National Institute of Allergy and Infectious Diseases, NIH: Volume 1: Frontiers in Research (Infectious Disease) (Infectious Disease)

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National Institute of Allergy and Infectious Diseases, NIH Volume 1

Frontiers in Research

Infectious Disease Vassil St. Georgiev

For other titles published in the series, go to www.springer.com / humana click on the series discipline click on the heading “Series” click on the name of the series

National Institute of Allergy and Infectious Diseases, NIH Volume 1

Frontiers in Research

Edited by Vassil St. Georgiev, PhD Karl A. Western, MD John J. McGowan, PhD National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

Editors Vassil St. Georgiev, PhD Karl A. Western, MD John J. McGowan, PhD National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

Series Editor Vassil St. Georgiev National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

ISBN 978-1-934115-77-0 e-ISBN 978-1-59745-569-5 DOI: 10.1007/978-1-59745-569-5 Library of Congress Control Number: 2007941162 © 2008 Humana Press, a part of Springer Science+Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Cover illustration: Adapted from Chapter 30, Fig. 30.3, showing the sCD4-17b bifunctional protein, which in turn is based on the atomic structure reported in Kwong et al., Nature, 393:648–659 (1998). Printed on acid-free paper 9 8 7 6 5 4 3 2 1 springer.com

Dedication

To the thousands of investigators who, for more than 50 years, have received the support of the National Institute of Allergy and Infectious Diseases (NIAID) and have dedicated their lives and careers to biomedical research.

RESEARCH IS NOT A SYSTEMATIC OCCUPATION BUT AN INTUITIVE ARTISTIC VOCATION Albert Szent-Györgyi

Preface

For more than 50 years, as part of the National Institutes of Health, the mission of the National Institute of Allergy and Infectious Diseases (NIAID) has been to conduct and support basic and applied research to better understand, treat, and prevent infectious, immunologic, and allergic diseases with the ultimate goal of improving the health of individuals in the United States and around the world. In recent years, NIAID has responded to new challenges including emerging and re-emerging infectious diseases, potential bioterrorism threats, and an increase in pediatric asthma prevalence. A cornerstone of NIAID-supported research also continues to be the discovery and improvement of vaccines focused on an array of infectious diseases with global public health importance. As part of its mission to foster biomedical discovery and to reduce the burden of human disease, NIH and NIAID in particular, are committed to encouraging the accelerated translation of biomedical discoveries into effective clinical care and public health practice throughout the world. In pursuit of this goal and its disease-specific scientific objectives, NIAID seeks to broaden research opportunities and collaborations involving scientists and institutions outside the United States. During 2006, special emphasis was given to fostering scientific collaboration between U.S. researchers and investigators in Central and Eastern Europe, the Baltic Region, Russia, Ukraine, and other newly independent states that were formerly part of the Soviet Union. Although the countries of Central and Eastern Europe have strong traditions in biomedical research, scientists from this region have been less successful than their Western European colleagues in competing for NIAID funding and in forming partnerships with U.S. scientists. To help address this situation, NIAID convened a research conference in Opatija, Croatia (June 24–30, 2006) so that U.S. and European scientists could explore shared research interests with a focus on microbiology and infectious diseases, HIV/AIDS, and basic and clinical immunology. In the field of microbiology and infectious diseases, major presentations at the conference focused on recent research developments in emerging and re-emerging infections (anthrax and other potential biological weapons, vector-borne infections, tuberculosis, and influenza). A number of presentations discussed ongoing research targeting the development of infectious disease prophylactics and therapeutics. One of the most serious problems worldwide that confronts efforts to control and treat infectious diseases is the increasing resistance of some pathogens to the current armamentarium of drugs. Microorganisms belonging to all four classes of infectious agents (bacteria, viruses, parasites, and fungi) have developed resistance to previously effective chemotherapeutics, thereby becoming serious threats to individual well-being and international public health. One striking example of drug resistance is the emergence of extensively drug-resistant tuberculosis. Several conference presentations were therefore focused on drug resistance. HIV/AIDS also remains a major infectious disease research priority and it was well addressed during the conference. Since the start of the HIV/AIDS pandemic in the early 1980s, nearly 20 million people worldwide have died of the disease. According to an estimate issued by the Joint United Nations Programme on HIV/AIDS (UNAIDS) by the end of 2003, about 38 million adults and children were living with HIV/AIDS and in many countries overall prevalence still is rising. Although much progress has been made in the treatment of AIDS and in understanding effective strategies to prevent HIV transmission, research is urgently needed on vaccines, microbicides, therapeutic agents, behavioral prevention strategies, and the management of HIVrelated co-morbidities. NIAID-funded research in basic and clinical immunology has led to significant discoveries that have guided the effective treatment of a host of immunological conditions. For example, “tolerance induction” research has enabled the selective blocking of inappropriate or destructive immune responses while leaving protective immune responses intact. Major presentations at vii

viii

Preface

the conference discussed various topics in immunomodulation, autoimmunity, infections and immunity, and vaccine development. Finally, two sessions at the research conference were designed to inform participants about NIAID’s research funding mechanisms and the NIH application process. With more than 100 participants, the 2006 NIAID Research Conference in Croatia clearly demonstrated NIAID’s commitment to a cutting-edge scientific exchange to help generate more research cooperation. Following the meeting, numerous research collaborations have been explored and numerous joint research applications have been prepared and submitted. NIAID is pleased to have supported this important and unusual meeting and it welcomes publication of the important scientific findings presented there. The future of science lies in cooperation across national borders. Therefore, it is particularly rewarding to see research partnerships grow between scientists from countries previously characterized by a lack of communication and mutual understanding. With a strong research base, talented investigators in the United States and abroad, and the availability of powerful new research tools, NIAID will continue to support scientists in the forefront of basic and applied infectious and immune-mediated disease research.

Vassil St. Georgiev Bethesda, MD

Acknowledgments

We would like to express our appreciation to Ms. Caroline Manganiello and the staff of technical writers for their help in the preparation of this volume.

ix

Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vassil St. Georgiev

vii

Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ix

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xv

PART I

INTRODUCTION

National Institute of Allergy and Infectious Diseases (NIAID): An Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Karl A. Western PART II

MICROBIOLOGY AND INFECTIOUS DISEASES

Section 1

Emerging and Re-Emerging Infections

1

Biotools for Determining the Genetics of Susceptibility to Infectious Diseases and Expediting Research Translation into Effective Countermeasures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Malak Kotb, Robert W. Williams, Nourtan Fathey, Mohamed Nooh, Sarah Rowe, Rita Kansal, and Ramy Aziz

3

13

2

Spore Surface Components and Protective Immunity to Bacillus anthracis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Patricia Sylvestre, Ian Justin Glomski, Evelyne Couture-Tosi, Pierre Louis Goossens, and Michèle Mock

19

3

New Candidate Anthrax Pathogenic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Serguei G. Popov

25

4

Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. L. Stevenson, N. Ismail, and D. H. Walker

37

5

Multiple Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) of Brucella spp. Identifies Species-Specific Markers and Insights into Phylogenetic Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . Lynn Y. Huynh, Matthew N. Van Ert, Ted Hadfield, William S. Probert, Bryan H. Bellaire, Michael Dobson, Robert J. Burgess, Robbin S. Weyant, Tanja Popovic, Shaylan Zanecki, David M. Wagner, and Paul Keim

6

Expression of the MtrC-MtrD-MtrE Efflux Pump in Neisseria gonorrhoeae and Bacterial Survival in the Presence of Antimicrobials. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William M. Shafer, Jason P. Folster, Douglas E. M. Warner, Paul J. T. Johnson, Jacqueline T. Balthazar, Nazia Kamal, and Ann E. Jerse

47

55

xi

xii

Contents

Section 2

Tuberculosis

7

What can Mycobacteriophages Tell Us About Mycobacterium tuberculosis? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Graham F. Hatfull

67

8

Clinical Mycobacterium tuberculosis Strains Differ in their Intracellular Growth in Human Macrophages . . . . . . . . Sue A. Theus, M. Donald Cave, and Kathleen D. Eisenach

77

9

Mechanisms of Latent Tuberculosis: Dormancy and Resuscitation of Mycobacterium tuberculosis . . . . . . . . . . . . . Galina Mukamolova, Elena Salina, and Arseny Kaprelyants

83

10

Separating Latent and Acute Disease in the Diagnosis of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T. Mark Doherty

91

11

Mutant Selection Window Hypothesis: A Framework for Anti-mutant Dosing of Antimicrobial Agents . . . . . . . . . . Karl Drlica and Xilin Zhao

101

Section 3 Avian Influenza 12

The NIAID Influenza Genome Sequencing Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lone Simonsen, Gayle Bernabe, Karen Lacourciere, Robert J. Taylor, and Maria Y. Giovanni

109

13

Lessons from the 1918 Spanish Flu Epidemic in Iceland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Magnús Gottfredsson

115

14

Control of Notifiable Avian Influenza Infections in Poultry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ilaria Capua and Stefano Marangon

123

15

Understanding the Complex Pathobiology of High Pathogenicity Avian Influenza Viruses in Birds . . . . . . . . . . . . . David E. Swayne

131

Section 4 Prophylactics and Therapeutics for Infectious Diseases 16 Development of Prophylactics and Therapeutics Against the Smallpox and Monkeypox Biothreat Agents . . . . . . . . Mark Buller, Lauren Handley, and Scott Parker 17 The Hierarchic Informational Technology for QSAR Investigations: Molecular Design of Antiviral Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. E. Kuz’min, A. G. Artemenko, E. N. Muratov, L. N. Ognichenko, A. I. Hromov, A. V. Liahovskij, and P. G. Polischuk

145

163

18

Antivirals for Influenza: Novel Agents and Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . William A Fischer, II and Frederick Hayden

179

19

Anti-Infectious Actions of the Proteolysis Inhibitor ε-Aminocaproic Acid (ε-ACA) . . . . . . . . . . . . . . . . . . . . . . . . . V. P. Lozitsky

193

20

A New Highly Potent Antienteroviral Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lubomira Nikolaeva-Glomb, Stefan Philipov, and Angel S. Galabov

199

Section 5

Russian Perspectives in Emerging and Re-Emerging and Infections Research

21

Reduction and Possible Mechanisms of Evolution of the Bacterial Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . George B. Smirnov

205

22

Interaction of Yersinia pestis Virulence Factors with IL-1R/TLR Recognition System . . . . . . . . . . . . . . . . . . . . . . . . Vyacheslav M. Abramov, Valentin S. Khlebnikov, Anatoly M. Vasiliev, Igor V. Kosarev, Raisa N. Vasilenko, Nataly L. Kulikova, Vladimir L. Motin, George B. Smirnov, Valentin I. Evstigneev, Nicolay N. Karkischenko, Vladimir N. Uversky, and Robert R. Brubaker

215

Contents

xiii

23

IS481-Induced Variability of Bordetella pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ludmila N. Sinyashina, Alisa Yu. Medkova, Evgeniy G. Semin, Alexander V. Chestkov, Yuriy D. Tsygankov, and Gennagiy I. Karataev

227

24

Microarray Immunophosphorescence Technology for the Detection of Infectious Pathogens . . . . . . . . . . . . . . . . . . Nikolay S. Osin and Vera G. Pomelova

233

25

Development of Immunodiagnostic Kits and Vaccines for Bacterial Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Valentina A. Feodorova and Onega V. Ulianova

241

Section 6

26

Perspectives in Emerging and Re-Emerging Infections—Research in Central Asia and Caucasus

Research in Emerging and Re-Emerging Diseases in Central Asia and the Caucasus: Contributions by the the National Institute of Allergy and Infectious Diseases and the National Institutes of Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Katherine T. Herz

27

Disease Surveillance in Georgia: Benefits of International Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lela Bakanidze, Paata Imnadze, Shota Tsanava, and Nikoloz Tsertsvadze

28

Epidemiology (Including Molecular Epidemiology) of HIV, Hepatitis B and C in Georgia: Experience From U.S.–Georgian Collaboration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tengiz Tsertsvadze

29

The National Tuberculosis Program in the Country of Georgia: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Archil Salakaia, Veriko Mirtskhulava, Shalva Gamtsemlidze, Marina Janjgava, Rusudan Aspindzelashvili, and Ucha Nanava

PART III

251 253

257 263

HUMAN IMMUNODEFICIENCY VIRUS AND AIDS

30

Virus Receptor Wars: Entry Molecules Used for and Against Viruses Associated with AIDS . . . . . . . . . . . . . . . . . . . Edward A. Berger

271

31

HIV Latency and Reactivation: The Early Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Guido Poli

279

32

HIV-1 Sequence Diversity as a Window Into HIV-1 Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Milloni Patel, Gretja Schnell, and Ronald Swanstrom

289

33

Human Monoclonal Antibodies Against HIV and Emerging Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dimiter S. Dimitrov

299

34

Biological Basis and Clinical Significance of HIV Resistance to Antiviral Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . Mark A. Wainberg and Susan Schader

309

35

NIAID HIV/AIDS Prevention Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . David N. Burns and Roberta Black

319

36

Epidemiological Surveillance of HIV and AIDS in Lithuania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Saulius Caplinskas

327

PART IV

IMMUNOLOGY AND VACCINES

Section 1

Immunomodulation

37

TACI, Isotype Switching, CVID, and IgAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Emanuela Castigli and Raif S. Geha

343

38

A Tapestry of Immunotherapeutic Fusion Proteins: From Signal Conversion to Auto-stimulation . . . . . . . . . . . . . . . Mark L. Tykocinski, Jui-Han Huang, Matthew C. Weber, and Michal Dranitzki-Elhalel

349

xiv

Contents

39

A Role for Complement System in Mobilization and Homing of Hematopoietic Stem/Progenitor Cells . . . . . . . . . . M. Z. Ratajczak, R. Reca, M. Wysoczynski, M. Kucia, and J. Ratajczak

40

Post-translational Processing of Human Interferon-γ Produced in Escherichia coli and Approaches for Its Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Maya Boyanova, Roumyana Mironova, Toshimitsu Niwa, and Ivan G. Ivanov

Section 2

357

365

Autoimmunity

41

B-cell dysfunctions in Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Moncef Zouali

377

42

A Model System for Studying Mechanisms of B-cell Transformation in Systemic Autoimmunity . . . . . . . . . . . . . . Wendy F. Davidson, Partha Mukhopadhyay, Mark S. Williams, Zohreh Naghashfar, Jeff X. Zhou, and Herbert C. Morse, III

385

43

Breach and Restoration of B-Cell Tolerance in Human Systemic Lupus Erythematosus (SLE) . . . . . . . . . . . . . . . . . Iñaki Sanz, R. John Looney, and J. H. Anolik

397

Section 3

Infection and Immunity

44

Dendritic Cells: Biological and Pathological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jacques Banchereau, John Connolly, Tiziana Di Pucchio, Carson Harrod, Eynav Klechevsky, A. Karolina Palucka, Virginia Pascual, and Hideki Ueno

409

45

Immunomic and Bioinformatics Analysis of Host Immunity in the Vaccinia Virus and Influenza A Systems . . . . . . Magdalini Moutaftsi, Bjoern Peters, Valerie Pasquetto, Carla Oseroff, John Sidney, Huynh Hoa-Bui, Howard Grey, and Alessandro Sette

429

46

Immunoreactions to Hantaviruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Alemka Markotic´ and Connie Schmaljohn

435

47

Innate Immunity to Mouse Cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Djurdjica Cekinovic´, Irena Slavuljica, Tihana Lenac, Astrid Krmpotic´, Bojan Polic´, and Stipan Jonjic´

445

Section 4

Vaccines

48

Research and Development of Chimeric Flavivirus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Simon Delagrave and Farshad Guirakhoo

459

49

Correlates of Immunity Elicited by Live Yersinia pestis Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vivian L. Braciale, Michael Nash, Namita Sinha, Irina V. Zudina, and Vladimir L. Motin

473

PART V

BUILDING A SUSTAINABLE PERSONAL RESEARCH PORTFOLIO

50

Strategies for a Competitive Research Career . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Hortencia Hornbeak and Peter R. Jackson

483

51

Selecting the Appropriate Funding Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Priti Mehrotra, Hortencia Hornbeak, Peter R. Jackson, and Eugene Baizman

487

52

Preparing and Submitting a Competitive Grant Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Peter R. Jackson and Hortencia Hornbeak

497

53

Identifying Research Resources and Funding Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Eugene Baizman, Hortencia Hornbeak, Peter R. Jackson, and Priti Mehrotra

507

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

519

Contributors

Vyacheslav M. Abramov • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia J. H. Anolik • Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA A. G. Artemenko • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Rusudan Aspindzelashvili • National Center for Tuberculosis and Lung Diseases / National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Ramy Aziz • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Eugene Baizman • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Lela Bakanidze • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Jacqueline T. Balthazar • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Jacques Banchereau • Baylor Institute for Immunology Research, Dallas, TX, USA Bryan H. Bellaire • Louisiana State University Health Science Center, Shreveport, LA, USA Edward A. Berger • Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Gayle Bernabe • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Roberta Black • Prevention Sciences Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Maya Boyanova • Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria Vivian L. Braciale • Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Robert R. Brubaker • Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA Mark Buller • Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, MO, USA Robert J. Burgess • Armed Forces Institute of Pathology, Washington, DC, USA

David N. Burns • Prevention Sciences Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Saulius Caplinskas • Lithuanian AIDS Center, Mykolas Romeris University, Vilnius, Lithuania Ilaria Capua • OIE/FAO Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy Emanuela Castigli • Division of Immunology, Children’s Hospital, Boston, MA, USA M. Donald Cave • Neurobiology and Developmental Science, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA Djurdjica Cekinovic´ • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Alexander V. Chestkov • State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia Evelyne Couture-tosi • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Wendy F. Davidson • Marlene and Stewart Greenebaum Cancer Center and Department of Microbiology and Immunology, and the Center for Vascular and Inflammatory Diseases, BioPark Building 1, University of Maryland, Baltimore, MD, USA Simon Delagrave • Acambis Inc., Cambridge, MA, USA Tiziana Di Pucchio • Baylor Institute for Immunology Research, Dallas, TX, USA Dimiter S. Dimitrov • Protein Interactions Group, Center for Cancer Research Nanobiology Program, National Cancer Institute, National Institutes of Health, Frederick, MD, USA Michael Dobson • Armed Forces Institute of Pathology, Washington, DC, USA T. Mark Doherty • Statens Serum Institut, Department of Infectious Disease Immunology, Copenhagen, Denmark Michal Dranitzki-Elhalel • Hadassah Medical Center, Ein Kerem, Israel Karl Drlica • Public Health Research Institute, Newark, NJ, USA Kathleen D. Eisenach • Departments of Pathology, Microbiology and Immunology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USA Valentin I. Evstigneev • Department of Biochemistry, Immunity, and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Nourtan Fathey • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA

xv

xvi Valentina A. Feodorova • Scientific and Research Department, Saratov State University, Saratov Russia, Russia William A Fischer, II • Johns Hopkins Hospital, Baltimore, MD; Global Influenza Program, World Health Organization, Geneva, Switzerland; and University of Virginia, Charlottesville, VA, USA Jason P. Folster • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Angel S. Galabov • Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Shalva Gamtsemlidze • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Raif S. Geha • Division of Immunology, Children’s Hospital, Boston, MA, USA Vassil St. Georgiev • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Maria Y. Giovanni • Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Magnús Gottfredsson • Department of Medicine, Landspitali University Hospital and University of Iceland School of Medicine, Reykjavik, Iceland Ian Justin Glomski • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Pierre Louis Goossens • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Howard Grey • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Farshad Guirakhoo • Senior Director, External Research & Development, Global Research and R&D, Sanofi Pasteur Acambis Inc., Cambridge, MA, USA Ted Hadfield • Armed Forces Institute of Pathology, Washington, DC, USA Lauren Handley • Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, MO, USA Carson Harrod • Baylor Institute for Immunology Research, Dallas, TX, USA Graham F. Hatfull • Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA Frederick Hayden • Johns Hopkins Hospital, Baltimore, MD; Global Influenza Program, World Health Organization, Geneva, Switzerland and University of Virginia, Charlottesville, VA, USA Katherine T. Herz • Office of Global Research, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA Huynh Hoa-bui • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Hortencia Hornbeak • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA A. I. Hromov • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Jui-han Huang • Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA Lynn Y. Huynh • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA

Contributors Paata Imnadze • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Ivan G. Ivanov • Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria Peter R. Jackson • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Marina Janjgava • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Ann E. Jerse • Veterans Affairs Medical Center, Decatur; and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Paul J. T. Johnson • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Stipan Jonjic • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Nazia Kamal • Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA Rita Kansal • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN Arseny Kaprelyants • Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Gennagiy I. Karataev • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Nicolay N. Karkischenko • Scientific Center of Biomedical Technologies RAMS, Russia Paul Keim • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Valentin S. Khlebnikov • Department of Biochemistry, Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Eynav Klechevsky • Baylor Institute for Immunology Research, Dallas, TX, USA, Technion–Israel Institute of Technology, Technion City, Haifa, Israel Igor V. Kosarev • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Malak Kotb • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Astrid Krmpotic´ • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia M. Kucia • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA Nataly L. Kulikova • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia V. E. Kuz’min • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Karen Lacourciere • Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Tihana Lenac • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia A. V. Liahovskij • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Contributors R. John Looney • Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA V. P. Lozitsky • Ukrainian I.I. Mechnikov Research Anti-Plague Institute, Odessa, Ukraine Stefano Marangon • OIE/FAO Reference Laboratory for Newcastle Disease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy Alemka Markotic´ • University Hospital of Infectious Diseases, Zagreb, Croatia John J. McGowan • National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Alisa Yu. Medkova • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Priti Mehrotra • Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Roumyana Mironova • Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria Veriko Mirtskhulava • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia and Emory University, Atlanta, GA, USA Michèle Mock • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Herbert C. Morse, III • Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA Vladimir L. Motin • Departments of Pathology/Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Magdalini Moutaftsi • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Galina Mukamolova • Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Partha Mukhopadhyay • Marlene and Stewart Greenebaum Cancer Center and Center for Vascular and Inflammatory Diseases, University of Maryland, Baltimore, MD, USA E. N. Muratov • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Ucha Nanava • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Michael Nash • Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Toshimitsu Niwa • Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Nagoya, Japan Lubomira Nikolaeva-glomb • Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria Mohamed Nooh • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Zohreh Naghashfar • Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA L. N. Ognichenko • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

xvii Carla Oseroff • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Nikolay S. Osin • Department of Biological Microanalysis, State Research Center, R&D Institute of Biological Engineering, Moscow, Russia A. Karolina Palucka • Baylor Institute for Immunology Research, Dallas, TX, USA Scott Parker • Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, MO, USA Virginia Pascual • Baylor Institute for Immunology Research, Dallas, TX, USA Valerie Pasquetto • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Milloni Patel • Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Bjoern Peters • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Stefan Philipov • Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria Guido Poli • AIDS Immunopathogenesis Unit, San Raffaele Scientific Institute, Milano, Italy Bojan Polic´ • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia P. G. Polischuk • A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine Vera G. Pomelova • Laboratory of Molecular Diagnostics, Department of Biological Microanalysis, State Research Center, R&D Institute of Biological Engineering, Moscow, Russia Serguei G. Popov • National Center for Biodefense and Infectious Disease, George Mason University, Manassas, VA, USA Tanja Popovic • United States Centers for Disease Control and Prevention, Atlanta, GA, USA William S. Probert • California State Department of Health Services, Microbial Diseases Laboratory, CA, USA J. Ratajczak • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA M. Z. Ratajczak • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA Sarah Rowe • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA Archil Salakaia • National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia Elena Salina • Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia Iñaki Sanz • Department of Medicine, Division of Clinical Immunology and Rheumatology. University of Rochester School of Medicine and Dentistry, Rochester, NY, USA Susan Schader • McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada Connie Schmaljohn • U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA Gretja Schnell • Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

xviii Evgeniy G. Semin • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Alessandro Sette • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA John Sidney • Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA Lone Simonsen • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Namita Sinha • Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA Ludmila N. Sinyashina • Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia Irena Slavuljica • Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia George B. Smirnov • The Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia William M. Shafer • Department of Microbiology and Immunology and Laboratories of Microbial Pathogenesis, Emory University School of Medicine, Atlanta, GA, USA H. L. Stevenson • Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA Ronald Swanstrom • Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA David E. Swayne • Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Athens, GA, USA Patricia Sylvestre • Unité Toxines et Pathogénie Bactériennes, Institut Pasteur, Paris, France Robert J. Taylor • Office of the Director, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Sue A. Theus • Department of Pathology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA Shota Tsanava • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Nikoloz Tsertsvadze • National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia Tengiz Tsertsvadze • Infectious Diseases, AIDS, and Clinical Immunology Research Center, Tbilisi, Republic of Georgia Yuriy D. Tsygankov • State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia Mark Tykocinski • Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

