both by virtue of its relatively benign nature and the fact that lysogeny in nature usually occurs between like species. Inside smegmatis lay a host of TM4, the Greek soldiers within that horse. Of paramount interest was that neither smegmatis nor TM4 alone was able, in and of themselves, to kill virulent mycobacteria, but combined, they did.16 Thus, when the Russians, in a 1996 WHO inspired program for tuberculosis vaccine development, used M. smegmatis in mice without phage,22 pure M. smegmatis did not possess the sought after protective activity against tuberculosis, though, significantly, it did not have morbidity towards its subjects either.

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Results

In vitro both M. avium and M. tuberculosis are killed by phage TM4 outside the macrophage (Fig. 1). However, extracellular phage TM4 by itself could not kill intracellular mycobacteria lodged within. This was because phage therapy of any intracellular infection would require the internalization of the phages to the site where the pathogen is located within the cell. Both Figure 2 and Figure 3 show the dramatically increased killing of M. avium (fowel tuberculosis) when phage (TM4) was added to M.smegmatis and used to target M. avium. In the first case (Fig. 2) M. avium was given 24 hours to infect the macrophage before treatment with TM4 infected M. smegmatis was initiated. Whereas in Figure 3, 48 hours was given for M. avium to establish tissue infection before phage treatment was initiated. In both cases the result is exponential killing of M. avium. Similarly, in Figure 4, while neither M. smegmatis nor phage TM4 treatment by themselves was associated with killing intracellular Mycobacterium tuberculosis, the combination of M. smegmatis and phage TM4 was dramatic, with a 100-fold killing of intracellular tuberculosis after only 4 days. In Figure 5A, we see the actual fusing of a virulent avian tuberculosis with a phage laden M. smegmatis just prior to M. avium’s termination. Mycobacteria, such as tuberculosis do not like and are not able to thrive in acidic environments. To establish that after M. smegmatis/TM4 treatment that this intolerable acidity was established inside the mycobacterial milieu, acridine orange, a dye that concentrates specifically in acidic compartments and stains acidic bacteria green, was employed. In Figure 5B, where phage therapy with M. smegmatis/TM4 is already underway, hints of the acid

Figure 1. Effect of TM4 phage on Mycobacterium avium and Mycobacterium tuberculosis viability M. avium and M. tuberculosis were incubated with TM4 for 1 h and then the number of viable bacteria was determined over time. There was a rapid killing of M. avium following exposure to the phage (P < 0.05),compared with the unexposed control. An even more significant bactericidal effect (P < 0.01) was obtained when TM4 was incubated with M. tuberculosis. The number of M. avium and M. tuberculosis untreated with TM4 remained 1 ± 0.1 × 109 cfu for the 4 h of the experiment. (Reproduced from reference 16, © 2002 the Infectious Diseases Society of America 0022-1899/2002/18608-0014).

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Figure 2. Effect of treatment of RAW 264.7 macrophages infected with Mycobacterium avium for 24 h with Mycobacterium smegmatis carrying the TM4 lytic phage. RAW 264.7 macrophage monolayers were infected with M. avium for 24 h and later were treated with M. smegmatis or M. smegmatis carrying TM4. P < 0.05, macrophages treated with M. smegmatis/TM4 vs untreated macrophages at 2 and 4 days. P < 0.05, M. smegmatis/TM4 vs M. smegmatis treatments. (Reproduced from reference 16, © 2002 the Infectious Diseases Society of America 0022-1899/2002/18608-0014).

Figure 3. Effect of treatment of Mycobacterium avium-infected RAW 264.7 macrophages with Mycobacterium smegmatis carrying the TM4 lytic phage. Macrophage monolayers were infected with M. avium 109 for 48 h and then coinfected with M. smegmatis carrying TM4 or M. smegmatis alone. The number of viable intracellular bacteria was determined after 2 and 4 days. P < 0.05, untreated monolayers vs monolayers treated with M. smegmatis/ TM4 for 2 and 4 days. P > 0.05, monolayers treated with M. smegmatis vs M. smegmatis/ TM4. (Reproduced from reference 16, © 2002 the Infectious Diseases Society of America 0022-1899/2002/18608-0014).

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Figure 4. Effect of treatment of RAW 264.7 macrophages infected with Mycobacterium tuberculosis for 24 h with Mycobacterium smegmatis carrying the TM4 lytic phage. Macrophage monolayers were infected with M. tuberculosis H37Rv for 24 h and then coinfected with M. smegmatis carrying TM4 or M. smegmatis alone. The number of viable intracellular bacteria was determined after 2 and 4 days following coinfection. P < 0.05, all the comparisons vs treatment with M. smegmatis/TM4. (Reproduced from reference 16, © 2002 the Infectious Diseases Society of America 0022-1899/2002/18608-0014).

dye already being picked up by both mycobacteria are in evidence; whereas in Figure 5C non-acidic M. avium, not under M. smegmatis/TM4 attack, does not turn green with acridine orange. Finally, in Figure 5D both phage infected M. smegmatis and its intended victim, M. avium, turn green in the destructive acidic environment created by a M. smegmatis/TM4 attack.

Conclusion and Implications

It is clear that with regards to resistant, disseminated, intracellular infections such as M. avium and other mycobacteria that the newer antibiotics, including the macrolides, do not usually achieve bacterial killing in deep tissues such as the bone marrow and inside the body’s macrophage and monocyte defense.23 Recently Sleator and Hill concluded that given the increasing emergence of drug resistance and the inability of antibiotics to kill dormant organisms, that the Trojan horse approach to phage therapy represented yet another successful application of the patho-biotechnology approach.24 What then was this approach? It was a novel method devised to serve as a template to successfully kill an intracellular pathogen with bacteriophage. Although it centered on an attack against virulent M. avium and M. tuberculosis, it was by no means limited to these alone and a similar approach could be employed to destroy other drug resistant pathogens. Since phages, in contrast to antimicrobials, are not diffusible across membranes and at the same time vulnerable to body defenses and constitution, a strategy needed to be devised to deliver phage to intracellular pathogens. The study’s results showed that, when delivered to the site where the pathogenic bacteria resided inside the macrophage, TM4 was effective in lysing M. avium and, even more significantly; M.

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Figure 5. Fusion of Mycobacterium smegmatis and Mycobacterium avium phagosomes. A) Time lapse video microscopy showing fusion of M. avium and M. smegmatis vacuoles after 2 days of coinfection. B) Acridine orange staining of M. smegmatis in RAW 264.7 macrophages after 2 days of infection. Bacteria are observed incorporating the dye. C) M. avium (48 h)-infected macrophages stained with acridine orange. No bacterium incorporating the dye is seen, indicating that M. avium is in a nonacidic environment. D) RAW 264.7 macrophages infected with M. avium for 24 h and subsequently with M. smegmatis for 48 h. The figure shows M. smegmatis (long rods) and M. avium (short rods; arrowheads) stained with acridine orange, indicating that both are in acidic environment. (Reproduced from reference 16, © 2002 the Infectious Diseases Society of America 0022-1899/2002/18608-0014).

tuberculosis. In fact, the decrease in bacterial numbers after 4 days was similar to or better than the anti-M. avium effect obtained with macrolides clinically used in anti-tuberculous drugs.25 Similar but more compelling results were observed with M. tuberculosis. Because not all M. avium vacuoles fused with M. smegmatis vacuoles after 4 days (Fig. 5A), it is probable that a longer period of observation would have led to still increased, if not total, killing. The use of live attenuated ‘atypical mycobacteria’ systemically for vaccine, if not cure, is not new as evidenced by the widespread use of the even more virulent, dilute M. bovis in BCG vaccination. BCG is still extensively used and four of six studies have shown a relatively high degree of immunization against future TB infections.17 Although M. smegmatis is not considered a human pathogen, it can very occasionally cause skin or soft tissue disease which is sensitive to a variety of antibiotics.26 However M. smegmatis found in our genital secretions, is an uncommon pathogen in humans27 and disease from it is a rarity.28 Furthermore, although MTB is far more virulent than M. smegmatis, it cannot confer it’s virulence to M. smegmatis.29 There is much evidence in the literature that antibiotic resistant bacteria evolved from interspecies transfer of phages and that the germs Mankiewicz, for example, found in lung cancer were as a result of gene swapping through phages among the Actinomycetales to which the mycobacteria

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belong.30 Likewise in AIDS, multi-drug-resistant (MDR) and Extremely-Drug-Resistant (XTB) M. tuberculosis and M. avium can be traced to probable intraspecies phage transfer. In fact, many of man’s deadly bacterial epidemics were and are a byproduct of bacteria infecting other bacteria with their viral phages.31It is when such an attack does not kill, cripple or maim the opponent that antibiotic-resistant pathogens are born for mankind to cope with. And the proper way to deal with this is to learn from nature how bacteria kill one another—and then, recruit relatively benign bacteria to deliver lytic phage to destroy otherwise incurable infection through lysogeny.

References

1. Lewis S. Arrowsmith. New York: New American Library,1961. 2. Eaton MD. Bayne-Jones S bacteriophage therapy. Council on Pharmacy and Chemistry 1934, JAMA. 3. Dubos RJ. Straus JH multiplication of bacteriophages in vivo and its protective effect against an experimental infection with shigella dysenteriae. J Experiment Medic 1943; 78(3):161-168. 4. Waksman SA. The Conquest of Tuberculosis. Berkeley: University of California Press, 1964. 5. Salazar L. Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis. Mol Microbiol 1966; 20(2):283-93. 6. WHO Surveillance TWIGP Anti-tuberculosis drug resistance in the world Geneva: World Health Organization Global Tuberculosis Programme 1977. 7. Falkinham JO III. Epidemiology of infection by nontuberculosis mycobacteria. Clin Microbiol Rev 1996; 9:177-215. 8. Bloom B. Tuberculosis: Pathogenesis, Protection and Control. Washington, DC: American Society for Microbiology Press, 1995. 9. Levin BR, Bull JJ. Phage therapy revisited. The population biology of a bacterial infection and its treatment. Am Nat 1996; 147(6):881. 10. Sula L. Therapy of experimental tuberculosis in guinea pigs with mycobacterial phages DS-6A, GR-21T, MY-327. Czech Med 1981; 4(4):209-214. 11. Iseman MD. Evolution of drug resistant tuberculosis: A tale of two species. Proc Nat Acad Sci USA 1994; 91:2428-29. 12. Naik G. Agency to Unveil A Joint Assault on TB and HIV, 2002 Wall Street Journal. 13. Sula L, Sulova J, Stolcpartova M. Therapy of experimental tuberculosis in guinea pigs with mycobacterial phages DS-6A, GR-21 T, My-327. Czech Med 1981; 4:209-14. 14. Brenner S, Cesaveni G. Phasmids: Hybrids between CO1E1 plasmids and E. coli bacteriophage lambda. Gene 1982; 17:27-44. 15. Lessel EF. Stedman’s Medical Dictionary. 22nd Edition. Baltimore: The Williams & Wilkins Company, 1972:733. 16. Broxmeyer L, Sosnowska D, Miltner E et al. Killing of Mycobacterium avium and Mycobacterium tuberculosis by a Mycobacteriophage delivered by a nonvirulent Mycobacterium: A model for phage therapy of intracellular bacterial pathogens. J Infect Dis 2002; 186(8):1155-60. 17. Youmans GP, ed. Tuberculosis. Philadelphia: WB Saunders Company, 1979. 18. Rich AR. The Pathogenisis of Tuberculosis. 2nd ed. Springfield: Charles C. Thomas Publisher, 1946. 19. Pethe K, Puech V. Mycobacterium smegmatis lamin-binding glycoprotein shares epitopes with Mycobacteria tuberculosis heparin-binding haemagglutin. Mol Microbiol 2001; 39(1):89. 20. David HL, Jones WD. Biosynthesis of a Lipase by Mycobacterium smegmatis ATTC 607 infected by Mycobacteriophage D29. Am Rev Respir Dis, 1970; 102(5):818-20. 21. Ford ME, Stenstrom C. Mycobacteriophage TM4: Genome structure and gene expression. Tuber Lung Dis 1998; 79(2):63-73. 22. Eremeev VV, Maiorov KB. An experimental analysis of Mycobacterium smegmatis as a possible vector for the design of a new tuberculosis vaccine. Probl Tuberk 1996; 1:49-51. 23. Hafner B, Inderlied CB, Peterson DM et al. Correlation of quantitative bone marrow and blood cultures in AIDS patients with disseminated Mycobacterium avium complex infection. J Infect Dis 1999; 180:438-47. 24. Sleator RD, Hill C. Patho-biotechnology: Using bad bugs to do good things. Curr Opin Biotech 2006; 17:211-216. p.215. 25. Bermudez LE, Young LS. New drugs for the therapy of mycobacterial infections. Curr Opinion Infect Dis 1995; 8:428-38. 26. Wallace RJ Jr. Human disease due to Mycobacterium smegmatis. J Infect Dis 1988; 58(1):52-9. 27. Newton JA Jr, Weiss PJ. Soft-tissue infection due to Mycobacterium smegmatis: Report of two cases. Clin Infect Dis 1993; 16(4):531-3.

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28. Schreiber J, Burkhardt U. Non-tubercular mycobacterial infection of the lungs due to Mycobacterium smegmatis. Pneumologue 2001; 55(5):238-243. 29. Bange FC, Collins FM. Survival of mice infected with Mycobacterium smegmatis containing large DNA fragments from Mycobacterium tuberculosis. Tuber Lung Dis 1999; 79(3):171-80. 30. Mankiewicz E. Bacteriophares that lyse mycobacteria and corynebacteria and show cytopathogenic effect on tissue cultures of renal cells of cercopithecus aethiops. Canadian Med Assn J 1965; 92:31-33. 31. Redmond WB. Mycobacterial variations as influenced by phage and other genomic factors. Pneumonologie/Pneumonology 1970; 142:191-197.

Chapter 4

Bacterial Ghosts as Vaccine and Drug Delivery Platforms

Ulrike Beate Mayr, Verena Juliana Koller, Petra Lubitz and Werner Lubitz*

Abstract

T

he Bacterial Ghost (BG) Vaccine Platform Technology represents a particulate carrier system for protein subunit or DNA-encoded antigens endowed with intrinsic adjuvant properties. By all its biological background BG vaccines alert the immune system with signals for a bacterial infection and induce innate and adaptive immune responses against the antigens. Presentation of subunit vaccines within the BG complex is of advantage for the recognition of the target antigens by the immune system. Delivered as particle, to facilitate the uptake by professional antigen presenting cells (APC), BG satisfy the requirement of naturally furnished adjuvant particles for submit vaccine candidates. Such BG particles have a surface make-up which is not denatured and their surface adhesins are fully functional for the interaction with cellular receptors of APCs to induce the release of natural danger signals and cytokines characteristic for infections with real pathogens. The specificity for targeting tissues or cells, the easy method of production and the versatility in entrapping and packaging various compounds in different compartments of BG can be used for the creation of Advanced Drug Delivery Systems (ADDS). The original targeting functions of BG enable them to bind to and/or are being taken up by specific cells or tissues of animal, human or plant origin. The BG system represents a platform technology for creating new qualities in nonliving carriers which can be used for the specific targeting of drugs, DNA or other active compounds such as tumour cytostatics to overcome toxic or non desired obstacles. The new system is an alternative to liposomes and may have an advantage to its higher specificity for targeting different tissues, its easy way of production and its versatility in entrapping and packaging various compounds in different compartments of the carriers.

Introduction

In analogy to empty erythrocyte ghosts which are devoid of cytoplasmic content and to bacteriophage ghosts which are free of nucleic acids the empty envelope of Gram-negative bacteria produced by PhiX174 gene E-mediated lysis has been named Bacterial Ghost (BG). BG will be used in the following for a single bacterial ghost as well as for multiple bacterial ghosts. BG is already a common abbreviation for any type of Gram-negative cell envelope produced by expression of controlled expression of cloned PhiX174 gene E.1 BG have been produced from different bacteria including E. coli C, different E.coli K12 laboratory strains, enterotoxigenic E. coli (ETEC) and enterohemorrhagic E. coli (EHEC), Salmonella enterica serovar Typhimurium, S. enterititis, Shigella flexneri, Vibrio cholerae O1 and O139, Helicobacter pylori, Neisseria meningititis, Bordetella bronchiseptica, Actinobacillus pleuropneumoniae, Pasteurella multocida, Mannheimia haemolytica and Francisella tularensis LVS and Pectobacterium cypripedii. Except of E. coli C and K12 derivatives *Corresponding Author: Werner Lubitz—Department of Medicinal Chemistry, University of Vienna, Vienna, Althanstr. 14, UZA2 2B522, 1090 Vienna, and BIRD-C GmbH&CoKEG, Kritzendorf, Austria. Email: [email protected]

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and Pectobacterium cypripedii all bacterial strains used are pathogenic strain causing severe diseases in animals or in man. This disproportion of the number of BG produced from pathogenic versus nonpathogenic Gram-negative bacteria reflects one of the major applications of BG as vaccines. Investigations with E. coli C and K12 strains were mostly done for genetic engineering work to establish the BG system and with the plant-specific P. cypripedii for plant pesticide targeting.2 The other major application of BG in Advanced Drug Delivery Systems (ADDS) is in part an extension of our basic investigations how to produce and to modify BG and to use the empty cell envelops for biomedical applications. BG production is characterized by the introduction of a small hole in the envelope of the bacteria and release of the cytoplasmic content driven by the osmotic pressure difference between the cytoplasma and the outside growth medium of the bacteria. The process of E-mediated lysis of bacteria is driven by gene E expression and the cotranslational integration of the E-polypeptide into the inner membrane followed by conformational changes of E and represents an endogenous genetic intracellular method to inactivate bacteria.3 Gram-positive bacteria are simply killed by E causing break down of the membrane potential without loss of cytoplasmic material.4 BG have multiple applications in biomedicine as vaccines, as adjuvants, as DNA transfection vehicle for DNA vaccines or somatic gene transfer, as drug carrier for tumour therapy, miniature bioreactors, artificial bacterial life forms and last but not least as construction modules for molecular machines in micro- and nanotechnology. All these different applications are based on our knowledge how to produce and modify BG for specific purposes. In the following examples a focus is given to applications of the BG platform technology in vaccines and ADDS where the products are based on common components and production processes. More information on the BG vaccination trials can be found in several recent reviews where most of the original literature is cited1,5-8 as well as for the application as ADDS with schematic illustrations.9,10

Characterization of BG

Studies of the E-lysis process of Gram-negative bacteria emerged from basic science addressing both the lysis mechanism of bacteriophage PhiX174 after infection of Escherichia coli and, the specific mode of action of the cloned lysis gene E of the phage11-15 State of the art BG production requires the transformation of the host bacterium with a plasmid which carries the gene E under an inducible promoter. Since the gene E product is highly lethal the recipient bacteria it is necessary that the corresponding repressor system is either carried on the chromosome or on a plasmid encoding a repressor system which is able to raise a critical repressor concentration in the bacterium to silence gene E expression. Thus, the proper establishment of the genetic repression/expression system in a given Gram-negative bacterium determines the success of BG production. A series of plasmids have been developed which carry the gene E under various inducible expression control systems. The most elaborate system is derived from the phage Lambda left or right promoter/operator system with expression control by the thermosensitive cI857 repressor or derivatives providing the growth of bacteria up to 28, 36 and 39˚C and E-mediated lysis at any temperature 2˚C above the maximal repression temperature up to 42-44˚C for enterobacteriacae and most other bacteria.16 The loss of nucleic acids due to E-mediated lysis greatly minimizes the risk of horizontal gene transfer of pathogenic islands or antibiotic resistance genes which have been used for the plasmid system(s) used. As DNA can unspecifically adsorb to the inner membrane of BG “superclean” BG can be produced by expression and activation of the cloned Staphylococcus aureus nuclease (SNUC) which degrades DNA and RNA.2 The remaining DNA level of such BG preparations is below the real-time-PCR detection level and sets new quality criteria for inactivated vaccines diminishing the risk of horizontal gene transfer. A short characterization of protein E and SNUC is given in Table 1. Minimal amounts of protein E are required to lyse Gram-negative bacteria17 therefore the challenge in BG production is to assure the complete repression of gene E before induction of gene E expression. Induction of gene E, however, is not enough to achieve proper E-mediated lysis. There are additional requirements such as active growth and functional control elements of

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Table 1. Short characterization of PhiX174 protein E and Staphylococcus aureus nuclease A (SNUC) Protein E

Very hydrophobic 91 aa membrane-active structural polypeptide with no enzymatic activity

Mode of action

Fusion of inner and outer membrane of Gram-negative bacteria for release of progeny phages in PhiX174 infected cells or transmembrane tunnel formation for production of BG

SNUC

149 aa cytoplasmic protein from Staphylococcus aureus strain Foggi

Mode of action

Phosphodiesterase degrades ssDNA, dsDNA and RNA; addition of Ca-/Mg-ions stimulate DNAse activity for degradation of chromosomal and plasmid DNA in course of BG production when at least 99.9% of bacteria are converted to BG

cell division and of autolytic activity of the host bacteria. Membrane adhesion sites, FtsZ protein in the septosome,18 cis-trans proline isomerases for conformational change of protein E,19 chaperones, the strength of the membrane potential,20 the activity of the autolytic system,21 the ph and osmotic strength of the medium22 and other factors influence the E-lysis process. A reasonable BG production rate of a growing culture is approximately 99.9-99.99% and depends largely on exponential growth of the bacterial culture. Only growing bacteria can be lysed, bacteria entering stationary phase are phenotypically resistant to lysis.23 Although it seems to be trivial to E-lyse bacteria a good and efficient E-lysis needs experience and stringent process control. Investigations of the molecular mechanism of E-mediated lysis showed that protein E fuses the inner and outer membranes of Gram-negative bacteria, thereby forming a transmembrane lysis tunnel in the bacterial envelope through which the cytoplasmic content is released. High resolution transmission electron micrographs clearly show the intact structure of the bacterial envelope and of the continuity of the inner and outer membranes forming the border of the E-specific transmembrane.13 On average, the diameter of the E-specific transmembrane tunnel varies between 40 and 80 nm.13 The variation in size and irregular tunnel structures indicate that the E-specific transmembrane tunnel structure is dynamically formed by the strong force ejecting the cytoplasmic content through the E-lysis hole which is most probably formed by E-oligomers.24 Under normal bacterial growth conditions the osmotic pressure difference between the total solutes of the cytoplasma and the outside growth medium is more than 1 bar. Due to the integration of protein E in the inner membrane the paracrystaline peptidoglycan net located in the periplasmic space between the inner and outer membrane of the cell envelope structure of Gram-negative bacteria exhibits a higher turn-over rate at potential sites of lysis tunnel formation. As a consequence, the borders of the E-lysis tunnel are determined by the local mash size of the peptidoglycan which is the shape determining rigid structure of the bacteria. Except for the hole BG resemble their natural mother bacteria and in the outer surface of BG pili, flagella and lipopolysaccharide are well retained. The inner side of the BG envelope complex corresponds to the inside of the cytoplasmic membrane and its associated products which are not released by E-mediated lysis. As there is a hole in the envelope the membrane potential of BG is no longer existing whereas membrane integrated enzymes like the ATPases are still functional.20,25 The space between both membranes is the periplasmic space (PPS) which by its nature is a gel like environment rich in membrane derived oligosaccharides, specific enzymes, proteins and peptidoglycan. It should be mentioned that because of the fusion of the inner and outer membranes at the E-specific transmembrane lysistunnel the PPS is sealed.13,20 Again, enzymes specific for the PPS like alkaline phosphatase and beta-lactamase are still active. The outer membrane and all its appendices are functionally preserved which is of major importance for their targeting and adhesion properties. Thus, BG are highly sophisticated designed

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like their mother bacteria for adhesion and eventually uptake by cells or tissues and better equipped than any engineered liposome, which can be artificially produced. As the nature and the architecture of BG are important for their functions in the following sections two applications of BG namely BG as vaccines and BG as ADDS are discussed.

BG as Vaccines

There is no doubt that functional vaccines are the most effective medical interventions to safe lives and reduce costs in healthcare. Some of the traditional vaccines still in use do not meet the following requirements: (i) to be safe and immunogenic in young children, the adult and the elderly, (ii) include multiple serotypes/species, (iii) be inexpensive (less than a Euro per dose), easy to produce, stable without refrigeration and amenable for needle-free administration and, last but not least, (iv) should confirm robust immunity with three or less doses. As far as tested and calculated, BG vaccines meet most the criteria listed and provide improvement and substitution for existing vaccines and follow well the paradigm shift from single subunit vaccines to multiple antigen vaccines. BG are also excellent alternatives to vaccines which use chemicals, heat or irradiation to inactivate the pathogen. All these methods denature essential structural components of the bacteria whereas the E-lysis process for BG production is a genetic/biochemical method leading to a superior preservation of the antigenic properties. In the simplest form BG vaccines consist of a freeze dried powder of BG particles without any additions of stabilizers or adjuvant. The latter is very important as some of the adverse reactions against vaccine preparations originate from the adjuvants or stabilizers used. The BG system, however, is a potent vaccine delivery system with intrinsic adjuvant properties. Due to the particulate nature of BG and the fact that they contain many well known immune stimulating compounds such as LPS, lipid A and peptidoglycan BG enhance immune responses without any further additions. The results of different in vivo and in vitro studies confirm the potential of bacterial ghosts as carrier and adjuvant in modern vaccine development addressing key immune cells, such as dendritic cells, macrophages or monocytes via toll-like receptors or opsonized antibody facilitated uptake.26 Oral, intra-nasal, intra-ocular, intra-vaginal, rectal and aerogenic are the preferred routes of needle-free BG vaccine delivery. The oral immunization of rabbits with V. cholerae BG induced protective immunity determined with the RITARD test and conferred cross protection between classical O1 and the recently emerging O139 strain.27 100% protection levels against heterologous lethal challenge with EHEC was achieved in mice after two oral immunizations with EHEC BG.28 The same result could be obtained with a single rectal immunization of mice with EHEC BG (Mayr and Lubitz, to be published elsewhere). Here, it should be emphasized that rectal immunization could have a high value for vaccine delivery in children as accurate dosing is given by the supensorium and parents normally feel comfortable and are familiar with fever reducing supensorium. Intra-nasal immunizations with BG in mice gave good immune protection against Shigella infections. Aerosol immunization of pigs with Actinobacillus pleuropneumoniae BG induced sterile immunity against lethal bacterial challenge.29 BG have an ideal aerosol diameter of 1-3 um and can be prepared as dry or wet aerosols.30 The pathogen A. pleuropneumoniae and the experimental animal model (pigs) used for the former mentioned study are good models for human lung immunizations or for the aerosol delivery of drugs deep into the alveolar space.

BG with Extended Properties

The BG system offers a construction kit with a modular concept where different molecular parcels/bricks are assembled within a living cell by genetic engineering before BG formation is induced (Fig. 1). For specific needs and purposes genetic engineering of the host bacteria which are candidates for BG production can be used to modify their cell envelope to carry foreign proteins and/or to use such recombinant proteins to bind other proteins, DNA or active substances. Some of the applications of such modified BG are in vaccines whereas other find their application in BG as carrier of active substances or in more technical applications. It should be mentioned here that one of our future aims is the development of therapeutic BG combinations for drug targeting

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and vaccines in a single BG. In Table 1 vaccine applications and ADDS properties of engineered BG are discussed together. The following modules are in our construction kit for BG vaccine and BG carrier design:

i. Plain BG Vaccines

The BG envelope is either derived from a pathogen or from a nonpathogenic organism (Fig. 1). In the first case the envelope itself is sufficient to induce a relevant immune response in the host. In the latter case the BG can serve to stimulate the innate immune system.

ADDS

The BG from pathogens are also excellent targeting vehicles for primary immune cells, endothelial cells of blood vessels or intestine, or to recognize tumour tissue by specific ligand receptor interaction.

ii. BG as Carrier of Additional Proteins Vaccines

The BG envelope can be derived from a nonpathogenic bacterium and serves as carrier and adjuvants in regard to the target antigens, or can be derived from a pathogenic organism carrying out the same functions but representing a combination vaccine. The additional protein represents always multi-epitope antigens which can be presented in several ways, e.g., anchoring of the foreign protein to the inside of cytoplasmic membrane by fusing it to a N-, C- or N- and C-terminal membrane anchor (Fig. 1B). Fusion of the target antigens with the maltose binding protein or fusing it to an export sequence are methods to export foreign protein or polypeptide constructs to the periplasmic space. For the insertion of target antigens on the surface of the outer membrane OmpA-fusion can be used. Foreign or homologous pili can be inserted in the BG envelope which Table 2. Properties of BG and engineered BG in vaccines, ADDS and technical applications Vaccine

i. Plain BG

BG envelope vaccine; BG carrier of external loaded substances which Stimulants of innate have a natural affinity immune system to lipid membranes; water-soluble substances or polymers in cytoplasmic lumen

Micrometer carrier with 250 ft volume in cytoplasmic lumen; E-specific lysis tunnel with diameter of 40-100 nm

ii. BG as carrier BG as carrier of subunit vaccine; of additional combination vaccine; protein(s) BG piggy-back carrier of membrane vesicles from other bacteria

BG carrier of enzymes; BG carrier with Streptavidin as anchoring device for biotinylated products and vice versa

Micro enzyme reactor; BG with membrane vesicles attached at E-lysis tunnel influencing release properties; BG carrier of nano vesicle

iii. BG as carrier of nucleic acids

BG DNA delivery vehicle BG microparticle with for somatic gene transfer inside DNA architecture and/or for siRNA

BG DNA vaccine

ADDS

Micro-/Nano-/Pico-/ Femtotech/Other

Type of BG

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can either act as subunit vaccine or to broaden the specific receptor recognition repertoire of the BG. S-layer protein matrices formed by SbsA or SbsB can be modified to carry foreign inserts.31 As both proteins form sheet like self-assembly structures they are not expelled together with the cytoplasma and remain in the inner cytoplasmic lumen after E-mediated lysis. When SbsA or SbsB fusions are exported as maltose-binding protein fusion to the periplasmic space they still retain their self assembly capacity and fill this space with sheets carrying target antigens.

ADDS

If the foreign protein is an enzyme or a functional active polypeptide the same methods for internalization of the target protein as described above for antigens can be used. In addition, if streptavidin is used as foreign protein other biotinylated ligands can be added to the BG protein/ polypeptide carrier (Fig. 1B).32 In one of the application examples biotinylated dextran has been coupled to the cytoplasmic lumen of BG exposing membrane-anchored streptavidin (Fig. 1C). Such polymers themselves can be substituted with drugs and represent a slow release system. In a specific formulation with in vivo biotinylated E or E-Streptavidin fusions which are still lysis active the ligand receptor moieties of the fusion constructs are exposed on the BG surface surrounding the E-specific lysis tunnel (Fig. 1D). Any streptavidin or biotinylated new compound, respectively, can be directed to bind to this crown like ligand ring (Fig. 1E-1C). For example, small vesicles have been bound to the BG carrier piggy-backing inside out vesicles of other bacteria. It is also feasible to construct net-like streptavidin-biotin chimneys of variable length on top of the E-lysis tunnel structure to form nano tubular structures (Fig. 1F) which can modify the release properties of the BG carrier filled with water soluble substances (Fig. 1G).

BG as Carrier of Nucleic Acids Vaccines

BG are derived from nonpathogenic or pathogenic bacteria and serve as carrier of DNA and provide the DNA vaccine targeting functions for antigen-presenting cells and a highly efficient intracellular DNA release from the endosome-lysosome compartment. The high transformation and target gene expression rates of dendritic cells (75%) and for macrophages (60%) with DNA-BG carriers can compete with commercial transfection systems. The method for filling BG with DNA is rather simple and in its standard version freeze dried BG are resuspended in a DNA solution and after washing off the excess of DNA which is not bound to the inside of the cytoplasmic membrane, the DNA-BG can either be used immediately for gene transfer experiments or can be stored after freeze drying for later applications. The number of plasmids per BG depends on the concentration of the DNA solution used and as greater the DNA concentration the greater the DNA loaded per BG. Loading of BG with DNA is very efficient as more than 3,000 copies of a medium sized plasmid can be bound per BG.33 A more sophisticated version of in vivo loading BG with DNA uses the specific interaction of an inner membrane anchored DNA binding protein with the corresponding operator region on target plasmid (Fig. 1H). This allows a one-step process for creation of DNA loaded BG vaccines.28

ADDS

For the application of DNA in somatic gene transfer it is desirable to use DNA constructs devoid of antibiotic resistance cassettes and origin of replication of the production plasmid and to design minimal minicircles including an eukaryotic promoter for the gene of interest with poly A tail and transcription stop sequences. The improved version of our self-immobilisation plasmids encodes the par A resolvase which is a specific DNA recombination enzyme recognizing homologous sequences on the plasmid. After expression of the par A resolvase and the inner membrane anchored DNA binding protein only minicircle DNA is bound to the inner membrane. By E-mediated lysis BG are produced with in vivo loaded minicircle DNA.34 In addition to the functional aspects of DNA encoding genes or siRNA for regulation of gene expression, DNA can also be considered as pure

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Figure 1, legend viewed on following page.

structural element for construction purposes to architecture for instance the inner lumen of BG and to serve as docking molecule for other DNA or RNA tailed molecules.

BG as Carrier Vehicles for DNA and/or Cytostatic Drugs in Tumour Therapy

DNA and/or drugs can be used as active substances for tumour treatment and investigations have shown that human melanoma and colon carcinoma cells can be targeted with BG for delivery of DNA or drugs, respectively. In a recent study, eight different human melanoma cell lines which have many functions in common with antigen presenting cells35,36 have been investigated for their

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Figure 1. Schematic line drawings of different entities from the BG construction kid. A) Empty BG, inner line corresponds to the inner membrane and outer line to the outer membrane, the space between both lines corresponds to the periplasmic space. B) BG with inner memor in vivo biotinylation C—terminal amino acid sequence brane anchored streptavidin C) BG as carrier of membrane immobilized polymers: , inner membrane anchored , biotinylated polymere, blue and green line represent different polymers, streptavidin; e.g., dextran and polyhydroxybutyric acid. D) BG closed with vesicles attached by specific streptavidin—biotin interaction at E—specific transmembrane lysis tunnel: , protein E with in vivo C-terminal biotinylation site; , inner membrane anchored streptavidin on the outside of inside-out membrane vesicles from Gram-negative bacteria. E) Detail view of in side out vesicles closing the E—specific lysis tunnel. Membrane anchored streptavidin or biotinylated membrane anchor are either expressed on the in side out vesicle or at the E—lysis protein. F) Streptavidin—biotin net-like chimneys of variable length which connect the in side out vesicle with the E—lysis tunnel. G) BG as carrier of water-soluble active substances, water space with . H) BG as carrier of inner membrane anchored solubilized substances leaking out of BG, . DNA binding protein, e.g., LacI binding operator site of plasmid or minicircle DNA,

capacity to bind and phagocytise BG.38 Bowes cells exhibited roughly 80% expression level of BG delivered marker gene indicating that the BG system is suitable as vehicle for the transfer of DNA encoded functional RNA such as siRNA, enzymes for pro-drug conversion or inducer of other activities with therapeutic effects for the host. Recent investigations of binding polyphenolic compounds to BG confirm the finding that molecules with organic ring structures bind unspecifically to the membrane compartments of BG and add a group of new compounds to the list of active therapeutic substances for intracellular delivery. Treatment of the human colon cancer cell line CaCo2 with doxorubicin (DOX) loaded BG showed an effect on proliferation inhibition by two log difference compared to the free drug.39 In extended investigations it was determined that DOX BG reduced the growth inhibitory concentrations of the drug up to more than 300 times compared to the EC50 of the free drug and enhanced the intracellular DOX concentrations up to 42 times compared to the free drug (Mader and Lubitz, to be published elsewhere). These results are promising as DOX for instance has accumulating side effects on heart functions and the tumour therapy often has to be terminated once a critical maximal dose is reached. It is not envisaged to apply DOX BG intravenously but rather topically in combination with surgery by short term rinsing or flushing of the colon to deliver the therapeutic cargo. We hope that with this procedure the time window for DOX therapy can be considerably extended and can also contribute to an increased quality of life for the patients as side reactions to the drug are also less severe. The concept to specifically target tumour tissue in combination with surgery could probably also be applied to other tumours such as head and neck cancer and brain tumours as certain BG have a surface make-up that allow them to bind to and being taken up by the tumour cells. The important benefit of BG drug delivery is the increased bioavailability due to the bioadhesive nature of BG envelope surface targeting tumour tissue and the unique intracellular release mechanism of the drug.

Acknowledgment

The authors thank Christine Sladek for artwork.

References

1. Walcher P, Mayr UB, Azimpour-Tabrizi C et al. Antigen discovery and delivery of subunit vaccines by nonliving bacterial ghost vectors. Expert Rev Vaccines 2004; 3(6):681-91. 2. Hatfaludi T, Liska M, Zellinger D et al. Bacterial ghost technology for pesticide delivery. J Agric Food Chem 2004; 52(18):5627-34. 3. Schön P, Schrot G, Wanner G et al. Two-stage model for integration of the lysis protein E of phi X174 into the cell envelope of Escherichia coli. FEMS Microbiol Rev 1995; 17(1-2):207-12. 4. Halfmann G, Gotz F, Lubitz W. Expression of bacteriophage PhiX174 lysis gene E in Staphylococcus carnosus TM300. FEMS Microbiol Lett 1993; 108(2):139-43.

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5. Jalava K, Eko FO, Riedmann E et al. Bacterial ghosts as carrier and targeting systems for antigen delivery. In: Dietrich G, Goebel W, eds. Vaccine Delivery Strategies. Wymondham: Horizon Scientific Press, 2002;163-184. 6. Jalava K, Hensel A, Szostak M et al. Bacterial ghosts as vaccine candidates for veterinary applications. J Control Release 2002; 85(1-3):17-25. 7. Jalava K, Eko FO, Riedmann E et al. Bacterial ghosts as carrier and targeting systems for mucosal antigen delivery. Expert Rev Vaccines 2003; 2(1):45-51. 8. Mayr UB, Walcher P, Azimpour C et al. Bacterial ghosts as antigen delivery vehicles. Adv Drug Deliv Rev 2005; 57(9):1381-91. 9. Tabrizi CA, Walcher P, Mayr UB et al. Bacterial ghosts—Biological particles as delivery systems for antigens, nucleic acids and drugs. Curr Opin Biotechnol 2004; 15(6):530-7. 10. Paukner S, Stieol T, Kuoela P et al. Bacterial ghosts as a novel advanced targeting system for drug and DNA delivery. Expert Opin Drug Deliv 2006; 3(1):11-22. 11. Blasi U, Henrich B, Lubitz W. Lysis of Escherichia coli by cloned phi X174 gene E depends on its expression. J Gen Microbiol 1985; 131(Pt 5):1107-14. 12. Henrich B, Lubitz W, Plapp R. Lysis of Escherichia coli by induction of cloned phi X174 genes. Mol Gen Genet 1982; 185(3):493-497. 13. Witte A, Wanner G, Bläsi U et al. Endogenous transmembrane tunnel formation mediated by phi X174 lysis protein E. J Bacteriol 1990; 172(7):4109-14. 14. Witte A, Wanner G, Lubitz W et al. Effect of phi X174 protein E-mediated lysis on murein composition of Escherichia coli. FEMS Microbiol Lett 1998; 164(1):149-57. 15. Haidinger W, Mayr UB, Szostak MP et al. Escherichia coli ghost production by expression of lysis gene E and staphylococcal nuclease. Appl Environ Microbiol 2003; 69(10):6106-13. 16. Jechlinger W, Szostak MP, Witte A et al. Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli. FEMS Microbiol Lett 1999; 173(2):347-52. 17. Witte A, Wanner G, Lubitz W et al. Phi X174 protein E-mediated lysis of Escherichia coli. Biochimie, 1990; 72(2-3): 191-200. 18. Witte A, Brand E, Mayrhofer P et al. Mutations in cell division proteins FtsZ and FtsA inhibit phiX174 protein-E-mediated lysis of escherichia coli. Arch Microbiol 1998; 170(4):259-68. 19. Witte A, Schrot G, Schön P et al. Proline 21, a residue within the alpha-helical domain of phiX174 lysis protein E, is required for its function in Escherichia coli. Mol Microbiol 1997; 26(2):337-46. 20. Witte A, Lubitz W, Bakker EP. Proton-motive-force-dependent step in the pathway to lysis of Escherichia coli induced by bacteriophage phi X174 gene E product. J Bacteriol 1987; 169(4):1750-2. 21. Halfmann G. Different sensitivity of autolytic deficient Escherichia coli mutants to the mode of induction. FEMS Microbiology Letters 1984; (24):205-208. 22. Lubitz W, Halfmann G, Plapp R. Lysis of Escherichia coli after infection with phiX174 depends on the regulation of the cellular autolytic system. J Gen Microbiol 1984; 130(Pt 5):1079-87. 23. Witte A, Lubitz W. Biochemical characterization of phi X174-protein-E-mediated lysis of Escherichia coli. Eur J Biochem 1989; 180(2):393-8. 24. Blasi U, Linke RP, Lubitz W. Evidence for membrane-bound oligomerization of bacteriophage phi X174 lysis protein-E. J Biol Chem 1989; 264(8):4552-8. 25. Witte A, Wanner G, Sulzner M et al. Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli. Arch Microbiol 1992; 157(4):381-8. 26. Riedmann EM, Kyd JM, Cripps AW et al. Bacterial ghosts as adjuvant particles. Expert Rev Vaccines 2007; 6(2):241-53. 27. Eko FO, Schukovskaya T, Lotzmanova EY et al. Evaluation of the protective efficacy of Vibrio cholerae ghost (VCG) candidate vaccines in rabbits. Vaccine 2003; 21(25-26):3663-74. 28. Mayr UB, Haller C, Haidinger W et al. Bacterial ghosts as an oral vaccine: A single dose of Escherichia coli O157:H7 bacterial ghosts protects mice against lethal challenge. Infect Immun 2005; 73(8):4810-7. 29. Hensel A, van Leengoed LA, Szostak M et al. Induction of protective immunity by aerosol or oral application of candidate vaccines in a dose-controlled pig aerosol infection model. J Biotechnol 1996; 44(1-3):171-81. 30. Katinger A, Lubitz W, Szostak MP et al. Pigs aerogenously immunized with genetically inactivated (ghosts) or irradiated Actinobacillus pleuropneumoniae are protected against a homologous aerosol challenge despite differing in pulmonary cellular and antibody responses. J Biotechnol 1999; 73(2-3):251-60. 31. Kuen B, Lubitz W. Analysis of S-lyer proteins and genes. in Crystalline Bacterial Cell Surface Proteins 1996; 77-102.

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32. Huter V, Szostak MP, Gampfer J et al. Bacterial ghosts as drug carrier and targeting vehicles. J Control Release 1999; 61(1-2):51-63. 33. Paukner S, Kudela P, Kohl G et al. DNA-loaded bacterial ghosts efficiently mediate reporter gene transfer and expression in macrophages. Mol Ther 2005; 11(2):215-23. 34. Jechlinger W, Azimpour Tabrizi C, Lubitz W et al. Minicircle DNA immobilized in bacterial ghosts: in vivo production of safe nonviral DNA delivery vehicles. J Mol Microbiol Biotechnol 2004; 8(4):222-31. 35. Brady MS, Lee F, Petrie H et al. CD4(+) T-cells kill HLA-class-II-antigen-positive melanoma cells presenting peptide in vitro. Cancer Immunol Immunother 2000; 48(11):621-6. 36. Curiel-Lewandrowski C, Demierre MF. Advances in specific immunotherapy of malignant melanoma. J Am Acad Dermatol 2000; 43(2 Pt 1):167-85; quiz 186-8. 38. Kudela P, Paukner S, Mayr UB et al. Effective gene transfer to melanoma cells using bacterial ghosts. Cancer Lett 2007; Sec 28 PMID-18164809 ahead of print. 39. Paukner S, Kohl G, Lubitz W. Bacterial ghosts as novel advanced drug delivery systems: Antiproliferative activity of loaded doxorubicin in human caco2 cells. J Control Release 2004; 94(1):63-74.

Chapter 5

In Vivo Remote Control of Bacterial Vectors for Prophylaxis and Therapy Holger Loessner* and Siegfried Weiss

Abstract

T

he use of live attenuated bacteria as prophylactic vaccines has a proven track record in human and veterinary medical praxis. In addition, bacteria-based medicines are currently developed for the treatment of diseases such as cancer, gene deficiencies, autoimmunity and allergy. Treatment of these diseases often requires an individual therapeutic approach for each patient. Hence, bacterial vectors have to be designed in a way that allows the delivery of therapeutic molecules in a spatial, temporal and quantitative deliberate manner. In order to achieve this goal, we and others have recently established the concept of in vivo remote control (IVRC) of bacterial vectors. IVRC is based on regulatory systems that mediate tight inducible expression of therapeutic molecules by the bacterial vector residing in cells or tissues of the patient upon external stimulation by physical means or administration of inducer drugs. In addition, the development of systems that allow IVRC expression of homologous or heterologous genes in bacteria constitute valuable tools for the functional analysis of host-pathogen interactions.

Introduction

The human body provides many niches for nonpathogenic and pathogenic bacteria. For instance, commensal bacteria of the gastrointestinal tract exceed the number of all cells of the human body. On the other hand, pathogenic bacteria have evolved a plethora of mechanisms to invade niches provided by the host, for example by diverting defense reactions of the immune system. The great potential of microorganisms to be used for the benefit of man has been recognized long time ago and is exploited in medical praxis today. Thus, the use of live attenuated bacteria as vaccines or the use of commensal bacteria as nutritional supplement, so called probiotics, is well established.1,2 However, such applications were worked out often intuitively without knowledge of the underlying mechanisms. Now, the elucidation of the complex interactions between microorganisms and corresponding hosts progresses rapidly. This will eventually provide a new base for the frequent uses of live microbial medicines. In contrast to homogenous bacteria the medical use of engineered bacteria as delivery systems for heterologous proteins or nucleic acids is still in its infancy. A multitude of applications for bacterial vectors has been investigated preclinically and in some instances such vectors have been tested in clinical studies.3 Most work in this regard was focused on the development of recombinant live bacterial vaccines. However, another exciting example is the use of tumor targeting bacteria for the delivery of anticancer drugs as well as for diagnostic purposes to detect cancerous tissue.4-6 In addition, a proficiency of several bacteria to deliver nucleic acids to host cells have been discovered. Therefore, the use of bacteria as vectors for therapeutic genes, for the delivery of DNA vaccines or even therapeutic RNA molecules holds great promise.7-9 Furthermore, commensal bacteria *Corresponding Author: Holger Loessner—Department of Molecular Immunology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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have been equipped with modules for the expression of enzymes or the secretion of therapeutic molecules, thus providing new options for the amelioration of metabolic disorders, allergies or autoimmune diseases.10-13 Reasons for the slow introduction of live recombinant bacterial medicines into clinical praxis are primarily safety concerns.14,15 Nevertheless, several other shortcomings need to be addressed in addition: (i) At present, neither adjustment of bacterial effector functions nor monitoring of bacterial colonization is possible as soon as the therapeutic bacteria are administered to the patient. ii) Bacterial vectors are mostly derived from pathogenic bacteria by attenuation. Risks associated with residual virulence of such strains often remain, e.g., for immunocompromised patients. iii) In order to produce heterologous proteins, carrier strains are often equipped with plasmids for constitutive expression (Fig. 1A). Upon administration to the recipient animal or patient, such carrier strains due to the metabolic burden of constitutive expression usually display a reduced colonization capability.14 iv) In absence of antibiotic selection in vivo conventional plasmids are often quickly lost.16 As a solution so called in vivo inducible (IVI) promoters have been introduced into vaccine strains in order to reduce heterologous gene expression during the administration phase (Fig. 1B).17-19 IVI promoters are derived from genes of the carrier strain, which are induced at defined stages during the course of host invasion and colonization. However, IVI promoters cannot be regulated deliberately from the outside. The requirement for external control of therapeutic gene expression becomes most evident in case of anti-cancer bacteria. For tumor eradication, bacteria should be designed in a way that allows deliberate adjustment of therapeutic gene expression during ongoing treatment. Moreover, in case of complications clinicians should have the option to remove the anti-cancer bacteria without time delay. Antibiotics are not sufficient in this case since the time required for successful bacterial removal might be to long. In order to achieve this goal, the concept of in vivo remote control (IVRC) of bacterial vectors has been established.20-22 An IVRC bacterial vector contains an appropriate inducible promoter that is used to regulate the expression of heterologous genes in a temporal and quantitative deliberate manner after the vector strain has already colonized the target host tissue (Fig. 1C). Promoters suitable for IVRC can either be regulated by physical means or the administration of an inducer drug. Such physical means or inducer drugs ideally should fulfill the following prerequisites: (i) they should be safe and not cause any adverse effects, (ii) they should be non-immunogenic and applicable in a repetitive manner, (iii) they should be sufficiently persistent in order to induce sustained gene expression, (iv) they should disseminate throughout the colonized tissue in order to reach all bacteria, (v) they should be of high patient compliance and (vi) their use should be clinically feasible. The choice of a suitable genetic switch for IVRC of bacterial vectors depends on the colonized body niche and the specific requirements of the medical intervention. In this respect, the different properties of physical means or inducer drugs have to be carefully considered (Table 1). Moreover, compatibility of regulatory systems has to be ensured when multiple IVRC modules are implemented within the same bacterial vector strain (Fig. 1D). In the subsequent sections we will review properties of regulatory systems, which were either used or which have the potential for IVRC of bacterial vectors to be applied for various biomedical applications, in particular tumor therapy.

Remote Control of Bacterial Vectors by Physical Means

Bacteria have evolved regulatory systems in order to sense different environmental stimuli or stress of physical nature, e.g., radiation, temperature, magnetism, light or ultrasound. Such systems mediate adaptive responses of the bacteria affecting their physiologic state, DNA repair proficiency, expression of virulence factors, motility or other functions.23 The dissection of these systems has led to the characterization of individual promoters, which can be induced by physical means in a defined manner. Radiation inducible eukaryotic promoters have been studied extensively and have been tested for targeted cancer gene therapy in clinical trials.24 Similarly, in bacteria a number of radiation inducible promoters are known. The use of such promoters for IVRC of

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anti-cancer bacteria was pioneered by Nuyts et al.20,25 They found the promoter of the recA gene (PrecA) to be suited for IVRC of therapeutic gene expression by tumor targeted Clostridia. RecA is a prominent member of the SOS-repair system.26 DNA damage caused by ionizing radiation increases the proteolytic activity of RecA giving rise to the cleavage of the LexA repressor protein. As a result, transcription from LexA regulated promoters increases, including PrecA. Nuyts et al initially constructed recombinant Clostridia in which they linked PrecA with genes encoding mouse TNF-alpha or Escherichia coli (E. coli) cytosine deaminase. In tumor therapy, the cytokine TNF-alpha has been shown to exert cytotoxicity on tumor cells, to activate cellular immune responses and to destruct tumor microvasculature,27 while the prodrug-converting enzyme cytosine deaminase acts by converting the nontoxic compound 5-fluorocytosine (5-FC) into the cytotoxic agent 5-fluorouracil (5-FU).28 The treatment was started by intravenous administration of about 108 recombinant Clostridia endospores to tumor bearing mice (Fig. 2A). Endospores germinate within the necrotic core of the solid tumor. Subsequent rapid vegetative proliferation ensues until ∼109 bacteria have accumulated within one gram of tumor tissue. Importantly, only the tumor provides a growth niche for obligate anaerobic Clostridia due to the hypoxic conditions in the necrotic inner core. Moderate targeted irradiation of colonized tumors, at a dose of two Gray, was sufficient to elevate expression of therapeutic molecules by the bacteria (Fig. 2A). In addition to the direct therapeutic effects exerted by TNF-alpha or 5-FU, both molecules are known to sensitize tumor cells for irradiation.24 Therefore, synergy between irradiation and bacteria-mediated tumor therapy might be achievable. The development of magnetic nanoparticles for cancer treatment and diagnosis is intensively explored presently.29,30 Upon systemic administration such particles can be guided to tumors either by covalently linked targeting moieties, e.g., antibodies, or, alternatively, by a directed magnetic

Figure 1. Conventional and IVRC expression strategies of heterologous protein by bacterial vectors in vivo. A) Conventionally, heterologous antigen (Ag) expression by the bacteria is driven by a constitutive promoter throughout the observation period. The time point of administration of recombinant bacteria to the recipient host is indicated by the dotted line. B) IVI promoters initiate heterologous antigen expression when bacteria have invaded their specific target cellular compartment or tissue of the recipient. Bacteria are not metabolically weakened by antigen expression during the colonization phase. C) An IVRC regulatory system in conjunction with the deliberate application of the corresponding inducer stimuli (indicated by arrow). It allows a heterologous protein to be repeatedly expressed by bacterial vectors residing in host tissue at defined time points and in a dosed manner. D) The use of several IVRC regulatory systems in parallel enables the establishment of multifunctional bacterial vectors in which several factors can be induced independently.

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Table 1. Comparison of general properties of physical means and inducer drugs suitable for IVRC Properties of Inducer

Physical Means e.g., Ionizing Radiation, Magnetism

Inducer Drugs e.g., L-Arabinose, Xylan, Aspirin

Tissue damage

Damage of healthy tissue by ionizing Non-toxic in appropriate dosage irradiation, exact targeting and dosing necessary

Immunogenicity

Non-immunogenic, repetition possible

Depending on drug: non-immunogenic, immunogenic or immunosuppressive

Targeting

Spational and quantitative dosing feasible

Mucosal and systemic administration possible, distribution via circulation, site-specific direct injection possible

Persistence

Non-persistent

Short or prolonged persistent depending on the rate of drug degradation and/or excretion

Tissue permeation

Varying degree of tissue penetration depending on the nature of the physical means

Varying degree of tissue penetration depending on the properties of the drug

Patient compliance

Irradiation low, others high

Varying degree, high for oral administration of inducer drug

Feasibility

High cost, specialist clinicians and high-tech equipment required

Low cost, easy to administer and low-tech equipment sufficient

field. Natural occurring magnetotactic bacteria are able to orient and migrate along magnetic field lines.31 Such bacteria contain intracellular magnetic structures, so called magnetosomes, which comprise nanometer-sized, membrane-enclosed crystals of the magnetic iron minerals magnetite (Fe3O4) or greigite (Fe3S4). Potentially, engineered bacteria harbouring magnetosomes could be used for the delivery of anti-cancer drugs.32 Such bacteria could be manipulated and monitored in several ways in an IVRC-like manner. First of all, targeting of anti-cancer bacteria could be directed by an external magnetic field to the tumor. Exposure of the colonized tumor to hyperthermia could excite bacterial magnetosomes which could mediate a toxic effect on tumor cells.33 In a similar way, the bacteria could be disrupted and cleared from the tissue which would constitute an attractive safety measure. Finally, magnet resonance tomography (MRT) could be applied to visualize bacteria during ongoing treatment within the patient. However, magnetotaxis is a complex phenotype with involvement of genes of yet unknown function.34,35 Therefore, the engineering of remote controlled magnetotactic bacterial vectors requires further investigation and at this point can only be envisioned. Moreover, many regulatory systems responsive to various physical conditions such as temperature, ultrasound, light etc. have been studied in the past. Their suitability for IVRC has yet to be established.36-38

Drug-Mediated Remote Control of Bacterial Vectors

Like physical conditions, a great variety of substances can be sensed by bacterial regulatory systems. A number of bacterial promoters inducible by various substances have been characterized in detail and have been found useful for the regulated expression of heterologous proteins in vitro.39 However, in order to use these promoters for IVRC of bacterial vectors in medical applications the respective inducer substance has to fulfill ideally all the prerequisites mentioned before (see

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Figure 2. IVRC of tumor targeted bacterial vectors. A) Radiation-mediated IVRC of recombinant anti-cancer Clostridia. Recombinant Clostridia harbor an expression plasmid for a therapeutic molecule, e.g., TNF-alpha, under control of the radiation inducible promoter PrecA. Upon intravenous (i.v.) administration at sufficient numbers, metabolically inactive endospores emigrate passively from blood into the solid tumor (T). The necrotic area (N) of the tumor provides a hypoxic sanctuary for vegetative bacterial proliferation. Targeted irradiation of colonized cancerous tissue gives rise to PrecA-mediated expression of the therapeutic molecule (♦) which should perfuse the whole tumor including the outer rim of viable tissue (V). B) Sugar-mediated IVRC of recombinant anti-cancer S. typhimurium. Recombinant S. typhimurium harbor an expression plasmid for a diagnostic marker or a therapeutic molecule under control of the promoter PBAD inducible by the monosaccharide sugar L-arabinose. Upon i.v. administration at sufficient numbers invasive bacteria emigrate from blood into the solid tumor. Both, the viable region and the necrotic area of the tumor provide niches for bacterial colonization and growth. Systemically administered L-arabinose perfuses the whole body of the recipient mouse including cancerous tissue. Uptake of L-arabinose by the bacteria residing within the tumor gives rise to PBAD-mediated expression of the diagnostic marker () or therapeutic molecule.

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introduction section). Depending on the niche in the body where the bacteria reside, the conditions affecting penetration and degradation of the inducer substance may vary and have to be taken into account. The evaluation of potential IVRC promoters in conjunction with its respective inducer substance for individual medical applications represents a wide field of investigation. Recently, proof of principal studies have demonstrated the feasibility of drug-induced IVRC of bacterial vectors localized in mucosal and systemic compartments of mice.21,40,41 The importance of commensal gut bacteria for human health is well established.42 Such bacteria have been successfully engineered to produce and secrete cytokines, e.g., interleukin-10 (IL-10), IL-2 and IL-6, which could be used for the amelioration of chronic gut disorders.43 However, the uncontrolled constitutive expression of immunologically active molecules may have serious adverse effects. In order to address this potential problem Farrar et al suggest an IVRC approach for the delivery of IL-2 by the gut commensal bacterium Bacteroides ovatus.13 These authors inserted the coding sequence of murine IL-2 in conjunction with a bacterial secretion signal sequence into the xylanase operon of the bacterial chromosome. Upon addition of the polysaccharide xylan to the medium of cultured recombinant bacteria, secretion of biologically active IL-2 was observed. The implementation of this system to the in vivo situation has yet to be demonstrated. Xylan is a cell wall component of plants and algae. Thus, diets could be formulated that would stimulate cytokine release from the recombinant, gut colonizing commensal bacteria in a controlled fashion. In addition to obligate anaerobic tumor targeting bacteria, several facultative anaerobic bacteria have been shown to colonize and proliferate inside solid tumors. From these bacteria, attenuated strains of Salmonella enterica serovar Typhimurium (S. typhimurium) were used most often as tumor targeting vectors,44-46 but also strains of E. coli,40 Salmonella choleraesuis,47 Shigella flexneri40 and Vibrio cholera48 were successfully tested. These nonsporulating bacteria were administered systemically in a metabolically active form at doses ranging between 105 to 107 cells. Consequently, until bacteria accumulate inside the tumor they are distributed via circulation within the whole body. Therefore, constitutive expression of therapeutic molecules by the bacteria may damage healthy tissue during this early phase after administration. A solution to this problem was offered by the elegant development of the tumor-specific promoter PHIP-1 for expression of therapeutic molecules by a S. typhimurium vector strain.49 PHIP-1 was generated by engineering the promoter region of gene pepT that is known to be induced during anaerobic growth.50 When tested in culture PHIP-1 upregulated reporter gene expression up to 200-fold under hypoxic conditions. More important, this promoter indeed mediated tumor-restricted constitutive gene expression in mice. However, such tumor-specific constitutive promoters are not suited for the external adjustment of therapeutic gene expression in a deliberate manner, which is often necessary. In order to establish an IVRC approach for tumor targeted bacteria, we tested several drug-inducible promoters linked to suitable reporter genes. As bacterial vector for tumor targeting in mice, we used the aroA-attenuated S. typhimurium strain SL7207.51 This strain colonizes spleen and liver of mice in considerable numbers in addition to the tumor. It is therefore not suitable for tumor therapy on the long run.46 However, it allowed us to compare IVRC of reporter gene expression in organs and the tumor in parallel. This initial screening led us to the conclusion that promoter PBAD derived from the E. coli arabinose operon,52 in conjunction with its activator protein AraC and the inducer substance L-arabinose is very well suited for IVRC of the S. typhimurium vector.21 106 bacteria harboring a reporter cassette under control of PBAD were administered to mice and received two days later a systemic dose of L-arabinose (Fig. 2B). About 5 hours after induction reporter gene activity peaked in tumors (Fig. 3A,B). Thereafter, reporter activity ceased probably due to the removal of L-arabinose from the circulation by host enzymes.53 Tight control of PBAD allowed the linkage to the suicide gene E of bacteriophage phiX174. No interference with growth, invasiveness or tumor colonization of bacteria was noticed in the uninduced state.21 Induction of gene E expression by administration of L-arabinose to mice, removed the majority of bacteria from the tumor. Therefore, this IVRC suicide module constitutes a safety measure for the use in tumors instead of antibiotics.

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Figure 3. IVRC expression of firefly luciferase by tumor targeted S. typhimurium. BALB/c mice, bearing a subcutaneous CT26 tumor, were administered with an i.v. dose of ∼106 Salmonella SL7207 harboring plasmid pHL259.21 2 days post infection mice received intraperitoneally (i.p.) 100 mg L-arabinose. At indicated time points mice were subjected to non-invasive in vivo bioluminescence imaging. A) Pseudocolored low light images of one representative mouse are displayed. B) Light intensities, corresponding to the expression level of luciferase, were quantified with the software Xenogen Living Image 2.6.1.. n = number of animals in one experiment of several.

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L-arabinose apparently fulfills all criteria of a versatile drug for IVRC of bacterial vectors. It is a low molecular weight, biocompatible compound which is able to penetrate host tissues. However, bacteria localized in spleen and liver seem to be less accessible for L-arabinose compared to bacteria residing within the tumor. This was suggested by different levels of reporter gene expression of individual bacteria. One plausible explanation could be that most bacteria inside the tumor are not confined to host cells as was evident from histology.21 In contrast, S. typhimurium in spleen and liver reside within phagosomes of host cells,54 a fact that might limit the supply of L-arabinose to the bacteria in these organs. In addition, the physiological state of the bacteria might differ between organs and tumor. Nevertheless, even within the host cells bacteria are accessible to L-arabinose mediated IVRC. This provides new options for the design of efficient bacterial carriers and could be extended to targeting antigen-presenting cells for heterologous bacterial vaccines.22 IVRC of a reporter gene linked to PBAD was also obtained when the probiotic strain E. coli Nissle 1917 was used for tumor colonization.40 In this study L-arabinose was administed via the systemic and oral route. In both cases reporter gene expression reached nearly the same level, although expression was delayed in case of oral IVRC. This result represents proof of principle that diet-mediated IVRC of tumor-targeted bacterial vectors is feasible. Since E. coli Nissle 1917 was shown to exclusively colonize the tumor40 and has a well established safety record as a probiotic strain12,55 it constitutes an attractive candidate strain for tumor therapy in patients. Many alternative regulatory systems in different bacteria have been described. Their potential for IVRC of bacteria to be used in prophylaxis and therapy has not been studied so far. However, the discovery of new drug-inducible genetic switches suitable for IVRC can be envisaged. One step into this direction was the evaluation of an acetylsalicylic acid (aspirin) responsive promoter for IVRC expression of the prodrug-converting enzyme cytosine deaminase by S. typhimurium in tumor-bearing mice.41 The use of aspirin as IVRC inducer is attractive since it is one of the most widely used analgesic and anti-inflammatory drug in medical praxis.56 5-FC was converted into the toxic drug 5-FU by the recombinant Salmonella within tumors when mice received aspirin systemically. This became evident from retardation of tumor growth. In contrast, no tumor therapeutic effect was observed when a tetracycline responsive promoter was tested in the same setting. However, molarity of administered inducer substances were different. Thus, no direct comparison of the promoters was possible. Careful dose response studies are needed in order to determine the optimal amount of inducer substances for IVRC of gene expression in bacteria residing in different niches of the body. Nevertheless, this indicates that in future it might be possible to combine several of such modules in order to generate a versatile IVRC bacterial vector with exquisite safety profiles.

Conclusions

The development of bacterial vectors for particular medical applications is a complex task. Until recently, efficient genetic tools for the manipulation of bacterial strains and modules for the synthesis and secretion of heterologous molecules were limited. However, this situation is changing dramatically. The rise of synthetic and systems biology based on high speed sequencing and omics technologies will make an increasing number of bacterial building blocks with defined functions and new tools for genetic modification available. Thus, the computer-aided design of microorganisms becomes feasible. Eventually a new class of bacterial vectors will emerge, which are assembled from components of various origins and are programmed to fulfill all requirements of a safe and efficacious treatment. In this context the implementation of IVRC circuits into bacterial vectors will be of central importance. The discovery of further IVRC switches will create new options for the establishment of multifunctional bacterial vectors to be extremely useful in a number of biomedical applications. Engineering of tightly regulated promoters and corresponding inducer substances will enlarge the range and sensitivity of bacterial IVRC. Finally, IVRC will make an important contribution in research allowing minute assessment of virulence factors during defined stages of infection processes.

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Acknowledgement

The authors gratefully acknowledge support by the DFG, the BMWi, the BMBF as well as the Deutsche Krebshilfe.