Contributors Hideki Ueno • Baylor Institute for Immunology Research, Dallas, TX, USA Onega V. Ulianova • Scientific and Research Department, Saratov State University, Saratov, Russia Vladimir N. Uversky • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Matthew N. Van Ert • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Anatoly M. Vasiliev • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia Raisa N. Vasilenko • Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia David M. Wagner • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Mark A. Wainberg • McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec, Canada Matthew C. Weber • Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA D. H. Walker • Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA Douglas E. Warner • Veterans Affairs Medical Center, Decatur; and Department of Microbiology and Immunology, USA Karl A. Western • Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA Robbin S. Weyant • United States Centers for Disease Control and Prevention, Atlanta, GA, USA Mark S. Williams • Department of Microbiology and Immunology, University of Maryland School of Medicine, and Center for Vascular and Inflammatory Diseases, University of Maryland, Baltimore, MD, USA Robert W. Williams • The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis TN, USA M. Wysoczynski • Stem Cell Biology Program, University of Louisville, Louisville, KY, USA M. Shaylan Zanecki • Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA Xilin Zhao • Public Health Research Institute, Newark, NJ, USA Jeff X. Zhou • Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA Moncef Zouali • Inserm, Paris, University of Paris, France Irina V. Zudina • Department of Pathology, University of Texas Medical Branch, Galveston, TX, USA

National Institute of Allergy and Infectious Diseases (NIAID): An Overview Karl A. Western

The National Institute of Allergy and Infectious Diseases (NIAID) of the U.S. National Institutes of Health (NIH) is within the U.S. Department of Health and Human Services (DHHS; Figure 1). The NIH is the DHHS agency responsible for biomedical research and research training. In the U.S. federal system, health is considered primarily a local and state responsibility, with the federal government providing support and assistance as required. Biomedical research, however, is viewed as a federal responsibility. For that reason, the NIH size and budget have resulted in its becoming the largest of the DHHS agencies. The NIH consists of 27 institutes and centers, 24 of which carry out and fund biomedical research and three that support the NIH biomedical research endeavor (Figure 2). Each institute consists of two major components: the extramural and the intramural. Intramural programs consist of NIH scientists working in NIH government laboratories. Intramural research constitutes of about 10 to 20% of each institute’s research effort and budget. Intramural researchers select scientists to come to their laboratories for research training and conduct international research using the funding available to their laboratory. The extramural program of each institute is approximately 80 to 90% of its total funding and operates through both unsolicited and solicited research applications for grants, collaborative agreements, and contracts. Applications are submitted to the NIH Center for Scientific Review, which assigns each application to the appropriate initial review group for scientific peer review and to an institute according to the scientific content of the application and the research mission of the institute. NIH is unique among national biomedical research agencies in that nearly one-half of the intramural scientists are not U.S. citizens and that foreign scientists are eligible to apply directly or as a partner in extramural awards. From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

NIAID is similar in its organization to other NIH institutes in that it has three intramural divisions and five extramural divisions (Figure 3). The Division of Intramural Research heavily emphasizes basic biomedical research, while the Vaccine Research Center’s mission includes the discovery and early development of vaccine products. The Division of Clinical Research was established in 2006 to set up domestic and international sites to carry out human subject studies on new or improved diagnostic tests, drugs, vaccines and other prevention products. The Division of Microbiology and Infectious Diseases is responsible for all infectious and parasitic diseases except for the human acquired immunodeficiency syndrome (AIDS). The Division of AIDS is responsible for AIDS and related conditions. The Division of Allergy, Immunology, and Transplantation is concerned with the human immune system. The Division of Extramural Activities provides support to the other three extramural divisions through NIAID-organized initial review groups, grant and contract management, and award databases. The NIAID mission is to understand, treat, and ultimately prevent infectious, immunological, and allergic diseases that affect or threaten U.S. populations and hundreds of millions of people worldwide. The major areas of NIAID investigation currently are (in alphabetical order): AIDS; acute respiratory infections, including influenza; antimicrobial drug resistance, asthma and allergic diseases; civilian biodefense; emerging infectious diseases; enteric infections; genetics, transplantation, and immune tolerance; immune disorders; malaria and other tropical diseases; sexually transmitted diseases; tuberculosis, and vaccine development and evaluation. The evolution of the NIAID budget is summarized in Figure 4. Prior to the recognition of AIDS, NIAID was the seventh largest NIH Institute. As a result of its research responsibilities in infectious diseases and immunology, funding for AIDS and AIDS-related research rose to become one-half of the NIAID budget. Subsequent to the anthrax attacks in 2001, NIAID was given lead responsibility for the U.S. Civilian Biodefense Research Initiative. At the present time, NIAID is the second 3

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K. A. Western

U.S. Department of Health and Human Services The Secretary Deputy Secretary

Administration for Children and Families (ACF)

Administration on Aging (AoA)

Food and Drug Administration (FDA)

Centers for Medicare and Medicaid Services (CMS)

Agency for Health Care Policy and Research (AHCPR)

Indian Health Services (IHS)

Centers for Disease Control and Prevention (CDC)

Agency for Toxic Substances and Disease Registry (ATSDR)

Substance Abuse and Mental Health Services Administration (SAMHSA)

Health Resources and Services Administration (HRSA)

National Institutes of Health (NIH)

Program Support Center (PSC)

Figure 1. (See Color Plates).

largest institute after the National Cancer Institute. NIAID research funding is approximately one-third AIDS, one-third civilian biodefense, and one-third non-AIDS/non-biodefense. Following a Congressional mandate to double the NIH budget in the 1990s, the NIH budget has been flat for the past several years, resulting in overall inflation-adjusted negative growth. During this period, NIAID funding for international research has maintained a slow and steady growth (Figure 5) so that international research now accounts for 10% of the total NIAID budget. This remarkable sustainability is due to the globalization of health problems, the relevance of health conditions globally to domestic U.S. health problems, humanitarian objectives, and the economic development, political stability, and increasing investment in international health on the part of key international partners such as Brazil, China, and India. This sustained interest and growth in international research is not seen across NIH. One major factor that fuels NIAID’s global research activities is that our mission in infectious diseases necessitates that we partner with countries that have heavier burdens of disease and/or different risk factors in the development of clinical sites and the evaluation of new or improved diagnostic tests, treatment modalities, or prevention products.

NIAID operates under five guiding principles in Global Health Research. First, every effort is made to target collaborative research efforts to the needs of the partner country or region. Second, it strives to develop collaborative relationships that begin with collaboration in basic research and discovery so that intellectual property can be shared and proceed through product development, the design of human subject studies, and the conduct of rigorous clinical trials that generate data resulting in approval of the product by regulatory agencies. Third, to achieve multidisciplinary research collaboration, research capacity must be built and sustained in the host country. Fourth, NIAID strives to stimulate scientific collaboration and global multi-sector partnerships. Finally, NIAID international collaboration must develop training, communication, and outreach programs. NIAID uses six approaches to support its international research. The first is through the NIAID intramural research divisions for pre- and postdoctoral research training. This research training frequently results in sustained collaboration once the visiting scientists have returned to their home countries. Intramural collaboration is limited by the resources available in each laboratory but has the advantages of being

NIAID: An Overview

5

National Institutes of Health Office of the Director

National Institute on Aging

National Institute on Alcohol Abuse and Alcoholism

National Institute of Allergy and Infectious Diseases

National Institute of Arthritis and Musculoskeletal and Skin Diseases

National Cancer Institute

National Institute of Child Health and Human Development

National Institute on Deafness and Other Communication Disorders

National Institute of Dental and Craniofacial Research

National Institute of Diabetes and Digestive and Kidney Diseases

National Institute on Drug Abuse

National Institute of Environmental Health Sciences

National Eye Institute

National Institute of General Medical Sciences

National Heart, Lung, and Blood Institute

National Human Genome Research Institute

National Institute of Mental Health

National Institute of Neurological Disorders and Stroke

National Institute of Nursing Research

National Institute of Biomedical Imaging and Bioengineering

National Center for Complementary and Alternative Medicine

Fogarty International Center

National Center for Research Resources

National Library of Medicine

National Center on Minority Health and Health Disparities

Clinical Center

Center for Information Technology

Center for Scientific Review


decentralized and scientifically driven, and it provides the opportunity to establish long-term collaboration with the NIAID laboratory and other researchers who have trained there. Because about 50% of NIH intramural scientists are from outside the United States and only 10% of intramural scientists become tenured, the intramural research training experience provides an opportunity to become part of a global network linking trainees and their home institutions with NIAID-tenured scientists, U.S. scientists who take academic or private sector appointments or join other U.S. agencies, and foreign scientists who return home to continue their research careers. Foreign investigators are encouraged to partner with U.S. extramural investigators in the submission of investigatorinitiated research applications or in response to solicited program announcements (PAs) and requests for applications (RFAs). This is how NIAID supports the bulk of its international research. If the collaboration is between U.S. scientists

and scientists in another industrialized country, there may be no NIAID funding involved. On the other hand, if the collaborating overseas scientist is from a middle- or lower-income country and/or does not have his or her own funding, NIAID will provide the U.S. investigator with research funds to support the overseas component. NIH is unique among national domestic research agencies in that foreign investigators are eligible to apply directly for investigator-initiated research awards. Foreign scientists and institutions are also eligible to apply for most solicited grant and collaborative agreement solicitations. There are no international set-aside funds, and foreign investigators must compete against experienced U.S. investigators. All unsolicited foreign applications with a competitive score must also be approved by the National Allergic and Infectious Diseases Council before funding. Because of the intense competition and grantsmanship required, NIAID does not encourage foreign investigators to apply directly unless their ideas are

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K. A. Western

NIAID Divisions and Offices (simplified) Board of Scientific Counselors Associate Director for Management & Operations

Office of Financial Management

Office of Management for New Initiatives

Office of Human Resources Management

Office of Administrative Services

Office of Technology Information Systems

Office of Policy Analysis

Office of the Director

National Advisory Allergy & Infectious Disease Counselors

Office of Biodefense Research

Office of Global Research

Office of Clinical Research

Office of Ethics Office of Technology Development

Division of Acquired Immunodeficiency Syndrome

Office of Communications & Public Relations

Division of Allergy, Immunology & Transplantation

Division of Microbiology & Infectious Diseases

Office of Equal Employment Opportunities

Division of Extramural Activities

Dale & Betty Bumpers Vaccine Research Center

Division of Intramural Research


truly novel and the investigator has considerable experience preparing NIAID grant applications. NIH is obligated to follow U.S. contracting laws, so that foreign institutions can be funded in response to requests for proposals only if there is a prior determination that there is no viable U.S. source, or the foreign application is clearly superior to responses from U.S. institutions. NIAID also participates in a number of bilateral programs with foreign governments and institutions. These agreements may be developed at the Presidential, State Department, DHHS, or NIH levels in science and technology, health, or biomedical research. In the majority of cases, these agreements have no NIAID funding associated with them and collaborative activities must be undertaken with resources currently at hand in intramural laboratories or using extramural funding mechanisms. NIH intramural scientists are encouraged to collaborate with counterparts at other U.S. government agencies such as the Centers for Disease Control and Prevention, the Food and

Drug Administration, and the U.S. Army or Navy. U.S. Government scientists, however, may not compete for NIH extramural research funds. When there is mutual interest, however, NIH may negotiate interagency agreements with these and other agencies such as the State Department or the U.S. Agency for International Development that serve as a contractual mechanism to transfer funds and resources between the participating agencies. Finally, NIAID collaborates with multilateral agencies such as the World Health Organization (WHO), the Pan American Health Organization, and the Joint United Nations Program on HIV/AIDS through consultation, serving on advisory boards, and participation in technical meetings. NIAID has provided targeted funding to the WHO/World Bank/UNDP Special Program for Research and Training in Tropical Disease Research. NIAID also has a Congressional mandate to provide funding to the Global Fund to Combat AIDS, Tuberculosis, and Malaria.

NIAID: An Overview

7

Figure 5. (See Color Plates). Figure 4. (See Color Plates).


8

The NIAID strategy to respond globally to new or emerging infectious diseases and scientific opportunity has been first to encourage the intramural research community to turn their talent and attention to the new or underserved research area. The second step is to encourage extramural investigators working in relevant research areas to submit supplemental research proposals. The third step is to alert the more general scientific community about NIAID’s research priorities and interests in the area through notices, PAs, and RFAs in the NIH Guide for Grants and Contracts. Foreign investigators are ordinarily eligible to partner with U.S. applicants and, if they prefer, apply directly for NIAID funding. The result of these solicitations is to increase the research, the research training base, and eventually the pool of investigators in the targeted area. NIAID fulfills the need for directed activities in support of research in the targeted area through contracts to build the infrastructure and to provide research reagents and repositories. After a critical mass of individual extramural awards has been reached, NIAID usually puts out an RFA to establish multidisciplinary centers of excellence in the field. These centers of excellence provide further opportunities for research training of U.S. and foreign scientists. The centers of excellence are usually encouraged to engage in international research and/or carry out research training through the center award and/or independent research and research training awards. Examples of NIAID centers of excellence programs include the Sexually Transmitted Disease Research Centers, the Tropical Disease Research Units, the Centers for AIDS Research, the Tuberculosis Research Unit, the

K. A. Western

Regional Centers for Emerging Infectious Diseases, and the recently announced Centers for Influenza Research and Surveillance. Once the domestic centers of excellence are established, the next phase is the establishment of special programs to link the domestic network to international partners. RFAs are published to solicit applications for collaboration with one or more foreign partners. This is the time when the NIH Fogarty International Center solicits applications from U.S. institutions for international research training in the targeted area. Examples of linkage programs include the International Collaboration in Infectious Disease Research Program, the HIV Vaccine Trials Network, HIV Prevention Trials Network, the NIAID International Centers of Excellence, and the International Emerging Infectious Disease Research and Training Program. The third phase is reached when the linkage programs are mature and international partners have developed the capacity to carry out and account for their own research. NIAID develops solicitations open to foreign institutions to apply directly to NIAID in the targeted area. Examples of mechanisms to support foreign researchers include the Tropical Medicine Research Centers, the Multilateral Initiative on Malaria, the Comprehensive International Program for Research on AIDS, and the International Research in Infectious Diseases Program. Further information on NIAID and NIH international grants and funding opportunities may be found at http://grants1.nih. gov/grants; http://www.niaid.nih.gov/ncn/; and http://www. niaid.nih.gov/ncn/grants/int/default.htm.

Chapter 20 A New Highly Potent Antienteroviral Compound Lubomira Nikolaeva-Glomb, Stefan Philipov, and Angel S. Galabov

20.1

Introduction

The enteroviruses are widely spread viruses associated with diverse clinical syndromes and diseases, ranging in severity from minor febrile disorders to severe and potentially lifethreatening conditions. They may affect various organs and systems: the central nervous system, the respiratory system, the skin, the heart, the pancreas, and the eye. The enteroviruses are the most common etiological agent of viral meningitis. They may also cause encephalitis. In addition, these viruses can cause summer colds, herpangina, pleurodynia, hemorrhagic conjunctivitis, uveitis, and chronic fatigue syndrome. They are implicated in cardiac infections such as myocarditis and pericarditis that both in some cases may lead to dilated cardiomyopathy, where the singular option of recovery is the heart transplantation. Enteroviruses have also been implicated to play a role in the development of juvenile-onset (type 1) insulin-dependent diabetes mellitus (1). Antienteroviral therapy until now has had certain limitations. To date, there is no enterovirus-specific drug available for clinical use. Indeed, a great number of enterovirus inhibitors have been described so far, but only a few of them have shown effectiveness in vivo and none has been approved for clinical use yet. Thus, etiological therapy remains elusive, and there is a clear need for continued development of new and effective inhibitors of enteroviral replication.

20.2

Oxoglaucine

In a pilot study performed by our research group, a series of aporphinoid alkaloids were isolated from Glaucium flavum Crantz (yellow horn poppy) or obtained synthetically have been tested in vitro for their antiviral activity against viruses From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

belonging to several taxonomic groups, including picorna-, orthomyxo-, paramyxo-, and herpesviruses. One of the compounds, oxoglaucine, has manifested a well-pronounced inhibitory effect against the replication of poliovirus type 1 (Mahoney), of the Picornaviridae family. The antiviral effects in the preliminary screening tests have been evaluated by the semi-quantitative agar-diffusion test (2). Oxoglaucine is isolated from the epigean parts of Glaucium flavum Crantz (3) and it can also be obtained synthetically from the main plant alkaloid glaucine (4). The cytotoxic effect of oxoglaucine has been tested in two experimental procedures. The first was a microscopic evaluation of the effect of different concentrations of oxoglaucine on the morphology of the cell monolayer resulting in determination of the maximal tolerated (nontoxic) concentration (MTC). The second involved tracing the growth curve of the cell culture in the presence of different concentrations of the compound followed by determination of the concentration that reduces the number of viable cells by 50%, the cell growth inhibitory concentration 50 (CGIC50). The concentration of oxoglaucine, which produces no visible cytotoxic effect on monolayer FL cells, the MTC, is 6.4 µg/mL, and the concentration reducing their growth by 50% (CGIC50) is 4 µg/mL (Figure 20.1). The antienteroviral spectrum of oxoglaucine has been tested against 16 enteroviruses—poliovirus type 1 (Mahoney), poliovirus type 1 (LSc-2ab), and a series of viruses belonging to human enterovirus B. The following enteroviruses have been included in the test: poliovirus type 1 (PV-1), coxsackievirus (CV)-A9, the six coxsackie B viruses (CV-B1, CV-B2, CV-B3, CV-B4, CV-B5, and CV-B6), and echovirus (EV)-2, EV-4, EV-6, EV-9, EV-13, EV-15, and EV-19. The endpoint dilution method in the multi-cycle cytopathic effect (CPE)inhibition setup in FL cells and the plaque-inhibition test has been used for determining the antiviral effect. Oxoglaucine reveals a marked inhibitory effect on all of the tested viruses. Oxoglaucine concentrations that reduce virus titer by 1, 1.67, and 2 lg as compared to that of virus control 199

number of viable cells as % of the untreated control

200

L. Nikolaeva-Glomb et al.

100

50

0 0.1

1

10

100

concentration (µg / ml)

Figure 20.1. Effect of oxoglaucine on the growth of Fl cells.

Cells are seeded in a growth medium containing various concentrations of the compound. After formation of cell monolayer in the untreated control (no compound in the growth medium), cells are trypsinized and viable cells counted. The number of viable cells in each sample is compared with the number of viable cells in the control and is presented as percent of the untreated control.

of oxoglaucine. On the opposite end, the highest dose of oxoglaucine was required to inhibit the replication of CV-B1. The antiviral effects of oxoglaucine against the replication of CVs and the EVs included in the investigation are presented on Figure 20.2 and Figure 20.3, respectively. On the basis of the results presented in Table 20.1 and the cytotoxicity parameters of oxoglaucine, the selectivity index (SI) has been calculated as the ratio of MTC and the inhibitory concentration 50 (IC50). The results are shown on Table 20.2. As expected, oxoglaucine exerts the highest selectivity against CV-B4, followed by EV-9, EV-13, and EV-19 as well as by CV-B3 and CV-A9. In general, oxoglaucine reveals a broad-spectrum antienteroviral activity accompanied by high selectivity. This conclusion is supported by the results obtained in the plaque-inhibition test, which is considered the “gold standard” in experimental in vitro antienteroviral chemotherapy. The plaque-inhibition test has been carried out for poliovirus type 1 (Mahoney), poliovirus type 1 (LSc-2ab), and the six coxsackie B viruses. The concentration that reduces the number of plaques by 50% relative to the control with no inhibitor present in the agar overlay (IC50) was determined and SI calculated. Results are presented in Table 20.3. From the tested viruses, CV-B4 is again the most sensitive one to the antiviral effect of oxoglaucine demonstrating SI approximating 400.

Table 20.1. Antienteroviral effect of oxoglaucine determined in the CPE-inhibition test.

6

IC (µg/mL) ∆ lg = 1

∆ lg = 1.67

∆ lg = 2

PV-1 (LSc-2ab) PV-1 (Mahoney) EV-2 EV-4 EV-6 EV-9 EV-13 EV-15 EV-19 CV-B1 CV-B2 CV-B3 CV-B4 CV-B5 CV-B6 CV-A9

0.0007 n.d.

0.018 n.d. 0.01 0.12 0.003 0.06 0.09 0.2 0.12 0.25 n.d. 0.07 0.03 0.04 n.d. 0.12

0.33 n.d. 0.2 0.17 0.06 0.08 0.1 0.25 0.15 0.30 n.d. 0.1 0.04 0.06 n.d. 0.15

0.03 0.04 0.03 0.14 0.03 0.17 n.d. 0.04 0.01 n.d. 0.04

n.d., not done The antiviral effect is determined in the endpoint dilution method according to the CPE-inhibition procedure and the antiviral effect is presented as the difference of titers (∆ lg) of the untreated virus control and the oxoglaucine-treated samples

(with no oxoglaucine in the maintenance medium) have been determined in the CPE-inhibition test and results are shown on Table 20.1. CV-B4 and CV-B5, followed by CV-B3, CVA9, and EV-9 were the most susceptible to the antiviral effect

4

∆ lg

Virus type

5

CAV-9 CBV-1 CBV-3 CBV-4 CBV-5

3

2

1

0

0.001

0.01

0.1

1

concentration(µg/ml)

Figure 20.2. Antiviral effect of oxoglaucine against the replication of CV-A9, CV-B1, CV-B3, CV-B4, and CV-B5 in FL cells. Monolayer FL cells in 96-well plates are inoculated with 0.1 mL virus suspension containing 100 000, 10 000, 1000, 100, 10, and 1 CCID50 (or 320 000, 32 000, 3 200, 320, 32, and 3 CCID50). After an hour for virus adsorption, the excessive virus is discarded and cells are inoculated with 0.2 mL of maintenance medium containing 0.5 lg concentrations of the tested compound. The antiviral effect is scored according to the appearance of the cytopathic effect on the 48th hour p.i., virus titer in the presence or absence of the compound is determined and the defference of titers (∆ lg) of the untreated virus control and the oxoglaucine-treated samples is calculated.