References

1. Curtiss R III. Bacterial infectious disease control by vaccine development. J Clin Invest 2002; 110:1061-1066. 2. Reid G, Jass J, Sebulsky MT et al. Potential uses of probiotics in clinical practice. Clin Microbiol Rev 2003; 16:658-672. 3. Roland KL, Tinge SA, Killeen KP et al. Recent advances in the development of live, attenuated bacterial vectors. Curr Opin Mol Ther 2005; 7:62-72. 4. Pawelek JM, Low KB, Bermudes D. Bacteria as tumour-targeting vectors. Lancet Oncol 2003; 4:548-556. 5. Mengesha A, Dubois L, Chiu RK et al. Potential and limitations of bacterial-mediated cancer therapy. Front Biosci 2007; 12:3880-3891. 6. Jain RK, Forbes NS. Can engineered bacteria help control cancer? Proc Natl Acad Sci USA 2001; 98:14748-14750. 7. Loessner H, Weiss S. Bacteria-mediated DNA transfer in gene therapy and vaccination. Expert Opin Biol Ther 2004; 4:157-168. 8. Daudel D, Weidinger G, Spreng S. Use of attenuated bacteria as delivery vectors for DNA vaccines. Expert Rev Vaccines 2007; 6:97-110. 9. Schoen C, Stritzker J, Goebel W et al. Bacteria as DNA vaccine carriers for genetic immunization. Int J Med Microbiol 2004; 294:319-335. 10. de Vrese M, Stegelmann A, Richter B et al. Probiotics-compensation for lactase insufficiency. Am J Clin Nutr 2001; 73:421S-429S. 11. Magliani W, Conti S, Frazzi R et al. Engineered commensal bacteria as delivery systems of anti-infective mucosal protectants. Biotechnol Genet Eng Rev 2002; 19:139-156. 12. Westendorf AM, Gunzer F, Deppenmeier S et al. Intestinal immunity of Escherichia coli NISSLE 1917: a safe carrier for therapeutic molecules. FEMS Immunol Med Microbiol 2005; 43:373-384. 13. Farrar MD, Whitehead TR, Lan J et al. Engineering of the gut commensal bacterium Bacteroides ovatus to produce and secrete biologically active murine interleukin-2 in response to xylan. J Appl Microbiol 2005; 98:1191-1197. 14. Galen JE, Levine MM. Can a ‘flawless’ live vector vaccine strain be engineered? Trends Microbiol 2001; 9:372-376. 15. Lewis GK. Live-attenuated Salmonella as a prototype vaccine vector for passenger immunogens in humans: are we there yet? Expert Rev Vaccines 2007; 6:431-440. 16. Bauer H, Darji A, Chakraborty T et al. Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids. Gene Ther 2005; 12:364-372. 17. Chatfield SN, Charles IG, Makoff AJ et al. Use of the nirB promoter to direct the stable expression of heterologous antigens in Salmonella oral vaccine strains: Development of a single-dose oral tetanus vaccine. Biotechnology 1992; 10:888-892. 18. Hohmann EL, Oletta CA, Loomis WP et al. Macrophage-inducible expression of a model antigen in Salmonella typhimurium enhances immunogenicity. Proc Natl Acad Sci USA 1995; 92:2904-2908. 19. Bumann D. Regulated antigen expression in live recombinant Salmonella enterica serovar Typhimurium strongly affects colonization capabilities and specific CD4(+)-T-cell responses. Infect Immun 2001; 69:7493-7500. 20. Nuyts S, Van Mellaert L, Theys J et al. Radio-responsive recA promoter significantly increases TNFalpha production in recombinant Clostridia after 2 Gy irradiation. Gene Ther 2001; 8:1197-1201. 21. Loessner H, Endmann A, Leschner S et al. Remote control of tumour-targeted Salmonella enterica serovar Typhimurium by the use of L-arabinose as inducer of bacterial gene expression in vivo. Cell Microbiol 2007; 9:1529-1537. 22. Loessner H, Endmann A, Leschner S et al. Improving live attenuated bacterial carriers for vaccination and therapy. Int J Med Microbiol 2007; 2007.07.005. 23. Cases I, de Lorenzo V. Expression systems and physiological control of promoter activity in bacteria. Curr Opin Microbiol 1998; 1:303-310. 24. Mezhir JJ, Smith KD, Posner MC et al. Ionizing radiation: A genetic switch for cancer therapy. Cancer Gene Ther 2006; 13:1-6. 25. Nuyts S, Theys J, Landuyt W et al. Increasing specificity of anti-tumor therapy: Cytotoxic protein delivery by nonpathogenic Clostridia under regulation of radio-induced promoters. Anticancer Res 2001; 21:857-861.

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26. Cox MM. Regulation of bacterial RecA protein function. Crit Rev Biochem Mol Biol 2007; 42:41-63. 27. van Horssen R, Ten Hagen TL, Eggermont AM. TNF-alpha in cancer treatment: Molecular insights, antitumor effects and clinical utility. Oncologist 2006; 11:397-408. 28. Mullen CA, Kilstrup M, Blaese RM. Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc Natl Acad Sci USA 1992; 89:33-37. 29. Wagner E. Programmed drug delivery: Nanosystems for tumor targeting. Expert Opin Biol Ther 2007; 7:587-593. 30. Duguet E, Vasseur S, Mornet S et al. Magnetic nanoparticles and their applications in medicine. Nanomed 2006; 1:157-168. 31. Schuler D, Frankel RB. Bacterial magnetosomes: Microbiology, biomineralization and biotechnological applications. Appl Microbiol Biotechnol 1999; 52:464-473. 32. Plank C, Schillinger U, Scherer F et al. The magnetofection method: Using magnetic force to enhance gene delivery. Biol Chem 2003; 384:737-747. 33. Hilger I, Hergt R, Kaiser WA. Use of magnetic nanoparticle heating in the treatment of breast cancer. IEE Proc Nanobiotechnol 2005; 152:33-39. 34. Ullrich S, Kube M, Schubbe S et al. A hypervariable 130-kilobase genomic region of Magnetospirillum gryphiswaldense comprises a magnetosome island which undergoes frequent rearrangements during stationary growth. J Bacteriol 2005; 187:7176-7184. 35. Richter M, Kube M, Bazylinski DA et al. Comparative genome analysis of four magnetotactic bacteria reveals a complex set of group-specific genes implicated in magnetosome biomineralization and function. J Bacteriol 2007; 189:4899-4910. 36. Whitworth DE, Bryan SJ, Berry AE et al. Genetic dissection of the light-inducible carQRS promoter region of Myxococcus xanthus. J Bacteriol 2004; 186:7836-7846. 37. Vollmer AC, Kwakye S, Halpern M et al. Bacterial stress responses to 1-megahertz pulsed ultrasound in the presence of microbubbles. Appl Environ Microbiol 1998; 64:3927-3931. 38. Chao YP, Chern JT, Wen CS et al. Construction and characterization of thermo-inducible vectors derived from heat-sensitive lacI genes in combination with the T7 A1 promoter. Biotechnol Bioeng 2002; 79:1-8. 39. Hannig G, Makrides SC. Strategies for optimizing heterologous protein expression in Escherichia coli. Trends Biotechnol 1998; 16:54-60. 40. Stritzker J, Weibel S, Hill PJ et al. Tumor-specific colonization, tissue distribution and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. Int J Med Microbiol 2007; doi 10.1016/ j.ijmm.2007.01.008. 41. Royo JL, Becker PD, Camacho EM et al. In vivo gene regulation in Salmonella spp. by a salicylatedependent control circuit. Nat Methods 2007; doi 10.1038/nmeth1107. 42. Kelly D, King T, Aminov R. Importance of microbial colonization of the gut in early life to the development of immunity. Mutat Res 2007; 622:58-69. 43. Steidler L, Rottiers P. Therapeutic drug delivery by genetically modified Lactococcus lactis. Ann N Y Acad Sci 2006; 1072:176-186. 44. Pawelek JM, Low KB, Bermudes D. Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 1997; 57:4537-4544. 45. Low KB, Ittensohn M, Le T et al. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat Biotechnol 1999; 17:37-41. 46. Forbes NS, Munn LL, Fukumura D et al. Sparse initial entrapment of systemically injected Salmonella typhimurium leads to heterogeneous accumulation within tumors. Cancer Res 2003; 63:5188-5193. 47. Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther 2005; 12:175-184. 48. Yu YA, Shabahang S, Timiryasova TM et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat Biotechnol 2004; 22:313-320. 49. Mengesha A, Dubois L, Lambin P et al. Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated Salmonella. Cancer Biol Ther 2006; 5. 50. Lombardo MJ, Lee AA, Knox TM et al. Regulation of the Salmonella typhimurium pepT gene by cyclic AMP receptor protein (CRP) and FNR acting at a hybrid CRP-FNR site. J Bacteriol 1997; 179:1909-1917. 51. Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella typhimurium are nonvirulent and effective as live vaccines. Nature 1981; 291:238-239. 52. Guzman LM, Belin D, Carson MJ et al. Tight regulation, modulation and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 1995; 177:4121-4130.

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53. Seri K, Sanai K, Matsuo N et al. L-arabinose selectively inhibits intestinal sucrase in an uncompetitive manner and suppresses glycemic response after sucrose ingestion in animals. Metabolism 1996; 45:1368-1374. 54. Richter-Dahlfors A, Buchan AM, Finlay BB. Murine salmonellosis studied by confocal microscopy: Salmonella typhimurium resides intracellularly inside macrophages and exerts a cytotoxic effect on phagocytes in vivo. J Exp Med 1997; 186:569-580. 55. Altenhoefer A, Oswald S, Sonnenborn U et al. The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol Med Microbiol 2004; 40:223-229. 56. Weissmann G. Aspirin. Sci Am 1991; 264:84-90.

Chapter 6

Genetic Immunization:

Bacteria as DNA Vaccine Delivery Vehicles Pablo Daniel Becker, Miriam Noerder, Carlos Alberto Guzmán*

Abstract

T

he so-called DNA vaccination represents one of the most notable tools under development in the field of vaccinology. The concept of administering the gene coding for any given protective antigen and make responsible vaccinee’s own cells to produce the protein appeals as too simple to be true. Indeed, the implementation of this approach for mass vaccination should overcome several bottlenecks, such as need of high dosages and poor immunogenicity. In this context, the use of live attenuated bacteria as delivery system for plasmid DNA has emerged as a promising alternative to overcome many of those pitfalls. In addition, this approach is not only amenable for mucosal administration, but allows to specifically target professional antigen presenting cells. This results in their transfection, as well as in their activation and maturation, due to their built-in adjuvant properties resulting from the stimulation of pattern recognition receptors. This chapter discusses the specific features that should be taken into consideration when designing a plasmid vector, current candidate bacterial carriers for DNA delivery and main safety issues.

Introduction

In the early 1990s, DNA vaccination has emerged as a promising strategy to induce major histocompatibility complex (MHC) class I-restricted immune responses, in particular CD8+ T-cell mediated cytotoxicity. However, seminal work suggesting that this would be feasible approach was carried out much earlier. In 1960, Ito demonstrated that papilloma was induced by injecting nucleic acid of the Shope rabbit papilloma virus.1 In 1962, studies performed by Atanasiu demonstrated that polyoma virus DNA from infected cells injected into Hamsters lead to anti-polyoma antibodies and tumors.2 Later, in 1979, Fried showed that polyoma virus DNA delivered by plasmids or phages was able to transform murine fibroblasts.3 However, the real potential of these observations went through unnoticed, until the 1990s, when serendipity brought Wolff to observe in controls of transfection studies using cationic lipids that naked nucleic acid transfer in muscles also led to the expression of several reporter genes.4 This and the subsequent proof-of-principle provided by Ulmer for the promotion of protective immunity against flu in 1993,5 triggers a literal explosion in the field of nucleic acid vaccination with literally thousands of patents being filed in a few years. The DNA vaccination strategy is associated with many advantages. The biosynthetic machinery of the host’s cells is responsible for the production of the antigen. Therefore, optimal glycosylation and folding is warranted. In addition, production and purification of individual antigens is not necessary.4 Furthermore, since cultivation is not required, vaccines can be developed against pathogens for which non appropriate cultivation techniques exist. Interestingly, the use of this approach may help to overcome maternal immunity, thereby facilitating the development of vaccines for newborns. Furthermore, the stability of DNA renders nucleic acid vaccines particularly robust *Corresponding Author: Carlos Alberto Guzmán—Department of Vaccinology, Helmholtz Centre for Infection Research, Inhoffenstraße 7, D-38124, Braunschweig Germany. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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in terms of cold-chain requirements. Finally, DNA vaccines exert immune stimulatory activities, due to the presence of pathogen associated molecular patterns (PAMPs), which are recognized by receptors from cells of the innate immune system (i.e., Toll-like receptors; TLR). By manipulating the genes coding for selected antigens (e.g., ubiquitination), it is possible to further enhance their delivery into the MHC class I-restricted pathway.6,7 An important issue to be taken into consideration is which cell subset is responsible for gene expression. By naked DNA vaccination, in which the plasmid is usually given intramuscularly, two different cell types are transfected, namely somatic cells (e.g., myocytes) and professional antigen presenting cells (APCs). However, injected DNA is degraded by extracellular nucleases, making necessary the use of high amounts of DNA to ensure expression.8,9 The studies performed in 1980 by Walter Schaffner demonstrating that cloned genes can be directly transferred from bacteria to mammalian cell provided the rational for using bacteria as a DNA delivery system.10 In fact, three independent groups exploited the ability of different intracellular Gram-negative bacteria to transfer plasmids encoding eukaryotic expression systems to infected cells.11-13 Later, it was also demonstrated that Gram-positive bacteria, such as self-destructing Listeria monocytogenes is able to deliver plasmid DNA.14 The delivery by live attenuated bacteria has several advantages over the normal naked DNA. There is no need for the purification of plasmid DNA, but rather a simple fermentation. The bacterial carrier can specifically target and transfer main APCs, such as dendritic cells (DCs).15,16 Furthermore, after bacterial uptake by DCs,17 they act as natural adjuvants due to the presence of PAMPs that modulate early innate immune responses, thereby promoting robust and long-lasting adaptive responses.18,19 Most carriers tested up to now as bactofection vectors are human intestinal pathogens (e.g., Salmonella spp., Shigella spp., Listeria spp., Escherichia coli). This is particularly advantageous due to their ability to infect human colonic mucosa following oral administration. After mucosal delivery of the carrier, both mucosal and systemic immune responses can be stimulated.20 Besides, epithelial cells in the gut or hepatocytes could serve as antigen sources for cross-priming after infection.

Plasmid Design Promoters and Enhancers

Expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a functional eukaryotic promoter. Viral promoters are very often used with this purpose, providing greater gene expression in vivo than other eukaryotic promoters. In particular, the cytomegalovirus (CMV) immediate early enhancer promoter (PCMV ) has been shown to direct the highest levels of transgene expression in eukaryotic tissues when compared with other promoters. Other frequently used viral promoters are: (i) the early promoter from SV40 (PSV40), (ii) the polyhedrin promoter from baculovirus (PPH), (iii) the thymidine kinase promoter from Herpes simplex virus (HSV)-1 (PTK), (iv) the 5΄LTR promoter from human immunodeficiency virus (HIV; PLTR) and (v) the promoter of the Rous sarcoma virus (PRSV ). Since eukaryotic and bacterial promoters have different consensus sequences, it is regarded as unlikely that promoter sequences functional in eukaryotic cells would be able to direct efficient transcription initiation in bacteria.21,22 However, it was demonstrated that not only eukaryotic promoters but any type of eukaryotic DNA has a high probability to initiate transcription after transfer into bacteria. In fact, PCMV and PSV40 carry structural features required by the bacterial RNA polymerase to initiate transcription,23 such as sequences with homology to the bacterial -10 and -35 promoter consensus motifs which induce expression as strong as the promoter from the bacterial TEM-β-lactamase gene.23 Therefore, expression of a foreign gene by a heterologous promoter must be taken into consideration when defining safety, especially when toxic products are expressed. Additionally, bacterial expression can be silenced by insertion of a transcription terminator, deletion or site-direct mutagenesis of the bacterial ribosome binding site downstream from the promoter, in such a way that only eukaryotic cells would be able to express the gene of interest. Translation from the prokaryotic machinery

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can also be avoided by adding introns, thereby ensuring that expression is only possible through a eukaryotic system. It is also important to consider that some widely used virally-derived promoters, such as PCMV and PSV40, may not be suitable for some applications, since treatment or endogenous induction of interferon (IFN)-γ or tumor necrosis factor (TNF)-α may silence them, thereby inhibiting transgene expression from DNA vaccines containing these promoters.24,25 Thus, other promoter systems are currently under investigation, such as tissue specific, mammalian, synthetic and hybrid (e.g., hybrid CMV enhancer—nonviral promoter) promoters.26-31

Polyadenylation, Kozak Sequence and Codon Usage

Another regulatory element is the polyadenylation (polyA) sequence necessary for transcriptional termination, stability, processing and translation of eukaryotic mRNA. Since the rate of transcription initiation is generally high due to the use of strong promoters/enhancers, the rate of transcriptional termination may become rate-limiting.32 In addition, the efficiency of primary RNA transcripts processing an polyA is known to vary between the polyA sequences of different genes. Thus, the polyA sequence should be selected or, alternatively, a second enhancer could be added downstream of the polyA sequence to increase expression levels.33 In addition to the polyA sequence, most eukaryotic genes possess sequences flanking the AUG initiator codon, the so-called “kozak” consensus sequences. They are responsible for mRNA recognition by eukaryotic ribosomes, thereby affecting expression levels.34,35 It is important to consider that mammalian codon usage differs from microorganisms. Many groups have demonstrated that altering the codon use pattern may provide substantial increases in expression levels and improvements in antigenicity.36-39 Several studies have showed that optimization of the codon usage of bacterial, parasitic and viral sequences to that of highly expressed mammalian genes significantly increased both the gene-expression levels and the immunogenicity of the DNA vaccine.40-42 Thus, it is useful to examine the sequence looking for clusters of rare codons, as they may retard ribosomes during translation due to exhaustion of rare tRNAs, promoting ribosomal frame-shifting and premature truncation of the protein.43 A remarkable example of codon optimization is the case of the gene gag of HIV, its optimization resulted in a mutational inactivation of an inhibitory sequences (AU-rich sequences responsible for the dependence of gag expression on the viral Rev protein) allowing the expression of gag in a Rev-independent manner.37,42,44

Customizing Antigen Coding Sequence to Vaccination Needs

In addition to codon optimization, antigen coding genes may be modified to produce a protein that is secreted, membrane bound, cytosolic or associated with an organelle. Therefore, by altering the cellular localization it can be influenced the immune response. A protein that is naturally secreted can be modified to enhance the rate of secretion by replacement of its signal sequence with one of a highly expressed protein. A common signal used with this purpose is the tissue-type plasminogen activator (t-PA).45-48 The t-PA signal sequence of 27 amino acids can be fused to the N-terminus of an antigen, however, the addition of a secretion signal does not warrant its secretion. Some proteins have additional motifs, such as nuclear localization signals or membrane anchors, which should be modified or deleted to achieve efficient secretion. Therefore, the modification of proteins to optimize their secretion is a good strategy to increase antibody production in vaccinated subjects.49 Likewise, if the signal sequence is removed, a naturally secreted protein can be retained in the cytoplasm.50 Targeting of gene products for degradation also represents a useful modification. Enhanced degradation reduces protein levels, however, the number of peptides available for presentation by antigen presenting cells in the context of MHC class I molecules is considerably increased. Thus, targeting to proteasomes or endosomes using ubiquitin or LAMP-1 fusions38,51 is an useful strategy when induction of cytotoxic T-lymphocytes (CTL) is required.52,53 It is also important to consider that some genes from pathogens encode products which are immunosuppressive or possess potentially harmful properties. Thus, biological inactivation may be essential. For example, the reverse

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transcriptase and the Nef proteins of HIV exhibit enzymatic54 and immunosuppressive55 activities, whereas the human papillomavirus E7 protein represents an example of transforming activity.56 Often, it is necessary to deliver more than one gene to trigger a protective immune response (e.g., two antigens or one antigen and one cytokine). This can be achieved by the use of multiple plasmids or the generation of a bi- or multicistronic expression system by using an internal ribosome entry site (IRES) to initiate translation of the second gene.57 The use of polyepitopes represents a valid alternative to the expression of whole genes. Thus, a single plasmid can contain multiple expression cassettes or a large number of known epitopes linked together to be expressed as a single polypeptide with optimized flanking sequences to ensure proteosomal degradation.

Stimulatory Motifs in the Backbone Plasmid and Genetic Adjuvants

Bacterial DNA, but not eukaryotic DNA, stimulates the immune system when administered to mammals. This effect is mediated through unmethylated phosphodiester linked cytosine and guanine (CpG) motifs flanked by species-specific bases58-60 which act through TLR-961 to induce a series of immune stimulatory cytokines. This in turn leads to activation of B-cells, monocytes, macrophages, DCs and natural killer cells enhancing both nonspecific and antigen-specific responses.62 There are different types of CpG that carry varying amounts of immune stimulatory motifs. They activate different types of immune cells and induce different cytokines and chemokines production.63,64 On the other hand, it was demonstrated that polyG or GC-rich encoded on oligodeoxynucleotides suppress the immunostimulatory effect of bacterial CpG.65,66 Thus, it seems to be advantageous to define the backbone’s CpG motifs and to understand exactly their functional activity in the host cell. Species-specific CpG motifs should be taken into consideration. Undefined CpG motifs and bacterial DNA sequences that may have suppressive or undesirable immunomodulatory activities should be avoided. Another attractive pathogen motif is the double-stranded RNA (dsRNA) molecules associated with or derived from viral infection which are absent in normal eukaryotic cells. Such dsRNA molecules are recognized by TLR-3 resulting in the production of IFN-β, inflammatory cytokines (TNF-α, interleukine [IL]-6 and IL-12) and the maturation of DCs, which help to shape the overall immune response.67-69 Furthermore, dsRNA can directly activate IRF-3, which in turn results in a transcriptional activation of IFN-α and IFN-β promoters and the IFN-α/β-responsive genes.70 It is also well established that dsRNA can activate dsRNA-dependent protein kinase and oligoadenylate synthetase function to shut down protein translation and induce cell apoptosis.18,71-73 Thus, plasmid-derived hairpin RNA molecules with various stem lengths were recently evaluated. Short-stem (40 bp) RNA molecules showed slight effect on cell apoptosis and reporter gene expression level, but were able to stimulate significantly enhanced antigen-specific cellular immune responses. In contrast, DNA vaccines encoding long-stem (750 bp) RNA induce vigorous cell apoptosis, but no significant improvement in cell-mediated immune responses in immunized mice.74 These results showed that the delivered plasmid and resulting transcript are not only gene encoding sequences, but they are valuable tools for immunoregulation. In addition, DNA plasmids can be designed to contain interference RNA (RNAi). This approach can be exploited to silence genes that might affect the overall efficiency of the stimulated immune response and/or divert from optimal response pattern. Recently, a novel strategy to deliver genetic material into eukaryotic cells using bacteria was described. Instead of delivering DNA, a self-destructing L. monocytogenes strain is used to release translation-competent mRNA directly into the cytosol.75 The simplicity of this elegant approach is based on the genetic fusion at the 5΄-end of the mRNA to an IRESEMCV element. Thus, eukaryotic translation is achieved in 5΄cap-independent manner by the infected host.76 This system may circumvent the bottleneck of limited access to the nuclear compartment of nondividing cells. Furthermore, it was demonstrated that when mRNA is delivered, translation starts earlier than when DNA plasmid is delivered.75 Genes encoding cytokines, chemokines and other molecules have been investigated for their capacity to improve the immune responses elicited against a plasmid encoded-antigen (see Table 1).

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Thus, the integration of potential molecules with immunomodulatory properties into the plasmid represents a “no cost” strategy for DNA vaccine optimization.

Plasmid Stabilization

During bacterial growth it is crucial to maintain plasmid among daughter cells. The well-studied recombination site cer from the E. coli ColE1 plasmid allows multimers resolve to monomers by the XerCD recombinase.77 A promoter within cer initiates synthesis of a short transcript (Rcd) in multimer-containing cells. The Rcd checkpoint hypothesis proposes that Rcd delays cell division until multimer resolution is complete. Thus, inclusion of the cer sequence in the plasmid backbone may assist in plasmid maintenance.78 Besides the origin of replication, antibiotic-resistance genes are part of the standard plasmid. Antibiotic-resistance genes are widely used as selection marker to increase plasmid stability and bacterial selection per se. However, the use of antibiotics may keep the selective pressure in vitro during biotechnological process, but high copy number plasmids are often unstable in vivo. Furthermore, there are other problems associated with antibiotic-selection markers, namely the risk of horizontal gene transfer of the antibiotic-resistance cassettes harbored by the plasmids and the alteration of the gene-expression profile in mammalian cells.66,79 Thus, several strategies have emerged to achieve plasmid stabilization avoiding the use of antibiotics. Biological stabilization systems have been developed based on active or passive approaches. Passive systems rely on metabolic auxotrophies or other gene defects, such as DNA precursor, amino acid and cell wall biosynthetic pathways, which are supplemented with the intact gene. An illustrative example of a passive containment system is the use of asd mutant strains. The asd gene coding for aspartate-β-semialdehyde dehydrogenase is involved in the biosynthesis of diaminopimelic acid (DAP). Since DAP is not prevalent in the animal host and is a key constituent of peptidoglycan in the Gram-negative and Gram-positive bacterial cell wall, essentially 100% of the surviving avirulent bacteria (e.g., Salmonella spp. asd mutant) recovered from an immunized animal host still contain the recombinant plasmid and express the foreign antigen.80-83 It has been shown that an attenuated S. enterica serovar Typhi asd mutant is safe in humans.84,85 Nevertheless, it is important to keep in mind bacterial physiology when vaccine strains with chromosomal gene mutations are complemented in trans by a multicopy plasmid (pBR or pUC ori) containing the entire gene with its promoter. Using once more the example of the asd gene, when a high copy number vector (e.g., pUC-like) coding for the asd gene is used, the Asd protein is produced in 200-300 fold excess than necessary,82 which in turns result in a slightly increased generation time and a 10-fold increase of LD50 in mice when compared to the same strain complemented with a low-copy (pSC101 ori) or intermediate copy (p15A ori) number plasmid.82 Thus, when designing the plasmid, it should be consider the protein level necessary to complement the system to obtain a more balanced stabilization of the expression plasmid. Other examples of metabolic auxotrophies or gene defects are: (i) the thymidylate synthase (thyA), an essential gene involved in de novo synthesis of the DNA precursor thymidine monophosphate,86-91 (ii) the adenylosuccinate lyase (purB) which catalyzes an essential step in the de novo synthesis of adenosine monophosphate involved in the purine biosynthetic pathway,92,93 (iii) the glutamine synthetase (glnA) required for glutamine synthesis, which serves together with glutamate as primary nitrogen source in bacterial metabolism,94 (iv) the tryptophanyl-tRNA synthetase (trpS)50 and (v) the translation initiation factor 1 (infA), a small intracellular protein essential for cell viability.95 On the other hand, active stabilization provides control through the conditional production of a toxic compound whose expression is tightly controlled by an environmental factor or suppressed by internal elements. Toxin-antitoxin systems, also known as postsegregational cell killing, ensure plasmid maintenance during replication by conferring an advantage on plasmid-retaining cells due to reduced competitiveness of their plasmid-free counterparts. The hok-sok system from the E. coli plasmid R1 is one of best-characterized toxin-antitoxin systems. In this two-component system, hok encodes the lethal pore forming Hok protein, whose translation is blocked by binding of the sok

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Table 1. The use of live attenuated bacteria as delivery systems for DNA vaccines and therapeutic/modulatory genes

Bacteria (Strain)

Therapeutic/ Modulatory Gene Antigen

S. typhimurium ΔaroA (SL5000)

Human CD40L

S. typhimurium ΔaroA (SL7207)

Animal Model

Mouse Strain

Ref.

-

B-cell lymphoma

BALB/c

161

VEGFR-2 (FLK-1)

-

Melanoma, colon and lung carcinoma

BALB/c and C57Bl/6

162

S. typhimurium ΔaroA (SL7207)

IFN-γ

-

Immunocompromised

IFN-γ–/– BALB/c 121

S. typhimurium ΔaroA (SL7207)

GM-CSF

Cruzipain

Trypanosoma cruzi infection

C3H/HeN

120

S. typhimurium ΔaroA (SL7207)

IL-10

gD and gB from HSV

HSV-2 infection

BALB/c

113

S. typhimurium ΔaroA (SL7207)

Antigen fused to ubiquitin

gp10025-35 Melanoma and TRP2181-188 epitopes

C57Bl/6J, 163 C57Bl/6J-Cd8a tm1 Mak and C57Bl/6-Cd4 tm1 Mak

S. typhimurium ΔaroA (SL7207)

PRE

Tyrosine hydroxylase

Neuroblastoma

A/J

S. typhimurium ΔaroA (SL7207)

CD40L

CEA

Colon adenocarcinomas

CEA-transgenic 165 C57Bl/6J

S. typhimurium ΔaroA, Δdam (Re88)

CCL21

Survivin

Lung cancer

C57Bl/6J

166

S. typhimurium (SL3261)

IL-4 and IL-18 -

Echinococus granulosus infection

BALB/c

118

164

S. choleraesuis

murine PrT

gD from PsRV

Pseudorabies infection BALB/c

167

S. choleraesuis

Porcine PrT

-

S. choleraesius infection

BALB/cJ and C3H/HeJ

168

S. choleraesuis

Thrombospondin-1

-

Melanoma

C57Bl/6

169

L. monocytogenes IL-12 (EGD)

-

Leishmania major infection

BALB/c and C57Bl/6

170

S. typhimurium ΔaroA (SL7207)

IL-2

ureB

Helicobacter pylori infection

C57Bl/6

171

E. coli TG-1

PrT

gD from PsRV

Pseudorabies infection BALB/c

172

Abbreviations: CEA: carcinoembryonic antigen; gD: glycoprotein D; HSV-2: herpes simplex virus type 2; PRE: posttranscriptional regulatory elements; PrT: prothymosin α; PsRV: pseudorabies virus; TRP: tyrosine-related protein; VEGFR: vascular endothelial growth factor receptor.

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transcript. However, sok mRNA is highly susceptible to degradation by nucleases and its protective intracellular concentration must be maintained at a critical level by constitutive transcription from a resident plasmid carrying the hok-sok locus.96

Safety Concerns Related with Bacterial Delivered DNA

Beside the aforementioned aspects, regulatory agencies strongly recommend total avoidance of antibiotic-resistance genes and removal of all unnecessary backbone DNA in plasmids used for vaccination or gene therapy. As for naked DNA, one of the major biosafety concerns for bacterial delivered DNA is the potential risk of chromosomal integration, particularly at potentially dangerous locations, such as oncogenes or regulatory regions. Integration of plasmid sequences into the host genome could occur, since in vitro transfection with plasmid DNA almost always results in integration of the plasmid, even without selection. It is clear that live bacterial vectors (LBV)-delivered plasmids enter the host cell nucleus. Thus, not surprisingly, plasmid integration into the genome has been found after LBV-mediated vaccine delivery under in vitro conditions in several studies.11,14,97 However, these findings have not yet been extended to in vivo studies. Chromosomal integration after intramuscular vaccination with naked DNA in mice has been found to be negligible.98 In any case, careful design of the plasmid vectors is needed to minimize the risk of integration. The nucleotide sequence of the entire plasmid should be known and a careful analysis for homologies to the human genome should be performed. From a regulatory standpoint, analysis of the integration potential of a new plasmid must be carried out before any clinical trial, particularly when the backbone vector has not been tested before. Other issues related to vaccination with DNA are the spread to and long-term persistence of the plasmid in multiple tissues (biodistribution) and the potential induction of tolerance to the immunizing antigen. Adequate guidelines and regulations concerning these issues have been published by regulatory agencies.99,100 In addition the duration of expression of the antigen, as well as the use of costimulatory molecules, such as cytokines and CpG DNA motifs, must be carefully evaluated since they pose further risks.101

Live Attenuated Bacterial Carriers

Currently, only three live attenuated bacterial vaccines are licensed for human use, S. enterica serovar Typhi Ty21a strain against typhoid fever, Vibrio cholera CVD 103-HgR strain against cholera and Mycobacterium bovis bacillus Calmette-Guerin (BCG) against tuberculosis. In addition, there are several promising live attenuated bacterial strains which have been tested in clinical trials and can potentially be exploited as delivery system for DNA vaccines (Table 2). From the point of view of DNA vaccination they should be classified in extracellular pathogens (e.g., E. coli and Yersinia spp.), intraphagosomal pathogens (e.g., Salmonella spp.) and intracytosolic pathogens (e.g., L. monocytogenes and Shigella spp.) (Fig. 1). Gene transfer from bacteria to the eukaryotic cell is the most important parameter for the success of the DNA vaccination process. Thus, many bacteria used as DNA vectors were engineered or attenuated in such a way that they lyse upon cellular entry; such as Shigella flexneri13 and invasive E. coli,102 as a result of impaired cell wall synthesis, or L. monocytogenes,14,103 due to the production of a phage lysine (Fig. 1). For example, a DAP auxotroph of E. coli K12, which undergoes lysis upon entry into the cytosol, is able to transfect a higher percentage of eukaryotic cells than its autotrophic counterpart.102 Nevertheless, lysis of the carrier does not seem to be an absolute requirement for plasmid delivery, since immunization with S. flexneri ΔicsA, which is impaired in cell-to-cell spread and does not lyse, still induced a protective immune response.104 Once the plasmid is released from the carrier into the cytosol, it has to enter the nuclear compartment for transcription. Thus, an important obstacle for efficient gene transfer may be the import of plasmid DNA into the nucleus. It has been shown that nondividing cells are only hardly, if at all, transfectable with cationic lipids.105-107 This indicates that in the case of naked plasmid DNA the nuclear membrane represents a major barrier to efficient transfer to the nucleus. However, many reports showed that using live attenuated bacteria is possible to achieve efficient

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Table 2. Live attenuated bacterial strains used as vaccines in humans Attenuating Mutation

Immunization Route

Safety

Immune Responses

Reference

S. flexneri 2a (CVD1204)

ΔguaBA

Oral

+

Humoral

173

S. flexneri 2a (CVD1208(S))

ΔguaBA, Δset, Δsen

Oral

+++

Humoral and cellular

173,174

S. flexneri 2a (CVD1207)

ΔguaBA, ΔvirG, Δset, Δsen

Oral

+++

Humoral and cellular

142

S. flexneri Y (SFL124)

ΔaroD

Oral

+++

Humoral

175-177

S. flexneri 2a (SFL1070)

ΔaroD

Oral

++

Humoral

178

S. flexneri 2a (CVD1203)

ΔaroA, ΔvirG

Oral

+

Humoral (weak)

141

S. flexneri 2a (SC602)

ΔvirG, ΔiucA

Oral

++

Humoral

139,140

S. sonnei (WRSS1)

ΔvirG

Oral

++

Humoral and cellular

179,180

S. typhi (Ty21a)

ΔgalE, ΔrpoS, ΔilvD

Oral

+++

Humoral and cellular

181

S. typhi CDC10-80 (PBCC211)

ΔaroA, ΔaroD

Oral

+

Humoral (weak)

182

S. typhi CDC10-80 (PBCC222)

ΔaroA, ΔaroD, ΔhtrA

Oral

+

Humoral (weak)

182

Humoral

183

Bacteria (Strain)

ΔphoP, ΔphoQ

Oral

S. typhi Ty2 (Ty445)

ΔphoP, ΔphoQ, ΔaroA

Oral

+++

Humoral (weak)

184

S. typhi Ty2 (ZH9)

ΔaroC, ΔssaV

Oral

+++

Humoral

185

S. typhi Ty2 (M01ZH09)

ΔaroC, ΔssaV, ΔSPI-2

Oral

+++

Humoral and cellular

186,187

S. typhi ISP1820 (CVD906)

ΔaroC, ΔaroD

Oral

+

Humoral

188

S. typhi Ty2 (CVD908)

ΔaroC, ΔaroD

Oral

++

Humoral)

189,190

S. typhi Ty2 (CVD908-htrA)

ΔaroC, ΔaroD, ΔhtrA

Oral

+++

Humoral and cellular

135,190,191 continued on next page

Patho-Biotechnology

S. typhi Ty2 (Ty800)

+++

Attenuating Mutation

Immunization Route

Safety

Immune Responses

S. typhi Ty2 (CVD909)

ΔaroC, ΔaroD, ΔhtrAa

Oral

+++

Humoral and cellular

190,192

S. typhi Ty2 (X3927)

Δcya, Δcrp

Oral

+

Humoral

188

S. typhi Ty2 (X4073)

Δcya, Δcrp, Δctd

Oral

++

Humoral

85

S. typhi Ty2 (X4632)

Δcya, Δcrp, Δctd, Δasd (complemented with plasmid bearing asd gene)

Oral

++

Humoralb

85

S. typhimurium TML (WT05)

ΔaroC, ΔssaV

Oral

+++

Humoral (variable)

185

Bacteria (Strain)

Reference

S. typhimurium (LH1160)

ΔphoP, ΔphoQ, ΔpurB

Oral

++

Humoral

93

S. typhimurium (VNP20009)

YS72 (ΔpurI, Δxyl), ΔmsbB

i.v.

++

Humoral

193

L. monocytogenes (LH1169)

ΔactA, ΔplcB

Oral

++

Humoral (weak) and cellular

144 194,195

i.d.

++

ND

V. cholerae O1 El Tor Inaba (CVD103-HgR)

ΔctxA, ΔhlyA

Oral

+++

Humoral (also tested in children) 181,196

V. cholerae O1 El Tor Ogawa (638)

ΔctxΦ hapA::celA

Oral

+++

Humoral

F. tularensis (NDBR101)

M. bovis BCG V. cholerae O1 El Tor Inaba (Peru-15)

i.d. ΔctxΦ recA::ctxB

Oral

+++

Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles

Table 2. Continued

197

Cellular (variable)

200-202

Humoral (weak in children, strong in adults)

198,199

a promoter P tviA in Salmonella enterica serovar Typhi vaccine CVD908-htr was replaced with the constitutive promoter Ptac; bno detectable immune response against the recombinant expressed fusion protein HBc-preS; BCG: Bacillus Calmette-Guérin; i.d.: intradermal; i.p.: intraperitoneal; i.v.: intravenous; ND: not determined; + poorly tolerated; ++ modestly tolerated; +++ well tolerated. Licensed vaccines are shown in bold.

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Figure 1. Schematic representation of plasmid delivery by bacterial carriers. I) Salmonella spp. and other intraphagosomal pathogens usually promote their own uptake into phagocytic cells. Salmonella are able to survive and deliver their DNA cargo to the nucleus. Whether this plasmid DNA is released into the cytoplasm or directly to the nucleus, as well as the underlying molecular mechanisms involved in such a process still remain to be elucidated. Attenuated strains with mutations in genes of metabolic pathways usually lyse within phagosomes, thereby improving plasmid release. II) After transcytosis through M cells of the intestine Listeria spp. and Shigella spp. (intracytosolic pathogens) are able to invade APCs and other cellular subtypes present at the mucosa, escaping to the cytosol. IIa) A common strategy to optimize delivery is to provide the strain with self-destructing capacities. Thus, after escape bacteria induce their own lysis, leading to plasmid release. Despite the fact that the plasmid might be efficiently released into the cytosol, the transport into the nucleus is not as efficient as in Salmonella. A hypothesis is that the plasmid released by Listeria to the cytosol does not contain the appropriate proteins to (e.g., histone-like) to allow an efficient transportation, however, there is still not direct evidence for this. On the other hand, it might be also possible that Salmonella resides in a particular compartment, which has preferential access to the nucleus. IIb) There is an engineered L. monocytogenes that has a T7 RNA polymerase under the control of a cytosolic promoter, therefore, when bacteria invade the cytoplasm, not only induce their own lysis but also produce mRNA that will be translated directly in the cytosol, avoiding the transfer of the genetic material to the nucleus. The translated proteins can be directed to MHC class I restricted antigen presentation pathway in order to activate CD8+ T-cell. Alternatively, it can be released by the cell through lysis or by adding secretory signals to the protein. This results in protein access to the MHC class II-restricted antigen presentation pathway of bystander APCs, which in turn leads to stimulation of antibody production and CD4+ T helper responses.

transfer of plasmid to the nucleus in non dividing cells.12,16,108 It was speculated that, as in many DNA viruses, retroviruses75 and Agrobacterium tumefaciens,109 the genetic material is not merely naked, but associated with molecules that prevent degradation and may even facilitate its transfer to the nucleus.110

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In 2005 it was licensed the first DNA vaccine for veterinary use. This vaccine, which protects horses from the West Nile encephalitis virus, has reached the market after only a few years of research. This demonstrated that the development of nucleic acids-base formulations is much faster than antigen-based vaccines. There are other DNA vaccines licensed for veterinary use and clinical trials (www.clinicaltrials.gov) are running in humans at the moment (e.g., influenza DNA vaccine from PowerMed). Surprisingly, despite the large number of preclinical studies using attenuated bacteria as DNA delivery vectors, no clinical trials have been performed in humans. In the next section, we describe the most promising bacterial vectors under preclinical evaluation, which are expected to be translated in phase I studies in the near future.

Salmonella spp. Strains Preclinical Studies

The S. enterica serovar Typhimurium purine auxotrophic strain 22-11 was assessed as vector for the oral delivery of a DNA vaccine encoding the major outer membrane protein of the respiratory pathogen Chlamydia trachomatis. This vaccine led to partial protection against pulmonary challenge with C. trachomatis in a murine model, demonstrating that plasmid delivery through the gut could elicit immune responses able to confer protection at a distant mucosal surface, namely the lung.111 Bacterial DNA delivery against viral diseases has also been assessed (Table 3). For example, infection with HSV-2 can be controlled by strong T-cell responses in the genital mucosa. Oral immunization with S. enterica serovar Typhimurium ΔaroA carrying DNA plasmids encoding the HSV-2 glycoproteins D (gD) or B (gB) resulted in strong systemic and mucosal (vaginal) T-cell responses, including vaginal memory T-cells and conferred protection against a vaginal challenge with HSV, demonstrating clear superiority to intramuscular injection of the same plasmid.112,113 Furthermore, when a plasmid coding for the hepatitis B surface antigen (HBsAg) was delivered using S. enterica ΔaroA by oral route, it induced, as naked plasmid DNA, stronger CTL response than the classical alum-adjuvanted recombinant HBsAg protein, which induces mainly antibody production.114 These results suggest that live attenuated bacteria are a promising delivery system for therapeutic DNA vaccines against hepatitis B infection. S. enterica ΔaroA was also evaluated as delivery system of a DNA vaccine against the hepatitis C virus (HCV) encoding the NS3 antigen. This vaccination protocol resulted in the induction of strong CD8+ T-cell responses and protection against a challenge with a recombinant HCV-NS3 vaccinia virus.115 Most of the studies done so far in the area of tumor DNA vaccine delivery were performed in mice using Salmonella spp. as carrier (Table 3). Paglia et al16 were the first to demonstrate that DNA delivery using S. enterica ΔaroA as a carrier is able to induce effective cellular and humoral immune responses against a highly aggressive fibrosarcoma expressing β-galactosidase as operational tumor associated antigen. Interestingly, this report demonstrated that by using this approach it is possible to achieve a direct targeting and transfection of DCs. The natural adjuvant effect of live attenuated bacteria was also demonstrated by the fact that the S. enterica ΔaroA vector was able per se to induce partial protection, thereby highlighting the import role played by the activation of the innate immune system in the overall anti-tumor response. As mentioned above, an important issue to take into consideration is the DNA release from bacteria to the cytosol. To improve release several approaches have been pursued, such as the expression of the lambda bacteriophage S and R genes under the control of an arabinose inducible promoter.116 Another approach that could be exploited for this purpose is a recently described salicylate-dependent regulatory control circuit, which would enable tightly-regulated in vivo expression.117 Using this system, the live bacterial carrier could express an autolysin after induction using aspirin at therapeutic concentrations.117 On the other hand, S. enterica ΔaroA has been widely used in different experimental systems to deliver cytokines encoding genes in the context of tumor therapy, such as IL-12, IL-4, IL-18 and granulocyte-macrophage colony-stimulating factor (GM-CSF).118,119 This resulted in cytokine expression and partial protection against unrelated tumors.118,119 The same bacterial vector was used to deliver soluble mediators able to modulate immune response against codelivered antigens.

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Table 3. Preclinical studies using live attenuated bacteria as delivery system for nucleic acid vaccine Attenuating

Antigen

Bacteria (Strain)

Mutation

(Pathogen)

Animal Model

Immunization Route

Immune Responses

S. flexneri 2a (CVD1203)

ΔaroA, ΔiscA

gp120 (HIV-1)

BALB/c mouse

S. flexneri 2a (CVD1204)

ΔguaBA

Tetanus toxin frag. C

S. flexneri 2a (CVD1208)

ΔguaBA, Δset, Δsen

HA (measles virus)

S. flexneri 2a (15D)

Δasd

gp120 (HIV-1)

BALB/c mouse

i.n.

+

+

ND

205

S. flexneri 2a (15D)

Δasd

Fusion protein, HA, NP (measles virus)

BALB/c, BALB/c-scid and IFN-γ–/– C57Bl/6 mice

i.n.

+

+

ND

160

S. flexneri 2a (SA100)

rfbF::Tn5

gag (HIV-1)

CB6F1 mouse

i.n.

+

ND

ND

206

S. typhi Ty21a

ΔgalE, ΔrpoS, ΔilvD

gp120 (HIV-1)

BALB/c mouse

i.n.

+

+

ND

205

S. typhi Ty21a

ΔgalE, ΔrpoS, ΔilvD

NP (measles virus)

BALB/c mouse

i.p.

+

+

ND

160

S. typhi (CVD915)

ΔguaBA

Tetanus toxin frag. C

BALB/c mouse

i.n.

+

+

ND

207

S. typhi (CVD908-htrA)

ΔaroC, ΔaroD, ΔhtrA

HA (measles virus)

Cotton rat

i.n.

+

+

+

204

S. typhimurium (SL7207)

ΔaroA

gp120 (HIV-1)

BALB/c mouse

i.n.

+

+

ND

205

Protectiona Ref

Cellular

Ab

i.m.

+

ND

+b

104

Guinea pig

i.n.

ND

+

–

203

Cotton rat

i.n.

+

+

+

204

Patho-Biotechnology

continued on next page

Attenuating

Antigen

Immunization

Bacteria (Strain)

Mutation

(Pathogen)

Animal Model

Route

S. typhimurium (SL7207)

ΔaroA

env (HIV-1)

BALB/c mouse

S. typhimurium (SL7207)

ΔaroA

ActA, listeriolysin (L. monocytogenes)

S. typhimurium (SL7207)

ΔaroA

S. typhimurium (SL7207)

ΔaroA

Immune Responses Protectiona Ref

Cellular

Ab

i.g.

+

ND

ND

BALB/c mouse

oral

+

+

+

HBsAg (HBV)

BALB/c mouse

oral

+

+

ND

NS3 region (HCV)

AAD mousec

oral

+

ND

+d

208 12 108, 114 115

S. typhimurium (SL7207)

ΔaroA

Mp1p (P. marneffei)

BALB/c mouse

oral

+

+

+/–

209

S. typhimurium (SL7207)

ΔaroA

cruzipain (T. cruzi)

C3H/HeN mouse

oral

+

+

+

120

S. typhimurium (SL7207)

ΔaroA

ureB (H. pylori)

C57Bl/6 mouse

oral

ND

ND

+

171

S. typhimurium (SL7207)

ΔaroA

H. pylori neutrophil activating protein (H. pylori)

C57Bl/6 mouse

oral

ND

+

ND

210

S. typhimurium (SL7207)

ΔaroA

MDR-1

C57Bl/6 and BALB/c mice

oral

+

ND

+e

211

S. typhimurium (SL7207)

ΔaroA

gp100

C57Bl/6 mouse

oral

+

ND

–f

212

S. typhimurium (SL7207)

ΔaroA

gp100 and TRP-2g

C57Bl/6J and C57Bl/ 6J scid/scid mice

oral

+

ND

+h

213

S. typhimurium (SL7207)

ΔaroA

tyrosine hydroxylase

A/J mouse

oral

+

ND

+i

S. typhimurium (SL7207)

ΔaroA

β-galactosidase

BALB/c and Scid mice

oral

+

+

+/–

S. typhimurium (SL7207)

ΔaroA

β-galactosidase

BALB/c mouse

oral

+

+

+k

Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles

Table 3. Continued

214 j

215 16

83

continued on next page

84

Table 3. Continued Attenuating

Antigen

Bacteria (Strain)

Mutation

(Pathogen)

Animal Model

Immunization Route

Immune Responses

S. typhimurium (SL7207)

ΔaroA

human gp100

C57Bl/6 mouse

oral

+

ND

+l

216

S. typhimurium (RE88)

ΔaroA, Δdam

endoglin

BALB/c mouse

i.g.

+

ND

+m

217

S. typhimurium (22-11)

ΔrepA

Cellular

Ab

Protectiona Ref

MOMP (C. trachomatis)

BALB/c mouse

oral

ND

ND

+/–

111

S. choleraesuis

glycoprotein D (Pseudorabies virus)

BALB/c mouse

oral

+

+

+

167

E. coli (TG-1)

glycoprotein D (Pseudorabies virus)

BALB/cBYJ mouse

i.m

+

ND

+

172

L. acidophilus (SW1)

VP1 gene (Foot-and-mouth disease virus)

BALB/c mouse

i.m., i.p., i.n., oral

+

+

ND

218

L. monocytogenes Δ2

ΔactA, Δmpl and ΔplcB

Ag85a, Ag85b and MPB/ C57Bl/6 and BALB/c MPT51 (M. tuberculosis) mice

i.p.

+

ND

+

145

M. smegmatis

rephigh

gp120 (HIV-1)

BALB/c mouse

i.p.

+

ND

ND

219

BFR (B. abortus) P39 (B. abortus)

BALB/c mouse

i.g.

+ +

+/+

+ +

155

Y. enterocolitica O:9

Patho-Biotechnology

Abbreviations: Ab: antibodies; BFR: bacterioferritin; HA: hemagglutinin; i.g.: intragastric; i.m.: intramuscular; i.n.: intranasal; i.p.: intraperitoneal; i.v.: intravenous; MDR-1: multidrug resistance protein (colon/lung carcinoma); MOMP: major outer membrane protein; Mp1p: mannoprotein from Penicillium marneffei; ND: not determined; NP: nucleoprotein; TRP: tyrosinase related protein. a Unless otherwise stated protection is defined as survival after challenge with the wild type organism; bchallenge with vaccinia-env virus; ctransgenic mouse expressing the alpha 1 and 2 domains of human HLA-A2.1 and the alpha 3 domain of murine H-2Dd; dchallenge with vaccinia-NS3 virus; echallenge with either MDR-1 expressing CT-26 colon carcinoma cells or MDR-1 expressing Lewis lung carcinoma cells; fchallenge with the melanoma cell line B16F1; ggenes fused to a murine ubiquitin gene; hchallenge with the melanoma cell line B16G3.26; ichallenge with NXS2 tumor cells; jchallenge with murine renal carcinoma line transfected with the lacZ gene; kchallenge with the cell line F1.A11 transduced with the lacZ gene; lchallenge with B16-hgp100 tumor cells; mchallenge with the murine D2F2 breast cancer cell line.

Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles

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For example, immunization with a S. enterica ΔaroA carrying a vector coding for cruzipain (Cz) from Trypanosoma cruzi alone or in combination with a strain harboring a plasmid coding for GM-CSF resulted in the stimulation of immune responses able to control T. cruzi infection, thereby reducing parasite loads and subsequent damage to muscle tissues.120 Another application of Salmonella-mediated bactofection was in the context of gene therapy, in order to restore a genetic deficiency at systemic level. Paglia et al demonstrated that oral administration of a S. enterica ΔaroA bearing a plasmid encoding IFN-γ was able to restore the expression of IFN-γ in macrophages from IFN-γ –/– mice, thereby restoring the resistance of these immunocompromised animals against bacterial infections.121 Other promising attenuated derivatives of S. enterica serovar Typhimurium are those carrying the sopB mutantion. The SopB protein is encode in the Salmonella pathogenicity island (SPI)-5 and is secreted and translocated into the cytosol through the SPI-1 encoded type III secretion system. SopB has an inositol phosphate phosphatase activity which affects cytoskeletal rearrangement, host cell invasion and chloride homeostasis.122 The inactivation of SopB reduced inflammatory responses and fluid secretion into the intestinal lumen.123 However, they have the same tissue distribution and promote similar chemokine/cytokine profiles as wild-type strains.124 Recent studies have demonstrated that SopB is responsible for immune escape mechanisms which can be subverted to optimize carrier performance. In fact, SopB derivatives are less toxic for DCs, which are more efficient in their capacity to process and present antigens expressed by the carrier.122

Attenuated Salmonella Strains Tested in Humans

S. enterica serovar Typhi Ty21a strain is a licensed oral vaccine against typhoid fever, which was isolated after chemical and UV mutagenesis and screened for a deficiency in the galE-encoded enzyme uridine diphosphate (UDP)-galactose-4-epimerase.125 Further untargeted mutations have later been detected in the vaccine strain (e.g., deficiency in the production of the acidic Vi capsule polysaccharide and in the biosynthesis of the amino acids isoleucine and valine due to the ilvD locus mutation).126 It also harbors mutations at the rpoS locus, encoding the stationary phase-specific sigma factor S.127,128 It is genetically stable and extremely safe.126,129 The attenuated Ty21a strain, accordingly to the directive 2001/18/EC, is not considered as a genetically modified organism due to the methodology used for its generation. Nevertheless, it has an excellent biosafety profile, both for humans and the environment. The shedding by human volunteers was shown to be minimal or inexistent130,131 and the mutated rpoS locus affect bacterial resistance to environmental stresses.127,132 Ty21a, as well as the widely use attenuated S. enterica serovar Typhimurium aroA strain SL7207133 die within the phagosomal compartment of phagocytes, where they release their plasmid cargo. However, it is still unclear by which mechanism the plasmid DNA reaches the cytosol and/or the nucleus in order to be transcribed. The S. enterica serovar Typhi CVD 908-htrA strain is a derivative of the strain Ty2 in which aroC and aroD (involved in the biosynthetic pathway to aromatic metabolites) were deleted, as well as gene htrA (involved in the cellular response to stress).134 The presence of three attenuating mutations minimizes the risk of reversion to full-blown wild-type virulence. In clinical trials, CVD 908-htrA was strongly immunogenic and in contrast with the parental strain CVD 908 (Ty2 aroC aroD), CVD 908-htrA was not detectable in the blood of vaccinees.85,135 Although the duration of excretion was short, the relatively high numbers of organisms excreted per gram of stool warranted microcosm studies. The results suggested that is unlikely cells released in water or soil survive for long period of time.126 This strain represents a promising DNA carrier for human studies.

Shigella Strains Preclinical Studies

Shigella spp. entry seems to be restricted in the gut mucosa to M cell-rich sites overlying Peyer’s patches.136 Following transcytosis across M cells, Shigella may invade macrophages, DCs and colonic mucosal cells. In contrast to Salmonella spp., Shigella spp. rapidly escape from endocytic vacuoles to the cytosol, where it may undergo lysis, thereby releasing the DNA cargo (Fig. 1). S. flexneri 2a ΔaroA

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ΔiscA was employed to deliver a DNA vaccine encoding gp120 of HIV-1.104 Intranasal immunization of mice elicited strong CD8+ T-cell responses comparable to those induced by intramuscular injection of the plasmid or intraperitoneal immunization with a recombinant vaccinia-env vector. The immune responses mediated by the Shigella-based DNA vaccine were protective against a vaccinia-env challenge in a mouse model (Table 3).104

Attenuated Shigella Strains Tested in Humans

The S. flexneri 2a SC602 strain is the vaccine candidate developed at the Pasteur Institut, by deleting the plasmid-encoded icsA/virG virulence gene and the chromosomal iucA-iut siderophore aerobactin locus involved in iron uptake by the bacterial cells.137 This strain is substantially attenuated and it has been tested in several clinical studies.138-140 The obtained results demonstrated that this strain is able to evoke protection against the most severe symptoms of shigellosis. From a regulatory point of view, the residual reactogenicity of the strain at a 4-log dose may preclude routine use in humans, since there are concerns regarding potential spread to household and workplace contacts. In addition, there are no published data focused on environmental risk assessment. Several other attenuated S. flexneri vaccine strains have been generated at the Center for Vaccine Development in Baltimore USA (e.g., CVD 1204, CVD 1207, CVD 1208) by deletion of the chromosomal guaBA locus. Testing performed in human volunteers showed that the CVD 1208 strain, which harbors also a deletion in the plasmid-encoded sen and set toxin genes is the most promising one.138,141,142 This strain was well-tolerated and triggered strong immune responses, self limited fever was only observed at the higher dosages.138

Listeria monocytogenes Strains

L. monocytogenes is the most used Gram-positive carrier for the delivery of DNA vaccines. This intracellular pathogen is capable of invading a broad spectrum of host cell types through the intestinal mucosal surface, including macrophages, DCs, hepatocytes, intestinal epithelial cells or the endothelium of the blood-brain barrier.143 Like Shigella spp., Listeria multiply within the cytosol of host cells and disseminate through tissues by intracellular spread. Dietrich et al14 created a L. monocytogenes Δ2 mutant harboring deletions of the genes mpl, actA and plcB. This mutant is still able to invade eukaryotic cells entering the cytosol, but has lost its ability to spread intracellularly and from cell-to-cell, exhibiting a high degree of attenuation in animal models14 and humans.144 Further modifications, such as the engineering for an active lysis of intracellular bacteria upon escape from the phagosome, further facilitate DNA delivery.14 Recently, a L. monocytogenes attenuated mutant, which is defective in actA and plcB, was evaluated in a Phase I clinical trials for safety and immunogenicity. Some volunteers had abnormal liver functions, which were temporally associated with the inoculation. Therefore, further studies will be needed to evaluate this particular strain, before it can be widely implemented as conventional carrier and/or delivery system for DNA vaccines.

Preclinical Studies

Vaccination with a self-destructing attenuated L. monocytogenes Δ2 strain carrying eukaryotic expression plasmids encoding antigen 85 complex (Ag85A and Ag85B) and MPB/MPT51 (mycobacterial proteins secreted by M. bovis BCG/M. tuberculosis) molecules induced M. tuberculosis-specific cellular immunity (e.g., foot pad reactions, proliferative responses and IFN-γ production), which conferred comparable levels of protection to those evoked by the Mycobacterium bovis BCG vaccine strain (Table 3).145 Administration of a recombinant Listeria vaccine strain called LM-LLO-E7, which secrete the HPV-16 E7 protein fused to a nonhemolytic form of LLO, resulted in complete regression of tumors in a well-characterized mouse model of cervical cancer (TC-1 tumor cell line). Even when the strategy worked, Listeria cannot secrete highly hydrophobic proteins, presumably due to their difficulty in translocating across the membrane or cell wall. Therefore, a Listeria-based DNA vaccine which contains the actA-ply118 suicide release system was tested in the same tumor mouse experimental model. The results demonstrated that the system was able to promote the stimulation

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87

of E7-specifc CD8+ effector T-cells that home to and penetrate the tumor, resulting in significant reduction of tumor growth, but it was not sufficient to induce tumor regression.146 On the other hand, L. monocytogenes can be used to deliver mRNA encoding antigens directly into the cytosol of host cells because of its intracellular lifestyle (Fig. 1). In fact, vaccination of mice with self-destructing L. monocytogenes carriers delivering mRNA that encodes a nonsecreted form of ovalbumin (OVA) resulted in a significant OVA-specific CD8+ T-cell response.76

Vibrio cholera Strains

Vibrio cholera is an extracellular pathogen, therefore, no studies have been performed until now to evaluate V. cholera as DNA delivery system. However, due to the availability of attenuated strains for human use, including the actual licensed attenuated vaccine, it is reasonable to conceive that as it was done with E. coli, V. cholera can be engineered to invade specific eukaryotic cells and deliver DNA plasmid.116

Vibrio cholerae CVD 103-HgR

The classical Inaba recombinant cholera vaccine strain CVD 103-HgR was constructed at the Center for Vaccine Development in Baltimore, MD, USA.147,148 Since 1994, it is the first and only licensed live oral vaccine prepared by recombinant DNA techniques. Therefore, it has served as a standard for the registration of genetically modified organism-based vaccines. The CVD 103-HgR was constructed by deletion of 94% of the ctxA active subunit gene of the cholera toxin operon and the deletion of the haemolysin gene (hlyA), wherein it is inserted the mercury resistance operon from the NR1 plasmid of S. flexneri.149 This vaccine strain is extremely safe and protective efficacy up to 100% against homologous challenge was observed after challenge in human volunteers.150 The strain is genetically stable during production and during transit through the gut of vaccinees. On the other hand, less than 200 CVD103-HgR live bacteria are shed per gram faeces by 20-30% of vaccinees from non-endemic countries (maximum of 7 days), which is remarkable lower in comparison with a wild-type strain (>107 vibrios per g faeces).150,151 The vaccine strain dies within 20 days under various environmental conditions.149

Vibrio Cholerae Peru-15

The O1 El Tor Inaba vaccine strain was constructed by deletion of the entire cholera toxin locus (CTXΦ) and subsequent integration of the ctxB gene within recA locus- This disables the recombinational capability of the vaccine while preserving expression of the CT-B subunit. The final strain was also selected for motility deficiency and a nonfilamentous phenotype.152 In clinical trials this vaccine strain was well tolerated, promoting 100% protection against moderate to severe diarrhea following homologous challenge.153 However, Peru-15 was excreted at higher levels and for longer periods of time than CVD 103-HgR.154

Other Bacterial Vectors

An attenuated Yersinia enterocolitica strain was tested as delivery system for a DNA vaccine against Brucella abortus. Preliminary studies have shown that animals immunized with a Yersinia strain expressing the lipopolysaccharide of the serotype O:9 (cross reacting with the Brucella) showed partial protection against challenge, but not control animals immunized with a strain expressing the serotype O:3. Although anti-lipopolysaccharide antibodies play an important role in protective immunity against Brucella, it is also known that a Th1 response seems to be critical to eliminate intracellular residing pathogens. This brought to the notion that the Yersinia strains O:9 and O:3 could be used as DNA delivery vectors for plasmids coding for the B. abortus antigens bacterioferritin and P39 (Table 3). Both prototypes induced a strong Th1 response with IFN-γ production. However, the best protection was achieved using Yersinia O:9 harboring the plasmid encoding for P39.155 This confirms that both cellular and humoral responses are necessary for achieving optimal protection against this pathogen. Attenuated Yersinia pseudotuberculosis strains have also been used to deliver DNA in vitro and in vivo. However, due to safety reasons, it will probably be more difficult to implement Yersinia strains vaccine delivery vehicles.155,156

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Recently, a Lactobacillus acidophilus SW1 strain harboring a vector encoding the structural protein VP1 of the foot-and-mouth disease virus (FMDV) was used as DNA delivery vector (Table 3). The strongest antibody responses against VP1 were observed after intramuscular administration, followed by those of animals receiving the strain by intraperitoneal, intranasal and oral routes. In addition, immunization with L. acidophilus was able to generate moderate cellular immune responses. The results clearly demonstrate that the use of Lactobacillus as a carrier is a promising approach for DNA vaccination. Other bacteria that have been used as DNA delivery systems are Bifidobacterium longum,157 which is a nonpathogen bacterium found in the lower small intestine of humans and Agrobacterium tumefaciens, a soil pathogen that elicit neoplastic growths on host plant species.158 In nature, Agrobacterium introduces several oncogenic genes into the host plant, leading to formation of tumors, being widely used in the laboratory for plant genetic engineering.158,159 Agrobacterium infection requires the presence of two genetic components located on the bacterial tumor-inducing (Ti) plasmid: the transferred DNA (T-DNA), which is introduced into the plant cell genome and the virulence region encoding most components of the protein apparatus for T-DNA transfer. Kunik et al demonstrated that Agrobacterium is also able to introduce T-DNA into mammalian cells. Therefore, it would be possible to deliver DNA for noninvasive bacteria into intact mammalian cells.158

Conclusions and Outlook

The development of DNA vaccines is considerably faster than their subunit counterpart since major bottlenecks, such as difficulties in antigen expression and purification, lack of posttranslational modifications, poor stability and batch-to-batch variability are solved. Thus, it is expected that due to their cost- and time-efficient development, they will be rapidly transfer into the clinical development pipeline. In fact, the first clinical trials of DNA vaccines are now paving the road for their transfer into the field. However, there are still major drawbacks associated with the use of DNA vaccines, such as the need of extremely high DNA dosages and poor immunogenicity. Therefore, considerable efforts are being invested in the establishment of efficient alternatives for the delivery of DNA vaccines. The use of live attenuated bacterial as carries for DNA vaccines is a very promising approach. There are several benefits over other systems, like no need for plasmid purification, built-in immune stimulatory properties and amenability for mucosal administration. Furthermore, many of the bacterial vectors coming into consideration as carriers for DNA vaccines allow specific targeting of plasmids to APCs. Albeit pre-existing immunity to the live carrier may affect the efficacy DNA delivery, it has been shown that not necessarily this has important detrimental effects.160 However, before live bacterial vector-mediated DNA delivery reaches clinical trials, some roadblocks should be overcome. It is essential to elucidate the underlying molecular events leading to DNA transfer from bacteria to the cytosol and to the nucleus, which will probably differ between different carries. This is indeed a prerequisite to establish strategies to maximize DNA release. In fact, most of the attempts for vector improvement were based on empiric approaches up to now. This can in turn explain the high variability observed between studies performed using different experimental settings, as well as the fact that already planned clinical trials have been consistently delayed. It would be also of interest to develop bacterial carriers able to target specific cellular subsets in vivo, in order to fine-tune the elicited responses. In conclusion, bactofection appeals as a prominent strategy in the field of DNA vaccination, which in the coming years should prove its worth in clinical trials.

References

1. Ito Y. A tumor-producing factor extracted by phenol from papillomatous tissue (Shope) of cottontail rabbits. Virology 1960; 12:596-601. 2. Atanasiu P, Orth G, Dragonas P. Delayed specific antitumoral resistance in the hamster immunized shortly after birth with the polyoma virus. C R Hebd Seances Acad Sci 1962; 254:2250-2252. 3. Fried M, Klein B, Murray K et al. Infectivity in mouse fibroblasts of polyoma DNA integrated into plasmid pBR322 or lambdoid phage DNA. Nature 1979; 279(5716):811-816.

Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles

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4. Wolff J, Malone R, Williams P et al. Direct gene transfer into mouse muscle in vivo. Science 1990; 247:1465-1468. 5. Ulmer JB, Donnelly JJ, Parker SE et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993; 259(5102):1745-1749. 6. Lelouard H, Ferrand V, Marguet D et al. Dendritic cell aggresome-like induced structures are dedicated areas for ubiquitination and storage of newly synthesized defective proteins. J Cell Biol 2004; 164(5):667-675. 7. Lelouard H, Gatti E, Cappello F et al. Transient aggregation of ubiquitinated proteins during dendritic cell maturation. Nature 2002; 417(6885):177-182. 8. Kawabata K, Takakura Y, Hashida M. The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake. Pharm Res 1995; 12(6):825-830. 9. Lew D, Parker SE, Latimer T et al. Cancer gene therapy using plasmid DNA: Pharmacokinetic study of DNA following injection in mice. Hum Gene Ther 1995; 6(5):553-564. 10. Schaffner W. Direct transfer of cloned genes from bacteria to mammalian cells. Proc Natl Acad Sci USA 1980; 77(4):2163-2167. 11. Courvalin P, Goussard S, Grillot-Courvalin C. Gene transfer from bacteria to mammalian cells. C R Acad Sci III 1995; 318(12):1207-1212. 12. Darji A, Guzman CA, Gerstel B et al. Oral somatic transgene vaccination using attenuated S. typhimurium Cell 12 1997; 91(6):765-775. 13. Sizemore DR, Branstrom AA, Sadoff JC. Attenuated Shigella as a DNA delivery vehicle for DNA-mediated immunization. Science 1995; 270(5234):299-302. 14. Dietrich G, Bubert A, Gentschev I et al. Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nat Biotechnol 1998; 16(2):181-185. 15. Levine MM, Sztein MB. Vaccine development strategies for improving immunization: The role of modern immunology. Nat Immunol 2004; 5(5):460-464. 16. Paglia P, Medina E, Arioli I et al. Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma. Blood 1998; 92(9):3172-3176. 17. Mellman I, Steinman RM. Dendritic cells: Specialized and regulated antigen processing machines. Cell 2001; 106(3):255-258. 18. Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol 2002; 20:197-216. 19. Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity. Nat Immunol 2004; 5(10):971-974. 20. Walker RI. New strategies for using mucosal vaccination to achieve more effective immunization. Vaccine 1994; 12(5):387-400. 21. Bertolla F, Simonet P. Horizontal gene transfers in the environment: Natural transformation as a putative process for gene transfers between transgenic plants and microorganisms. Res Microbiol 1999; 150:375-384. 22. Nielsen K, Bones A, Smalla K et al. Horizontal gene transfer from transgenic plants to terrestrial bacteria—a rare event? FEMS Microbiol Rev 1998; 22:79-103. 23. Lewin A, Mayer M, Chusainow J et al. Viral promoters can initiate expression of toxin genes introduced into Escherichia coli. BMC Biotechnol 2005; 5:19. 24. Harms J, Oliveira S, Splitter G. Regulation of transgene expression in genetic immunization. Braz J Med Biol Res 1999; 32:155-162. 25. Qin L, Ding Y, Pahud D et al. Promoter attenuation in gene therapy: Interferon-gamma and tumor necrosis factor-alpha inhibit transgene expression. Hum Gene Ther 1997; 8:2019-2029. 26. Loirat D, Li Z, Mancini M et al. Muscle-specific expression of hepatitis B surface antigen: no effect on DNA-raised immune responses. Virology 1999; 260(1):74-83. 27. Bojak A, Hammer D, Wolf H et al. Muscle specific versus ubiquitous expression of Gag based HIV-1 DNA vaccines: a comparative analysis. Vaccine 2002; 20(15):1975-1979. 28. Cazeaux N, Bennasser Y, Vidal PL et al. Comparative study of immune responses induced after immunization with plasmids encoding the HIV-1 Nef protein under the control of the CMV-IE or the muscle-specific desmin promoter. Vaccine 2002; 20(27-28):3322-3331. 29. Vanniasinkam T, Reddy ST, Ertl HC. DNA immunization using a nonviral promoter. Virology 2006; 344(2):412-420. 30. Tornoe J, Kusk P, Johansen TE et al. Generation of a synthetic mammalian promoter library by modification of sequences spacing transcription factor binding sites. Gene 2002; 297(1-2):21-32. 31. Garg S, Oran AE, Hon H et al. The hybrid cytomegalovirus enhancer/chicken beta-actin promoter along with woodchuck hepatitis virus posttranscriptional regulatory element enhances the protective efficacy of DNA vaccines. J Immunol 2004; 173(1):550-558.