20. Antienteroviral Compound

201 Table 20.3. Antienteroviral effect of oxoglaucine determined in the plaque-inhibition test.

9

EV-2 8

EV-4 EV-6

7

EV-9 EV-13

6

EV-15 EV-19

Virus type

IC50 (µg/mL)

SI (MTC/ IC50)

0.15 0.041 0.03 0.017 0.038 0.017 0.02 0.042

42 156 213 376 168 376 320 152

PV-1 (LSc-2ab) PV-1 (Mahoney) CV-B1 CV-B2 CV-B3 CV-B4 CV-B5 CV-B6

∆ lg

5 4 3 2

Table 20.4. Direct virucidal effect of oxoglaucine. 1

Virus titer lg CCID50 /mL 0.01

0.1

1

T°C 4°C

concentration (µg / ml)

Figure 20.3. Antiviral effect of oxoglaucine against the replication of EV-2, EV-4, EV-6, EV-9, EV-13, EV-15, and EV-19 in FL cells. Table 20.2. Selectivity of oxoglaucine determined according to the CPE-inhibition test. SI (MTC/IC) Virus type

∆ lg = 1

∆ lg = 1.67

∆ lg = 2

PV-1 (LSc-2ab) PV-1 (Mahoney) EV-2 EV-4 EV-6 EV-9 EV-13 EV-15 EV-19 CV-B1 CV-B2 CV-B3 CV-B4 CV-B5 CV-B6 CV-A9

9 142 n.a. — 213 — 160 213 45 213 38 n.a. 160 640 — n.a. 160

355 n.a. 640 53 2 133 107 71 32 53 26 n.a. 91 213 160 n.a. 53

19 n.a. 32 38 107 80 64 26 43 21 n.a. 64 160 107 n.a. 43

n.a., not applicable

Room temperature 37°C

Oxoglaucine control Oxoglaucine control Oxoglaucine control

0 min 15 min 30 min 60 min

6h

24 h

— — — 8.0 — —

8.0 8.5 8.5 8.0 8.0 8.0

8.5 8.5 8.5 8.0 8.5 8.5

7.5 8.0 7.5 8.5 8.0 7.5

7.5 8.0 7.5 8.0 8.5 8.5

8.0 8.0 8.0 7.5 8.5 8.0

Experiments have been carried out on undiluted poliovirus type 1 (LSc-2ab; 108.25 CCID50/mL) by the virucidal quantitative suspension test in the presence of 5 µg/mL and virus samples have been titrated by the endpoint dilution method in FL cells

9

VC 0h

8

infectious virus titer (CCID50/ml)

0 0.001

1h 2h

7

3h 4h

6

5h 5

6h

4

3

2 0

1

2

3

4

5

6

7

8

hours post infection

Research on the mode of action of oxoglaucine is in progress. In a preliminary stage of the research, the direct virucidal effect of the compound has been tested in a quantitative suspension test at three temperature regimens (4°C, room temperature, and 37°C). The results obtained indicate undoubtedly that oxoglaucine possesses a virus-specific mode of antiviral action and its effect is not due to the direct inactivation of extracellular virions (Table 20.4).

Figure 20.4. Timing-of-addition study on the mode of antiviral action of oxoglaucine. Monolayer FL cells in test-tubes are inoculated with 0.1 mL of poliovirus type 1 (LSc-2ab) at a multiplicity of infection 50 and cells are incubated at 37°C. Oxoglaucine in a concentration of 1 µg/mL has been added in the maintenance medium on hours 0, 1, 2, 3, 4, 5, and 6 p.i. Virus samples are frozen and thawed on hour 3, 4, 6 and 8 p.i. followed by titration by the endpoint dilution method.

202

Initial studies on the specific mode of action of oxoglaucine have been carried out in the timing-of-addition study in the one-step replication cycle of poliovirus type 1 (LSc-2ab) at high multiplicity of infection. The study reveals that the susceptible period to the antiviral effect of oxoglaucine is the latent and lag phase of the virus replication cycle (Fig 20.4). The following conclusions may be drawn out from the results obtained so far: (i) oxogalaucine possesses a strong antiviral effect in vitro against a broad spectrum of eneteroviruses, (ii) a high selectivity ratio is observed for all tested viruses exceeding 100 in most cases, and (iii) the latent and the lag phase of the virus replication cycle is the susceptible period to the effect of oxoglaucine.

L. Nikolaeva-Glomb et al.

References 1. Galabov AS, Angelova A (2006) Antiviral agents in the prevention and treatment of virus-induced diabetes. Anti-Infective Agents in Medicinal Chemistry 5:293-307. 2. Galabov AS, Nikolaeva L, Philipov S (1995) Aporphinoid alkaloid glaucinone: a selective inhibitor of poliovirus replication. Antivir Res 26:A347. 3. Kuzmanov BA, Philipov SA, Deligiozova-Gegova IB (1992) Comparative photochemical and chemosystematic research of populations of Glaucinum flavum Crantz in Bulgaria. Fitologia 52-57. 4. Philipov S, Ivanovska N, Nikolova P (1998) Glaucine analogues as inhibitors of mouse splenocyte activity. Die Pharmazie 53:694-698.

Chapter 19 Anti-Infectious Actions of Proteolysis Inhibitor e-Aminocaproic Acid (e-ACA) V. P. Lozitsky

19.1

Introduction

For various proteins, proteolytic cleavage represents the universal mechanism of activation. The activation of proteolysis plays an important role in the pathogenesis of many diseases. So, our supposition about antiviral activity of the proteolytic inhibitors (1) has been well founded. Previous research data has made it possible to formulate the “vicious circle” concept of viral virulence. That is: the virus activates the proteolytic systems, which in turn assists in the development, generalization, and aggravation of the infectious process at the expense of influence on the etiologic and pathogenesis factors (see Scheme 19.1; refs. 1 and 2). The inhibitors of proteolysis may prevent the forming of or destroy this “vicious circle.” The antiviral action of proteolytic inhibitors was discovered on all levels from subcellular to whole organism in both the experiment stage and the clinic. Furthermore, the antiviral therapeutic and prophylactic action of the proteolytic inhibitors was demonstrated against a wide spectrum of RNA and DNA viruses, such as the influenza A and B, herpes, HIV, Newcastle disease viruses (NDVs), and the adenoviruses (3). In this study, we present results on anti-infectious action of the proteolytic inhibitor Σ-aminocaproic acid (Σ-ACA).

19.2

Materials and Methods

1. Σ-ACA manufactured by pharmaceutical company “Zdorov’ya” (Kharkiv, Ukraine) was used. 2. Patients: sick children with influenza and other ARVI, adult patients with genital herpetic infection. 3. Laboratory animals: inbred white mice, white rats.

From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

4. Viruses: human influenza A viruses H1N1, H2N2, H3N2; avian influenza A viruses H5N3 and H7N3; influenza B virus; NDV; adenovirus; HSV-1; and HIV. 5. Tissue and cell cultures: tissue cultures of chorioallantoic membranes (CCM) of 12- to 14-day-old chicken embryos, cell culture Hep-2. 6. Bacterial agents of emerging and nosocomial infections: vaccine strain 15 and virulent strain 29 of Francisella tularensis; strains of Vibrio cholerae: Vibrio cholerae cholerae strain 569; Vibrio cholerae El-Tor strains 754 and 878; Vibrio cholerae non-01 strain 146/11; hospital isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. 7. Anti-influenza and anti-NDV activities in vitro were studied by inhibition of virus replication in tissue cultures CCM. CCM was infected with 1000 TID50 (tissue infection dose) of virus. Samples contained Σ-ACA were called “experimental” and those without inhibitor were named “control.” Control and experimental samples were tested on viral infective titers after incubation (24 hours, 37°C for influenza A viruses and NDV, and 32°C for influenza B viruses). At least five experiments were carried out of the investigation of each compound. Anti-influenza activity is expressed in log10 TID50 and reflected suppression of viral replication in “experimental” samples to “control.” 8. Anti-influenza activity of Σ-ACA in vivo was studied in mouse models of models lethal and non-lethal experimental influenza infections (2, 4). 9. Antiherpetic action of Σ-ACA was tested using cyto-morphological method. Hep-2 cells were infected with HSV-1 strain US in dose 5 IFU/cell. The cells were incubated at 37°C during 48 hours in maintenance medium that contained Σ-ACA (experimental samples) or without its (control samples). Then, cells were fixed with 96% ethanol and stained with 0.01% acridine orange solution. The amount of infected cells with DNAcontaining virus inclusion bodies was counted by fluorescent microscopy. Anti-HSV activity of compounds was estimated as the ratio of the percentage of infected cells in treated to percentage of infected cells in untreated cell cultures. 193

194

V. P. Lozitsky

Scheme 19.1. The participation of proteolytic system during development of influenza infection and etiopathogenetic action of protease inhibitors (x-show the points of inhibitors action).

19.3

Results and Discussion

Usually Σ-ACA is used for hemostasis when fibrinolysis contributes to bleeding. Σ-ACA is a low toxic drug. The intravenous and oral LD50 values of Σ-ACA were 3.0 and 12.0 g/kg, respectively, in the mouse and 3.2 and 16.4 g/kg, respectively, in the rat. An intravenous infusion dose of 2.3 g/kg was lethal in the dog. Σ-ACA prevented the enhancement of proteolysis during the interaction of virions with cell membranes and decreased the penetration of virions into sensitive cells. It brought down the proteolytic cleavage of the HA precursor to HA-1 and HA-2, and reduced the infectious virus harvest. High levels of Σ-ACA anti-influenza efficacy were shown by us in vitro on the H1N1, H2N2, and H3N2 subtypes of influenza A human viruses, influenza B viruses (2, 4), and on H5N3 and H7N3 subtypes of avian influenza viruses (5). The obtained results showed that while both H5N3 and H7N3 avian influenza viruses are sensitive to Σ-ACA, the H5 subtype was more sensitive (Figure 19.1). Further results have shown that ε-ACA prevented enhancement of the alkaline proteases activity in lungs of mice infected with influenza virus in addition to exhibiting therapeutic and prophylactic effects (4, 6, 7). Σ-ACA intensified the production of specific antibodies, increased cell immunity, prevented vessels’ permeability and hemorrhagic phenomena, and decreased the destruction of bronchial epithelium. Mice treated by ε-ACA during infection were more protected from re-infection with influenza virus (7). ε-ACA, when used in the treatment of influenza, decreased the virus reproduction in lungs and also enhanced the humoral immune response (Figure 19.2). The antibody titers on day 21 postinfection were significantly higher in the treated animals.

On day 30 after challenge with the homologous strain A/ Hong Kong/1/68 (H3N2), the virus reproduced to low level in the lungs of untreated convalescent mice; however, no virus was detected in the lungs of mice (except one animal) that had been treated with ε-ACA during the primary infection (Figure 19.3A). A marked increase of the antibody level was found in such mice (Figure 19.3B). Upon challenge with lethal doses of the virulent strain A/Leningrad/49/32 (H1N1), the protection was significantly higher among animals treated with ε-ACA during the primary infection with sublethal virus dose. We believe that the immunomodulatory action of ε-ACA may play an important role in the increased resistance to challenge exhibited by treatment. The reproduction of influenza virus in the lungs was reduced in half 10 days after a single application of ε-ACA, and 4 weeks after 5-day prophylactic course (6). This correlated with the ability of the proteolysis inhibitor to stimulate the early production of specific serum antibodies. The favorable effect of prophylactic administration of ε-ACA was especially significant in experimental lethal influenza (Figure 19.4). A significant protection was observed from days 3 to day 14 post-infection. The prophylactic effects produced by different types of antiviral preparations, such as inactivated vaccine and ε-ACA, used separately or in combination in experimental lethal infection induced by influenza virus A/Leningrad/49/32 (H1N1) in mice were compared (8). The quantitative evaluation of the antiinfluenza effect was carried out by using the method of multifactor analysis after the optimum second-order plan based on the mathematical theory of experiment. This made it possible to determine the best combination of the preparations and their doses to establish the time of the formation of reliable protection from influenza in mice. The results of study on combined use of inactivated vaccine and ε-ACA condition for prevention of lethal experimental influenza in mice are exhibited on Figure 19.5.

19. Anti-Infectious Actions of Proteolysis Inhibitor ε-Aminocaproic Acid (ε-ACA)

195

4,5 control

4

30 mg/ml E-ACA

3,5

20 mg/ml E-ACA 15 mg/ml E-ACA

- log10 TID50

3 2,5 2 1,5 1 0,5 0 1

2

Figure 19.1. The influence of E-ACA on avian influenza viruses H5N3 and H7N3 replication in tissue culture of chorio-allantoic membranes of chicken embryos. A

1,4

mice treated with E-ACA control group

1,2 5 4,5 4 3,5 3 2,5 2 1,5 1 0,5 0

mice treated with E-ACA control group

-log10 TID50

-log10 TID50

A

0,8 0,6 0,4 0,2 0 31

1

2

3

4

5

33

35

37

40

44

Days of experiment

6 7 8 9 10 11 12 13 14 Days of experiment

B

1400 250

Titres of antibodies

B

Titres of antibodies

1

control group mice treated with E-ACA

200 150 100 50

1200

5

7

10

14

21

mice treated with E-ACA

800 600 400 200 0

0

control group

1000

30

31

33

35

37

40

44

Days of experiment

Days of experiment

Figure 19.2. Influence of therapeutic E-ACA usage on (A) titers of infections influenza A/Hong Kong/1/68(H3N2) in murine lungs and (B) titers of anti-hemagglutinins in their serum.

Figure 19.3. Influence of treatment with E-ACA of primary infection on (A) titers of infections influenza virus A/Hong Kong /1/68(H3N2) in murine lungs and (B) titers of anti-hemagglutinins in their serum after re-infection on day 30 of the experiment.

The Ukrainian Ministry of Public Health has allowed applying Σ-ACA as an antiviral agent for the treatment of influenza and other acute respiratory viral infections (ARVIs) in

children on the basis of our experimental results and clinical trials. Instructions for dosage and administration of ε-ACA for treatment of influenza and other ARVIs are as follows:

-Log10 LD50

196

V. P. Lozitsky

7

mice pretreated with E-ACA

6

control

5 4 3 2 1 0 21

30

42

Days of infection after beginning of E-ACA application course

Figure 19.4. Action of prophylactic usage of E-ACA on death of infected with influenza virus A/PR/8/34(H1N1) mice. 6 5

log10 LD50

4 3 2 1 0 1

2

3

4

1 - control; 2 - immunization with inactivated influenza virus (512 HAU); 3 - E-ACA (30 mg); 4 - immunization + E-ACA

Figure 19.5. Influence of E-ACA on efficacy of mice immunization with inactivated influenza virus.

●

● ●

●

ε-ACA is used orally, intravenously, nasally, or in inhalations during days 3 to 7. ε-ACA is given orally 25 to 75 mg/kg gid. Besides that, nasal instillation every 4 hours or inhalation (twice a day) of 5% ε-ACA solution are expedient. The daily dose may be up to 0.5 to 1.0 g/kg, and half of this dose may be given intravenously to individuals with toxic course of disease.

Following is an account of results of some clinical studies of efficacy of ε-ACA in the treatment of influenza and other ARVIs in children. We have determined that proteolytic activity (pH 7-6) in the blood of hospitalized patients has been statistically higher than such activity in blood of healthy children, therefore necessitating the use of the proteolytic inhibitor ε-ACAof primary importance. Including ε-ACA in the therapeutic complex for treatment of influenza and other ARVIs in children resulted in a decrease of duration of intoxication symptoms (in 2 to 3 days), fever (in 1.5 to 3 days), and catarrhal symptoms (on 1.5 to 2 days). The level of complications is reduced to 17%. The results of our studies on the anti-influenza action of ε-ACA have validated the permission to use it as prophylaxis

and treatment of influenza in both human and veterinary medicine. ε-ACA has shown significant antiviral activity not only toward avian influenza viruses but also toward NDV (9). We believe that a combined use of proteolytic and neuraminidase inhibitors may be a highly effective way for influenza prevention and treatment because while the proteolytic inhibitors would prevent the influenza viruses of entering into sensitive cells, the neuraminidase inhibitors will prevent them from exiting the infected cells. We are open for collaboration in such joint projects. Furthermore, we have determined that ε-ACA displays not only anti-influenza activity but antiherpetic efficacy as well. Thus, ε-ACA inhibits the HSV-1 replication in cell cultures, and when applied at a concentration of 13 µg/mL on cell culture Hep-2 during infection with HSV-1, it succeeded in protecting the cells by 52%. Dr. J. Drach from Michigan University had studied by our request the anti-HSV activity of this preparation on his cell models and confirmed its activity. ε-ACA also showed protective efficacy after intranasal or intraperitoneal infection of mice by HSV, as well as its therapeutic efficacy in a rabbit model of herpetic keratoconjunctivitis. Immunization of experimental animals by inactivated influenza viruses, HSV, and adenoviruses combined with ε-ACA was more effective than immunisation by each of these viruses without ε-ACA (10). The antiherpetic activity of ε-ACA may be explained in connection with the discovery of serine protease in HSV that plays an important role in the viral physiology. In further studies, we have examined the effectiveness of ε-ACA in the treatment of patients with recurrent genital herpes (11). Patients of the control group received vitamins of the B-group and the antiviral drugs Chelpinum and Megasinum. Chelpinum was administrated orally at a dose of 125 mg, 4 times daily for 7 to10 days. Megasinum was administrated topically as 3% liniment three times daily. ε-ACA was administrated orally (1 tablespoon of 5% solution) 4 times daily for 7 to 10 days. 100 mL of a 5% ε-ACA solution was administrated intravenously once a day for 5 days if the patient’s condition was serious. These patients also received 1 tablespoon of ε-ACA solution orally twice daily during the same days. Then, these patients received ε-ACA only orally. ε-ACA was also administrated topically as powder or solution for the treatment of lesions. The improvement of the patients’ conditions can be summarized as follows: ●

●

periods of time between remissions were from 1.5 to 2.5 times (2.0 ± 0.5) longer recurrences are from 2 to 4 days shorter (3.0 ± 1.0).

Improvement can be viewed as non-significant when frames of solving recurrences are lightly shortened while number of recurrences stayed the same. Twenty patients (out of 50) treated with ε-ACA (50%) had significant improvement, 15 patients (37.5%) had improvement, and five patients (12.5%) had non-significant

19. Anti-Infectious Actions of Proteolysis Inhibitor ε-Aminocaproic Acid (ε-ACA)

improvement. In the control group of 30 patients, 11 (36.3%) had significant improvement, 13 (43%) had improvement and 6 (20.7%) had non-significant improvement. The overall results of this study showed that the synthetic protease inhibitor ε-ACA has higher therapeutic effectiveness during recurrent genital herpes as compared to such antiviral drugs as Chelpinum and Megasinum, thereby underlining the necessity to use it for the treatment and prevention of most largescale acute (influenza) and chronic (herpes) viral infections. In addition, our studies have shown antiviral activity of ε-ACA against adenoviruses and HIV. The possibility for blocking the transformation of the precursor of the structural core protein (polypeptide p-VII) into structural protein (polypeptide VII) by ε-ACA has been demonstrated in Hep-2 cells infected by adenovirus type 2 (12). A significant inhibition of the formation of intranuclear inclusion bodies and virus yield in the treated cells was also detected. The obtained results showed that thisrocess can be one of the targets for adenovirus reproduction inhibition. ε-ACA in a dose of 35 ég/mL inhibited the replication of HIV-1 (strain-IIIB) by 50% of C8166 cultural cells. At a concentration of 920 g/mL, ε-ACA inhibited the HIV-1 (strainIIIB)s replication by 80% (13). It is known that proteolytic inhibitors may increase efficacy of antibiotics against some microorganisms (14). We have studied the influence of ε-ACA on the sensitivity of some emerging pathogens and agents of nosocomial infections to various antibiotics (15). Thus, we have studied the antimicrobial action of 18 representatives of 10 antibiotics classes combined with addition of 5 % Σ-ACA to the nutrition medium. Two strains of Francisella tularensis [vaccine strain 15 (FT 15) and virulent strain 29 (FT 29)], and four strains of Vibrio cholerae [Vibrio cholerae cholerae strain 569 (VCC 569); Vibrio cholerae El-Tor strain 754 (VCEl 754) and strain 878 (VCEl 878): Vibrio cholerae non-01 strain 146/11 (VCNon-01 146/11)] were used as pathogens

197

of emerging infections. We have discovered that all studied infectious pathogens had been resistant to Σ-ACA when used alone without antibiotics. Furthermore, the F. tularensis FT 15 strain was resistant to carbenicillin and ofloxacin. Growth inhibition zones around the discs containing these antibiotics were totally absent. However, the addition of Σ-ACA led to the increase of FT 15 sensitivity to antibiotics. The diameter of inhibition zones was equal to 16 mm in the case of carbenicillin and to 28 mm in the case of ofloxacin. Strain FT 29 was totally non-sensitive to cefotaxim and ceftriaxone. The addition of ε-ACA resulted in FT 29 becoming highly sensitive to these antibiotics. Furthermore, at low concentration (1%) ε-ACA increased the antibacterial action of streptomycin against FT 15 and of tetracycline against FT 29. The addition of ε-ACA transformed VCNon-01 146/11 strain-resistant to cefotaxim, ceftriaxone, erythromycin, norfloxacin and tetracycline into highly sensitive one to these antibiotics (Figures 19.6 and Figures 19.7). Zones of growth inhibition for VCNon-01 146/11 due to the mentioned antibiotics combined with ε-ACA were in the 11- to 25-mm range. It has been shown that the addition of ε-ACA increased this strain’s sensitivity to kanamycin, ofloxacin, and lomefloxacin. Addition of ε-ACA caused increase in the sensitivity of the classic cholere strain VCC 569 to ampicillin, gentamicin, sisomycin, vancomycin, and kanamycin (Figure 19.8). This strain was totally resistant to erythromycin. After ε-ACA addition the growth inhibition zone diameter was increased up to 28 mm (Figure 19.6). Strain VCEl 754 became much more sensitive to penicillin antibiotics, aminoglycosides, norfloxacin, and tetracycline after combined application with ε-ACA (Figures 19.7 and Figures 19.9). It also increased the antibacterial action of gentamicin against VCEl 878. ε-ACA can enhance activity of antibiotics some agents of nosocomial infections. It increased the antibacterial action of ciprofloxacin, oxacillin, and cefuroxim against isolate 1329

Diameter of growth inhibition zone (mm)

25 28 20 15 10

11

5 0

Diameter ofgrowth inhibition zone (mm)

20 30

18

20

20

16 14 12 10

10

8 6 4 2

0

0 1

erythromycin

2

erythromycin and 5% E-ACA

Figure 19.6. The influence of E-ACA to susceptibility to erythromycin of V. cholerae cholerae 569(1) and V. cholerae non 01 146/11(2) strains.

1 tetracycline

2 tetracycline with 5% E-ACA

Figure 19.7. The influence of E-ACA on susceptibility of V. cholerae non 01 146/11 (1) and V. cholerae E1 Tor 754 (2) strains to tetracycline.

Diameter of growth inhibtion zone (mm)

198

V. P. Lozitsky

References

40 39

35 30

30

25 20 15

22

28

26

24

16

16

10 5 0 1

2

3

4

1-ampicillin; 2-sizomycin; 3-canamycin; 4-vancomycin antibiotics

antibiotics with 5% E-ACA

Diameter of growth inhibition zone (mm)

Figure 19.8. The influence of E-ACA on antibacterial action of some antibiotics in relation to V. cholerae cholerae 569 strain.

30 30

30 28

25 24

25

20

21

20

15 10

12

5 0 1 2 3 4 1-ampicillin; 2-carbenicillin; 3-sizomycin; 4-norfloxacin antibiotics

antibiotics with 5% E-ACA

Figure 19.9. The influence of E-ACA on antibacterial action of some antibiotics in relation to V. cholerae EI Tor 754 strain.

of S. aureus, of ciprofloxacin against an isolate of P. aeruginosa, and of cephazoline against an isolate of E. coli.

19.4

Conclusions

1. The results of our long-term research have demonstrated that the use of the proteolysis inhibitor Σ-ACA in human and veterinary medicine is a rational and well-warranted approach for prevention and treatment of viral and bacterial infections. 2. A further development of methods and schemes for combined usage of protease and neuraminidase inhibitors for prevention and therapy of influenza is very promising. 3. Proteolysis inhibitors represent a very promising group of drugs for future research as anti-infective agents.

Acknowledgments. This work was partially supported by the Science & Technology Center in Ukraine (STCU project #3147).

1. Lozitsky VP, Polyak RYa (1982) The role of proteolysis in reproduction of human and animal viruses and antiviral activity of proteases inhibitors. Uspekhi sovremennoi biologii 93:352–362. 2. Lozitsky VP, Fedchuk AS, Puzis LE, Buiko VP, Bubnov VV, Girlia YuI (1987) The participation of proteolysis system in realization of influenza virus virulent potential and in development of infectious process. Antiviral action of proteases inhibitors. Voprosi virusologii 32:413–419. 3. Lozitsky VP, Fedchouk AS, Girlya YuI, Puzis LE, Sudakov AYu, Buyko VP (1996) Proteolysis inhibition as the mechanism of antiviral action of various agents. Xth International Congress of Virology. Jerusalem, p. 152. 4. Lozitsky VP, Polyak RYa, Parusou VN (1979) Participation of proteolysis system in experimental infection and anti-influenza action of protease inhibitors. In Antiviral Activity and Mechanism of Action of Different Chemical Compounds. Riga, “Zinatne,” pp. 352–362. 5. Lozitsky VP, Gridina TL, Fedchuk AS, Boschenko YuA, Grigorasheva IN (2006) Antiviral action of officinal medicines e-aminocaproic acid and unithiolum toward avian influenza viruses. Odes’kyi medychnyi zhurnal 3:4–8. 6. Puzis LE, Lozitsky VP, Polyak RYa (1986) Effectiveness of prophylactic administration of epsilon-aminocaproic acid during influenza in mice. Acta Virol 30:58–62. 7. Lozitsky VP, Puzis LE, Polyak RYa (1988) Resistance of mice to reinfection after E-aminocaproic acid treatment of primary influenza virus infection. Acta Virol 32:117–123. 8. Lozitsky V, Puzis L, Razoryonov G, Polyak R (1996) Effectiveness of the combined use of inactivated vaccine (IV) and the inhibitor of proteolysis E-aminocaproic acid (E-ACA) in prevention of experimental influenza. Antiviral Res 30:53. 9. Lozitsky VP, Fedchuk AS, Mulyak SV, Sozinov VA (1994) Antiviral action of synthetic proteolysis inhibitors toward Neweastle Disease Virus. Veterenariya 1:34–35. 10. Lozitsky VP, Puzis LE,Grigorasheva IN, Tokolova SS (1985) Stimulation of antiviral immunity by synthetic fibrinolysis inhibitors. In Mekhanizmy Immunostimulyatsii Kiev, pp. 131–132. 11. Fedchouk AS, Veveritsa PG, Lozitsky VP, Girlia YuI (1999) Medical cure of recidiving herpes simplex virus infection by means of proteolysis inhibitors. Antiviral Res 41:67. 12. Nosach L, Dyachenko N, Zhovnovataya V, Lozinsky M, Lozitsky V (2002) Inhibition of proteolytic processing of adenoviral proteins by E-aminocaproic acid and ambenum in adenovirusinfected cells. Acta Biochim Polonika 49:1005–1012. 13. Lozitsky V, Ershov F, Scheglovitova O, Fedchuk A, Girlia Yu, Sudakov O, Novitsky V, Kolomiets N, Kolomiets A, Buiko V, Sozinov V, Muliak S (1995) Antiviral action of officinal medicines E-aminocaproic acid and unithiolum. Abstracts of First National Conference on Problems HIV/AIDS With Participation of Foreign Experts. Kiev, pp. 113–114. 14. Pel’kis PS, Shevchenko LI, Lozinsky MO, Kutsenko TA, Shamrai AE, Ckoroded TM (1986) Synthetic inhibitors of fibrinolysis. Kiev, Naukova Dumka, p. 172. 15. Boschenko YuA, Dronova IYu, Lozitsky VP, Pushkina VA, Yurdanova AN, Fedchuk AS, Man’kovskaya NN, Shitikova LI (2002) Ability of proteolysis inhibitor E-aminocaproic acid to amplify antimicrobial action of antibiotics against emerging infections diseases agents. International Conference on Emerging Infectious Diseases, Program and Abstracts Book. Atlanta, GA, p. 130.

Chapter 18 Antivirals for Influenza: Novel Agents and Approaches William A. Fischer, II and Frederick Hayden

18.1

Introduction

Influenza viruses are global pathogens that infect approximately 10% of the world’s population each year and cause epidemics of excess hospitalizations and deaths (http://www. who.int/mediacentre/factsheets/fs211/en/) (1). Unpredictable antigenic shifts in influenza A viruses can cause pandemic disease and in 1918 resulted in the death of more than 50 million people. Currently, the highly pathogenic avian influenza H5N1 is causing a epizootic in poultry populations, as well as sporadic human infections associated with high mortality and threatening to ignite an influenza pandemic. Annual immunization currently remains the developed world’s principle defense against the impact of seasonal influenza epidemics. Each year candidate vaccine viruses are selected on the basis of global surveillance conducted through the World Health Organization’s Global Influenza Surveillance Network. Seasonal vaccines do not provide immunity against a novel variant such as the H5N1 virus. Moreover, vaccine utility is limited by timely manufacturing of sufficient vaccine, potential for antigenic mismatches with circulating strains, and by reduced immunogenicity and efficacy in certain important target populations experiencing high morbidity and sometimes mortality from seasonal influenza (infants and young children, the elderly, and those with underlying medical conditions or compromised immune systems) (1). Because vaccine production, including manufacturing, testing, and distribution, requires at least 6 months, a rapid response to a novel virus is not possible at the present time. In addition, the global manufacturing capacity for egg-grown influenza vaccines is about 350 million doses of the standard trivalent vaccine. Various candidate human vaccines for H5N1 appear safe and immunogenic, but initial studies indicate the need for two doses and the use of high levels of hemagglutinin (HA) From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

antigen and/or adjuvants (2–4). In addition, the virus continues to evolve antigenically, so that multiple vaccine candidate strains are necessary (http://www.who.int/csr/disease/avian_ influenza/guidelines/h5n1virus/en/index.html). Historically, antiviral drugs for influenza have been perceived as an adjunct to vaccination. Given the current lack of an approved H5N1 vaccine and uncertainties regarding vaccine availability for a pandemic virus in general, antivirals have emerged as an important pharmacologic response to both sporadic human H5N1 infections and to a possible pandemic event. The range of antiviral use strategies includes individual case management, short-term chemoprophylaxis of at-risk contacts, long-term prophylaxis of selected populations, and potentially geographically targeted mass chemoprophylaxis to extinguish or delay an emerging pandemic virus (4a–9). Currently, two classes of antiviral drugs are licensed for use against influenza: adamantanes and neuraminidase (NA) inhibitors (10, 11). Adamantanes, specifically amantadine and rimantadine, block the M2 transmembrane ion channel that is integral to the uncoating of the ribonucleoprotein complex early in influenza replication (12–14). Despite documented efficacy as a prophylactic agent in seasonal and pandemic influenza, the utility of the adamantanes is limited by adverse central nervous system and gastrointestinal effects, a spectrum of activity limited to influenza A, and the emergence of drug-resistant isolates (10). Recent studies have documented the presence of a particular mutation in the M2 protein (Ser31Asn) that confers high-level resistance to adamantanes in increasing proportions of community H3N2 isolates. Increased resistance was first documented in isolates from China and Hong Kong in 2003 and reached more than 90% of such isolates in North America in 2005–2006 (15, 16). An increasing prevalence of this resistance mutation has also been reported recently in community H1N1 viruses. Furthermore, all clade 1 and some clade 2 H5N1 viruses harbor this same, or less often other mutations, that confer resistance to both amantadine and rimantadine (17). Such observations make this antiviral class unreliable as a therapeutic 179

W. A. Fischer and F. Hayden

180 Table 18.1. Representative studies of the in vitro activity of antiviral agents against differing strains of influenza. Study Virus Ref. 56a H1N1 H3N2 B Ref. 57 H1N1 H3N2 H2N2 H6N2 B Ref. 56b

Oseltamivir carboxylate EC50 (ug/mL)

Zanamivir IC50 (nM)

Peramivir

Number of strains tested

IC50 (nM)

EC50 (ug/mL)

5 6 8

0.69–2.24 0.27–0.45 5.33–18.33

0.73–1.05 1.85–3.13 2.00–3.10

0.26–0.43 0.47–0.87 1.08–1.95

6 4 4 1 8

0.69–2.2 0.21–0.56 0.01–1.5 0.84 6.4–24.3

0.3–0.8 0.68–2.3 0.76–1.8 1.07 1.5–17.0

0.09–0.81 0.14–0.83 0.17–1.4 1.08 0.6–10.8

0.22 to > 100 < 0.01–0.65 0.2–0.22 0.03–1.3

IC50 (nM)

EC50 (ug/mL)

A-315675 EC50 (ug/mL)

T-705 EC50 (ug/mL)

H1N1

5

0.17 to >100

0.09–21

H3N2 H5N1 B Ref. 62 H1N1

12 2 5

< 0.01–0.50 0.22–0.26 0.11–3.0

8

≤ 0.01–0.7

≤ 0.015–1.25

H3N2 B Ref. 67 H1N1 H2N2 H3N2

4 9

≤ 0.01–5.1 ≤ 0.01–0.125

≤ 0.03–5.1 ≤ 0.01

5 4 4

0.029–0.2 0.013–0.3 0.078–0.48

B

3

0.002–0.0096 0.00017–0.68 0.00049– 0.003 0.0063–0.031

C

3

> 100

0.03–0.57

< 0.01–0.19 0.01–0.02 0.06–3.2

0.039–0.089

EC50 values determined by cell culture assays; IC50 values determined by neuraminidase inhibition assays

or prophylactic agent in both seasonal influenza and in H5N1 infections in the absence of local susceptibility data. NA inhibitors, zanamivir and oseltamivir, comprise the principle class for pharmacotherapy of influenza infections today. Products of rational drug design, NA inhibitors target the active enzymatic site of NA, a highly conserved region in influenza A and B viruses and inhibit NA’s cleavage of terminal sialic acid residues on receptors for viral HA (Table 18.1; ref. 18). Oral oseltamivir and orally inhaled zanamivir are highly effective as prophylactic agents against human influenza A and B infections (18–23). When used as early treatment of uncomplicated influenza, both reduce lower respiratory complications, and oseltamivir also reduces hospitalizations and appears to reduce all-cause mortality after influenza infection (21, 24–28). In contrast to the adamantanes, emergence of community isolates of influenza resistant to the NA inhibitors has been low (29–31). Emergence of oseltamivir resistance has been documented in less than 1 to 4% of adults and in 5.5 to 18% of children during or shortly after treatment for human influenza (32–34). These drugs are generally well-tolerated, but inhaled zanamivir has been associated with serious bronchospasm and oseltamivir with gastrointestinal, cutaneous, and possibly neuropsychiatric side effects. Despite activity against the virus in vitro and in animal models (35), the therapeutic value of oseltamivir in human H5N1

disease is unproven and the optimal dose regimen is uncertain (4a, 36). Resistance has already been documented in three H5N1 patients treated with oseltamivir and appeared to be associated with prolonged viral replication and fatal outcome in two (37, 38). Inhaled zanamivir is unstudied in human H5N1 infections and there are serious concerns about the tolerability and antiviral efficacy of inhaled zanamivir in H5N1 patients with progressive viral pneumonia, in part because it may not reach sites of replication in affected alveoli, distal airways, or extra-pulmonary sites (4a, 15, 17). In general, there is a need for a parenterally administered antiviral agent that provides reliable delivery of drug to affected sites in seriously ill influenza patients. Given the paucity and limitations of currently available agents, the purpose of this chapter is to review the properties of investigational antiviral agents for influenza viruses. We selectively discuss mechanisms of antiviral action and resistance, pre-clinical activity, human pharmacology, and, when available, data about tolerability and antiviral efficacy in humans. Our focus is on interventions that are nearing or are presently in early clinical development, particularly ones that may provide new options for influenza management in terms of improved pharmacology, alternative routes of delivery, or novel antiviral mechanisms of action that provide the potential for activity against viruses resistant to other classes and for use in combination therapies (39–42).

18. Antivirals for Influenza: Novel Agents and Approaches

The reader is referred to a recent review that discusses potential influenza antiviral targets and a number of novel agents that have shown activity in pre-clinical testing (43).

18.2 18.2.1

NA Inhibitors Intravenous Zanamivir

Zanamivir (5-acetamido-4-guanidino-6-(1,2,3-trihydroxypropyl)-5,6-dihydro-4H-pyran-2-carboxylic acid) is a potent NA inhibitor in vitro and is approved in many countries for both treatment and prophylaxis of influenza infections by oral inhalation (15, 18, 44). Controlled clinical trials leading to its regulatory approval demonstrated the importance of drug delivery to the tracheobronchial tree, as contrasted with intranasal application alone (15, 45, 46). Of note, the commercial device currently used for zanamivir administration requires a cooperative patient and may not be appropriate for younger children, cognitively impaired persons, or very frail persons. Regarding potential pandemic threat strains of influenza, topically applied zanamivir protected mice against lethal challenge with H5N1 and chickens from H7N7 (47). Despite the ability to generate strains of influenza resistant to zanamivir in the laboratory (48), only one clinical case of resistance in an immunocompromised patient has been reported during treatment (49). In vitro characterization of virus with the mutation (Arg152) causing resistance to zanamavir demonstrated attenuation of the virus due to altered NA activity (50). Of particular importance, zanamivir retains inhibitory activity against most oseltamivir-resistant variants, including the His274Tyr mutation that confers high-level oseltamivir resistance in N1 NAs (Table 18.2; refs. 38, 51, and 52). Consequently, systemic zanamivir has the potential to be useful in treating patients with severe influenza infections due to oseltamivir-resistant variants, including H5N1. Initial human studies examined the pharmacokinetics of zanamivir by differing routes of administration. Although all routes of administration were well tolerated, oral bioavailability was only approximately 2%. Repeated inhalation of 10 mg of the dried powder formulation led to maximal serum concentration (Cmax) of 39 to 54 ng/mL at 1 to 2 hours post-dosing with an elimination half-life of 4 to 5 hours (53, 54). In comparison, intravenous zanamivir at doses of 600 mg resulted in a Cmax of 32,000 to 39,000 ng/mL achieved in 30 minutes and led to levels in the respiratory tract many-fold higher than required

181

to inhibit viral NA (54). In comparison, oral oseltamivir at a standard dose of 75 mg twice daily gives a carboxylate Cmax of about 350 ng/mL (54a). The plasma elimination half-life of intravenous zanamivir averages about 2 hours. In a small, randomized, placebo-controlled, double-blind study, intravenous zanamivir prophylaxis (600 mg iv bid × 5 days starting 4 hours before viral inoculation) prevented influenza seroconversion (86% efficacy) and viral shedding (100% efficacy) in subjects experimentally infected with A/ Texas/36/91 (55). In this trial, intravenous zanamavir also demonstrated significant reductions in clinical measurements of disease, including fever, respiratory illness, cough, and myalgias compared to placebo (55). Zanamavir was detected in nasal washes on day 2 and 4, consistent with effective respiratory tract distribution after intravenous administration. Additional phase I studies of intravenous zanamivir are in progress at present, and it is hoped that intravenous zanamivir will progress to phase II clinical trials in the near future.

18.2.2

Peramivir

Peramivir (ethyl (5S,3R,4R)-4-(acetylamino)-5-amino-3(ethylpropoxy)cyclohex-1-enecarboxylate) is a cyclopentane derivative that targets the active site of NA in both influenza A and B strains. It has a positively charged guanidino group, a negatively charged carboxylate group, and lipophilic side chains that lead to some differences in antiviral spectrum and pharmacology compared to available NA inhibitors (47, 56). In vitro studies indicate that peramivir is as active as oseltamivir against human influenza A viruses and even more effective against influenza B viruses (57). Peramivir also demonstrated greater activity against H2N2 and H3N2 viruses and comparable activity against Trfluenza B Viruses when compared with zanamivir in vitro (Table 18.1; ref. 57). For avian strains, all nine NA subtypes were susceptible in enzyme inhibition assays with IC50 values ranging from 0.9 to 4.3 nM compared with zanamivir 2.2 to 30.1 nM and oseltamivir carboxylate 1.9 to 69.2 nM (57). Studies in lethal murine models have demonstrated the in vivo efficacy of oral peramivir against human and avian strains of influenza. Complete protection against the H5N1 and H9N2 strains related to human cases in Hong Kong in 1997 and 1999, respectively, was achieved with peramivir 1 or 10 mg/kg/day; oral peramivir was not significantly different from oral oseltamivir (47).

Table 18.2. NA inhibitor resistance profiles. Susceptibility in the NAI assay (fold change) NA mutation

NA type/subtype

Oseltamivir

Zanamivir

Peramivir

A-315675

E119V

A/N2

R (>50)

S (1)

S (1)

S (1)

R292K

A/N2

R (>1000)

S (4–25)

R (40–80)

S (8)

H274Y

A/N1

R (>700)

S (1)

R (40–100)

S (3)

R152K

B

R (>30–750)

R (10–100)

R (>400)

R (150)

Data taken from refs. 51 and 52 R= resistant, S = susceptible. The fold change compared to value for wile-type NA is indicated


182

Peramivir remains active against some strains resistant to oseltamivir and/or zanamivir. However, it shows substantial loss of inhibitory activity for certain oseltamivir-resistant variants including the Arg292Lys in N2 and His274Tyr in N1 Ns (Table 18.2). Attempts at selecting influenza strains resistant to peramivir in vitro have resulted in variants with mutations at Lys189Glu, a change that appears to be associated with reduced fitness. Parallel efforts designed to select resistant strains in mice were unsuccessful in at least one study (58), and resistance was not detected in the context of experimental human infections (59). Several randomized, double-blind, placebo-controlled trials evaluated oral peramivir’s activity as a prophylactic and therapeutic agent. In volunteers inoculated with an H1N1 virus (A/Texas/36/91), oral peramivir treatment initiated at 24 hours after infection showed dose-related antiviral effects; peramivir 400 mg q24h resulted in a 73% reduction in viral shedding compared with placebo (59). Similarly, subjects inoculated with an influenza B virus and treated with 800 mg q24h of peramivir had 61% decreased viral shedding. However, peramivir prophylaxis did not demonstrate a significant decrease in viral shedding at doses less than to 400 mg q day compared with placebo (59). Furthermore, oral peramivir failed to significantly reduce the time to relief of symptoms in phase III human clinical trials, likely due to its poor oral bioavailability (less than or equal to 3%), and thus is no longer being developed (59a, 60). The gastrointestinal tolerability of oral peramivir appears better than oseltamivir (59) and no doselimiting end-organ toxicities have been recognized to date. Parenterally delivered peramivir overcomes the limitation of low oral bioavailability and has demonstrated promising results in animal models (60). One-time dosing is effective in animal models because of the prolonged plasma T1/2elim and also the prolonged time of dissociation between the NA and the drug (measured as Kioff). A single intramuscular injection of 10 mg/kg of peramivir was comparable to a 5-day course of oral oseltamivir in preventing mortality from H3N2 and H1N1 in lethal murine influenza models (60). A single intramuscular injection of 30 mg/kg was comparable to oral oseltamivir in a murine model of A/Vietnam/1203/04 (H5N1) infection, and repeated intramuscular doses of 30 mg/kg/day were active in a ferret infected with this virus (61). In recently completed human studies, intravenous peramivir was well-tolerated up to doses of 8 mg/kg/day for as long as 10 days and demonstrated predictable, dose-dependent pharmacokinetics (J Beigel, unpublished observations). Intravenous doses of 4 mg/kg provide mean Cmax of 20,492 ng/mL and are associated with a prolonged plasma T1/2elim of about 20 hours (J. Alexander, unpublished observations). Intramuscular peramivir at doses of 300 mg also provided drug exposure, based on plasma levels over time, comparable to an intravenous dose of 4 mg/kg. The high plasma concentrations and prolonged plasma T1/2elim of peramivir offers the possibility of oncedaily intravenous dosing in seriously ill patients and possibly single-dose intramuscular therapy in outpatients. Phase II studies of intravenous peramivir in patients hospitalized with

serious influenza and of intramuscular peramivir in outpatients were initiated during the 2006–2007 season.

18.2.3

A-315675

A-315675 (D-proline, 5-[(1R,2S)-1-(acetylamino)-2-methoxy2-methylpentyl]-4-(1Z)-1-propenyl-, (4S,5R)-) is a novel pyrrolidine-based NA inhibitor with documented in vitro and in vivo activity against influenza A and B (Table 18.1; ref. 62). In enzymatic ligand-binding assays, A-315675 had superior potency against influenza B and A/N2 NAs (Ki of 0.14–0.31 nM and 0.19, respectively) compared with oseltamivir carboxylate (Ki of 1.1–2.1 nM and 1.3, respectively) and comparable potency against N1 and N9 enzymes (62). A-315675’s potency in the binding assays can be attributed to a delayed dissociation of the inhibitor from the NA (Kioff of A-315675 is 18 times slower than that of oseltamivir) with a T1/2 of approximately 10 to 12 hours, compared with 30 to 60 minutes seen with oseltamivir carboxylate for representative NAs (62). In cell culture assays, A-315675 exhibited greater antiviral activity than oseltamivir against the majority of clinical influenza B strains and greater than or equal activity against a wide range of N1- and N2-containing viruses (Table 18.1) (62). One important feature of A-315675’s antiviral spectrum is inhibitory activity against most oseltamivir-resistant variants (Table 18.2). In particular, it shows minimal or no loss of activity against the most common oseltamivir-resistant variants in N1and N2-containing viruses. Oral A-322278, the prodrug of A315675 is comparably active to oseltamivir in murine models of H2N2 infection, although both agents are associated with resistance emergence in immunocompromised mice (62a). This feature and the compound’s oral bioavailability make it an interesting development candidate. However, it has not yet progressed to human testing.