90

Patho-Biotechnology

32. Proudfoot N. How RNA polymerase II terminates transcription in higher eukaryotes. Trends Biochem Sci 1989; 14:105-110. 33. Xu Z-L, Mizuguchi H, Ishii-Watabe A et al. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene 2001; 272:149-156. 34. Kozak M. At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J Mol Biol 1987; 196:947-950. 35. Kozak M. Recognition of AUG and alternative initiator codons is augmented by G in position +4 but is not generally affected by the nucleotides in positins +5 and +6. EMBO J 1997; 16:2482-2492. 36. Andre S, Seed B, Eberle J et al. Increased Immune Response Elicited by DNA Vaccination with a Synthetic gp120 Sequence with Optimized Codon Usage. J Virol 1998; 72(2):1497-1503. 37. zur Megede J, Chen MC, Doe B et al. Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene. J Virol 2000; 74(6):2628-2635. 38. Liu WJ, Zhao K-N, Gao FG et al. Polynucleotide viral vaccines: codon optimisation and ubiquitin conjugation enhances prophylactic and therapeutic efficacy. Vaccine 2001; 20(5-6):862-869. 39. Grosjean H, Fliers W. Preferential codon usage in prokaryotic genes: The optimal codon-anticodon interaction energ y and the selective codon usage in efficiently expressed genes. Gene 1982; 18:299-209. 40. Ko HJ, Ko SY, Kim YJ et al. Optimization of codon usage enhances the immunogenicity of a DNA vaccine encoding mycobacterial antigen Ag85B. Infect Immun 2005; 73(9):5666-5674. 41. Narum DL, Kumar S, Rogers WO et al. Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice. Infect Immun 2001; 69(12):7250-7253. 42. Deml L, Bojak A, Steck S et al. Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein. J Virol 2001; 75(22):10991-11001. 43. Kane JF. Effects of rare codon clusters on high-level expression of heterologous proteins in Escherichia coli. Curr Opin Biotechnol 1995; 6(5):494-500. 44. Schwartz S, Campbell M, Nasioulas G et al. Mutational inactivation of an inhibitory sequence in human immunodeficiency virus type 1 results in Rev-independent gag expression. J Virol 1992; 66(12):7176-7182. 45. Li Z, Howard A, Kelley C et al. Immunogenicity of DNA vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences. Infect Immun 1999; 67(9):4780-4786. 46. Leitner WW, Seguin MC, Ballou WR et al. Immune responses induced by intramuscular or gene gun injection of protective deoxyribonucleic acid vaccines that express the circumsporozoite protein from Plasmodium berghei malaria parasites. J Immunol 1997; 159(12):6112-6119. 47. Burke RL, Pachl C, Q uiroga M et al. The functional domains of coagulation factor VIII:C. J Biol Chem 1986; 261(27):12574-12578. 48. Chapman BS, Thayer RM, Vincent KA et al. Effect of intron A from human cytomegalovirus (Towne) immediate-early gene on heterologous expression in mammalian cells. Nucleic Acids Res 1991; 19(14):3979-3986. 49. Haddad D, Liljeqvist S, Stahl S et al. Comparative study of DNA-based immunization vectors: effect of secretion signals on the antibody responses in mice. FEMS Immunol Med Microbiol 1997; 18(3):193-202. 50. Ashok MS, Rangarajan PN. Protective efficacy of a plasmid DNA encoding Japanese encephalitis virus envelope protein fused to tissue plasminogen activator signal sequences: Studies in a murine intracerebral virus challenge model. Vaccine 2002; 20(11-12):1563-1570. 51. Cheng W-F, Hung C-F, Hsu K-F et al. Enhancement of Sindbis Virus Self-Replicating RNA Vaccine Potency by Targeting Antigen to Endosomal/Lysosomal Compartments doi:10.1089/1043034015021 8387. Human Gene Therapy 2001; 12(3):235-252. 52. Wu Y, Kipps TJ. Deoxyribonucleic acid vaccines encoding antigens with rapid proteasomedependent degradation are highly efficient inducers of cytolytic T-lymphocytes. J Immunol 1997; 159(12):6037-6043. 53. Rodriguez F, Zhang J, Whitton JL. DNA immunization: Ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. J Virol 1997; 71(11):8497-8503. 54. Isaguliants MG, Petrakova NN, Zuber B et al. DNA-encoding enzymatically active HIV-1 reverse transcriptase, but not the inactive mutant, confers resistance to experimental HIV-1 challenge. Intervirology 2000; 43(4-6):288-293. 55. Liang X, Fu TM, Xie H et al. Development of HIV-1 Nef vaccine components: Immunogenicity study of Nef mutants lacking myristoylation and dileucine motif in mice. Vaccine 2002; 20(27-28):3413-3421.

Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles

91

56. Smahel M, Sima P, Ludvikova V et al. Modified HPV16 E7 Genes as DNA Vaccine against E7-Containing Oncogenic Cells. Virology 2001; 281(2):231-238. 57. Ertl PF, Thomsen LL. Technical issues in construction of nucleic acid vaccines. Methods Nucleic Acid Vaccines 2003; 31(3):199-206. 58. Krieg AM, Yi AK, Matson S et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995; 374(6522):546-549. 59. Bauer S, Kirschning CJ, Hacker H et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA 2001; 98(16):9237-9242. 60. Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B-cells. J Immunol 2000; 164(2):944-953. 61. Hemmi H, Takeuchi O, Kawai T et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408(6813):740-745. 62. Krieg AM, Yi A-K, Hartmann G. Mechanisms and therapeutic applications of immune stimulatory CpG DNA. Pharmacol Ther 1999; 84(2):113-120. 63. Klinman DM. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat Rev Immunol 2004; 4(4):249-258. 64. Krieg AM. CpG motifs in bacterial DNA and their immune eff ects. Annu Rev Immunol 2002; 20:709-760. 65. Yamada H, Gursel I, Takeshita F et al. Effect of suppressive DNA on CpG-induced immune activation. J Immunol 2002; 169(10):5590-5594. 66. Krieg AM, Wu T, Weeratna R et al. Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs. Proc Natl Acad Sci USA 1998; 95(21):12631-12636. 67. Matsumoto M, Funami K, Oshiumi H et al. Toll-like receptor 3: A link between toll-like receptor, interferon and viruses. Microbiol Immunol 2004; 48(3):147-154. 68. Cella M, Salio M, Sakakibara Y et al. Maturation, activation and protection of dendritic cells induced by double-stranded RNA. J Exp Med 1999; 189(5):821-829. 69. Karpala AJ, Doran TJ, Bean AG. Immune responses to dsRNA: Implications for gene silencing technologies. Immunol Cell Biol 2005; 83(3):211-216. 70. Yoneyama M, Suhara W, Fukuhara Y et al. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J 1998; 17(4):1087-1095. 71. Gil J, Esteban M. Induction of apoptosis by the dsRNA-dependent protein kinase (PKR): Mechanism of action. Apoptosis 2000; 5(2):107-114. 72. Bergmann-Leitner ES, Leitner WW. Danger, death and DNA vaccines. Microbes Infect 2004; 6(3):319-327. 73. Castelli J, Wood KA, Youle RJ. The 2-5A system in viral infection and apoptosis. Biomed Pharmacother 1998; 52(9):386-390. 74. Li D, Liu Y, Zhang Y et al. Adjuvant effects of plasmid-generated hairpin RNA molecules on DNA vaccination. Vaccine 2007; 25(39-40):6992-7000. 75. Schoen C, Stritzker J, Goebel W et al. Bacteria as DNA vaccine carriers for genetic immunization. Int J Med Microbiol 2004; 294(5):319-335. 76. Loeffler DI, Schoen CU, Goebel W et al. Comparison of different live vaccine strategies in vivo for delivery of protein antigen or antigen-encoding DNA and mRNA by virulence-attenuated Listeria monocytogenes. Infect Immun 2006; 74(7):3946-3957. 77. Chatwin HM, Summers DK. Monomer-dimer control of the ColE1 Pcer promoter. Microbiology 2001; 147(11):3071-3081. 78. Chant EL, Summers DK. Indole signalling contributes to the stable maintenance of Escherichia coli multicopy plasmids. Mol Microbiol 2007; 63(1):35-43. 79. Hartikka J, Sawdey M, Cornefert-Jensen F et al. An improved plasmid DNA expression vector for direct injection into skeletal muscle. Hum Gene Ther 1996; 7(10):1205-1217. 80. Curtiss R 3rd, Galan JE, Nakayama K et al. Stabilization of recombinant avirulent vaccine strains in vivo. Res Microbiol 1990; 141(7-8):797-805. 81. Zhang T, Stanley SL Jr. Oral immunization with an attenuated vaccine strain of Salmonella typhimurium expressing the serine-rich Entamoeba histolytica protein induces an antiamebic immune response and protects gerbils from amebic liver abscess. Infect Immun 1996; 64(5):1526-1531. 82. Kang HY, Srinivasan J, Curtiss R 3rd. Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar Typhimurium vaccine. Infect Immun 2002; 70(4):1739-1749. 83. Wyszynska A, Raczko A, Lis M et al. Oral immunization of chickens with avirulent Salmonella vaccine strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral immune response associated with protection against challenge with wild-type Campylobacter. Vaccine 2004; 22(11-12):1379-1389.

92

Patho-Biotechnology

84. Nardelli-Haefliger D, Kraehenbuhl JP, Curtiss R 3rd et al. Oral and rectal immunization of adult female volunteers with a recombinant attenuated Salmonella typhi vaccine strain. Infect Immun 1996; 64(12):5219-5224. 85. Tacket CO, Kelly SM, Schodel F et al. Safety and immunogenicity in humans of an attenuated Salmonella typhi vaccine vector strain expressing plasmid-encoded hepatitis B antigens stabilized by the Asd-balanced lethal vector system. Infect Immun 1997; 65(8):3381-3385. 86. Morona R, Yeadon J, Considine A et al. Construction of plasmid vectors with a non-antibiotic selection system based on the Escherichia coli thyA+ gene: Application to cholera vaccine development. Gene 1991; 107(1):139-144. 87. Tacket CO, Forrest B, Morona R et al. Safety, immunogenicity and efficacy against cholera challenge in humans of a typhoid-cholera hybrid vaccine derived from Salmonella typhi Ty21a. Infect Immun 1990; 58(6):1620-1627. 88. Attridge SR, Dearlove C, Beyer L et al. Characterization and immunogenicity of EX880, a Salmonella typhi Ty21a-based clone which produces Vibrio cholerae O antigen. Infect Immun 1991; 59(7):2279-2284. 89. Bumann D, Metzger WG, Mansouri E et al. Safety and immunogenicity of live recombinant Salmonella enterica serovar Typhi Ty21a expressing urease A and B from Helicobacter pylori in human volunteers. Vaccine 2001; 20(5-6):845-852. 90. Liang W, Wang S, Yu F et al. Construction and evaluation of a safe, live, oral Vibrio cholerae vaccine candidate, IEM108. Infect Immun 2003; 71(10):5498-5504. 91. Fu X, Xu JG. Development of a chromosome-plasmid balanced lethal system for Lactobacillus acidophilus with thyA gene as selective marker. Microbiol Immunol 2000; 44(7):551-556. 92. DiPetrillo MD, Tibbetts T, Kleanthous H et al. Safety and immunogenicity of phoP/phoQ-deleted Salmonella typhi expressing Helicobacter pylori urease in adult volunteers. Vaccine 1999; 18(5-6):449-459. 93. Angelakopoulos H, Hohmann EL. Pilot study of phoP/phoQ-deleted Salmonella enterica serovar Typhimurium expressing Helicobacter pylori urease in adult volunteers. Infect Immun 2000; 68(4):2135-2141. 94. Ryan ET, Crean TI, Kochi SK et al. Development of a DeltaglnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae. Infect Immun 2000; 68(1):221-226. 95. Hagg P, de Pohl JW, Abdulkarim F et al. A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli. J Biotechnol 2004; 111(1):17-30. 96. Gerdes K, Gultyaev AP, Franch T et al. Antisense RNA-regulated programmed cell death. Annu Rev Genet 1997; 31:1-31. 97. Hense M, Domann E, Krusch S et al. Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Cell Microbiol 2001; 3(9):599-609. 98. Ledwith BJ, Manam S, Troilo PJ et al. Plasmid DNA vaccines: Assay for integration into host genomic DNA. Dev Biol (Basel) 2000; 104:33-43. 99. Cichutek K. DNA vaccines: development, standardization and regulation. Intervirology 2000; 43(4-6):331-338. 100. Spreng S, Viret JF. Plasmid maintenance systems suitable for GMO-based bacterial vaccines. Vaccine 2005; 23(17-18):2060-2065. 101. Robertson JS, Griffiths E. Assuring the quality, safety and efficacy of DNA vaccines. Methods Mol Med 2006; 127:363-374. 102. Grillot-Courvalin C, Goussard S, Huetz F et al. Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol 1998; 16(9):862-866. 103. Pilgrim S, Stritzker J, Schoen C et al. Bactofection of mammalian cells by Listeria monocytogenes: improvement and mechanism of DNA delivery. Gene Ther 2003; 10(24):2036-2045. 104. Shata MT, Hone DM. Vaccination with a Shigella DNA vaccine vector induces antigen-specifi c CD8(+) T-cells and antiviral protective immunity. J Virol 2001; 75(20):9665-9670. 105. Escriou V, Carriere M, Bussone F et al. Critical assessment of the nuclear import of plasmid during cationic lipid-mediated gene transfer. J Gene Med 2001; 3(2):179-187. 106. Fasbender A, Zabner J, Zeiher BG et al. A low rate of cell proliferation and reduced DNA uptake limit cationic lipid-mediated gene transfer to primary cultures of ciliated human airway epithelia. Gene Th er 1997; 4(11):1173-1180. 107. Mortimer I, Tam P, MacLachlan I et al. Cationic lipid-mediated transfection of cells in culture requires mitotic activity. Gene Ther 1999; 6(3):403-411. 108. Zheng B, Woo PC, Ng M et al. A crucial role of macrophages in the immune responses to oral DNA vaccination against hepatitis B virus in a murine model. Vaccine 2001; 20(1-2):140-147.

Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles

93

109. Zupan JR, Citovsky V, Zambryski P. Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells. Proc Natl Acad Sci USA 1996; 93(6):2392-2397. 110. Weiss S, Chakraborty T. Transfer of eukaryotic expression plasmids to mammalian host cells by bacterial carriers. Curr Opin Biotechnol 2001; 12(5):467-472. 111. Brunham RC, Zhang D. Transgene as vaccine for chlamydia. Am Heart J 1999; 138(5 Pt 2): S519-522. 112. Flo J, Tisminetzky S, Baralle F. Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus-2 induced disease in mice. Vaccine 2001; 19(13-14):1772-1782. 113. Elias F, Flo J. Modulation of the immune response mediated by oral transgene administration of IL-10. Cell Immunol 2002; 216(1-2):73-81. 114. Woo PC, Wong LP, Zheng BJ et al. Unique immunogenicity of hepatitis B virus DNA vaccine presented by live-attenuated Salmonella typhimurium. Vaccine 2001; 19(20-22):2945-2954. 115. Wedemeyer H, Gagneten S, Davis A et al. Oral immunization with HCV-NS3-transformed Salmonella: induction of HCV-specific CTL in a transgenic mouse model. Gastroenterology 2001; 121(5):1158-1166. 116. Jain V, Mekalanos JJ. Use of lambda phage S and R gene products in an inducible lysis system for Vibrio cholerae- and Salmonella enterica serovar Typhimurium-based DNA vaccine delivery systems. Infect Immun 2000; 68(2):986-989. 117. Royo JL, Becker PD, Camacho EM et al. In vivo gene regulation in Salmonella spp. by a salicylatedependent control circuit. Nat Methods 2007; 4(11):937-942. 118. Rosenkranz CD, Chiara D, Agorio C et al. Towards new immunotherapies: Targeting recombinant cytokines to the immune system using live attenuated Salmonella. Vaccine 2003; 21(7-8):798-801. 119. Yuhua L, Kunyuan G, Hui C et al. Oral cytokine gene therapy against murine tumor using attenuated Salmonella typhimurium. Int J Cancer 2001; 94(3):438-443. 120. Cazorla SI, Becker PD, Frank FM et al. Oral vaccination with Salmonella as cruzipain-DNA delivery system confers protective immunity against Trypanosoma cruzi. Infect Immun 2007. 121. Paglia P, Terrazzini N, Schulze K et al. In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene Ther 2000; 7(20):1725-1730. 122. Link C, Ebensen T, Standner L et al. An SopB-mediated immune escape mechanism of Salmonella enterica can be subverted to optimize the performance of live attenuated vaccine carrier strains. Microbes Infect 2006; 8(8):2262-2269. 123. Zhang S, Santos RL, Tsolis RM et al. The Salmonella enterica serotype Typhimurium effector proteins SipA, SopA, SopB, SopD and SopE2 Act in concert to induce diarrhea in calves. Infect Immun 2002; 70(7):3843-3855. 124. Reis BP, Zhang S, Tsolis RM et al. The attenuated sopB mutant of Salmonella enterica serovar Typhimurium has the same tissue distribution and host chemokine response as the wild type in bovine Peyer’s patches. Vet Microbiol 2003; 97(3-4):269-277. 125. Germanier R, Fuer E. Isolation and characterization of Gal E mutant Ty 21a of Salmonella typhi: A candidate strain for a live, oral typhoid vaccine. J Infect Dis 1975; 131(5):553-558. 126. Favre D, Viret JF. Biosafety evaluation of recombinant live oral bacterial vaccines in the context of European regulation. Vaccine 2006; 24(18):3856-3864. 127. Robbe-Saule V, Coynault C, Norel F. The live oral typhoid vaccine Ty21a is a rpoS mutant and is susceptible to various environmental stresses. FEMS Microbiol Lett 1995; 126(2):171-176. 128. Coynault C, Robbe-Saule V, Norel F. Virulence and vaccine potential of Salmonella typhimurium mutants deficient in the expression of the RpoS (sigma S) regulon. Mol Microbiol 1996; 22(1):149-160. 129. Gentschev I, Spreng S, Sieber H et al. Vivotif—A ‘magic shield’ for protection against typhoid fever and delivery of heterologous antigens. Chemotherapy 2007; 53(3):177-180. 130. Gilman RH, Hornick RB, Woodard WE et al. Evaluation of a UDP-glucose-4-epimeraseless mutant of Salmonella typhi as a liver oral vaccine. J Infect Dis 1977; 136(6):717-723. 131. Wahdan MH, Serie C, Cerisier Y et al. A controlled field trial of live Salmonella typhi strain Ty 21a oral vaccine against typhoid: Three-year results. J Infect Dis 1982; 145(3):292-295. 132. Rozen Y, Belkin S. Sur vival of enteric bacteria in seawater. FEMS Microbiol Rev 2001; 25(5):513-529. 133. Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella typhimurium are nonvirulent and effective as live vaccines. Nature 1981; 291(5812):238-239. 134. Levine MM, Galen J, Barry E et al. Attenuated Salmonella as live oral vaccines against typhoid fever and as live vectors. J Biotechnol 1996; 44(1-3):193-196. 135. Tacket CO, Sztein MB, Wasserman SS et al. Phase 2 clinical trial of attenuated Salmonella enterica serovar typhi oral live vector vaccine CVD 908-htrA in US volunteers. Infect Immun 2000; 68(3):1196-1201.

94

Patho-Biotechnology

136. Philpott DJ, Edgeworth JD, Sansonetti PJ. The pathogenesis of Shigella flexneri infection: lessons from in vitro and in vivo studies. Philos Trans R Soc Lond B Biol Sci 2000; 355(1397):575-586. 137. Barzu S, Fontaine A, Sansonetti P et al. Induction of a local anti-IpaC antibody response in mice by use of a Shigella flexneri 2a vaccine candidate: implications for use of IpaC as a protein carrier. Infect Immun 1996; 64(4):1190-1196. 138. Riddle MS, Sanders JW, Putnam SD et al. Incidence, etiology and impact of diarrhea among long-term travelers (US military and similar populations): A systematic review. Am J Trop Med Hyg 2006; 74(5):891-900. 139. Katz DE, Coster TS, Wolf MK et al. Two studies evaluating the safety and immunogenicity of a live, attenuated Shigella flexneri 2a vaccine (SC602) and excretion of vaccine organisms in North American volunteers. Infect Immun 2004; 72(2):923-930. 140. Coster TS, Hoge CW, VanDeVerg LL et al. Vaccination against shigellosis with attenuated Shigella flexneri 2a strain SC602. Infect Immun 1999; 67(7):3437-3443. 141. Kotloff KL, Noriega F, Losonsky GA et al. Safety, immunogenicity and transmissibility in humans of CVD 1203, a live oral Shigella flexneri 2a vaccine candidate attenuated by deletions in aroA and virG. Infect Immun 1996; 64(11):4542-4548. 142. Kotloff KL, Noriega FR, Samandari T et al. Shigella flexneri 2a strain CVD 1207, with specific deletions in virG, sen, set and guaBA, is highly attenuated in humans. Infect Immun 2000; 68(3):1034-1039. 143. Vazquez-Boland JA, Kuhn M, Berche P et al. Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 2001; 14(3):584-640. 144. Angelakopoulos H, Loock K, Sisul DM et al. Safety and shedding of an attenuated strain of Listeria monocytogenes with a deletion of actA/plcB in adult volunteers: A dose escalation study of oral inoculation. Infect Immun 2002; 70(7):3592-3601. 145. Miki K, Nagata T, Tanaka T et al. Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51. Infect Immun 2004; 72(4):2014-2021. 146. Souders NC, Verch T, Paterson Y. In vivo bactofection: Listeria can function as a DNA-cancer vaccine. DNA Cell Biol 2006; 25(3):142-151. 147. Levine MM, Kaper JB, Herrington D et al. Safety, immunogenicity and efficacy of recombinant live oral cholera vaccines, CVD 103 and CVD 103-HgR. Lancet 1988; 2(8609):467-470. 148. Ketley JM, Michalski J, Galen J et al. Construction of genetically marked Vibrio cholerae O1 vaccine strains. FEMS Microbiol Lett 1993; 111(1):15-21. 149. Viret JF, Dietrich G, Favre D. Biosafety aspects of the recombinant live oral Vibrio cholerae vaccine strain CVD 103-HgR. Vaccine 2004; 22(19):2457-2469. 150. Tacket CO, Cohen MB, Wasserman SS et al. Randomized, double-blind, placebo-controlled, multicentered trial of the efficacy of a single dose of live oral cholera vaccine CVD 103-HgR in preventing cholera following challenge with Vibrio cholerae O1 El tor inaba three months after vaccination. Infect Immun 1999; 67(12):6341-6345. 151. Cryz SJ Jr, Kaper J, Tacket C et al. Vibrio cholerae CVD103-HgR live oral attenuated vaccine: construction, safety, immunogenicity, excretion and nontarget effects. Dev Biol Stand 1995; 84:237-244. 152. Kenner JR, Coster TS, Taylor DN et al. Peru-15, an improved live attenuated oral vaccine candidate for Vibrio cholerae O1. J Infect Dis 1995; 172(4):1126-1129. 153. Cohen MB, Giannella RA, Bean J et al. Randomized, controlled human challenge study of the safety, immunogenicity and protective efficacy of a single dose of Peru-15, a live attenuated oral cholera vaccine. Infect Immun 2002; 70(4):1965-1970. 154. Killeen K, Spriggs D, Mekalanos J. Bacterial mucosal vaccines: Vibrio cholerae as a live attenuated vaccine/vector paradigm. Curr Top Microbiol Immunol 1999; 236:237-254. 155. Al-Mariri A, Tibor A, Lestrate P et al. Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen. Infect Immun 2002; 70(4):1915-1923. 156. Dietrich G, Spreng S, Favre D et al. Live attenuated bacteria as vectors to deliver plasmid DNA vaccines. Curr Opin Mol Ther 2003; 5(1):10-19. 157. Yazawa K, Fujimori M, Nakamura T et al. Bifidobacterium longum as a delivery system for gene therapy of chemically induced rat mammary tumors. Breast Cancer Res Treat 2001; 66(2):165-170. 158. Kunik T, Tzfira T, Kapulnik Y et al. Genetic transformation of HeLa cells by Agrobacterium. Proc Natl Acad Sci USA 2001; 98(4):1871-1876. 159. Bevan M. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res 1984; 12(22):8711-8721. 160. Fennelly GJ, Khan SA, Abadi MA et al. Mucosal DNA vaccine immunization against measles with a highly attenuated Shigella flexneri vector. J Immunol 1999; 162(3):1603-1610.

Genetic Immunization: Bacteria as DNA Vaccine Delivery Vehicles

95

161. Urashima M, Suzuki H, Yuza Y et al. An oral CD40 ligand gene therapy against lymphoma using attenuated Salmonella typhimurium. Blood 2000; 95(4):1258-1263. 162. Niethammer AG, Xiang R, Becker JC et al. A DNA vaccine against VEGF receptor 2 prevents eff ective angiogenesis and inhibits tumor growth. Nat Med 2002; 8(12):1369-1375. 163. Niethammer AG, Xiang R, Ruehlmann JM et al. Targeted interleukin 2 therapy enhances protective immunity induced by an autologous oral DNA vaccine against murine melanoma. Cancer Res 2001; 61(16):6178-6184. 164. Pertl U, Wodrich H, Ruehlmann JM et al. Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma. Blood 2003; 101(2):649-654. 165. Xiang R, Primus FJ, Ruehlmann JM et al. A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T-cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice. J Immunol 2001; 167(8):4560-4565. 166. Xiang R, Mizutani N, Luo Y et al. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res 2005; 65(2):553-561. 167. Shiau AL, Chen YL, Liao CY et al. Prothymosin alpha enhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis. Vaccine 2001; 19(28-29):3947-3956. 168. Shiau AL, Chen CC, Yo YT et al. Enhancement of humoral and cellular immune responses by an oral Salmonella choleraesuis vaccine expressing porcine prothymosin alpha. Vaccine 2005; 23(48-49):5563-5571. 169. Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther 2005; 12(2):175-184. 170. Shen H, Kanoh M, Liu F et al. Modulation of the immune system by Listeria monocytogenes-mediated gene transfer into mammalian cells. Microbiol Immunol 2004; 48(4):329-337. 171. Xu C, Li ZS, Du YQ et al. Construction of recombinant attenuated Salmonella typhimurium DNA vaccine expressing H pylori ureB and IL-2. World J Gastroenterol 2007; 13(6):939-944. 172. Shiau AL, Chu CY, Su WC et al. Vaccination with the glycoprotein D gene of pseudorabies virus delivered by nonpathogenic Escherichia coli elicits protective immune responses. Vaccine 2001; 19(23-24):3277-3284. 173. Kotloff KL, Pasetti MF, Barry EM et al. Deletion in the Shigella enterotoxin genes further attenuates Shigella flexneri 2a bearing guanine auxotrophy in a phase 1 trial of CVD 1204 and CVD 1208. J Infect Dis 2004; 190(10):1745-1754. 174. Kotloff KL, Simon JK, Pasetti MF et al. Safety and immunogenicity of CVD 1208S, a live, oral deltaguaBA deltasen deltaset Shigella flexneri 2a vaccine grown on animal-free media. Hum Vaccin 2007; 3(6). 175. Li A, Pal T, Forsum U, Lindberg AA. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in volunteers. Vaccine 1992; 10(6):395-404. 176. Li A, Cam PD, Islam D et al. Immune responses in Vietnamese children after a single dose of the auxotrophic, live Shigella flexneri Y vaccine strain SFL124. J Infect 1994; 28(1):11-23. 177. Li A, Karnell A, Huan PT et al. Safety and immunogenicity of the live oral auxotrophic Shigella flexneri SFL124 in adult Vietnamese volunteers. Vaccine 1993; 11(2):180-189. 178. Karnell A, Li A, Zhao CR et al. Safety and immunogenicity study of the auxotrophic Shigella flexneri 2a vaccine SFL1070 with a deleted aroD gene in adult Swedish volunteers. Vaccine 1995; 13(1):88-99. 179. Orr N, Katz DE, Atsmon J et al. Community-based safety, immunogenicity and transmissibility study of the Shigella sonnei WRSS1 vaccine in Israeli volunteers. Infect Immun 2005; 73(12):8027-8032. 180. Kotloff KL, Taylor DN, Sztein MB et al. Phase I evaluation of delta virG Shigella sonnei live, attenuated, oral vaccine strain WRSS1 in healthy adults. Infect Immun 2002; 70(4):2016-2021. 181. Dietrich G, Griot-Wenk M, Metcalfe IC et al. Experience with registered mucosal vaccines. Vaccine 2003; 21(7-8):678-683. 182. Dilts DA, Riesenfeld-Orn I, Fulginiti JP et al. Phase I clinical trials of aroA aroD and aroA aroD htrA attenuated S. typhi vaccines; effect of formulation on safety and immunogenicity. Vaccine 2000; 18(15):1473-1484. 183. Hohmann EL, Oletta CA, Killeen KP et al. phoP/phoQ-deleted Salmonella typhi (Ty800) is a safe and immunogenic single-dose typhoid fever vaccine in volunteers. J Infect Dis 1996; 173(6):1408-1414. 184. Hohmann EL, Oletta CA, Miller SI. Evaluation of a phoP/phoQ-deleted, aroA-deleted live oral Salmonella typhi vaccine strain in human volunteers. Vaccine 1996; 14(1):19-24. 185. Hindle Z, Chatfield SN, Phillimore J et al. Characterization of Salmonella enterica derivatives harboring defined aroC and Salmonella pathogenicity island 2 type III secretion system (ssaV) mutations by immunization of healthy volunteers. Infect Immun 2002; 70(7):3457-3467.

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186. Kirkpatrick BD, Tenney KM, Larsson CJ et al. The novel oral typhoid vaccine M01ZH09 is well tolerated and highly immunogenic in 2 vaccine presentations. J Infect Dis 2005; 192(3):360-366. 187. Kirkpatrick BD, McKenzie R, O’Neill JP et al. Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC-ssaV-) M01ZH09, with a defined mutation in the Salmonella pathogenicity island 2, as a live, oral typhoid vaccine in human volunteers. Vaccine 2006; 24(2):116-123. 188. Tacket CO, Hone DM, Curtiss R 3rd et al. Comparison of the safety and immunogenicity of delta aroC delta aroD and delta cya delta crp Salmonella typhi strains in adult volunteers. Infect Immun 1992; 60(2):536-541. 189. Tacket CO, Hone DM, Losonsky GA et al. Clinical acceptability and immunogenicity of CVD 908 Salmonella typhi vaccine strain. Vaccine 1992; 10(7):443-446. 190. Tacket CO, Levine MM. CVD 908, CVD 908-htrA and CVD 909 live oral typhoid vaccines: A logical progression. Clin Infect Dis 2007; 45 Suppl 1:S20-23. 191. Salerno-Goncalves R, Wyant TL, Pasetti MF et al. Concomitant induction of CD4+ and CD8+ T-cell responses in volunteers immunized with Salmonella enterica serovar typhi strain CVD 908-htrA. J Immunol 2003; 170(5):2734-2741. 192. Wahid R, Salerno-Goncalves R Tacket CO et al. Cell-mediated immune responses in humans aft er immunization with one or two doses of oral live attenuated typhoid vaccine CVD 909. Vaccine 2007; 25(8):1416-1425. 193. Toso JF, Gill VJ, Hwu P et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 2002; 20(1):142-152. 194. Fuller CL, Brittingham KC, Porter MW et al. Transcriptome analysis of human immune responses following live vaccine strain (LVS) Francisella tularensis vaccination. Mol Immunol 2007; 44(12):3173-3184. 195. Hepburn MJ, Purcell BK, Lawler JV et al. Live vaccine strain Francisella tularensis is detectable at the inoculation site but not in blood after vaccination against tularemia. Clin Infect Dis 2006; 43(6):711-716. 196. Suharyono, Simanjuntak C, Witham N et al. Safety and immunogenicity of single-dose live oral cholera vaccine CVD 103-HgR in 5-9-year-old Indonesian children. Lancet 1992; 340(8821):689-694. 197. Garcia L, Jidy MD, Garcia H et al. The vaccine candidate Vibrio cholerae 638 is protective against cholera in healthy volunteers. Infect Immun 2005; 73(5):3018-3024. 198. Qadri F, Chowdhury MI, Faruque SM et al. Peru-15, a live attenuated oral cholera vaccine, is safe and immunogenic in Bangladeshi toddlers and infants. Vaccine 2007; 25(2):231-238. 199. Sack DA, Sack RB, Shimko J et al. Evaluation of Peru-15, a new live oral vaccine for cholera, in volunteers. J Infect Dis 1997; 176(1):201-205. 200. Kamath AT, Fruth U, Brennan MJ et al. New live mycobacterial vaccines: The Geneva consensus on essential steps towards clinical development. Vaccine 2005; 23(29):3753-3761. 201. Colditz GA, Brewer TF, Berkey CS et al. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. JAMA 1994; 271(9):698-702. 202. Calmette A PH. Protective inoculation against tuberculosis with BCG. Am Rev Tuberc 1929; 19:567-572. 203. Anderson RJ, Pasetti MF, Sztein MB et al. DeltaguaBA attenuated Shigella flexneri 2a strain CVD 1204 as a Shigella vaccine and as a live mucosal delivery system for fragment C of tetanus toxin. Vaccine 2000; 18(21):2193-2202. 204. Pasetti MF, Barry EM, Losonsky G et al. Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats. J Virol 2003; 77(9):5209-5217. 205. Vecino WH, Morin PM, Agha R et al. Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T-cells against HIV. Immunol Lett 2002; 82(3):197-204. 206. Xu F, Hong M, Ulmer JB. Immunogenicity of an HIV-1 gag DNA vaccine carried by attenuated Shigella. Vaccine 2003; 21(7-8):644-648. 207. Pasetti MF, Anderson RJ, Noriega FR et al. Attenuated deltaguaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses. Clin Immunol 1999; 92(1):76-89. 208. Shata MT, Reitz MS Jr, DeVico AL et al. Mucosal and systemic HIV-1 Env-specific CD8(+) T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector. Vaccine 2001; 20(3-4):623-629. 209. Wong LP, Woo PC, Wu AY et al. DNA immunization using a secreted cell wall antigen Mp1p is protective against Penicillium marneffei infection. Vaccine 2002; 20(23-24):2878-2886. 210. Sun B, Li ZS, Tu ZX et al. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity. World J Gastroenterol 2006; 12(43):7042-7046.

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211. Niethammer AG, Wodrich H, Loeffler M et al. Multidrug resistance-1 (MDR-1): a new target for T-cell-based immunotherapy. FASEB J 2005; 19(1):158-159. 212. Weth R, Christ O, Stevanovic S et al. Gene delivery by attenuated Salmonella typhimurium: comparing the efficacy of helper versus cytotoxic T-cell priming in tumor vaccination. Cancer Gene Ther 2001; 8(8):599-611. 213. Xiang R, Lode HN, Chao TH et al. An autologous oral DNA vaccine protects against murine melanoma. Proc Natl Acad Sci USA 2000; 97(10):5492-5497. 214. Lode HN, Pertl U, Xiang R et al. Tyrosine hydroxylase-based DNA-vaccination is effective against murine neuroblastoma. Med Pediatr Oncol 2000; 35(6):641-646. 215. Zoller M, Christ O. Prophylactic tumor vaccination: Comparison of effector mechanisms initiated by protein versus DNA vaccination. J Immunol 2001; 166(5):3440-3450. 216. Cochlovius B, Stassar MJ, Schreurs MW et al. Oral DNA vaccination: Antigen uptake and presentation by dendritic cells elicits protective immunity. Immunol Lett 2002; 80(2):89-96. 217. Lee SH, Mizutani N, Mizutani M et al. Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer. Cancer Immunol Immunother 2006; 55(12):1565-1574. 218. Li YG, Tian FL, Gao FS et al. Immune responses generated by Lactobacillus as a carrier in DNA immunization against foot-and-mouth disease virus. Vaccine 2007; 25(5):902-911. 219. Mo Y, Quanquin NM, Vecino WH et al. Genetic alteration of Mycobacterium smegmatis to improve mycobacterium-mediated transfer of plasmid DNA into mammalian cells and DNA immunization. Infect Immun 2007; 75(10):4804-4816.

Chapter 7

Bacteria Mediated Gene Therapy Strategies Sophie Conchon* and Georges Vassaux

Abstract

T

he recent discovery that genes carried by bacterial vectors can be functionally transferred to mammalian cells has led to the utilization of various bacterial strains in gene therapy. Genetically modified, nonpathogenic bacteria that have been used include attenuated strains of Salmonella, Shigella, Listeria, Yersinia, Clostridium or Bifidobacterium. Their potential as tumor-targeting agents, as well as their ability to deliver therapeutic molecules have been demonstrated in vitro and in vivo in experimental models. Therapeutic benefits have been observed in immunotherapy against cancer, vaccination against infectious diseases and topical delivery of immunomodulatory cytokines in inflammatory bowel disease. The studies presented in this review show that bacteria can be considered as a promising therapeutic vector against various pathologies.

Introduction

Gene therapy strategies consist in the delivery of genetic material in a given organ or cell type. These strategies require efficient and relevant transfer vectors that are usually classified into two main groups: viral and nonviral vectors. Viral vectors, replicating or not, are derived from various viruses (adenoviruses, retroviruses, poxviruses, parvoriruses, herpexviruses) in which all or part of the viral genome has been replaced by a transgene and all the regulatory sequences required for its expression in the target cells. Nonviral gene transfer usually consists in the association of naked DNA with chemically defined compounds such as liposomes, which increase the efficiency of DNA penetration across the cell membrane.1 Bacteria can be considered as an alternative vector for gene therapy. The transfer of plasmid DNA from bacteria to a variety of recipient cells including yeast2 and plants3 has been known for many years and the functional gene transfer from bacteria to mammalian cells has been studied and reported by several groups in the last two decades.4-10 This review describes the mechanism of bacteria-mediated gene transfer, gives some examples of therapeutic applications and of emerging technologies based on this strategy.

Mechanism of Bacteria-Mediated Gene Transfer

Gene transfer with bacterial vector is achieved through entry of the whole bacterium in the target cell. Whereas bacteria play a passive role during penetration of professional phagocytic cells (macrophages, dendritic cells), they are the key active player in the complex mechanisms of entry in nonphagocytic cells.11 Depending on the bacterial strain, there are two major strategies to gain entry into eucaryotic cells (Table 1): *Corresponding Author: Sophie Conchon—INSERM CIC04, Biothérapies Hépatiques, Université de Nantes, Nantes Atlantique Universités, EE0502 Institut des Malades de l’Appareil Digestif—IMAD CHU Hotel Dieu, Nantes, F-44000 France. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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• The zipper mechanism used by Yersinia or Listeria. The bacteria express a surface protein (invasin and internalin, respectively) able to bind eukaryotic surface receptors often involved in cell-matrix or cell-cell adherence (β1 integrin and E-cadherin, respectively). Expression of this protein leads to the formation of a vacuole that engulfs the bacterium through a zipper-like process. • The trigger mechanism used by Salmonella and Shigella. Contact between bacteria and cells is mediated by the type III secretory system which allows direct delivery of bacterial invasion proteins in the eukaryotic cell, leading to the activation of components of the cytoskeleton. These massive rearrangements trigger the formation of a macropinocytic pocket and the bacterial uptake. Once inside the cell (phagocytic or non phagocytic), the bacteria are in the phagosomal vacuoles and are targeted for degradation. Escape from this compartment must be achieved to allow the plasmid to be delivered to the nucleus. The process of phagosomal escape is well characterized for bacterial vectors that replicate in the cytosol, such as Listeria monocytogenes.5,12 After phagocytosis, these bacteria secrete Lysteriolysin O (LLO), which can create pores in the vacuolar membrane allowing the bacteria to escape into the cytosol where they can replicate.13 An attenuated L. monocytogenes strain has been engineered to produce a phage endopeptidase (or lysin) under the control of the Pact promoter, which is preferentially activated after the bacteria have entered the host cytoplasm.5 These bacteria underwent self-destruction in the cell cytoplasm and were able to deliver a plasmid with the chloramphenicol acetyltransferase gene (CAT) or part of the chicken ovalbumine gene (ova) under the control of the CMV promoter into the P388D macrophage cell line. Functional gene transfer was in the range of 0.2% and CAT activity and presentation of ova epitopes were detected. Some events of genomic integration were detected at a frequency of 10–7. Another bacterial vector taking advantage of the pore-forming activity of the LLO protein is the invasive E.coli.6,7 Both the inv locus encoding the invasin from Yersinia pseudotuberculosis and the hly locus from Lysteria monocytogenes encoding the LLO have been inserted into these bacteria. As mentioned earlier, binding of invasin to β1-integrin at the surface of mammalian cells is necessary and sufficient for entry of the whole bacterium in the cell.14 Therefore, invasin expression restricts the tropism of these bacteria to β1-integrin expressing nonphagocytic cells15 and recombinant E.coli that do not express invasin can be used to target professional phagocytic cells.16,17 In the invasive E. coli strain, LLO is modified to remain intra-bacterial.6,7,15,18 Once internalized, the bacteria are degraded, releasing LLO in the lysosomal compartment and the bacterial content (including the plasmid to be delivered) can escape in the cytosol via the pores formed by LLO. Much less is known about the mechanism of plasmid DNA escape for bacterial vectors that remain in the phagocytic compartment, such as Salmonella enterica serovar Typhimurium (S. typhimurium).8 It has been suggested that some eukaryotic promoters would remain partially active in the bacteria, leading to protein rather that gene transfer to the host cell.19 The final step of gene transfer is the journey from cytosol to nucleus. Since there is no active mechanism of transport, a likely way for the plasmid to reach the nucleus is disruption of the nuclear membrane during mitosis. Table 1. General characteristics of the main bacterial vectors Organism

Gram

Anaerobe

Invasion Mechanism

Intracellular Lifestyle

Salmonella Shigella Yersinia Listeria

– – – +

Facultative Facultative Facultative Facultative

Trigger Trigger Zipper Zipper

Vacuole Cytosol Vacuole Cytosol

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Bacteriolysis of Tumors Introduction

For many years anaerobic bacteria have been known to infiltrate, replicate and preferentially accumulate in tumors.20-23 The abnormal vascular supply found in tumors is responsible for the combination of poor nutrient supply and oxygen starvation, which result in nonproliferating hypoxic-anoxic cells. These necrotic/hypoxic centers promote growth of anaerobic and facultative anaerobic bacteria, by providing them with necessary nutrient (e.g., purines) and by protecting them from immune clearance.24,25 Three main strains of bacteria are being investigated to improve their tumor selectivity, their tumor specific toxicity and their lack of immunogenicity. Two of these are obligate anaerobes, Clostridium and Bifidobacterium and one is facultative anaerobe, Salmonella (Table 2).

Obligate Anaerobes

These bacteria can only replicate in anoxic environments and thus preferentially proliferate in necrotic/anoxic regions of solid tumors, leaving the well-oxygenated tissues, including the outer rim of the main tumor and small metastasis unaffected.26 Investigators have attempted to use the tumor-targeting properties of Clostridium for the selective delivery of pro-drug converting enzymes. In these studies, the bacterial enzymes cytosine deaminase27-29 or nitroreductase30 were expressed in Clostridium and were shown to convert the nontoxic pro-drugs 5-fluorocytosine and CB1954, respectively, into cytotoxic compounds capable of diffusing in the tumors. The large bystander effect of the activated drugs kills the cancer cells even outside the hypoxic fraction. The effect of multiple consecutive treatment cycles with antibiotic bacterial clearance between cycles has been tested with a Clostridium strain expressing nitroreductase. When the pro-drug was added, these repeated cycles of bacterial treatment had a significant anti-targeting effect on human colon carcinoma tumors in nude mice.31 Using a similar principle, a Clostridia strain was engineered in which a radio-responsive promoter drives the expression of TNFα.32 A strain of C. novyi devoid of its lethal toxin was engineered (C. novyi-NT) and when its spores were administered to nude mice bearing HCT116 tumors in combination with a vascular targeting agent, dolastatin 10 and the chemotherapeutic drug mitomycin C, necrosis of tumors often developed within 24 hours, resulting in significant and prolonged antitumor effect.33 Encouraging antitumor activities in preclinical targeting models were also observed when C. novyi-NT was administered in combination with anti-microtubules agents34 or associated with radiation therapy.35 Very recently, Cheong et al showed that combined treatment of mice bearing large established tumors with C. Novyi-NT plus a single dose of liposomal doxorubicin could lead to the eradication of these tumors. In this approach, the hemolytic properties of the bacterium are exploited to increase the release of liposome-encapsulated doxorubicin within the targeting.36 Finally, in immunocompetent animals with syngeneic tumors, this bacteriolysis was shown to trigger a long-lasting immune response amplifying the bacteriolytic action of C. novyi-NT in absence of any additional treatment.37 The gram-positive anaerobic bacteria Bifidobacterium represent an alternative to Clostridium and have been shown to colonize large tumors. In contrast to Clostridia, Bifidobacteria are nonpathogenic, nonspore-forming and are found naturally in the digestive tract of humans and other mammals. Following systemic injection, selective localization of Bifidobacteria to various preclinical models of solid tumors has been reported.38-40 B. adolescentis carrying a plasmid encoding the endostatin gene was shown to target subcutaneously implanted liver tumors in Balb/c mice, which led to inhibition of both angiogenesis and growth of the targeting.41 The same group has also reported that the oral administration of B. longum carrying the endostatin gene was efficient in the same targeting model. This effect was amplified by co-administration of selenium, thought to act through an improved activity of NK and T-cells.42 Bifidobacteria may therefore represent a safer alternative to Clostridia.

Organism Clostridium Gram + Obligate anaerobe

Tumor Model Mammary carcinoma s.c. (BalB/c mice) Colon, renal carcinoma s.c. (BalB/c mice), liver tumor VX2 (rabbit) Human colon carcinoma s.c. (nu/nu mice) Human colon carcinoma s.c. (nu/nu mice) Human colon carcinoma s.c. (nu/nu mice) Rhabdomyosarcoma (rat) Various human tumor cell lines s.c. (nu/nu mice) Human colon carcinoma s.c. (nu/nu mice), colon carcinoma s.c. (BalB/c mice)

Combined Treatment – – – dolastatin10+mitomycinC radiation therapy combretastatin anti microtubules agents liposomal doxorubicin

Reference 30 37 31 33 35 29 34 36

Bifidobacterium Gram— Obligate anaerobe

Ehrlich ascites (mice) Melanoma, lung cancer s.c. (C57Bl/6 mice) Mammary tumors (rat) Liver tumor s.c. (nu/nu mice)

– – – Selenium

38 40 39 41,42

– x ray – – – – – –

47 53 46 48 57 49 50,51 56

Syngeneic melanoma, bladder and liver cancer s.c. (mice)

–

55

Syngeneic lung and liver cancer s.c. (mice)

cisplatin

54

Salmonella Melanoma s.c. (C57Bl/6 mice), human melanoma and colon carcinoma s.c. (SCID mice) Gram— Syngeneic melanoma s.c. (mice) Facultative anaerobe Spontaneous neoplasia (dog) Melanoma, renal carcinoma (human) Solid tumors (human) Human prostate cancer s.c. (nu/nu mice) Human breast and prostate cancer, orthotopic (nu/nu mice) Melanoma s.c. (C57Bl/6 mice)

Bacteria Mediated Gene Therapy Strategies

Table 2. Clostridia, Bifidobacteria and Salmonella as vectors for bacteriolysis of tumors (s.c.: subcutaneous)

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Gram-negative Salmonella have also been proposed as oncolytic agents. In contrast to obligate anaerobic bacteria such as Clostridia and Bifidobacteria, Salmonella are facultative anaerobic bacteria and have the potential to colonize oxygenated small metastatic lesions as well as large tumors with a hypoxic center. The first demonstration of the potential of Salmonella was provided in 1997, when Salmonella auxotrophs injected into tumor-bearing animals were shown to preferentially replicate in tumors leading to a therapeutic effect.43 However, a major drawback for the therapeutic exploitation of Salmonella is that it is highly immunogenic and induces TNFα-mediated sceptic shock. To reduce this possibility, lipid-A-modified Salmonella auxotrophs (purI-) were developed by mutation of the waaN (previously msbB) gene.44,45 The antitumor activity of the Salmonella strain VNP20009 was tested in dogs with spontaneous neoplasia, in the context of a phase I dose escalation trial.46 VNP20009 is a genetically modified strain of S. typhimurium that includes genetically stable attenuated virulence (purI-), reduction of septic shock potential (waaN-) and antibiotic susceptibility.47 A significant antitumor activity, with four complete responses out of 41 animals treated was reported.46 The same strain was administered to 24 patients with metastatic melanoma and 1 patient with metastatic renal carcinoma.48 The results established a maximum-tolerated dose of 3 × 108 cfu/m2 and dose-limiting toxicity was observed in patients receiving 109 cfu/m2 with symptoms that included thrombocytopenia, anemia, persistent bacteraemia, hyperbilirubinaemia, diarrhea and vomiting. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines. Tumor colonization occurred in three patients but no objective tumor regression was observed. This clinical trial highlights the need for the generation of strains with reduced toxicities and improved tumor colonization properties. Other auxotrophic Salmonella have successfully targeted and induced regression of model tumors in mice. S. typhimurium A1 which has a requirement for amino acids Leu and Arg was shown to grow specifically in tumor xenografts but to be rapidly eliminated from normal tissues.49 More recently the same group reported the isolation of an A1-R strain, from a human colon tumor growing in a nude mice injected with S. typhimurium A1. Treatment with this S. typhimurium A1-R strain resulted in highly effective tumor targeting and significant tumor regression in mice with human breast and prostate cancer xenografts.50,51 To increase the potential of attenuated Salmonella, Mengesha et al52 developed a hypoxia-inducible promoter (HIP-1) to limit gene expression specifically to the tumor. They demonstrated that HIP-1 could drive hypoxia-mediated gene expression in bacteria that have colonized human tumor xenografts in nude mice. Expression of fluorescent proteins driven by HIP-1 demonstrated a 15-fold increase relative to a constitutive promoter, when the tumors were made hypoxic. Moreover, the use of the constitutive promoter resulted in reporter gene expression in both tumors and normal tissue, whereas reporter gene expression with HIP-1 was confined to the tumors. The antitumor effect mediated by Salmonella was also shown to be enhanced by radiation53 and low-doses of cisplatin.54 To amplify the antitumor effect, strains of genetically engineered Salmonella armed with therapeutically relevant genes have been produced. They include strains capable of delivering the herpes simplex thymidine kinase protein (hsv-tk)43,45 as well as the genes encoding for endostatin55 and thrombospondin-1.56 A clinical trial involved the intratumoral injection of attenuated Salmonella expressing the E. coli cytosine deaminase gene (3 × 106 to 3 × 107 cfu/m2) in three refractory cancer patients, followed by administration of 5-fluorocytosine. No significant adverse events related to the treatment were observed. Two patients had evidence of bacterial colonization of the tumor that persisted for at least 15 days after the initial injection. Conversion of 5-fluorocytosine to 5-fluorouracil as a result of cytosine deaminase expression was demonstrated in these two patients.57 In addition, diagnostic imaging is an unexpected and potentially powerful application of tumor-targeting Salmonella.58,59 The strategy involves the ability of bacteria expressing the hsv-tk to phosphorylate radiolabelled nucleoside analogue. In this way, the radiotracer becomes trapped in the bacteria and as Salmonella accumulates in tumors, the radioactive signal that can be monitored by positron emission tomography provide information on the localization of tumor sites.

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Bacterial Vectors for Immunotherapy (Table 3)

The technique using attenuated bacteria for the direct delivery of plasmid-encoded antigens under the transcriptional control of eukaryotic promoters has been used successfully in vaccination. The methodology relies on the delivery of the plasmid in which a eukaryotic promoter drives the expression of the antigen into macrophages and/or dendritic cells. At the same time, bacteria naturally possess biological components that activate antigen-presenting cells. These pathogen-associated molecular patterns (PAMPs) include compounds such as lipopolysaccharide (LPS),60,61 bacterial DNA containing unmethylated Cytosine-phosphate-Guanine (CpG) dinucleotides,62 flagellin63 or bacterial lipoproteins.64 PAMPs are specific ligands for receptors of the Toll-like receptor (TLR) family present in the plasma membrane of APC.65 The binding of PAMPs to TLRs activates the APC and triggers an innate immune response with a production of reactive oxygen, pro-inflammatory cytokines and nitrogen species as well as an up-regulation of costimulatory molecules. These responses promote maturation and migration of dendritic cells to secondary lymph nodes66 and in this way, PAMPs amplify the immune response against the antigen and act as adjuvant.

Cancer Immunotherapy

Cancer immunotherapy relies on the recognition and destruction of cells presenting specific target antigens. Two kinds of antigens can be distinguished: tumor antigens, which are expressed by the tumor cells and antigens expressed by cells from the tumor stroma, such as endothelial cells, fibroblasts. It is mostly Salmonella strains that have been used as bacterial vectors to deliver DNA encoding these antigens to APC. “Model” tumor antigens such as β-Galactosidase or human gp100 (hgp100) were encoded in eukaryotic expression vectors carried by strains of Salmonella. Oral administration of these bacterial strains protected the mice against challenges with fibrosarcoma,9 renal carcinoma67 and melanoma68 cells expressing the relevant model antigen. The efficacy of oral administration of Salmonella DNA carrier was also assessed against autologous tumor antigens also expressed in normal tissues. Successful protection were observed with transgenes including the murine gp100 (mgp100) fused to the invariant chain,69 epitopes of mgp100 and TRP2 fused to ubiquitin,70 a minigene encoding epitopes of the tyrosine hydroxylase (TH) fused to ubiquitin,71 the complete TH coding sequence linked to a virally derived posttranscriptional regulatory element,72 human carcinoembryonic antigen (hCEA) in a hCEA mouse transgenic model73 and alpha-fetoprotein in mouse subcutaneous models of liver and colon cancer.74 As all these antigens were self-antigens, these observations strongly suggest that Salmonella-mediated DNA vaccination can break immunological tolerance. In many cases, the protection observed was strongly improved by co-administration of IL-2.69,72,73 The vaccination against a tumor antigen can be amplified by codelivery of another plasmid encoding a cytokine or chemokine. This strategy was successfully illustrated by the vaccination of mice with a Salmonella strain carrying eukaryotic expression plasmids encoding the transcription factor Fos-related antigen 1 (Fra-1) over expressed in an aggressive murine breast carcinoma Table 3. Bacteria as gene therapy for vaccination. Bibliographic references for different types of antigens Organism

Tumor Antigens

Salmonella

9,67,68,69,70,71, 72,73,74,75

Shigella

Tumor Associated Antigens 76,77,78,79,80

Viral Antigens

Bacterial Antigens

81,82,83,84,85, 86,88,93,95

4,8,100,101,102, 103,104

88,89,90,91,92

10,97

Yersinia Listeria

98 96

99

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model and IL-18, a cytokine known to suppress angiogenesis and to stimulate INF-γ production by T and NK cells [75]. Another example is the oral delivery of a DNA vaccine encoding the anti apoptotic protein survivin over-expressed in many cancers and the angiostatic chemokine CCL21. This treatment was effective against established pulmonary metastases of nonsmall cell lung carcinoma.76 There is growing evidence that the stromal environment plays a crucial role in tumorigenesis and invasion. Genetic vaccination mediated by Salmonella has been used to target proteins expressed by endothelial cells such as vascular endothelium growth factor receptor 2 or endoglin (CD105).77-79 Recently, fibroblast-activating protein (FAP), which is over-expressed by tumor-associated fibroblasts, has been targeted by an oral vaccination.80 A doubly attenuated strain of S. typhimurium (AroA-dam-) containing a eukaryotic vector encoding the murine FAP was tested both in prophylactic and therapeutic setting against colon and breast carcinoma models in BalB/c mice. This oral vaccine elicited a cytotoxic T-lymphocyte (CTL) immune response against FAP and, when combined with doxorubicin, it was able to inhibit tumor growth and lead to complete tumor rejection in 50% of mice. Tumor tissue of FAP-vaccinated mice had markedly decreased level of collagen type I expression and up to 70% greater uptake of doxorubicin.80

Vaccination against Viral Antigens

Viral infections have also been targeted through DNA vaccination mediated by bacteria. Oral vaccination with S. typhimurium encoding an hepatitis B virus surface antigen (HbsAg) proved successful at inducing CTLs in Balb/c mice81 and a single administration of these bacteria to transgenic mice expressing HbsAg in the liver led to the loss of expression of the antigen in hepatocytes.82 This single administration of the recombinant bacteria induced CTLs, Th1 T-cells as well as HbsAg-specific IgG2 subclass of antibodies, as a further proof that immune tolerance against the viral antigen had been broken. A similar S. typhimurium was also used in vaccination against the nonstructural region 3 (NS3) of hepatitis C virus in HLA-A2.1 transgenic mice.83 Recombinant S. typhimurium administered orally was also reported to induce an immune response against herpes simplex virus 284 and human papilloma virus 16.85,86 Vaccination against HIV was attempted using attenuated strains of Shigella and Salmonella carrying eukaryotic expression plasmids encoding HIV env and gp120, respectively.87,88 Oral administration of recombinant Salmonella as well as intranasal vaccination with the recombinant Shigella led to the activation of antigen specific CD8 T-cell responses. In addition, Shigella HIV gp120 DNA vaccination conferred some protection against a challenge with a vaccinia virus expressing env.87 A study comparing the ability of a Shigella strain to different Salmonella strains suggested that the induction of antigen-specific CD8 T-cell responses was superior with Shigella.89 An attenuated Shigella defective in LPS-O-antigen synthesis and carrying a HIV gag DNA vaccine was used as a boost, after priming by intramuscular DNA injection. This strategy resulted in a dramatic increase of T-cell response in the lungs.90 Other DNA vaccinations mediated by bacteria include vaccination against measles virus91 and influenza92 with a recombinant strain of S. flexneri, vaccination against pseudo rabies virus using either nonpathogenic E. coli administered intramuscularly93 or swine-adapted strain of S. choleraesuis94 or a live attenuated S. typhimurium,95 vaccination against feline immunodeficiency syndrome with a recombinant strain of Listeria monocytogenes.96

Vaccination against Bacterial Antigens

Strains of Shigella flexneri mutated in a gene essential for cell wall synthesis can deliver plasmids in vitro resulting in the β-galactosidase expression in mammalian cells10 and nasal inoculation of these bacteria in mice led to the induction of β-galactosidase-specific humoral and cellular responses.97 Oral administration in mice of an attenuated Yersinia enterocolitica carrying a eukaryotic expression plasmid encoding Brucella genes elicited humoral and cellular responses. This immune response, in conjunction with potential cross-reacting antibodies against LPS of both Yersinia and Brucella induced a protection against a Brucella challenge.98

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Inoculation with attenuated self-destructing Listeria monocytogenes harboring plasmid DNA vaccines for a subset of proteins from Mycobacterium tuberculosis induced a specific cellular immune response and conferred a certain degree of protective immunity against TB to the vaccinated mice.99 Another set of studies exploited Salmonella typhimurium as a gene delivery vector.4 When β-galactosidase was used as a transgene, specific CTLs and T helper (Th) cells, mainly of the Th1-type, as well as specific antibodies could be detected after a single oral vaccination. A very similar immune response was elicited when two virulence factors of L. monocytogenes (LLO and ActA) were encoded in S. typhimurium vectors.100 Protection of mice against a lethal challenge with L. monocytogenes was observed.4,101 The route of administration appears to affect these responses. Oral administration appeared more efficient than nasal inoculation to obtain a T-cell response in spleen.8,101 Independently, partial protective responses against Chlamydia were obtained in the lung of mice after oral administration of Salmonella encoding the major outer membrane protein of Chlamydia trachomatis.8 Salmonella-mediated vaccination has essentially been achieved with auxotrophic strains of S. typhimurium such as SL7207,102 but intranasal vaccination with S. typhi was also reported.103 Finally, a recent study performed with the same antigen, tetanus toxin C-fragment, investigated the influence of plasmid stability on the immune response induced by oral administration of S. typhimurium. Comparison of oral vaccination with S. typhimurium loaded with different plasmids which all contain the same expression cassette consisting of the CMV promoter followed by the sequence encoding the C fragment led to the demonstration that plasmid copy number impacts both the plasmid stability in S. typhimurium and the induction of an antigen-specific immune response.104

Other Examples of Bacterial Gene Therapy Strategies

Bacterial vectors for gene transfer have been investigated in the field of monogenic diseases such as cystic fibrosis. Preliminary attempts using invasive E. coli18 or Lysteria monocytogenes105 were performed on cultured cells. Using a GFP-CFTR (cystic fibrosis transmembrane conductance regulator) fusion as transgene, a thorough analysis demonstrated that the mechanism of gene transfer with L. monocytogenes in various cell lines was very inefficient and poorly understood. Further optimization is needed before in vivo application in the field of inherited disorders.106 Inflammatory bowel disease (IBD) is a significant health-care problem characterized by diarrhea, pain, other intestinal symptoms and lifelong relapses. The pathogenesis is complex and relies on the interaction between genetic susceptibility, intestinal bacteria and the gut mucosal immune response. The lack of organ specificity is one of the limitations shared by most strategies that use immunomodulation and results in unpleasant side effects. To address these limitations, a recombinant Lactoccocus lactis strain was engineered to produce the anti-inflammatory cytokine IL-10.107 Administration of this strain led to the local production of the anti-inflammatory cytokine and prevented the development of colitis in different mouse models.107, 108 Following the demonstration of the proof of principle, further studies are now focusing on the characterization of the best possible transgene (reviewed in ref. 109). Another bacterial strain used in the treatment of experimental IBD is the invasive E. coli,7 expressing invasin from Yersinia and LLO from Listeria and carrying a eukaryotic expression plasmid in which the constitutive CMV promoter drives the expression of the immunomodulatory cytokine TGF-β1.110 Oral administration of these bacteria known to allow gene transfer in vitro7,15 led to a significant reduction of the severity of experimental colitis in mice, with vector-specific transcripts detected in colonic and extra-colonic tissues such as the lungs, liver and spleen. To avoid expression in these extracolonic tissues, the CMV promoter was replaced by the inflammation-inducible IL-8 promoter. This substitution led to the restriction of expression of TGF-β1 in the inflamed colon without affecting the therapeutic effects,110 demonstrating that targeted gene expression and therapeutic benefits can be obtained using bacterial gene transfer in the gut.

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Emerging Technologies Bacterial Minicells

Concerns about adverse reactions to the bacterial vectors can be lessened through modifications made to the bacterial strains or the form of administration. Another attempt involves the use of bacterial minicells for DNA, protein or drug delivery. Minicells are produced due to premature septation after inactivation of bacterial genes that control normal cell division. They are typically 100 to 400 nm achromosomal vesicles that do not divide. Minicells have been used to develop a tumor targeted drug delivery system. They were loaded with chemotherapeutic drugs and targeted towards tumor cells using bispecific antibodies. This minicell drug delivery could induce inhibition/regression of tumor growth in several mice, dogs and pigs models, with a delivery of amounts of drugs that were markedly smaller than those required with systemic delivery of free drugs.111 Minicells have also been used to efficiently deliver DNA and protein to antigen presenting cells. This delivery combination was able to induce high levels of IgG in the sera after introduction by several different routes, including intramuscular, intranasal and oral administration.112

Transfer of Large DNA Molecules and Artificial Chromosomes

The utilization of bacteria to transfer large DNA molecules to mammalian cells would simplify the procedure and reduce the possibility of mechanical breakage. The delivery of bacterial artificial chromosomes was first demonstrated into HeLa cells using an invasive E. coli.113 Direct DNA transfer of up to around 1Mb was demonstrated and as the bacterial vector is equipped with an inducible recombination system, modifications of the bacterial artificial chromosome sequences should be possible. More recently, efficient transfer of an alpha-satellite DNA cloned into a P1-based artificial chromosome was stably delivered into HT1080 cell line and efficiently generated human artificial chromosomes de novo.114 A 160 kb construct containing the CFTR gene was also transferred in the same cells where it was transcribed and correctly spliced.114 In a study in mice, large DNA molecules carrying the viral genome of the murine cytomegalovirus (MCMV) were transferred using E. coli and S. typhimurium as delivery vectors.115 This transfer led to a productive virus infection that resulted in elevated titers of specific anti-MCMV antibodies, protection against lethal MCMV challenge and strong expression of additional genes introduced into the viral genome. Thus, the reconstitution of infectious virus from live attenuated bacteria presents a novel concept for multivalent virus vaccines launched from bacterial vectors.

Transfer of Small Interfering RNA (siRNA)

The utilization of double-stranded RNA to silence target genes (RNA interference: RNAi) has potential therapeutic applications that are widely acknowledged.116 Bacteria-mediated induction of RNAi was established by demonstrating target-specific gene silencing after transfer of double-stranded RNA from E. coli in the nematode Caenorhabditis elegans.117,118 A first report of the use of an attenuated strain of S. typhimurium as a delivery system for siRNA-based tumor therapy has been published. In this study, the invasive recombinant S. typhimurium carrying a plasmid vector that express Stat3-specific siRNA was compared with the bacteria alone for its ability to inhibit growth of an orthotopic model of prostate cancer in C57Bl/6 mice. The expression of the Stat3 siRNA led to a 4 and 11 fold higher tumor suppressive effect compared to S. typhimurium alone and buffer control, respectively. This effect was obtained on the primary tumors as well as on the development of metastases.119

Conclusion

Live attenuated bacteria are promising vectors for gene therapy. Preclinical studies have demonstrated the value of this strategy in oncology, where these bacteria can be used for tumor targeting and immunotherapy, or in infectious diseases where they can be used as vectors for vaccination. New developments are still appearing, improving their tolerability and/or efficacy, such as the combination of bacterial tumor-targeting with siRNA technology, which will hopefully be translated into clinical benefit for the patients.