18.2.4

Long-acting NA Inhibitors

The term long-acting NA inhibitors (LANI) refers to several chemically diverse molecules that have been designed to maximize lung retention times after topical application to the respiratory tract. One of these molecules, CS-8958 (or R-125489) undergoes enzymatic activation in the respiratory tract. Others that are multivalent zanamivir constructs, also known as Flunet and exemplified by “compound 8,” that transcend typical monovalent binding and show improved antiviral potency and compound retention in the lung (63, 64).

18.2.4.1

Multivalent LANIs

Compound 8, a dimeric derivative of zanamivir connected via a 7-carbamate group to a linker of 16 atoms, exhibited potency (EC50s, 0.07–0.1 ng/mL) at least 100 times greater than zanamivir (EC50s, 17–40 ng/mL) in cytopathic effect inhibition assays (63). In plaque reduction assays, this derivative demonstrated 1,000-fold more inhibitory activity (EC50, 55 pM) than zanamivir (EC50s, 11,000–50,000 pM; ref. 63). The increased potency of this dimeric zanamivir derivative is believed to be


secondary to its multivalency, which is postulated to promotes inter-NA and inter-virion binding. Enzymatically, compound 8 manifested a Kioff six times longer than zanamavir (6 × 10–4 and 1.1 × 10–4, respectively), demonstrating increased binding of the drug to the active site in addition to significant compound retention in the lung (63). Rats sacrificed 168 hours post-dosing exhibited a 100-fold greater concentration of compound 8 compared with zanamivir (63). Remarkably, one intranasal dose 7 days prior to an infectious challenge in the murine model led to a significant decrease in the amount of virus remaining in the lungs 24 hours post-challenge compared to zanamivir and at a fraction of the dose (63). In lethal murine models, early polyvalent zanamivir compounds based on a dextran polymer backbone demonstrated animal protection over a 2-week period following administration of a single dose of 12.5 mg/kg (S. Tucker, 15th International Conference on Antiviral Research, 2002; S. Tucker, personal communication). Several smaller multimers, including dimeric zanamivir compounds like compound 8, were found to share long lung retention profiles ranging from 10- to more than 100- fold longer than seen with zanamivir (S. Tucker, personal communication, ref. 64). Such observations raise the possibility of a one-dose treatment or once weekly prophylaxis. A topically applied zanamivir dimer is expected to enter initial human testing in 2008.

18.2.4.2

CS-8958

CS-8958 is a LANI with single-dose efficacy as both a prophylactic and therapeutic agent in animal models. Through hydrophobic interactions, the long hydrophobic acyl chain allows CS-8958 to colocalize with airway epithelial cells for an extended duration. Once in the airways it is cleaved by esterases into an active antiviral hydrophilic molecule that is retained locally (65). In a murine model, 100% of mice treated with 0.5 µmol/kg and then infected with A/PR/8/34 (H1N1), A/Aich/2/68 (H3N2), or B/Hong Kong/5/72 4 days later survived compared with 20% of zanamavir-treated mice and 0% of mice receiving saline as a placebo (65). In studies of prophylactic efficacy, almost all mice given a single dose of 0.4 µmol/kg CS-8958 24 hours prior to infectious challenge survived, compared with less than 10% of mice protected with a similar dose of zanamivir (66). This molecule has progressed through single-dose tolerability studies in healthy volunteers and is anticipated to move into phase II dose-ranging efficacy studies in natural influenza in Japan soon (Biota Annual Report 2006).

18.3 18.3.1

Nucleoside Analogs T-705

T-705 (6-fluoro-3-hydroxy-2-pyrazinecarboxamide) represents a novel therapeutic agent with an expanded antiviral spectrum including not only influenza A and B viruses, but also influenza C and several other RNA viruses (67). T-705 is a purine analog that undergoes intracellular metabolism to a nucleo-

183

side triphosphate that selectively inhibits influenza viral RNA polymerase (68). Antiviral inhibition by T-705 can be reversed through addition of purines and purine nucleosides but not pyrimidines (68). The monophosphate inhibits cellular IMP dehydrogenase to a limited extent, but this probably does not contribute to its anti-influenza activity as more selective inhibitors of this cellular target do not inhibit influenza replication in vitro (69). T-705’s novel antiviral mechanism of action results in inhibitory action against influenza strains resistant to the amantadanes or NA inhibitors and offers the potential for combination antiviral therapy. In plaque reduction assays T-705 demonstrated less potency than oseltamivir against most strains of influenza A and B strains (Table 18.1). However, T-705 is active against influenza C (EC50, 0.095 µg/mL), whereas oseltamivir is not (EC50 >100 µg/mL; ref. 67). T-705 has inhibitory effects for poliovirus, rhinovirus, and RSV replication in vitro at 10- to 100-fold higher concentrations than for influenza. No T-705-resistant influenza viruses have been reported to date, although further selection studies are in progress. In time-of-removal experiments, production of progeny virus was not observed after removal of T-705 from cell culture media in contrast to NA inhibitors (67). Whether this effect might play a role in limiting the development of T-705 resistance during clinical use, particularly in the setting of non-compliance or suntherapeutic drug exposure, remains to be determined. Murine models have demonstrated dose-dependent inhibitory effects with complete survival from A/PR/8/34 infection at 200 mg/kg/day T-705 orally and 85.7% protection at 100 mg/kg/day (67). Furthermore, lung virus titers decreased in a dose-dependent fashion with undetectable viral lung levels in 50% of mice treated with 100 mg/kg/day and 80% treated with 200 mg/kg/day (67). Significant survival benefit was seen in mice treated orally with 200 to 400 mg/kg/day T-705 (100% survival in both groups) compared with 200 and 400 mg/kg/day oseltamivir (7 and 21% survival, respectively) after challenge with A/PR/8/34 at more than 1000-fold LD50 (70). More recently, oral T-705 has shown protective effects against H5N1 infections in mice, even with once-daily dosing and when dosing was initiated upto 60 hours after infection (70a). T-705 represents an orally bioavailable, broad spectrum anti-influenza agent that appears to target viral RNA polymerase and thus has a novel mechanism of action. This agent to entered phase I human testing in 2007.

18.3.2

Viramidine and Ribavirin

Ribavirin has long been recognized as an inhibitor of influenza A and B virus replication in vitro, and oral ribavirin has been approved for influenza treatment in some countries. Only high doses have shown evidence of clinical benefit in uncomplicated influenza (71). Intravenous or aerosolized ribavirin has been used in severely ill or immunocompromised patients (72), and aerosolized ribavirin showed modest clinical benefits in children hospitalized with influenza (73). No influenza resistance to ribavirin has been recognized.


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Systemic ribavirin use has been limited by a narrow therapeutic index with documented adverse events including haemolytic anemia, electrolyte disturbances and potential teratogenesis. However, intravenous ribavirin is an option for study in combination therapy with NA inhibitors for severe influenza (42). Viramidine, a carboxamidine analog of ribavirin, is a second purine analog with documented activity against influenza A and B and was better tolerated than ribavirin in phase I human trials (74). Viramidine is thought to act as a prodrug of ribavirin and requires conversion to its active form by adenosine deaminase (74). Viramidine exhibits less cellular cytotoxicity but also decreased anti-influenza potency in vitro compared to ribavirin (EC50s, 2.1–32 µg/mL and 0.6–5.5 µg/mL, respectively; refs. 75 and 76). Overall, the selectivity index as measured by the ratio of CC50 (concentration of drug which causes 50% decrease in cell growth)/EC50 remained lower for viramidine than ribavirin (75). Viramidine inhibits H5N1 virus replication in vitro (75). In animal studies, treatment with viramidine via oral gavage conferred 100% survival among mice infected with A/NWS/33 at doses of 125 to 500 mg/kg/day, A/Victoria/3/75 at 62.5 to 250 mg/kg/day, B/Hong Kong/5/72 at 125 mg/kg/day, and B/Sichuan/379/99 at 80 mg/kd/day (75). When initiation of therapy was delayed to 48 hours post-infection, both ribavirin and viramidine remained effective (75). Clinical trials in chronic hepatitis C have confirmed the superior tolerability of viramidine over ribavirin but also found less efficacy at the doses tested. Further evaluations of its safety profiles and dosing requirements are needed before testing in human influenza.

18.4

HA and Attachment Inhibitors

Viral attachment to a host cell, an integral step in viral pathogenesis, has recently emerged as a potential site of targeted antiviral activity. Cell surface receptors bearing terminal sialic acid residues serve as primary sites for influenza binding. The preferred receptors for avian and equine influenza viruses vary from those for human viruses by the type of linkage connecting sialic residues to the penultimate galactose of carbohydrate side chains, alpha 2,3-linked and alpha 2,6-linked, respectively. In vitro studies demonstrate decreased influenza infection by over 90% when these sialic acid receptors are removed (77). Furthermore, sialic acid polyacrylamide conjugates, or sialylglycopolymers, have been shown to be inhibitory for influenza virus replication in vitro and in murine models (78). Topical delivery to the respiratory tract is required for these agents, and significant hurdles exist with respect to safe and effective delivery in humans.

18.4.1

DAS 181

DAS 181 also termed Fludase is a recombinant fusion protein consisting of a human epithelium-anchoring domain,

called amphiregulin, linked to a bacterial salidase derived from Actinomycosis viscosus (77). The construct is expressed in Escherichia coli and has a molecular weight of about 44.8 kDa. The exosialidase mediates the removal of sialic acids from glycoconjugates (77). In cell protection assays and viral replication assays, it demonstrated significant viral inhibition (EC50 values of 0.1–15.6 nM) and cell protection with multiple strains of influenza A and B (77). The presence of the epithelial binding domain was found to increase inhibitory effects (77). Additionally, pre-treatment of cells 24 hours prior to viral exposure yielded results that mirrored those with viral exposure immediately following cell treatment. Murine studies have demonstrated increased survival and improved lung function when DAS 181 was administered topically to the nose as prophylaxis before influenza challenge (77). Additionally, when DAS 181 was used as a therapeutic agent (25–30 units/treatment intranasally), a significant survival benefit was noted, even when initiation of treatments was delayed to 48 hours post-infection with A/NWS/33 virus (77). A precursor molecule, designated DAS178, was evaluated in the ferret model of influenza; intranasal dosing demonstrated decreased viral shedding and increased protection against nasal inflammatory responses compared with control (77). In this study, only 3 of 12 animals shed virus on day 1 compared with 8 of 8 in the vehicle-treated group. Intranasal DAS 181 at doses of 1 mg/kg/d was recently found to be protective against lethal H5N1 infections in mice and showed therapeutic effects upto 72 hours post infection (77a). No serious safety problems or enhancement of bacterial infection have been found in limited pre-clinical studies to date (77). DAS 181 represents a novel mechanism of antiviral activity that targets virus receptors. It has demonstrated efficacy as both a prophylactic and therapeutic agent in vivo and is anticipated to entry initial human phase I testing in 2007.

18.4.2

Cyanovirin-N

Cyanovirin-NA is a protein with potent anti-human immunodeficiency virus (HIV) activity that also shows inhibitory activity against influenza A and B viruses. Cyanovirin-NA inhibits HIV by blocking high-mannose oligosaccharides on the HIV glycoproteins gp120 and gp41. For influenza, incubation with whole virus lysates suggested viral HA as the likely target of cyanovirin-N. Almost all strains of influenza A and B, including NA inhibitor-resistant strains, were inhibited in vitro by cyanovirin-NA with EC50 values ranging from 0.005 to 0.2 µg/mL for influenza A and 0.02 to 1.3 µg/mL for influenza B viruses (79). Two strains, however, PR/8/34 and NWS/33, displayed high levels of natural resistance to cyanovirin-NA (79), probably due to differences in glycosylation patterns in these highly passaged laboratory strains of influenza. Cyanovirin-N’s activity against a number of influenza A and B strains via an anti-HA mechanism is encouraging but in vivo studies are needed.


18.4.3

Entry Blocker (EB)

EB is a novel 20-amino-acid peptide, derived from the signal sequence of human fibroblast growth factor 4, which inhibits influenza virus attachment and replication (80). EB is believed to inhibit viral attachment by binding directly to viral HA. Pre-treatment of H5N1 virus with EB decreased cell death in vitro with a mean EC50 of 4.5 µM. Inhibition of the hemagglutinating activity of other influenza A and B viruses occurrs at 3 to 10 µM EB. However, the peptide shows cellular cytotoxicity at concentrations of about 50 µM, so that its therapeutic margin is narrow in vitro. When EB 2 mM was delivered intranasally with H5N1 virus, 100% of mice survived and lung titers were decreased. In contrast, daily EB administration starting at 6 hours after virus inoculation failed to prevent mortality and demonstrated non-significant effects on lung virus titers (80). EB shows initial promise as a lead molecule for a topically applied viral attachment inhibitor, but further confirmatory testing is needed.

18.5

Protease Inhibitors

HA is translated as a single protein, HA0, and requires cleavage by a protease for activation into its infectious conformation. The liberation and conformational alignment of two protein domains, HA1 and HA2, from this cleavage step are essential for viral fusion activity and further replication (81–83). Both endogenous and exogenous protease inhibitors, most notably aprotinin (82, 83), inhibit influenza replication, and agents like ambroxol that stimulate endogenous inhibitor secretion are active in murine models (81). Aprotonin and camostat mesilate were shown to inhibit influenza A (mean EC50 values of 170 and 2.2 µg/ml, respectively) and B in vitro (mean EC50s, 260 and 5.8 µg/ml, respectively) at levels significantly below their CC50 (84). No studies to date have evaluated whether a therapeutic level of camostat mesilate is achievable with an oral formulation, as doses used for pancreatitis in the past have not been sufficient. Animal studies demonstrated a 50% protection rate against lethal doses of influenza when treated with inhalational aprotinin at 6 µg/day (85). One human study reported that inhaled aprotinin decreased indices of disease in 52 human subjects during natural influenza infection (83). The inhibition of the influenza protease is yet another potential target of viral activity that to date has been relatively unexplored.

18.6

Serotherapy

Passive immunoprophylaxis has been shown to be efficacious against a variety of viral illnesses including hepatitis A, hepatitis B, rabies, and RSV (86, 87). Passive immunotherapy with anti-HA antibodies is effective in various animal models of

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influenza, including immunocompromised mice (88–90). A retrospective meta-analysis evaluating the use of convalescent blood products for pneumonia patients during the1918 influenza pandemic found a clinically important reduction in casefatality rates between treated (16% or 54 of 336) and untreated groups (37% or 452 of 1219; ref. 91). Early administration (< 4 days after onset) appeared to be important for benefit. Several different antibody preparations have been reported to show anti-H5 activity in animal models. Fab fragments derived from horses immunized with an H5N1 vaccine were shown to provide dose-dependent protection against lethal challenge in a murine model (92). However, such equine products are limited by potential for immune reactions, particularly with repeated dosing. Use of convalescent or postimmunization blood products has not been rigorously studied in H5N1 disease to date. Humanized monoclonal antibodies to HA have been shown to be inhibitory for H5N1 viruses in vitro and in lethal murine models (87). These chimeric antibodies, the most effective of which is called VN04-2-huG1, are composed of variable domains derived from mice infected with H5N1 fused with constant domains of the human kappa light chain and IgG1 heavy chain. VN04-2-huG1 shows neutralizing activity in vitro for a clade 1 H5N1 virus and is believed to be directed against epitopes on the 140 loop of H. When given to mice 24 hours prior to lethal virus inoculation, doses of 5 to 10 mg/kg protected mice from disease (87). Additionally, when VN04-2-huG1I was given one day following challenge, 80% of mice treated with 1 mg/kg and all of those receiving 5 or 10 mg/kg survived; treatment with 10 mg/kg beginning 3 days following inoculation also conferred complete survival (87). In another approach, B cells isolated from convalescent H5N1 patients in Vietnam have been immortalized to produce virus-neutralizing antibodies that are protective in vitro and in vivo against infection with H5N1 virus (92a). Two such human monoclonal antibodies, 3F3 and 5F12, not only neutralized the homologous strain of H5N1 from clade 1, but also exhibited activity against viruses from clade 2. However these antibodies were not active against a H3N2 virus, suggesting a H5-specific protective profile (92a). When used as therapeutic agents, 5F12, and to a lesser extent 3F3, protected mice from a lethal challenge with A/Vietnam/1203/04 even when treatment was initiated 18 hours later. Mice treated with either monoclonal antibody manifested 10-100-fold lower titres of virus in the lung and undetectable viral levels in the brain (92a). Passive immunotherapy with these monoclonal antibodies not only blocks initial adhesion of viral particles but also reduces viral burden in lungs and subsequent dissemination to other organs. However, passive immunotherapy may be limited by cost, production, and antigenic variability. The constraints of providing large scale immunotherapy in the setting of a pandemic are likely to be substantial. The specificity of monoclonal antibodies and the potential for escape mutants to emerge will


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likely require use of panels of monoclonal antibodies. However, this type of approach offers new possibilities for both prevention and treatment of severe influenza that could move rapidly into clinical development.

efficacy, resistance patterns, safety, and delivery mechanisms and routes are needed.

18.7

Interferons are critical mediators of host cell defense against viral infection and are currently used as the standard of care in some viral infections like pegylated α interferon in the treatment of chronic hepatitis C. Interferons are inhibitory for influenza viruses in vitro but very limited contemporary information is available for human influenza. Several clinical trials evaluating the use of intranasal interferon failed to demonstrate prophylactic benefit against seasonal influenza (103–105). In a community-based, placebo-controlled trial during an H3N2 outbreak, intranasal interferon at 1.5, 3, or 6 MU twice a day for 28 days displayed no protective benefit over placebo (105). A second trial evaluating prophylaxis with intranasal α-2b interferon also failed to significantly protect the treated group from those receiving placebo (106). Intranasal interferon are clearly ineffective against natural human influenza and use longer than 1 to 2 weeks causes dose-dependent respiratory mucosal irritation, consisting primarily of nasal stuffiness and epistaxis. Whether alternative routes of administration, specifically systemic or perhaps inhalational, would be safe and effective for treatment of influenza remains to be determined. While interferon responses are brisk in uncomplicated human influenza, limited evidence suggests that they might be depressed (107) or possibly suboptimal (108) in highly pathogenic H5N1 infections. Absent lung interferon levels were reported in patients dying of influenza viral pneumonia by the discoverer of the interferon system, Aleck Isaacs (109), and a recent primate study with the reconstituted 1918 virus also demonstrated a failure of interferon responses (110). If such observations are confirmed in H5N1-infected patients, systemic administration of interferon would warrant consideration for study as part of an antiviral treatment regimen.

RNA Interference

RNA interference represents an innovative approach to drug design and virus inhibition. Originally described in Caenorhabditis elegans, RNA interference is a mechanism by which double stranded RNA modulates sequence specific degradation of mRNA in order to control gene expression (93, 94). This process is directed by the dsRNA-specific endonuclease called Dicer-RDE-1 and it can be reproduced by artificially introducing synthetic duplex strands of RNA (21–25 nt in length) into a cell (95, 96). Treatment with siRNA targeting sequences coding for influenza nucleocapsid protein (NP) and a component of the RNA transcriptase complex (PA) both pre- and post-infection demonstrated inhibitory activity in vitro (96). Because of the central role of these protein products in viral RNA synthesis, inhibition was not limited to the mRNA for NP or PA, but involved all viral RNAs without significantly altering RNA transcription levels of the host cell. Other sequences including the matrix 1 protein have been targeted in vitro with mixed results (97–99). In animal models, the same siRNA fragments targeting NP delivered by inhalation and intravenous modes led to a significant reductions of lung virus titers in infected mice (9, 11, 21, and 56-fold, when challenged with H7N7, H5N1, H9N2, and H1N1 viruses, respectively) and protected mice from a lethal challenge with clinically relevant strains, including H5N1 (100). Additional in vivo experiments demonstrated a 1000fold decrease in lung titer when mice were treated with 120 µg of siRNA to NP intravenously 5 hours post H1N1 challenge (98, 101). The importance of target sequence homology of the siRNA for the infecting strain of influenza as was seen with failure to decrease lung virus titers for a B strain with only 50 to 70% homology (100). Phosphorodiamidate morpholino oligomers (PMOs) are single-stranded anti-sense agents that cause steric interference with mRNA transcription and/or translation. One study found that pre-treatment with micromolar concentrations of PMOs targeting the PB1 transition start region and the 3' terminal region of NP vRNA significantly decreased viral titers in a cell culture assays against a wide range of influenza viruses including H1N1, H3N2, H3N8, H7N7, and H5N1 (102). Similar to other studies using RNA interference, the efficacy of the PMOs is limited by sequence homology, as oligomers with more than 1 basepair discordance demonstrated a significant loss of inhibitory activity. PMOs also appear to have a narrow therapeutic index in vitro (102). In general, siRNA and related technologies remain in their infancy with regard to its human applicability and further pre-clinical studies of

18.8

18.9

Interferons

Host Cellular Targets

Influenza infection provokes a multitude of intracellular signal transduction events that lead to the activation of type I interferons (α and β), interleukins, and other pro-inflammatory cytokines and chemokines that contribute to host antiviral responses. One synthetic human Toll-like receptor 7/8 receptor agonist and cytokine inducer, designated 3M-001, showed antiviral activity after intranasal delivery in a rat influenza model that correlated with the compound’s ability to stimulate type I interferons and other cytokines (111). Intranasal application of liposomal poly ICLC, a TLR3 agonist, or of synthetic lipid A mimetics that stimulate TLR4 receptors are also protective in murine models (111a, 111b). However, drugs that inhibit innate immune responses like gemfibrizol have also been reported to be beneficial in such models (111c).


Viruses have evolved gene products to interfere with these antiviral responses, and the NS1 protein of influenza that appears to be central in suppressing the induction and effects of type I interferons (112–114). However, influenza viruses also possess the capability to exploit these signaling cascades to benefit replication. Two signaling pathways have been recently identified as necessary to influenza viral replication, specifically the IKK-nuclear factor-κB (NF-κB) and Raf/MEK/ERK cascades (115, 116). Consequently, targeting these host cell pathways is another potential arena for influenza chemotherapy. For example, an inhibitor of the upstream MAPK/ERK kinase, designated U1026, has been shown to impair influenza viral growth by inhibiting the export of viral ribonucleoprotein complexes without apparent host cell cytotoxicity (117, 118). Influenza infection also activates NF-κB, which promotes the gene expression of pro-inflammatory and antiviral cytokines such as IFN-α/β and tumor necrosis factor (TNF)-α. Interestingly, recent studies have demonstrated decreased viral titers when influenza viruses have infected cells with impaired NF-κB signaling (118), suggesting that this pathway can be manipulated by the influenza virus into a virus-supporting cascade. It has been proposed that the NF-kB pathway is responsible for promoting the expression of TNF-related apoptosis-inducing ligand and Fas, which induce caspase activation and thus facilitate the release of RNP complexes from the nucleus via a caspase-mediated nuclear pore complex disruption (116). The NF-κB inhibitor SC75741 shows dose-related effects on influenza replication in vitro and on survival in experimentally infected mice (O. Planz, VIRGIL Annual International Symposium on Antiviral Drug Resistance, Lyon, May 23, 2007). Targeting intracellular signaling cascades as opposed to viral gene products represents a novel approach to antiinfluenza chemotherapy but one in which safety concerns will need close attention, as modulating the host antiviral and inflammatory responses to achieve the optimal balance will be challenging in different stages and severities of influenza infection. While short-term use of an agent directed against host responses (i.e., for treatment of a severe infection) might be acceptable, longer-term use for chemoprophylaxis would likely be more problematic.