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References

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1. Glover DJ, Lipps HJ, Jans DA. Towards safe, nonviral therapeutic gene expression in humans. Nat Rev Genet 2005; 6:299-310. 2. Heinemann JA, Sprague GF Jr. Bacterial conjugative plasmids mobilize DNA transfer between bacteria and yeast. Nature 1989; 340:205-209. 3. Lessl M, Lanka E. Common mechanisms in bacterial conjugation and Ti-mediated T-DNA transfer to plant cells. Cell 1994; 77:321-324. 4. Darji A, Guzman CA, Gerstel B et al. Oral somatic transgene vaccination using attenuated S. typhimurium. Cell 1997; 91:765-775. 5. Dietrich G, Bubert A, Gentschev I et al. Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nat Biotechnol 1998; 16:181-185. 6. Grillot-Courvalin C, Goussard S, Courvalin P. Bacteria as gene delivery vectors for mammalian cells. Curr Opin Biotechnol 1999; 10:477-481. 7. Grillot-Courvalin C, Goussard S, Huetz F et al. Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol 1998; 16:862-866. 8. Loessner H, Weiss S. Bacteria-mediated DNA transfer in gene therapy and vaccination. Expert Opin Biol Ther 2004; 4:157-168. 9. Paglia P, Medina E, Arioli I et al. Gene transfer in dendritic cells, induced by oral DNA vaccination with Salmonella typhimurium, results in protective immunity against a murine fibrosarcoma. Blood 1998; 92:3172-3176. 10. Sizemore DR, Branstrom AA, Sadoff JC. Attenuated Shigella as a DNA delivery vehicle for DNAmediated immunization. Science 1995; 270:299-302. 11. Cossart P, Sansonetti PJ. Bacterial invasion: The paradigms of enteroinvasive pathogens. Science 2004; 304:242-248. 12. Grillot-Courvalin C, Goussard S, Courvalin P. Wild-type intracellular bacteria deliver DNA into mammalian cells. Cell Microbiol 2002; 4:177-186. 13. Portnoy DA, Chakraborty T, Goebel W et al. Molecular determinants of Listeria monocytogenes pathogenesis. Infect Immun 1992; 60:1263-1267. 14. Isberg RR, Leong JM. Multiple beta 1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells. Cell 1990; 60:861-871. 15. Critchley RJ, Jezzard S, Radford KJ et al. Potential therapeutic applications of recombinant, invasive E. coli. Gene Ther 2004; 11:1224-1233. 16. Radford KJ, Higgins DE, Pasquini S et al. A recombinant E. coli vaccine to promote MHC class I-dependent antigen presentation: Application to cancer immunotherapy. Gene Ther 2002; 9:1455-1463. 17. Radford KJ, Jackson AM, Wang JH et al. Recombinant E. coli efficiently delivers antigen and maturation signals to human dendritic cells: Presentation of MART1 to CD8+ T-cells. Int J Cancer 2003; 105:811-819. 18. Fajac I, Grosse S, Collombet JM et al. Recombinant Escherichia coli as a gene delivery vector into airway epithelial cells. J Control Release 2004; 97:371-381. 19. Goussard S, Grillot-Courvalin C, Courvalin P. Eukaryotic promoters can direct protein synthesis in Gram-negative bacteria. J Mol Microbiol Biotechnol 2003; 6:211-218. 20. Carey RW, Holland JF, Whang HY et al. Clostridial oncolysis in man. Eur J Cancer 1967; 3:37-46. 21. Engelbart K, Gericke D. Oncolysis by Clostridia. V. Transplanted tumors of the hamster. Cancer Res 1964; 24:239-243. 22. Mose JR, Mose G. Oncolysis by Clostridia. I. Activation of Clostridium bytiricum (M-55) and other nonpathogenic Clostridia against the Ehrlich sarcoma. Cancer Res 1964; 24:212-216. 23. Thiele EH, Arison RN, Boxer GE. Oncolysis by Clostridia. III. Effects of Clostridia and chemotherapeutic agents on rodent tumors. Cancer Res 1964; 24:222-231. 24. Yu YA, Shabahang S, Timiryasova TM et al. Visualization of tumors and metastases in live animals with bacteria and vaccinia virus encoding light-emitting proteins. Nat Biotechnol 2004; 22:313-320. 25. Yu YA, Timiryasova T, Zhang Q et al. Optical imaging: Bacteria, viruses and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals. Anal Bioanal Chem 2003; 377:964-972. 26. Theys J, Landuyt W, Nuyts S et al. Improvement of Clostridium tumour targeting vectors evaluated in rat rhabdomyosarcomas. FEMS Immunol Med Microbiol 2001; 30:37-41. 27. Fox ME, Lemmon MJ, Mauchline ML et al. Anaerobic bacteria as a delivery system for cancer gene therapy: in vitro activation of 5-fluorocytosine by genetically engineered clostridia. Gene Ther 1996; 3:173-178. 28. Liu SC, Minton NP, Giaccia AJ et al. Anticancer efficacy of systemically delivered anaerobic bacteria as gene therapy vectors targeting tumor hypoxia/necrosis. Gene Ther 2002; 9:291-296.

108

Patho-Biotechnology

29. Theys J, Landuyt W, Nuyts S et al. Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum. Cancer Gene Ther 2001; 8:294-297. 30. Lemmon MJ, van Zijl P, Fox ME et al. Anaerobic bacteria as a gene delivery system that is controlled by the tumor microenvironment. Gene Ther 1997; 4:791-796. 31. Theys J, Pennington O, Dubois L et al. Repeated cycles of Clostridium-directed enzyme prodrug therapy result in sustained antitumour effects in vivo. Br J Cancer 2006; 95:1212-1219. 32. Nuyts S, Van Mellaert L, Theys J et al. Radio-responsive recA promoter significantly increases TNFalpha production in recombinant clostridia after 2 Gy irradiation. Gene Ther 2001; 8:1197-1201. 33. Dang LH, Bettegowda C, Huso DL et al. Combination bacteriolytic therapy for the treatment of experimental tumors. Proc Natl Acad Sci USA 2001; 98:15155-15160. 34. Dang LH, Bettegowda C, Agrawal N et al. Targeting vascular and avascular compartments of tumors with C. novyi-NT and anti-microtubule agents. Cancer Biol Ther 2004; 3:326-337. 35. Bettegowda C, Dang LH, Abrams R et al. Overcoming the hypoxic barrier to radiation therapy with anaerobic bacteria. Proc Natl Acad Sci USA 2003; 100:15083-15088. 36. Cheong I, Huang X, Bettegowda C et al. A bacterial protein enhances the release and efficacy of liposomal cancer drugs. Science 2006; 314:1308-1311. 37. Agrawal N, Bettegowda C, Cheong I et al. Bacteriolytic therapy can generate a potent immune response against experimental tumors. Proc Natl Acad Sci USA 2004; 101:15172-15177. 38. Kimura NT, Taniguchi S, Aoki K et al. Selective localization and growth of Bifidobacterium bifidum in mouse tumors following intravenous administration. Cancer Res 1980; 40:2061-2068. 39. Sasaki T, Fujimori M, Hamaji Y et al. Genetically engineered Bifidobacterium longum for tumortargeting enzyme-prodrug therapy of autochthonous mammary tumors in rats. Cancer Science 2006; 97:649-657. 40. Yazawa K, Fujimori M, Amano J et al. Bifidobacterium longum as a delivery system for cancer gene therapy: selective localization and growth in hypoxic tumors. Cancer Gene Ther 2000; 7:269-274. 41. Li X, Fu GF, Fan YR et al. Bifidobacterium adolescentis as a delivery system of endostatin for cancer gene therapy: selective inhibitor of angiogenesis and hypoxic tumor growth. Cancer Gene Ther 2003; 10:105-111. 42. Fu GF, Li X, Hou YY et al. Bifidobacterium longum as an oral delivery system of endostatin for gene therapy on solid liver cancer. Cancer Gene Ther 2005; 12:133-140. 43. Pawelek JM, Low KB, Bermudes D. Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 1997; 57:4537-4544. 44. Khan SA, Everest P, Servos S et al. A lethal role for lipid A in Salmonella infections. Mol Microbiol 1998; 29:571-579. 45. Low KB, Ittensohn M, Le T et al. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat Biotechnol 1999; 17:37-41. 46. Thamm DH, Kurzman ID, King I et al. Systemic administration of an attenuated, tumor-targeting Salmonella typhimurium to dogs with spontaneous neoplasia: Phase I evaluation. Clin Cancer Res 2005; 11:4827-4834. 47. Clairmont C, Lee KC, Pike J et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 2000; 181:1996-2002. 48. Toso JF, Gill VJ, Hwu P et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 2002; 20:142-152. 49. Zhao M, Yang M, Li XM et al. Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci USA 2005; 102:755-760. 50. Zhao M, Geller J, Ma H et al. Monotherapy with a tumor-targeting mutant of Salmonella typhimurium cures orthotopic metastatic mouse models of human prostate cancer. Proc Natl Acad Sci USA 2007; 104:10170-10174. 51. Zhao M, Yang M, Ma H et al. Targeted therapy with a Salmonella typhimurium leucine-arginine auxotroph cures orthotopic human breast tumors in nude mice. Cancer Res 2006; 66:7647-7652. 52. Mengesha A, Dubois L, Lambin P et al. Development of a flexible and potent hypoxia-inducible promoter for tumor-targeted gene expression in attenuated Salmonella. Cancer Biol Ther 2006; 5:1120-1128. 53. Platt J, Sodi S, Kelley M et al. Antitumour effects of genetically engineered Salmonella in combination with radiation. Eur J Cancer 2000; 36:2397-2402. 54. Lee CH, Wu CL, Tai YS et al. Systemic administration of attenuated Salmonella choleraesuis in combination with cisplatin for cancer therapy. Mol Ther 2005; 11:707-716. 55. Lee CH, Wu CL, Shiau AL. Endostatin gene therapy delivered by Salmonella choleraesuis in murine tumor models. J Gene Med 2004; 6:1382-1393.

Bacteria Mediated Gene Therapy Strategies

109

56. Lee CH, Wu CL, Shiau AL. Systemic administration of attenuated Salmonella choleraesuis carrying thrombospondin-1 gene leads to tumor-specific transgene expression, delayed tumor growth and prolonged survival in the murine melanoma model. Cancer Gene Ther 2005; 12:175-184. 57. Nemunaitis J, Cunningham C, Senzer N et al. Pilot trial of genetically modified, attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Ther 2003; 10:737-744. 58. Soghomonyan SA, Doubrovin M, Pike J et al. Positron emission tomography (PET) imaging of tumor-localized Salmonella expressing HSV1-TK. Cancer Gene Ther 2005; 12:101-108. 59. Tjuvajev J, Blasberg R, Luo X et al. Salmonella-based tumor-targeted cancer therapy: Tumor amplified protein expression therapy (TAPET) for diagnostic imaging. J Control Release 2001; 74:313-315. 60. Poltorak A, He X, Smirnova I et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: Mutations in Tlr4 gene. Science 1998; 282:2085-2088. 61. Poltorak A, Ricciardi-Castagnoli P, Citterio S et al. Physical contact between lipopolysaccharide and toll-like receptor 4 revealed by genetic complementation. Proc Natl Acad Sci USA 2000; 97:2163-2167. 62. Hemmi H, Takeuchi O, Kawai T et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000; 408:740-745. 63. Gewirtz AT, Navas TA, Lyons S et al. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol 2001; 167:1882-1885. 64. Aliprantis AO, Yang RB, Mark MR et al. Cell activation and apoptosis by bacterial lipoproteins through toll-like receptor-2. Science 1999; 285:736-739. 65. Hoebe K, Janssen E, Beutler B. The interface between innate and adaptive immunity. Nat Immunol 2004; 5:971-974. 66. Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392:245-252. 67. Zoller M, Christ O. Prophylactic tumor vaccination: Comparison of effector mechanisms initiated by protein versus DNA vaccination. J Immunol 2001; 166:3440-3450. 68. Cochlovius B, Stassar MJ, Schreurs MW et al. Oral DNA vaccination: Antigen uptake and presentation by dendritic cells elicits protective immunity. Immunol Lett 2002; 80:89-96. 69. Weth R, Christ O, Stevanovic S et al. Gene delivery by attenuated Salmonella typhimurium: Comparing the efficacy of helper versus cytotoxic T-cell priming in tumor vaccination. Cancer Gene Ther 2001; 8:599-611. 70. Xiang R, Lode HN, Chao TH et al. An autologous oral DNA vaccine protects against murine melanoma. Proc Natl Acad Sci USA 2000; 97:5492-5497. 71. Lode HN, Pertl U, Xiang R et al. Tyrosine hydroxylase-based DNA-vaccination is effective against murine neuroblastoma. Med Pediatr Oncol 2000; 35:641-646. 72. Pertl U, Wodrich H, Ruehlmann JM et al. Immunotherapy with a posttranscriptionally modified DNA vaccine induces complete protection against metastatic neuroblastoma. Blood 2003; 101:649-654. 73. Xiang R, Primus FJ, Ruehlmann JM et al. A dual-function DNA vaccine encoding carcinoembryonic antigen and CD40 ligand trimer induces T-cell-mediated protective immunity against colon cancer in carcinoembryonic antigen-transgenic mice. J Immunol 2001; 167:4560-4565. 74. Chou CK, Hung JY, Liu JC et al. An attenuated Salmonella oral DNA vaccine prevents the growth of hepatocellular carcinoma and colon cancer that express alpha-fetoprotein. Cancer Gene Ther 2006; 13:746-752. 75. Luo Y, Zhou H, Mizutani M et al. Transcription factor Fos-related antigen 1 is an effective target for a breast cancer vaccine. Proc Natl Acad Sci USA 2003; 100:8850-8855. 76. Xiang R, Mizutani N, Luo Y et al. A DNA vaccine targeting survivin combines apoptosis with suppression of angiogenesis in lung tumor eradication. Cancer Res 2005; 65:553-561. 77. Feng KK, Zhao HY, Qiu H et al. Combined therapy with flk1-based DNA vaccine and interleukin-12 results in enhanced antiangiogenic and antitumor effects. Cancer Lett 2005; 221:41-47. 78. Lee SH, Mizutani N, Mizutani M et al. Endoglin (CD105) is a target for an oral DNA vaccine against breast cancer. Cancer Immunol Immunother 2006; 55:1565-1574. 79. Niethammer AG, Xiang R, Becker JC et al. A DNA vaccine against VEGF receptor 2 prevents eff ective angiogenesis and inhibits tumor growth. Nat Med 2002; 8:1369-1375. 80. Loeffler M, Kruger JA, Niethammer AG et al. Targeting tumor-associated fibroblasts improves cancer chemotherapy by increasing intratumoral drug uptake. J Clinical Investigat 2006; 116:1955-1962. 81. Woo PC, Wong LP, Zheng BJ et al. Unique immunogenicity of hepatitis B virus DNA vaccine presented by live-attenuated Salmonella typhimurium. Vaccine 2001; 19:2945-2954. 82. Zheng BJ, Ng MH, Chan KW et al. A single dose of oral DNA immunization delivered by attenuated Salmonella typhimurium down-regulates transgene expression in HBsAg transgenic mice. Eur J Immunol 2002; 32:3294-3304.

110

Patho-Biotechnology

83. Wedemeyer H, Gagneten S, Davis A et al. Oral immunization with HCV-NS3-transformed Salmonella: induction of HCV-specific CTL in a transgenic mouse model. Gastroenterology 2001; 121:1158-1166. 84. Flo J, Tisminetzky S, Baralle F. Oral transgene vaccination mediated by attenuated Salmonellae is an effective method to prevent Herpes simplex virus-2 induced disease in mice. Vaccine 2001; 19:1772-1782. 85. Baud D, Benyacoub J, Revaz V et al. Immunogenicity against human papillomavirus type 16 virus-like particles is strongly enhanced by the PhoPc phenotype in Salmonella enterica serovar Typhimurium. Infect Immun 2004; 72:750-756. 86. Baud D, Ponci F, Bobst M et al. Improved efficiency of a Salmonella-based vaccine against human papillomavirus type 16 virus-like particles achieved by using a codon-optimized version of L1. J Virol 2004; 78:12901-12909. 87. Shata MT, Hone DM. Vaccination with a Shigella DNA vaccine vector induces antigen-specific CD8(+) T-cells and antiviral protective immunity. J Virol 2001; 75:9665-9670. 88. Shata MT, Reitz MS Jr, DeVico AL et al. Mucosal and systemic HIV-1 Env-specific CD8(+) T-cells develop after intragastric vaccination with a Salmonella Env DNA vaccine vector. Vaccine 2001; 20:623-629. 89. Vecino WH, Morin PM, Agha R et al. Mucosal DNA vaccination with highly attenuated Shigella is superior to attenuated Salmonella and comparable to intramuscular DNA vaccination for T-cells against HIV. Immunol Lett 2002; 82:197-204. 90. Xu F, Hong M, Ulmer JB. Immunogenicity of an HIV-1 gag DNA vaccine carried by attenuated Shigella. Vaccine 2003; 21:644-648. 91. Pasetti MF, Barry EM, Losonsky G et al. Attenuated Salmonella enterica serovar Typhi and Shigella flexneri 2a strains mucosally deliver DNA vaccines encoding measles virus hemagglutinin, inducing specific immune responses and protection in cotton rats. J Virol 2003; 77:5209-5217. 92. Vecino WH, Q uanquin NM, Martinez-Sobrido L et al. Mucosal immunization with attenuated Shigella flexneri harboring an influenza hemagglutinin DNA vaccine protects mice against a lethal influenza challenge. Virology 2004; 325:192-199. 93. Shiau AL, Chu CY, Su WC et al. Vaccination with the glycoprotein D gene of pseudorabies virus delivered by nonpathogenic Escherichia coli elicits protective immune responses. Vaccine 2001; 19:3277-3284. 94. Shiau AL, Chen YL, Liao CY et al. Prothymosin alpha enhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis. Vaccine 2001; 19:3947-3956. 95. Eo SK, Yoon HA, Aleyas AG et al. Systemic and mucosal immunity induced by oral somatic transgene vaccination against glycoprotein B of pseudorabies virus using live attenuated Salmonella typhimurium. FEMS Immunol Med Microbiol 2006; 47:451-461. 96. Stevens R, Lavoy A, Nordone S et al. Pre-existing immunity to pathogenic Listeria monocytogenes does not prevent induction of immune responses to feline immunodeficiency virus by a novel recombinant Listeria monocytogenes vaccine. Vaccine 2005; 23:1479-1490. 97. Sizemore DR, Branstrom AA, Sadoff JC. Attenuated bacteria as a DNA delivery vehicle for DNAmediated immunization. Vaccine 1997; 15:804-807. 98. Al-Mariri A, Tibor A, Lestrate P et al. Yersinia enterocolitica as a vehicle for a naked DNA vaccine encoding Brucella abortus bacterioferritin or P39 antigen. Infect Immun 2002; 70:1915-1923. 99. Miki K, Nagata T, Tanaka T et al. Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51. Infect Immun 2004; 72:2014-2021. 100. Bauer H, Darji A, Chakraborty T et al. Salmonella-mediated oral DNA vaccination using stabilized eukaryotic expression plasmids. Gene Ther 2005; 12:364-372. 101. Darji A, zur Lage S, Garbe AI et al. Oral delivery of DNA vaccines using attenuated Salmonella typhimurium as carrier. FEMS Immunol Med Microbiol 2000; 27:341-349. 102. Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella typhimurium are nonvirulent and effective as live vaccines. Nature 1981; 291:238-239. 103. Pasetti MF, Anderson RJ, Noriega FR et al. Attenuated deltaguaBA Salmonella typhi vaccine strain CVD 915 as a live vector utilizing prokaryotic or eukaryotic expression systems to deliver foreign antigens and elicit immune responses. Clin Immunol 1999; 92:76-89. 104. Gahan ME, Webster DE, Wesselingh SL et al. Impact of plasmid stability on oral DNA delivery by Salmonella enterica serovar Typhimurium. Vaccine 2007; 25:1476-1483. 105. Krusch S, Domann E, Frings M et al. Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells. J Gene Med 2002; 4:655-667.

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106. Zelmer A, Krusch S, Koschinski A et al. Functional transfer of eukaryotic expression plasmids to mammalian cells by Listeria monocytogenes: a mechanistic approach. J Gene Med 2005; 7:1097-1112. 107. Steidler L, Hans W, Schotte L et al. Treatment of murine colitis by Lactococcus lactis secreting interleukin-10. Science 2000; 289:1352-1355. 108. Steidler L, Neirynck S, Huyghebaert N et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 2003; 21:785-789. 109. Shanahan F. Making microbes work for mankind—Clever trick or a glimpse of the future for IBD treatment? Gastroenterology 2004; 127:667-668. 110. Castagliuolo I, Beggiao E, Brun P et al. Engineered E. coli delivers therapeutic genes to the colonic mucosa. Gene Ther 2005; 12:1070-1078. 111. MacDiarmid JA, Mugridge NB, Weiss JC et al. Bacterially derived 400 nm particles for encapsulation and cancer cell targeting of chemotherapeutics. Cancer Cell 2007; 11:431-445. 112. Giacalone MJ, Sabbadini RA, Chambers AL et al. Immune responses elicited by bacterial minicells capable of simultaneous DNA and protein antigen delivery. Vaccine 2006; 24:6009-6017. 113. Narayanan K, Warburton PE. DNA modification and functional delivery into human cells using Escherichia coli DH10B. Nucleic Acids Res 2003; 31:e51. 114. Laner A, Goussard S, Ramalho AS et al. Bacterial transfer of large functional genomic DNA into human cells. Gene Ther 2005. 115. Cicin-Sain L, Brune W, Bubic I et al. Vaccination of mice with bacteria carrying a cloned herpesvirus genome reconstituted in vivo. J Virol 2003; 77:8249-8255. 116. Karagiannis TC, El-Osta A. RNA interference and potential therapeutic applications of short interfering RNAs. Cancer Gene Ther 2005; 12:1559-1572. 117. Timmons L, Court DL, Fire A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 2001; 263:103-112. 118. Timmons L, Fire A. Specific interference by ingested dsRNA. Nature 1998; 395:854. 119. Zhang L, Gao L, Zhao L et al. Intratumoral delivery and suppression of prostate tumor growth by attenuated Salmonella enterica serovar Typhimurium carrying plasmid-based small interfering RNAs. Cancer Res 2007; 67:5859-5864.

Chapter 8

Use of Intracellular Bacteria for the Development of Tools for Tumor Therapy and the Detection of Novel Antibacterial Targets Christoph Schoen,* Jochen Stritzker, Thilo M. Fuchs, Stephanie Weibel, Ivaylo Gentschev, Aladar A. Szalay and Werner Goebel

Abstract

I

ntracellularly replicating bacteria are suitable tools for a combined immunological and drug therapy of tumors. For example, virulence-attenuated strains of L. monocytogenes, Salmonella spp., Shigella spp. and enteroinvasive Escherichia coli (EIEC) have already been used to develop live vaccines against tumors. Here, we show that the type 1 secretion system (T1SS) of E. coli (α-hemolysin) can be applied to secrete microbial or tumor antigens into mammalian cells and that autolysing Listeria monocytogenes strains enables the transfer of DNA or RNA encoding functional enzymes into tumor cells. Furthermore, the knowledge of the metabolic processes essential for intracellular replication of these bacteria may help to optimize the construction of virulence-attenuated strains and to detect novel targets that permit the screening for new antibacterial drugs.

Introduction

In the past years several aspects concerning molecular mechanisms of virulence of many bacterial pathogens were extensively investigated by many groups including our own.1,2 Among other topics, our studies addressed the characterization of virulence factors from extraintestinal pathogenic E. coli strains such as the α-hemolysin3,4 as well as those from L. monocytogenes like the listerial cytolysin called listeriolysin.5 The α-hemolysin from E. coli is the prototype of a protein that is secreted by a T1SS. This rather simple protein transport system can also successfully be applied for efficient secretion of heterologous microbial and tumor antigens and thus represents an interesting tool for the development of recombinant live vaccines.6 Especially Salmonella spp., Shigella spp. and EIEC are appropriate bacterial carriers for the development of such T1SS-based live vaccines as these facultative intracellular bacterial pathogens induce mainly cellular immune responses, e.g., CD4 and CD8 T-cells, which are needed not only for the elimination of intracellular microbial pathogens but also of tumor cells. Salmonella spp. replicate intracellularly in a specialized membrane-surrounded vacuole, whereas Shigella (or EIEC) and L. monocytogenes multiply in the cytosol of their host cells.1,5 While intracellular bacteria generally induce efficient cellular immune responses against the delivered antigens, *Corresponding Author: Christoph Schoen—Institut für Hygiene und Mikrobiologie, Universität Würzburg, Josef-Schneider-Str. 2 97074 Würzburg, Germany. Email: [email protected]

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cytosolically replicating intracellular bacteria are particularly potent in stimulating cytotoxic T-cells (CTL) against suitable antigens. An enhanced CTL response can even be obtained against Salmonella-delivered antigens by arming the Salmonella carrier strain with the ability to produce listeriolysin7 and a similar immunological effect by listeriolysin was recently described for the tuberculosis vaccine strain BCG.8 This cytolysin of L. monocytogenes acts as a potent membrane opener and is mainly responsible for the release of this intracellular pathogen from the primary phagosome into the cytosol.1,5 Furthermore, the strength of the immune responses can be significantly enhanced if the antigens are secreted by the bacterial carriers,9 a prerequisite that can be achieved for most antigens by the use of the E. coli T1SS. For the application of carrier strains, the virulence of these intracellular pathogenic bacteria has to be properly attenuated. The optimal virulence attenuation is obtained when the invasiveness of the bacterial carrier strains is essentially retained but the capacity of intracellular multiplication is strongly contained. For the design of such virulence-attenuated intracellular carrier strains, the precise knowledge of the bacterial metabolism inside mammalian host cells is crucial. Although this latter topic still belongs to the least understood aspects of bacterial pathogenicity, recent studies indicate that the in vivo metabolism of bacterial pathogens, especially the intracellular ones, may be quite different from the metabolism known from previous in vitro studies in defined culture media.10 In this chapter we concentrate on the tools for the development of live recombinant vaccines developed in our group, on bacterial growth within solid tumors in mice and on some recent aspects concerning bacterial metabolism inside mammalian host cells. The data reported may be of importance for the further development of recombinant live tumor vaccines based on virulence-attenuated intracellular bacteria as well as for the identification of new evaluated bacterial targets allowing the screening for novel antimicrobial agents.

Secretion of Microbial and Tumor Antigens in Salmonella enterica Serovar Typhi Ty21 by T1SS

α-hemolysin is a protein exotoxin which is frequently found in extraintestinal pathogenic E. coli strains, particularly in uropathogenic E. coli (UPEC).2 The secretion system of this virulence factor represents the prototype T1SS of Gram-negative bacteria.3 It consists of the three proteins HlyB, HlyD and TolC11,12 which form a transport channel that allows the simultaneous, sec-independent protein transport across both membranes of the Gram-negative cell envelope (Fig. 1A). The secretion signal (HlyAs), essential but also sufficient for transport by this transport system, is located at the carboxy-terminal end of α-hemolysin. Vector plasmids have been constructed previously13 which allow the easy connection of HlyAs to the C-terminus of any heterologous protein, including microbial and tumor antigens14 (Fig. 1B). These recombinant plasmids can be introduced into S. enterica serovar Typhi Ty21, the vaccine strain against typhoid fever and into EIEC strains (Fig. 1C).

Bactofection of Tumor Cells with Bacterial Carriers Introducing DNA and RNA for Antigens and Prodrug-Converting Enzymes

Recently, we constructed autolysing L. monocytogenes carrier strains15 that are able to introduce DNA16 or RNA17 encoding functional proteins into the cytosol of mammalian cells (Fig. 2), an approach that has also been termed bactofection. The use of bacteria as plasmid DNA carriers for genetic immunization is an already well established approach. So far, different bacterial species have been tested as bacterial DNA-vaccine carriers, including amongst others S. flexneri, Salmonella spp. and L. monocytogenes (for recent reviews see. 18,19) Our studies focused on the improvement of L. monocytogenes mediated bactofection in vitro using different cell types16 as well as in vivo applying a murine model system.20 In particular, this approach was already successfully applied for introducing into B16 melanoma cells (Fig. 3) as well as into 4T1 breast tumor cells DNA encoding enzymes that convert nontoxic prodrugs, such as 5-fluorocytosine or 6-methylpurine deoxyribose, into the cell-toxic drugs 5-fluorouracil and

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Figure 1, legend viewed on following page.

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Figure 1, figure viewed on previous page. Construction and secretion of heterologous hemolysin fusion proteins via a T1SS in Gram-negative bacteria. A) A topological model for secretion of heterologous hemolysin proteins. The assembled T1SS spans the inner (IM) and outer (OM) membrane as well as the intervening periplasmic space. The active transporter complex consists of the translocase HlyB which belongs to the ATP-binding cassette (ABC) superfamily of transporters and two auxiliary proteins. HlyD is a member of the membrane fusion protein (MFP) family and anchored via a short N-terminal hydrophobic sequence in the inner membrane. The extended hydrophilic region of HlyD is protruding into the periplasm and interacting with the second auxiliary component TolC, which forms a channel across the periplasmic space and the outer membrane. The secretion signal, here referred as HlyAs is located within the last C-terminal 50-60 amino acids of hemolysin (HlyA). The heterologous proteins or part thereof are marked by a dotted line. Modified figure according to ref. 13. For more details on T1SS see refs. 13, 30 and 31. B) Construction of Salmonella and E. coli strains expressing and secreting a c-Raf-HlyAs fusion protein. C-Raf encodes for a serine-threonine kinase of the Raf family (A-Raf, B-Raf, C-Raf). These proteins are central players in cellular signal transduction and thus often causally involved in the development of cancer when mutated or over-expressed. Phly indicates the hemolysin promoter region. C) Identification of a c-Raf-HlyAs fusion protein by immunodetection with anti-HlyA antibodies. Cultures of S. typhi Ty21a (lane 1) and E. coli (EIEC) EDL 1284 (lane 2) strains transformed with pMOhly-cRaf plasmid14 were grown to an optical density at 600nm of 1.0 and subsequently used for isolation and analysis of supernatant proteins. As can be seen, the fusion protein is successfully produced and secreted via the T1SS in both carrier systems.

6-methylpurine, respectively. It was shown that the tumor cells bactofected with such DNA were completely killed in vitro (Stritzker et al, submitted). Preliminary data indicate that this procedure is also active in the mouse tumor model (Stritzker, unpublished observation). To circumvent the nuclear compartment necessary for the expression of antigen-encoding plasmid-DNA and thus to achieve an earlier onset of antigen-expression particularly in antigen-presenting dendritic cells, a novel L. monocytogenes-based carrier system was developed for the delivery of translation competent messenger RNA directly into the cytosol of the infected host cell.17 Accordingly, Loeffler and colleagues could demonstrate that the infection of mice with self-destructing L. monocytogenes carriers delivering mRNA that encodes a nonsecreted form of the chicken ovalbumin (OVA) used as model antigen resulted in a significant OVA-specific CD8 T-cell response.20

Growth of E. coli in Solid Tumors Transplanted into Mice

It is well known that anaerobic or facultative anaerobic bacteria introduced intravenously into tumor-bearing mice will reach and multiply within the tumor.21 We have used a tumor-mouse model with transplanted 4T1 solid tumors to study in detail the fate of E. coli Nissle 1917 and E. coli K-12 colonizing the tumor after injection of these bacteria into the tail vein and the effect of the bacterial colonization on the tumor microenvironment.22 The histological analysis of the colonized tumor shows extensive necrosis of tumor tissues and total destruction of vasculature in the inner section of the tumor (Weibel et al, submitted). This seems to be caused by bacteria-mediated high induction of tumor necrosis factor-α and matrix mettaloproteinase-9. Massive bacterial colonization occurs in the outer part of this necrotic zone which is shielded off the peripheral proliferating tumor cells by a zone of macrophages. A similar observation was made when EIEC or S. enterica serovar Typhimurium were injected (Goetz and Stritzker, unpublished results). Nevertheless, despite the initial necrosis of the inner part of the tumor, the bacterial colonization alone does not lead to the reduction of tumor growth—at least in the investigated aggressive 4T1 breast cancer model system. A highly significant reduction in tumor growth was, however, observed when the mice were vaccinated with S. enterica serovar Typhi Ty21 secreting tumor antigens fused to CtxB, a cholera toxin subunit, via T1SS,23 indicating that tumor-associated T-lymphocytes might be activated by this vaccination.

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Figure 2. Model of the delivery of antigens (A), antigen-encoding plasmid DNA (B) or mRNA (C) to antigen presenting cells by intracellular replicating bacteria such as L. monocytogenes. A) Protein delivery. After invasion of the antigen presenting cell (1) and escape from the phagosome (2) the antigen being favorably produced in a secreted form by the bacterial carrier (3) is degraded by the proteasome (4). The resulting peptides are loaded via the TAP transporter on MHC-I molecules (5) which present them to CD8 + T-cells (6). Alternatively, the secreted protein antigen can also be presented to CD4 + T-helper cells (2). B) DNA delivery. After invasion (1), escape from the phagosome (2) and autolysis of the bacterial carrier in the cytosol (3a) the plasmid DNA is released and transported into the nucleus of the antigen presenting cell (3b) where it is transcribed into mRNA. After export and translation of the mRNA (3d) the protein is presented via the MHC-I as in antigen delivery (4-6). In contrast to the delivery of protein antigens synthesized by the bacterial carrier, DNA delivery allows also for the synthesis and consecutive processing of posttranslationally modified proteins that are specific for eukaryotic cells. C) mRNA delivery. In contrast to the delivery of plasmid DNA as depicted in panel B, the delivered antigen-encoding mRNA can immediately be transcribed and translated in the host cell cytosol (3b) thus circumventing the nuclear compartment of the host cell. For further details see reference 32.

Metabolic Pathways as Evaluated Targets for Virulence Attenuation and Screening of Antimicrobials

The use of intracellularly replicating bacteria such as S. enterica (serovar Typhimurium or Typhi) or L. monocytogenes as carriers for the development of bacteria-based recombinant live vaccines allows the induction of strong cellular immune responses (Th1 and/or CTL) against the heterologous secreted antigens.24 However, suitable virulence-attenuated strains are indispensable for the application of these pathogenic bacteria as vaccine carriers. Optimally attenuated strains seem to be those which still invade and survive within host cells, but which replicate slower, because those virulence attenuated utants still lead to a good cellular immune response. Recently, Stritzker and colleagues have shown that the deletion of genes in the basic branch of the aromatic amino acid biosynthesis pathway of L. monocytogenes lead to strong virulence attenuation.25 Nevertheless, these attenuated strains still retain the ability to elicit a strong, protective cellular immune response against L. monocytogenes wild-type strains as well as heterologous antigens25 (Weibel et al, submitted). The aroA mutant performs a predominant anaerobic metabolism due to the lack of menaquinone which is the only quinone biosynthesized in L. monocytogenes. Due to this metabolic switch the aroA mutant shows a highly enhanced production of internalin A (InlA) and to a lesser extent of InlB, which leads to increased internalization of the aroA mutant into nonphagocytic cells.26 But despite the increased invasiveness, the aroA mutant is strongly attenuated in virulence due to the strongly impaired intracellular replication and cell-to-cell spread.26 Furthermore, we studied more systematically the metabolism of intracellularly growing L. monocytogenes by screening of a mutant library and by13 C-isotopologue analysis.27,28 In general, these studies revealed a high metabolic flexibility of this pathogen that are adapted to uptake and utilize host cell metabolites for efficient replication. Particularly, it appeared that mainly C3-components deriving from the intermediary catabolic pathways of the host cells and glucose-6-phosphate are major carbon sources to sustain the listerial metabolism within host cells (Schauer and Eylert, in preparation). Several metabolic reactions such as carboxylation of pyruvate to oxaloacetate by pyruvate carboxylase (PycA) and the synthesis of branched chain amino acids also revealed to be required for intracellular growth of L. monocytogenes. In addition, we identified a set of transporters responsible for the uptake of sugars, oligopeptides and vitamins that are essential for intracellular replication but not necessarily for extracellular growth. Deletion of the genes involved in these steps also led to growth attenuation in epithelial cells without interfering with adhesion, invasion and intracellular survival. Those metabolic pathways can not only be exploited to construct appropriately attenuated carrier strains, but also be applied for antimicrobial drug screening against this pathogen which is frequently occurring as major risk factor in food industry.29 Promising target candidates are those

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Figure 3. Use of bacteria carrying a eukaryotic expression plasmid for the synthesis of prodrug converting enzymes in experimental tumor therapy. A) Principle of a prodrug-drug-system for the conversion of non-toxic prodrugs into cytotxic compounds. In the example, 6-methylpurine deoxyribose (MePdR) is converted into the cell toxic active drug 6-methylpurine (MeP) by purine nucleoside phosphorylase (PNP) encoded by the deoD gene from E. coli. B) Melanoma B16 cells are killed upon L. monocytogenes (Lm) – mediated transfer of the deoD gene encoding PNP (triangles) after treatment with the prodrug MePdR. B16 cells infected with control bacteria were not killed by prodrug therapy (squares). The deoD gene was placed under the transcriptional control of the cytomegalovirus promoter (Pcmv).

enzymes and transporters involved in the metabolic reactions described above that are essential for in vivo virulence, e.g., in a mouse infection model. In combination with similar metabolic studies to be performed with other relevant intracellular pathogens, we expect the identification

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of pathogen-specific as well as general metabolic targets for the detection of drugs against bacterial pathogens.

Conclusions

The Gram-negative intracellular Enterobacteriaceae, especially Salmonella and Shigella/EIEC are well-suited as carriers for the delivery of heterologous antigens via T1SS. In vivo, this delivery can occur directly in antigen-presenting cells (APC), thus inducing efficient cellular immune responses. Properly manufactured antigens can also be secreted into the cytosol of APC by autolysing L. monocytogenes. Bactofection of mammalian cells, i.e., the bacteria-mediated transfer of DNA and RNA that are directly expressed by these cells, has also been demonstrated using these bacteria. Optimized virulence attenuation especially of L. monocytogenes and EIEC carrier strains may be best accomplished by blocking specific metabolic reactions essential for their intracellular replication. Furthermore, EIEC strains already synthesize purine nucleoside phosphorylase (PNP) whose production can be further improved and adapted to specific cells. PNP has been shown to be a potent prodrug-drug converting enzyme. In addition, these bacterial carriers can be equipped with DNA encoding cytotoxic agents that may be directly expressed by the invaded host cells. In summary, virulence-attenuated EIEC and similarly Salmonella spp. and L. monocytogenes may serve as excellent tools for a combined bacteria-mediated tumor therapy which includes immune responses as well as cytotoxic drug reactions specifically directed against the tumor cells. The identification of specific metabolic targets in these intracellular bacterial pathogens will also support the screening of novel antimicrobials against these pathogens that represent major risk factors in food.

References

1. Miller VL, Kaper JB, Portnoy DA, eds. Molecular Genetics of Bacterial Pathogenesis. Washington DC: ASM Press, 1994. 2. Hacker J, Heesemann J, eds. Molecular Infection Biology: Interactions Between Microorganisms and Cells. New York: Wiley & Sons, 2002. 3. Holland IB, Schmitt L, Young J. Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review). Mol Membr Biol 2005; 22(1-2):29-39. 4. Goebel W, Royer-Pokora B, Lindenmaier W et al. Plasmids controlling synthesis of hemolysin in Escherichia coli: molecular properties. J Bacteriol 1974; 118(3):964-973. 5. Kreft J, Vazquez-Boland JA, Altrock S et al. Pathogenicity islands and other virulence elements in Listeria. Curr Top Microbiol Immunol 2002; 264(2):109-125. 6. Spreng S, Dietrich G, Goebel W et al. The Escherichia coli haemolysin secretion apparatus: A potential universal antigen delivery system in gram-negative bacterial vaccine carriers. Mol Microbiol 1999; 31(5):1596-1598. 7. Gentschev I, Sokolovic Z, Mollenkopf HJ et al. Salmonella strain secreting active listeriolysin changes its intracellular localization. Infect Immun 1995; 63(10):4202-4205. 8. Hess J, Miko D, Catic A et al. Mycobacterium bovis Bacille Calmette-Guerin strains secreting listeriolysin of Listeria monocytogenes. Proc Natl Acad Sci USA 1998; 95(9):5299-5304. 9. Hess J, Gentschev I, Miko D et al. Superior efficacy of secreted over somatic antigen display in recombinant Salmonella vaccine induced protection against listeriosis. Proc Natl Acad Sci USA 1996; 93(4):1458-1463. 10. Munoz-Elias EJ, McKinney JD. Carbon metabolism of intracellular bacteria. Cell Microbiol 2006; 8(1):10-22. 11. Wandersman C, Delepelaire P. TolC, an Escherichia coli outer membrane protein required for hemolysin secretion. Proc Natl Acad Sci USA 1990; 87(12):4776-4780. 12. Wagner W, Vogel M, Goebel W. Transport of hemolysin across the outer membrane of Escherichia coli requires two functions. J Bacteriol 1983; 154(1):200-210. 13. Gentschev I, Dietrich G, Goebel W. The E. coli alpha-hemolysin secretion system and its use in vaccine development. Trends Microbiol 2002; 10(1):39-45. 14. Gentschev I, Fensterle J, Schmidt A et al. Use of a recombinant Salmonella enterica serovar Typhimurium strain expressing C-Raf for protection against C-Raf induced lung adenoma in mice. BMC Cancer 2005; 5:15. 15. Dietrich G, Bubert A, Gentschev I et al. Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes. Nat Biotechnol 1998; 16(2):181-185.

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16. Pilgrim S, Stritzker J, Schoen C et al. Bactofection of mammalian cells by Listeria monocytogenes: Improvement and mechanism of DNA delivery. Gene Ther 2003; 10(24):2036-2045. 17. Schoen C, Kolb-Maurer A, Geginat G et al. Bacterial delivery of functional messenger RNA to mammalian cells. Cell Microbiol 2005; 7(5):709-724. 18. Daudel D, Weidinger G, Spreng S. Use of attenuated bacteria as delivery vectors for DNA vaccines. Expert Rev Vaccines 2007; 6(1):97-110. 19. Schoen C, Stritzker J, Goebel W et al. Bacteria as DNA vaccine carriers for genetic immunization. Int J Med Microbiol 2004; 294(5):319-335. 20. Loeffler DI, Schoen CU, Goebel W et al. Comparison of different live vaccine strategies in vivo for delivery of protein antigen or antigen-encoding DNA and mRNA by virulence-attenuated Listeria monocytogenes. Infect Immun 2006; 74(7):3946-3957. 21. Mengesha A, Dubois L, Chiu RK et al. Potential and limitations of bacterial-mediated cancer therapy. Front Biosci 2007; 12:3880-3891. 22. Stritzker J, Weibel S, Hill PJ et al. Tumor-specific colonization, tissue distribution and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. Int J Med Microbiol 2007; 297(3):151-162. 23. Fensterle J, Bergmann B, Yone C et al. Cancer immunotherapy based on recombinant Salmonella enterica serovar Typhimurium aroA strains secreting prostate-specific antigen (PSA) and cholera toxin subunit B (CtxB). Cancer Gene Ther 2007; In press. 24. Loessner H, Weiss S. Bacteria-mediated DNA transfer in gene therapy and vaccination. Expert Opin Biol Ther 2004; 4(2):157-168. 25. Stritzker J, Janda J, Schoen C et al. Growth, virulence and immunogenicity of Listeria monocytogenes aro mutants. Infect Immun 2004; 72(10):5622-5629. 26. Stritzker J, Schoen C, Goebel W. Enhanced synthesis of internalin A in aro mutants of Listeria monocytogenes indicates posttranscriptional control of the inlAB mRNA. J Bacteriol 2005; 187(8):2836-2845. 27. Eisenreich W, Slaghuis J, Laupitz R et al. 13C isotopologue perturbation studies of Listeria monocytogenes carbon metabolism and its modulation by the virulence regulator PrfA. Proc Natl Acad Sci USA 2006; 103(7):2040-2045. 28. Joseph B, Przybilla K, Stuhler C et al. Identification of Listeria monocytogenes genes contributing to intracellular replication by expression profiling and mutant screening. J Bacteriol 2006; 188(2):556-568. 29. Vazquez-Boland JA, Kuhn M, Berche P et al. Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 2001; 14(3):584-640. 30. Koronakis V. TolC—the bacterial exit duct for proteins and drugs. FEBS Lett 2003; 555(1):66-71. 31. Hahn HP, von Specht BU. Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters. FEMS Immunol Med Microbiol 2003; 37(2-3):87-98. 32. Schoen C, Loeffler DI, Frentzen A et al. Listeria monocytogenes as novel carrier system for the development of live vaccines. Int J Med Microbiol 2008;298(1-2):45-58.

Chapter 9

Bacterial Vectors for RNAi Delivery Thu Nguyen and Johannes H. Fruehauf*

Abstract

R

NA interference (RNAi) is a recently discovered powerful research tool which allows the targeted “silencing” of particular genes. RNAi is also thought to have immense therapeutic potential to treat and prevent a wide range of diseases from inflammation to cancer and to target genes which have formerly been considered “undruggable”. However, the advancement of RNAi technology as a means of therapy is hindered by the challenges of delivering RNAi into the cytoplasm of target cells. As a versatile gene vector, bacteria have been employed and shown to be an effective, safe and inexpensive measure for delivering RNAi to mammalian cells. Two systems that exploit the therapeutic benefits of bacteria to deliver RNAi are transkingdom RNA interference (tkRNAi) and bacteria-mediated RNA interference (bm-RNAi). Both systems are effective in eliciting gene silencing both in vitro and in vivo and they suggest an important role for bacteria in future RNAi therapeutics. RNA interference (RNAi) is a highly conserved, potent gene-silencing mechanism whereby endogenous genes of interest are down-regulated by a targeted enzymatic cleavage of messenger RNA (mRNA) in a highly specific and efficient manner. This phenomenon had been observed in plants in the 1990s1,2 and its mechanism was elucidated in animals in 1998 by Drs Fire and Mello,3 who published their findings on RNA induced silencing in the nematode worm C. elegans, work for which they were awarded the Nobel Prize for Physiology or Medicine in 2006.The RNAi pathway is conserved in many species and has likely evolved as innate immunity against viral infection. This discovery of RNAi gained even more importance after it was demonstrated that RNAi is also active in mammalian cells.4 Since then, RNAi has been found to be a highly useful tool for basic research as it helps to rapidly elucidate gene function through targeted knockdown studies. Furthermore, RNAi has also spawned a novel field of drug research, RNAi-based therapeutics, which has become a quickly growing area of drug development. RNAi-based therapy attracts a lot of interest, since—at least theoretically—every disease caused by an upregulation of a known gene could be targeted using RNAi-based drugs. Therapeutic applications could range from viral infection to cancer treatment. The classical RNAi pathway is triggered by the introduction of short interfering RNA (siRNA) to the cytoplasm. siRNA consists of short (19-21 bp) double stranded RNA with two nucleotide overhangs at each 3' end.5 One of the strands (the sense strand) is homologous to an area in the mRNA of the gene to be silenced. Alternatively, the RNAi pathway can also be triggered by the introduction of siRNA precursors such as double-stranded RNA (dsRNA) or short hairpin RNA (shRNA), which may be transcribed from eukaryotic plasmids inside the target cell or from viral vectors. Both shRNA and dsRNA can be processed by Dicer, an enzyme with RNAse III activity, to become siRNA and induce RNAi-mediated gene silencing in the cytoplasm of mammalian cells or live animals.6,7 shRNA molecules can function as siRNA following the cleavage of the loop by Dicer *Corresponding Author: Johannes H. Fruehauf—Skip Ackerman Center for Molecular Therapeutics, GI Cancer Laboratory, DANA 607, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave, Boston, MA 02215, USA. Email: [email protected]

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while long dsRNA can be cleaved by the same cytoplasmic enzyme into short, double-stranded siRNA fragments of 19-21bp with 2-4 nucleotide overhangs at the 3' ends. The siRNA duplexes are then unwound via an ATP-dependent mechanism and the antisense or guide strand is bound to a multi-protein structure called the RNA-induced silencing complex (RISC).7 This silencing complex catalyzes the cleavage of a homologous mRNA within the cytoplasm to inhibit translation and thereby prevent protein expression of the gene of interest. Although RNAi technology holds great promise for the advancement of new gene directed therapies, the difficulty of delivering siRNA to target cells has impeded the rapid expansion of RNAi-based therapies. The use of viral vectors, nanoparticles, liposomes and chemical modifications to siRNA have been exploited to deliver RNAi6,8 and delivery of therapeutic RNA interference has become a field of its own right in the biotechnology industry. Currently (Fall 2007), few RNAi-therapeutic compounds are in clinical trials for target organs such as the eye (direct injection of modified siRNA), the pulmonary epithelium (inhalation of unmodified siRNA) or the liver (systemic injection of liposomes or nanoparticles carrying siRNA). However, these delivery methods may not be suitable for a number of other organs or cell types, particularly in the gastrointestinal or genitourinary tract, where numerous potential medical indications exist which could be treated with RNAi based therapeutics, but for which targeting and delivery is more difficult. Bacteria are highly versatile and useful tools for biotechnology and have helped drive most of the recent rapid growth in our understanding of genes and gene function. Bacteria are increasingly recognized for their vast potential as therapeutics and efforts are being made in various areas of therapeutic drug development based on live attenuated or otherwise modified bacteria. Other chapters in this book cover the use of bacteria as delivery agents for vaccines, chemicals, therapeutic proteins, or even as gene therapy vectors. Bacteria have also been found to be useful for the delivery of short interfering RNA. Unlike some viral vectors, bacteria do not integrate genetic material into the host genome and they can be controlled with antibiotics or engineered to increase safety using nutrient auxotrophy. Bacteria are versatile gene vectors and have been shown to be an effective, safe and inexpensive measure for delivering RNAi to target cells with high selectivity and high specificity.6 In a concept called transkingdom RNA interference (tkRNAi), it was shown that laboratory strains of E.Coli can be harnessed to produce and deliver therapeutic short interfering RNA.9

Transkingdom RNA Interference

Transkingdom RNA Interference (tkRNAi) is an example of a bacteria-mediated RNAi delivery system that is successful in eliciting efficient gene silencing both in vitro and in vivo, primarily targeting epithelial cells. tkRNAi was developed at the Beth Israel Deaconess Medical Center in Boston, MA and uses genetically modified bacteria, e.g., Escherichia coli, to produce and deliver short hairpin RNA (shRNA). In the prototype concept, this is achieved through the use of a transkingdom RNA interference plasmid (TRIP). The TRIP plasmid enables the bacteria to produce shRNA, invade target cells, release shRNA into the cells’ cytoplasm and activate the RNAi pathway to induce gene silencing. The TRIP prototype plasmid consists of three main components: 1. A shRNA expression cassette, which drives the expression of a short hairpin RNA under the control of the T7RNA polymerase promoter and terminator. The cassette is flanked by two characteristic restriction enzyme sites (Sal 1 and BamH1) which allow for a rapid exchange of the shRNA sequence to develop TRIP vectors against different targets. shRNA is produced after induction of the T7RNA polymerase which causes shRNA to accumulate inside the bacteria and becomes released after the bacteria dies inside the host cell. 2. The inv gene on the TRIP encodes for the expression of invasin protein on the bacterial surface (derived from Yersinia pseudotuberculosis),10 which enables bacterial entry into the target (host) cell. Invasin interacts in a highly efficient manner with beta(1)integrins which are expressed on the surface of epithelial cells. This leads to the rearrangement of

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host cell cytoskeleton, formation of a groove and uptake of the invasin-expressing bacteria into an endosome. 3. The hly gene on the TRIP encodes for listeriolysin O.11,12 This protein is derived from Listeria Monocytogenes, a pore-forming toxin which is released upon bacterial lysis inside the endosome and ruptures the endosomal membrane. This results in the release of the accumulated shRNA into the cytoplasm of the host cell. The theoretic steps required for tkRNAi are described in Figure 1. The successful entry of the bacteria into epithelial cells is dependent upon the expression of invasin on the bacterial surface, encoded by the inv gene from Yersinia pseudotuberculosis. Invasin endows the bacteria with the capability to invade mammalian cells containing beta-1 integrin receptors. Meanwhile, the hly gene encodes for the production of listeriolysin O, a pore-forming toxin, which accumulates

Figure 1. Transkingdom RNA interference (tkRNAi): mechanism of action.

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in the bacterial cytoplasm and periplasma. Listeriolysin is released into the endosome of the host cell after invasion when the bacterial cell wall starts to disintegrate due to nutrient deficiency and host cell lysosomal action. Listeriolysin O will result in the formation of pores and rupture of the endosomal membrane, allowing the bacterial content (which includes the shRNA) to reach the cytoplasm of the host cell. Once shRNA has escaped from the lysed bacteria into the cytoplasm, it is processed by the host cell machinery. The protein Dicer cleaves off the loop structure of the hairpin RNA which results in the formation of a short double stranded RNA of 21 paired nucleotides (siRNA). The guide strand of the siRNA is then integrated into a multi protein complex called RISC (RNA induced silencing complex) which retrieves mRNA with perfectly homologous regions. In the presence of a perfect match between the guide strand and the mRNA, cleavage of the mRNA is induced by the ago-2, a component of the RISC complex. This results in the rapid degradation of the remaining mRNA fragments. Thereby, the production of the protein encoded by this particular mRNA will not take place (prevention of translation). The RISC complex containing the guide strand of the siRNA continues to target other copies of the mRNA resulting in a very efficient process of degradation of the specific mRNA of the targeted gene. To demonstrate the effects of tkRNAi technology, a TRIP plasmid was constructed against the human colon cancer oncogene CTNNB1 (catenin β-1) and cloned into a competent strain of nonpathogenic E. coli, BL21DE3. The transformed strain of BL21DE3 was shown to successfully down-regulate the beta-catenin level in human colon cancer cells (SW480) in vitro with a clear dose-dependent effect.9 tkRNAi was also shown to be effective in inducing local gene silencing in the intestinal epithelium of mice that were treated orally with E. coli expressing shRNA against mouse Ctnnb1. In addition, tkRNAi is also successful in eliciting systemic gene silencing in mice with xenografts of human colon cancer that were treated intravenously with E. coli encoding shRNA against human CTNNB1. tkRNAi is currently being developed as a novel method for the delivery of RNAi-based therapeutics with applications ranging from colon cancer prevention to treatment of Human Papilloma Virus infection and inflammatory bowel disease (IBD) by Cequent Pharmaceuticals of Cambridge, MA (more information at www.cequentpharma.com).

Bacteria-Mediated RNA Delivery

Bacteria-mediated RNA interference (bmRNAi) delivery through the use of invasive bacteria such as Salmonella typhimurium is another approach that employs naturally invasive bacteria to deliver RNA interference. Unlike the tkRNAi delivery system, carrier bacteria in the bmRNAi system do not produce shRNA, but rather they transfer shRNA expression plasmids to the host cell which then utilizes its own transcriptional machinery to produce shRNA in the nucleus. Some speculate that this approach may induce more sustained silencing since the siRNA is constantly produced by the host cell and may be more stable than that produced by the bacteria and released into the host cell cytoplasm. Attenuated S. typhimurium has successfully been used in the past as a means of delivery for a wide variety of therapeutic payloads from proteins to DNA for vaccine or gene therapy applications.13-16 One widely used version of attenuated S. typhimurium is SL720717 in which the aroA gene is inactivated. It requires aromatic amino acids for survival, which it cannot find after invasion into a host cell. This results in rapid lysis of the bacteria after host cell invasion, liberation of their payload and a lowered toxicity compared with wildtype S. typhimurium. S. typhimurium have been used successfully to deliver shRNA expression plasmids for the treatment of a mouse prostate cancer model.18 In our own work, we have seen consistent and robust gene silencing in vitro and in vivo using attenuated S. typhimurium as carriers for shRNA expression plasmids for various indications (Fruehauf et al, unpublished data). Bacteria have evolved as an amazingly versatile tool in the last 20 years which drives the genomic revolution and the development of biotechnology. The recent discovery that bacteria can be useful carriers for RNAi is just one more step in our understanding of this kingdom of life. While there are still a host of obstacles to overcome, such as the avoidance of the innate immune system,

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activation of cellular and humoral immune responses to therapeutically administered bacteria, the remaining risk of bacterially-induced disease and others, we believe that bacteria will soon become much more acceptable as carriers of therapeutic payloads and as little bio-robots to fulfill useful tasks inside a host organism.

References

1. Napoli C, Lemieux C, Jorgensen R. Introduction of a chimeric chalcone synthase gene into petunia results in reversible cosuppression of homologous genes in trans. Plant Cell 1990; 2:279-289. 2. Romano N, Macino G. Q uelling: Transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 1992; 6:3343-3353. 3. Fire A et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998; 391:806-811. 4. Elbashir SM et al. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411:494-498. 5. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev 2001; 15:188-200. 6. Li CX et al. Delivery of RNA interference. Cell Cycle 2006; 5(18):2103-9. 7. Zamore PD, Tuschl T, Sharp PA et al. RNAi: Double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101:25-33. 8. de Fougerolles A, Vornlocher HP, Maraganore J et al. Interfering with disease: A progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007; 6:443-453. 9. Xiang S, Fruehauf J, Li CJ. Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nat Biotechnol 2006; 24:697-702. 10. Isberg RR, Voorhis DL, Falkow S. Identification of invasin: A protein that allows enteric bacteria to penetrate cultured mammalian cells. Cell 1987; 50:769-778. 11. Courvalin P, Goussard S, Grillot-Courvalin C. Gene transfer from bacteria to mammalian cells. C R Acad Sci III 1995; 318:1207-1212. 12. Grillot-Courvalin C, Goussard S, Huetz F et al. Functional gene transfer from intracellular bacteria to mammalian cells. Nat Biotechnol 1998; 16:862-866. 13. Hense M et al. Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells. Cell Microbiol 2001; 3:599-609. 14. Weiss S, Chakraborty T. Transfer of eukaryotic expression plasmids to mammalian host cells by bacterial carriers. Curr Opin Biotechnol 2001; 12:467-472. 15. Bermudes D, Zheng LM, King IC. Live bacteria as anticancer agents and tumor-selective protein delivery vectors. Curr Opin Drug Discov Devel 2002; 5:194-199. 16. Paglia P, Terrazzini N, Schulze K et al. In vivo correction of genetic defects of monocyte/macrophages using attenuated Salmonella as oral vectors for targeted gene delivery. Gene Ther 2000; 7:1725-1730. 17. Hoiseth SK, Stocker BA. Aromatic-dependent Salmonella typhimurium are nonvirulent and effective as live vaccines. Nature 1981; 291:238-239. 18. Zhang L et al. Intratumoral delivery and suppression of prostate tumor growth by attenuated Salmonella enterica serovar Typhimurium carrying plasmid-based small interfering RNAs. Cancer Res 2007; 67:5859-5864.

Chapter 10

Viral Pathogens as Therapeutic Delivery Vehicles Helen O’Shea*

Abstract

T

he term patho-biotechnology describes the exploitation of pathogenic bacteria for beneficial applications in food and biomedicine. We propose extending this definition to include viruses, for several reasons. Viruses, as well as providing a threat to human and animal health, can be used for benefit, via the generation of recombinant viruses. Recent advances in molecular biology have made it possible to manipulate and introduce DNA into cells and organisms. An extension of this approach is to use this technology to manipulate viruses to carry ‘foreign’ genes (transgenes) into defective cells, in order to correct defects. Several different viruses have been used as viral vectors (mainly Retroviruses, Adenoviruses, Adeno-Associated Virus, Herpes Simplex virus and Vaccinia virus). All of these viruses have advantages and disadvantages, due to their inherent properties (i.e., size constraints, cell tropism, risk of cell transformation, toxic products and immune response generated). The biology of each virus, how it interacts with the host and the generation of the immune response, are all factors that must be taken into account when considering a particular viral vector. Viral vectors have enormous potential for the delivery of therapeutic genes into diseased tissues. However, further development, especially regarding safety issues, will be required in many instances, before gene therapy becomes standard.

Introduction

Virology is a rapidly developing area and disease prevention is a major goal for virologists. Advances in recombinant DNA technology e.g., Polymerase Chain Reaction (PCR), Real Time PCR (RT-PCR), have contributed enormously to the area, as well as improved diagnostic methods, many developed as a result of use of monoclonal antibody technology (e.g., agglutination tests, ELISAs). Many challenges remain e.g., complete understanding of the Immune System, virus-host interactions, but new technologies are being constantly developed, which will contribute to increased understanding of how viruses can manipulate their host cells. Recent advances in molecular biology have made it possible to manipulate and introduce DNA into cells and organisms. This technology can be used to introduce genes (transgenes) into a viral genome, infect target cells with the recombinant (vector) virus and express the genes, thus allowing viruses to be used for human benefit. The term patho-biotechnology describes the exploitation of pathogenic microorganisms for beneficial applications in biotechnology and biomedicine.1,2 This definition can be expanded to include viruses, for several reasons. Pathogenic bacteria have pathogenic traits [e.g., resistance to environmental factors, to Gastro Intestinal Tract (GIT) specific stresses, translocation, phagosomal vacuolar escape, systemic spread to various organs] which can be exploited for potential applications [e.g., to improve industrially utilised strains, to maximise delivery to the GIT, to specific organs and to the Immune System.1-4 In the same way, the pathogenic *Helen O’Shea—Department of Biological Sciences, Cork Institute of Technology, Rossa Avenue, Bishopstown, Cork, Ireland. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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traits of viruses [e.g., the ability to adsorb onto specific cell types, to integrate into host DNA, to hijack the host cell machinery and produce large amounts of virus-encoded proteins, and to lyse cells], can be exploited [e.g., to target specific cell types, to introduce foreign DNA into host cells, to transcribe and translate large amounts of the transgene-encoded proteins, to specifically kill or alert the immune system to cancer cells]. Many different viruses have been used as viral vectors—mainly DNA viruses e.g., Adenoviruses (AV), Adeno-Associated Virus (AAV), Herpes Simplex virus (HSV), Vaccinia virus (VV), but also Retroviruses (RV, which are RNA viruses).5-8 These vectors will be subsequently discussed. There has also been some success with other RNA viruses, bacteriophages, baculoviruses and plant viruses, but discussion of these vectors is beyond the scope of this chapter, which concentrates on those used in current clinical trials or at advanced preclinical stages. All of these viruses have advantages and disadvantages, due to their inherent properties (i.e., ease of manipulation of the vector, type of nucleic acid contained in the genome, complexity of the virus, generation of products causing cell toxicity, size constraints, cell tropism, survival within host cells, risk of cell transformation, immune response generated). There is an enormous diversity of disease targets which are being considered for gene therapy and no single vector is suitable for all applications. The biology of each virus, it’s interactions with host cells and the host organism in it’s entirety, also the generation of the immune and inflammatory responses, are all factors that must be taken into account when considering a particular viral vector. Viral vectors have enormous potential for the delivery of therapeutic genes into diseased tissues. However, further development, especially regarding standardisation of methods and safety issues, will be required in many instances before gene therapy becomes commonplace. Early clinical trials using an adenoviral vector in patients with ornithine transcarbamylase (OTC) and X-linked SCID-XI syndrome, where patients suffered fatal or adverse effects have led to restricted use of gene therapy.8-17 Ethical issues are also worth mentioning, as gene therapy cannot be carried out on germ line cells, but is, by law, confined to the somatic cells of a patient.

Gene Therapy

Gene therapy is the introduction of DNA sequences to the cells of humans for therapeutic purposes.6 One of the original applications was the restoration of normal function in patients with inherited single gene defects e.g., cystic fibrosis.18 The rationale is that, by introducing a normal copy of the gene into the patient’s cells, the disease would thus be eliminated. The use of viral vectors has expanded and broadened enormously, with a variety of potential applications, particularly in the areas of cancer therapy, where the aim is to try to kill cancer cells, (or cause them to become apoptotic) but not normal cells, cardiovascular disease, neurodegenerative disorders and infectious diseases.8,19 There are many different ways to introduce DNA into target cells, but viruses represent a ‘natural’ vector system, as viruses are obligate intracellular parasites, existing solely for the purpose of introducing their nucleic acid into a suitable host cell. Many different viruses have been used as viral vectors (Retroviruses, Adenoviruses, Adeno-Associated Virus, Herpes Simplex virus, Vaccinia virus) for infecting vertebrates. The most successful viruses used to date are either DNA viruses or retroviruses (which include generation of a cDNA copy of the RNA genome during the replication cycle). These vectors will be subsequently discussed. However, many other viruses, including RNA viruses, bacteriophages, baculoviruses and plant viruses have also been used as vectors and this area is a rapidly growing area of virology research.

Viruses as Vectors

In order for gene therapy to succeed, the DNA has to enter the target cell. This involves crossing the cell membrane and entering the nucleus. Naked nucleic acid is susceptible to degradation within the host cell, thus ideally, should be protected. Using viral vectors circumvents these problems. Viruses are obligate intracellular parasites and are highly evolved vectors for the transfer of genetic

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Figure 1. Overview of a generalised strategy for generating recombinant viral vectors. The vector DNA contains the transgene (gene of interest) and the noncoding regions (NCR, including the packaging sequence, psi,ϕ). The helper DNA must contain the genes essential for viral replication that are absent in the vector e.g., DNA replication proteins and/or structural proteins. For small viruses e.g., retroviruses, most/all of the viral genome is replaced by the transgene, while the larger viruses e.g., HSV, will frequently have non-essential gene deletions (see text). The helper DNA can be provided by a helper virus, plasmid or can be stably integrated into the DNA of the packaging cell. It is sometimes integrated in the packaging cell line in two distinct segments, to minimize the risk of recombination with the vector (see Fig. 2 and text). The viral proteins required to replicate the vector DNA and assemble progeny virus particles are produced, the vector is thus replicated, amplified and packaged. The helper, which lacks the packaging sequence (ϕ) is not packaged. SP = structural proteins.

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Figure 2. Transduction of the target cell. The viral vector, containing the transgene, binds to the target cell, usually via binding of a viral protein to it’s cell surface receptor(s). The nucleic acid is uncoated, transcription and translation occurs, thus the therapeutic protein, introduced via the recombinant vector, is produced.

information into cells. They infect host cells, introduce their own nucleic acid into the infected host, with the sole purpose of producing new progeny virus particles. A recombinant virus, carrying a foreign gene (transgene), will thus be able to deliver the gene into the cell. An ideal viral vector will use the infection pathway but will not express viral genes that lead to replication and toxicity. No virus is perfect as a gene therapy vector and all of the viruses in common use have advantages and disadvantages. Generally, several features are necessary in a virus to enable its use as a vector.5,6 i. The recombinant virus must be safe to use in patients. In particular, the virus should not be able to replicate in the host. This usually involves rendering the virus replication-defective e.g., by deleting functions essential for replication (Fig. 1). The therapeutic gene must be delivered without a high level of toxicity or harmful side-effects. ii. The virus must recognise receptors on the target cell (Fig. 2). As different viruses infect different cell types, due to the presence/absence of the receptor, this will govern the suitability of the candidate vector for a particular cell type. Widespread dissemination of the

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vector is not desirable. Problems can also arise if a therapeutic gene is toxic or antigenic when expressed in an inappropriate tissue. iii. It should be possible to grow the recombinant virus to high titres in the laboratory. When viral vectors are produced and manipulated in vitro, this may result in inefficient packaging of vector genomes compared to wild type virus. iv. The virus should be able to deliver DNA to the nucleus. Thus, DNA viruses or viruses that use a cDNA intermediate, are potentially the most useful (and will be mainly discussed in this chapter). v. The recombinant virus should have sufficient space in its genome to accommodate the transgene.

Table 1. Major advantages and disadvantages of the vector systems discussed Viral Vector

Advantages

Disadvantages

Retrovirus (Oncoretroviruses) family retroviridae small, single stranded RNA virus.

Capable of integration, transgene passed to daughter cells. Suitable if stable integration in dividing cells is required. Can be pseudotyped to broaden host range and stabilize particles.

Small genome—size constraints. Danger of tumour formation (insertional mutagenesis). Growth to low titres. Only actively dividing cells infected. Prolonged expression difficult.

Lentivirus family retroviridae small, single stranded RNA virus.

Frequently pseudotyped. Some based on HIV. Persistent gene transfer. Biosafety increased by development of SIN vectors.

Small genome—size constraints.

Adenovirus family adenoviridae medium, double stranded DNA virus.

Can be grown to high titres. Broad host range. Infect dividing and nondividing cells. Low pathogenicity. Good expression of transgene. Low risk of integration into host DNA.

Early vectors—potential of recombination between vectors and E1 sequence. Toxicity. Transient expression. Broad host range—possibility of binding to cells which are not targets. Immune response develops.

Adeno-associated viruses family parvoviridae small, single stranded DNA virus.

Broad host range. Can transduce nondividing cells. Ease of viral manipulation. Low immunogenicity and toxicity. Possibility of pseudotyping capsids.

Small genome—size constraints. Difficult to scale up. Pre-existing immunity to AAVs.

Herpes virus family herpesviridae medium, double stranded DNA virus.

Large vector capacity. Can transduce both dividing and nondividing cells. Many grow to high titre. Life threatening disease rare and effective anti-HSV drugs available. Potential especially for neurons.

Little data re recombinant HVs in patients. Difficulties re long-term transgene expression in some tissues e.g., brain. Difficulties re vector targeting, as attachment and entry is complex.

Vaccinia virus family poxviridae large, double stranded DNA virus.

Extensive clinical knowledge available. Large vector capacity. Broad cell tropism. High level of transgene expression. Growth to high titres.

Not suitable for long-term gene delivery. Population generally immune due to immunisation programme for smallpox (future potential). Problems with immunodeficient patients.

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vi. If therapy is for the lifetime of the host, the transgene must persist in the cells and be inherited by daughter cells e.g., retroviral vectors, which can integrate safely into the chromosome. vii. The vector and/or its therapeutic gene products should not induce an immune response, or autoimmunity or break tolerance. Each viral vector system (Table 1) is characterised by properties that affect its suitability for specific applications. For some conditions e.g., genetic disorders, long term expression from a small proportion of cells would restore function, while in the case of cancer cells, gene transfer into a large number of affected cells would be necessary.

Making Recombinant Viruses

In order to generate a recombinant virus, the wild type or parental virus must be cloned.6,7 DNA viruses can be cloned directly, while, in the case of RNA viruses, cDNA must first be generated. The cloned nucleic acid can then be modified e.g., by removal of segments and replacement with foreign DNA (transgenes) of interest. Thus, in the case of smaller viruses, the whole genome can be cloned e.g., into a bacterial plasmid, or in the case of larger viruses, segments of the genome can be cloned. Subsequent generation of infectious virus particles is more difficult, with different techniques used for different types of virus. Some viruses (e.g., many RNA viruses) have proved very difficult to manipulate, thus more information exists concerning the use of DNA viruses and retroviruses (which generate a cDNA copy of the genome as part of the replication cycle). Generally, the vector contains the DNA of interest (transgene). The sequences (usually the terminal repeat sequences, TRs) required for cis functions e.g., packaging or integration of the vector, are also retained in the vector. As discussed, genes essential for viral replication have been deleted, to ensure that the vector does not replicate (Fig. 1). Thus, helper DNA must provide genes essential for viral replication (in trans). This can be in the form of a plasmid, helper virus or inserted into the host chromosomal DNA of the packaging cells. In some cases the helper DNA is split into different DNA molecules for safety reasons (e.g., to lessen the risk of recombination). The helper DNA lacks the packaging sequence (psi, ϕ), thus cannot be packaged into a viral particle. Thus, the viral replication proteins and viral structural proteins are produced by the helper. The viral cis-acting sequences, linked to the transgene, are introduced into the packaging cells, along with the helper, thus the vector genome is copied and replication-defective particles are produced. The viral structural proteins recognise the vector (psi positive) nucleic acid and only the vector genome is packaged. The recombinant vector particles are then purified and quantified. Issues and concerns at this stage are the high costs (some procedures are labour intensive, with low yields of vector), ease of scale up for large-scale industrial production, reduction of damage to the vector particles and titre and risk of contamination with defective particles. One problem with engineered vector systems is that the regulatory changes in the intact genome are interfered with e.g., interactions between the genome and translated products. This can result in e.g., inefficient packaging, defective vectors etc. There are many uses for recombinant viruses, not confined to their uses as viral vectors for gene therapy. These viruses can be used to study viral replication, different cell processes, as well as for viral vaccines. However, one of the most exciting areas in recent years has been the exploitation of recombinant viruses as gene therapy agents. The following viral vectors will be discussed—Retroviruses, Adenoviruses, Adeno-Associated Virus, Herpes Simplex virus, Vaccinia virus. These are sometimes classified into two groups—1. Those whose genomes integrate into host chromosomes (e.g., retroviruses and lentiviruses) and 2. Those that persist in the cell as extrachromosomal elements (e.g., Adenoviruses ).

Retroviral Vectors

Retroviruses are small, single-stranded, enveloped RNA viruses, which replicate by producing a DNA (cDNA) intermediate, due to the action of viral-encoded reverse transcriptase.20 Following

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Figure 3. Retroviral vectors and packaging system. The (wild-type or parental) retroviral genome codes for at least three genes—gag (group associated glycoprotein, coding for structural/core proteins), pol (polymerase i.e., reverse transcriptase) and env (coding for the viral envelope protein). Each end of the genome has long terminal repeats (LTRs, which encode promoter/enchancer regions and sequences involved with integration). Generally, in the retroviral vector, the viral genes are replaced by the gene of interest (transgene) and sometimes an exogenous promoter (*P). Some vectors use the retroviral promotor or enhancer sequence located in the 5’LTR. A *marker gene is frequently inserted downstream from the transgene. Viral proteins are supplied in trans, frequently from two separate constructs (gag-pol and env). The packaging sequence (ψ) is absent so that the constructs cannot be packaged and produce viable retrovirus. The ‘env’ gene can either encode parental envelope protein, or another retroviral protein or vesicular-stomatitis virus (VSV-G) to broaden the target cell population (‘pseudotyping’). The principle of vector construction with lentiviruses is similar to that used for retroviral vectors.

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infection, the retroviral genome is reverse transcribed into double stranded DNA (provirus), which is integrated into the host genome. The genome is transcribed and translated by the cell. The viral genome (Fig. 3) codes for at least three genes—gag (group associated glycoprotein, coding for structural/core proteins), pol (polymerase i.e., reverse transcriptase) and env (coding for the viral envelope protein). Each end of the genome has long terminal repeats (LTRs, which encode most of the cis-acting sequences—i.e., promoter/enchancer regions and sequences involved with integration) and the viral genes (gag-pol, encoding capsid proteins and env, encoding the viral glycoprotein) are transcribed using the LTR promoter and polyadenylation signal. There are sequences required for packaging the viral genome (ϕ) and RNA splice sites in the env gene.5,16 Some retroviruses (e.g., Rous sarcoma viruses, RSV) contain protooncogenes, which, when mutated, can cause cancers, but in the production of recombinant vectors, these are removed. However, due to the contribution made to the area of oncology by the study of retroviruses, these were among the earliest vectors studied. Retrovirus vectors were the first class of viral vector developed and have been extensively used in clinical trials.8 The most commonly used retroviral vectors are from murine leukaemia virus (Moloney murine leukaemia virus, Mo-MLV). Mo-MLV is an amphotrophic virus, capable of infecting murine and human cells, thus enabling development in mouse models and treatment of human cells. Retroviral vectors have all their normal coding sequences (gag, pol and env) removed and replaced with a cDNA (transgene) of interest. Up to 8 kb of exogenous DNA can be inserted and expressed in place of the viral genes (Fig. 3). The most commonly used method to produce recombinant virus is to grow the virus in a ‘packaging’ cell line, which is engineered to provide the viral proteins required to form particles containing the recombinant genome, but not expressing packageable helper viral RNA. Nowadays, in order to minimise the risk of recombination between the vector and packaging genomes, ‘split genome’ packaging cells lines, in which the gag-pol and env genes are expressed from separately transfected transcription units, neither of which include the packaging sequence, are used.5-8 The virus particles reverse-transcribe their genome and integrate the DNA copy (provirus) randomly into the host cell genome using enzymes (viral reverse transcriptase and integrase). However, as all viral genes are absent from the particle, no further events in the infectious cycle can occur. Thus, the transgene, which the vector has carried into the cell, is present for the life of the cell and will be passed on to progeny daughter cells at cell division. By replacing the env gene with that of another virus, the host range of the recombinant virus can be expanded. This technique is called pseudotyping e.g., vectors pseudotyped with the G glycoprotein of vesicular stomatitis virus (VSV-G), can infect most cells.8 To enable retroviruses to integrate and express viral genes, cell division in the target cells is necessary. Also, retroviral vectors generally grow only to relatively low concentrations. These factors limit use of gene therapy to proliferating cells in vivo or ex-vivo, where cells are removed from the patient, replication is stimulated and the cells are transduced with the retroviral vector, then reimplanted. Thus, these vectors can be used to treat cells of the lymphoid system and haematopoietic cells, as these cells can be explanted and subsequently reimplanted.6,7 Also, integration is required, as lymphoid cells are constantly being replaced with progenitor cells. Two inherited immunodeficiencies, both of which result in a lack of T-lymphocytes and thus a failure of both the humoral and cell mediated immune response, have been treated with retrovirus-mediated gene therapy; The first human gene therapy involved removal of peripheral blood cells from children with adenosine deaminase (ADA) deficiency, in vitro transduction with a retroviral vector encoding ADA and reintroduction of these cells into the children. Another example is X-linked severe combined immunodeficiency (X-SCID).9 Here, the problem is mutation in the gene encoding the γ-subunit of several cytokine receptors. Haemopoietic stem cells were transduced with a retroviral vector containing the γ subunit gene. In both examples, there was evidence of clinical improvement in the patients, but in the case of X-SCID patients, some children developed T-cell proliferative disease, due to the ability of retroviruses to integrate near protoncogenes and subsequently effect the expression of host growth control genes.8,13-16 Thus, the safety of retroviral vectors needs to be improved.17

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Figure 4. Adenovirus vectors. The adenovirus genome can be divided into Immediate early (E1A), early (E1B, E2A, E2B, E3, E4), Intermediate (IX, Iva2) and Late (L1-L5) genes. There are four early (E) transcriptional units (E1, E2, E3 and E4), which have regulatory functions and a later (L) transcript, which codes for structural proteins. Early (first generation) vectors had a deletion in either E1 or E3, which made these vectors replication-deficient under noncomplementing conditions. The deleted gene was supplied in trans by either a helper virus, plasmid, or integrated into a helper cell genome. Later, ‘second generation’ vectors used an E2A temperature sensitive mutant or an E4 deletion, in addition to E1 and E3. The most recent ‘gutless’ (‘third generation’) vectors contain only the inverted repeats, in order to increase the capacity of the vector and to decrease the host immune response. The cis-acting elements required for viral replication and packaging sequence are the only elements remaining. The necessary genes are provided by a helper (intact) virus.

Lentiviral Vectors

A major disadvantage of early retroviral vectors is that they are not capable of replication in nonproliferating cells. Lentiviruses are a subclass of retroviruses (with a slightly more complex genome), which are able to infect both proliferating and nonproliferating cells, therefore, gene therapy based on lentivirus systems could overcome this problem. Early results with vectors based on HIV-1 have demonstrated the ability to infect neurons.7 The principle of vector construction is similar to that used for retroviral vectors, but there has been some success in broadening the target cells by incorporating other viral glycoproteins (instead of env) into the packaging cell line. This process is called ‘pseudotyping’ e.g., VSV-G pseudotyped retroviral vectors, where the G glycoprotein of vesicular stomatitis virus was used to produce murine leukaemia-derived vectors and the resulting recombinant vectors were able to infect a wider range of cell types than the paternal virus, due to the presence of the replaced envelope glycoprotein.5,8 Recent reports where equine infectious anaemia virus (EIAV) has been pseudotyped with rhabdoviruses and LCMV, injected into rodents and their transduction patterns analysed, suggest that this approach has potential for designing therapeutic strategies for neurological diseases e.g., Parkinson’s disease.21 Vector safety has also been increased by the development of self-inactinating (SIN) vectors, containing deletions of the regulatory elements in the downstream LTR—thus the risk of recombination is reduced.22

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Adenoviruses (Ad) are medium-sized, non-enveloped, double stranded DNA viruses.23 There are over 50 Adenovirus serotypes (classified into 6 subgroups, A-F), but the serotype 5, a mild respiratory pathogen, is most commonly used as a vector.23,24 The life cycle does not normally involve integration into the host genome, but the viruses replicate as episomal (i.e., extrachromosomal) elements in the nucleus of the host cell. Adenoviruses encode a larger number of proteins than retroviruses—approximately 35 kb, of which up to 30 kb can be replaced with foreign DNA. The genome (Fig. 4) can be divided into Immediate early (E1A), early (E1B, E2A, E2B, E3, E4), Intermediate (IX, Iva2) and Late (L1-L5) genes. The early (E) transcriptional units (E1, E2, E3 and E4) have regulatory functions and the later (L) transcript, which codes for structural proteins, is expressed following viral DNA synthesis.5,6,23 Ad vectors have been used in many clinical studies, including trials involving treatment of monogenic diseases, vaccines and cancer. The strategies used to generate AV vectors are reviewed by Shen and Post.5 Early (first generation) vectors had a deletion in either E1 or E3, (where the transgene was inserted) which made these vectors replication-deficient under noncomplementing conditions (Fig. 3). The deleted gene was supplied in trans by either a helper virus, plasmid, or integrated into a helper cell genome. Problems with these early vector systems arose because cells, which were transduced with these vectors, expressed other adenoviral genes at low levels and induced strong cytotoxic T-cell responses, thereby eliminating transgene expression.5,7,8 Later, ‘second generation’ vectors used an E2A temperature sensitive mutant or an E4 deletion, in addition to E1 and E3. The most recent ‘gutless’ (‘third generation’) vectors contain only the inverted repeats, in order to increase the capacity of the vector and to decrease the host immune response. The cis-acting elements required for viral replication and packaging sequence are the only elements remaining. The necessary genes are provided by a helper (intact) virus. Second and third generation vectors that contain additional deletions in other early genes have shown reduced toxicity in animal models.25-27 Due to the large size of this virus (compared to retroviral vectors), it is not yet possible to make packaging cells that will allow the growth of vectors where all of the viral genes have been deleted (‘gutless vectors’). Most vectors used to date have the majority of their viral genes retained to enable them to grow in cells that complement their gene deficiencies e.g., a vector lacking E1A and E1B genes, which encode proteins required for the expression of other viral genes and E3, which encodes several proteins not required for growth in vitro but which modulate the host response to infection. High titres of virus particles can be produced, suitable for direct use in humans. The major disadvantages with these vectors are the development of an immune response, eliminating cells that have acquired the transgene, also contamination with the helper viruses.28,29 The immune response is generated, in part, due to the expression of some viral genes remaining on the vector, but this is an advantage in some areas e.g., in the treatment of vascular and coronary artery disease, where transient expression is an advantage, also in the area of cancer gene therapy, which involves targeting the virus to cancer cells, and immunogenicity could enhance anti-tumour effects.30-32 For other areas, transient immunosuppressive therapies or induction of tolerance have been attempted. The development of ‘gutless’ vectors, containing no viral sequences, has resulted in more prolonged expression, but may still cause problems to due the immune response to the ‘stuffer’ DNA. Other approaches include using different serotypes or nonhuman adenoviruses to avoid problems with pre-existing immunity.7 Adenoviral vectors have been used for the treatment of cystic fibrosis (CF), where the cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered to CF patients. Complications arose in early studies with first generation Ad vectors due to inflammatory responses.33,34 Subsequent trials using second generation and gutless vectors have had some success in animal models, but long term expression of Ad vectors in dividing cells is not possible due to the immune response generated. Another application is the use of Ad vectors as vaccine delivery vectors. The vigorous immune response to Ad is an advantage when using Ad vectors as vaccine

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Figure 5. Adeno-associated virus (AAV) vectors and packaging system. The Adeno-associated virus (AAV) genome consists of two genes; rep, coding for proteins which control viral replication, structural gene expression and integration into the host genome and cap, which codes for capsid structural proteins. At either end of the genome are inverted terminal repeats (TRs), which play a role in integration and rescue of the AAV genome. When used as a vector, the rep and cap genes are replaced by the transgene, its associated regulatory sequences and a poly A site. Production of the recombinant vector requires that rep and cap are provided in trans, along with helper virus products (from the adenovirus genome). One method is to cotransfect two plasmids, one for the vector and the second for rep and cap, but this method has drawbacks (see text). More modern methods remove all adenovirus structural genes and use rep resistant plasmids or conjugate a rep expression plasmid to the mature virus prior to infection.

delivery vectors, however and several investigators have reported success in the generation of immunity against HIV, Ebola virus and Plasmodium.35-37 The use of adenoviral (as well as many other viral) vectors for cancer therapy, where the ultimate aim is to bring about the death of the cell, is being explored.38-41 Two approaches are being examined. The first involves deletion of genes necessary for replication in healthy cells but not necessary for neoplastic cells (e.g., ONYX-015, which replicates in and kills a broad range of cancer cells, but with reduced replication in normal cells).5,38 The second approach involves placing viral genes essential for replication under the control of promoters preferentially expressed in tumours (e.g., CV706, where expression of E1A is controlled by prostate-specific antigen (PSA) gene promoter and thus will replicate only in prostate cancer cells).5,38 Ad vectors are being used as vehicles for the delivery of tumour-specific anti-cancer genes. Current strategies to improve Ad vectors involve using transductional targeting to redirect the virus to a novel receptor, genetic modification to increase tropism and use of alternative serotypes, including nonhuman Ad.38 Again, safety issues arise e.g., due to toxicity and host immune response.

Adeno-Associated Viral Vectors

Adeno-associated viruses (AAVs) are small, non-enveloped, single stranded DNA viruses, belonging to the parvovirus family.42 Most AAV vectors are derived from AAV2 but different AAV serotypes, capable of infecting different cell types have been identified.43 This is useful, as pseudotyping AAV2 with capsids from other serotypes can be carried out. AAVs are dependent

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on a helper virus, usually adenovirus (or herpesvirus) to replicate. In the absence of a helper virus, AAVs integrate into (a specific location in) the genome, establishing a latent infection. The DNA genome consists of two genes; rep, coding for proteins which control viral replication, structural gene expression and integration into the host genome and cap, which codes for capsid structural proteins. At either end of the genome are inverted terminal repeats (ITRs/TRs), which play a role in integration and rescue of the AAV genome and are required in cis for viral DNA replication and encapsidation. When used as a vector, the rep and cap genes are replaced by the transgene and its associated regulatory sequences (Fig. 5). Production of the recombinant vector requires that rep and cap are provided in trans, along with helper virus products (from the adenovirus genome). One method is to cotransfect two plasmids, one for the vector and the second for rep and cap, but this method has drawbacks—low yields, contamination with adenovirus and wild type AAV. More modern methods remove all adenovirus structural genes and use rep resistant plasmids or conjugate a rep expression plasmid to the mature virus prior to infection.42,44 However, this method is labour intensive. Another approach is to use hybrid helper viruses, which provide helper virus functions, expression of Rep and Cap and delivery of rAAV DNA.5,45,46 AAV is defective in the absence of a helper virus and is noncytotoxic, evokes a mild immune response and is nonpathogenic. All of these features make AAV a desirable vector. It is possible to administer high (and repeated) doses of vector, without eliciting acute inflammatory responses or toxic side effects. AAVs can infect a broad range of cell types, including nondividing cells and it’s genome is easily manipulated.5,7,47 However, due to the small genome, size constraints exist, which is a major limitation. There have been several reports of safe and long-term transgene expression in animal models when rAAVs espressing the CFTR, Blood Clotting Factor IX (FIX) were studied.48-50 Clinical studies with CF and haemophila B patients, receiving CFTR- and FIX—AAVs showed

Figure 6. Categories of herpesivral (HSV) vectors. 1. Replicating HSV (or replication-conditional) vectors are made by replacing non-essential genes with foreign genes. Their replication is restricted to certain cell types e.g., HSV vectors with mutations or deletions in genes encoding thymidine kinase. 2. Nonreplicating HSV vectors are those where deletion of some of the (essential) IE genes prevents the lytic cycle from occurring. Other IE proteins, which can be toxic to host cells, are also deleted but the resultant viruses can be difficult to grow, thus complementation of the essential and nonessential genes is required. 3. Amplicon vectors are similar to gutless adenovirus vectors. Only the essential cis-acting DNA is in the vector. The transgene cassette is placed in a plasmid which contains the viral packaging or cleavage sequence (pac) and origin of replication (Ori-s). The essential viral gene functions must be provided in trans, via a helper virus genome—but this can result in contamination with replication-competent virus or cytotoxic virus particles (see text).

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some promise, but problems with transient expression, toxicity, immune response, suggests that AAV vector development continue.51,52

Herpes Simplex Viral Vectors

Herpes viruses are a large family of enveloped, double stranded DNA viruses.53 A key feature of the Herpes virus family is their ability to persist, in a latent state, without causing disease, following primary infection. The viruses have a broad host range and the large genome size also lends itself to a greater vector capacity than many other viral vectors. The prototype is herpes simplex virus type 1 (HSV-1), which has been used as a vector for gene delivery, but gammaherpesviruses, including Epstein Barr virus and herpes saimiri have also been utilised as vectors.5 Non-essential genes frequently encode functions involved in virus-host interactions in vivo and may be deleted in the generation of vectors, enabling the delivery of large transgenes (up to 30 kb). HSV-1 has a broad host range but many applications have focused on it’s use as a vector for gene transfer to the nervous system. The life cycle can be lytic or latent, where the virus persists for the lifetime of the host. Latently infected neurons do not activate the immune response and, although the latent virus is not transcriptionally very active, it possesses neuron specific promoters, which are capable of functioning during latency.5,53 The viral genome contains two unique sequences, long and short (UL and US), which are linked in either orientation by internal repeat sequences (IRL and IRS). At the nonlinker end of the unique regions are terminal repeats (TRL and TRS). There are about 90 genes and approximately half of these are not essential for replication in vitro. Thus, non-essential genes can be deleted and about 40-50 kb of foreign DNA can be packaged in the viral vector. There are 3 main classes of HSV-1 genes—immediate-early (IE or alpha) genes, early (E or beta) genes and late (L or gamma) genes. Following lytic infection, transcription of IE genes commences, allowing production of early genes, responsible for DNA replication. The products of these genes, in turn, activate late genes, leading to the production and release of progeny virus and cell lysis. Alternatively, the genome may enter a latent state, where the genome persists as a stable episomal element. Only a set of nontranslated RNA species, latency associated transcripts (LATs), are synthesised during latency. LATS play a role in establishment of, and reactivation from, latency.5,53 Similar to adenovirus, HSV DNA is infectious. There are three main of HSV vectors (Fig. 6)—(1) replicating HSV vectors, (2) nonreplicating HSV vectors, (1 and 2 are sometimes referred to as recombinant HSV-2 viruses) and (3) amplicon vectors. Replicating HSV vectors are made by replacing non-essential genes with foreign genes. These vectors are often referred to as replication—conditional vectors as their replication is restricted to certain cell types e.g., HSV vectors with mutations or deletions in genes encoding thymidine kinase.54 Nonreplicating HSV vectors are those where deletion of some of the (essential) IE genes prevents the lytic cycle from occurring. Other IE proteins, which can be toxic to host cells, are also deleted, but the resultant viruses can be difficult to grow, thus complementation of the essential and nonessential genes is required.55,56 Amplicon vectors are similar to gutless adenovirus vectors. Only the essential cis-acting DNA is in the vector. The transgene cassette is placed in a plasmid, which contains the viral packaging or cleavage sequence (pac) and origin of replication (Ori-s). The essential viral gene functions must be provided in trans, via a helper virus genome—but this can result in contamination with replication-competent virus or cytotoxic virus particles. There are solutions to this problem are e.g., use of a helper virus in which the genome packaging sequence is flanked by loxP sites.57 When such a virus infects Cre-expressing cells with an amplicon, both the helper and amplicon are replicated, but Cre-loxP-mediated deletion of the packaging sequence from the helper virus results in only the amplicon being packaged.57 HSV has a natural tropism for the nervous system, which raises the possibility of using HSV vectors to treat neuropathological disorders (e.g., Parkinson’s disease).58 Another application is its use as an oncolytic virus (for a review see ref. 33). Vectors have been tested for tumor-specific delivery of antineoplastic gene products e.g., angiogenesis inhibitors. Today, many HSV vectors

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(e.g., OncoVEX and the HF10 vector), which have been deleted for the main neurovirulence gene (γ34.5) and contain other modifications to avoid generation of WT-HSV and restore replication efficiency, are undergoing clinical trials.33,59 Human vaccination does not exist but several live animal herpesvirus vaccines (e.g., using Pseudorabies and equineherpes viruses as vectors) have been explored.61-63 It is also worth mentioning the use of gammaherpesviruses, particularly Epstein-Barr virus (EBV), which is related to, but quite different from the HSVs. This virus can cause a latent infection in B-cells. Vectors derived from EBV were capable of delivering foreign DNA to B-cells.64 More recently, hybrid vectors, capable of transforming mammalian cells (see future developments) have been constructed.65,66

Vaccinia Viral Vectors

Vaccinia virus (VV) is a member of the poxvirus family and is a large, double stranded DNA virus.67 It is a complex virus, encoding a large number of proteins, including enzymes necessary for transcription and replication and is practically independent of the host cellular machinery, replicating (unusually for DNA viruses) in the cytoplasm of the infected cell. VV has been used as a vaccine for smallpox for many years and the virus is very well studied, thus, although other poxviruses have also been used as vectors, vaccinia is the most extensively utilised.5,67,68 VV can infect many mammalian cells types. An efficient method of introducing transgenes is homologous recombination between viral DNA and cotransfected plasmids. The most common site of integration is the TK gene. Insertion therefore eliminates TK function, thus the virus is attenuated in normal tissues in vivo.69 As VV contains its own specific polymerase, it is necessary to use promoters of poxvirus origin for gene expression. Uses of recombinant VV vectors include protein expression in cultured mammalian cells, vaccine carriers and oncolytic agents. Genetically altered VV vectors that do not replicate in mammalian cells but retain the capacity for high level expression of transgenes exist. Another, well utilised approach is the transfection of plasmids containing the transgene, followed by infection with inactivated VV. Vaccinia virus has been modified to express antigens from several infectious agents e.g., rabies.70 This vaccine has been used to immunise wild animals against rabies, resulting in a reduced incidence of rabies.70 Vaccinia has also been engineered to express tumor antigens, with success in both animal models and clinical trials. Antitumor activities have been demonstrated in VV expressing antitumor antigens (e.g., melanoma-associated antigens, also when combinations of tumor antigens and immunoregulatory molecules e.g., cytokines were used.71,72 However, safety issues exist, esp in infants and immunosuppressed individuals.73 As regards use as an oncolytic agent, the observations that VV replicates, lyses and spreads well in tumors make it an attractive prospect. The Western Reserve (WR) strain of VV has been used as an oncolytic virus.74 The strategy used (similar to that for HSVs) is to delete gene functions critical for viral replication in normal cells but not in tumor cells.71 Thus, VV have potential as delivery vectors for therapeutic gene products e.g., antiangiogenesis factors.

RNA Viral Vectors

RNA virus vectors are not used as extensively as DNA virus vectors and one important safety consideration is their high mutaton rate, but recently, some advances have been made.5,75 Advantages of RNA viruses in general, include the relative simplicity of many RNA viruses and the fact that replication occurs in the cytoplasm of the infected cell, without a DNA intermediate, thus reducing the risk of cell transformation. Positive sense, single stranded RNA viruses used include the alphaviruses—Sindbis, Semliki Forest and Venezuelan equine encephalitis (VEE) viruses.5,75,76 Some studies have also been carried out using poliovirus and coronavirus.77,78 In the alphavirus vectors, the structural proteins are replaced with the transgene and helper virus is used to enable vector genomic packaging to occur.5,75 Negative strand RNA viral vectors include Newcastle Disease Virus, a paramyxovirus. The PV701 strain has been reported to cause clinical regression of tumor xenografts following intravenous administration.79 Measles virus, another paramyxvirus, has potential as an oncolytic

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agent, while Sendai virus and parainfluenza virus have been used as vectors.5 Influenza virus, an orthomyxovirus has potential for use as an oncolytic vector. Rhabdoviruses, e.g., VSV has potential as an oncolytic agent, while rabies virus has also been developed as a vector. Human reovirus type 3, which is a double stranded RNA virus has been used as an oncolytic virus.80-82

Nonviral Methods of DNA Transfer

Viruses are natural vectors for the transfer of DNA into cells but they have several disadvantages. Due to their small genome size (in comparison to a eukaryotic cell), their capacity is limited. Also, viruses will generate an immune response, which may interfere with vectors which are required to be administered repeatedly. There is also the issue of safety, due to e.g., toxicity and the risk of insertional mutagenesis. Thus, nonviral methods of DNA transfer are also being explored and merit a brief discussion. These methods require a small number of proteins, will not be either infectious or capable of insertional mutagenesis, but can also be used for large scale production. There are three methods of nonviral DNA transfer—naked DNA, liposomes and molecular conjugates. Naked DNA (in the form of a plasmid) has been injected into cells or attached to gold particles and ‘shot’ into the tissue. This has led to the development of DNA vaccines which are useful where there is currently no existing vaccine or where the current vaccine is not effective. Liposomes are lipid bilayers, containing associated DNA, which can interact with the cell membrane, thus gaining entry into the cell. Molecular conjugates consist of protein or synthetic ligands to which a DNA binding agent has been attached.

Comparison of Vectors

There are very few studies comparing different delivery systems. The ideal vector should have a relatively simple preparation, result in high vector concentrations, express the transgene efficiently for the desired length of time, with no significant host immune or imflammatory response. The absence of an immune response would allow transgene expression to be prolonged, avoiding readministration. Another alternative is integration into the host, ensuring that the transgene is not lost. None of the vectors discussed have all of these requirements, but the systems are being improved and the weaknesses/disadvantages in each system addressed e.g., the genome sizes of the vectors are being reduced to contain only the transgene and essential genes. Table 1 outlines the major advantages and disadvantages with the main vector systems discussed above.

Strategies for Gene Therapy

Gene therapy can be applied ex vivo or in vivo. A disadvantage with ex vivo is that surgery is required both to obtain and replace cells. For some diseases, a particular organ is affected and must be treated e.g., cystic fibrosis in the lungs. Other diseases are systemic e.g., haemophilia and for these types of diseases, specific sites for therapy e.g., liver, gut, muscle are chosen for ease of access, size and metabolic activity. Many gene therapy treatments exist for cancer.3,4,33 All cancers involve acquired mutations and generally, as cancers progress, they accumulate more mutations and become less differentiated. Generally, cancers have at least one mutation changing a protoocogene to an oncogene and another to a tumor suppressor gene, which allows uncontrolled proliferation. Thus, a range of cancers and mutations exist, facilitating different strategies for gene therapy—e.g., immunopotentiation (to enhance the immune response to cancers), oncogene inactivation (frequently the approach used is to inactivate the oncogene promoter or to prevent transport and translation of the oncogene mRNA), tumor suppressor gene replacement (with the aim of causing apoptosis and preventing further tumor growth), molecular chemotherapy (which involves the transduction of the gene coding for a toxic product) and drug resistance genes. Two main approaches exist. One involves the use of replication-defective vectors to deliver e.g., anti-angiogenic factors, tumor suppressor genes. The other approach is to use oncolytic viral vectors to find and destroy malignant cells. Viruses induce changes in cellular metabolism e.g., p53

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inactivation. Vectors can have genes that are necessary for replication in normal cells deleted, thus the vector can replicate only in tumor cells. Several such viruses have been developed.5,8,33

Future Development of Viral Vectors for Gene Therapy

The insertion of a transgene into the vector may give rise to potential adverse effects, dependant on the transgene, the type of vector used and the host involved. These effects include the residual virulence of vector, the effect of the inserted sequence—toxic effects of the novel protein and the risk of autoimmune disease (AID) if self antigen is being encoded by the transgene. Generally, if a choice of vectors is available, the vector with the most restricted host range should be choosen, to prevent accidental spread of the vector. Other risks include that of recombination between the vector and related viruses present in the host, immune and inflammatory responses, cell transformation, particularly with oncogenic viruses and pre-existing immunity against the vector. Improving the safety of the virus vectors is a crucial area and vectors are constantly being improved upon, e.g., development of site-specific integrating vectors. The use of new techniques e.g., RNA interference to silence genes will possible be useful in combination with viral vectors to develop therapies for several diseases.8 A major limitation in gene therapy is the fact that it is not possible to deliver genes to specific target cell populations. The ideal vector should attach to and enter the target cells only. This is an area of ongoing investigation via techniques e.g., optimization of tissue-specific promoters, genetic modification of the proteins involved in binding to the host cell receptor, use of different virus serotypes and the use of strategies to retarget receptor-ligand binding to broaden the host cell range.5,8,40,41 The control of the immune (and inflammatory) response is also important, particularly when treating disease, as responses can be generated against both vector and products. As outlined, adenovirus vectors are strong inducers of cytotoxic T-lymphocyte (CTL) responses, where responses against both viral and transgene products are elicited. The capsid can also evoke humoral and cytokine-mediated responses. Advances have been made in the area of creation of new systems for less immunogenic vectors.8 Recently, blends of vectors (hybrid vectors), in order to combine properties of both vectors have been attempted.45 Examples of these hybrid vectors include AV and AAV vectors and retroviral genomes contained within adenovirus.46 This, more novel approach, needs further characterisation. It is likely that combinations of different viruses, novel viruses and nonviral methods of DNA transfer will continue to be investigated in the future.5,7,8 A huge number of current gene-therapy trials are for cancer.8 Replication-defective vectors are used to deliver anti-angiogenic factors, tumor suppressor genes, immunostimulatory genes etc.8 Viruses are also used to replicate in and lyse cancer cells.33 Viral vectors expressing antiangiogenic factors are also being used to treat vascular and coronary artery disease.8

Conclusions

The ideal viral vector should be safe, efficiently delivered by non-invasive routes, have no toxic effects, elicit no immune response, target only the necessary cells, express the transgene in a regulated fashion, for the required amount of time. This scenario is still a long way in the future.

Acknowlegements

I thank Maria Karcz Marczyk, of the Centre for Food Safety, NUI, Dublin, for assistance with literature searches. I thank Catherine Dawson, of the Department of Biological Sciences, CIT, for practical, artistic and secretarial assistance in the preparation of this chapter.

References

1. Sleator R, Hill C. Patho-biotechnology: Using bad bugs to do good things. Curr Opin Biotechnol 2006; 17:211-216. 2. Sleator R, Hill C. Patho-biotechnology: Using bad bugs to make good bugs better. Science Prog 2007; 90(1):1-14.

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3. Sleator R, Hill C. Improving probiotic function using a patho-biotechnology approach. Gene Ther Mol Biol 2007; 11:269-274. 4. Sleator R, Hill C. Probiotics as therapeutics for the developing world. J Infect Develop Count 2007; 1(1):7-12. 5. Shen Y, Post L. Viral vectors and their applications. In: Knipe D, Howley P, eds. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:539-604. 6. Dimmock N, Easton A, Leppard K. Horizons in human virology. In: Dimmock N, Easton A, Leppard K, eds. Introduction to Modern Virology. 6th ed. Oxford: Blackwell, 2007:422-428. 7. Kay M, Glorioso J, Naldini L. Viral vectors for gene therapy: The art of turning infectious agents into vehicles of therapeutics. Nat Med 2001; 7:33-41. 8. Thomas C, Ehrhardt A, Kay M. Progress and problems with the use of viral vectors for gene therapy. Nature 2003; 4:346-357. 9. National Institutes of Health Recombinant DNA Advisory Committee. Assessment of adenoviral vector safety and toxicity. Human Gene Ther 2002; 13:3-13. 10. Marshall E. Gene therapy death prompts review of adenovirus vector. Science 1999; 286:2244-2245. 11. Schnell MA, Zhang Y, Tazelaar J et al. Activation of innate immunity in nonhuman primates following intraportal administration of adenoviral vectors. Mol Ther 2001; 3:708-722. 12. Bostanci A. Blood test flags agent in death of Penn subject. Science 2002; 295:604-605. 13. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288:669-672. 14. Hacein-Bey-Abina S, von Kalle C, Schmidt M et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348:255-256. 15. Check E. Gene therapy: A tragic setback. Nature 2002; 420:116-118. 16. Kaiser J. Seeking the cause of induced leukaemias in X-SCID trial. Science 2003; 299:457-607. 17. Check E. Cancer risk prompts US to curb gene therapy. Nature 2003; 420:116-118. 18. Kay MA, Woo SL. Gene therapy for metabolic disorders. Trends Genet 1994; 10:253-257. 19. Lowenstein P. Why are we doing so much cancer gene therapy? Disentangling the scientific basis from the origins of gene therapy. Gene Ther 1997; 4:755-756. 20. Goff S. Retroviridae: The retroviruses and their replication. In: Knipe D, Howley P, eds. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:1999-2070. 21. Kordower JH, Emborg ME, Bloch J et al. Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 2000; 290:767-773. 22. Zufferey R, Dull T, Mandel RJ et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998; 72:9873-9880. 23. Shenk T. Adenoviridae: The viruses and their replication. In: Knipe D, Howley P, eds. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:2355-2394. 24. Lennon G, Cashman O, Lane K et al. Prevalence and characterisation of enteric adenoviruses in the south of Ireland J Med Virol 2007; 79:1518-1426. 25. Lusky M, Christ M, Rittner K et al. In vitro and in vivo biology of recombinant adenovirus vectors with E1, E1/E2A, or E1/E4 deleted. J Virol 1998; 72:2022-2032. 26. O’Neal WK, Zhou H, Morral N et al. Toxicological comparison of E2a-deleted antitrypsin after systemic delivery. Hum Gene Ther 1998; 9:1587-1598. 27. Andrews J, Kadan M, Gorziglia M et al. Generation and characterisation of E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII. Mol Ther 2001; 3:329-336. 28. Lozier JN, Csako G, Mondoro TH et al. Toxicity of a first generation adenoviral vector in rhesus macaques. Hum Gene Ther 2002; 13:113-124. 29. Zhang Y, Chirmule N, Gao GP et al. Acute cytokine response to systemic adenoviral vectors in mice is mediated by dendritic cells and macrophages. Mol Ther 2001; 3:697-707. 30. Isner JM. Myocardial gene therapy. Nature 2002; 415:234-239. 31. Alemany R, Balague C, Curiel DT. Replicative adenoviruses for cancer therapy. Nature Biotechnol 2000; 18:723-727. 32. Isner J, Asahara T. Angiogenesis and vasculogenesis as therapeutic strategies for postnatal neovascularisation. J Clin Invest 1999; 103:1231-1236. 33. Crystal R, Jaffe A, Brody S et al. A phase 1 study, in cystic fibrosis patients, of the safety, toxicity and biological efficacy of a single administration of a replication deficient recombinant adenovirus carrying the cDNA of the normal cystic fibrosis transmembrane conductance regulator gene in the lung. Hum Gene Ther 1995; 6:643-666. 34. Crystal R, Mastrangeli A, Sanders A et al. Evaluation of repeat administration of a replication deficient, recombinant adenovirus containing the normal cystic fibrosis transmembrane conductance regulator cDNA to the airways of individuals with cystic fibrosis. Hum Gene Ther 1995; 6:667-703.

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35. Bruna-Romero O, Gonzalez-Aseguinolaza G, Hafalla JC et al. Complete, long-lasting protection against malaria of mice primed and boosted with two distinct viral vectors expressing the same plasmodial antigen. Proc Natl Acad Sci USA 2001; 98:11491-11496. 36. Shiver JW, Fu TM, Chen L et al. Replication-incompetent adenoviral vaccine vector elicits effective anti-immunodeficiency virus immunity. Nature 2002; 415:331-335. 37. Sullivan NJ, Sanchez A, Rollin PE et al. Development of a preventive vaccine for Ebola virus infection in primates. Nature 2000; 408:605-609. 38. Vaha-Koskela M, Heikkila J, Hinkkanen A. Oncolytic viruses in cancer therapy. Cancer Lett 2007; 254:178-216. 39. Dobbelstein M. Replicating adenoviruses in cancer therapy. Curr Top Microbiol Immunol 2004; 273:291-334. 40. Everts M, Curiel DT. Transductional targeting of adenoviral cancer gene therapy. Curr Gene Ther 2005; 12:141-161. 41. Havenga MJ, Lemckert AA, Ophorst OJ et al. Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease. J Virol 2002; 76:4612-4620. 42. Berns K Parrish C. Parvoviridae. In: Knipe D, Howley P, eds. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:2437-2478. 43. Lu Y. Recombinant adeno-associated virus as delivery vector for gene therapy—A review. Stem Cells Dev 2004; 13:133-145. 44. Xiao X, Li J, Samulski RJ. Production of high-titer recombinant adeno-assoicated virus vectors in the absence of helper adenovirus. J Virol 1998; 72:2224-2232. 45. Lieber A, Steinwaerder DS, Carlson CA et al. Integrating adenovirus-adeno-associated virus hybrid vectors devoid of all viral genes. J Virol 1999; 73:9314-9324. 46. Recchia A, Parks RJ, Lamartina S et al. Site-specific integration mediated by a hybrid adenovirus/ adeno-associated virus vector. Proc Natl Acad Sci USA 2000; 96:2615-2620. 47. Zaiss AK, Muruve DA. Immune responses to adeno-associated virus vectors. Curr Gene Ther 2005; 5:323-331. 48. Conrad CK, Allen SS, Afione SA et al. Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Ther 1996; 3:658-668. 49. Herzog RW, Yang EY, Couto LB et al. Long-term correction of canine haemophilia B by gene transfer of blood coagulation factor IX mediated by adeno-associated viral vector. Nat Med 1999; 5:56-63. 50. Snyder RO, Miao C, Meuse L et al. Correction of haemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med 1999; 5:64-70. 51. Aitken ML, Moss RB, Waltz DA et al. A phase I study using aerosolised administration of tgAAVCF to cystic fibrosis subjects with mild lung disease. Hum Gene Ther 2001; 12:1907-1916. 52. Manno CS, Chew AJ, Hutchison S et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe haemophilia B. Blood 2003; 101:2963-2972. 53. Pellet P Roizman B. The family: Herpesviridae. A brief introduction In: Knipe D, Howley P, eds. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:2479-2500. 54. Lou E. Oncolytic herpes viruses as a potential mechanism for cancer therapy. Acta Oncol 2003; 42:660-671. 55. Krisky DM, Wolfe D, Goins WF et al. Deletion of multiple immediate-early genes from herpes simplex virus reduces cytotoxicity and permits long-term gene expression in neurons. Gene Ther 1998; 5:1593-1603. 56. Samaniego LA, Neiderhiser L, DeLuca NA. Persistence and expression of the herpes simplex virus genome in the absence of immediate-early proteins. J Virol 1998; 72:3307-3320. 57. Oehmig A, Fraefel C, Breakefield XO. Update on herpes virus amplicon vectors. Mol Ther 2004; 10:630-643. 58. Song CK, Enquist LW, Bartness TJ. New developments in tracing neural circuits with herpesviruses. Virus Res 2005; 111:235-249. 59. Varghese S, Rabkin SD. Oncolytic herpes simplex virus vectors for cancer virotherapy. Cancer Gene Ther 2002; 9:967-978. 60. Nakao A, Kimata H, Imai T et al. Intratumoral injection of herpes simplex virus HF10 in recurrent breast cancer. Ann Oncol 2004; 15:988-989. 61. van Zijl M, Wensvoort G, de Kluyver E et al. Live attenuated pseudorabies virus expressing envelope glycoprotein E1 of hog cholera virus protects swine against both pseudorabies and hog cholera. J Virol 1991; 65:2761-2765. 62. Trapp S, von Einem J, Hofmann H et al. Potential of equine herpesvirus 1 as a vector for immunisation. J Virol 2005; 79:5445-5454. 63. Whitehouse A. Herpesvirus saimiri: A potential gene delivery vector [review]. Int J Mol Med 2003; 11:139-148.

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64. White RE, Wade-Martins R, James MR. Infectious delivery of 120-kilobase genomic DNA by an Epstein-Barr virus amplicon vector. Mol Ther 2002; 5:427-435. 65. Dorigo O, Gil JS, Gallaher SD et al. Development of a novel helper-dependent adenovirus-Epstein-Barr vvirus hybrid system for the stable stransformation of mammalian cells. J Virol 2004; 78:6556-6566. 66. Oehmig A, Fraefel C, Breakefield XO et al. Herpes simplex vector type 1 amplicons and their hybrid virus partners, EBV, AAV, retrovirus. Curr Gene Ther 2004; 4:385-408. 67. Moss B. Poxviridae: The viruses and their replication In: Knipe D, Howley P, eds. Field’s Virology. 5th ed. Philadelphia: Lippincott, Williams & Wilkins, 2007:2905-2946. 68. Vanderplasschen A, Pastoret PP. The uses of poxviruses as vectors. Curr Gene Ther 2003; 3:583-595. 69. Zeh HJ, Bartlett DL. Development of a replication-selective, oncolytic poxvirus for the treatment of human cancers. Cancer Gene Ther 2002; 9:1001-1012. 70. Brochier B, Costy F, Pastoret PP. Elimination of fox rabies from Belgium using a recombinant vaccinia-rabies vaccine: an update. Vet Microbiol 1995; 46:269-279. 71. Shen Y, Nemunaitis J. Fighting cancer with vaccinia virus: Teaching new tricks to an old dog. Mol Ther 2005; 11:180-195. 72. Kim JH, Oh JY, Park DE et al. Systemic armed oncolytic and immunologic therapy for cancer with JX-594, a targeted poxvirus expressing GM-CSF. Mol Ther 2006; 14:361-370. 73. Fulginiti VA, Papier A, Lane JM et al. Smallpox vaccination: A review, part II. Adverse events. Clin Infect Dis 2003; 37:251-271. 74. Guo ZS, Bartlett DL. Vaccinia as a vector for gene delivery. Expert Opin Biol Ther 2004; 4:901-917. 75. Yamanaka R. Alphavirus vectors for cancer gene therapy (review). Int J Oncol 2004; 24:919-923. 76. Jeromin A, Yuan LL, Frick A et al. A modified Sindbis vector for prolonged gene expression in neurons. J Neurophysiol 2003; 90:2741-2745. 77. de Haan CA, Haijema BJ, Boss D et al. Coronaviruses as vectors: Stability of foreign gene expression. J Virol 2005; 79:12742-12751. 78. Dobrikova EY, Florez P, Gromeier M. Structural determinants of insert retention of poliovirus expression vectors with recombinant IRES elements. Virology 2003; 311:241-253. 79. Pecora AL, Rizvi N, Cohen GI et al. Phase I trial of intravenous administration of PV701, an oncolytic virus, in patients with advanced solid cancers. J Clin Oncol 2002; 20:2251-2266. 80. Strayer DS. Gene therapy using SV40-derived vectors: What does the future hold? J Cell Physiol 1999; 181:375-384. 81. Wahlfors JJ, Zullo SA, Loimas S et al. Evaluation of recombinant α-viruses as vectors in gene therapy. Gene Ther 2000; 7:472-480. 82. Palese P, Zheng H, Engelhardt OG et al. Negative-strand RNA viruses: Genetic engineering and applications. Proc Natl Acad Sci USA 1996; 93:11354-11358.

Chapter 11

Promiscuous Drugs from Pathogenic Bacteria in the Post-Antibiotics Era Arsenio M. Fialho, Tapas K. Das Gupta and Ananda M. Chakrabarty*

Abstract

T

he era of the antibiotics, active against prokaryotic and lower eukaryotic infectious agents, is believed to be coming to an end, because of the rapid development of antibiotic resistance in susceptible organisms. Today’s drugs are mostly targeted to a single step to inhibit progression of a single disease, even though drugs, that target multiple steps in multiple diseases, are often known to be more efficacious with less side effects. We describe here the elaboration of potential protein drugs by pathogenic bacteria that are uniquely designed to bind and often inhibit key eukaryotic, including mammalian, proteins involved in diverse diseases. Such bacterial proteins have demonstrated efficacies in significantly inhibiting the critical steps that are known to be involved in the progression of these diseases. We believe that bacterially derived promiscuous drugs will likely be our next generation drugs in the post-antibiotics era, targeting both prokaryotic and eukaryotic agents of human diseases.

Introduction

Bacteria, particularly members of the Streptomyces species, are known to produce antibiotics which are currently used for the treatment of various bacterial and fungal infections. Antibiotics are not usually very effective against cancers, parasites and viruses because they are produced by soil bacteria to keep other competitors in soil in check. These competitors are mostly prokaryotes or lower eukaryotes and so antibiotics are quite effective against them but not against nonsoil-residing pathogens. Because antibiotics have been used for several decades now, many susceptible pathogenic bacteria have developed or are continuing to develop resistance against various antibiotics, making them nonfunctional.1,2 On the other hand, microbial pathogenic traits are continually evolving, giving rise to such diseases as AIDS, avian influenza (caused by viruses of subtype H5N1) and others that did not exist a few decades ago. The old as well as the emerging pathogens also appear to be able to cope with our attempts to develop vaccines against them by changing the nature of their surface components. Thus no effective vaccines exist today for old and new diseases such as tuberculosis, malaria and AIDS. A similar strategy for developing resistance against immune surveillance and drugs is also employed by cancers so that many forms of cancer become resistant to even newly-developed drugs. Against this backdrop, there is a dire need to develop not only new drugs but also drugs that will target multiple diseases at the same time and outsmart the disease agents to develop resistance. Given the fact that we know very little about the life style of many pathogens or how they cause various diseases, including cancers and how they subvert the host immune system, or how cancer cells subvert cellular regulatory mechanisms, we *Corresponding Author: Ananda M. Chakrabarty—Department of Microbiology and Immunology, University of Illinois College of Medicine, 835 South Wolcott Avenue, Chicago, IL 60612, USA. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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have had only limited success in developing new drugs against such diseases as cancers, malaria, tuberculosis or HIV/AIDS. While efforts have been directed in looking for new antibiotics from soil bacteria, or using genomics/proteomics/synthetic chemistry and high throughput screening for developing new drugs, very little attention has been given to a potential new source for our drugs. We are referring to pathogenic bacteria, some of which live in our body for years to cause chronic infections. These are not the commensal bacteria that live in our guts or other tissues but these are pathogenic bacteria that grow extremely slowly, often in a biofilm mode or forming a protective granule for latency and doing little harm to host normal cells. Bacteria, of course, have evolutionary wisdom because they were the first to appear on earth, preceding other higher forms of life and became organelle such as mitochondria in eukaryotes including mammals.3,4 It is no wonder, therefore, that many pathogenic bacteria take over host functions by directly injecting in the host cells some of their own proteins, known as effectors or virulence factors that mimic mammalian signaling or other cellular mechanisms to interfere in host defense.5 Once established in the host cells, some of the extracellular pathogens form biofilms on epithelial cells and can survive for long periods of time without causing significant damage to the host cells. Once settling down for long term residency, however, these pathogenic bacteria appear to also become protective of their turf, trying to prevent other intruders to get in and either dislodge the bacteria or kill the host, whereby the bacteria lose their sanctuary. Thus the preventive measures the resident pathogens take very much resemble the functions of the host innate or adaptive immune system, mediated through production of immunoglobulins. It now appears that some of the bacterial weapons, for example azurin from the extracellular pathogen Pseudomonas aeruginosa, not only demonstrate promiscuity in attacking different intruders and disarming them at multiple steps6 but also show interesting structural similarity with the variable domains of immunoglobulins.7 It is to be emphasized here that humans have large genomes of more than three billion nucleotides and therefore human immune systems can produce by a switch-recombination shuffling mechanism individual immunoglobulins that can target individual antigens present in invading pathogens. In contrast, bacteria have small genomes of a few million bases and do not possess the capability to design individual deterrents for individual target molecules in the invaders. On the other hand, as mentioned earlier, bacteria have evolutionary wisdom and a knowledge base of the cellular regulatory circuits and metabolic pathways of mammalian and other higher forms of life and can consequently design their weapons in such a way that such weapons are uniquely active against many target cellular components of parasites, viruses or even cancer, that are considered by the pathogenic bacteria as intruders in their habitat.6,7

Pathogenic Bacteria as Anticancer Agents

That pathogenic bacteria target cancers and allow their regression has been known for more than a hundred years and reviewed extensively.8-15 In general, bacteria belonging to genera such as Samonella, Shigella, Clostridia, Listeria, Yersinia and Bifidobacteria have been shown to preferentially target cancer cells and grow in the core hypoxic areas of the tumors that are normally resistant to radiation or chemotherapy. However, introduction of live, pathogenic or even nonpathogenic bacteria activates immune surveillance in animals and humans, leading to toxicity problems. Thus most vector bacteria are attenuated through introduction of various genetic mutations or deletions so that they cause minimal toxicity problems.11,15,16 In addition, to enhance the anticancer effect of the vector bacteria, various genes encoding cytokines, toxins, prodrug-converting enzymes or inhibitors of angiogenesis have been cloned in eukaryotic expression plasmids for allowing cancer regression.8,10,15 For example, Salmonella strains have been used as a delivery vehicle to express cloned E. coli cytosine deaminase gene in three patients where such genes were shown to be expressed without significant adverse effect.17 A combination of C. novyi-NT and anti-microtubule agents, that inhibited microtubule synthesis or stabilized microtubules, showed rapid or slow tumor regression.16 More importantly, cloned genes or DNA vaccines with antiangiogenic agents led to significant growth inhibition of tumor cells,18,19 suggesting that bacterial vectors can be usefully

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employed to specifically target tumor vasculature and growth. However, preclinical animal studies or phase I studies have shown limited bacterial tumor colonization and tumor regression17,20,21 suggesting that better tumor targeting and sustained colonization and antitumor activity are needed to apply live bacteria in useful cancer therapy. The only strain of live bacteria that is used today in cancer therapy is Mycobacterium bovis BCG. Although originally believed to allow regression of melanoma, leukaemia or prostate cancer, such effects have not been found to be consistent. The major application of M. bovis BCG today is against bladder cancer, which can give complete response in 80% of patients8 as an immunotherapeutic agent. Although Salmonella, Clostridia and other bacteria are believed to target tumors and grow in the hypoxic areas of the tumor, thereby depriving the tumor of their nutrients and allowing their regression, the antitumor actions of M. bovis and most other bacteria are believed to be due to their ability to activate the immune system that can in turn allow tumor regression. Thus bacterial antitumor activity is often described as immunotherapy.8,10,15

Immune Action Alone Does Not Explain Microbial Antitumor Activity

Bacteria are not the only infectious agents that allow tumor regression. Viruses and parasites can elicit similar regression as well. For example, when mice receiving B16 melanoma cells were infected with Toxoplasma gondii, an obligate intracellular parasite, B16 cells failed to form subcutaneous tumors.22 To investigate if immune system activation was responsible for the failure of the B16 melanoma cells to form tumors, Hunter et al22 used several mutant mice strains lacking iNOS or perforin where cytolytic functions of lymphocytes were severely impaired or scid-beige mice in which cytotoxic T-lymphocytes (CTL) are absent and NK cells are not cytotoxic. In all cases, T. gondii infections suppressed B16 melanoma tumor growth,10,22 demonstrating that cytotoxic immune action in tumor growth suppression was not primarily involved in T. gondii infection. Further experimentations led to the finding that T. gondii infections allowed production of soluble circulating antiangiogenic factors that prevented tumor vascularization and growth.22 It was unclear, however, whether the antiangiogenic factor(s) was a product of the parasite or possibly of host cell origin triggered by the parasitic infection. Interestingly, similar protective effect was also observed with other pathogens of bacterial and viral nature, leading to the conclusion that such antiangiogenic factors were likely produced by the host.10 The fact that soluble antiangiogenic factors are produced in mice during infections by parasites, bacteria or viruses does not necessarily mean that they are of host origin. It is possible, even likely, that the soluble antiangiogenic factor(s) observed by Hunter et al22 was encoded by T. gondii and secreted in response to the presence of the melanoma cell. This begs the question if T. gondii or various bacteria mentioned previously might allow tumor regression not only by targeting and growing in tumor cells but also by elaborating antiangiogenic or other agents that would actively prevent cancer cell growth by interfering in multiple steps by which cancer cells grow.

Bacterial Proteins as Anticancer Agents

Cancer is a multigenic disease where mutations in approximately 345 genes are implicated in the development of human cancer.23 This means that more than 1% of the human genes are involved in the causation and progression of cancer. Thus mutations that affect major cellular machinery for promoting growth and cell division such as growth factors, signaling kinases, tumor suppressor proteins, regulators such as NF-kB, the Bcl 2 protein family, etc, may all contribute to cancer development. Tumors also secrete growth factors to promote their own growth and spread to distant organs and ultimately to general circulation.24 They also temper adaptive immunity to sustain themselves,25 providing clear evidence that significant control of cancer growth can only be accomplished by interfering in some of these essential steps that are used by cancer cells for their growth and migration. The current industry trend is to develop drugs that target a specific step in cancer growth progression, usually an inhibitor of a kinase in a signaling pathway,26 or a monoclonal antibody

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(herceptin, avastin) that targets a key surface antigen important for and hyperexpressed in cancer cells. Such drugs may slow down the growth of the cancers but also allow cancer cells to rapidly become resistant because of the single target site. Interference in multiple steps in cancer progression is key to develop drugs that will not only substantially inhibit cancer cell growth but will interfere in resistance development because of the need and the difficulty for the cancer cells to mutate simultaneously in multiple sites to develop resistance.6,7 The initial observation that an extracellular pathogen such as P. aeruginosa elaborated in the growth medium a water soluble, copper containing redox protein azurin that had cytotoxic activity against a macrophage cell line J774 was made by Zaborina et al27, who also demonstrated the elaboration of another redox protein, cytochrome c551, which is believed to be a partner of azurin during electron transfer in vitro and showed similar, albeit greatly reduced, cytotoxicity to J774 cells.27 Unlike azurin, however, cytochrome c551 had very little ability to induce apoptosis in J774 cells, but demonstrated significant inhibition of cell cycle progression in such cells through enhancement of the intracellular level of tumor suppressor protein p16ink4a, a known inhibitor of cell cycle progression.28 Interestingly, similar to P. aeruginosa cytochrome c551, human cytochrome c protein can also enter J774 cells, which are murine reticulum cell sarcomas and induce cell cycle arrest at the G1 to S phase, as well as at the G2/M phase at higher concentrations, leading to apoptosis of such cells.29 Given the fact that cytochrome c is a known component of apoptosomes, has antioxidant activity and catalyzes the synthesis of long chain fatty acyl glycines which are lipid signaling molecules,30 it is clear that cytochrome c and the redox proteins in general are important for cellular growth regulation. A patent has been issued31 and another patent application published32 to cover the potential use of azurin and cytochrome c551 in cancer therapy. The ability of azurin and cytochrome c551 to enter and induce apoptosis or cell cycle arrest in J774 cells prompted us to look at azurin effect in both J774 and human cancer cells. In J774 cells, azurin was demonstrated to enter such cells and form a complex with the tumor suppressor protein p53, thereby stabilizing this normally labile protein and enhancing its intracellular concentration.33 Using apo-azurin devoid of copper and various site-directed redox-negative mutants, Goto et al34 demonstrated that complex formation with p53 and generation of reactive oxygen species (ROS), rather than azurin redox activity, were important in the cytotoxic action of azurin towards J774 macrophages.34 Most importantly, the ability of azurin to enter human cancer cells such as melanoma35 or breast cancer36 and to induce apoptosis in such cells was also reported. In both cases, azurin entered into the cytosol of the cancer cells, traveled to the nucleus and enhanced intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol, activating caspase cascade and initiating the apoptotic process35,36. Also, in both cases, in vivo injection of azurin in immunodeficient mice harboring xenografted human melanoma or breast cancer cells led to statistically significant regression of the tumors35,36 without any apparent toxicity to normal cells, suggesting potential application of azurin in cancer therapy. Using iso-thermal titration calorimetry, Apiyo and Wittung-Stafshede demonstrated azurin binding to the N-terminal domain of p53, four azurin molecules binding each p53 monomer with a K D value of 33 ± 12 nM per binding site.37 While wild-type azurin bound p53 in the N-terminal to central domain, a site-directed mutant azurin, M44KM64E azurin, formed a different complex with p53 affecting p53’s oligomerisation. Further, unlike wild-type azurin that induced apoptosis in J774 cells, the less hydrophobic M44KM64E mutant azurin caused strong inhibition of cell-cycle progression at the G1 to S phase and a higher level of transcription of the p21 gene.38 Corresponding to high p21 levels, the levels of cyclins and cyclin-dependent kinases were greatly lowered in M44KM64E mutant azurin-treated J774 cells. However, the mutant azurin failed to elicit this effect in human breast cancer MCF-7 cells, presumably because of mutation at the retinoblastoma tumor suppressor protein that allows functional E2F formation in MCF-7 cells even in the presence of high intracellular p21 level.38 Thus the wild-type azurin induces apoptosis but little inhibition of cell cycle progression while the M44KM64E mutant azurin causes the reverse effect, demonstrating how a single bacterial protein azurin, based on its hydrophobicity, modulates the nature of p53 complex formation and its transcriptional specificity in mammalian cells.38

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Azurin Inhibits Cancer Cell Growth by Interfering in Multiple Steps

Today’s anticancer drugs employ monoclonal antibodies or low molecular weight inhibitors that interfere in specific steps of the cancer pathway. Because cancer cells grow through multiple pathways, such drugs are often only partially effective. Since the tumor suppressor protein p53 is intracellular, shuttling between the cytosol and nucleus, an important question was the mode of entry of azurin in cancer or normal cells. Azurin was shown to be internalized in J774 or human cancer cells in a temperature dependent manner with preferential entry to cancer cells as compared to normal cells.39 An 28 amino acid fragment of azurin was shown to be able to enter cancer cells presumably by a receptor-mediated endocytic process and this fragment comprising of amino acids 50 to 77 of azurin could act as a protein transduction domain (PTD) and could be used as a vehicle to transport cargo proteins preferentially to cancer cells.39 What happens after azurin enters preferentially to cancer cells? How does it distinguish cancer cells from normal cells? It should be pointed out here that azurin does not have to enter in cancer cells to inhibit cancer cell growth. Receptor tyrosine kinases such as EphB2, that are surface exposed and bind their ligand ephrinB2, are known to initiate cell signaling leading to cancer growth. There are many ephrins that have been subdivided into two classes; ephrin A1 to A8 are cell membrane-linked whereas ephrin B1 to B6 possess highly conserved intracellular domains and a transmembrane domain. These ephrin ligands bind with a family of 14 extracellular receptor proteins known as Eph receptor tyrosine kinases, of which EphB2 is one.40 Azurin has structural similarity with ephrinB2, the ligand for EphB2. Although azurin binds with A-type receptors such as EphA6, EphA4 or EphA7, azurin’s strong binding with EphB2 interferes in the binding of the ligand ephrinB2 and in tyrosine phosphorylation in the kinase domain of EphB2,40 leading to inhibition of cancer growth (Fig. 1A). The equilibrium binding constants (KD values) of the binding of azurin or its truncated derivatives with EphB2 or the tumor suppressor protein p53 are shown in Figure 2. On entry inside the cancer cell, azurin is not only found in the cytosol but also in the nucleus, presumably being piggy-backed due to complex formation with p53, where azurin-mediated apoptosis leads to nuclear DNA fragmentation and ultimately to the death of the cancer cell (Fig. 1A). Azurin is a periplasmic protein in P. aeruginosa. Thus if P. aeruginosa uses azurin as a weapon to kill cancer cells, azurin must come out of the P. aeruginosa cells to attack cancer cells, either binding with extracellular receptor tyrosine kinases or going inside and inducing apoptosis. Is there a sensing mechanism in P. aeruginosa so that the bacteria will release azurin to the outside when they encounter their enemy? Indeed, Mahfouz et al41 have reported that on exposure to cancer cells, P. aeruginosa cells release azurin to the outside medium in an energy-independent manner. Most Figure 1, viewed on following page. Azurin and Laz as novel drug candidates effective against cancers and infectious agents, such as HIV-1 virus, P. falciparum and T. gondii parasites. The mode of action in the induction of cell death, prevention of adhesion and invasion and growth suppression is shown schematically in the four panels (A, B, C and D). Ribbon representation of azurin from P. aeruginosa and the 3D-model for Laz, a Neisserial modified azurin, is shown in the central part of the figure. A)—Azurin and Laz as anticancer agents: Azurin can bind to the surface-exposed receptor tyrosine kinase EphB2, interfering in its binding with the ligand ephrinB2 and thereby interfering in tyrosine phosphorylation in the kinase domain of EphB2, leading to inhibition of cancer growth.40 Once inside the cancer cell, azurin forms a complex with the tumor suppressor protein p53, stabilizing it and enhancing its intracellular level, which leads to apoptosis.35 B)—Azurin and Laz as anti-malarial agents: Azurin or Laz binds strongly to the merozoite surface protein MSP1 (MSP-19) and interferes in the P. falciparum growth in infected red blood cells (RBC).45 C)—Azurin and Laz as anti-HIV agents: Azurin and Laz can bind the envelope glycoprotein gp120 of the HIV-1 virus, as well as the ICAM-3 and the DC-SIGN proteins 45 leading to interference in the entry of HIV-1 to the T-cells. (D)—Azurin and Laz as anti-toxoplasma agents: azurin or Laz has structural similarity to both SAG1, a major surface antigen of T. gondii and to a monoclonal antibody to SAG1. Azurin or Laz binds strongly to SAG1 and interferes in the T. gondii adhesion to host cells.46 Laz had a much more pronounced effect.46

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Figure 1, legend viewed on previous page.