18.10

Combination Chemotherapy

Antiviral combination therapy, usually targeting different steps in the viral replication cycle, is an important approach that has been used in efforts to enhance antiviral efficacy, reduce emergence of resistant strains, and possibly decrease dosage and the toxicity of individual agents. This strategy has been applied with clinical success in HIV infections with highly active antiretroviral therapy and in chronic hepatitis C infections. The first study in influenza reported that interferons and amantadine combinations showed enhanced activity in vitro (119), and subsequent pre-clinical reports

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showed that effectiveness of various combinations of M2 inhibitors, ribavirin, interferons and NA inhibitors compared to monotherapy (39–42, 119–125). Studies evaluating oseltamivir and ribavirin in vivo found that certain combinations were superior to single agents alone in a lethal mouse model of influenza B infection, although not in influenza A (124). In cell culture studies, the combination of oseltamivir and rimantadine was associated with prevention of resistant variants emerging during serial passage (125). The only controlled clinical trial tested a combination of aerosolized zanamivir with rimantadine compared to rimantadine with placebo in patients hospitalized with serious influenza (126). This study was under-enrolled but was notable for the finding that the only M2 inhibitor-resistant variants were observed in the rimantadine monotherapy group. In a lethal murine model due to an amantadine-sensitive A(H5N1) virus, survival was improved from 30% with oseltamivir alone (10 mg/kg/day) to 90% when combined with amantadine (30 mg/kg/day), and greater reductions in lung and visceral viral titers were found with the combination compared to either monotherapy (127). Survival was not augmented by the combination compared to oseltamivir alone when mice were challenged with an amantadine-resistant strain of A(H5N1). No mutations in the HA, NA, and M2 proteins were detected with combination therapy. The high frequencies of pre-existing resistance to M2 inhibitors in circulating A(H3N2) and A(H1N1) strains, as well as in many A(H5N1) viruses, limits the applicability of this particular combination at present (16, 128). However, antiviral combination therapy or therapy with combinations of antivirals and immunomodulators represent important approaches, and further animal model studies that evaluate different combinations of chemotherapeutic agents are needed to identify the optimal ones for clinical testing.

Acknowledgments. The authors thank Dr. Nikki Shindo, Global Influenza Program, World Health Organization (WHO) for her helpful comments on the manuscript and for facilitating Dr. Fischer’s visit to the WHO. We also thank Drs. Robert Sidwell and Dale Barnard, Utah State University, Logan; John Beigel, National Institutes of Health, Bethesda; Yousuke Furuta, Toyama Pharmaceuticals, Japan; Jane Ryan, Biota, Melbourne; James Alexander, Biocryst, Birmingham, AL; Michael Ossi, GlaxoSmithKline, Research Triangle Park; Jackie Katz, Centers for Disease Control and Prevention, Atlanta, GA for sharing unpublished information. We also thank Diane Ramm, University of Virginia, for assistance in manuscript preparation.

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Chapter 1 Biotools for Determining the Genetics of Susceptibility to Infectious Diseases and Expediting Research Translation Into Effective Countermeasures Malak Kotb, Robert W. Williams, Nourtan Fathey, Mohamed Nooh, Sarah Rowe, Rita Kansal, and Ramy Aziz

1.1

Introduction

Infectious diseases, like most human diseases, are affected by complex polymorphic and nonpolymorphic interactive traits that influence host–pathogen interactions and modulate disease phenotype. It is well established that host genetic variability strongly affects susceptibility to infectious diseases and can significantly potentiate the severity of their clinical manifestations. The same individual could be highly susceptible to a particular infection yet completely resistant to another—ultimately these complex genetic variations ensure that some of us will be selected to survive catastrophic biological threats and help protect our species from extinction. As a result of global environmental, social and political changes, we are facing real danger that could result from major natural, deliberate, or accidental biological threats. The best means of protection against these impending threats is to be better prepared. To do so, we need to gain a deeper understanding of how our genotypes modulate our susceptibility and reaction to specific infectious agents, because this information helps us to better understand disease mechanism. Our research has been focused on linking specific genotypes to susceptibility phenotypes and delineating pathways and molecular events that modulate host resistance or susceptibility to specific infectious pathogens. Inasmuch as it is quite difficult to conduct certain infectious disease studies in humans, there has been a critical need for small animal models for infectious diseases. Appreciating the limitations of the existing models, we have developed several novel and complementary mouse models that can be used to gain a better understanding of complex disease mechanisms and reveal the interactive network(s) that lead to eradication of the infection or to serious pathology caused by our overzealous response to it. From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

Recombinant inbred (RI) mouse strains are a powerful tool for identifying quantitative trait loci (QTL) and interactive gene networks modulating infectious disease phenotypes. Data generated using the RI reference population provides a roadmap for the disease that helps focus hypotheses and expedite the process of discovery and forward research translation. Potential diagnostics, therapeutics, and vaccines suggested from the RI mice studies can also be tested in our fully humanized mouse model, where the mouse immune system has been replaced with a human immune system. Together, these models provide valuable preclinical information and allow the screening for vaccine efficacy or adverse effects, to focus and expedite the translation of research into effective countermeasures in major biological threats.

1.1.1 A Genetically Diverse, Genomically Well Defined Reference Mouse Panel Afford an Ideal Model for a Systems Biology Approach to Infectious Diseases Traditionally, most experimental models of infectious diseases have involved inbred rodents, including the most common 10 strains of inbred mice (i.e., A/J, BALB/c, CBA, C3H/He, C57BL/6J, DBA/2, NZB, and AKR). Whereas these models have been invaluable to scientists, their downfall is their limited genetic variability, where certain phenotypes may be suppressed or grossly exaggerated. A good analogy would be like conducting a clinical trial using the same eight people every time and expecting to generalize the results to the rest of the human population. Clearly, this is neither optimal nor representative of the variation seen in humans. For this reason, several groups have been generating panels of genetically diverse mice to study the genetics of susceptibility to various diseases. Of these, the RI mice are ideal for many reasons (1, 2). The RI strains are generated by crossing two inbred strains followed by ≥ 20 consecutive generations mating among siblings (1–4) (Figure 1.1). These RI mouse strains are a powerful tool for identifying QTL and interactive gene networks 13

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Figure 1.1. Derivation of BXD RI strains of mice. (A) RI strains are derived by systemic inbreeding beginning with the F2 generation of the cross of two pre-existing inbred progenitor strains (B6 and DBA/2J). Multiple independent strains are then derived without selection. Once inbred, such a set of RI strains can be thought of as a stable segregated population. (B) The RI strains are typed with respect to the numerous genetic differences that distinguish the progenitor strains. Each locus has a particular pattern of inheritance called the strain distribution pattern (SDP). Comparisons are made between SDPs. A significant excess of parental genotypes, with respect to two SDPs, signals the possibility of genetic linkage. From: Mountz JD, Van Zant GE, Zhang HG, Grizzle WE, Ahmed R, Williams RW, et al (2001) Genetic dissection of age-related changes of immune function in mice. Scand J Immunol 54:10–20 (See Color Plates).

modulating infectious disease phenotypes. The panel we have in Memphis is the advanced RI (ARI) mice derived from the parental C57BL/6J and DBA/2 strains, which are known to differ considerably in their susceptibility to a number of infectious agents. These ARI BXD strains contain roughly twice as many recombinations as standard RI strains (4, 5). The BXD ARI strains can therefore be used to map QTLs with twice the positional precision as can be achieved with the original BXDs (1, 2). Figure 1.1 illustrates the schema used for generating those strains. We currently have 80 BXD strains that are being extensively phenotyped and genotyped. Each BXD strain is genetically distinct from other strains, but all members of a given BXD strain are inbred (i.e., genetically identical). Thus, studies can be repeated on the same strain (individual) at different times, for as many times, and with a large number of biological replicas, thereby providing strong statistical power for the data. Another important feature of our BXD strains is that both parental strains have been sequenced and this greatly facilitates the identification of genes within mapped QTLs.

Prior to using the ARI mice for mapping and reverse genetics studies, we spend considerable time optimizing and standardizing the infection model. Once this has been accomplished, we basically infect mice from the ARI panel with the same dose of pathogen and measure different phenotypes (e.g., survival, weight loss, pathogen load in blood and dissemination to peripheral organs, etc.). The ARI mice are then ranked relative to each other for a given phenotype. These relative phenotype values are then analyzed in the context of the mouse genotype using WebQTL tools available on www.genenetwork.com, which provides the QTL mapping for phenotypes of interest. The bioinformatics tools allow us to inspect the single nucleotide polymorphism density within the mapped loci and to examine the genes within the loci in order to narrow down the number of candidate genes that should be further interrogated. The tools also allow us to identify interactive loci, through which we can discover interactive pathways modulating the measured phenotype. Data generated using the ARI reference population reveal polygenic and pleiotropic networks modulating disease

1. Genetics of Infectious Disease Susceptibility

phenotype and thereby providing a disease roadmap that helps focus hypotheses and expedite the process of forward systems discovery and research translation. The studies described in this chapter illustrate the utility of these mice in infectious disease studies.

1.1.2 Studies on the Genetics of Susceptibility to Invasive Group A Streptococcal (GAS) Sepsis Illustrate the Utility of RI Mice in Infectious Disease Research Severe forms of invasive GAS infections associated with high morbidity and mortality were prevalent during the 1918 flu pandemic, then virtually disappeared from 1920 to the 1980s, when suddenly severe invasive disease reemerged in many parts of the world, causing panic and leading the media to dub it “the flesh-eating disease” (6–10). The bacteria are considered an ideal model for studying the effect of host genetics on the infection outcome, because the same bacteria can cause a wide spectrum of diseases in different individuals. These diseases range from mild sore throat to deadly diseases, such as streptococcal toxic shock (STSS), necrotizing fasciitis (NF), rheumatic fever and rheumatic heart disease (RHD), glomerulonephritis, and neurological disorders. We and others have identified specific immunogenetic polymorphisms that predispose to particular forms of GAS diseases and determine the level of risk for the severe forms of these illnesses, including RHD, NF and STSS (11–13). Our STSS susceptibility studies have been ongoing since 1992 in collaboration with Dr. Donald E. Low and the Ontario Streptococcal Study Group. GAS are the richest known bacteria in superantigens (SAgs), with more than 13 identified SAgs to date (SpeA-C, SpeF-M, SSA, and SmeZ 1-24), with different GAS strains having different SAg repertoires. SAgs trigger excessive activation of T cells and MHC II-expressing cells, and cause massive release of inflammatory cytokines (e.g., TNF-β and IFN-γ). Responses of different individual to the same SAg can also vary quite drastically (11–13). Besides the SAgs, GAS possess many surface and secreted proteins that interact with the immune system (immune cells and complement proteins), e.g., M protein, C5a peptidase, SIC, and many streptodornases, which are involved in degrading neutrophil extracellular traps (14). In the first phase of our studies, we focused on genetic elements that may potentiate the host response to GAS SAgs. We identified specific HLA-II alleles and haplotypes that confer very strong resistance to STSS, and others that predispose to it . We validated our association studies, biologically, through both in vitro studies with human PBMC (different HLA types) and in HLA-tg mice carrying alleles of interest. The role of HLA-II variation in STSS susceptibility is logical because the GAS SAgs, which are pivotal mediators of STSS, utilize the HLA-II molecules as receptors through which they interact with TCRVβ elements and elicit potent inflammatory

15

responses leading to STSS in genetically predisposed high responders (Figures 1.2 and 1.3). Thus, we hypothesize that other host genetic elements might also modulate susceptibility to severe GAS sepsis and STSS, notably in earlier stages of infection controlled by the innate immune response of the host, and we are interested in finding pathways and networks rather than individual genes that are modulated by immune cells in response to GAS. To discover additional genetic variations and pathways that modulate the outcome of GAS sepsis, we turned to the ARI BXD mice described earlier. The data included in the following paragraph underscore the utility of these mice in the discovery process. Our initial studies showed that DBA/2J mice are more susceptible to severe GAS sepsis than C57BL/6J. Initially, we used approximately 300 mice from 20 BXD strains to optimize the infection dose and identify confounding nongenetic factors that need to be adjusted or included as significant covariates in the final analysis. An optimal dose of 1–3 × 107 CFU/100 µL per mouse of a virulent M1T1 GAS clinical isolate and BXD strains ages 40 to 120 days were used in this intravenous model of GAS sepsis. As shown in Figure 1.4, several BXD strains showed phenotypes outside the ranges of the parental strains, with several significantly more susceptible or resistant than their ancestors. In nine ensuing experiments, about 360 mice from 34 strains (32 BXDs and two ancestors) were infected intravenously with the optimal dose of the bacteria and survival was monitored every eight hours for seven days. Mice were weighed every 12 hours and weight loss was calculated. Bacterial load in blood (CFU/mL) was determined for all mice at 24 hours, and a bacteremia index was determined and corrected for covariates. All mice developed bacteremia, but with considerable differences in severity and survival rates (Figure 1.4).

Low protective Abs Increase risk for bacterial invasion T cell Group A streptococci Superantigen infection

HLA allelic variation modulate sepsis severity

TCR HLA class II (DRB1*15/DQB1*0602) Lower cytokine response protects against STSS

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Figure 1.2. HLA class II allelic variation modulate susceptibility to severe streptococcal sepsis (See Color Plates).

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Figure 1.4. A bar chart showing the mean values of corrected relative survival indices (cRSI) for 20 BXD strains, arranged in ascending order. Parental strains (C57Bl/6J and DBA/2J) are shown on the two extremities of the x-axis. Error bars represent the standard errors of the means. The total number of animals (n) used per strain is indicated. Data from Aziz RK, Kansal R, Abdeltawab NF, Rowe SL, Su Y, Carrigan D, et al (2007) Susceptibility to severe Streptococcal sepsis: use of a large set of isogenic mouse lines to study genetic and environmental factors. Genes and Immunity 8:404–415.

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Figure 1.3. Validation of HLA class II association with severe streptococcus sepsis using HLA-transgenic mice. (A) In vivo differential susceptibility of HLA-tg mice to M1T1 GAS sepsis. (5 × 106 CFU) live M1T1 bacteria in 250 µL PBS were injected intravenously into DQ6 (Σ) or DR4/DQ8 (□) mice. Mice survival was monitored twice daily and death was recorded over seven days. (B) IFN and TNF levels in plasma of HLA-tg mice, 24 hours postinfection. (C) Bacterial load in the blood, liver, and spleen of DQ6 (Σ) or DR4/DQ8 ( ) mice intravenously infected with 5 × 106 CFU bacteria. ∗p < 0.05; ∗∗p < 0.01. From: Nooh MM, El-Gengehi N, Kansal R, David CS, Kotb M (2007) HLA transgenic mice provide evidence for a direct and dominant role of HLA class II variation in modulating the severity of streptococcal sepsis. J Immunol 178:3076–3083.

Survival was recorded in day/day fraction post infection, and for each experiment the data was normalized to calculate a relative survival index (RSI). RSI for each strain and for each experiment were corrected for variables (mainly age) to generate a corrected index, cRSI. Using the Data Desk statistical program, we conducted multiple regression analyses for individual as well as for all nine combined experiments. Age was confirmed as a significant determinant of survival and bacterial spread, but the strongest factor influencing survival, as expected, was the genetic background of BXD strains (p ≤ 0.0001). These studies allowed us to map a strong QTL-modulating sepsis severity to a locus on chromosome 2. We are currently fine-tuning the mapping using additional BXD strains and interrogating genes of interest within the mapped QTL. We believe it will be quite informative to acquire systems information on GAS in the BXD strains in vitro and in vivo. We plan to compare the data to human in vitro responses and patient’s acute and convalescent plasma. This will provide a comprehensive mouse to human in vitro and in vivo correlation using a very well characterized set of samples.

References 1. Bailey DW (1971) Recombinant-inbred strains. An aid to finding identity, linkage, and function of histocompatibility and other genes. Transplantation 11:325–327.

1. Genetics of Infectious Disease Susceptibility 2. Taylor B (1978) Recombinant inbred strains: using gene mapping. In Origins of Inbred Mice (IIIHC M, ed.), pp 423–438Academic Press New York. . 3. Chesler EJ, Lu L, Shou S, Qu Y, Gu J, Wang J, Hsu HC, Mount JD, Baldwin NE, Langston MA, Threadgill DW, Manley KF, Williams RW (2005) Complex trait analysis of gene expression uncovers polygenic and pleiotropic networks that modulate nervous system function. Nat Genet 37:233–242. 4. Peirce JL, Lu L, Gu J, Silver LM, Williams RW (2004) A new set of BXD recombinant inbred lins from advanced intercross populations in mice. BMC Genet 5:7. 5. Shifman S, Bell JT, Copley RR, Taylor M, Williams RW, Mott R, Flint J (2006) A high-resolution single nucleotide polymorphism genetic map of the mouse genome. PLoS Biol 4:e395. 6. Cunningham MW (2000) Pathogenesis of group A streptococcal infections. Clin Microbiol Rev 13:470–511. 7. Cone LA, Woodard DR, Schlievert PM, Tomory GS (1987) Clinical and bacteriologic observations of a toxic shock-like syndrome due to streptococcus pyogenes. N Engl J Med 317:146–149. 8. Low DE (1997) The reemergence of severe group A streptococcal disease: an evolutionary perspective. In Emerging Infections I (Hughes WMSa, ed.), American Society for Microbiology Press Washington, D.C.

17 9. Stevens DL, Tanner MH, Winship J, Swarts R, Ries KM, Schlievert PM, Kaplan E (1989) Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. N Engl J Med 321:1–7. 10. Davies HD, McGeer A, Schwartz B, Green K, Cann D, Simor AE, Low DE (1996) Invasive group A streptococcal infections in Ontario, Canada. Ontario Group A Streptococcal Study Group. N Engl J Med 335:547–554. 11. Kotb M, Norrby-Teglund A, McGeer A, Green K, Low DE (2003) Association of human leukocyte antigen with outcomes of infectious diseases: the streptococcal experience. Scand J Infect Dis 35:665–669. 12. Kotb M, Norrby-Teglund A, McGeer A, El-Sherbini H, Dorak MT, Khurshid A, Green K, Peeples J, Wade J, Thomson G, Schwartz B, Low DE (2002) An immunogenetic and molecular basis for differences in outcomes of invasive group A streptococcal infections. Nat Med 8:1398–1404. 13. Kotb M (1995) Bacterial pyrogenic exotoxins as superantigens. Clin Microbiol Rev 8:411–426. 14. Buchanan JT, Simpson AJ, Aziz RK, Liu GY, Kristian SA, Kotb M, Feramisco J, Nizet V (2005) DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps. Current Biology 16:396–400.

Chapter 8 Clinical Mycobacterium tuberculosis Strains Differ in their Intracellular Growth in Human Macrophages Sue A.Theus, M. Donald Cave, and Kathleen D. Eisenach

8.1

Introduction

Tuberculosis (TB) remains a global public health crisis despite being a curable disease (1). Access to the complete genomic sequence of the Mycobacterium tuberculosis (MTB) laboratory strain H37Rv and clinical isolate CDC1551 has facilitated investigations into the pathogenicity of MTB. However, mycobacterial factors that contribute to the virulence of MTB or modulate the interaction of this pathogen with the human host are only beginning to be elucidated. Despite previous studies suggesting that MTB has an extremely low mutation rate, there are compelling reasons to suspect that strain-specific attributes contribute directly to virulence and disease outcome. Our studies of infections in THP-1 cells with epidemiologically distinct clinical MTB strains provide strong evidence that specific clinical MTB strains may be differentially pathogenic (2, 3). Clinical strains associated with TB outbreaks grow significantly faster in human macrophages than do non-outbreak strains. The rapid growth demonstrated by strains associated with outbreaks was highly correlated with rapid production of interleukin (IL)-10 and suppression of tumor necrosis factor (TNF)-α. These results suggest that the enhanced capacity of MTB to grow rapidly in human macrophages is a marker of virulence, and virulence of certain strains may be attributed to downregulation of the Th1type immune response. The ability to combine genomic information with pathogenesis studies employing diverse clinical strains will enable us to continue to unravel the molecular basis of MTB virulence and identify strain-specific attributes that influence clinical presentation, outcome (treatment failure, relapse, and development of drug resistance), and transmission of infection. Ultimately, having the ability to identify relevant strain characteristics could From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

potentially impact how we treat and control the spread of TB and elucidate whether strain virulence will impact the protection afforded by a new candidate vaccine.

8.2

TB Infection and Disease

MTB as a successful intracellular pathogen has evolved sophisticated strategies to infect and persist in host macrophages (4). In approximately 90% of immunocompetent people, the infection is asymptomatic and infecting bacilli are either killed or remain viable but latent. In the infected individuals who develop active disease, bacilli appear to evade or subvert the host’s protective cellular immune responses. Infection is established, bacilli continue to replicate in host tissues, and clinical symptoms are seen. Development of active TB is likely determined by multiple factors. First, evidence suggests that host genetic factors are involved in determining susceptibility or resistance of an individual to TB (5, 6). Also, naturally occurring mutations in the human genes for interferon (IFN)-γ receptor and IL-12 receptor have been shown to be associated with increased susceptibility to TB infection (7–10). Second, MTB itself may undergo genetic changes, modifying virulence (11). Third, the ability of a particular MTB strain to elicit a strong or weak host immune response may be important in determining development of disease. For example, the MTB clinical isolate CDC1551 induces an unusually high frequency of seroconversion in exposed persons, yet the rates of active disease are not unusually high (12).

8.3 8.3.1

Strain-specific MTB Pathogenesis Murine Models of Virulence

Few studies have investigated the differentially pathogenic nature of specific clinical MTB isolates; thus, the role of strain variability in outcome of infection remains uncertain. 77

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Lopez et al. investigated the influence of strain diversity on the course of experimental disease in BALB/c mice using MTB strains of major genotype families (defined on the basis of IS6110 restriction fragment-length polymorphism [RFLP] analysis and spoligotyping) found worldwide (13). Infection with the Beijing genotype induced accelerated bacterial multiplication, early and massive pneumonia, and death. Conversely, infection with the Canetti genotype induced a slowly progressive disease characterized by delayed bacterial multiplication, limited pneumonia, steadily increasing granulomas, and virtually no mortality. Similarly, Manca et al. examined the differential response of B6D2/F1 mice to infection with various outbreak clinical isolates (14, 15). One isolate associated with an unusually high proportion of active TB cases in Texas, HN878, was found to be hypervirulent as demonstrated by very early death of infected immunocompetent mice compared to infection with other isolates. Isolate HN878 failed to induce MTB-specific proliferation and IFN-γ production by spleen and lymph node cells from infected mice. In addition, lower levels of TNF-α. IL-6, IL-12, and IFN-γ mRNA levels were observed in lungs of HN878 infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated. In contrast, IFN-α mRNA levels were significantly higher in lungs of these mice. These results suggest that the hypervirulence of HN878 may be due to failure to stimulate Th1-type immunity. Conversely, other outbreak strains, CDC1551 and NHN5, induced very high cytokine levels in the lungs, levels associated with long-term survival in the mice. Subsequently, isolate HN878 was found to be a Beijing family strain and had the same IS6110 RFLP pattern as strain 210; CDC1551 and NHN5 are non-Beijing strains.