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Figure 2. Azurin as a promiscuous protein able to interact with unrelated targets such as the tumor suppressor protein p53,37 the receptor tyrosine kinase EphB2,40 the HIV-protein gp 120, the intercellular adhesion molecule ICAM-3, the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)45 and the parasite proteins MSP1-19 and SAG1, respectively from P. falciparum and T. gondii.45,46 The numbers indicated in the table represent the equilibrium binding constant (kD) values (nanomolar) of the binding of azurin or its truncated derivatives to the target proteins and are determined by surface plasmon resonance binding titrations. Abbreviation: ND: not determined.

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interestingly, a discrete piece of DNA high in cytosine content and rich in CpG dinucleotides, is also released from P. aeruginosa and exerts anti-tumor activity.41 This CpG-rich DNA fragment allows activation of the Toll-like receptor 9 (TLR9) which allows tumor regression by modulating various cytokine production.41 Entry of azurin in cancer cells, in addition to complex formation with p53 and induction of apoptosis, has other consequences such as inhibition of angiogenesis (blood vessel formation in cancer cells) as shown earlier for T. gondii infection.10,22 For example, the 28 amino acid PTD, termed P28, which can preferentially enter into cancer cells, has also been shown to allow inhibition of capillary tube formation of the human umbilical vein endothelial cells (HUVEC) in a dose dependent manner,42 suggesting that P28 inhibits the capillary tube formation step of angiogenesis. P28 also inhibited the migration of HUVEC cells on Matrigel in a scratch wound migration assay, indicating that P28 also inhibits the migration step of angiogenesis. P28 has also been reported to prevent normal angiogenesis-related developments in cytoskeleton in HUVEC cells growing on Matrigel, suggesting that azurin and its PTD, the P28 peptide, have direct effect on the morphological hallmarks of angiogenesis, leading to inhibition of growth of human melanoma cells both in vitro and in vivo.42 Thus azurin or peptides derived from it appear to interfere in cancer growth in multiple steps, i.e., inducing apoptosis in p53-positive cancer cells, slowing down angiogenesis and interfering in receptor tyrosine kinase or perhaps other signaling mechanisms that promote cancer growth. Such mode of action of azurin appears to differ from targeted drugs that inhibit usually a single step in cancer progression. Azurin also has a wide target range of cancers including bone cancer osteosarcomas,43 demonstrating its versatility as an anticancer agent. Azurin is not only produced by P. aeruginosa but also by other pathogenic bacteria including gonococci and meningococci.39 Thus meningitis-causing bacterium Neisseria meningitides harbors an azurin-like protein. Unlike other bacterial azurins where azurin is periplasmic, Neisserial azurin, termed Laz, is surface-exposed and harbors an additional 39 amino acid moiety in its N-terminal called an H.8 epitope44 which is also lipidated. While the P. aeruginosa azurin, termed Paz, was shown to be deficient in entering brain tumors such as glioblastomas and exhibited low cytotoxicity, Laz was much more proficient in entering glioblastoma cells and showed a higher level of cytoxicity.44 The lipidation of the H.8 moiety was shown to be important for the surface display of Laz but not for the cytoxicity, suggesting that the H.8 epitope plays an important role in disrupting entry barriers to glioblastoma cells. Cloning of the H.8 epitope in the N-terminal of Paz allowed the modified H.8-azurin to enter glioblastoma cells much more proficiently to exert cytotoxicity in glioblastoma cells, suggesting that perhaps the H.8 epitope might be useful in allowing other toxic drug candidates to enter into brain tumor cells for effective therapy.

Azurin as a Promiscuous Drug

One of the interesting features of azurin is its structural similarity with the variable domains of immunoglobulins, suggesting perhaps a common ancestry.7 For example, azurin shows structural similarity with an antibody fab fragment complexed with the parasite Plasmodium falciparum or the surface antigen SAG1 of the parasite Toxoplasma gondii or host proteins such as ephrinB2,40 ICAM-3, or DC-SIGN.45 This has raised the question if such structural similarity might have any physiological significance. The P. falciparum merozoite surface protein 1 (MSP1) is a major antigenic protein in the blood stage merozoites of all the erythrocyte invasive species. Since azurin has structural similarity with the antibody fab fragment that complexes with MSP1, can azurin form a complex with MSP1 and inhibit invasion? Both azurin and Laz inhibit the growth of the parasite during infections of red blood cells (RBC) with the schizont forms of P. falciparium.45 This inhibition is due to binding of the MSP1 with azurin or Laz45 (Fig. 1B) with KD values of 32.2 nM and 26.2 nM respectively (Fig. 2). The ability of GST-fused truncated azurin consisting of amino acids 36-89 or 36-128 to bind with MSP1 with KD values of 20 to 24.5 nM (Fig. 2) demonstrated that the N-terminal 35 amino acids of azurin are not necessary for such binding. Azurin not only shows structural similarity with the fab fragment complexing with MSP1-19, but also with the amino terminal domain of CD4 and the extracellular domain of two host proteins

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involved in HIV-1 binding and entry into host cells. Both azurin and Laz have been shown to strongly inhibit the growth of three subtypes of HIV-1,45 a clade B predominant in the US and Western Europe, a clade B African isolate and a clade C Indian isolate. Figure 1C provides a schematic diagram of how entry of HIV-1 in the susceptible host cells can be prevented by binding of azurin to either the virus envelope glycoprotein gp120, which binds the host receptor CD4, or the host proteins DC-SIGN and ICAM-3. Surface plasmon resonance studies have shown that azurin binds gp120, ICAM-3 or DC-SIGN avidly with KD values varying between 0.83 nM to 20 nM (Fig. 2). Interestingly, while GST-fused truncated azurin peptide harboring amino acids 36 to 89 did not show any binding with DC-SIGN, a corresponding peptide harboring amino acids 88 to 113, that previously showed strong binding to the receptor tyrosine kinase EphB240 showed significant binding with DC-SIGN,45 with a KD value of 5.98 nM (Fig. 2), suggesting that particular domains of azurin bind with particular proteins, as this peptide (azurin 88-113) showed no binding with MSP1-19 or SAG1 (Fig. 2). SAG1, as mentioned above, is a predominant surface antigen of the parasite Toxoplasma gondii, involved in parasite attachment to host cells.46 Azurin shows structural similarity to not only SAG1 but also to a monoclonal antibody to SAG1. Surface plasmon studies have shown that azurin binds SAG1 with a KD value of 12.8 nM, while Laz binds SAG1 with a higher affinity and a KD value of 4.8 nM. Interestingly, the GST fusion derivatives of truncated azurin harboring amino acids 36 to 128 did not show any binding, suggesting that the N-terminal 35 amino acids of azurin are important in SAG1 binding.46 While azurin inhibited T. gondii invasion of the human foreskin fibroblasts (HFF) by interfering in adhesion, Laz had a much more pronounced effect (Fig. 1D), suggesting that the H.8 epitope greatly facilitates azurin binding with many surface proteins.46 The ability of azurin or Laz to bind key proteins involved in the invasion and growth of cancers, parasites such as P. falciparum or T. gondii and viruses such as HIV-1, makes azurin or Laz, or specific peptides derived from them, potential promiscuous drug candidates.6 Promiscuous drugs such as clozapine, responsible for exceptionally beneficial actions in schizophrenia and related disorders, even with considerable side effects, are valued as antipsychotic drugs because of their effectiveness.47 Indeed, it is becoming clear that promiscuous drugs that hit many target diseases are better and more effective with less side effects and elicit less resistance than drugs that target a single step of a single disease.48,49 It remains to be seen if azurin or Laz will meet such criteria of promiscuity in humans and be therapeutically useful as anticancer, antiparasitic or antiviral drugs of choice.

Antiparasite Activity of Azurin and Laz

Parasitic diseases are rampant in developing countries and are occasional problems in industrialized countries. For example, Schistosomiasis japonica is a snail-mediated zoonotic parasitic disease in both humans and animals. In many parts in China, greater than 50 million persons are affected by this disease because of poor water quality.50,51 Water quality is also a factor in gastroenteritis-related infections caused by another parasite Cryptosporidium parvum which is resistant to chlorination and caused a major outbreak of cryptosporidiosis in Milwaukee in the spring of 1993.52 The most vulnerable population for this infection are the elderly, the children and immunocompromised patients. Such outbreaks also occur sporadically in UK and the human infecting variants are known as genotype 1 while those that infect both livestock and humans are referred to as genotype 2.53 Insect vectors are also known for parasitic diseases such as visceral, mucocutaneous and cutaneous leishmaniasis with skin lesions caused by the parasite Leishmania donovani or similar species such as L. tropica, L. infantum, L. chagasi, etc. A single strain such as L. donovani may not only cause visceral but also cutaneous leishmaniasis.54 By far, however, the most dangerous parasite that causes about 300 million infections and more than one million deaths a year is the malarial parasite Plasmodium falciparum,55 followed by another intracellular protozoan parasite Toxoplasma gondii which causes a less severe form of a disease called toxoplasmosis.56 Toxoplasmosis is a major cause of intrauterine infections in pregnant women leading to birth defects and in immunocompromised patients, as well as in AIDS patients. In HIV-infected individuals, latent asymptomatic infections can be activated leading the bradyzoite form of parasite cysts to replicate to form the more severe

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tachyzoite forms which in turn may lead to fatal or debilitating encephalitis.57,58 No drugs are available to treat the encysted bradyzoite forms and while pyrimethamine and sulfonamides are used to treat the infectious tachyzoite replication, they are mildly toxic which prevents their long term sustained use in AIDS patients.56 Thus new drugs are sorely needed for the treatment of T. gondii infections, particularly in AIDS patients. While the water-borne parasites cause major health-related problems, they are sporadic and mostly manageable through filtered clean water availability. Much more serious is malaria, the disease caused by the mosquito-borne parasites Plasmodium falciparum and P. vivax. According to WHO estimate, more than 1 million people, mostly children, are killed annually and 40% of the world’s population are at risk of being infected by this parasite. Similar to the lack of availability of effective vaccines against HIV infections, no effective antimalarial vaccine exists to prevent such infections. Additionally, resistance against most potent antimalarial drugs such as chloroquine, primaquine or even the recent drug atovaquone has developed during the last decades, making malaria difficult to treat. This problem has also been exacerbated by the development of insectide resistance in vector mosquito Anopheles gambiae so that the mosquito eradication has also been problematic.55,59,60 While major efforts are underway in genetically transferring the artemisinin biosynthetic genes from plants to bacteria to produce this antimalarial drug cheaply and throughout the year, there is a great need to come up with new, cheap and nontoxic antimalarial drugs effective against drug resistant Plasmodium species. Antimalarial drugs are needed not only to treat malaria but might also be useful in the treatment of AIDS patients infected with P. falciparum as well. Infection of AIDS patients with P. falciparium is known to contribute to enhanced HIV viral load.61 On the other hand, infection with the AIDS virus HIV-1 can contribute to severe forms of malaria62 as well as contributing to the failure of antimalarial drug treatments,63,64 essentially leading to high levels of morbidity and mortality, particularly in sub-Saharan Africa.65 The ability of azurin and Laz to significantly interfere in the entry and growth of the HIV-1 virus and parasitemia involving P. falciparum45 and T. gondii46 appears to suggest that they might be potentially useful in the treatment of AIDS, malaria, toxoplasmosis and in particular in mixed infections involving P. falciparum or T. gondii in AIDS patients. Given the anticancer activity of azurin and Laz as mentioned previously, the use of a single bacterial protein or peptides derived from it in the treatment of cancer, viral and parasitic diseases could be of great economic and health benefits for common people in both developing and developed countries. Since these bacterial proteins appear to inhibit cancer or HIV-1 growth by interfering in multiple steps, it is likely that resistance development against these potential protein drugs, should they be proven to be nontoxic and clinically efficacious, would be delayed for them to enjoy a longer drug life. Interference in multiple pathways by which cancer cells or HIV viruses invade and grow in human body may also lead to higher level of efficacy compared to drugs that target only a single step.

Azurin/Laz as Inhibitors of Entry/Growth of the HIV-1 Virus

The binding of azurin or Laz to both the HIV-1 envelope protein gp120 and the host proteins DC-SIGN or ICAM-3 to prevent entry of HIV-1 to host cells45 raised an interesting question if azurin or Laz targets similar viruses when P. aeruginosa infects the host animals and has retained the binding and growth inhibitory specificity for the human viruses as well. Not much is known about the ability of P. aeruginosa to cause natural infections in animals although mouse or similar animal models are routinely used for studying P. aeruginosa pathogenicity and its varied virulence factors. The ability of azurin or Laz to bind the viral or host proteins involved in the transport or entry of the HIV-1 virus in the CD4+ T-cells leads to inhibition of entry and viral growth in peripheral blood mononuclear cells (PBMCs) although it is not known if azurin or Laz may interfere in specific steps in viral replication or maturation.45 The entry of HIV-1 in host cells involves binding with the host receptor CD4 and any of the two or both cell membrane chemokine receptors CCR5 and CXCR4,66-68 followed by fusion of the viral envelope with the cytoplasmic membrane of the target cells.69 All these interactions are

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Table 1. Some inhibitors in specific steps of HIV-1 entry undergoing clinical trials Inhibitor/Company

Step in Inhibition

TNX-355 (Tanox, Inc., Houston, TX)

CD4-gp120 binding

CCR5 mAb004 (Human genome sciences, Rockville, MD) PRO140 (Progenics Pharmaceuticals, Tarrytown, NY) Maraviroc (UK-427,857) Pfizer, Sandwich, UK Vicriviroc (SCH-D) (Schering-Plough, Kenilworth, NJ) Enfuvirtide (Trimeris Inc, Morrisville, NC; Roche, Nutley, NJ)

CCR5

CCR5

CCR5 CCR5

Fusion

Mode of Action/Clinical Trial Status Monoclonal antibody against CD4 targeting gp120 (phase II) Monoclonal antibody targeting CCR5/ (phase I) Humanized monoclonal antibody against CCR5 (phase I/II) Binds the transmembrane domain of CCR5 (phase II/III) Binds the transmembrane domain of CCR5 (phase II) 36 amino acid synthetic peptide mimicking gp41 HR2 region inhibiting HR1-HR2 interaction and six-helix bundle formation (FDA approved)

mediated by the HIV-1 envelope which is synthesized as an inactive precursor protein gp160 which is cleaved in the golgi to the surface subunit gp120 and the membrane spanning gp41. Given the fact that about 4 million HIV-1 infections occur every year,70 there have been intense studies in understanding how both CCR5 and/or CXCR4-binding virus may enter the host cells and proliferate. It is generally believed that the R5 virus is the dominant one and may dominate any infection cycle.66 The macrophages and microglia may also be susceptible to the virus after extensive loss of CD4+ T-cells.67,71 An understanding of the various steps by which HIV type 1 virus enters and replicates inside the susceptible cells has led to many efforts in drug development involving both viral entry and proliferation. Indeed, most of the common antiretroviral drugs used or being developed today are either protease inhibitors, integrase inhibitors, or nucleoside/nonnucleoside reverse transcriptase inhibitors that directly target the viral replication/maturation machinery. It is becoming clear, however, that candidate drugs that inhibit entry of the virus could be of potential interest, when used either alone or in combination with the protease/reverse transcriptase inhibitors. A short list of such candidate drugs that are undergoing clinical trials or are FDA-approved such as enfuvirtide is given in Table 1. Many such entry inhibitors are currently undergoing development and clinical trials.72-75 Most notable among the entry inhibitory drugs is the FDA approved 36 amino acid peptide enfuvirtide that mimics gp41 HR2 region and inhibits fusion of the viral and host cell membrane by binding gp41 HR1 and interfering in the formation of fusogenic six-helix bundle necessary for fusion.76,77 Most interestingly, a synergy in the inhibitory activities of enfuvirtide and TNX-355, a humanized monoclonal antibody targeting a CD4 epitope, has been observed in significantly enhancing antiretroviral activity.78 Apart from peptides or monoclonal antibodies, small molecule inhibitors are also being designed to interfere in CCR5-mediated viral entry. Two such inhibitors, SCH-C and TAK779, that bind the transmembrane domain of CCR5, inhibit gp120 binding to CCR5. There is synergy between these small molecule inhibitors and monoclonal antibodies such as PA14 because they act at different steps in gp120-CCR5 interactions, thereby enhancing interference in envelope-mediated entry of CCR5-tropic HIV-1 viruses.79 Small molecule compounds that interfere in gp120 binding with CD4 by binding to gp120 prior

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Table 2. Therapeutic proteins Class

Protein

Trade Name

Company

Target Disease

Hormones

Glucagon Somatotropin (Human growth hormone) Somatostatin (Peptide hormone) Insulin Insulin Interferon alfa-2b

GlucaGen Nutropin

Novo Nordisk Genentech

Diabetes Growth hormone deficiency

Sandostatin

Novartis

Humalog Novolog Intron-A

Eli Lilly Novo Nordisk Schering-Plough

Interleukin-2 (IL-2)

Proleukin

Novartis

Tumor necrosis factor (TNF) blocker Epoetin alfa Darbepoetin alfa Human deoxyribonucleasel (rhDNase) Streptokinase

Enbrel

Amgen

Inhibitory growth hormone secretion Diabetes Diabetes Melanoma, Leukimia, Hepatitis B and C Melanoma, renal carcinoma Rheumatoid arthritis

Epogen Aranesp Pulmozyme

Amgen Amgen Genentech

Anemia Anemia Cystic Fibrosis

Streptase

ZLB Behring

Factor VIII Becaplermin

Aafact Regranex

Epidermal growth factor (EGF) Rituximab

Regen-D Rituxan

Sanquin Johnson & Johnson Bharat Biotech International Genentech

Myocardial infarction, Pulmonary embolism Hemophilia Diabetic foot ulcers

Trastuzumab Alemtuzumab Cetuximab

Herceptin CamPath Erbitux

Genentech Genzyme Merck

Bevacizumab Palivizumab

Avastin Synagis

Genentech MedImmune

Cytokines

Erythropoietins Enzymes

Blood factor Growth factors

Monoclonal antibodies

TNX-355

Tanox

Diabetic foot ulcers Non-Hodgkin's lymphoma Breast cancer Leukemia Colorectal/ Head cancers Colorectal cancer Respiratory Syncytial Virus HIV-1

to CD4 binding and the subsequent envelope-cell-membrane fusion are also known.80,81 Cyclam compounds such as AMD3465, a monomacrocyclic CXCR4 antagonist, has been shown to block entry of X4-tropic HIV-1 virus and is an improvement of biclams such as AMD3100.82 Maraviroc (UK-427,857), on the other hand, is a potent, orally bioavailable inhibitor of CCR5 that binds and shows clinical efficacy against a range of primary CCR5-tropic global isolates and synergize

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Figure 3. Azurin, a natural scaffolding protein. A)—Amino acid sequence of azurin from P. aeruginosa. The 28 amino acid PTD, an extended α-helix termed P28 (protein transduction domain; residues 50 to 77), responsible for preferential entry into cancer cells versus normal cells39 and the four loop regions thought to contain potential hot-spots for binding, are highlighted. B)—Azurin has a single antibody-like structure. Ribbon representation of azurin (1JZG_A) from P. aeruginosa showing the eight antiparallel strands that adopt a compact and structurally rigid β-sandwich core (immunoglobulin fold) connected at its open ends by a set of four surface loops of varying length that resemble the CDRs (complementary determining regions) of the antibodies. C)—Azurin, a nonantibody scaffold able to mediate specific high-affinity interactions (at nanomolar range) with unrelated target proteins such as the tumor suppressor protein p53,37 the receptor tyrosine kinase EphB2,40 the HIV-protein gp 120, the intercellular adhesion molecule ICAM-3, the DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN),45 and the parasite proteins MSP1-19 and SAG1, respectively from P. falciparum and T. gondii.45,46

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with other antiretroviral drugs including the fusion inhibitor enfuvirtide. Given the fact that CCR5 function is nonessential in humans since delta 32 homozygous genotype populations that lack CCR5 are healthy but protected against HIV-1, the development of maraviroc type of CCR5 antagonists83 may prove to be clinically useful anti-HIV-1 drug candidates of the future. While the drug candidates mentioned above are designed to prevent HIV infections when given orally or as injectables, attempts are also being made to develop entry inhibitors as microbicides for blocking HIV transmission via the cervicovaginal or rectal mucosa.74 The idea is to apply such microbicides topically in the vaginal or rectal mucosal surfaces for blocking HIV-1 binding and transport to the T-cells.84,85 A monoclonal antibody against CD4 binding site of gp120 has shown efficacy in vaginal challenge with the humanized simian virus SHIV-SF162P4.86 Similarly, a peptide derived from the gp41 sequence that interferes in envelope-cell fusion has shown some protection from vaginal challenge with the SHIV in the macaque model as shown for the monoclonal antibody.87 Proteins such as cyanovirin-N, produced by cyanobacteria Nostoc ellipsosporum and that can bind the mannose-rich carbohydrate moieties on the HIV envelope and interfere in HIV binding to host cells, have also shown efficacy.88 The use of lactic acid bacteria that normally colonize vaginal areas engineered to secrete cyanovirin for application in the treatment of HIV infection is also gaining ground.89 The increasing development of lactic acid bacteria or other commensal bacteria that secrete various proteins or peptides involved in interfering with HIV entry and growth90-92 is an encouraging sign for continued and effective drug development to combat AIDS, although some safety and efficacy concerns remain for using live bacteria in such a process.93 The bacterial proteins azurin and Laz that demonstrate significant inhibition of HIV growth45 differ from the entry inhibitors mentioned above in two significant respects. Azurin and Laz are not only effective against HIV-1 of different clades45 but also suppress growth of parasites such as the malarial parasite Plasmodium falciparum45 or Toxoplasma gondii46 which are often involved in coinfection with HIV. A single drug affecting the growth of multiple infectious agents could be of great value in the treatment of pregnant women or children suffering from AIDS and harboring co-infections.6,7,45 A second advantage appears to be the fact that azurin or Laz binds to multiple targets involved in HIV entry and propagation such as DC-SIGN, ICAM-3 and gp120.45 The binding with host proteins that are involved in HIV transport and viral envelope protein involved in entry might make it difficult for the HIV to mutate for developing resistance. It remains to be seen if azurin/Laz or peptides derived from them will demonstrate lack of toxicity or efficacy in preclinical or human clinical trials with AIDS patients.

Azurin/Laz as a Scaffolding Protein

Aside the questions of toxicity and efficacy in humans as a potential promiscuous drug, there are some important aspects of azurin or Laz that merit mention. As a protein, azurin or Laz will be susceptible to immune attack, although preliminary evidence indicates that azurin has low immunogenicity.6 Many proteins are used as drugs (Table 2) but they are mostly human proteins not likely to be recognized as immune system targets, although prolonged administration of such proteins may eventually lead to the induction of human anti-human antibodies.94 Unlike antibodies that are large molecules with four individual protein chains, azurin has a hydrophobic patch and is water soluble which should help in its tissue penetration and clearance from the blood stream. Covalent attachment to polyethyleneglycol (PEG) should facilitate azurin’s stability and longer life in the blood stream if prolonged use is desired. By far the most important property of azurin is its ability to bind various mammalian proteins of therapeutic importance as well as surface-proteins of pathogenic infectious agents (Fig. 1,2) conferring on it the property of a natural scaffold protein (Fig. 3).95,96 The scaffold proteins are nonantibody recognition proteins belonging to different and divergent families but demonstrate the immunoglobulin-like binding characteristics.96 Such binding properties have led to the use of many proteins such as human serum transferin giving rise to Trans-bodies, fibronectin type III as Monobodies, Z-domain of the IgG-binding Protein A from Staphylococcus aureus giving rise to Affibodies, etc, as scaffolding proteins for therapeutic purposes.95-97 The ability of the 28 amino acid PTD of azurin to act as a vehicle to carry cargo

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proteins inside cancer cells39 and azurin’s ability to bind many different proteins because of its unique structural features (Fig. 3) makes the azurin PTD an important natural scaffold protein both for therapeutic and diagnostic purposes.98 Unlike most of the other proteins described in Table 2, which are expensive to prepare,99 azurin or Laz can be easily hyperexpressed in E. coli,33,34,44 with a low production cost. A further development in the concept of “scaffold” implies the generation of azurin variants displaying multiple picomolar affinities to predefined targets relevant in cancers, HIV-1 infection, malaria and toxoplasmosis. This strategy can be reached by randomized substitution of amino acids or substitutions of only a few selected residues within preexisting binding sites, specifically the exposed α-helix (P28) and the four surface exposed loops (Fig. 3). This approach can be realized, for example, by genetic engineering procedures followed by selection of mutant proteins via phage display. The novel properties of the azurin engineered variants will be supported by an overall structural integrity of the β-sandwich core that will remain conserved. Neither is azurin the only bacterial protein to hit multiple target diseases. Other bacterial proteins such as arginine deiminase from Mycoplasma arginini have been shown to have anticancer and antiviral activities100-102 perhaps heralding a new era for drug discovery from bacteria in the post-antibiotics era.

References

1. Vicente M, Hodgson J, Massidda O et al. The fallacies of hope: Will we discover new antibiotics to combat pathogenic bacteria in time? FEMS Microbiol Rev 2006; 30:841-52. 2. Projan SJ. Why is big Pharma getting out of antibacterial drug discovery? Curr Opin Microbiol 2003; 6:427-30. 3. Blackstone NW. A units-of-evolution perspective on the endosymbiont theory of the origin of the mitochondrion. Evolution 1995; 49:785-96. 4. Punj V, Chakrabarty AM. Redox proteins in mammalian cell death: An evolutionarily conserved function in mitochondria and prokaryotes. Cell Microbiol 2003; 5:225-31. 5. Mota LJ, Cornelis GR. The bacterial injection kit: Type III secretion systems. Ann Med 2005; 37:234-49. 6. Fialho AM, Das Gupta TK, Chakrabarty AM. Designing promiscuous drugs? Look at what nature made! Lett Drug Design Discov 2007; 4:40-43. 7. Fialho AM, Stevens FJ, Das Gupta TK et al. Beyond host-pathogen interactions: Microbial defense strategy in the host environment. Curr Opin Biotechnol 2007; 18:279-86. 8. Cheadle EF, Jackson AM. Bugs as drugs for cancer. Immunology 2002; 107:10-19. 9. Chakrabarty AM. Microorganisms and cancer: Quest for a therapy. J Bacteriol 2003; 185:2683-86. 10. Thomas-Tikhonenko A, Hunter CA. Infection and cancer: The common vein. Cytokine and Growth Factor Rev 2003; 14:67-77. 11. Pawelek JM, Low KB, Bermudes D. Bacteria as tumor-targeting vectors. Lancet Oncol 2003; 4:548-56. 12. Minton NP. Clostridia in cancer therapy. Nat Rev Microbiol 2003; 1:237-42. 13. Ryan RM, Green J, Lewis CE. Use of bacteria in anti-cancer therapies. Bioessays 2006; 28:84-94. 14. Barbe S, Van Mellaert L, Anne J. The use of clostridial spores for cancer treatment. J Appl Microbiol 2006; 101:571-78. 15. Vassaux GE, Nitcheu J, Jezzard S et al. Bacterial gene therapy strategies. J Pathol 2006; 208:290-98. 16. Dang LH, Bettegowda C, Agrawal N et al. Testing vascular and avascular compartments of tumors with C. novyi-NT and anti-microtubule agents. Cancer Biol Ther 2004; 3:326-37. 17. Nemunaitis J, Cunningham C, Senzer N et al. Pilot trial of genetically modified attenuated Salmonella expressing the E. coli cytosine deaminase gene in refractory cancer patients. Cancer Gene Therap 2003; 10:737-44. 18. Fu GF, Li X, Hou YY et al. Biofidobacterium longum as an oral delivery system of endostatin for gene therapy on solid liver cancer. Cancer Gene Therap 2005; 12:133-40. 19. Feng KK, Zhao HY, Qiu H et al. Combined therapy with flk1-based DNA vaccine and interleukin-12 results in enhanced antiangiogenic and antitumor effects. Cancer Lett 2005; 221:41-7. 20. Thamm DH, Kurzman ID, King I et al. Systemic administration of an attenuated, tumor-targeting Salmonella typhimurium to dogs with spontaneous neoplasia: Phase I evaluation. Clin Cancer Res 2005; 11:4827-34. 21. Toso JF, Gill VJ, Hwu P et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 2002; 20:142-52.

160

Patho-Biotechnology

22. Hunter CA, Yu D, Gee M et al. Cutting edge: systemic inhibition of angiogenesis underlies resistance to tumors during acute toxoplasmosis. J Immunol 2001; 166:5878-81. 23. Futreal PA, Coin L, Marshall M et al. A census of human cancer genes. Nat Rev Cancer 2004; 4:177-83. 24. Saharinen P, Tammela T, Karkkainen MJ et al. Lymphatic vasculature: Development, molecular regulation and role in tumor metastasis and inflammation. Trends Immunol 2004; 25:387-95. 25. Blattman JN, Greenberg PD. Cancer Immuno-therapy: A treatment for the masses. Science 2004; 305:200-05. 26. Sawyers CL. Opportunities and challenges in the development of kinase inhibitor therapy for cancer. Genes Develop 2003; 17:2998-3010. 27. Zaborina O, Dhiman N, Chen ML et al. Secreted products of a nonmucoid Pseudomonas aeruginosa strain induce two modes of macrophage killing: external-ATP-dependant P2Z-receptor-mediated necrosis and ATP-independent caspase-mediated apoptosis. Microbiology 2000; 146:2521-30. 28. Hiraoka Y, Yamada T, Goto M et al. Modulation of mammalian cell growth and death by prokaryotic and eukaryotic cytochrome c. Proc Natl Acad Sci USA 2004; 101:6427-32. 29. Hiraoka Y, Granja AT, Fialho AM et al. Human cytochrome c enters murine J774 cells and causes G1 and G2/M cell cycle arrest and induction of apoptosis. Biochem Biophys Res Communs 2005; 338:1284-90. 30. Mueller GP, Driscoll WJ. In vitro synthesis of oleoylglycine by cytochrome C points to a novel pathway for the production of lipid signaling molecules. J Biol Chem 2007; 282:22364-9. 31. Chakrabarty AM, Das Gupta TK, Punj V et al. Cytotoxic factors for modulating cell death. US patent 7,084,105, 2006. 32. Chakrabarty AM, Das Gupta TK, Punj V et al. Cytotoxic factors for modulating cell death. US patent 2006/0292136, published, 2006. 33. Yamada T, Goto M, Punj V et al. The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53. Infect Immun 2002; 70:7054-62. 34. Goto M, Yamada T, Kimbara K et al. Induction of apoptosis in macrophages by Pseudomonas aeruginosa azurin: tumour-suppressor protein p53 and reactive oxygen species, but not redox activity, as critical elements in cytotoxicity. Mol Microbiol 2003; 47:549-59. 35. Yamada T, Goto M, Punj V et al. Bacterial redox protein azurin, tumor suppressor protein p53 and regression of cancer. Proc Natl Acad Sci USA 2002; 99:14098-103. 36. Punj V, Bhattacharyya S, Saint-Dic D et al. Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer. Oncogene 2004; 23:2367-78. 37. Apiyo D, Wittung-Stafshede P. Unique complex between bacterial azurin and tumor-suppressor protein p53. Biochem Biophys Res Communs 2005; 332:965-8. 38. Yamada T, Hiraoka Y, Ikehata M et al. Apoptosis or growth arrest: Modulation of tumor suppressor p53’s specificity by baterial redox protein azurin. Proc Natl Acad Sci USA 2004; 101:4770-75. 39. Yamada T, Fialho AM, Punj V et al. Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity. Cell Microbiol 2005; 7:1418-31. 40. Chaudhari A, Mahfouz M, Fialho AM et al. Cupredoxin-cancer interrelationship: Azurin binding with EphB2, interference in EphB2 tyrosine phosphorylation and inhibition of cancer growth. Biochemistry 2007; 46:1799-810. 41. Mahfouz M, Hashimoto W, Das Gupta TK et al. Bacterial proteins and CpG-rich extrachromosomal DNA in potential cancer therapy. Plasmid 2007; 57:4-17. 42. Mehta RR, Taylor BN, Yamada T et al. Compositions and methods to control angiogenesis with cupredoxins. US Patent Application Serial No. 11/488,693; PCT/US06/27940, 2006. 43. Yang DS, Miao XD, Ye ZM et al. Bacterial redox protein azurin induce apoptosis in human osteosarcoma U20S cells. Pharmacol Res 2005; 52:413-21. 44. Hong CS, Yamada T, Hashimoto W et al. Distrupting the entry barrier and attacking brain tumors: the role of the Neisseria H.8 epitope and the Laz protein. Cell Cycle 2006; 5:1633-41. 45. Chaudhari A, Fialho AM, Ratner D et al. Azurin, Plasmodium falciparum malaria and HIV/AIDS: inhibition of parasitic and viral growth by azurin. Cell Cycle 2006; 5:1642-8. 46. Naguleswaran A, Fialho AM, Chaudhari A et al. Azurin-like protein blocks invasion of Toxoplasma gondii through interactions with parasite surface antigen SAG1. Antimicrob Agents Chemotherap 2007; submitted. 47. Roth BL, Sheffler DJ, Kroeze WK. Magic shotguns versus magic bullets: Selectively nonselective drugs for mood disorders and schizophrenia. Nat Rev Drug Discov 2004; 3:353-9. 48. Hopkins AL, Mason JS, Overington JP. Can we rationally design promiscuous drugs? Curr Opin Struct Biol 2006; 16:127-36.

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49. Hampton T. “Promiscuous” anticancer drugs that hit multiple targets may thwart resistance. JAMA 2004; 292:419-22. 50. Xhou XN, Wang LY, Chen MG et al. The public health significance and control of schistosomiasis in China—Then and now. Acta Trop 2005; 96:97-105. 51. Liang S, Yang C, Zhong B et al. Re-emerging schistosomiasis in hilly and mountainous areas of Sichuan, China. Bull World Health Organ 2006; 84:139-44. 52. MacKenzie WR, Hoxie NJ, Proctor ME et al. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 1994; 331:161-7. 53. McLauchlin J, Amar C, Pedraza-Diaz S et al. Molecular epidemiologic analysis of Cryptosporidium spp. in the United Kingdom: Results of genotyping in 1,705 fecal samples from humans and 105 fecal samples from livestock animals. J Clin Microbiol 2000; 38:3984-90. 54. Sharma NL, Mahajan VK, Kanga A et al. Localized cutaneous leishmaniasis due to Leishmania donovani and Leishmania tropica: Preliminary findings of the study of 161 new cases from a new endemic focus in Himachal Pradesh, India. Am J Trop Med Hyg 2005; 72:819-24. 55. Fauci AS, Touchette NA, Folkers GK. Emerging infectious diseases: A 10 year perspective from the National Institute of Allergy and Infectious Diseases. Emerg Infect Dis 2005; 11:519-25. 56. Radke JR, Eibs CA, Fox PD. Host cell-directed interactions with Toxoplasma influence pathogenesis. Microbe 2007; 2:244-50. 57. Luft BJ, Remington JS. Toxoplasmic encephalitis in AIDS. Clin Infect Dis 1992; 15:211-22. 58. Gagne SS. Toxoplasmosis. Prim Care Update OB Gyn 2001; 8:122-6. 59. Kublin JG, Dzinjalamala FK, Kamwendo DD et al. Molecular markers for failure of sulfadoxine, pyrimethamine and chlorproguanil-dapsone treatment of Plasmodium falciparum malaria. J Infect Dis 2002; 185:380-8. 60. Labbe AC, Patel S, Crandall I et al. Molecular surveillance system for global patterns of drug resistance in imported malaria. Emerg Infect Dis 2003; 9:33-6. 61. Kublin JG, Patnaik P, Jere CS et al. Eff ect of Plasmodium falciparum malaria on concentration of HIV-1-RNA in the blood of adults in rural Malawi: A prospective cohort study. Lancet 2005; 365:233-40. 62. Chandramohan D, Greenwood BM. Is there an interaction between human immunodeficiency virus and Plasmodium falciparum? Int J Epidemiol 1998; 27:296-301. 63. Kamya MR, Kigonya CN, McFarland W. HIV infection may adversely affect clinical response to chloroquine therapy for uncomplicated malaria in children. AIDS 2001; 15:1187-8. 64. Birku Y, Mekonnen E, Bjorkman A et al. Delayed clearance of Plasmodium falciparum in patients with human immunodeficiency virus coinfection treated with artemisinin. Ethiop Med J 2002; 40:17-26. 65. Korenromp EL, Williams BG, de Vlas SJ et al. Malaria attributed to the HIV-1 epidemic, sub Saharan Africa. Emerg Infect Dis 2005; 11:1410-19. 66. Moore JP, Kitchen SG, Pugach P et al. The CCR5 and CXCR4 coreceptors—Central to understanding the transmission and pathogenics of human immunodeficiency virus type 1 infection. AIDS Res Hum Retroviruses 2004; 20:111-26. 67. Sodhi A, Montaner S, Grutkind JS. Viral hijacking of G-protein-coupled-receptor signaling networks. Nat Rev Mol Cell Biol 2004; 5:998-1012. 68. Wyatt R, Sodroski J. The HIV-1 envelope glycoproteins: Fusogens, antigens and immunogens. Science 1998; 280:1884-8. 69. Davenport MP, Zaunders JJ, Hazenberg MD et al. Cell turnover and cell tropism in HIV-1 infection. Trends Microbiol 2002; 10:275-8. 70. Centers for Disease Control and Prevention (CDC). The global HIV/AIDS pandemic, 2006. MMWR Morb Mortal Wkly Rep 2006; 55:841-4. 71. Igarashi T, Brown CR, Endo Y et al. Macrophages are the principal reservoir and sustain high virus loads in rhesus macaques after the depletion of CD4+ T-cells by a highly pathogenic simian immunodeficiency virus/HIV type 1 chimera (SHIV): Implications for HIV-1 infections of humans. Proc Natl Acad Sci USA 2001; 98:658-63. 72. Youle M. The 13th conference on retroviruses and opportunistic infections (CROI 2006). J Viral Entry 2006; 2:30-44. 73. Laskso MM, Doms RW. The molecular basis of HIV entry and its inhibition. J Viral Entry 2006; 2:4-12. 74. Klasse PJ. What is the role for entry inhibitors in microbicides for blocking transmission of HIV-1 via the cervicovaginal or rectal mucosa? J Viral Entry 2006; 2:67-72. 75. Lalezari J, Lewis S, Subramanian GM et al. Parenteral inhibitors of HIV entry in current development. J Viral Entry 2006; 2:48-56. 76. Kilby JM, Hopkins S, Venetta TM et al. Potent suppression of HIV-1 replication in humans by T-20, a peptide inhibitor of gp41-mediated virus entry. Nat Med 1998; 4:1302-7.

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77. Matthews T, Salgo M, Greenberg M et al. Enfuvirtide: The first therapy to inhibit the entry of HIV-1 into host CD4 lymphocytes. Nat Rev Drug Discov 2004; 3:215-25. 78. Zhang XQ, Sorensen M, Fung M et al. Synergistic in vitro antiretroviral activity of a humanized monoclonal anti-CD4 antibody (TNX-355) and enfuvirtide (T-20). Antimicrob Agents Chemother 2006; 50:2231-3. 79. Safarian D, Carnec X, Tsamis F et al. An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immuno-deficiency virus type 1 entry. Virology 2006; 352:477-84. 80. Si Z, Madani N, Cox JM et al. Small molecule inihibitors of HIV-1 entry block receptor-induced conformational changes in the viral envelope glycoproteins. Proc Natl Acad Sci USA 2004; 101:5036-41. 81. Madani N, Perdigoto AL, Srinivasan K et al. Localized changes in the gp120 envelope glycoprotein confers resistance to human immunodeficiency virus entry inhibitors BMS-806 and #155. J Virol 2004; 78:3742-52. 82. Hatse S, Princen K, De Clercq E et al. AMD 3465, a monomacrocyclic CXCR4 antagonist and potent HIV entry inhibitor. Biochem Pharmacol 2005; 70:752-61. 83. Dorr P, Westby M, Dodds S et al. Maraviroc (UK-427,857), a potent, orally bioavailable and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity. Antimicrob Agents Chemother 2005; 49:4721-32. 84. Stone A. Microbicidies: a new approach to preventing HIV and other sexually transmitted infections. Nat Rev Drug Discov 2002; 1:977-85. 85. Dhawan D, Mayer KH. Microbicides to prevent HIV transmission: Overcoming obstacles to chemical barrier protection. J Infect Dis 2006; 193:36-44. 86. Veazey RS, Shattock RJ, Pope M et al. Prevention of virus transmission to macaque monkeys by a vaginally applied monoclonal antibody to HIV-1 gp120. Nat Med 2003; 9:343-6. 87. Veazey RS, Klasse PJ, Schader SM et al. Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion. Nature 2005; 438:99-102. 88. Balzarini J. Inihibition of HIV entry by carbohydrate-binding proteins. Antiviral Res 2006; 71:237-47. 89. Pusch O, Boden D, Hannify S et al. Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin. J Acquir Immun Defic Syndr 2005; 40:512-20. 90. Liu X, Lagenaur LA, Simpson DA et al. Engineered vaginal lactobacillus strain for mucosal delivery of the human immunodeficiency virus inhibitor cyanovirin-N. Antimicrob Agents Chemother 2006; 50:3250-9. 91. Chang TL, Chang CH, Simpson DA et al. Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc Natl Acad Sci USA 2003; 100:11672-7. 92. Rao S, Hu S, McHugh L et al. Toward a live microbial microbicide for HIV: Commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci USA 2005; 102:11993-8. 93. Lagenaur LA, Berger EA. An anti-HIV microbicide comes alive. Proc Natl Acad Sci USA 2005; 102:12294-5. 94. Mirick GR, Bradt BM, Denardo SJ et al. A review of human anti-globulin antibody (HAGA, HAMA, HACA, HAHA) responses to monoclonal antibodies. Not four letter words. QJ Nucl Med Mol Imaging 2004; 48:251-7. 95. Stocks MR. Intrabodies: production and promise. Drug Discov Today 2004; 9:960-6. 96. Hey T, Fieldler E, Rudolph R et al. Artificial, nonantibody binding proteins for pharmaceutical and industrial applications. Trends Biotechnol 2005; 23:514-22. 97. Nygren PA, Skerra A. Binding proteins from alternative scaffolds. J Immunol Methods 2004; 290:3-28. 98. Chakrabarty A, Das Gupta T, Yamada T et al. Cupredoxin derived transport agents and methods of use thereof. US Patent 2006/0149037 A1 2006. 99. Werner RG. Economic aspects of commercial manufacture of biopharmaceuticals. J Biotechnol 2004; 113:171-82. 100. Wheatley DN. Controlling cancer by restricting arginine availability—Arginine catabolizing enzymes as anticancer agents. Anti-cancer Drugs 2004; 15:825-33. 101. Kubo M, Nishitsuji H, Kurihara K et al. Suppression of human immunodeficiency virus type 1 replication by arginine deiminase of Mycoplasma arginini. J Gen Virol 2006; 87:1589-93. 102. Izzo F, Montella M, Oriando AP et al. Pegylated arginine deiminase lowers hepatitis c viral titers and inhibits nitric oxide synthesis. J Gastroenterol Hepatol 2007; 22:86-91.

Chapter 12

Attack and Counter-Attack:

Targeted Immunomodulation Using Bacterial Virulence Factors Mohammed Bahey-El-Din, Brendan T. Griffin and Cormac G.M. Gahan*

Abstract

V

irulence factors of pathogenic bacteria by definition play a crucial role in infectivity and pathogenesis. Many of these factors have evolved to modulate the host immune response often in a manner that favours survival of the infectious agent. In particular, these immunomodulating virulence factors may specifically stimulate a T helper type 1 (Th1), a T helper type 2 (Th2) or a mixed Th1/Th2 response. Cholera toxin (CT), Escherichia coli heat-labile toxin (LT) and Bordetallapertussis filamentous haemagglutinin (FHA) usually favour a Th2 immune response. In contrast, Helicobacter pylori neutrophil-activating protein (HP-NAP) and listeriolysin O (LLO) of Listeria monocytogenes direct the immune response towards a polarised Th1 and cell-mediated immune response. Moreover, other virulence factors, such as Bordetella pertussis toxin (PT), adenylate cyclase toxin (CyaA) and Clostridium difficile toxin A, elicit a mixed Th1/Th2 response with some ability to shift the response between Th1 and Th2 depending upon the context. These immunomodulating properties suggest a potential use for these virulence factors, in a detoxified or modified form, to deliberately direct the immune response towards a desired state. Thus, certain Th1-directing or Th2-suppressing immunomodulators have potential applications in cancer therapy, allergic conditions and viral infections where a Th1 response is preferable. On the other hand, Th2-driving or Th1-suppressing immunomodulators may be used in conditions where the pathology is mainly due to an exaggerated inflammatory response as is the case in Crohn’s disease. Moreover, all of these immunomodulators are promising candidates as vaccine adjuvants when given with heterologous antigens where they can potentiate an appropriate immune response.

Introduction

Pathogenic bacteria are equipped with a variety of virulence factors by which they can infect, persist and establish overt disease. These virulence factors are the molecular tools utilised by the bacterium to enter or attach to the host, evade harsh physical and immunoprotective mechanisms and reach target host microenvironments to establish infection. In very general terms virulence factors allow the bacterium to breach innate and adaptive host responses. Thus bacteria can circumvent the innate immune system beginning with physical barriers such as the skin and respiratory cilia, chemical barriers such as gastric acidity and intestinal bile and finally the effector cells of the innate immune system (represented mainly by the polymorphonuclear (PMN) cells, monocytes and macrophages).1 Broadly speaking, the subsequent adaptive immune response against pathogens can be divided into two major types coordinated by CD4 T-lymphocyte subgroups; T helper type 1 (Th1) and T helper type 2 (Th2) responses. This categorisation depends mainly on the different cytokine *Corresponding Author: Cormac G.M. Gahan—Alimentary Pharmabiotic Centre, Department of Microbiology and School of Pharmacy, University College Cork, Cork, Ireland. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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Figure 1. Diagramatic representation of the different pathways of the adaptive immune response.

profiles produced in either case. The Th2 pathway is characterised by the secretion of high levels of the interleukins (ILs) IL-4, IL-5 and IL-10 by host immune cells.2 This profile favours the activation of specific B lymphocytes to proliferate and clonally expand and therefore promotes an antibody-mediated or humoral immune response. Alternatively, the Th1 response is mainly cell-mediated, involving the secretion of high levels of IL-12, interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α).2 Another important arm of the adaptive cell-mediated response is mediated by cytotoxic T-lymphocytes (CTL) which are mainly CD8+ lymphocytes. Upon activation, these cells have the ability to attack intracellularly infected cells or cancer cells.3 Figure 1 summarizes these major pathways of the adaptive immune response. The immune response towards pathogens may be Th1, Th2 or a mixed Th1/Th2 response depending on the production of various pathogen- and host-related factors. Pathogenic virulence factors in particular play a crucial role in stimulating the immune response in a direction that is most favourable for the pathogen. Recent advances in immunology and biotechnology have focused upon the characterization of many of these factors and the mechanisms by which many bacterial virulence factors modulate the immune system are now more clearly understood. In this chapter, a number of bacterial virulence factors are discussed that have the ability to modulate the host immune response. These immunomodulators have potential medical applications that are currently the subject of significant investigation. Here we focus specifically on eight virulence factors that have been characterized in some detail and provide significant potential for biomedical applications (summarized in Table 1).

Vibrio cholerae Toxin (CT) and Escherichia coli Heat-Labile Enterotoxin (LT)

Vibrio cholerae toxin, or more commonly Cholera toxin (CT), is a well documented immunomodulator and mucosal vaccine adjuvant. CT is composed of two subunits; subunit A which is responsible for the toxicity and possesses ADP-ribosyltransferase activity and subunit B (CTB)

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Table 1. Summary of common bacterial virulence factors that have immunomodulating properties Immunomodulator

Target Immune Pathway

References

Cholera toxin (CT)

Mostly Th2-stimulator, sometimes mixed Th1/Th2 stimulator Th2-stimulator Mixed Th1/Th2 stimulator Th1- and CTL-stimulator

5-7,9 9 10 16-18,20,21

Th1-suppressant

27,29

Th1/Th2 stimulator Th1- and CTL-directing, sometimes Th2-stimulator Neutrophil activator and mostly Th1-stimulator CTL-directing CTL-directing Th1-stimulator

30 32-35

Cholera toxin subunit B (CTB) E. coli heat-labile toxin (LT) Listeriolysin O (LLO) of Listeria monocytogenes B. pertussis filamentous haemagglutinin (FHA) B. pertussis pertussis toxin (PT) B. pertussis adenylate cyclase (CyaA) Clostridium difficile toxin A Pseudomonas toxin Anthrax toxin Helicobacter pylori neutrophilactivating protein (HP-NAP)

37-39 47 47 45,46

which has a pentameric structure. CTB is responsible for the initial binding of the toxin to its receptor which has been identified as the glycosphingolipid, GM1-ganglioside, on the surface of the intestinal epithelial cells. Subsequently, subunit A triggers a cascade of intracellular events leading to elevated cellular cyclic AMP (cAMP) and cell toxicity.4 Cholera toxin is known for its strong immunomodulating effects. It suppresses IL-12 production by monocytes and also inhibits the expression of the β1 and β2 chains of IL-12 T-cell receptor, with the net effect of a significant suppression of the Th1 immune response.5 Moreover, when CT is co-administered via the mucosal route with other antigens, it stimulates a Th2 response by enhancing the levels of IL-4, IL-5 and IL-10. Preferential production of high antigen-specific serum IgE and IgG1 (reflective of a Th2 response), rather than the IgG2a (Th1 response) isotype has also been reported.6,7 Binding of CTB to its receptor was found to be important for CT adjuvant activity. However, the nontoxic CTB when mixed with other antigens, was not as efficient as whole CT vaccine adjuvant. In most studies, antigens combined with CTB and CT adjuvants produced a Th2-biased response with its characteristic cytokine profile and IgG1 isotype. However, it was also reported in some cases that CT could produce specific CD8+ cytotoxic and Th1 responses.8,9 When dendritic cells were pulsed with ovalbumin (OVA) chemically linked to CTB (OVA-CTB) or CT (OVA-CT) and then injected into mice, significantly different immune outcomes were observed. While OVA-CTB favoured a Th2 response against OVA, OVA-CT produced both Th1 and Th2 responses as evidenced from the cytokine profile and IgG isotype.9 Escherichia coli heat-labile enterotoxin (LT) is similar in structure (80% homology) and action to CT but exhibits lower toxicity. It is secreted by enterotoxigenic E. coli, the causative organism of traveller’s diarrhoea. LT is reported to elicit a mixed Th1/Th2 response as shown from cytokine and antibody profiles.10 Since both CT and LT have high toxicity which prevents their use as adjuvants especially in mucosal vaccines, mutants of both toxins have been created. Several nontoxic or less-toxic mutants of LT and CT have been generated from which two LT-mutant candidates, LTK63 and LTR72, proved to be promising. It was found that LTK63, a nontoxic mutant, enhanced a mixed Th1/Th2

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response against co-administered antigens, while LTR72, a partially detoxified mutant, favoured a Th2 response with Th1 suppression.6 Significantly, LTK63 is currently in phase I clinical trials as a mucosal adjuvant in an intranasal influenza vaccine.11

Listeria monocytogenes Listeriolysin O (LLO)

Listeria monocytogenes is an intracellular Gram-positive bacterium responsible for abortion in pregnant women and meningitis in the newborn and immunocompromised adults. The pathogen is transmitted through ingestion of contaminated food and once infection is established mortality rates are alarmingly high (∼30%). L. monocytogenes has unique virulence factors which enable it to survive and escape from the cell phagolysosome allowing growth and muliplication in the host cell cytoplasm.12 The major virulence factor of L. monocytogenes is the haemolysin listeriolysin O (LLO). Upon internalisation of the bacterium inside the phagolysosome, rapid acidification (∼pH 5.5) activates LLO , where it interacts with the membrane cholesterol, oligomerises and produces pores in the phagolysosomal membrane permitting bacterial escape to the cytoplasm.13 Due to the intracellular location of the pathogen, immunity against L. monocytogenes, depends mainly on the development of cell-mediated immunity rather than humoral immunity. This protective cell-mediated immunity mainly comprises cytotoxic CD8+ T-lymphocytes which develop against potential listerial virulence factors like LLO and P60.14 The unique ability of LLO to facilitate the escape of the bacterium from the phagosomal compartment to the cytosol has been the focus of intensive research. This translocation to the cytosol allows the processing of bacterial antigens through the major histocompatibility complex (MHC) class I pathway which leads eventually to the development of antigen-specific cytotoxic CD8+ T-cells.15 In other words, LLO can direct the processing of colocalised antigens towards the MHC class I pathway by facilitating their escape to the cytosol where they are proteasome processed. Thereafter, processed antigens are transported by the transporter associated with antigen processing (TAP) protein to the endoplasmic reticulum where they combine with the MHC class I complex and translocate to the cell surface of antigen presenting cells for recognition by CD8+ T-cells.14 Given this potent ability to redirect host immune responses, attenuated mutant strains of L. monocytogenes secreting LLO have been examined as vaccine candidates against Listeria or other pathogens whose antigens are coexpressed by the Listeria mutant. For instance, studies have demonstrated that specific CD8+ responses are elicited against lymphocytic choriomeningitis virus (LCMV) and influenza virus type A when the corresponding antigens are expressed in an LLO+ L. monocytogenes background.16,17 When LLO was expressed in the mycobacterium bacille Calmette-Guérin (BCG) strain, it increased the protective efficacy of the vaccine strain against tuberculosis due to better antigen presentation through the cytosolic MHC class I pathway.18 This recombinant vaccine is planned to enter phase I clinical trials in late 2007.19 Listeriolysin O has also been incorporated into liposomal delivery systems along with other antigens. The bioactive properties of LLO succeeded in eliciting a CTL-response against coadministered ovalbumin (OVA) as a model antigen.20 Furthermore, a liposome-LLO system could produce specific CD8+ T-cells against the nucleoprotein (NP) antigen of LCMV when the antigen was contained in the same liposomal preparation.21 Another liposomal-LLO system could successfully deliver the anti-tumor toxin gelonin to the cytosolic compartment and achieved improved gelonin anti-cancer cytotoxicity both in vitro and in vivo.22 The specific immunomodulatory properties of LLO have also been exploited in the creation of DNA vaccines. The use of eukaryotic DNA vectors coding for the C-terminal part of LLO, responsible for membrane cholesterol binding and thus subsequent phagosomal permeabilization, fused to SIV Gag antigen or the envelope gp140 antigen of human immunodeficiency virus (HIV) elicited strong specific CD4+ and CD8+ immune responses.23,24 LLO also has a potential stimulatory effect on cell-mediated immunity and the Th1 response and induces high levels of IL-12, IL-18 and IFN-γ. These properties were beneficial in a murine

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ovalbumin allergic rhinitis model where LLO treatment suppressed nasal mononuclear production of IL-4 and IL-5. Furthermore, upon challenge with OVA, inhibition of anti-OVA IgE and allergic symptoms was observed.25 As seen from the data above, LLO has a wide variety of applications from live attenuated vaccine strains and acellular liposomal preparations to naked DNA vaccines. All these applications depend upon the capacity of LLO to permeabilize the phagosomal compartment, thus facilitating antigen escape to the cytosol to be presented through the MHC class I pathway. This valuable characteristic of LLO attracts substantial research investigations in areas where cell-mediated immunity is required including viral and intracellular bacterial infections, as well as cancer and allergic conditions.

Bordetella pertussis Virulence Factors

Bordetella pertussis is a Gram-negative bacterium responsible for the childhood respiratory disease whooping cough. Pathogenicity of B. pertussis depends on several virulence factors including the pertussis toxin (PT), the filamentous haemagglutinin (FHA) and adenylate cyclase (CyaA). Host immune clearance of B. pertussis infection relies mainly on a cell-mediated immune response rather than antibody-mediated immunity. The filamentous haemagglutinin (FHA) is an important virulence factor which is responsible primarily for adhesion of B. pertussis to respiratory cilia and macrophages by interaction with integrin receptors.26 However, FHA has also been found to be involved in the initial suppression of a Th1 response against B. pertussis. In vitro, FHA suppressed the level of the Th1-inducing cytokine IL-12 and increased the levels of IL-10 and IL-6 produced by macrophages after treatment with lipopolysaccharide (LPS) and IFN-γ. Moreover, when FHA was injected into mice, it suppressed serum levels of IL-12 and IFN-γ following LPS intravenous injection in a murine septic shock model.27 It is noteworthy that FHA was found to induce specific T regulatory type 1 (Tr1) cells which secreted high levels of IL-10.28 The aforementioned findings encouraged Braat H and coworkers29 to investigate the effect of FHA on inflammatory bowel conditions such as Crohn’s disease whose pathology depends on an exaggerated cell-mediated immune response. FHA was injected subcutaneously into severe combined immunodeficient (SCID) mice with induced-colitis. Colitis manifestations were significantly alleviated in FHA-treated mice when compared to the control group. FHA was also found to stimulate the production of IL-10 and transforming growth factor-beta (TGF-β) in serum, lymph nodes and peyer’s patches of FHA-treated BALB/c mice.29 Thus, the Th1-suppressing properties of FHA may establish a promising therapeutic modality for Crohn’s disease which is worthy of further investigation. Another important virulence factor of B. pertussis is the pertussis toxin (PT). It is one of a family of ADP-ribosyltransferase toxins which also includes CT and LT. These ADP-ribosyltransferase toxins have a similar structure with two subunits; subunit A, responsible for the ADP-ribosylating activity and toxicity and the pentameric subunit B responsible for toxin binding. Pertussis toxin (PT) is believed to stimulate both Th1 and Th2 immune responses against coadministered antigens. When keyhole limpet haemocyanin (KLH) or B. pertussis filamentous haemagglutinin (FHA) were injected with PT in a murine model a pronounced mixed Th1/Th2 response was observed.30 High levels of IFN-γ, IL-2, IL-4 and IL-5 were produced by T cells. Moreover, both antigen-specific IgG1 and IgG2a isotypes were observed against coadministered antigens suggesting both Th1 and Th2 responses. The production of the Th2 cytokines (IL-4, IL-5) was mainly due to the effect of the upregulated IL-1 on Th2 cells. Similar strong adjuvant activities were observed with a mutated PT lacking the ADP-ribosylating activity but with functional B subunit.30 The third immunomodulatory virulence factor of B. pertussis is adenylate cyclase (CyaA). Adenylate cyclase binds specifically to CD11b/CD18 on dendritic cells and delivers its catalytic domain into the cytosol which subsequently binds to calmodulin, leading eventually to exaggerated high levels of cyclic AMP (cAMP) and cell toxicity.31 This cytoplasmic delivery of the catalytic domain to the cytosol drew the attention to the possible use of CyaA to target MHC class I and

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even class II, presentation pathways by linking foreign antigens to the catalytic domain. CyaA was found to promote Th1 and CTL responses or a Th2 response dependent upon the accompanying antigen. When CyaA was coadministered with keyhole limpet hemocyanin (KLH), high levels of IL-10, IL-4 and IL-5 were produced by murine lymph node cells and the serum IgG1 isotype was dominant indicative of a Th2 response.32 On the other hand, production of human papilloma virus 16 (HPV16)-specific CD8+ and CD4+ cells was attained when the E7 oncoprotein or small segments from it were fused inside the catalytic domain of recombinant enzymatically inactive, i.e., nontoxic, CyaA. High levels of IFN-γ and specific cytotoxic T-cells (CTL) and low IL-5 levels were observed in treated mice. Moreover, eradication of established HPV16-E7-expressing tumors was accomplished upon injection of the fused CyaA-E7 protein in the murine model.33 Finally, Mascarell et al could detect a specific Th1 response and high affinity neutralising antibodies against the human immunodeficiency virus-1 (HIV-1) Tat protein when it was genetically fused to a detoxified CyaA mutant both in murine and nonhuman primate models.34,35 In summary, B. pertussis offers a collection of immunomodulators which are currently proving useful tools in redirecting the immune response and as vaccine adjuvants.

Clostridium difficile Toxin A

Clostridium difficile is an anaerobic Gram-positive bacterium responsible for antibiotic-associated diarrhoea and pseudomembranous colitis. C. difficile causes these disorders after extensive therapy with broad spectrum antibiotics, a situation which disrupts the normal gut microbiota and impairs colonization resistance.36 The pathogenicity of C. difficile relies mainly on two secreted toxins: toxin A and toxin B. Toxin A is a potent enterotoxin causing acute mucosal inflammation of the colon and severe diarrhoea. Toxin B is a cytotoxic and apoptotic protein mostly lacking the enterotoxic properties of toxin A. These toxins have significant structural homology and each is composed of three main domains; a C-terminus receptor binding domain, a middle hydrophobic translocation domain and an N-terminus enzymatic and catalytic domain.36 The mechanism of action of toxin A depends initially on binding to oligosaccharide residues on epithelial cell surfaces via its C-terminus which is composed of repetitive peptide elements. Subsequently, toxin A is translocated into the cytosol where the toxin binds to its intracellular targets which are mainly the Rho proteins. The Rho proteins are a group of GTPases responsible for the regulation of cell actin polymerisation and fibre assembly. As a consequence, depolymerisation of cellular actin, disruption of cell cytoskeleton and cell rounding occur.36 Also by disrupting the Rho functions, the gut epithelial tight-junctions are opened and gut permeability is significantly increased. A major feature of toxin A-mediated pathology is the significant neutrophil infiltration through the affected mucosa, resulting in pronounced inflammation of intestinal tissue. It was found that toxin A stimulates monocytes and macrophages to secrete IL-8 which is a potent neutrophil chemoattractant.37 Moreover, Rocha et al 38 found that toxin A induced the release of IL-1β and TNF-α from rat peritoneal macrophages resulting in high neutrophil migration which was reduced by prior treatment with anti-IL-1β and anti-TNF-α antibodies. This suggests that neutrophil migration is partly dependent on these macrophage-secreted cytokines. In addition, the toxin A-affected intestinal epithelial cells secrete high amounts of IL-8 and the Monocyte-Chemotactic Protein-1 (MCP-1) which are upregulated in these cells through the NF-κB activation pathway.39 Since toxin A has these potent immunostimulatory effects, it has been investigated as an immunomodulator and vaccine adjuvant. The homology between the C-terminus of toxin A and other known potential immuno-adjuvants like Cholera toxin (CT) and E. coli heat-labile enterotoxin subunit B (EtxB), encouraged Castagliuolo et al to investigate toxin A immunomodulating effects.40 The nontoxigenic peptide comprising amino acids 2394 to 2706 (TxAc314) of the C-terminus of toxin A was expressed in E. coli and purified by affinity chromatography. Thereafter, TxAc314 was coadministered with the weak antigens, keyhole limpet hemocyanin (KLH) and hen egg lysozyme (HEL), by the oral and nasotracheal routes respectively. A significant rise in specific serum IgG and IgA antibodies against KLH was observed when coadministered orally with

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TxAc314 as compared to KLH alone, KLH plus CT and KLH plus EtxB.40 Upon examining IgG subclasses, it was found that the presence of TxAc314 produced significant rise in both IgG1 and IgG2a titers against KLH with more pronounced effect on IgG2a. Therefore, when given orally TxAc314 stimulates both the Th1 and Th2 arms of immune response with a more profound effect on the Th1 response.40 This was further highlighted by the high levels of IFN-γ and IL-2 and low levels of IL-4 and IL-10 in culture supernatants of KLH-stimulated splenocytes obtained from KLH/TxAc314 immunized mice. Furthermore, when TxAc314 was given intranasally with hen egg lysozyme (HEL), it produced significantly higher titers of HEL-specific serum IgG (both IgG1 and IgG2a) and IgA than HEL alone with comparable titers to those produced by intranasal HEL plus CT and HEL plus EtxB.40 The current data about C. difficile toxin A suggest that it is a good mucosal adjuvant and immunomodulator worthy of further investigation and toxicity assessment. Moreover, being a stimulator of a mixed Th1/Th2 immune response with preference to the Th1 arm makes toxin A a potential adjuvant for vaccination against viral and intracellular pathogens.

Helicobacter pylori Neutrophil-Activating Protein (HP-NAP)

Helicobacter pylori is a Gram-negative bacterium which has been implicated in many cases of gastric ulcers and gastric cancer worldwide.41 Various virulence factors are produced by H. pylori which are collectively responsible for its pathogenicity including the vacuolating cytotoxin (VacA), CagA protein and HP-NAP.42,43 Of these virulence factors, H. pylori neutrophil-activating protein (HP-NAP) has recently been the focus of research due to its unique immunomodulating properties. It has been found that HP-NAP is a major factor responsible for the inflammatory and Th1 immune response elicited against H. pylori. As understood from its name, it acts as a neutrophil-chemotactic and neutrophil-activating protein which induces the neutrophils to produce high amounts of free oxygen radicals.44 Upon H. pylori infection, large numbers of polymorphonuclear (PMN) leucocytes, monocytes and lymphocytes migrate to the site of infection and infiltrate through the gastric mucosa. The degree of damage to the mucosa is largely proportional to the degree of neutrophil infiltration. HP-NAP was found to stimulate the production of IL-12 and IL-23 from both neutrophils and monocytes. This effect was due to a specific interaction with Toll-like receptor 2 (TLR2) leading to upregulation of both IL-12p35 and IL-12p40 which combine together to form active IL-12 and also IL-23p19 which combines with IL-12p40 to form active IL-23.45 These cytokines establish an environment that favours the Th1 arm of the immune response rather than the Th2 immune response. Not only does HP-NAP enhance the Th1 response but also it negatively affects the Th2 response. This was evidenced by the measurement of the Th1 and Th2 cytokines (IFN-γ and IL-4 respectively) produced by the Tetanus Toxoid (TT)-specific T-lymphocytes in the presence and absence of HP-NAP. Although the immune response against TT is known to be mainly a Th2 response characterized by a high level of IL-4 secretion by TT-specific CD4+ cells, HP-NAP could successfully switch this response to a mostly polarized Th1 response with high levels of IFN-γ secretion.45 A similar response was also observed with the house dust mite allergen where an appreciable shift from Th2 to Th1 response occurred upon conditioning of the allergen-specific clones with HP-NAP. Moreover, high levels of TNF-α were produced by allergen-specific T-cell clones conditioned with HP-NAP and the cytotoxic activity of these HP-NAP-conditioned cells was elevated as measured using the 51Cr-release assay with P815 murine mastocytoma cells as targets.45,46 These findings suggest that HP-NAP is a new, potentially important Th1-directing immunomodulator. Since certain pathological conditions (such as allergen atopy) are mainly due to an exaggerated Th2 response, the Th2-downregulating effect of HP-NAP may be of benefit to alleviate these conditions. Also the enhanced Th1 response produced by HP-NAP can be useful in conditions where Th1-mediated immunity is required as is the case in cancer immunotherapy and vaccination against intracellular pathogens.

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Figure 2. Diagram showing possible applications of immunomodulating bacterial virulence factors.