8.3.2

Macrophage Models of Virulence

To examine strain-specific pathogenesis, we determined intracellular growth rates for clinical MTB strains, as well as the virulent MTB strain H37Rv in IFN-γ-activated THP-1 cells (2). Macrophages infected for seven days showed that H37Rv and clinical isolate 282 grew significantly more rapidly than another clinical isolate 284. Isolates 282 and 284 were from an epidemiologic study of TB transmission in homeless shelters in Los Angeles, CA (16). IS6110 genotyping showed that 27% of the TB cases in Los Angeles were caused by strain 210 (isolate 282 being a 210 strain), whereas isolate 284 accounted for only one TB case. Subsequently, it was found that strain 210 is a member of the Beijing family and is associated with a high proportion of TB cases worldwide (17). Zhang et al. have reported that strain 210 grows significantly faster in human blood monocytes (18). The significance of the rapid-growth phenotype is further supported by the recent finding that the principal sigma factor sigA, which may regulate expression of virulence genes, is upregulated in macrophage-grown isolates of strain 210 relative to other clinical strains (19). We also tested 26 strains from TB cases in IS6110 RFLP clusters that have

persisted in Arkansas three years or more and six strains from TB cases with unique RFLP patterns (caused TB in only one patient; ref. 3). The persistent clustered strains had significantly faster growth rates in THP-1 cells than strains with unique RFLP patterns. Together, these findings indicate that clinical strains differ in their ability to grow intracellularly and those with enhanced intracellular growth capacity are at an advantage for either transmission or establishment of disease in humans. TNF-α, a proinflammatory cytokine produced primarily by monocytes and macrophages, is essential for protection against acute MTB infection (14, 15, 20). Studies have shown that avirulent or less virulent mycobacteria induce significantly more TNF-α production by macrophages than virulent mycobacteria. For example, the attenuated M. bovis BCG induced more TNF-α production than virulent M. leprae, and the nonpathogenic M. smegmatis elicited higher levels of TNF-α compared with virulent strains of M. avium and MTB (21–23). Thus, there appears to be an inverse correlation between mycobacterial virulence and TNF-α production. Our results concur with these findings (i.e., strains considered less virulent on the basis of slow intracellular growth rates induced significantly higher levels of TNF-α than more virulent strains in THP-1 infections; ref. 3). Most likely, the course of infection with the slow-growth phenotype is modulated by the rapid and robust TNF-α response, which restricts mycobacterial replication. In contrast, the rapid-growth phenotype suppresses TNF-α secretion, likely through induction of high levels of IL-10. In contrast to TNF-α, IL-10 is generally considered to be primarily anti-inflammatory. Not only has IL-10 been shown to suppress the Th1-type immune response to MTB infection, but also it downregulates the release of TNF-α from macrophages; its inhibitory effect depends on concentration (24, 25). We observed that strains considered highly virulent based on rapid intracellular growth rates induced significantly higher levels of IL-10 within the first 24 hours of THP-1 cell infection than do strains of less virulence (3). This suggests that a cell signaling pathway is blocked during the early interaction of highly virulent strains and macrophages, resulting in a rapid anti-inflammatory response from the infected cells, providing an increased chance of survival. Two reports linking strain diversity to innate immune responses provide additional evidence that clinical isolates with different epidemiologic parameters behave differently in macrophage models of virulence. In one study, significant differences were observed in the cytokine profiles triggered in RAW murine macrophage cells: a spectrum of high to low TNF-α levels was observed, with the lowest level being in infections with the highly transmissible isolates (26). In the other study, a strain associated with the largest recorded outbreak in United Kingdom was shown to induce less TNF-α and more IL-10 from human monocytes (MNs) than CDC1551 and H37Rv, suggesting that the high attack rate was related to the strain’s ability to skew the innate response toward a nonprotective phenotype (27).

8. M. tuberculosis: Intracellular Growth in Human Macrophages

8.4 Virulence Assessment of Household Transmitted Isolates To further demonstrate the validity of the macrophage model system for identifying strains with virulence potential, we examined another group of isolates thought to have an advantage for transmission (28). These isolates were from households in which there was evidence of TB transmission. For comparison, another group of isolates from households in which MTB infection was not transmitted was selected. In addition to using THP-1 cell cultures, growth rates and cytokine secretion in MNs were determined. Strains were isolated from patients enrolled in the National Institutes of Health TB Research Unit Household Contact Study conducted in Kampala, Uganda (29). The strains tested consisted of three pairs from matched household (HH) with infection (PPD+) and no infection (PPD), three pairs from matched HH with co-prevalent disease and no disease, and three pairs from matched HH with incident disease or no disease. For the infection set, a case HH was defined as a one in which at least one HH contact of the index case was PPD+ at baseline visit; control HH were free of PPD+ contacts. For the co-prevalent and incident sets, a case HH was defined as a HH in which a contact of the index case had disease with the same strain as the index case at baseline visit or develops disease with the same strain within six months post-baseline, respectively. Households were matched on the basis of clinical characteristics of the index cases (degree of smear positivity, duration of cough) and HH descriptions (number of residents, number of rooms). Fifteen strains had unique IS6110 patterns and two strains shared the same pattern; copy number ranged from 11 to 19. In matchedpair analyses of log growth ratios observed for the incident and co-prevalent strain pairs combined, there was an overall statistically significant difference in mean log growth ratio (day 7/day 0) between the case and control HH strains after adjusting for pairs effects for both culture systems (p < 0.001). The size of the difference between log growth ratios between the matched cases and controls was not identical across pairs. Growth rates obtained in THP-1 cell infections were comparable to those in primary MNs. Because the THP-1 and MN models both demonstrated differences in intracellular growth between MTB isolates from HH with co-prevalent disease and their matched HH, these six paired isolates were used to evaluate the correlation between TNF-α and IL-10 responses and intracellular growth. In the THP-1 model system, peak TNF-α levels for each isolate were observed 48 hours following infection. In each pair of MTB isolates, the organism from the transmission HH induced significantly less TNF-α than did the corresponding matched HH isolate. In MN cultures, peak concentrations of TNF-α were observed 24 hours after infection. Comparisons of the results of infection of MN from each of 10 donors with paired organisms indicated that two of the three co-prevalent transmission isolates tended to induce less TNF-α than the isolates from matched HH in which co-prevalent disease was not observed.

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Due to donor-to-donor variability, these results were not statistically significant. The third co-prevalent transmission isolate tended to induce more TNF-α than its matched isolate, although this finding was again not statistically significant. TNF-α data from 48 hours after infection showed similar patterns, but again the differences observed were not statistically significant. In the THP-1 model system, the kinetics of IL-10 production by isolates from transmission and matched HH were consistently distinct. Specifically, transmission isolates demonstrated rapid induction of IL-10 with peak levels observed 24 hours after infection, whereas matched isolates displayed more gradual induction of IL-10, resulting in peak levels at day 5. In contrast, induction of IL-10 within primary MN did not result in such distinct contrasts between isolates from transmission and matched HH. Indeed, in the comparisons of both two co-prevalent pairs, isolates from transmission HH induced less IL-10 than isolates from matched HH without co-prevalent disease, although these differences were not statistically significant at any of the time points studied. For the third pair of isolates, differences in IL-10 induction were also non-significant at all time points, although the isolate associated with co-prevalent disease did induce somewhat more IL-10 than did the isolate from its matched HH in which co-prevalent disease was not observed. Thus, the two infection models yielded different results in terms of the relationship between intracellular growth and cytokine profiles, as assessed in studies of the CP transmission and matched isolates. The results from the THP-1 cultures coincide with those reported for clinical strains associated with outbreaks, i.e., rapid production of IL-10 and suppression of TNF-α in the THP-1 model is highly correlated with the rapid growth phenotype (3). In the MN model, none of the comparisons of cytokine production were statistically significant, as is frequently observed given the wide variability of cytokine responses of donor cells. However, this was a second study in which intracellular growth of clinical MTB isolates correlated with epidemiologic evidence of strain virulence.

8.5 Virulence Assessment of Strains of the Beijing Family In the third assessment, isolates representing the Beijing strain family were tested in THP-1 macrophages. Because members of the Beijing family are widely distributed around the world and have been responsible for several large outbreaks, it has been suggested that this genotype may have a selective advantage over other MTB strains (30). One such example is isolate HN878, a 210 strain (designated 210 for having 21 copies of IS6110) and a member of the Beijing family, which caused outbreaks in Texas over a three-year period. Infection with HN878 induces a weak Th-1-associated cytokine response in the lungs of infected mice and results in early death. Similarly, isolate 282, also a strain 210 genotype and member

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of the Beijing family, was responsible for the majority of TB cases in Los Angeles homeless shelters (2, 16). As described earlier, isolate 282 grows rapidly in macrophages and modulates the macrophage response. To determine if the Beijing genotype has a growth advantage, growth rates of 14 strains belonging to the Beijing family were measured in the activated THP-1 cell culture system (30). Strains were genetically diverse on the basis of having many different IS6110 RFLP patterns, with IS6110 copy number ranging from 9 to 22, and variable number of single nucleotide polymorphisms (31). These strains demonstrated a full range of growth phenotypes, and again we observed the strong correlation between rapid growth and suppression of TNF-α. Four strains grew significantly slower, and three (one of which was a 210 genotype) grew significantly faster than the other strains. We tested an additional 11 isolates that were strain 210 genotype (several being variants; +1, –1, or shift 1 IS6110 band) and they all grew as rapidly as the other fast growing strains. The growth advantage of strain 210 is consistent with strain 210 having been present for many years in different geographic locations. Furthermore, these results indicate that strains of the Beijing family vary phenotypically and few are as virulent as the hypervirulent strain 210.

8.6

Summary

We have shown that the IFN-γ-activated THP-1 cell culture system is a reliable model for distinguishing clinical isolates on the basis of intracellular growth rates and cytokine production. Result.s with three groups of clinically and epidemiologically characterized strains demonstrate that strains at an advantage for either transmission or establishment of disease in humans can be readily distinguished from other clinical strains on the basis of these phenotypic differences. This model will be useful to elucidate differences in virulence among MTB isolates. Furthermore, isolates identified in this manner should be of interest for further studies aimed at clarifying virulence mechanisms of MTB.

References 1. Dye C, Scheele S, Dolin P, Pathania V, Raviglione MC (1999) Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country [consensus statement]. WHO Global Surveillance and Monitoring Project. JAMA 282:677–686. 2. Theus SA, Cave MD, Eisenach KD (2004) Activated THP-1 Cells: an attractive model for the assessment of intracellular growth rates of Mycobacterium tuberculosis isolates. Infect Immun 72:1169–1173. 3. Theus SA, Cave MD, Eisenach KD (2005) Intracellular macrophage growth rates and cytokine profiles of Mycobacterium tuberculosis strains with different transmission dynamics. J Infect Dis 191:453–460. 4. Russell DG (2001) Mycobacterium tuberculosis: here today, and here tomorrow. Nat Rev Mol Cell Biol 8:569–577.

5. Bellamy R, Ruwende C, Corrah T, McAdam KP, Whittle HC, Hill AV (1998) Assessment of the interleukin 1 gene cluster and other candidate gene polymorphisms in host susceptibility to tuberculosis. Tuber Lung Dis 79:83–89. 6. Bellamy R, Beyers N, McAdam KP, Ruwende C, et al (2000) Genetic susceptibility to tuberculosis in Africans: a genomewide scan. Proc Natl Acad Sci USA 97:8005–8009. 7. Altare F, Ensser A, Breiman A, Reichenbach J, et al (2001) Interleukin-12 receptor beta1 deficiency in a patient with abdominal tuberculosis. J Infect Dis 184:231–236. 8. de Jong R, Altare F, Haagen IA, Elferink DG, et al (1998) Severe mycobacterial and Salmonella infections in interleukin-12 receptor-deficient patients. Science 280:1435–1438. 9. Park GY, Im YH, Ahn CH, Park JW, et al (2004) Functional and genetic assessment of IFN-gamma receptor in patients with clinical tuberculosis. Int J Tuberc Lung Dis 8:1221–1227. 10. Fraser DA, Bulat-Kardum L, Knezevic J, Babarovic P, et al (2003) Interferon-gamma receptor-1 gene polymorphism in tuberculosis patients from Croatia. Scand J Immunol 57:480–484. 11. Sreevatsan S, Pan X, Stockbauer KE, Connell ND, et al (1997) Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination. Proc Natl Acad Sci USA 94:9869–9874. 12. Valway SE, Sanchez MP, Shinnick TF, Orme I, et al (1998)An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis. N Engl J Med 338:633–639. 13. Lopez B, Aguilar D, Orozco H, Burger M, et al (2003) A marked difference in pathogenesis and immune response induced by different Mycobacterium tuberculosis genotypes. Clin Exp Immunol 133:30–37. 14. Manca C, Tsenova L, Bergtold A, Freeman S, et al (2001) Virulence of a Mycobacterium tuberculosis clinical isolate in mice is determined by failure to induce Th1 type immunity and is associated with induction of IFN-α /β. Proc Natl Acad Sci USA 98:5752–5757. 15. Manca C, Tsenova L, Barry CE, Bergtold A, et al (1999) Mycobacterium tuberculosis CDC1551 induces a more vigorous host immune response in vivo and in vitro, but is not more virulent than other clinical isolates. J Immunol 162:6740–6746. 16. Barnes PF, Yang Z, Preston-Martin S, Pogoda JM, et al (1997) Patterns of tuberculosis transmission in Central Los Angeles. JAMA 278:1159–1163. 17. Glynn JR, Whiteley J, Bifani PJ, Kremer K, van Soolingen D (2002) Worldwide occurrence of Beijing/W strains of Mycobacterium tuberculosis: a systematic review. Emerg Infect Dis 8:843–849. 18. Zhang M, Gong J, Yang Z, Samten B, Cave MD, Barnes PF (1999) Enhanced capacity of a widespread strain of Mycobacterium tuberculosis to grow in human macrophages. J Infect Dis 179:1213–1217. 19. Wu S, Howard ST, Lakey DL, Kipinis A, et al (2004) The principal sigma factor sigA mediates enhanced growth of Mycobacterium tuberculosis in vivo. Mol Microbiol 51:1551–1562. 20. Kisich KO, Higgins M, Diamond G, Heifets L (2002) Tumor necrosis factor alpha stimulates killing of Mycobacterium tuberculosis by human neutrophils. Infect Immun 70:4591–4599. 21. Oliveira MM, Charlab R, Pessolani MC (2001) Mycobacterium bovis BCG but not Mycobacterium leprae induces TNF-α secretion in human monocytic THP-1 cells. Mem Inst Oswaldo Cruz 96:973–978.

8. M. tuberculosis: Intracellular Growth in Human Macrophages 22. Roach SK, Lee SB, Schorey JS (2005) Differential activation of the transcription factor cyclic AMP response element binding protein (CREB) in macrophages following infection with pathogenic and nonpathogenic mycobacteria and role for CREB in tumor necrosis factor alpha production. Infect Immun 73:514–522. 23. Yadav M, Roach SK, Schorey JS (2004) Increased mitogen-activated protein kinase activity and TNF-α production associated with Mycobacterium smegmatis- but not Mycobacterium aviuminfected macrophages requires prolonged stimulation of the calmodulin/calmodulin kinase and cyclic AMP/protein kinase A pathways. J Immunol 172:5588–5597. 24. Moore KW, de Waal MR, Coffman RL, O’Garra A (2001) Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19:683–765. 25. Sharma S, Bose M (2001) Role of cytokines in immune response to pulmonary tuberculosis. Asian Pac J Allergy Immunol 19:213–219. 26. Stefanidou M, Griffin R, Ponce de Leon A, Sifuentes-Osornio, J, et al (2005) Comparative study of induction of early cytokines by diverse clinical strains of Mycobacterium tuberculosis. Abstract 3074, Keystone Symposium; Tuberculosis: Integrating Host and Pathogen Biology.

81 27. Newton SM, Smith RJ, Wilkinson KA, Nicol MP, et al (2006) A deletion defining a common Asian lineage of Mycobacterium tuberculosis associates with immune subversion. Proc Natl Acad Sci USA 103:15,594–15,598. 28. Theus SA, Cave DM, Eisenach K, Walrath J, Lee H, Mackay W, Whalen C, Silver RF (2006) Differences in the growth of paired Ugandan isolates of Mycobacterium tuberculosis within human mononuclear phagocytes correlate with epidemiological evidence of strain virulence. Infect Immun 74:6865–6876. 29. Guwatudde D, Nakakeeto M, Jones-Lopez EC, Maganda A, Chiunda A, Mugerwa RD, Ellner JJ, Bukenya G, Whalen CC (2003) Tuberculosis in household contacts of infectious cases in Kampala, Uganda. Am J Epidemiol 158:887–898. 30. Theus S, Eisenach K, Fomukong N, Silver RF, Cave MD (2006) Beijing Family Mycobacterium tuberculosis strains differ in their intracellular growth in THP-1 macrophages. Int J Tuberc Lung Dis 10:1087–1093. 31. Filliol I, Motiwala AS, Cavatore M, Qi M, et al (2006) Global phylogeny of Mycobacterium tuberculosis based on single nucleotide polymorphism (SNP) analysis: insights into tuberculosis evolution, phylogenetic accuracy of other DNA fingerprinting systems, and recommendations for a minimal standard SNP set. J Bacteriol 188:759–772.

Chapter 14 Control of Notifiable Avian Influenza Infections in Poultry Ilaria Capua and Stefano Marangon

14.1

Introduction

Avian influenza (AI) represents one of the greatest public health concerns that has emerged from the animal reservoir in recent times. Over the past 5 years there has been a sharp increase in the number of outbreaks of AI in poultry compared with the previous 40 years. It has been calculated that the impact of AI on the poultry industry has increased 100-fold with 23 million birds affected in a 40-year period between 1959 and 1998 and more than 200 million from 1999 to 2004 (1). In fact, from the late 1990s, AI infections have assumed a completely different profile both in the veterinary and medical scientific communities. In recent times, some outbreaks have maintained the characteristic of minor relevance, while others, such as the Italian outbreak of 1999–2000, the Dutch outbreak of 2003, the Canadian outbreak of 2004, and the ongoing Eurasian epidemics, have led to devastating consequences for the poultry industry, negative repercussions on public opinion, and, in some cases, created significant human health issues, including the risk of generating a new pandemic virus for humans via the avian–human link. Influenza viruses are segmented, negative-strand RNA viruses that are placed in the family Orthomyxoviridae in three genera: Influenzavirus A, B, and C. Only influenza A viruses have been reported to cause natural infections of birds. Type A influenza viruses are further divided into subtypes based on the antigenic characteristics of the surface glycoproteins hemagglutinin (H) and neuraminidase (N). At present, 16 H subtypes (H1–H16) and nine N subtypes (N1–N9) have been identified. Each virus has one H and one N antigen, apparently in any combination; all subtypes and the majority of possible combinations have been isolated from avian species. Influenza A viruses infecting poultry can be divided into two distinct groups on the basis of the severity of the disease they cause. The very virulent viruses cause highly pathoFrom: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

genic AI (HPAI), a systemic infection, in which flock mortality in some susceptible species may be as high as 100%. These viruses have been restricted to strains belonging to the H5 and H7 subtypes, exhibiting a multi-basic cleavage site at the precursor of the H molecule, or exhibiting a cleavage site motif similar to other HPAI viruses (2). HPAI is a dead-end infection in certain domestic birds, e.g., chickens and turkeys, and has a variable clinical behavior in domestic waterfowl and in wild birds, in which it may or may not cause clinical signs and mortality. To date, the potential role of wild birds and waterfowl as reservoirs of infection has been described only for the Asian HPAI H5N1 virus. The ecological and epidemiological implications of this unprecedented situation are not predictable. To the contrary, viruses belonging to all subtypes (H1–H16) lacking the multi-basic cleavage site, are perpetuated in nature in wild bird populations. Feral birds, particularly waterfowl, represent the natural hosts for these viruses and are therefore considered an ever-present source of viruses. Following introduction into domestic bird populations, these viruses cause low pathogenicity AI (LPAI). This is a localized infection, resulting in a mild disease consisting primarily of respiratory disease, depression, and egg production problems in laying birds. Current theories suggest that HPAI viruses emerge from H5 and H7 LPAI progenitors by mutation or recombination (3–5), although there must be more than one mechanism by which this occurs. This is supported by phylogenetic studies of H7 subtype viruses, which indicate that HPAI viruses do not constitute a separate phylogenetic lineage or lineages, but appear to arise from non-pathogenic strains (6, 7) and the in vitro selection of mutants virulent for chickens from an avirulent H7 virus (8). It appears that such mutations occur only after the viruses have moved from their natural wild bird host to poultry. However, the mutation to virulence is unpredictable and may occur very soon after introduction to poultry, or after the LPAI virus has circulated for several months in domestic birds. This hypothesis is further strongly supported by a study by Munster et al., who demonstrated that there is minor genetic and antigenic diversity between H5 and H7 123

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LPAI viruses found in wild birds and those having caused HPAI outbreaks in domestic poultry in Europe (9). The scientific evidence collected in recent years leads to the logical conclusion that not only HPAI viruses must be controlled in domestic populations but also LPAI viruses of the H5 and H7 subtypes, as they represent HPAI precursors. For this reason, the World Organization for Animal Health (OIE) has adopted the definition “Notifiable Avian Influenza” (NAI) to define all viruses of the H5 or H7 subtype, regardless of their virulence for chickens, and all viruses which have an intravenous pathogenicity index of 1.2 or higher.

14.2

Prevention of AI

The primary introduction of AI viruses in domestic poultry occurs either through direct or indirect contact with infected birds. This may occur through movement of infected poultry, contaminated equipment, fomites, or vehicles and penetration of contaminated infectious organic material. Airborne transmission has not been demonstrated to date. For these reasons, it is clear that if biosecurity measures are implemented at the farm level AI infections can be prevented. Outbreaks that involve significant numbers of animals are characterized by the penetration of infection into the commercial circuit. This includes industrially reared poultry but also all other poultry that is traded, including those from semiintensive and backyard farms. Concepts of disease prevention that are applied to industrially raised poultry in theory should not differ from management strategies that should be applied to smaller holdings. In practice, however, things differ significantly as very basic biosecurity measures, such as preventing the introduction of animals of different origin into a flock and avoiding the contact between poultry and feral waterfowl, are sufficiently well respected in the industrial system, but find very little compliance in the semi-industrial or rural environment. For this reason, in certain parts of the world, particularly where mixed species are reared together and traded through the live-bird market system, rural poultry may become a never-ending source of virus, perpetuating virus circulation and resulting in the establishment of an endemic situation. Biosecurity (encompassing bioexclusion and biocontainment) represents the first and most important means of prevention. It follows that if biosecurity measures of a high standard are implemented and maintained, they can be a firewall against the penetration and perpetuation in the industrial circuit. However, breaches in biosecurity systems do occur. On one hand, the occurrence and extent of the breach should be evaluated and corrective measures should follow, but on the other they indicate the need for the establishment of warning systems to aid diagnosis at an

early phase of infection and the urge to implement additional control tools for AI.