Conclusions

Bacterial virulence factors enable pathogens to persist and adapt to the host environment, often with specific effects on the host immune response. The immunomodulatory potential of these factors suggests that they may be developed as precise molecular weapons to counter-attack other invading pathogens. Indeed, a variety of pathological conditions, including those of infectious or non-infectious aetiology, are due to an improper or imbalanced immune response resulting in a diseased state. The immunomodulating virulence factors discussed above provide excellent tools with which the immune response can be redirected to reach a point where disease symptoms are alleviated. Figure 2 summarizes the possible uses of these immunomoduloators based on their immune targets.

References

1. Rabinovitch M. Professional and nonprofessional phagocytes: An introduction. Trends Cell Biol 1995; 5:85-87. 2. Kidd P. Th1/Th2 balance: the hypothesis, its limitations and implications for health and disease. Altern Med Rev 2003; 8:223-246. 3. Groothuis TA, Neefjes J. The many roads to cross-presentation. J Exp Med 2005; 202:1313-1318. 4. Sanchez J, Holmgren J. Virulence factors, pathogenesis and vaccine protection in cholera and ETEC diarrhea. Curr Opin Immunol 2005; 17:388-398. 5. Braun MC, He J, Wu CY et al. Cholera toxin suppresses interleukin (IL)-12 production and IL-12 receptor beta1 and beta2 chain expression. J Exp Med 1999; 189:541-552. 6. Pizza M, Giuliani MM, Fontana MR et al. Mucosal vaccines: Non toxic derivatives of LT and CT as mucosal adjuvants. Vaccine 2001; 19:2534-2541. 7. Marinaro M, Staats HF, Hiroi T et al. Mucosal adjuvant effect of cholera toxin in mice results from induction of T helper 2 (Th2) cells and IL-4. J Immunol 1995; 155:4621-4629. 8. Porgador A, Staats HF, Faiola B et al. Intranasal immunization with CTL epitope peptides from HIV-1 or ovalbumin and the mucosal adjuvant cholera toxin induces peptide-specific CTLs and protection against tumor development in vivo. J Immunol 1997; 158:834-841.

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9. Eriksson K, Fredriksson M, Nordstrom I et al. Cholera toxin and its B subunit promote dendritic cell vaccination with different influences on Th1 and Th2 development. Infect Immun 2003; 71:1740-1747. 10. Takahashi I, Marinaro M, Kiyono H et al. Mechanisms for mucosal immunogenicity and adjuvancy of Escherichia coli labile enterotoxin. J Infect Dis 1996; 173:627-635. 11. Stephenson I, Zambon MC, Rudin A et al. Phase I evaluation of intranasal trivalent inactivated influenza vaccine with nontoxigenic Escherichia coli enterotoxin and novel biovector as mucosal adjuvants, using adult volunteers. J Virol 2006; 80:4962-4970. 12. Meng J, Doyle MP. Emerging issues in microbiological food safety. Annu Rev Nutr 1997; 17:255-275. 13. Vazquez-Boland JA, Kuhn M, Berche P et al. Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 2001; 14:584-640. 14. Pamer EG. Immune responses to Listeria monocytogenes. Nat Rev Immunol 2004; 4:812-823. 15. Dietrich G, Hess J, Gentschev I et al. From evil to good: A cytolysin in vaccine development. Trends Microbiol 2001; 9:23-28. 16. Goossens PL, Milon G, Cossart P et al. Attenuated Listeria monocytogenes as a live vector for induction of CD8+ T-cells in vivo: a study with the nucleoprotein of the lymphocytic choriomeningitis virus. Int Immunol 1995; 7:797-805. 17. Ikonomidis G, Portnoy DA, Gerhard W et al. Influenza-specific immunity induced by recombinant Listeria monocytogenes vaccines. Vaccine 1997; 15:433-440. 18. Grode L, Seiler P, Baumann S et al. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin. J Clin Invest 2005; 115:2472-2479. 19. Andersen P. Tuberculosis vaccines—An update. Nat Rev Microbiol 2007; 5:484-487. 20. Lee KD, Oh YK, Portnoy DA et al. Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes. J Biol Chem 1996; 271:7249-7252. 21. Mandal M, Kawamura KS, Wherry EJ et al. Cytosolic delivery of viral nucleoprotein by listeriolysin O-liposome induces enhanced specific cytotoxic T-lymphocyte response and protective immunity. Mol Pharm 2004; 1:2-8. 22. Provoda CJ, Stier EM, Lee KD. Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin. J Biol Chem 2003; 278:35102-35108. 23. Ye L, Bu Z, Skeen MJ et al. Enhanced immunogenicity of SIV Gag DNA vaccines encoding chimeric proteins containing a C-terminal segment of Listeriolysin O. Virus Res 2003; 97:7-16. 24. Bu Z, Ye L, Skeen MJ et al. Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listeriolysin O. AIDS Res Hum Retroviruses 2003; 19:409-420. 25. Yamamoto K, Kawamura I, Tominaga T et al. Listeriolysin O derived from Listeria monocytogenes inhibits the effector phase of an experimental allergic rhinitis induced by ovalbumin in mice. Clin Exp Immunol 2006; 144:475-484. 26. Relman D, Tuomanen E, Falkow S et al. Recognition of a bacterial adhesion by an integrin: Macrophage CR3 (alpha M beta 2, CD11b/CD18) binds filamentous hemagglutinin of Bordetella pertussis. Cell 1990; 61:1375-1382. 27. McGuirk P, Mills KH. Direct anti-inflammatory effect of a bacterial virulence factor: IL-10-dependent suppression of IL-12 production by filamentous hemagglutinin from Bordetella pertussis. Eur J Immunol 2000; 30:415-422. 28. McGuirk P, McCann C, Mills KH. Pathogen-specific T regulatory 1 cells induced in the respiratory tract by a bacterial molecule that stimulates interleukin 10 production by dendritic cells: A novel strategy for evasion of protective T helper type 1 responses by Bordetella pertussis. J Exp Med 2002; 195:221-231. 29. Braat H, McGuirk P, Ten Kate FJ et al. Prevention of experimental colitis by parenteral administration of a pathogen-derived immunomodulatory molecule. Gut 2007; 56:351-357. 30. Ryan M, McCarthy L, Rappuoli R et al. Pertussis toxin potentiates Th1 and Th2 responses to co-injected antigen: adjuvant action is associated with enhanced regulatory cytokine production and expression of the costimulatory molecules B7-1, B7-2 and CD28. Int Immunol 1998; 10:651-662. 31. El-Azami-El-Idrissi M, Bauche C, Loucka J et al. Interaction of Bordetella pertussis adenylate cyclase with CD11b/CD18: Role of toxin acylation and identification of the main integrin interaction domain. J Biol Chem 2003; 278:38514-38521. 32. Boyd AP, Ross PJ, Conroy H et al. Bordetella pertussis adenylate cyclase toxin modulates innate and adaptive immune responses: distinct roles for acylation and enzymatic activity in immunomodulation and cell death. J Immunol 2005; 175:730-738. 33. Preville X, Ladant D, Timmerman B et al. Eradication of established tumors by vaccination with recombinant Bordetella pertussis adenylate cyclase carrying the human papillomavirus 16 E7 oncoprotein. Cancer Res 2005; 65:641-649.

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34. Mascarell L, Fayolle C, Bauche C et al. Induction of neutralizing antibodies and Th1-polarized and CD4-independent CD8+ T-cell responses following delivery of human immunodeficiency virus type 1 Tat protein by recombinant adenylate cyclase of Bordetella pertussis. J Virol 2005; 79:9872-9884. 35. Mascarell L, Bauche C, Fayolle C et al. Delivery of the HIV-1 Tat protein to dendritic cells by the CyaA vector induces specific Th1 responses and high affinity neutralizing antibodies in non human primates. Vaccine 2006; 24:3490-3499. 36. Poxton IR, McCoubrey J, Blair G. The pathogenicity of Clostridium difficile. Clin Microbiol Infect 2001; 7:421-427. 37. Linevsky JK, Pothoulakis C, Keates S et al. IL-8 release and neutrophil activation by Clostridium difficile toxin-exposed human monocytes. Am J Physiol 1997; 273:G1333-1340. 38. Rocha MF, Maia ME, Bezerra LR et al. Clostridium difficile toxin A induces the release of neutrophil chemotactic factors from rat peritoneal macrophages: Role of interleukin-1beta, tumor necrosis factor alpha and leukotrienes. Infect Immun 1997; 65:2740-2746. 39. Kim JM, Lee JY, Yoon YM et al. NF-kappa B activation pathway is essential for the chemokine expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A. Scand J Immunol 2006; 63:453-460. 40. Castagliuolo I, Sardina M, Brun P et al. Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant. Infect Immun 2004; 72:2827-2836. 41. Malaty HM. Epidemiology of Helicobacter pylori infection. Best Pract Res Clin Gastroenterol 2007; 21:205-214. 42. Montecucco C, de Bernard M. Molecular and cellular mechanisms of action of the vacuolating cytotoxin (VacA) and neutrophil-activating protein (HP-NAP) virulence factors of Helicobacter pylori. Microbes Infect 2003; 5:715-721. 43. Shibata W, Hirata Y, Maeda S et al. CagA protein secreted by the intact type IV secretion system leads to gastric epithelial inflammation in the Mongolian gerbil model. J Pathol 2006; 210:306-314. 44. Evans DJ Jr, Evans DG, Takemura T et al. Characterization of a Helicobacter pylori neutrophil-activating protein. Infect Immun 1995; 63:2213-2220. 45. Amedei A, Cappon A, Codolo G et al. The neutrophil-activating protein of Helicobacter pylori promotes Th1 immune responses. J Clin Invest 2006; 116:1092-1101. 46. D’Elios MM, Amedei A, Cappon A et al. The neutrophil-activating protein of Helicobacter pylori (HP-NAP) as an immune modulating agent. FEMS Immunol Med Microbiol 2007; 50:157-164. 47. Goletz TJ, Klimpel KR, Leppla SH et al. Delivery of antigens to the MHC class I pathway using bacterial toxins. Hum Immunol 1997; 54:129-136.

Chapter 13

Cosmetic and Therapeutic Applications of Botulinum Toxin in the Head and Neck Nwanmegha Young and Andrew Blitzer*

Background

B

otulinum toxin is the most potent neurotoxin known to man. It is produced by the anaerobic bacteria Clostridium botulinum. In the early nineteenth century, it was responsible for large outbreaks of botulism , a systemic food poisoning. In the twentieth century, botulinum toxin has developed from man’s most powerful poison into a tool used to treat in a variety of disorders. In the early 21st century the understanding and clinical applications of botulinum toxin have exploded. It’s indications now span a wide range of medical and surgical specialties.

Pathophysiology

Botulinum toxin (BTX) is a polypeptide with a 100-kDa heavy chain joined by a disulfide bond to a 50-kDa light chain.1 There are at least seven serologically distinct toxin types, designated A through G. All inhibit the release of acetylcholine, although their intracellular target proteins, the characteristics of their actions and their potencies vary substantially.2 BTX-A has been the most widely studied toxin and has been found to be very successful for therapeutic purposes. BTX-B is also commercially available but its effects are less prolonged and much higher protein load may be are required.3-5 BTX mechanism of action of occurs at the presynaptic neuron. The synaptic vesicle contains neurotransmitters that are released into the synapse via exocytosis. Vesicular exocytosis is regulated by SNARE (Soluble NSF Attachment Receptor) proteins, which mediates fusion of vesicles with the cell membrane. The light chain of the toxin cleaves the SNARE protein complex. The light chain of each serotype cleaves different proteins in the SNARE complex. When the SNARE proteins are cleaved, its complex doesn’t move the vesicle to the plasma membrane to allow for exocytosis of acetyl choline into the neuromuscular junction This inhibition of acetylcholine release, interferes with nerve impulse transmission and causes flaccid paralysis of muscles affected. This has implications for its therapeutic applications in hyperactive muscle disorders. The SNARE complex is highly conserved and is involved with vesicular release in other systems besides the neuromuscular junction. In autonomic neurons, BTX also blocks acetylcholine release.6 These autonomic neurons include efferent neurons innervating exocrine glands, such as sweat glands, salivary glands, lacrimal glands and smooth muscles. This provides the rationale for the therapeutic use of BTX for focal hyperhidrosis, hypersialorrhea and hyperlacrimation.7 Acetylcholine is not the only neurotransmitter affected by BTX. BTX also affects the release of substance P, CGRP, glutamate ,neuropeptides involved in neurogenic inflammation and the genesis of pain disorders The release of these neuropeptides is also requires the SNARE protein activity.8,9 Inhibition of substance P release by BTX was demonstrated in the iris muscles of rabbits as well as in cultured dorsal root ganglion neurons.10,11 It also has also been shown to suppress the release of glutamate, *Corresponding Author: Andrew Blitzer—Head and Neck Surgical Group and NY Center for Voice and Swallowing Disorders, 425 West 59th Street, New York, NY 10019, USA. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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a neurotransmitter involved in nociception in the periphery and in the dorsal horn of the spinal cord.12,13 BTX typically works between 2-10 days, reaching a peak effect within 2 weeks. The typical duration of action is 3-4 months with considerable variation in individual responses.

Clinical Applications

There is an ever expanding diversity of disorders treated by BTX in the head and neck. Since BTX affects the regulation of exocytosis of neurotansmitters, therefore innervation, it can be used for a variety of purposes in the head and neck. The disorders of the head and neck that respond to BTX therapy include cosmetic, muscle hyperactivity, hypersecretory and nociceptive disorders.

Cosmetic Applications

Botulinum therapy for cosmetic purposes is very popular in present day society. Most of the use is to treat hyperdynamic lines or rhytids. Hyperdynamic lines or rhytids result from the combination of aging skin and increasingly active underlying musculature. The underlying aponeurosis of the facial muscles have a perpendicular attachment to the dermis of skin. The movements of these muscles cause wrinkles of the overlying skin. Over the years, repetitive wrinkling of the skin, in addition loss of elasticity, causes permanent creases or deep rhytids on the face. These are most prominent in the area around the large muscles of the eyes (Crow’s feet lines), glabella or frown lines, forehead, perioral lines and neck bands or platysma muscle. BTX causes weakness of the muscles allowing the superficial dermis to relax and relieve wrinkles. BTX has also been used to cause atrophy of muscles (such as the masseters) to change the overall shape of the face and to relieve involuntary twitching.

Uses in the Upper Face Glabella

Frown lines in the glabella region are caused by two different muscles. The vertical lines between the eyebrows result from contraction of the corrugator muscles, while the horizontal rhytids at the root of the nose are due to contraction of the procerus muscle. Usually 5 injections sites are used with a dose range average of 15-25 units. Side effects include levator ptsosis and bruising (Fig. 1A,B).

Periorbital Region

The orbicularis oculi is responsible for the lateral canthal lines (crows feet). Typically 2-3 injections are done per side on the orbital rim. Average dose 7.5-15 units. Side effects include lid ptosis, diplopia (from paresis of lateral rectus muscle) and bruising (Fig. 2A,B).

Forehead

Common rhytids of the aging forehead include the horizontal rhytids due to the action of the frontalis. Typically there 6-8 injection points 1.5-2 cm apart. Laterally, the line of the injections is raised away from the brow to maintain some function of expression in the lateral part of the brow. Average doses range from 14-20 units. Side effects include lid and brow ptosis, (avoid injecting to close to the brow) and cockeyed brow.

Uses in the Lower Face and Neck Perioral Lines

Vertical lines form around the lips, especially in smokers, from repetitive puckering and sucking. Very small doses should be used (4-10 units, maximum 4 sites) to help relieve these wrinkles. Often, injectable fillers are added to correct this cosmetic deformity. Side effects include asymmetric smile and oral incompetence if too much weakness occurs.

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Figure 1. A) Demonstrates vertical frown lines caused by the corrugator muscle. B) The same patient after injection of botulinum toxin in the muscle.

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Figure 2. A) Demonstrates lateral canthal lines produced by the action of the orbicularis oculi muscle. B) The same patient after treament with botulinum toxin.

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Mental Creases

Peau De Orange or popply chin is caused by the mentalis muscle. Injection should be above the tip of chin. Usually two injections per side used for an average dose of 5 units per muscle. Side effects asymmetry of mouth movement.

Platysma Bands

The lateral bands of the platysma muscle facilitate facial expression by lowering the corners of the lower lip. Its posterior fibers continue superiorly to join the superficial musculoaponeurotic system (SMAS) of the face. Age-related downward pull of the platysma muscle creates vertical fibrous bands. The skin laxity over the platysma muscle produces horizontal rhytids. After preparing the skin, ask the patient to contract the platysma muscle; this identifies its bands. Typically injections are along the length of a band at 1.0 to 1.5 cm intervals from the jaw line to the lower neck. Approximately 3-10 units may be injected, depending on the thickness of the platysmal band. Most patients require a total of 20-50 units per side and some require as many as 100 units. Side effects are minimal and may include transient edema and ecchymoses, hematoma formation, muscle soreness and mild neck weakness. If the injections are given too deep, or too high a dose, it is possible to get weakness of the underlying strap muscles, causing dysphagia (Fig. 3A,B).

Other Cosmetic Uses Masseter Hypertrophy

The etiology of benign masseteric and temporalis hypertrophy is generally unknown. Some believe it is due to work hypertrophy, such as in habitual jaw clenching or teeth grinding.14-17 Interestingly, benign masseteric hypertrophy is frequently found among Korean persons who favor chewing dried squid, a tough and chewy delicacy. Masseter hypertrophy is largely regarded as a cosmetic facial deformity though some report pain associated with this condition.18 Treatment with BTX produces decreased force on chewing and clenching and a reduced use muscle atrophy.19-21 Typically 25 units are injected per side. Side effects include dyshagia (Fig. 4).

Facial Synkinesis

Facial synkinesis is the involuntary movement of facial muscles that accompanies purposeful movement of some other set of muscles; for Facial synkinesis can follow any injury or condition causing injury of the facial nerve. The most common associated disorder is Bell’s palsy; about 40% of all individuals who are recovering from Bell’s palsy will experience facial synkinesis during recovery.22 Other conditions that may prompt the development of facial synkinesis include stroke, head injury, birth trauma, head injury, trauma following tumor removal (such as acoustic neuroma), infection, diabetes and multiple sclerosis. Treatment with BTX typically begins with small doses to the affected muscles; often the contralateral side must be treated as well to correct any asymmetry.

Hemifacial Spasm

This disorder is characterized by recurrent involuntary twitches of the muscles innervated by the facial nerve. It is typically unilateral and started in the orbicularis oculi and spreads to other muscles over a long period of time. The most common cause is vascular compression of the nerve by an aberrant arterial loop.23 Management with BTX starts with minimal doses targeted to the affected muscles groups with individualized adjustment with subsequent treatments.

Therapeutic Applications

Though the cosmetic uses of BTX generate the most interest, globally therapeutic uses of the toxin predominates. Therapeutic use of BTX can be divided into hyperfunctioning muscle, hypersecretory and nociceptive disorders.

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Hyperfunctioning Muscle Disorders

These include the dystonias and other disorders of hyperactive muscle function.

Head and Neck Dystonias

Dystonia is a chronic neurological disorder of central motor processing dominated by involuntary muscle contractions that may by sustained (tonic), spasmodic (rapid or clonic), irregular or repetitive. Dystonia can involve any voluntary muscle, systemic or limited to a specific region of the body. In the head and neck dystonias tend to be localized. There are several options for patients with dystonia. All provide symptomatic relief only.24 Most focal dystonias are now treated effectively with local injections of outline toxin.

Blepharospasm

This disorder is characterized by involuntary forceful eye closure or excessive blinking. It is usually symmetric but occasionally there is marked asymmetry. It most commonly presents as a focal dystonia (essential blepharospasm) but occasionally is associated with oromandibular, laryngeal or cervical dystonia (Meige syndrome).25-27 BTX is dramatically effective and currently the treatment of choice for blepharospasm. The dose and injections are tailored to each individual patient. Typically BTX-A doses start at a total of 20 units at a concentration of 2.5 units/ml per site. Five to six subcutaneous injection sites in the orbital part of oribularis oculi are given on each side. Side effects include bruising, ptsosis and dry eye.

Oromandibular Dystonia

OMD is a dystonia involving the muscles of mastication, lower face and tongue. It produces spasms and deviation of the jaw.28 The spasms are usually provoked by voluntary action. Thorough knowledge of jaw anatomy is essential for the treatment of these patients. There are 3 categories of OMD based on predominate clinical features; jaw opening, jaw closing or jaw deviation dystonias. Patients may have involuntary tongue movements. Therapy is as always individualized. Based on

Figure 3. A) Patient with platysma banding.

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Figure 3. B) The same patient after multiple injections in the platysma muscle.

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Figure 4. Female with unilateral masseter hypertrophy.

the patients predominate features BTX is targeted to specific muscle groups. Tongue dystonia is not typically treated with BTX (Fig. 5).

Cervical Dystonia

Cervical dystonia can be isolated or part of segmental or generalized dystonia. It is characterized by painful involuntary posturing of the head away from its normal central position.29 There are over 50 muscles which influence head and neck posture. Therefore there clinical spectrum of abnormal head and neck posture is broad and depends on which muscles are involved and the type of activity the dystonia presents. Many open and randomized controlled trials have confirmed that BTX is highly effective for symptomatic treatment for cervical dystonia. When treating CD with BTX the most important step is to evaluate which pattern of muscles to inject. Total BTX dose depends on the severity of CD and can range from an average of a 100 units to over 400 units for the most severe cases. Side effects include dysphagia, neck muscle weakness and dry mouth.

Laryngeal Dystonia

Laryngeal dystonia is usually localized. The vast majority effects connected speech though there are a few rare cases of breathing dystonias.31 The intrinsic laryngeal muscles can be characterized in 2 groups; the abductors which move the vocal cords away from midline and enlarge the airway, giving a breathy voice and the adductors which move the vocal cord toward midline and close the glottis, giving a “strain-strangled” voice. Laryngeal dystonia primarily affects the adductor muscles of the larynx. This causes inappropriate glottal closure during connected speech, which manifests clinically as strangled breaks. Approximately 10-15% of LD affects the abductor muscles of the larynx.31 This causes inappropriate glottal opening, that manifest as breathy breaks on connected speech. BTX along with speech therapy is the standard for treatment of this disorder. For ADSD

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Figure 5. Man with oromandibular dystonia attempting to open his mouth.

BTX is typically injected into the thyroarytenoid muscle via the cricothyroid membrane. The average dose is approximately 1 unit and it is typically given bilaterally. It can also unilateral or bilateral injections staged depending on the needs of the individual patient. For ABSD the posterior cricoarytenoid muscle is typically injected. The average dose is usually 3.75 and typically only one side is injected at a time. If the other side is to be injected the clinician should wait 2 weeks and evaluate vocal mobility endoscopic to estimate a safe dose for the contralateral side. This prevents the potential life threatening possibility of airway obstruction. Other side effects include dysphagia and a breathy or weak voice.

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Essential Voice Tremor

Tremor is a involuntary rhythmical oscillation that causes changes in voice which vary in severity. The key features is persistent rhythmic variations in loudness and pitch. The tremor can be caused by the intrinsic muscles of the larynx (glottal). It can also be caused by the extrinsic laryngeal muscles which are responsible for elevating and depressing the larynx. BTX therapy is targeted to the muscles responsible for the tremor glottal (thyroarytenoid) or extraglottal (strap) muscles. Side effects include a breathy voice and dysphagia.

TMD

Temporomandibular disorders (TMD) are a collection of conditions affecting the temporomandibular joint (TMJ) and/or the muscles of mastication and contiguous tissue components. It is estimated that more than 10 million people suffer from this condition in the USA.32-34 Due to the complex relationship between the muscles of mastication and the TMJ there are a wide variety of clinical presentations for these disorders. The term TMD as a clinical label is used primarily to designate conditions that present with pain in the face or jaw area. It also encompasses the clinical syndrome of headaches, earaches, trismus, masticatory muscle hypertrophy and/or clicking and popping of the TMJ.35 The muscles injected were determined by the patients reporting of location of pain, muscle tenderness to palpation and observed functional abnormalities. Patients were generally started with 25 units in each masseter and 12.5 to 25 units in each temporalis muscle (in patients who primarily clenched their teeth) and 7.5 units in each external pterygoid (for patients with bruxing and grinding). Additional muscles were added, depending on symptoms and clinical exam. The patient returned at 2 weeks for a re-evaluation. If they continued to have pain, a booster dose was given to the persistently painful muscles. In reviewing the first 200 patients, we found a 70% response rate, with a response defined as 50% or more reduction in pain and/or frequency of pain. We noted that many of the patients who still had pain, had both a decrease in resting pain and a decrease in the amount of daily medication to control the pain. After repeated injections, there was a trend for increased response and increased duration of benefit. This suggests a central increase in pain threshold. Side effects were minimal. Less than 10% of the patients (usually those with doses of 50 U or more in the masseter muscles) complained of a decreased ability to chew solid foods. A number of the patients could not chew food before treatment due to pain. Two patients reported a change in their smile, due to slight weakness from diffusion of toxin to the levator angli oris and zygomaticus muscles. Slight tenderness at the site of injection and 3 patients with skin ecchymosis were also reported.

Cricopharngeal Spasm

The cricopharyngeus muscle functions as the upper esophageal sphincter. At rest it is activated and contracted. During swallowing it is relaxed allowing for the passage of the food bolus into the esophagus. In cricopharyngeal spasm this muscles fails to relax causing swallowing difficulties. This spasm can be caused by a variety of neurological conditions or could be idiopathic. BTX permits a nonsurgical alternative of upper esophageal spasm. Several studies have shown improvement of swallowing with removal of feeding tubes and weight gain.36,37 The typically doses range from 10-20 units. Side effects including bruising and rarely stridor (from diffusion of BTX to the PCA muscles above).

Palatal Myoclonus

Characterized by regular contraction of the soft palate muscle which may produce a clicking noise that is usually audible to the patient. It is usually idiopathic, but can be associated with head trauma or other neurological conditions. Injections are performed transorally with doses 5-10 units. Side effects include velvopharyngeal insufficiency and eustachian tube dysfunction.

Hypersecretory Disorders

The recognized ability of BTX to block not only cholinergic transmission at the neuromuscular junction but also the release of acetylcholine from cholinergic postganglionic sympathetic neurons

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has revolutionized the treatment of autonomic hypersecretory disorders.38 Since eccrine sweat glands are innervated by sympathetic nerve fibers and stimulated via cholinergic neurotransmission, BTX blockade of cholinergic autonomic nerve endings that innervate the eccrine sweat glands reduces hypersecretion of sweat. We will discuss three of these hypersecretion disorders below.

Frey Syndrome

Frey syndrome or gustatory sweating usually seen after parotidectomies or facial trauma. It is caused by the redistribution of parasympathetic fibers to the skin, which with produce sweating while eating. Area of problem can be localized with iodine solution. After localizing the area is then typically injected with 2.5 units of BTX 2 cm apart subcutaneously. Side effects include bruising, asymmetric smile.

Sialorrhea

Sialorrhea (excessive drooling or salivation) is a common and socially disabling symptom of many disease such as cerebral palsy, Parkinson’s disease, poststroke, ALS and other neurologic conditions. Sialorrhea is not only socially disabling but can also lead to choking, aspiration and chest infection. It usually is caused by swallowing dysfunction as opposed to increased salivary secretion rate. A number of studies evaluated the clinical efficacy of BTX in children with cerebral palsy.39,40 All showed a reduction of drooling and were associated with fewer and less serious side effects then scopolamine treatment. Several small, open-label studies of BTX-A therapy in adults with PD and amyotrophic lateral sclerosis demonstrated improvement in drooling with therapy.41-43 Injections are usually ultrasound guided. The parotid glands are usually treated first with an average of 10-20 units per gland. If there needs to be additional treatment, the submaxillary glands are also treated usually at 5-10 units a gland. Side effects include dry mouth and local muscle weakness.

Rhinorhea

Vasomotor rhinitis is characterized by excessive parasympathetic activity.44 One double-blind, placebo-controlled study assessed the effect of BTX therapy in chronic rhinitis patients.45 Each nasal cavity was injected with 4 U BTX type A. Rhinorhea was found to improve significantly by subjective rating and was associated with a 50% reduction in the number of paper tissues used.

Pain/Nociceptive Disorders Pathophysiology of Pain

The transmission of pain is dependent on signals from the periphery to the cortex. Unmyelinated C fibers and A-delta thinly myelinated fibers relay cutaneous and visceral noxious stimuli to the spinal cord or brain stem. From there they reach higher levels of the central nervous system. The C-fibers release neuropeptides, (e.g., substance P) whereas A-delta-fibers release glutamate. The functions of these peptides are incompletely understood, but they presumably mediate slow, modulatory synaptic activity in the dorsal horn neurons. Sensitization a major component of chronic pain, may develop either through peripheral mechanisms or as a consequence of alterations in nociceptive pathways in the spinal cord or forebrain. Peripheral sensitization can occur in nociceptor nerve terminals when repeated stimulation lowers the depolarization threshold. The peripheral sensitization model of pain proposes that peripheral silent afferents become activated after injury by the release of peripheral nociceptive mediators.46 In response to muscle inflammation, substance P, calcitonin gene-related peptide and glutamate are released from primary sensory nerve terminals in the injured area.47 These neuropeptides sensitize pain receptors, providing a feedback circuit sustaining inflammation, muscle pain and allodynia.48 The central sensitization model proposes that repetitive nociceptive stimulation involving substance P, glutamate, aspartate, calcitonin gene-related peptide and nitric oxide induces neuroplastic changes in the dorsal horn.49 Peripheral and central sensitization is likely to occur simultaneously in chronic pain disorders.50,51

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Subcutaneous injection of BTX produces a significant, long-lasting and dose-related inhibition of the sensitized pain response without apparent muscle weakness.52 The observed inhibition was attributed to a reduction of glutamate release in the periphery and expression of c-fos in the dorsal horn with an associated reduction in the nociceptive activity of wide-dynamic-range neurons in the dorsal horn . Therefore it is thought that BTX reduces pain by directly inhibiting the peripheral sensitization produced by local glutamate release from primary sensory neurons, which then leads to an indirect reduction in central sensitization.

Headache Tension Headache

Injections into the neck muscles to treat tension type headaches have yielded controversial results. This is because of the varying quality of the studies and methodological differences in treatment protocols. The major problem with the published studies to date is the lack of understanding of the pathophysiology of tension headache. The theory that muscle contraction is a factor for the pain in tension headaches is controversial. Some argue that there is little evidence that muscle hyperactivity plays a role in the etiology of tension-type headache (Rollnik et al 2000, Bendtsen 2000). Until the mechanism of tension headaches is further elucidated it will be difficult to design a study to determine the efficacy of BTX with a very selective mode of action.

Migraine Headache

There have been several studies suggesting that BTX can improve the symptoms of migraine.55,56 An open-label study of patients with migraine treated with duration of complete response of 2.6 months. Following an open label study, 123 patients took part in a prospective, randomized, double-blind, placebo-controlled study. The study was carried out of 12 centers in the US and had design similar to most pharmacological trials of migraine prophylaxis the results were inconclusive. Though the frequency and severity of migraine was reduced the difference from placebo was relatively minor. The results resemble early successes seen with acupuncture.

Trigeminal Neuralgia

Trigeminal neuralgia is a disorder characterized by sudden, severe, stabbing, or shock-like pain usually felt on one side of the jaw or cheek. The pain lasts several seconds and could be repeated in a series of attacks. Activities like talking, brushing teeth, or swallowing can trigger an attack. Also called tic douloureux, the disorder is more common in women than in men and typically affects those older than 50. There is currently an ongoing study examining the effect of BTX and trigeminal neuralgia.

Discussion

BTX is a well recognized treatment for wide spectrum of conditions in the head and neck ranging from the hyperdynamic frown lines of the aging face to the control of sialorhhea of cerebral palsy patients. One of the most important aspects of the newly realized uber medicine is the ability to selectively target focal areas with few if any systemic side effects. BTX is rarely a cure. It is a symptomatic treatment that will in most patients often need to be repeated on a regular basis. As more is understood about this toxin and newer serotypes become clinically available the potential of disorders that can be treated by this toxin will multiply rapidly.

References

1. Hackett R, Kam PC. Botulinum toxin: Pharmacology and clinical developments: A literature review. Med Chem 2007; 3(4):333-45. Review. 2. Dolly JO, Aoki KR. The structure and mode of action of different botulinum toxins. Eur J Neurol 2006; Suppl 4:1-9. Review. 3. Callaway JE. Botulinum toxin type B (Myobloc): Pharmacology and biochemistry. Clin Dermatol 2004; 22(1):23-8. Review.

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4. Ghei M, Maraj BH et al. Effects of botulinum toxin B on refractory detrusor overactivity: A randomized, double-blind, placebo controlled, crossover trial. J Urol 2005; 174(5):1873-7; discussion 1877. 5. Dressler D, Eleopra R. Clinical use of non-A botulinum toxins: Botulinum toxin type B. Neurotox Res 2006; 9(2-3):121-5. Review. 6. Robertshaw K, Watson L, Parkin T et al. Botulinum toxin A dosage: Autonomic function as a measure of side effects. Dev Med Child Neurol 2005; 47(11):792. 7. Expanding use of botulinum toxin. J Neurol Sci 2005; 235(1-2):1-9. Review. 8. Schiavo G, Matteoli M, Montecucco C. Neurotoxins affecting neuroexocytosis. Physiol Rev 2000; 80:717-766. 9. Li JY, Jahn R, Dahlstrom A. Synaptotagmin I is present mainly in autonomic and sensory neurons of the rat peipheral nervous system. Neuroscience 1994; 63:837-850. 10. Ashton AC, Dolly JO. Characterization of the inhibitory action of botulinum neurotoxin type A on the release of several transmitters from rat cerebrocortical synaptosomes. J Neurochem 1988; 50:1808-1816. 11. Marqueze B, Boudier JA, Mizuta M et al. Cellular localization of synaptotagmin I, II and III mRNAs in the central nervous system and pituitary and adrenal glands of the rat. J Neurosci 1995; 15:4906-4917. 12. Verderio C, Coco S, Rossetto O et al. Internalization and proteolytic action of botulinum toxins in CNS neurons and astrocytes. J Neurochem 1999; 73:372-379. 13. Washbourne P et al. Genetic ablation of the t-SNARE SNAP-25 distinguishes mechanisms of neuroexocytosis. Nat Neurosci 2002; 5:19-26. 14. Choe SW, Cho WI, Lee CK et al. Effects of botulinum toxin type A on contouring of the lower face. Dermatol Surg 2005; 31(5):502-7; discussion 507-8. 15. Ahn J, Horn C, Blitzer A. Botulinum toxin for masseter reduction in Asian patients Botulinum toxin for masseter reduction in Asian patients. Arch Facial Plast Surg 2004; 6(3):188-91. 16. Kim HJ, Yum KW, Lee SS et al. Effects of botulinum toxin type A on bilateral masseteric hypertrophy evaluated with computed tomographic measurement. Dermatol Surg 2003; 29(5):484-9. 17. Von Lindern JJ, Niederhagen B, Appel T et al. Type A botulinum toxin for the treatment of hypertrophy of the masseter and temporal muscles: an alternative treatment. Plast Reconstr Surg 2001; 107(2):327-32. 18. Rokadiya S, Malden NJ. Variable presentation of temporalis hypertrophy—A case report with literature review. Br Dent J 2006; 201(3):153-5. 19. Finn S, Ryan P, Sleeman D. The medical management of masseteric hypertrophy with botulinum toxin. J Ir Dent Assoc 2000; 46(3):84-6. 20. Moore AP, Wood GD. The medical management of masseteric hypertrophy with botulinum toxin type A. Br J Oral Maxillofac Surg 1994; 32(1):26-8. 21. Smyth AG. Botulinum toxin treatment of bilateral masseteric hypertrophy. Br J Oral Maxillofac Surg 1994; 32(1):29-33. 22. Wolf SM, Wagner JH, Davidson S et al. Treatment of Bell palsy with prednisone: A prospective, randomized study. Neurology 1978; 28(2):158-61. 23. Auger RG. Hemifacial spasm: clinical and electrophysiologic observations. Neurology 1979; 29(9 Pt 1):1261-72. 24. Greene P, Shale H, Fahn S. Analysis of open-label trials in torsion dystonia using high dosages of anicholinergics and other drugs. Mov Discord 1988; 3:46-60. 25. Tolosa E, Marti MJ. Blepharospasm-oromandibular dystonia syndrome (Meige’s syndrome): clinical aspects. Adv Neurol 1988; 49:73-84. 26. Jordan DR, Patrinely JR, Anderson RL et al. Essential blepharospasm and related dystonias. Surv Ophthalmol 1989; 34:123-132. 27. Tolosa ES. Clinical features of Meige’s disease (idiopathic orofacialo dystonia). A report of 17 cases. Arch Neurol 1981; 38:147-151. 28. Sutcher HD, Underwood RB, Beatty RA et al. Orofacial dyskinesia: A dental dimension. JAMA 1971; 216:1459-1463. 29. Jankovic J. Botulinum toxin therapy for cervical dystonia. Neurotox Res 2006; 9(2-3):145-8. Review. 30. Panegyres PK, Hillman D, Dunne JW. Laryngeal dystonia causing upper airway obstruction in progressive supranuclear palsy. J Clin Neurosci 2007;14(4):380-1. 31. Brin MF, Blitzer A, Stewart C. Laryngeal dystonia (spasmodic dysphonia): Observations of 901 patients and treatment with botulinum toxin. Adv Neurol 1998; 78:237-52. 32. Dworkin SF, Huggins KH, LeResche L et al. Epidemiology of the signs and symptoms in temporomandibular disorders: clinical signs in cases and controls. J Am Dent Assoc(1990); 120:273-281. 33. Carlsson GE. Epidemiology and treatment need for temporomandibular disorders. J Orofac Pain 1999; 13:232-237.

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34. Epidemiology. In: McNeill C, ed. Temporomandibular Disorders: Guidelines for Claasification, Assessment and Management. Chicago: Q uintessence Publishing Co., 1993:19-22. 35. Carlsson GE, LeResche L. Epidemiology of Temporomandibular Disorders in Temporomandibular Disorders and Related Pain Conditions. In: Sessle BJ, Bryant PS, Dionne RA, eds. Seattle: IASP Press, 1995. 36. Blitzer A, Brin MF. Use of botulinum toxin for diagnosis and management of cricopharyngeal achalasia. Otolaryngol Head Neck Surg 1997; 116(3):328-30. 37. Haapaniemi JJ, Laurikainen EA, Pulkkinen J et al. Botulinum toxin in the treatment of cricopharyngeal dysphagia. 38. Naumann M, Jost W. Botulinum toxin treatment of secretory disorders. Mov Disord 2004; 19 Suppl 8:S137-41. Review. 39. Suskind DL, Tilton A. Clinical study of botulinum-A toxin in the treatment of sialorrhea in children with cerebral palsy. Laryngoscope 2002; 112(1):73-81. 40. Jongerius PH, Rotteveel JJ, van Limbeek J et al. Botulinum toxin effect on salivary flow rate in children with cerebral palsy. Neurology 2004; 63(8):1371-5. 41. Bhatia KP, Munchau A, Brown P. Botulinum toxin is a useful treatment in excessive drooling in saliva. J Neurol Neurosurg Psychiatry 1999; 67(5):697. 42. O’Sullivan JD, Bhatia KP, Lees AJ. Botulinum toxin A as treatment for drooling saliva in PD. Neurology 2000; 55(4):606-7. 43. 43 Pal PK, Calne DB, Calne S et al. Botulinum toxin A as treatment for drooling saliva in PD. 44. Bentivoglio AR, Albanese A. Botulinum toxin in motor disorders. Curr Opin Neurol 1999; 12(4):447-56. Review. 45. Kim KS, Kim SS, Yoon JH. The effect of botulinum toxin type A injection for intrinsic rhinitis J Laryngol Otol 1998; 112(3):248-51. 46. Michaelis M, Habler HJ, Jaenig W. Silent afferents: A separate class of primary afferents? Clin Exp Pharmacol Physiol 1996; 23(2):99-105. Review. 47. Graven-Nielsen T, Mense S. The peripheral apparatus of muscle pain: Evidence from animal and human studies. Clin J Pain 2001; 17(1):2-10. Review. 48. Graven-Nielsen T, Arendt-Nielsen L. Peripheral and central sensitization in musculoskeletal pain disorders: an experimental approach. Curr Rheumatol Rep 2002; 4(4):313-21. Review. 49. Mense S. Group III and IV receptors in skeletal muscle: Are they specific or polymodal? Prog Brain Res 1996; 113:83-100. Review. 50. Malick A, Burstein R. Peripheral and central sensitization during migraine. Funct Neurol 2000; 15 Suppl 3:28-35. Review. 51. Strassman AM, Raymond SA, Burstein R. Sensitization of meningeal sensory neurons and the origin of headaches. Nature 1996; 384(6609):560-4. 52. Cui M, Khanijou S, Rubino J et al. Subcutaneous administration of botulinum toxin A reduces formalin-induced pain. Pain 2004; 107(1-2):125-33. 53. Rollnik JD, Tanneberger O, Schubert M et al. Treatment of tension-type headache with botulinum toxin type A: a double-blind, placebo-controlled study. Headache 2000; 40(4):300-5. 54. Bendtsen L. Central sensitization in tension-type headache—Possible pathophysiological mechanisms. Cephalalgia 2000; 20(5):486-508. Review. 55. Silberstein S, Mathew N, Saper J et al. Botulinum toxin type A as a migraine preventive treatment. For the BOTOX Migraine Clinical Research Group. Headache 2000; 40(6):445-50. 56. Binder WJ, Brin MF, Blitzer A et al. Botulinum toxin type A (BOTOX) for treatment of migraine headaches: an open-label study. Otolaryngol Head Neck Surg 2000; 123(6):669-76.

Chapter 14

The Use of Recombinant Phage Lysins for the Control of Bacterial Pathogens Marianne Horgan, Aidan Coffey,* R. Paul Ross, Jim O’Mahony, Gerald F. Fitzgerald and Olivia McAuliffe

Abstract

E

ndolysins are bacteriophage-encoded peptidoglycan hydrolases that accumulate in the cytosol of phage-infected bacterial cells, resulting in eventual cell lysis at the end of the lytic cycle. In view of the prevalence of antibiotic-resistant bacteria, these enzymes represent a truly novel and effective class of antimicrobials in the fight against infection. By recognising unique receptors present in the bacterial cell wall, they exhibit highly specific targeting of their host cell while leaving the normal microflora intact. An interesting feature of endolysins is their modular structure which lends itself to being manipulated by exchanging domains to alter the host spectrum or to improve the catalytic activity. Knowledge of the three dimensional structure is central to determining which domains would be favourable for the creation of designer lysins, tailored for biotechnological applications. Numerous in vitro and in vivo trials have yielded highly encouraging insights into the lytic capabilities and benefits provided by endolysins. Clearly, these potent enzymes possess many advantageous properties which make them suitable for exploitation as antimicrobials. Studies have demonstrated their potential to prevent pathogenic colonization of mucosal membranes and to control bacterial infections, as well as their use as tools in the control of potential biological weapons such as anthrax, in the biocontrol of bacteria in food and feed and to protect plants from phytopathogenic bacteria. These promising results warrant further investigation into the therapeutic potential of endolysins, conceivably leading to their eventual development as antimicrobial tools.

Introduction

Since the independent discovery of bacteriophage by Twort in 1915 and d’Herelle in 1917, the potential of bacteriophage therapy has only partially been realized.1,2 Indeed, with the advent of antibiotics in the 1940s, phage therapy was largely abandoned in the west. However, the emergence of multi-antibiotic-resistant bacteria such as Methicillin-Resistant Staphylococcus aureus (MRSA), Vancomycin-Resistant S. aureus (VRSA) and vancomycin-resistant enterococci (VRE) and the reduction in the profitability and approval of novel antibiotics,3 has placed renewed interest in phage therapy. In 2001, the hydrolytic enzymes of bacteriophages, known as endolysins, were first exploited in vivo as alternative antimicrobials.4 These enzymes demonstrate highly potent and specific targeted killing of pathogenic bacteria while leaving the normal microflora intact. Crucially, they can be easily modified through genetic engineering to create novel enzymes with increased lytic activity and a broader spectrum of inhibition.5-8 Most nonfilamentous bacteriophages with large double-stranded DNA genomes implement the holin-endolysin system to achieve bacterial lysis.9,10 This process is commonly called “lysis *Corresponding Author: Aidan Coffey—Department of Biological Sciences, Cork Institute of Technology, Bishopstown, Cork, Ireland. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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from within”.11 During the lytic cycle, endolysins accumulate in the cytosol and hydrolyse bacterial peptidoglycan to release phage virions at the terminal stage of the cycle.12 Typically, this action is tightly regulated by other phage proteins called holins which form nonspecific lesions in the cell membrane at a genetically determined time. Endolysins can pass through these lesions and attack the bacterial peptidoglycan,10 culminating in rapid cell lysis. When the holin-endolysin system is executed by the phage and endolysins act from within the bacterial cell, the holin proteins are crucial for lysis. However, the holin becomes redundant when lysins are used as recombinant enzymes and directly applied exogenously to bacterial cells, as no cell membrane is present.13 In this case, lysins act as exolysins and have direct access to bacterial peptidoglycan which leads to very rapid and potent lysis.14 This ability, known as “lysis from without”, is the basis of their potential as antimicrobials. Exolysis is generally only observed with Gram-positive bacterial cells since the outer membrane in Gram-negative cells acts as a barrier, preventing access to the peptidoglycan layer.4,13 However, the lipopolysaccharide layer of Gram-negative cells can be first disrupted by pretreatment with a detergent (e.g., Triton X-100) or with ethylenediamine tetraacetic acid (EDTA), so as to allow lysins to gain access the peptidoglycan.13 This was previously demonstrated with a Bacillus amyloliquefaciens broad range endolysin which possessed strong lytic activity against EDTA-treated Gram-negative bacteria Pseudomonas aeruginosa PAO1, Escherichia coli JM109 (DE3) and E. coli W3110.15 Endolysins usually retain a very narrow spectrum of inhibition identical to that of their homologous phage, whether it be species-specific16-18 or genus-specific.19,20 This enables selective killing of specific pathogenic bacteria, while leaving the surrounding commensal microflora virtually intact. On rare occasions, the range of the endolysin may exceed that of the phage. The Streptococcus agalactiae B30 phage for instance is able to lyse only a small number of type III Group B streptococci (GBS) strains, while its lysin demonstrated lysis against all tested β-haemolytic streptococci.21 In a similar manner, the PlyV12 endolysin of phage phi1 which was isolated originally from Enterococcus faecalis V12 also infects numerous other E. faecalis strains, as well as Enterococcus faecium strains and numerous pathogenic streptococcal and staphylococcal strains.22 Due to the worldwide proliferation of antibiotic-resistant pathogens,23,24 it is now crucial that alternative antimicrobials such as endolysins are investigated. MRSA is currently one of the most common problematic antibiotic-resistant pathogen in healthcare settings25 with rates of over 40% in some Southern European countries.26 From 2001 to 2005, the figures for reported cases of VRE have significantly increased in a number of European countries.26 Antibiotic resistance has so far led to reduced profitability, prolonged hospital stays and higher mortality rates.27 New multi-drug resistant bacteria are also rapidly emerging,28 emphasizing the urgent need for the exploitation of alternative antimicrobials. Endolysins represent an important progression in the fight against antibiotic-resistant bacteria and they offer many beneficial qualities that endorse their potential as new age antimicrobials (Table 1). Studies involving both in vitro13,29,30 and in vivo preclinical trials4,16,17,31-35 (Table 2) have revealed their efficacy for the prevention and elimination of pathogen growth and these are reviewed here in detail. This review focuses on the use of these enzymes as antimicrobials and their potential applications.

Endolysin Structure Modular Structure of Endolysins

The majority of phage endolysins display a modular structure, usually comprising of at least one catalytic domain which attacks bacterial peptidoglycan and a cell wall binding domain which is thought to target the lytic domain to its site of hydrolysis.36-38 The N-terminal domain is usually responsible for the catalytic activity which is characterized into four different groups, depending on the ability to target one of the five different major bonds in the peptidoglycan (Fig. 1), the main constituent of bacterial cell walls. Catalytic activities includes N-acetylmuramidases (lysozymes) (1, Fig. 1) and N-acetyl-β-D-glucosaminidases (2, Fig. 1) which attack the sugar moiety, L-alanoyl-D-glutamate endopeptidases (4, Fig. 1) and interpeptide bridge-specific endopeptidases (5, Fig. 1) which attack

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The Use of Recombinant Phage Lysins for the Control of Bacterial Pathogens

Table 1. Antibiotics versus bacteriophage endolysins. Adapted from Veiga-Crespo et al (102) Property

Antibiotics

Bacteriophage Endolysins

Resistance Specificity Immunogenicity Prophylaxis tools Risk of secondary infections due to treatment Ability to kill non-growing bacterial cells Application as sanitizing agents Costs Spectrum of inhibition

Yes No No No Yes No No High Broad

No Yes Yes Yes No Yes Yes Low Narrow

the peptide moiety and N-acetylmuramoyl-L-alanine amidases (3, Fig. 1) which cuts the amide bond connecting both moieties.12,39 The L-alanoyl-D-glutamate endopeptidases have only been reported in bacteriophage of Listeria species to date,40 while the interpeptide bridge-specific endopeptidases are present in numerous phages of Staphylococcus aureus.13,41-43 Muramidases and amidases are the most common types of activity reported in phage endolysins.13 Amidases attack a broader range of bacteria, due to the common presence of the amide bond between N-acetylmuramic acid and

Figure 1. The catalytic activity of endolysins at the different bonds in the peptidoglycan of Gram-positive bacteria. The interpeptide bridge varies in length and amino acid content and is indicated by X. The cleavage sites of the different activities are indicated: 1) N-acetylmuramidases; 2) N-acetyl-β-D-glucosaminidases; 3) N-acetylmuramoyl-L-alanine amidases; 4) L-alanoyl-D-glutamate endopeptidases; and 5) interpeptide bridge-specific endopeptidase. Abbreviations: MurNAc, N-acetylmuramic acid; GlcNAc, N-acetylglucosamine.

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Table 2. Summary of phage endolysin treatment in murine sepsis models Endolysin Phage

Endolysin Activity

Bacteria

Where

Results from in Vivo Studies

Reference

Cpl-1 Phage Cp-1

Muramidase

S. pneumoniae

Nasopharynx, mucosa, bloodstream

•

Treatment of infected mice one hour after infection, led to a 100% survival rate, compared with an 80% death rate in untreated mice Multiple doses required, since mice with advanced bacteraemia eventually succumbed to death Cpl-1 specific antibodies reduced but did not inhibit activity Half life of enzyme: 20.5 min No evident toxic effect on mice

(34, 103)

100% effective in the prevention of acute otitis media No evident toxic effect on mice

(35, 103)

•

• • • Muramidase

S. pneumoniae

Subjects were treated intranasally to prevent middle ear infection

•

Cpl-1 Phage Cp-1

Muramidase

S. pneumoniae

Aortic valve

• Elimination of pneumococci from blood within 30 minutes after high dosage treatment • No lysin resistance detected in persistent bacteria after low dosage treatment • Rapid bacterial lysis resulted in increased cytokine production, compared with vancomycin

(32, 103, 104)

Pal-1 Phage Dp-1

Amidase

S. pneumoniae

Nasopharynx, oropharynx

• •

(16, 105)

•

• •

No bacteria detected, five hours after one dose Prevention of recolonization by surviving pneumococci No lysin resistance detected after extensive exposure Nondisruptive towards normal microflora

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Patho-Biotechnology

Cpl-1 Phage Cp-1

Endolysin Phage

Endolysin Activity

Bacteria

Where

Results from in Vivo Studies

Reference

1. Cpl-1 Phage Cp-1 2. Pal-1 Phage Dp-1

1. Muramidase 2. Amidase

S. pneumoniae

Intraperitoneal

•

Complete bacterial clearance after a single dose of 200 μg • Synergistic cell wall destruction • No evident toxic effect on mice

(33, 103, 105)

PlyGBS Phage NCTC11261

Endopeptidase and muramidase (Putative)

Group B streptococci

Vagina, oropharynx

• Significant reduction of GBS colonization by a single dose • Bacterial numbers increased twenty-four hours after treatment • Multiple doses may be necessary

(31)

C1 Phage C1

Amidase

Group A streptococci (also Group C and E streptococci)

Oral mucosa, Nasal mucosa

• Eliminates pharyngeal colonization • Recolonization after treatment was not due to lysin-resistant bacteria • Immunogenicity is unlikely, as only nanogram quantities are required • Nondisruptive towards normal microflora • No evident toxic effect on mice

(4, 106, 107)

PlyG Phage γ

Putative amidase

*B. anthracis

Intraperitoneal

•

(17)

• •

Treatment of infected mice with 150U of enzyme 15 minutes after infection led to a 76.9% survival rate, compared with a 100% death rate in untreated mice No lysin-resistant bacteria No evident toxic effect on mice

The Use of Recombinant Phage Lysins for the Control of Bacterial Pathogens

Table 2. Continued

*Because of the limitations in working with B. anthracis, in vivo studies were performed on a genetically similar strain, B. cereus.

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L-alanine.43 Bifunctional endolysins which possess two catalytic domains are also quite common. Interestingly, an endopeptidase domain is usually accompanied by an amidase or muramidase, as is the case for LysK,19 LysWMY,44 PlyGBS,31 Ply18745 and Phi11.43 The C-terminal domain is usually responsible for cell wall binding activity to a specific substrate and involves noncovalent binding to a carbohydrate ligand in the cell wall which is different to a bacteriophage receptor.38 The different binding domains include SH3b, GW repeats, choline-binding domains, LysM domains and peptidoglycan-binding domains. The crystal structures of free and choline bound states of the pneumococcal phage endolysin Cpl-1 suggested that the choline-binding domain may assist in the correct orientation of the catalytic domain to enable efficient hydrolysis.46 It has been suggested that the C-terminal domain may also be involved in lytic activity inhibition in the absence of the target bacteria.13,30 While the C-terminal domain appears to be necessary for “lysis from without” in some endolysins,18,38,47 it is not always essential. The removal of the cell binding domain of some endolysins can actually significantly increase lysis compared to the wild type enzyme.42,45,48-50 Interestingly, the presence of the cell wall binding domain in the Bacillus anthracis endolysin, PlyL, had an inhibitory effect on its activity against several Bacillus species, except in the case of Bacillus cereus where its inhibitory effect is minimal. It has been proposed that the cell wall binding domain inhibits lytic activity through intramolecular interactions that are relieved upon binding to the bacterial cell wall.51 There are some exceptions to the modular organization of endolysins, where some endolysins have an N-terminal substrate binding domain and a C-terminal cell binding domain. For example both phage phiKZ and phage EL of the Gram-negative bacteria, P. aeruginosa, possess endolysins KZ144 and EL188, respectively with altered domain positions.52 In addition, some endolysins such as the T7 lysozyme have only single domain globular proteins, instead of the typical multidomain organization.53 It should also be noted that although some central catalytic domains and cell wall binding domains are not required for “lysis from without”, these domains may have critical functions when used by the phage in conjunction with holins to releasing phage progeny. The ability to engineer improved endolysins is an important feature when considering their use as antimicrobials, as it allows catalytic and binding activities to be potentially combined and optimized.

Creating Designer Lysins

It is generally preferable that endolysins target a wide range of strains within a bacterial species or genus when considering them as the potential basis for therapeutic treatment.54 If such an endolysin cannot be found, protein engineering may provide an alternative to develop fully active chimeric enzymes via domain swapping/addition. In this respect, the modular structure of endolysins potentially facilitates the construction of engineered hybrid enzymes with new features, customized for therapeutic applications. These designer lysins could be designed to target any pathogen and to have different catalytic activities and broader host ranges. Indeed, modular recombination between different bacteriophages and bacterial species is one of the driving forces in evolution to allow phage to adapt to new environments.55 This theory was supported when the N-terminus of the lactococcal phage Tuc2009 lysin and the C-terminus of a pneumococcal autolysin were fused to create an enzymatically active lysin.8 In a similar approach, a tripartite pneumococcal hydrolase was constructed by the fusion of domains from two different pneumococcal phage endolysins. The lysozyme domain of the Cpl-1 endolysin was ligated to the N-terminus of Hbl3, an endolysin possessing amidase activity and a choline-binding domain. The resulting enzyme exhibited 1.5- and 14-times more activity than the original Cpl-1 lysozyme and Hbl3 amidase, respectively.7 In addition, chimeric proteins have also been shown to exhibit more potent lytic activity compared to the wild-type form. When the N-terminal domain of the Klebsiella phage K11 lysin was fused to the C-terminal domain of the E. coli phage T7 lysin, amidase activity was approximately 45% greater than the wild type and the novel lysin was also more thermostable.5 It was suggested that the chimera may have adopted a more favourable structure for amidase activity.5 In a similar manner, superior activity was also seen when a chimeric protein was constructed with

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a pneumococcal autolysin and a phage lysozyme.6 Additionally, domain swapping studies may reveal which cell binding domains improve or hinder the activity of specific catalytic domains. Crystallization studies aimed at determining the three dimensional protein structure would determine which domains. Additionally it may act as ideal candidates for designer lysins, by revealing the structural relationships between different domains and might also inform on the structural ramafications of combining different domains at particular sites. Knowledge of domain structure, would lead to the construction of more efficient chimeric lysins, which would have more extensive consequences in the control of pathogens, some of which may be difficult to treat with antibiotics. A number of phage endolysins have been crystallized to date, including T4 lysozyme,56,57 T7 amidase,53 the B. anthracis phage endolysins, PlyB (catalytic domain of muramidase)58 and PlyL (amidase),51 the Listeria monocytogenes phage amidase PlyPSA59 and the pneumococcal phage lysozyme, Cpl-1.46 A recent report which documented the crystal structures of Cpl-1 in complex with three peptidoglycan analogues have given unparalleled insights into how peptidoglycan is degraded by endolysins and the recognition events that lead up to hydrolysis. As more crystal structures are being determined, knowledge into how exactly phage endolysins attack bacterial cell wall peptidoglycan is increasing.60

Important Properties of Phage Lysins

Novel drug development warrants the thorough undertaking of a number of preclinical trials, which may or may not justify clinical trials and potential market release. Some of the major factors to be investigated are drug toxicity, immunogenicity, efficacy and bacterial resistance. A number of in vitro and in vivo trials have previously been conducted on phage lysins to evaluate their usefulness as antimicrobials.

Toxicity

Bacteriophages are considered to be the most common biological systems on earth. They are found in our food, oceans, lakes, soil, on our skin and even within our gut cavity,61 so in theory endolysin treatment should not be toxic or harmful to humans. Nonetheless, the discovery of some phage-encoded toxin genes on host genomes has revealed their role in bacterial pathogenesis, emphasizing the need for phage to be screened prior to their use,62 to ensure that they are not toxic. Phage elements that could potentially increase bacterial pathogenicity must also be avoided, such as regulatory factors that increase virulence gene expression.63,64 However, in general, this problem only arises when the phage as a whole is being exploited as an antimicrobial, instead of individual lytic proteins. Since peptidoglycan bonds which are the targets of lysins are only present in bacteria, lysins do not attack eukaryotic cells and are therefore potentially harmless to humans and animals. This is further endorsed by the preclinical trials conducted in mice (Table 2),4,17,33-35 which are encouraging for the treatment of systemic infections with endolysins, but further investigations must be made to evaluate their safety and optimize dosage for therapeutic use in humans.

Endolysin Immunogenicity

Endolysins are large proteins that are capable of stimulating a humoral immune response, especially when used intravenously to treat systemic infections.30,65,66 Essentially, this reaction could strongly decrease their catalytic activity. To address this possibility, in vivo and in vitro experiments were conducted. During in vitro trials, rabbit hyperimmune serum was raised against the pneumococcal-specific endolysin, Cpl-1. After treatment of pneumococci with 2,000 μg of Cpl-1 exposed to hyperimmune serum, a reduction of killing efficiency by 0.8log10 was observed.34 To support these significant findings in vivo, six mice received three sequential intravenous doses of 1,000 μg, 2,000 μg or 4,000 μg of Cpl-1 and five of these mice tested positive for Immunoglobulin G (IgG) against the enzyme. Four weeks later, endolysin treatment of a single bolus of 200 μg of Cpl-1 reduced the bacteremic titre by log100.57 of the control naïve mice after 15 minutes. The bacteremic titre of Cpl-1-immunized mice was reduced by log100.4, lending support to the in vitro trials.34 The reason for this is partially explained by another study performed with two L.

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monocytogenes phage endolysins, Ply118 and Ply500. It was shown that the affinity of the cell wall binding domain of an endolysin for its receptor is in the nanomolar range (3 × 108 to 6 × 108) which is comparable to the affinity an IgG molecule has for its antigen.38 Similar results were seen with in vitro trials using B. anthracis and S. pyogenes specific endolysins.30 A “high continuous” dose of Cpl-1 also significantly increased cytokine production, compared to that of the vancomycin treatment. It is likely this was due to very rapid bacterial lysis and immediate release of the pathogens cell wall fragments caused by the high doses of the enzyme. However, regulated lower doses could significantly reduce immunogenicity.32 PEGylation which involves conjugating the protein to polyethylene glycol (PEG) would also reduce the immunogenicity of the enzyme by reducing antibody binding, as was shown with lysostaphin.67 However, pathogen killing was also reduced with the PEGylated enzymes, but this was compensated for by the dramatically enhanced pharmacokinetics.67 Therefore, like all proteins, endolysins stimulate an immune response, but since the affinity of a lysin for its recognised pathogen is comparable to the affinity of antibodies for their antigens,38 lytic activity is only slowed and not completely inhibited.34 These studies lend support for the potential use of phage lysins in the treatment of systemic infections.

Resistance

The emergence of endolysin-resistant bacteria with altered reinforced cell wall structure is unlikely,30 since phage have been constantly evolving in nature with bacteria over millions of years. Endolysins have been refined for the exact purpose of efficiently lysing bacterial cells, to release progeny virions. It has also been suggested that the cell wall binding domains may have evolved to bind to molecules in the cell wall that are essential for bacterial viability, making resistance unlikely.30 In fact, the cell wall receptor for pneumococcal endolysins is choline which is essential for pneumococcal viability.16,68 Another molecule, polyrhamnose, shown to be important for streptococcal growth is essential for cell wall binding of Group A streptococcal endolysins.69,70 To verify in vitro that resistance is a rare occurrence, Streptococcus pneumoniae was repeatedly exposed to low and high concentrations of the pneumococcal endolysin, Pal, on agar plates and in liquid cultures. No resistant mutants were found16 and similar results were reported when Bacillus was exposed repeatedly to the endolysin, PlyG on agar plates. In addition, a susceptible B. cereus isolate was subjected to methanesulphonic acid ethyl ester mutagenesis and while 1,000 to 10,000-fold increases in antibiotic resistance were observed, no PlyG-resistant mutants were found.17 In another study involving a murine sepsis model, streptococcal cells recolonized the oral mucosa and aorta valve after endolysin treatment, but this was thought to be due to internalization of the streptococci and not due to resistant cells (Table 2).4,32 Nevertheless, it must be noted that endolysins act almost immediately in vitro, lysing dense cultures within seconds or minutes13 and it is unlikely that resistance will occur in this situation. However, it should be considered that antibodies will slow catalytic activity in vivo,34,3 perhaps making resistance more likely in this situation in comparison to the in vitro trials. Therefore, although no resistance has been so far reported in vivo (Table 2), additional trials are required to confirm this theory. These promising results find particular relevance with the current escalating crisis of antibiotic-resistant bacteria.

Synergy

The potential of the combined use of different phage endolysins has also been demonstrated with a muramidase and an amidase, Cpl-1 and Pal, respectively. The simultaneous peptidoglycan cleavage by these two potent enzymes resulted in the synergistic lysis of pneumococcal cells in vitro71 and in vivo.33 In vitro results revealed that in thirty seconds, one unit per ml of either Pal or Cpl-1 reduced the bacteremic titre of five S. pneumoniae strains, three of which are penicillin-resistant by a median of 1.34log10 colony forming units per mililiter (cfu/ml) and 0.83log10 cfu/ml, respectively. However, crucially, the titre was reduced by 2.40log10 cfu/ml when both enzymes were used in combination, indicating synergy.71 In a recent study, this beneficial effect from the combined use of Pal and Cpl-1 was further confirmed when synergy was observed in the lysis of three out of four S. pneumoniae strains, two of which showed antibiotic resistance.72 Similarly, the combined

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treatment of 2.5 μg of both Cpl-1 and Pal completely rescued mice one hour after being challenged with 5 × 107 cfu. In contrast, a single injection of 5 μg of either Cpl-1 or Pal was not sufficient to protect mice and all died by day three.33 The basis of such encouraging results may be explained by improved access to the enzyme cleavage sites by the action of the opposite endolysin or the destructive effect of a two-dimensional hydrolysis in the three-dimensional peptidoglycan.71 An additional advantage of using two endolysins with different cleavage activities is that the likelihood of resistant clones emerging is further decreased.33 A synergistic effect was also found when Cpl-1 was used in combination with conventional antibiotics. Interestingly, synergy of Cpl-1 with gentamicin increased with a decreasing penicillin minimal inhibitory concentration (MIC), while Cpl-1 with penicillin shows increasing synergy with an increasing penicillin MIC. No synergy was observed with Cpl-1 and penicillin in the penicillin-susceptible and -intermediate strains, whereas more than a 100-fold lower titre was observed in the penicillin-resistant strain.73 The synergistic effects of combining Cpl-1 with antibiotics, cefotaxime or moxifloxacin were also observed against cefotaxime-resistant and moxifloxacin-resistant pneumococcal strains, respectively. However, when Pal enzyme was used in the combination, instead of Cpl-1, no synergy was apparent.72 Moreover, the combined use of Cpl-1 with antibiotics, levofloxacin or azithromycin did not produce a synergistic effect either.73 Although, the results demonstrate that synergy is dependent on suitable lysin and antibiotic combinations, this is an important advancement in the treatment of antibiotic-resistant pathogens, especially where increasing drug dosage is not an option due to toxicity effects.73 Also, combination therapy could alleviate the limitations of phage endolysins, such as their poor half-life in vivo, which is typical for most proteins.34,73

Potential Applications of Phage Endolysins Preclinical Studies

Endolysins were originally investigated as potential therapeutics and prophylactic tools with a view to killing bacteria infecting mucous membranes.4 Since then, many more successful trials have demonstrated their capabilities for the prevention of pathogenic colonization of the mucosa.4,16,31,32,34,35 Their potential use as antimicrobials against systemic diseases has also been demonstrated in a number of instances.17,33,34 While antibiotics often kill bacteria indiscriminately, endolysins possess high specificity and the normal microflora is relatively undisturbed. Moreover, antibiotics such as penicillin and cephalosporin function by inhibiting peptidoglycan synthesis, thereby lysing only dividing cells. On the other hand, endolysins destroy the peptidoglycan directly, destroying both growing and nongrowing cells.74 Environments such as hospitals and nursing homes which suff er from high numbers of antibiotic-resistant pathogens would benefit extensively from endolysin treatment.30 Effective lysin antimicrobial activity was demonstrated in murine sepsis models for the prophylaxis and elimination of pharyngeal, vaginal and peritoneal colonization, as well as experimental infective endocarditis in rats.4,16,17,31-35 A series of endolysin trials conducted in vivo are reviewed below and are summarized in Table 2.

Streptococcus pneumoniae

S. pneumoniae is a common cause of pneumonia, acute otitis media (AOM), septicaemia, bronchitis and meningitis,75 resulting in millions of deaths each year, primarily in young and old people.26 The rate of penicillin-resistance among pneumococcal strains has reached crisis proportions76,77 and the emergence of multidrug-resistant pneumococcal strains is also starting to become a major health concern.78 Preclinical trials are underway to investigate the antimicrobial properties of two pneumococcal phage endolysins, Cpl-1 and Pal which effectively lyse S. pneumoniae despite the presence of a polysaccharide capsule that acts as a virulence factor for the bacterial cell. Since pneumococci normally gain entry into the bloodstream from the nasopharynx, the overall aim of these trials

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was to prevent or control nasopharyngeal colonization and effectively protect animals from bacteraemia and death. Cpl-l was shown to be effective for intravenous treatment and topical nasal application in disease prevention. Significantly, a single 2,000 μg dose of Cpl-1 given one hour after intravenous injection of pneumococci, led to a 100% survival rate of treated mice after 48 hours, compared with an 80% death rate in untreated mice. In advanced bacteremic mice, two doses of Cpl-1 given five hours apart were capable of considerably prolonging the survival of mice, but all of these mice eventually succumbed to the infection and died. This may be explained by the short half life of 20.5 minutes of Cpl-1 in the blood of mice, which would suggest that optimum treatment would be through constant intravenous infusion for a determined time period.34 As a follow-up trial by the same group, the outcomes of different dose ranges of continuous infusions of Cpl-1 were examined. An initial decrease in pneumococcal titres was seen after low dose treatment of an intravenous bolus of 10 mg/kg, followed by uninterrupted infusion of 5 mg/kg/h for six hours. However, counts were comparable to the untreated controls at the sixth hour. Highly promising results were observed after high dose treatment of an intravenous bolus of 250 mg/kg, followed by uninterrupted infusion of 250 mg/kg/h for six hours. In this case, no pneumococci were detected 30 minutes after therapy and almost a complete eradication was seen for the following six hours.32 In a recent study, Cpl-1 treatment was shown to be 100% effective in the prevention of acute otitis media (AOM),35 which occurs when a viral infection which modifies Eustachian tube function or disrupts the mucosal membranes allows bacteria to ascend into the middle ear.79 After prior infection with Pneumococcus seven days earlier, mice were treated twice intranasally four hours apart with 1,000 μg of Cpl-1, before infection with the influenza virus. Fundamentally, the Cpl-1 lysin was effective in preventing AOM, a secondary bacterial infection resulting from influenza infection. All treated mice survived, while 80% of the control mice succumbed to the disease,35 lending highly promising support to the protective capabilities of endolysins. Furthermore, such encouraging results were also seen for another pneumococcal lysin, Pal, when low and high single dose treatments were examined for the control of pneumococcal colonization of the nasopharynx. Forty-two hours after intranasal bacterial challenge, mice were treated with a low dosage of 700 units of Pal lysin. Pneumococci were not detected in 62.5% of the mice and titres were considerably decreased in the remaining mice. Moreover, a high dosage of 1400 units of Pal resulted in undetectable pneumococcal titres, even five hours after the treatment. Significantly, this single dose treatment was also successful in preventing recolonization.16 In light of the promising results from previous trials, the effects of the intraperitoneal therapy of Cpl-1 or Pal enzyme were examined. A single injection of 200 μg of either enzyme, one hour after injection with antibiotic-resistant pneumococci was shown to protect mice from bacteraemia. Results of different lysin dosages, given ten minutes after bacterial challenge, were also examined and 10 μg was determined to be the minimal effective dose. When an evaluation of time delays in treatment was conducted, it was also determined that 200 μg of either lysin was sufficient to cure mice up to two hours after bacterial injection. Moreover, pneumococcal numbers failed to re-establish themselves and recolonization did not occur in the six-to-eight day follow-up period. However, when mice were treated four hours later, they survived significantly longer than untreated mice but all of these eventually succumbed and died.33

Group A and B Streptococci

Group A streptococci (GAS) are the most common cause of streptococcal pharyngitis80 and are also responsible for serious human infections, such as necrotizing fasciitis and cellulitis. Group B streptococci (GBS) are involved with sepsis and meningitis in neonates81 and can also result in extensive morbidity and mortality in adults.82 Since approximately one fifth of pregnant woman are vaginally colonized with GBS during delivery, a high percentage of babies are thus born colonised with the organism and may develop potentially fatal infections.83 Two important lysins are instrumental in the prevention and control of GAS and GBS, C1 and PlyGBS, respectively.

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Although the original host of the C1 bacteriophage is group C streptococci, its lysin is effective in the lysis of groups A and E, as well as group C streptococci when used for exolysis.84 In vitro trials revealed that just 1,000 units (10 ng) of C1 lysin were sufficient to eliminate approximately 107 cfu of GAS cells within five seconds.4 Three different in vivo trials were performed to further evaluate the lytic capabilities of the potent enzyme against GAS. Firstly, 1,000 units of lysin was mixed with streptococci and given orally and nasally to mice. Colonization was successfully prevented and even after one week of the treatment, only a single colony was found in 20 swabs.4 The second study also looked at the prevention of streptococcal colonization and 250 units of lysin were given orally to nine mice, prior to infection with 107streptococci. Colonization in untreated mice was reported to be 70.5%, compared to colonization of 28.5% in mice that underwent lysin treatment. As a final study, mice that were heavily colonized for four days were given an oral dose of 500 units of lysin. No streptococci were detected two hours after lysin treatment, but within 48 hours, one mouse died and two others were recolonized with streptococci. Since no resistant isolates were detected from these recolonized mice, the bacteria may have become internalized in mammalian cells where endolysins are usually too large to enter and thus, streptococci would have been protected from lysin action.4 Notwithstanding this, C1 may still be used as an effective antibacterial agent to eliminate or reduce streptococci from carriers or infected individuals.4 Endolysin therapy would still significantly reduce pathogen numbers, so that the immune system is able to cope and take over. If infections involving internalized cells need to be rapidly eradicated, a powerful strategy could be implemented using endolysins in conjunction with antibiotics.73 PlyGBS is also an effective anti-streptococcal agent. One day after bacterial challenge of the vaginal cavities with 106cfu of GBS, mice were treated with a single dose of PlyGBS which led to a substantial reduction of 3-logs in bacterial titre, even two to four hours after lysin treatment.31 However, twenty-four hours after treatment, streptococcal counts significantly increased again indicating again that multiple doses may be needed for total pathogen eradication. Single dose treatment may still be useful in the prevention of GBS transmission to newborns from a colonized mother. Treatment could be given four hours prior to delivery to reduce colonization by GBS or to decontaminate newborns, to effectively prevent neonatal meningitis and sepsis.31 Mice were also successfully treated orally and nasally to efficiently reduce colonization in the upper respiratory tract.31

Bacillus anthracis

Phage endolysins are suitable therapeutic candidates for the treatment and prophylaxis of potential antibiotic-resistant biological warfare pathogens. Inhalational anthrax occurs as a result of Bacillus anthracis85 which is an ideal pathogen for bioterrorism.86 The efficacy of a B. anthracis lysin, PlyG which attacks most B. anthracis isolates was demonstrated with in vivo studies. Since anthrax is highly lethal and toxic to humans, a genetically related B. cereus strain, lacking the B. anthracis virulence plasmid was used for experimental studies.87 Mice were infected with 1 × 106 cfu of a streptomycin-resistant B. cereus strain 4342, followed by injection with 150 units of PlyG 15 minutes later. This resulted in a 76.9% recovery rate. This is in stark contrast to the 100% death rate within five hours of untreated infected mice. Both vegetative cells and germinating spores were susceptible to this potent bactericidal action. The lytic ability of PlyG could also function as a very rapid and sensitive spore-detection method for B. anthracis after intentional germination. As was seen in many human cases, the current culture detection methods are often too slow for successful treatment. Novel methods involving DNA extraction and specialized instrumentation are currently under investigation, but it is not as feasible to employ these outside the lab.88 A research group at Rockefeller University have exploited the specificity of phage lysins to develop a hand-held luminometer, which detects ATP release from lysed cells after the application of PlyG. A signal was detected within 10 minutes of germinant addition and only 5 minutes after PlyG was added.17 However, since PlyG lyses both virulent and nonvirulent B. anthracis strains, it would have to be used as an initial rapid test on site, before later confirmation in the laboratory.88 Another recent study, also reported on using

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only the binding domain of PlyG, PlyGB as a detection tool and methods to reduce the assay time and cell detection limit are now in progress.89

Staphylococcus aureus and MRSA

The prevalence of antibiotic resistance among S. aureus strains is a major problem and MRSA is now the most frequently reported antibiotic-resistant bacteria in clinical settings in Europe, America, North Africa and the Middle- and Far-East. In some countries, the MRSA proportion rate in intensive care unit patients is over 60%.26 Furthermore, MRSA carriage is increasingly being reported in animals, especially pigs.90 S. aureus is associated with endocarditis, toxic shock syndrome, septicaemia, impetigo, among others.91 Such a prevalence of antibiotic resistance warrants the need for an alternative antimicrobial against S. aureus. Two staphylococcal phage endolysins, LysK (Fig. 2) and phi11 have been reported to lyse a broad range of live staphylococci including MRSA and glycopeptide-intermediate S. aureus (GISA) strains and mastitis-causing coagulase-negative staphylococcus (CoNS).19,92 Although no animal trials have been published to date on either lysin, they are both highly active antimicrobials with significant potential. In a kill curve experiment with crude LysK lysate, a 99% reduction in MRSA numbers was observed within one hour.19 Phi11, which has been shown to be active in a milk environment,92 is also efficient at eliminating S. aureus biofilms. This ability has substantial clinical relevance, since phi11 could be used to eradicate biofilm formation on medical devices such as catheters.47 Studies on LysK and phi11 lend major support to their use as antimicrobials in clinical settings as well as in the community.

Enterococcus faecalis and Enterococcus faecium

Enterococcal infections are becoming more challenging to treat, since vancomycin resistance in enterococcal strains has increased enormously especially in Ireland, Germany, the Czech Republic, Greece and Israel over the past four years.26 Normally, enterococcal colonization has no negative effect on the host. However, when the normal microflora environment environment is disturbed, enterococci can become invasive, leading to nosocomial infections,93 such as endocarditis, meningitis and urinary tract infections. An alternative to antibiotics is urgently required and the E. faecalis bacteriophage lysin, PlyV12 may provide the solution. As well as lysing its host strain, it lysed all strains of E. faecalis and E. faecium tested, including vancomycin-resistant strains. PlyV12 also lysed other pathogenic bacteria such as Streptococcus pyogenes and groups B, C, E and G streptococci and had little effect on the normal microflora with the exception of Streptococcus gordonii.22 Significantly, when enterococcal cells were exposed to 2.5 units of PlyV12 for 15 minutes in vitro, a 4-log reduction in viability was observed. Such encouraging results emphasize the need for further investigation into this rare broad range lysin.

Food Pathogens

In the United States alone, over 76 million cases of illnesses due to foodborne pathogens are reported, at a cost of seven to ten billion dollars annually.94 In addition, much like the situation in human medicine, the incidence of antibiotic-resistant pathogens in the food industry is rapidly increasing. Consequently, an obvious application for endolysins is their use as bioprotectants in food products. The purified enzymes could be applied directly to the raw product to prevent contamination by pathogens,13 or alternatively they could be cheaply produced by recombinant food-grade bacteria. The Listeria phage endolysin Ply511 was effectively produced by Lactococcus lactis under the control of the lactococcal promoter, P32, to develop dairy starter cultures with food biopreservation properties against L. monocytogenes contamination. Although high yields of Ply511 were generated by this system, the enzyme accumulated in the cytoplasmic cell fractions. However, after fusion of the ply511 gene with the Lactobacillus brevis S-layer protein signal sequence, high levels of fully active enzyme were successfully secreted to kill L. monocytogenes in the medium. A truncated derivative of the endolysin which exhibited strongly increased lytic activity is currently being investigated to control contaminations during soft cheese ripening.49

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Figure 2. Schematic representation of the production of recombinant lysin, LysK. Phage K forms plaques of 1-1.5 mm in diameter on various staphylococcal strains (A). Phage K is a member of the Myoviridae family (B), with a genome size of 127,395 base pairs (C) harbouring a variety of open reading frames (ORFs) (108). The lysin gene is interrupted by an intron, I-KsaI which is represented by the dotted lines (D) (108). When LysK is recombinantly expressed using the nisin expression vector pNZ8048, in L. lactis NZ9800, it shows activity against numerous staphylococcal strains (19), as seen in this zymogram plate (E).

Lactobacilli have the advantage of being highly colonizing organisms and are found on mucosal membranes, such as the oral cavity, the gastrointestinal tract and the vagina. The persistence of

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Figure 3. Schematic representation of the potential antimicrobial uses of endolysins.

membrane-colonizing commensal lactobacilli means they are ideal candidates for the expression and production of lysins in relevant animal models.95,96 To exploit the colonising properties of lactobacilli, the promoter and signal sequence of the Sep protein from Lactobacillus fermentum BR11 was used to express Ply511 in different probiotic, animal and food isolates of lactobacilli, as well as L. lactis.97 In this case lactic acid bacteria secreting Ply511 were demonstrated to have a high peptidoglycan hydrolytic activity against autoclaved Listeria cells, but it was unable to control L. monocytogenes growth. This was surprising since an identical system for the expression and export of lysostaphin was effective in the control of MRSA. Since the bactericidal activity of Ply511 is dependent on the growth rate of Lactobacillus, methods could be developed to increase its growth rate, thereby increasing the production rates of Ply511. Efforts are currently underway to improve this expression system97. The endolysin Ply3626 is also being investigated to control the growth of Clostridium perfringens, a common source of food poisoning and prevent its growth in poultry intestines or on processed raw poultry.18,98 Phages commonly reside in the gastrointestinal (GI) system of food animals,99,100 so it is unlikely the use of endolysins as antimicrobials would have a negative impact on animal health. Nevertheless, the effect of endolysins on the GI system and the function of endolysins in that environment must be further evaluated, although it is expected that these enzymes would be rapidly broken down in such a low pH and proteolytic environment.

Conclusion

In a period where antibiotic-resistance is emerging at an unprecedented rate, the use of recombinant bacteriophage lysins represents a truly novel and highly effective method of potentially controlling bacterial infectious diseases. Endolysins are highly flexible enzymes with a modular structure and they utilize a novel mode of antimicrobial action in that they directly cleave specific peptidoglycan bonds.12 As described in this review, these modular enzymes can be easily engineered to develop specific activities, especially tailored for antimicrobial applications. Improved bioinformatic tools and the crystallization of additional enzymes to decipher their three-dimensional structure will further aid in the development of designer lysins. Since most infections initiate from the pathogenic colonization of mucosal membranes, it is crucial to develop a tool that would effectively prevent this from happening in the first place. In light of the results obtained

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from the murine sepsis models (Table 2), endolysins show massive potential as antimicrobials. They are nontoxic, highly specific, powerful antimicrobials which are capable of killing even antibiotic-resistant pathogens. Despite stimulating an immune response, numerous experiments have demonstrated their bactericidal action holding promise for their use in the treatment of systemic infections. However, more preclinical trials are necessitated to establish if phage lysins will be effective antimicrobials when used intravenously as well as topically. Secondary infections such as Clostridium difficile, resulting from antibiotic use101 are also prevented since the normal microflora are left relatively undisturbed. In addition, significant quantities can be easily and cheaply produced.17 The use of antibiotics and vaccination would be too costly and time-consuming to prevent and control epidemics in refugee camps but treatment with endolysins could provide the perfect solution. Moreover, hospitals, nursing homes, day care centres, farms and even the community at large, which normally act as reservoirs for bacteria warrant the development of such antimicrobials that could be used to prevent infections, as well as to treat them. The results from the animal trials are encouraging, although many more studies are required to look at the effects of their long-term use. It should also be noted that endolysins represent a more environmentally friendly approach than food preservatives and antibiotics for the biocontrol of bacteria in food and medicine. The application of harsh chemical sprays to protect plants from phytopathogenic bacteria could be reduced, as well as the use of undesirable antibiotics to treat infections in farms animals. The regular use of harsh disinfectants to decontaminate food-processing plants could be substituted for endolysins which are naturally produced by bacteriophages, the most common biological entities on earth (Fig. 3). In light of the rapidly increasing numbers of multi-antibiotic-resistant bacteria, one of the foremost goals for the future should be to further increase our scientific knowledge of these remarkable bacteriophage enzymes with the eventual intention of their exploitation as potential antimicrobials.

References

1. d’Herelle FH. Sur un microbe invisible antagoniste des bacilles dysenteriques. Les Comptes rendus de l’Académie des sciences 1917; 165:373-375. 2. Twort FW. An investigation on the nature of ultra-microscopic viruses. Lancet 1915; ii:1241-1246. 3. Infectious Disease Society of America (IDSA). Bad Drugs, No Bugs. As Antibiotic Discovery Stagnates … A Public Health Crisis Brews 2004:1-35. 4. Nelson D, Loomis L, Fischetti VA. Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme. Proc Natl Acad Sci USA 2001; 98(7):4107-12. 5. Alcantara EH, Kim DH, Do SI et al. Bi-functional Activities of Chimeric Lysozymes Constructed by Domain Swapping between Bacteriophage T7 and K11 Lysozymes. J Biochem Mol Biol 2007; 40(4):539-46. 6. Croux C, Ronda C, Lopez R et al. Interchange of functional domains switches enzyme specificity: construction of a chimeric pneumococcal-clostridial cell wall lytic enzyme. Mol Microbiol 1993; 9(5):1019-25. 7. Sanz JM, Garcia P, Garcia JL. Construction of a multifunctional pneumococcal murein hydrolase by module assembly. Eur J Biochem 1996; 235(3):601-5. 8. Sheehan MM, Garcia JL, Lopez R et al. Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling. FEMS Microbiol Lett 1996; 140(1):23-8. 9. Young I, Wang I, Roof WD. Phages will out: Strategies of host cell lysis. Trends Microbiol 2000; 8(3):120-8. 10. Young R, Blasi U. Holins: Form and function in bacteriophage lysis. FEMS Microbiol Rev 1995; 17(1-2):191-205. 11. Delbruck M. The growth of bacteriophage and lysis of the host. J Gen Physiol 1940; 23:643-660. 12. Young R. Bacteriophage lysis: Mechanism and regulation. Microbiol Rev 1992; 56(3):430-81. 13. Loessner MJ. Bacteriophage endolysins—Current state of research and applications. Curr Opin Microbiol 2005; 8(4):480-7. 14. Loessner MJ, Schneider A, Scherer S. A new procedure for efficient recovery of DNA, RNA and proteins from Listeria cells by rapid lysis with a recombinant bacteriophage endolysin. Appl Environ Microbiol 1995; 61(3):1150-2.

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15. Morita M, Tanji Y, Mizoguchi K et al. Antibacterial activity of Bacillus amyloliquefaciens phage endolysin without holin conjugation. J Biosci Bioeng 2001; 91(5):469-73. 16. Loeffler JM, Nelson D, Fischetti VA. Rapid killing of Streptococcus pneumoniae with a bacteriophage cell wall hydrolase. Science 2001; 294(5549):2170-2. 17. Schuch R, Nelson D, Fischetti VA. A bacteriolytic agent that detects and kills Bacillus anthracis. Nature 2002; 418(6900):884-9. 18. Zimmer M, Vukov N, Scherer S et al. The murein hydrolase of the bacteriophage phi3626 dual lysis system is active against all tested Clostridium perfringens strains. Appl Environ Microbiol 2002; 68(11):5311-7. 19. O’Flaherty S, Coffey A, Meaney W et al. The recombinant phage lysin LysK has a broad spectrum of lytic activity against clinically relevant staphylococci, including methicillin-resistant Staphylococcus aureus. J Bacteriol 2005; 187(20):7161-4. 20. Loessner MJ, Maier SK, Daubek-Puza H et al. Three Bacillus cereus bacteriophage endolysins are unrelated but reveal high homology to cell wall hydrolases from different bacilli. J Bacteriol 1997; 179(9):2845-51. 21. Pritchard DG, Dong S, Baker JR et al. The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30. Microbiology 2004; 150(Pt 7):2079-87. 22. Yoong P, Schuch R, Nelson D et al. Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium. J Bacteriol 2004; 186(14):4808-12. 23. Kunin CM. Resistance to antimicrobial drugs—A worldwide calamity. Ann Intern Med 1993; 118(7):557-61. 24. Neu HC. The crisis in antibiotic resistance. Science 1992; 257(5073):1064-73. 25. Karchmer AW. Nosocomial bloodstream infections: Organisms, risk factors and implications. Clin Infect Dis 2000; 31 Suppl 4:S139-43. 26. European Antimicrobial Resistance Surveillance System. EARSS Annual Report 2005 2006:1-147. 27. Cosgrove SE, Sakoulas G, Perencevich EN et al. Comparison of mortality associated with methicillinresistant and methicillin-susceptible Staphylococcus aureus bacteremia: A meta-analysis. Clin Infect Dis 2003; 36(1):53-9. 28. Alekshun MN. New advances in antibiotic development and discovery. Expert Opin Investig Drugs 2005; 14(2):117-34. 29. Borysowski J, Weber-Dabrowska B, Gorski A. Bacteriophage endolysins as a novel class of antibacterial agents. Exp Biol Med (Maywood) 2006; 231(4):366-77. 30. Fischetti VA. Bacteriophage lytic enzymes: Novel anti-infectives. Trends Microbiol 2005; 13(10):491-6. 31. Cheng Q, Nelson D, Zhu S et al. Removal of group B streptococci colonizing the vagina and oropharynx of mice with a bacteriophage lytic enzyme. Antimicrob Agents Chemother 2005; 49(1):111-7. 32. Entenza JM, Loeffler JM, Grandgirard D et al. Therapeutic effects of bacteriophage Cpl-1 lysin against Streptococcus pneumoniae endocarditis in rats. Antimicrob Agents Chemother 2005; 49(11):4789-92. 33. Jado I, Lopez R, Garcia E et al. Phage lytic enzymes as therapy for antibiotic-resistant Streptococcus pneumoniae infection in a murine sepsis model. J Antimicrob Chemother 2003; 52(6):967-73. 34. Loeffler JM, Djurkovic S, Fischetti VA. Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia. Infect Immun 2003; 71(11):6199-204. 35. McCullers JA, Karlstrom A, Iverson AR et al. Novel strategy to prevent otitis media caused by colonizing Streptococcus pneumoniae. PLoS Pathog 2007; 3(3):e28. 36. Garcia P, Garcia JL, Garcia E et al. Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Gene 1990; 86(1):81-8. 37. Khosla C, Harbury PB. Modular enzymes. Nature 2001; 409(6817):247-52. 38. Loessner MJ, Kramer K, Ebel F et al. C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates. Mol Microbiol 2002; 44(2):335-49. 39. Lopez R, Garcia E. Recent trends on the molecular biology of pneumococcal capsules, lytic enzymes and bacteriophage. FEMS Microbiol Rev 2004; 28(5):553-80. 40. Loessner MJ, Wendlinger G, Scherer S. Heterogeneous endolysins in Listeria monocytogenes bacteriophages: a new class of enzymes and evidence for conserved holin genes within the siphoviral lysis cassettes. Mol Microbiol 1995; 16(6):1231-41. 41. Kakikawa M, Yokoi KJ, Kimoto H et al. Molecular analysis of the lysis protein Lys encoded by Lactobacillus plantarum phage phig1e. Gene 2002; 299(1-2):227-34. 42. Loessner MJ, Gaeng S, Wendlinger G et al. The two-component lysis system of Staphylococcus aureus bacteriophage Twort: a large TTG-start holin and an associated amidase endolysin. FEMS Microbiol Lett 1998; 162(2):265-74.

The Use of Recombinant Phage Lysins for the Control of Bacterial Pathogens

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43. Navarre WW, Ton-That H, Faull KF et al. Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity. J Biol Chem 1999; 274(22):15847-56. 44. Yokoi KJ, Kawahigashi N, Uchida M et al. The two-component cell lysis genes holWMY and lysWMY of the Staphylococcus warneri M phage varphiWMY: cloning, sequencing, expression and mutational analysis in Escherichia coli. Gene 2005; 351:97-108. 45. Loessner MJ, Gaeng S, Scherer S. Evidence for a holin-like protein gene fully embedded out of frame in the endolysin gene of Staphylococcus aureus bacteriophage 187. J Bacteriol 1999; 181(15):4452-60. 46. Hermoso JA, Monterroso B, Albert A et al. Structural basis for selective recognition of pneumococcal cell wall by modular endolysin from phage Cp-1. Structure 2003; 11(10):1239-49. 47. Sass P, Bierbaum G. Lytic activity of recombinant bacteriophage phi11 and phi12 endolysins on whole cells and biofilms of Staphylococcus aureus. Appl Environ Microbiol 2007; 73(1):347-52. 48. Cheng Q, Fischetti VA. Mutagenesis of a bacteriophage lytic enzyme PlyGBS significantly increases its antibacterial activity against group B streptococci. Appl Microbiol Biotechnol 2007; 74(6):1284-91. 49. Gaeng S, Scherer S, Neve H et al. Gene cloning and expression and secretion of Listeria monocytogenes bacteriophage-lytic enzymes in Lactococcus lactis. Appl Environ Microbiol 2000; 66(7):2951-8. 50. Horgan M, O’Flynn G, Fitzgerald GF et al. Deletion analysis of LysK, a bacteriophage-derived protein with anti-MRSA activity. 17th European Congress of Clinical Microbiology and Infectious Diseases, ICC, Munich, Germany 2007; Abstract number: 1732-298. 51. Low LY, Yang C, Perego M et al. Structure and lytic activity of a Bacillus anthracis prophage endolysin. J Biol Chem 2005; 280(42):35433-9. 52. Briers Y, Volckaert G, Cornelissen A et al. Muralytic activity and modular structure of the endolysins of Pseudomonas aeruginosa bacteriophages phiKZ and EL. Mol Microbiol 2007; 65(5):1334-44. 53. Cheng X, Zhang X, Pflugrath JW et al. The structure of bacteriophage T7 lysozyme, a zinc amidase and an inhibitor of T7 RNA polymerase. Proc Natl Acad Sci USA 1994; 91(9):4034-8. 54. Merril CR, Scholl D, Adhya SL. The prospect for bacteriophage therapy in Western medicine. Nat Rev Drug Discov 2003; 2(6):489-97. 55. Garcia E, Garcia JL, Garcia P et al. Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages. Proc Natl Acad Sci USA 1988; 85(3):914-8. 56. Matthews BW, Remington SJ. The three dimensional structure of the lysozyme from bacteriophage T4. Proc Natl Acad Sci USA 1974; 71(10):4178-82. 57. Sagermann M, Matthews BW. Crystal structures of a T4-lysozyme duplication-extension mutant demonstrate that the highly conserved beta-sheet region has low intrinsic folding propensity. J Mol Biol 2002; 316(4):931-40. 58. Porter CJ, Schuch R, Pelzek AJ et al. The 1.6 A crystal structure of the catalytic domain of PlyB, a bacteriophage lysin active against Bacillus anthracis. J Mol Biol 2007; 366(2):540-50. 59. Korndorfer IP, Danzer J, Schmelcher M et al. The crystal structure of the bacteriophage PSA endolysin reveals a unique fold responsible for specific recognition of Listeria cell walls. J Mol Biol 2006; 364(4):678-89. 60. Perez-Dorado I, Campillo NE, Monterroso B et al. Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1. J Biol Chem 2007; 282(34):24990-9. 61. Chibani-Chennoufi S, Bruttin A, Dillmann ML et al. Phage-host interaction: An ecological perspective. J Bacteriol 2004; 186(12):3677-86. 62. Wagner PL, Waldor MK. Bacteriophage control of bacterial virulence. Infect Immun 2002; 70(8):3985-93. 63. Guan S, Bastin DA, Verma NK. Functional analysis of the O antigen glucosylation gene cluster of Shigella flexneri bacteriophage Sf X. Microbiology 1999; 145(Pt5):1263-73. 64. Spanier JG, Cleary PP. Bacteriophage control of antiphagocytic determinants in group A streptococci. J Exp Med 1980; 152(5):1393-406. 65. Nilsson JB, Nilsson TK, Jansson JH et al. The effect of streptokinase neutralizing antibodies on fibrinolytic activity and reperfusion following streptokinase treatment in acute myocardial infarction. J Intern Med 2002; 252(5):405-11. 66. Squire IB, Lawley W, Fletcher S et al. Humoral and cellular immune responses up to 7.5 years after administration of streptokinase for acute myocardial infarction. Eur Heart J 1999; 20(17):1245-52. 67. Walsh S, Shah A, Mond J. Improved pharmacokinetics and reduced antibody reactivity of lysostaphin conjugated to polyethylene glycol. Antimicrob Agents Chemother 2003; 47(2):554-8. 68. Fernandez-Tornero C, Lopez R, Garcia E et al. A novel solenoid fold in the cell wall anchoring domain of the pneumococcal virulence factor LytA. Nat Struct Biol 2001; 8(12):1020-4. 69. Fischetti VA. Novel method to control pathogenic bacteria on human mucous membranes. Ann N Y Acad Sci 2003; 987:207-14. 70. Yamashita Y, Shibata Y, Nakano Y et al. A novel gene required for rhamnose-glucose polysaccharide synthesis in Streptococcus mutans. J Bacteriol 1999; 181(20):6556-9.

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71. Loeffler JM, Fischetti VA. Synergistic lethal effect of a combination of phage lytic enzymes with different activities on penicillin-sensitive and -resistant Streptococcus pneumoniae strains. Antimicrob Agents Chemother 2003; 47(1):375-7. 72. Rodriguez-Cerrato V, Garcia P, Del Prado G et al. In vitro interactions of LytA, the major pneumococcal autolysin, with two bacteriophage lytic enzymes (Cpl-1 and Pal), cefotaxime and moxifloxacin against antibiotic-susceptible and -resistant Streptococcus pneumoniae strains. J Antimicrob Chemother 2007;60(5):1159-62. 73. Djurkovic S, Loeffler JM, Fischetti VA. Synergistic killing of Streptococcus pneumoniae with the bacteriophage lytic enzyme Cpl-1 and penicillin or gentamicin depends on the level of penicillin resistance. Antimicrob Agents Chemother 2005; 49(3):1225-8. 74. Matsuzaki S, Rashel M, Uchiyama J et al. Bacteriophage therapy: A revitalized therapy against bacterial infectious diseases. J Infect Chemother 2005; 11(5):211-9. 75. Brown PD, Lerner SA. Community-acquired pneumonia. Lancet 1998; 352(9136):1295-302. 76. Fenoll A, Jado I, Vicioso D et al. Evolution of Streptococcus pneumoniae serotypes and antibiotic resistance in Spain: update (1990 to 1996). J Clin Microbiol 1998; 36(12):3447-54. 77. Sibold C, Wang J, Henrichsen J et al. Genetic relationships of penicillin-susceptible and -resistant Streptococcus pneumoniae strains isolated on different continents. Infect Immun 1992; 60(10):4119-26. 78. Klugman KP. Pneumococcal resistance to antibiotics. Clin Microbiol Rev 1990; 3(2):171-96. 79. Heikkinen T, Chonmaitree T. Importance of respiratory viruses in acute otitis media. Clin Microbiol Rev 2003; 16(2):230-41. 80. Schappert SM, Nelson C. National Ambulatory Medical Care Survey: 1995-96 summary. Vital Health Stat 13 1999(142):i-vi, 1-122. 81. Baker CJ, Barrett FF. Transmission of group B streptococci among parturient women and their neonates. J Pediatr 1973; 83(6):919-25. 82. Farley MM, Harvey RC, Stull T et al. A population-based assessment of invasive disease due to group B Streptococcus in nonpregnant adults. N Engl J Med 1993; 328(25):1807-11. 83. Regan JA, Klebanoff MA, Nugent RP et al. Colonization with group B streptococci in pregnancy and adverse outcome. VIP Study Group. Am J Obstet Gynecol 1996; 174(4):1354-60. 84. Maxted WR. The active agent in nascent phage lysis of streptococci. J Gen Microbiol 1957; 16(3):584-95. 85. Mock M, Fouet A. Anthrax. Annu Rev Microbiol 2001; 55:647-71. 86. Inglesby TV, O’Toole T, Henderson DA et al. Anthrax as a biological weapon, 2002: Updated recommendations for management. JAMA 2002; 287(17):2236-52. 87. Turnbull PC. Definitive identification of Bacillus anthracis—A review. J Appl Microbiol 1999; 87(2):237-40. 88. Rosovitz MJ, Leppla SH. Virus deals anthrax a killer blow. Nature 2002; 418(6900):825-6. 89. Fujinami Y, Hirai Y, Sakai I et al. Sensitive detection of Bacillus anthracis using a binding protein originating from gamma-phage. Microbiol Immunol 2007; 51(2):163-9. 90. Voss A, Loeffen F, Bakker J et al. Methicillin-resistant Staphylococcus aureus in pig farming. Emerg Infect Dis 2005; 11(12):1965-6. 91. Lowy FD. Staphylococcus aureus infections. N Engl J Med 1998; 339(8):520-32. 92. Donovan DM, Lardeo M, Foster-Frey J. Lysis of staphylococcal mastitis pathogens by bacteriophage phi11 endolysin. FEMS Microbiol Lett 2006; 265(1):133-9. 93. Jett BD, Huycke MM, Gilmore MS. Virulence of enterococci. Clin Microbiol Rev 1994; 7(4):462-78. 94. Mead PS, Slutsker L, Dietz V et al. Food-related illness and death in the United States. Emerg Infect Dis 1999; 5(5):607-25. 95. Chang TL, Chang CH, Simpson DA et al. Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc Natl Acad Sci USA 2003; 100(20):11672-7. 96. Kruger C, Hu Y, Pan Q et al. In situ delivery of passive immunity by lactobacilli producing single-chain antibodies. Nat Biotechnol 2002; 20(7):702-6. 97. Turner MS, Waldherr F, Loessner MJ et al. Antimicrobial activity of lysostaphin and a Listeria monocytogenes bacteriophage endolysin produced and secreted by lactic acid bacteria. Syst Appl Microbiol 2007; 30(1):58-67. 98. Granum PE. Clostridium perfringens toxins involved in food poisoning. Int J Food Microbiol 1990; 10(2):101-11. 99. El-Shibiny A, Connerton PL, Connerton IF. Enumeration and diversity of campylobacters and bacteriophages isolated during the rearing cycles of free-range and organic chickens. Appl Environ Microbiol 2005; 71(3):1259-66. 100. Klieve AV, Bauchop T. Morphological diversity of ruminal bacteriophages from sheep and cattle. Appl Environ Microbiol 1988; 54(6):1637-41.

The Use of Recombinant Phage Lysins for the Control of Bacterial Pathogens

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101. Dancer SJ. How antibiotics can make us sick: The less obvious adverse effects of antimicrobial chemotherapy. Lancet Infect Dis 2004; 4(10):611-9. 102. Veiga-Crespo P, Ageitos JM, Poza M et al. Enzybiotics: A look to the future, recalling the past. J Pharm Sci 2007; 96(8):1917-24. 103. Garcia JL, Garcia E, Arraras A et al. Cloning, purification and biochemical characterization of the pneumococcal bacteriophage Cp-1 lysin. J Virol 1987; 61(8):2573-80. 104. Fluckiger U, Moreillon P, Blaser J et al. Simulation of amoxicillin pharmacokinetics in humans for the prevention of streptococcal endocarditis in rats. Antimicrob Agents Chemother 1994; 38(12):2846-9. 105. Garcia P, Lopez R, Ronda C et al. Mechanism of phage-induced lysis in pneumococci. J Gen Microbiol 1983; 129(2):479-87. 106. Fischetti VA, Gotschlich EC, Bernheimer AW. Purification and physical properties of group C streptococcal phage-associated lysin. J Exp Med 1971; 133(5):1105-17. 107. Raina JL. Purification of Streptococcus group C bacteriophage lysin. J Bacteriol 1981; 145(1):661-3. 108. O’Flaherty S, Coffey A, Edwards R et al. Genome of staphylococcal phage K: A new lineage of Myoviridae infecting gram-positive bacteria with a low G+C content. J Bacteriol 2004; 186(9):2862-71.

Chapter 15

Engineered Pharmabiotics with Improved Therapeutic Potential Roy Sleator* and Colin Hill

Abstract

A

lthough described for over a century, scientists and clinicians alike are only now beginning to realise the significant medical applications of probiotic cultures. Given the increasing commercial and clinical relevance of probiotics, improving their stress tolerance profile and ability to overcome the physiochemical defences of the host is an important biological goal. Patho-biotechnology describes the application of pathogen derived (ex vivo and in vivo) stress survival strategies for the design of more technologically robust and effective probiotic cultures with improved biotechnological and clinical applications as well as the development of novel vaccine and drug delivery platforms.

Introduction

While much information is available concerning the exploitation of attenuated pathogens and their associated virulence factors, considerably less information is available concerning the so called bio-engineering of nonpathogenic or probiotic strains for improved therapeutic effect.1 Herein, we review the most recent advances in this aspect of the patho-biotechnology concept. This strategy can be further divided into three distinct approaches. The first tackles the issue of probiotic storage and delivery by cloning and expression of pathogen specific stress survival mechanisms (facilitating improved survival at extremes of temperature and water availability), thus countering reductions in probiotic numbers which can occur during manufacture and storage of delivery matrices (such as foods and tablet formulations). The second approach aims to improve host colonisation by expression of host specific survival strategies (or virulence associated factors) thereby positively affecting the therapeutic efficacy of the probiotic. The final approach involves the development of so called ‘designer probiotics’; strains which specifically target invading pathogens by blocking crucial ligand-receptor interactions between the pathogen and host cell.2

Improving Probiotic Stress Tolerance Ex Vivo

Perhaps the most important stresses encountered during food production and/or tablet formulation (the most common probiotic delivery matrices) are temperature (both low and high) and water availability (aw).3 The ability to overcome such stresses is thus a particularly desirable trait in the selection of commercially important probiotic strains. A common strategy employed by both prokaryotes and eukaryotes to overcome low aw and temperature stress involves the accumulation of protective compounds, termed compatible solutes (examples of which include betaine and trehalose), which stabilise protein structure and function at low temperatures and prevent water loss from the cell and plasmolysis under low aw conditions.4 *Corresponding Author: Roy Sleator—Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland. Email: [email protected]

Patho-Biotechnology, edited by Roy Sleator and Colin Hill. ©2008 Landes Bioscience.

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Improving a strain’s ability to accumulate compatible solutes is thus an obvious step in the development of more robust probiotic strains. Bacteria (as well as higher organisms) have evolved several sophisticated mechanisms for compatible solute accumulation. Indeed, the foodborne pathogen Listeria monocytogenes (probably the best studied pathogen in terms of compatible solute accumulation5) possesses three distinct uptake systems (BetL, Gbu and OpuC), the simplest of which (in genetic terms, since it is encoded by a single gene) is the secondary betaine transporter BetL.6-8 By cloning the betL gene under the transcriptional control of the nisin inducible promoter PnisA we were able to assess the ability of BetL and thus betaine accumulation, to contribute to probiotic survival under a variety of stresses likely encountered during food and/or tablet manufacture.9 Our probiotic of choice, Lactobacillus salivarius UCC118, while already in possession of an endogenous betaine uptake system,10 exhibits significantly lower accumulation levels than L. monocytogenes and is correspondingly less physiologically robust than the pathogen. Thus, we proposed that expressing betL in L. salivarius might increase betaine accumulation, thereby improving the strains stress tolerance profile. As expected, following nisin induction, the betL complemented L. salivarius strain showed a significant increase in betaine accumulation compared to the wild type (Fig. 1A). Indeed, sufficient BetL was produced to confer increased salt tolerance, with growth of the transformed construct at significantly higher salt concentrations (7% NaCl) than the parent strain (Fig. 1B), or indeed any other lactobacilli. In addition to increased osmotolerance the BetL+ strain showed significantly improved resistance to both chill and cryotolerance (2 logs greater survival than the control at −20˚C and 0.5 logs greater at −70˚C) as well as freeze-drying (36% survival as compared to 18% for the control strain) and spray-drying (1.4% compared to 0.3%); common stresses encountered during food and/or tablet formulation. Furthermore, the presence of BetL resulted in a significant improvement in barotolerance (Fig. 1C). This is particularly significant given that high pressure processing is gaining increasing popularity as a novel nonthermal mechanism of food processing and preservation.11,12 As well as compatible solute uptake systems, recent studies have shown that heterologously expressed compatible solute syntheses systems may also offer similar protective effects. For example Termont et al,13 recently demonstrated that Lactococcus lactis expressing the trehalose synthesizing genes (ostAB) from Escherichia coli (under the transcriptional control of PnisA) retained almost 100% viability after freeze-drying, together with a markedly prolonged shelf life. Thus, improving the stress tolerance profile of probiotic cultures significantly improves tolerance to processing stress and prolongs survival during subsequent storage. This in turn contributes to a significantly larger proportion of the administered probiotic reaching the desired location (e.g., the gastrointestinal tract) in a bioactive form.

In Vivo

In addition to the ex vivo stresses encountered during food/tablet manufacture and storage, probiotic bacteria must also overcome the physiological defences of the host in order to survive within the gastrointestinal tract in sufficient numbers to exert a therapeutic effect. Recently we demonstrated that cloning betL into Bifidobacterium breve UCC2003, significantly improved the tolerance of the probiotic to gastric juice.14 Interestingly, in support of this observation Termont et al,13 also reported improved tolerance to gastric juice in a L. lactis strain expressing the E. coli trehalose synthesis genes, thus suggesting a novel protective role for compatible solutes in the gastric environment. Genes encoding carnitine uptake, which we have shown to contribute to the gastrointestinal survival of L. monocytogenes15 are other obvious candidates for this approach. Once free of the stomach bacteria enter the upper small intestine where they are exposed to low aw conditions (equivalent to 0.3 M NaCl). Consistent with our previous observations with L. salivarius UCC118,9 a significant osmoprotective effect was observed following the introduction of betL into B. breve, facilitating growth of the probiotic in conditions similar to those encountered in vivo (1.5% NaCl and 6% sucrose, both of which approximate the osmolarity of the gut). Furthermore, whilst stable colonisation of the murine intestine was achieved by oral administration of B. breve UCC2003, strains harbouring BetL were recovered at significantly higher levels in the faeces,

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intestines and caecum of inoculated animals. Finally, in addition to improved gastric transit and intestinal persistence, the addition of BetL improved the clinical efficacy of the probiotic culture; mice fed B. breve UCC2003 (BetL+) exhibited significantly lower levels of systemic infection compared to the control strain following oral inoculation with L. monocytogenes (Fig. 2). This is, to the best of our knowledge, the first clear evidence of an enhanced therapeutic effect following precise bio-engineering of a probiotic strain.

‘Designer Probiotics’

In addition to improving their physiological stress tolerance, recent studies have lead to the development of ‘designer probiotics’ which specifically target enteric infections by blocking crucial ligand-receptor interactions between the pathogen and host cell.2 Many of the pathogens responsible for the major enteric infections exploit oligosaccharides displayed on the surface of host cells as receptors for toxins and/or adhesins, enabling colonization of the mucosa and entry of the pathogen or secreted toxins into the host cell. Blocking this adherence prevents infection, while toxin neutralization ameliorates symptoms until the pathogen is eventually overcome by the immune system. ‘Designer probiotics’ have been engineered to express receptor-mimic structures on their surface.16 When administered orally these probiotics bind to and neutralize toxins in the gut lumen and interfere with pathogen adherence to the intestinal epithelium (Fig. 3). One such construct consists of an E. coli strain expressing a chimeric lipopolysaccharide (LPS) terminating in a shiga toxin (Stx) receptor. 1 mg dry weight of this recombinant strain has been shown to

Figure 1. A) (14C)glycine betaine uptake in the Lactobacillus salivarius wild type (light bar) and the BetL complemented strain UCC118-BetL+ (dark bar). B) Growth of L. salivarius wild type (light circles) and UCC118-BetL+ (dark circles) in MRS broth with 7% added NaCl. C) High-pressure-induced inactivation of L. salivarius wild type (light bars) and UCC118-BetL+ (dark bars). Adapted from Sheehan et al9. A color version of this image is available online at www.eurekah.com

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Figure 2. A) Recovery of Bifidobacterium breve UCC2003-BetL+ (dark circles) and UCC2003 wild type (light circles) from female BALB/c mice over 32 days of analysis. Faeces for bacteriological analysis were obtained from five mice in each treatment group and viable counts of B. breve UCC2003 derivatives were determined. B) Listerial infection in the spleens of BALB/c mice. Animals were fed ~109 CFU ml-1 of either UCC2003-BetL+ or UCC2003 wild type for three consecutive days. On the fourth day, all animals were infected with ~1011 CFU ml-1 Listeria monocytogenes EGD-e. Three days post infection the animals were sacrificed and the numbers of Listeria were determined. Adapted from Sheehan et al,14 (2007). A color version of this image is available online at www.eurekah.com.

neutralize >100 μg of Stx1 and Stx2.16 Paton et al,17,18 have also constructed probiotics with receptor blocking potential against Enterotoxigenic E. coli (ETEC) toxin LT and cholera toxin (Ctx). As well as treating enteric infections, ‘designer probiotics’ have also been recruited to combat HIV. Rao et al,19 recently described the construction of a probiotic strain of E. coli, engineered to secrete HIV-gp41-haemolysin A hybrid peptides, which block HIV fusion and entry into target cells. When administered orally or as a rectal suppository, this ‘live microbicide’ colonizes the gut mucosa and secretes the peptide in situ, thereby providing protection in advance of HIV exposure for up to a month.20 Other anti-HIV probiotics currently in development include a genetically engineered Streptococcus gordonii which produces cyanovirin-N, a potent HIV-inactivating protein originally isolated from cyanobacterium and a natural human vaginal isolate of Lactobacillus jensenii modified to secrete two-domain CD4 which inhibits HIV entry into target cells.21 In addition to in infection control probiotics (and other nonpathogenic bacteria) are also being engineered to function as novel vaccine delivery vehicles which can stimulate both innate and acquired immunity but lack the possibility of reversion to virulence which exists with more conventional pathogenic platforms. Guimarães et al,22 recently described the construction of a L. lactis strain expressing inlA, encoding internalin A, a surface protein related to invasion in L. monocytogenes. In this instance the otherwise non-invasive L. lactis strain is now capable of invading the small intestine and delivering molecules (DNA or protein) into mammalian epithelial cells, making it a safer and more attractive alternative to attenuated L. monocytogenes as an antigen delivery vehicle. Furthermore, the authors suggest that the addition of hlyA (encoding listeriolysin) to L. lactis inlA+ may promote phagosomal escape within the macrophage and induction of an immune response comparable to that of the intracellular pathogen. Probiotic vaccine carriers administered by the mucosal route mimic the immune response elicited by natural infection and can lead to long lasting protective mucosal and systemic responses.23 Mucosal vaccine delivery (those administered orally, anally or by nasal spray) also offers significant technological and commercial advantages over traditional formulations including: reduced pain and the possibility of cross contamination associated with intramuscular injection as well as the lack of a requirement for medically trained personnel to administer the vaccine.

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Figure 3. Receptor mimic strategy employed by ‘designer probiotics’. Probiotic bacteria (in blue) engineered to express surface host-receptor mimics that can bind to and neutralise toxins (stars) in the gut lumen or interfere with the adherence of pathogens/antigens (circles) to the intestinal epithelium. A color version of this image is available online at www.eurekah.com.

Biological Containment

The use of genetically modified organisms in medicine raises legitimate concerns about their survival and propagation in the environment and the host and about the dissemination of antibiotic markers or other genetic modifications to other microorganisms. At least some of these concerns might be allayed by the implementation of stringent bio-containment measures. Biological containment systems can be subdivided into active and passive forms. Active containment provides control through the conditional production of a compound which is toxic to the cells. Examples of this type of control include the phage T7 lysozome and colicin E3 in E. coli.24 Passive containment on the other hand is dependent on complementation of an auxotrophy by supplementation with either an intact gene or the essential metabolite. Perhaps the most elegant biological containment strategy devised to date targets the essential thymidylate synthase (thyA) gene, replacing it with a transgene (encoding the desired stress survival trait).25 Since the thyA gene is essential for growth; mutant strains only grow in the presence of added thymidine or thymine. Thymine auxotrophy involves activation of the SOS repair system and DNA fragmentation, thereby constituting an indigenous suicide system. Thymine and thymidine growth dependence differs from most other auxotrophies in that absence of the essential component is bactericidal in the former and bacteriostatic in the latter. The choice of thyA as a target gene thus combines the advantages of passive and active containment systems with the end result being that thyA-deficient bacteria cannot accumulate in the environment. This approach addresses biosafety concerns on several levels. Firstly, no resistance marker is required to guarantee stable inheritance of the transgene, thus overcoming any potential problems associated with dissemination of antibiotic resistance to the commensal populations or opperyunistic pathogens. Second, accumulation of the genetically modified organism in the environment

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is highly unlikely given that rapid death occurs upon thymidine starvation. Finally should an intact thyA be acquired from closely related bacteria by means of homologous recombination then the transgene would be lost as a result of the double cross over.

Conclusions

Although conventional molecular medical research continues to provide effective therapeutic and prophylactic compounds, their application is often complicated by in vivo sensitivity and rising production costs. Engineered probiotics thus provide an effective means of circumventing the short half-life and fragility of conventional therapeutics, providing a cost effective alternative which will ultimately contribute to health and social gain particularly in developing countries.26 However while consumer acceptance of genetically engineered ‘designer probiotics’ still remains a significant challenge this obstacle should eventually be overcome by the application of rigorous scientific controls, such as adequate biological containment and proper risk-benefit analysis of the potential advantages of such a strategy.

Acknowledgements

Dr Roy Sleator is a Health Research Board Principal Investigator. The authors also wish to acknowledge the continued financial assistance of the Alimentary Pharmabiotic Centre, funded by Science Foundation Ireland under the Centres for Science, Engineering and Technology Research Programme. This chapter is based on an earlier review “Improving probiotic function using a pathobiotechnology approach” Gene Ther Mol Biol 2007; 11:269-274.

References

1. Sleator RD, Hill C. ‘Bioengineered bugs’—A Patho-biotechnology approach to probiotic research and applications. Med Hypotheses 2007; 70:167-169. 2. Paton AW, Morona R, Paton JC. Designer probiotics for prevention of enteric infections. Nat Rev Microbiol 2006; 4:193-200. 3. Hill C, Cotter P, Sleator RD et al. Bacterial stress response in Listeria monocytogenes: Jumping the hurdles imposed by minimal processing. Int Dairy J 2002; 12:273-283. 4. Sleator RD, Hill C. Bacterial osmoadaptation: The role of osmolytes in bacterial stress and virulence. FEMS Microbiol Rev 2002; 26:49-71. 5. Sleator RD, Gahan CGM, Hill C. A postgenomic appraisal of osmotolerance in Listeria monocytogenes. Appl Environl Microbiol 2003; 69:1-9. 6. Sleator RD, Gahan CGM, Abee T et al. Identification and disruption of BetL, a secondary glycine betaine transport system linked to the salt tolerance of Listeria monocytogenes LO28. Appl Environl Microbiol 1999; 65:2078-2083. 7. Sleator RD, Gahan CGM, O’Driscoll B et al. Analysis of the role of betL in contributing to the growth and survival of Listeria monocytogenes LO28. Int J Food Microbiol 2000; 60:261-268. 8. Sleator RD, Wood JM, Hill C. Transcriptional regulation and posttranslational activity of the betaine transporter BetL in Listeria monocytogenes is controlled by environmental salinity. J Bacteriol 2003; 185:7140-7144. 9. Sheehan VM, Sleator RD, Fitzgerald GF et al. Heterologous expression of BetL, a betaine uptake system, enhances the stress tolerance of Lactobacillus salivarius UCC118. Appl Environ Microbiol 2006; 72:2170-2177. 10. Claesson MJ, Li Y, Leahy S et al. Multireplicon genome architecture of Lactobacillus salivarius. Proc Natl Acad Sci USA 2006; 103:6718-6723. 11. Smiddy M, Sleator RD, Patterson MF et al. Role for compatible solutes glycine betaine and L-carnitine in listerial barotolerance. Appl Environ Microbiol 2004; 70:7555-7557. 12. Smiddy M, O’Gorman L, Sleator RD et al. Greater high-pressure resistance of bacteria in oysters than in broth. Inn Food Sci Emerg Tech 2005; 6:83-90. 13. Termont S, Vandenbroucke K, Iserentant D et al. Intracellular accumulation of trehalose protects Lactococcus lactis from freeze-drying damage and bile toxicity and increases gastric acid resistance. Appl Environ Microbiol 2006; 72:7694-7700. 14. Sheehan V, Sleator RD, Fitzgerald G et al. Improving gastric transit, gastrointestinal persistence and therapeutic efficacy of the probiotic strain Bifidobacterium breve UCC2003. Microbiol 2007; 153:3563-3571.

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15. Sleator RD, Gahan CGM, Abee T et al. Analysis of the role of OpuC, an Osmolyte Transport system, in salt tolerance and virulence potential of Listeria monocytogenes. Appl Environ Microbiol 2001; 67:2692-2698. 16. Paton AW, Morona R, Paton JC. A new biological agent for treatment of Shiga toxigenic Escherichia coli infections and dysentery in humans. Nat Med 2000; 6:265-270. 17. Paton AW, Morona R, Paton JC. Neutralization of shiga toxins Stx1, Stx2c and Stx2e by recombinant bacteria expressing mimics of globotriose and globotetraose. Infect Immun 2001; 69:1967-1970. 18. Paton AW, Jennings MP, Morona R et al. Recombinant probiotics for treatment and prevention of enterotoxigenic Escherichia coli diarrhea. Gastroenterology 2005; 128:1219-28. 19. Rao S, Hu S, McHugh L et al. Toward a live microbial microbicide for HIV: Commensal bacteria secreting an HIV fusion inhibitor peptide. Proc Natl Acad Sci USA 2005; 102:11993-11998. 20. Laurel AL, Berger EA. An anti-HIV microbicide comes alive Proc Natl Acad Sci USA 2005; 102:12294-12295. 21. Chang TL-Y, Chang CH, Simpson DA et al. Inhibition of HIV infectivity by a natural human isolate of Lactobacillus jensenii engineered to express functional two-domain CD4. Proc Natl Acad Sci USA 2003; 100:11672-11677. 22. Guimarães VD, Gabriel JE, Lefèvre F et al. Internalin-expressing Lactococcus lactis is able to invade small intestine of guinea pigs and deliver DNA into mammalian epithelial cells. Microbes Infect 2005; 7:836-844. 23. Holmgren J, Czerkinsky C. Mucosal immunity and vaccines. Nat Med 2005; 11:S45-53. 24. Torres B, Jaenecke S, Timmis KN et al. A dual lethal system to enhance containment of recombinant micro-organisms. Microbiol 2003; 149:3595-3601. 25. Steidler L, Neirynck S, Huyghebaert N et al. Biological containment of genetically modified Lactococcus lactis for intestinal delivery of human interleukin 10. Nat Biotechnol 2003; 21:785-9. 26. Sleator RD, Hill C. Probiotics as therapeutics for the developing world. J Infect Develop Countries 2007; 1:7-12.

Index A Acetylcholine 173, 182 Active compound 50 Adaptive immunity 147 Adeno-associated virus 126, 127, 131, 136 Adenovirus 130, 134-138, 141 Adhesin 33, 50, 208 Adjuvant 3, 50, 51, 53, 54, 71, 72, 74, 81, 103, 163-169 Advanced drug delivery system (ADDS) 50, 51, 53, 54 AIDS 3, 42, 43, 48, 145, 146, 153, 154, 158 Anopheles gambiae 26, 154 Anti-angiogenic 140, 141 Antibiotic resistance 24, 25, 51, 55, 145, 188, 194, 198, 210 Antigen presenting cell (APC) 50, 71-73, 80, 88, 103, 106, 117, 119, 166 Arginine deiminase 159 Autoimmune disease 61, 141 Azurin 146, 148, 149, 151-154, 157-159

B Bacillus anthracis 2, 192, 197 Bacillus Calmette-Guérin (BCG) 47, 77, 79, 86, 113, 147, 166 Bacillus sphaericus 23, 26, 28 Bacillus thuringiensis 15, 18-22, 28 Bacteria 1-3, 5-7, 9, 11, 15, 16, 27, 29, 30, 33, 35, 41-48, 50-55, 57, 60-65, 67, 71, 72, 74-84, 86-88, 98-100, 102-106, 112, 113, 115, 117-119, 121-126, 145-147, 149, 152, 154, 158, 159, 163, 173, 187-198, 200, 201, 207, 209-211 Bacterial ghost (BG) 50-57 Bacterial interference 3, 8, 10, 50 Bacterial vector 60-65, 67, 77, 81, 87, 88, 98, 99, 103, 105, 106, 121, 146 Bacteria-mediated RNA delivery 124 Bacteriocin 5-10 Bacteriocin immunity 7 Bactofection 72, 85, 88, 113, 119 Baculovirus 16-20, 72

Beauveria bassiana 29 Betaine 206-208 Betl 207-209 Bifidobacterium 3, 88, 98, 100-102, 146, 207, 209 Bifidobacterium breve 207, 209 Biological warfare 197 Biomedicine 51, 126 Blepharospasm 178 Bordetella pertussis 163, 167 Bordetella pertussis toxin (PT) 163, 165, 167 Botulinum toxin 173, 175, 176 Budded virus 16, 17

C C1 191, 196, 197 Cancer 47, 57, 60-64, 76, 84, 86, 98, 100-104, 106, 115, 121, 124, 127, 131, 133, 135, 136, 140, 141, 145-149, 152-154, 156, 157, 159, 163, 164, 166, 167, 169 Cancer gene therapy 61, 135 Cancer therapy 127, 136, 147, 148, 163 Cathepsins 18, 19 CD4 76, 80, 112, 117, 152-156, 158, 163, 166, 168, 169, 209 CD8 71, 80, 81, 86, 87, 104, 112, 115, 117, 164-166, 168 Chitinases 18, 35 Clinical trial 9, 10, 61, 77, 81, 85-88, 102, 122, 127, 133, 139, 155, 158, 166, 193 Clostridia 62, 64, 100-102, 146, 147 Clostridium difficile 163, 165, 168, 201 Clostridium perfringens 200 Colonization 2, 3, 8-11, 61, 62, 64, 65, 67, 102, 115, 147, 168, 187, 191, 195-198, 200, 208 Commensal 2, 4, 5, 8, 60, 65, 146, 158, 188, 200, 210 Compatible solute 206, 207 Cosmetic 173, 174, 177 Cpl-1 190-196 Cricopharyngeal spasm 182

214

Crow’s feet 174 Cystic fibrosis transmembrane conductance regulator (cftr) gene 105, 106, 135, 137

D Delivery system 20, 34, 35, 43, 50, 51, 53, 60, 71, 72, 76, 77, 81, 82, 86-88, 106, 122, 124, 140, 166 Dendritic cell 55, 72 Designer probiotics 206, 208-211 DNA vaccine 51, 54, 55, 60, 71-77, 81, 86-88, 104, 105, 140, 146, 166, 167 Drug 2, 41-43, 46-48, 50, 51, 53, 55-57, 60, 61, 63, 65, 67, 100, 106, 112, 113, 117-119, 121, 122, 130, 140, 145-149, 152-155, 158, 159, 188, 193, 195, 206 Dystonia 178, 180, 181

E Early genes 135, 138 Effector 1-5, 9, 10, 35, 61, 87, 146, 163 E-mediated lysis 50-52, 55 Enteric infection 208, 209 Enterococcus faecalis 188, 198 Ex vivo 1, 140, 206, 207

F Facial synkinesis 177 Frown line 174, 175, 184

G Gastrointestinal tract (GI tract) 60, 199, 207 Gene therapy 77, 85, 98, 103, 106, 122, 124, 126, 127, 129, 131, 133-135, 140, 141 Genetic adjuvant 74 Gene vector 121, 122 Gram-negative cell envelope 50, 113 Granulovirus 16, 20 Group a streptococci 191, 196 Group b streptococci 188, 191, 196

Patho-Biotechnology

H Headache 182, 184 Helicobacter pylori 50, 76, 163, 165, 169 Helper virus 128, 131, 134-139 Hemifacial spasm 177 Herpes simplex virus 72, 76, 104, 138 HIV 72-74, 82-84, 86, 104, 130, 134, 136, 146, 149, 151-159, 166, 168, 209 Holin 187, 188, 192 Hybrid vector 139, 141

I IL-4 76, 81, 164, 165, 167-169 IL-12 74, 76, 81, 164-167, 169 Immune system 3, 33, 50, 54, 60, 72, 74, 81, 124, 126, 127, 145-147, 158, 163, 164, 197, 208 Immunoglobulin 146, 152, 157, 158, 193 Immunomodulator 157, 158, 163-165, 168, 169 Immunotherapy 98, 103, 106, 147, 169 Indigenous microbiota 1-4 Insect-pathogenic virus 35 Interleukin 65, 156, 164 Interpeptide bridge-specific endopeptidase 188, 189 Intracellular persistence 7 In vivo remote control 60, 61

L Lactobacillus 3, 88, 198, 200, 207-209 Lactobacillus salivarius 207, 208 L-alanoyl-D-glutamate endopeptidase 188, 189 Laryngeal dystonia 180 Laz 149, 152-154, 158, 159 Listeria 72, 80, 86, 98, 99, 103-105, 112, 123, 146, 163, 165, 166, 189, 193, 198, 200, 207, 209 Listeria monocytogenes 72, 86, 99, 104, 105, 163, 165, 166, 193, 207, 209 Listeriolysin 83, 112, 113, 123, 124, 163, 165, 166, 209 Live microbicide 209 Lysin 99, 187, 188, 190-201

215

Index

M Malaria 15, 26, 30, 145, 146, 154, 159 Mucosal delivery 72 Mycobacterium 41-47, 77, 86, 105, 147, 166 Mycoplasma arginini 159

Pseudomonas aeruginosa 146, 188 Pseudotyping 130, 132-134, 136

R

N-acetylmuramidase 188, 189 Neisseria meningitides 152 Neutrophil-activating protein (NAP) 163, 165, 169 Novel antibacterial target 112 Nucleopolyhedrovirus 16, 18

Recombinant microbial insecticide 36 Recombinant strain 25-28, 104, 208 Recombinant virus 20, 35, 126, 129- 131, 133 Replacement therapy 4, 5, 8, 9, 11, 121, 122, 124 Retrovirus 98, 126-128, 130-35 RNA delivery 124 RNAi delivery 121, 122 RNA interference (RNAi) 2, 3, 74, 106, 121-124, 141

O

S

Oncolytic 102, 138-140 Oromandibular dystonia 178, 181

Safety 16, 20, 27-29, 33, 61, 63, 65, 67, 71, 72, 77-79, 86, 87, 122, 126, 127, 131, 133, 134, 136, 139-141, 158, 193 Salmonella 50, 65-67, 72, 75, 77, 79-81, 85, 98-105, 112, 113, 115, 119, 124, 146, 147 Shigella 50, 53, 65, 72, 77, 80, 85, 86, 98, 99, 103, 104, 112, 119, 146 Short hairpin RNA (shRNA) 121-124 Sialorrhea 183 Small interfering RNA (siRNA) 55, 57, 106, 121, 122, 124 S. mutans BCS3-L1 9 Staphylococcus aureus 2, 51, 52, 158, 187, 189, 198 Streptococcus pneumoniae 10, 194, 195 Streptococcus salivarius 3 Structure-function relationship 67, 105, 112, 113, 146, 154, 195 Subunit vaccine 50, 53-55

N

P Pain 105, 173, 177, 182-184, 209 Pal 190, 191, 194-196 Palatal myoclonus 182 Particulate carrier system 50 Patho-biotechnology 46, 126, 206 Pathogen 1-5, 8-11, 15, 16, 30-35, 41-44, 46-48, 50, 53, 54, 60, 71-74, 77, 80-84, 86-88, 99, 100, 102-105, 112, 113, 117-119, 126, 135, 145-148, 163, 164, 166, 169, 170, 187, 188, 192-195, 197, 198, 201, 206-210 Pathogenesis 21, 33, 105, 163, 193 Pbad 64, 65, 67 Phage 8, 41-48, 51, 52, 71, 77, 99, 159, 187-195, 197-201, 210 Pharmabiotic 206, 211 Plasmid design 72 Platysmal bands 177 Ply3626 200 Ply511 198, 200 PlyGBS 191, 192, 196, 197 PlyL 192, 193 Preca 62, 64 Preclinical study 81, 82, 85, 86, 106, 195 Probiotic 2-4, 6, 10, 60, 67, 200, 206-211 Protease 19, 21, 32, 35, 155 Protein drug 145, 154

T Temporomandibular disorder 182 Terminal repeat 132, 133, 136-138 Th1 87, 104, 105, 117, 163-169 Th2 163-169 Titre 130, 131, 135, 193-197 TkRNAi 121-124 Toll-like receptor 53, 72, 103, 152, 169

216

Toxin 16, 18-21, 23-28, 30, 32-35, 62-67, 71, 73, 75, 81, 82, 84, 86-88, 98, 100, 105, 115, 122, 123, 146, 163-169, 173, 175-178, 182, 184, 193, 208-210 Toxin A 163, 165, 168, 169 Toxoplasma gondii 147, 152, 153 Toxoplasmosis 153, 154, 159 Transduction 115, 129, 133, 134, 140, 149, 157 Transgene 16, 18, 32-34, 72, 73, 98, 103, 105, 126-133, 135-141, 210, 211 Transkingdom RNA interference 121-123 Tropism 99, 126, 127, 130, 136, 138 Tumor 63-67, 71, 73, 81, 84, 86-88, 98, 100-104, 106, 112, 113, 115, 118, 119, 130, 135, 136, 138-141, 146-149, 151, 152, 156, 157, 164, 166, 168, 177 Tumor therapy 51, 56, 57, 61, 62, 65, 67, 81, 106, 112, 118, 119 Tumour cytostatics 50

Patho-Biotechnology

V Vaccination 98, 103-106, 115, 139, 169, 201 Vaccine 43, 47, 50, 51, 53-55, 60, 61, 67, 71-79, 81, 82, 85-88, 97, 104-106, 112, 113, 117, 122, 124, 131, 135, 139, 140, 145, 146, 154, 163-168, 206, 209 Vaccine platform 50 Vaccinia virus 81, 104, 126, 127, 130, 131, 139 Vibrio cholerae 50, 87, 164 Viral vector 98, 121, 122, 126-131, 133, 136, 138-141 Virulence factor 154, 163-167, 169, 170, 195, 206

Y Yersinia 87, 98, 99, 103-105, 122, 123, 146

Biotechnology intelligence unit

The chapters in this book, as well as the chapters of all of the five Intelligence Unit series, are available at our website.

Roy Sleator and Colin Hill

INTELLIGENCE UNITS Biotechnology Intelligence Unit Medical Intelligence Unit Molecular Biology Intelligence Unit Neuroscience Intelligence Unit Tissue Engineering Intelligence Unit

Sleator • Hill

BIOTECHNOLOGY Intelligence Unit

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Patho-Biotechnology

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Patho-Biotechnology