14.3

Vaccination for AI

Between December 1999 and April 2003, more than 50 million birds died or were depopulated following HPAI infection in the European Union alone (1), causing significant economic losses to the private and public sectors. The pre-emptive slaughter and destruction of great numbers of animals is also questionable from an ethical point of view. This would suggest that the strategies and control measures utilized to combat the disease at the European level require improvement both from a disease control and animal welfare perspective. Until recent times, infections caused by NAI viruses of the H5 and H7 subtype occurred rarely and vaccination was discouraged as stamping out was the recommended control option. Primarily for this reason, the knowledge of AI vaccinology has not grown at the same rate as that generated for other infectious diseases of animals. Data on experimental and field research in the field of AI vaccinology are currently being generated but there are still areas of uncertainty concerning the rather complex task of vaccinating poultry in different farming and ecological environments. The issue of disease control in developing countries has been addressed on several occasions following the spread of H5N1 in Southeast Asia. Guidelines on disease prevention and control have been issued as joint OIE/Food and Agriculture Organization of United Nations/World Health Organization recommendations in the recent meetings in Rome (February 3–4, 2004), Bangkok (February 26–28, 2004), and Ho-Chi Min City (February, 23–25 2005; ref. 10). These recommendations, however, need to be put into practice in a variety of different field situations, and the applicability of one system rather than another in a given situation must be evaluated bearing in mind the benefits of a successful result but also the drawbacks of a failure. Vaccination has been shown to be a powerful tool to support eradication programmes if used in conjunction with other control methods. Vaccination has been shown to increase resistance to field challenge, reduce shedding levels in vaccinated birds and reduce transmission dynamics (11–13). Both these occurrences contribute to controlling AI. However, previous experiences have indicated that to be successful in controlling and ultimately in eradicating the infection vaccination programmes must be part of a wider control strategy, which includes monitoring the evolution of infection and biosecurity. To eradicate AI, the vaccination system must allow the detection of field exposure in the vaccinated flock. This can be achieved both by using conventional inactivated vaccines and recombinant vector vaccines. Conventional inactivated vaccines containing the same viral subtype as the field virus allow the detection of field exposure by regularly testing unvaccinated sentinels left in the flock.

14. Avian Influenza Infections in Poultry

This system is applicable in the field but is rather impracticable, especially for the identification of sentinel birds in premises that contain floor-raised birds. An encouraging system based on the detection of anti-NS1 antibodies has been recently developed, and is in a concept applicable with all inactivated vaccines provided they have the same H subtype as the field virus (14). This system is based on the fact that the NS1 protein is synthesized only during active viral replication and is therefore not significantly present in inactivated vaccines. Birds that are vaccinated with such vaccines will develop antibodies to the NS1 only following field exposure. Full and field validation under different circumstances of this system is still in progress and should be made available before this system is recommended (15). To date, the only system that enables the detection of field exposure in a vaccinated population that has been used successfully and has resulted in eradication is a DIVA (Differentiating Infected from Vaccinated Animals) system based on heterologous vaccination. This system was developed to support the eradication programs against several introductions of LPAI viruses of the H7 subtype (1, 11). Briefly, a vaccine containing a virus possessing the same H but a different N to the field virus is used. This vaccination strategy enables the detection of field exposure in a vaccinated population through the detection of antibodies to the N antigen of the field virus. For the sake of clarity, a vaccine containing an H7N3 virus can be used against a field virus of the H7N1 subtype. Antibodies to H7 are cross-protective, thus ensuring clinical protection, increased resistance to challenge and reduction of shedding, while antibodies to the N of the field virus (in this case N1) can be used as a natural marker of infection. Experimental data on the quantification of the effect of vaccination on transmission within a flock using this system have been generated, indicating that the reproduction ratio can be reduced to ALT) and a marked decrease in the frequency of CD4+ lymphocytes in the spleen, which may correlate with the lymphopenia observed in severe cases of HME (52, 53). Apoptosis of hepatocytes and sinusoidal lining cells occurs as early as day 5. By day 9 post-infection, a focal hepatic necrosis is observed, mainly in the midzone of the hepatic lobules. The lung pathology is also similar to that in HME with the interstitium focally thickened and an infiltration of monocytes present on day 9 post-infection. Ehrlichial morulae are not present within the cytoplasm of apoptotic cells as determined by double staining of the TUNEL preparations with anti-Ehrlichia antibodies. IOE has also been used to study secondary challenge infections in C57BL/6 mice (54). In this study, mice were given low-dose IOE and survived the initial infection; however, when given an additional inoculation with the same dose four weeks later, mice succumbed to the disease. The fatal outcome was CD8+ T cell-dependent and associated with high levels of serum TNF-α and CCL2. These observations further support the hypothesis that pathogenic CD8+ T cells play a role in fatal ehrlichiosis. The development of the E. muris and IOE models is a significant advance in the field, enabling the study of differences between mild and severe disease; however, the antigenic and genetic differences in the ehrlichiae cannot be ignored when

H. L. Stevenson et al.

interpreting the results. Recently, our laboratory reported that IOE when inoculated intradermally also resulted in mild disease with characteristics similar to E. muris that all mice inoculated intradermally survived an infectious dose that would be fatal had mice been inoculated intraperitoneally (53). This route of inoculation also better mimics natural ehrlichial transmission because the intradermal (i.d.) location is the inoculation site for ehrlichiae by feeding ticks. Furthermore, i.d. inoculation of IOE resulted in a lower systemic bacterial burden and much less inflammation and apoptosis in the liver when compared to i.p. inoculation of mice with the same dose of organisms. The hepatic lesions observed in these three mouse models (Figure 4.4) reflect the difference in the clinical severity. Mild forms of the disease initiated by either i.p. inoculation with E. muris or i.d. inoculation with IOE, resulted in a more localized, contained inflammatory response with low levels of serum TNF-α and IL-10, and relatively little apoptosis. To the contrary, i.p. inoculation with IOE produced dispersed hepatic inflammation and extensive apoptosis of both hepatocytes and mononuclear cells, which correlates with an uncontrolled inflammatory response leading to high levels of serum TNF-α and IL-10 (53, 55). At this time, IOE has not been associated with human disease; however, its ability to infect both invertebrate and vertebrates hosts in Japan and its striking virulence in mice, warrants further investigation of potential human involvement.

Figure 4.4 Comparison of liver pathology in three mouse models of human ehrlichiosis. On day 7 post-infection, E. muris inoculated intraperitoneally (A) and IOE inoculated intradermally (B) both result in mild disease characterized by perivascular lymphohistiocytic infiltration. In contrast, IOE inoculated intraperitoneally results in severe apoptosis (C) and multi-focal hepatic necrosis (D) (See Color Plates).

4. Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology

4.6 Characteristics of the Protective and Detrimental Immune Responses to Ehrlichiae Both cell-mediated and humoral immunity are required for complete protection against Ehrlichia. Cell-mediated immunity, orchestrated mainly by CD4+ Th1 cells, is required for protection against severe disease (46, 56). Interestingly, although Ehrlichia are obligately intracellular organisms, anti-Ehrlichia antibodies provide passive protection against i.p. inoculation of IOE (52, 56–60). Mouse models of HME also allow the determination of the factors that lead to the development of severe ehrlichiosis, which include high concentrations of TNF-α and IL-10 in the serum, a high frequency of TNF-α-producing CD8+ T cells in the spleen, low IL-12 levels in the spleen, and a 40-fold decrease in the number of IFN-γ-producing CD4+ T cells (52). The decrease in IL-12, one of the main Th1-promoting cytokines, may be due to multiple factors including intracellular repression of IL-12 by the organism itself (36) or apoptosis of host macrophages, one of the main antigen-presenting cells involved in early IL-12 production. In several different mouse strains, induction of Th1 immunity has been essential to host survival, and when CD4+ T cell help is compromised, the immune response becomes dysregulated and destructive (52). Overall, the pathogenesis of ehrlichial disease appears to be multifactorial as in other septic conditions, which explains the inability to control disease severity by inhibiting one component such as TNF-α (55). Whether features of the defective immune response that have been characterized in mice, such as high levels of serum TNF-α and IL-10, occur in humans needs to be determined by performing more clinical studies, particularly in regions where potential vectors of HME are prevalent.

4.7 Tick Vectors, Ecology, and Ehrlichial Transmission As previously mentioned, the distribution of the arthropod vectors and vertebrate hosts correlates with the presence of the diseases that they transmit (11, 61–63). A prototypical example of this tight vector-host relationship is that of HME cases and the distribution of its primary vector (Amblyomma americanum) in the United States. This relationship also correlates with the regions in which the reservoir host is found; however, this association is not as striking for HME because the main reservoir, the white-tailed deer (Odocoileus virginianus) is extremely widely distributed in North America. The transmission cycle for E. chaffeensis (Figure 4.5) is representative of all of the ticktransmitted human ehrlichioses. In addition, E. chaffeensis has been identified in several other mammals including canids, goats, raccoons, and opossums (19). Other vectors of E. chaffeensis include other Metastriata ticks in the Ixodidae family such as Ixodes pacificus (64), Ixodes ricinus (65), Haemaphysalis yeni (66), Amblyomma testudinarium (66), possibly Amblyomma maculatum (67), and Dermacentor variabilis (68). In addition,

43

Wen et al. (11) reported the presence of E. chaffeensis DNA by PCR in several tick species including A. testudinarium, H. yeni, I. ovatus, I. persulcatus, Boophilus microplus, and Dermacentor silvarum, in both northern and southern China; previously this bacterium was thought to exist only in North America and possibly Thailand (13). If E. chaffeensis is successfully transmitted by these various metastriata tick vectors, the distribution of HME may be more widespread than predicted. However, many of the previously mentioned reports used only serology and/or PCR for detection of either anti-Ehrlichia antibodies or ehrlichial DNA, which may limit interpretation of the results due to crossreactivity or genomic similarities among ehrlichial species, respectively. Other more conclusive studies that include isolation of the bacteria and further genetic characterization are necessary. The occurrence of HME-like illness described in patients with febrile illness in Russia, along with the presence of I. persulcatus ticks throughout the country (9), makes this region ideal for studying of not only vector–host ecology, but also of its relationship with the incidence of human disease. Ehrlichiae have not been isolated from I. persulcatus; even though genetically they appear to be the mildly virulent E. muris, the agent deserves further study including isolation and experimental infection of mice to confirm its pathogenicity. The Anan and IOE (formerly H565 strain) strains differed by only three nucleotides of 1,449 base pairs in the 16S rRNA gene; however, when inoculated intraperitoneally into mice, these strains caused 0 and 100% fatalities, respectively (50). The main reservoir for E. muris in Japan is wild mice (Apodemus spp.), which makes this vector–host relationship feasible for manipulation in the laboratory (12). The range of I. persulcatus ticks extends contiguously from Eastern Europe to Korea and Japan, an area comprising nearly one-fifth of the global population (48). Further studies in these regions are necessary to determine if this species is a competent vector of a HME. I. ovatus ticks, the species from which IOE was isolated, are found throughout Japan which also provides an ideal environment for future ecological studies. Although significant advances have been made in our understanding of the host immune responses to ehrlichiosis, our knowledge could be further expanded if the effect of natural tick transmission and salivary components could be determined. Experimental transmission studies utilizing these organisms and their corresponding vectors have not been reported.

4.8

Conclusions

Ehrlichiae have evolved many mechanisms to survive within their arthropod and mammalian hosts, including immune evasion strategies such as the absence of identified PAMPs (e.g., LPS and peptidoglycan), the ability to vary surface antigens by differential expression of bacterial glycoproteins, and the inhibition of phagolysosome fusion involving two-component regulatory systems. In addition, ehrlichiae are able to synthesize several nucleotides, vitamins and cofactors to aid in their

44


Figure 4.5. A life cycle of E. chaffeensis. Noninfected larvae obtaining blood from a bacteremic vertebrate reservoir host (e.g., white-tailed deer [shaded]) become infected and maintain ehrlichiae to the nymphal stage. Infected nymphs may transmit E. chaffeensis to susceptible reservoir hosts (unshaded) or to humans during acquisition of blood. Infected adult ticks, having acquired ehrlichiae either by transstadial transmission from infected nymphal stage or during blood meal acquisition as noninfected nymphs on infected deer, may also pass E. chaffeensis to humans or other susceptible reservoirs. Transovarial transmission has not been demonstrated, and eggs and unfed larvae are presumably not infected (19).

intracellular survival, and have genes that encode enzymes to counteract host defenses. Continued comparison of the genomes of these pathogens and further characterization of encoded proteins will provide valuable insight into the pathogenesis initiated by these unique bacteria. The availability of the different animal models will facilitate our understanding of the host response to these agents. The range of clinical manifestations seen even in immunocompetent hosts proves that host factor differences cannot be ignored when studying these diseases. Furthermore, the ability of organisms that lack PAMPs (such as LPS and peptidoglycan) to cause toxic shock-like syndrome will provide valuable information about novel mechanisms of inducing immune-mediated pathology. Human studies investigating these immune phenomena are much needed and will help to confirm many of the intriguing mechanisms elucidated using animal models. Finally, the emergence of these infectious diseases and increasing reports of human cases worldwide necessitates

further studies of potential vector–host relationships. As awareness of these diseases and surveillance increases, climate change and ecological disturbances continue, and effective methods to detect these pathogens are more readily available, the prevalence of human ehrlichioses throughout the world will continue to challenge current concepts.

References 1. Rodriguez-Vivas RI, Albornoz RE, Bolio GM (2005) Ehrlichia canis in dogs in Yucatan, Mexico: seroprevalence, prevalence of infection and associated factors. Vet Parasitol 127:75–79. 2. Gongora-Biachi R, Zavala-Velazquez JE, Castro-Sansores CJ, Gonzalez-Martinez P (1999) First case of human ehrlichiosis in Mexico. Emerg Infect Dis 5:481. 3. Calic SB, Galvao MA, Bacellar F, Rocha CM, Mafra CL, Leite RC, Walker DH (2004) Human ehrlichioses in Brazil: first suspect cases. Braz J Infect Dis 8:259–262.

4. Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology 4. Aguirre E, Sainz A, Dunner S, Amusategui I, Lopez L, RodriguezFranco F, Luaces I, Cortes O, Tesouro MA (2004) First isolation and molecular characterization of Ehrlichia canis in Spain. Vet Parasitol 125:365–372. 5. Loftis AD, Reeves WK, Szumlas DE, Abbassy MM, Helmy IM, Moriarity JR, Dasch GA (2006) Rickettsial agents in Egyptian ticks collected from domestic animals. Exp Appl Acarol 40:67–81. 6. Ndip LM, Ndip RN, Esemu SN, DIckmu VL, Fokam EB, Walker DH, McBride JW (2005) Ehrlichial infection in Cameroonian canines by Ehrlichia canis and Ehrlichia ewingii. Vet Micrbiol 111:59–66. 7. Wielinga PR, Gaasenbeek C, Fonville M, de Boer A, de Vries A, Dimmers W, Op Akkerhuis GJ, Schouls LM, Borgsteede F, van der Giessen JW (2006) Longitudinal analysis of tick densities and Borrelia, Anaplasma and Ehrlichia infection of Ixodes ricinus ticks in different habitat areas in the Netherlands. Appl Environ Microbiol 72:7594–7601. 8. Alekseev AN, Dubinina HV, Pol I, Van DeSchouls LM (2001) Identification of Ehrlichia spp. and Borrelia burgdorferi in Ixodes ticks in the Baltic regions of Russia. J Clin Microbiol 39:2237–2242. 9. Rar VA, Fomenko NV, Dobrotvorsky AK, Livanova NN, Rudakova SA, Fedorov EG, Astanin VB, Morozova OV (2005) Tickborne pathogen detection, Western Siberia, Russia. Emerg Infect Dis 11:1708–1715. 10. Shpynov SN, Rudakov NV, Iastrebov VK, Leonova GN, Khazova TG, Egorova NV, Borisova ON, Preider VP, Bezrukov GV, Fedorov EG, Fedianin AP, Sherstneva MB, Turyshev AG, Gavrilov AP, Tankibaev MA, Fournier PE, Raoult D (2004) [New evidence for the detection of ehrlichia and anaplasma in ixodes ticks in Russia and Kazakhstan]. Med Parazitol (Mosk) 2:10–14. 11. Wen B, Cao W, Pan H (2003) Ehrlichiae and ehrlichial diseases in China. Ann NY Acad Sci 990:45–53. 12. Kawahara M, Rikihisa Y, Lin Q, Isogai E, Tahara K, Itagaki A, Hiramitsu Y, Tajima T (2006) Novel genetic variants of Anaplasma phagocytophilum, Anaplasma bovis, Anaplasma centrale, and a novel Ehrlichia sp. in wild deer and ticks on two major islands in Japan. Appl Environ Microbiol 72:1102–1109.. 13. Heppner DG, Wongsrichanalai C, Walsh DS, McDaniel P, Eamsila C, Hanson B, Paxton H (1997) Human ehrlichiosis in Thailand. Lancet 350:785–786. 14. Parola P, Cornet JP, Sanogo YO, Miller RS, Thien HV, Gonzalez JP, Raoult D, Telford III Sr, Wongsrichanalai C (2003) Detection of Ehrlichia spp., Anaplasma spp., Rickettsia spp., and other eubacteria in ticks from the Thai-Myanmar border and Vietnam. J Clin Microbiol 41:1600–1608. 15. Suksawat J, Xuejie Y, Hancock SI, Hegarty BC, Nilkumhang P, Breitschwerdt EB (2001) Serologic and molecular evidence of coinfection with multiple vector-borne pathogens in dogs from Thailand. J Vet Intern Med 15:453–462. 16. Olano JP, Masters E, Hogrefe W, Walker DH (2003) Human monocytotropic ehrlichiosis, Missouri. Emerg Infect Dis 9:1579–1586. 17. Kovats RS, Campbell-Lendrum DH, McMichael AJ, Woodward A, Cox JS (2001) Early effects of climate change: do they include changes in vector-borne disease? Philos Trans R Soc Lond B Biol Sci 356:1057–1068. 18. Olano JP, Hogrefe W, Seaton B, Walker DH (2003) Clinical manifestations, epidemiology, and laboratory diagnosis of human monocytotropic ehrlichiosis in a commercial laboratory setting. Clin Diagn Lab Immunol 10:891–896. 19. Paddock CD, Childs JE (2003) Ehrlichia chaffeensis: a prototypical emerging pathogen. Clin Microbiol Rev 16:37–64.

45 20. Lin M, Rikihisa Y (2003) Ehrlichia chaffeensis and Anaplasma phagocytophilum lack genes for lipid A biosynthesis and incorporate cholesterol for their survival. Infect Immun 71:5324–5331. 21. Zhang JZ, Sinha M, Luxon BA, Yu XJ (2004) Survival strategy of obligately intracellular Ehrlichia chaffeensis: novel modulation of immune response and host cell cycles. Infect Immun 72:498–507. 22. McBride JW, Yu X-J, Walker DH (2000) Glycosylation of homologous immunodominant proteins of Ehrlichia chaffeensis and Ehrlichia canis. Infect Immun 68:13–18. 23. Lin M, Rikihisa Y (2004) Ehrlichia chaffeensis downregulates surface toll-like receptors 2/4, CD14 and transcription factors PU.1 and inhibits lipopolysaccharide activation of NF-κB, ERK 1/2 and p38 MAPK in host monocytes. Cell Microbiol 6:175–186. 24. Rikihisa Y (2006) Ehrlichia subversion of host innate responses. Curr Opin Microbiol 9: 95–101. 25. Mavromatis K, Doyle CK, Lykidis A, Ivanova N, Francino MP, Chain P, Shin M, Malfatti S, Larimer F, Copeland A, Detter JC, Land M, Richardson PM, Yu XJ, Walker DH, McBride JW, Kyrpides NC (2006) The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies. J Bacteriol 188:4015–4023. 26. Nethery K, Doyle C, Elsom B, Herzog N, Popov V, McBride J (2005) Ankyrin repeat containing immunoreactive 200 kDa glycoprotein (gp200) orthologs of Ehrlichia chaffeensis and E. canis are translocated to the nuclei of infected monocytes, abstr. O-60. Fourth International Conference on Rickettsiae and Rickettsial Diseases, Logrono, Spain, June 18–21. 27. Hotopp JC, Lin M, Madupu R, Crabtree J, Angiuoli SV, Eisen J, Seshadri R, Ren Q, Wu M, Utterback TR, Smith S, Lewis M, Khouri H, Zhang C, Niu H, Lin Q, Ohashi N, Zhi N, Nelson W, Brinkac LM, Dodson RJ, Rosovitz MJ, Sundaram J, Daugherty SC, Davidsen T, Durkin AS, Gwinn M, Haft DH, Selengut JD, Sullivan SA, Zafar N, Zhou L, Benahmed F, Forberger H, Halpin R, Mulligan S, Robinson J, White O, Rikihisa Y, Tettelin H (2006) Comparative genomics of emerging human ehrlichiosis agents. PLoS Genet 2:e21. 28. Popov VL, Han VC, Chen SM, Dumler JS, Feng HM, Andreadis TG, Tesh RB, Walker DH (1998) Ultrastructural differentiation of the genogroups in the genus Ehrlichia. J Med Microbiol 47:235–251. 29. Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski S, Cascales E (2005) Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu Rev Microbiol 59:451–485. 30. Cheng Z, Kumagai Y, Lin M, Zhang C, Rikihisa Y (2006) Intraleukocyte expression of two-component systems in Ehrlichia chaffeensis and Anaplasma phagocytophilum and effects of the histidine kinase inhibitor closantel. Cell Microbiol 8:1241–1252. 31. Kumagai Y, Cheng Z, Lin M, Rikihisa Y (2006) Biochemical activities of three pairs of Ehrlichia chaffeensis two-component regulatory system proteins involved in inhibition of lysosomal fusion. Infect Immun 74:5014–5022. 32. Kanehisa M, Goto S, Hattori M, oki-Kinoshita KF, Itoh M, Kawashima S, Katayama T, Araki M, Hirakawa M (2006) From genomics to chemical genomics: new developments in KEGG. Nucleic Acids Res 34:D354–D357. 33. Zahrl D, Wagner M, Bischof K, Koraimann G (2006) Expression and assembly of a functional type IV secretion system elicit extracytoplasmic and cytoplasmic stress responses in Escherichia coli. J Bacteriol 188:6611–6621. 34. Zhang JZ, Popov VL, Gao S, Walker DH, Yu XJ (2007) The developmental cycle of Ehrlichia chaffeensis in vertebrate cells. Cell Microbiol 9:610–618.

46 35. Popov VL, Yu X, Walker DH (2000) The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry. Microb Pathog 28:71–80. 36. Zhang J-Z, McBride JW, Yu X-J (2003) L-selectin and E-selectin expressed on monocytes mediating Ehrlichia chaffeensis attachment onto host cells. FEMS Microbiol Let 227:303–309. 37. Singu V, Liu H, Cheng C, Ganta RR (2005) Ehrlichia chaffeensis expresses macrophage- and tick cell-specific 28-kilodalton outer membrane proteins. Infect Immun 73:79–87. 38. Nandi B, Winslow G (2006) Identification of T cell epitopes Ehrlichia outer membrane proteins that elicit protective immunity, abstr. 124. 20th Meeting of The American Society for Rickettsiology in conjunction with the 5th International Conference on Bartonella as Emerging Pathogens, Asolimar, CA, September 2–7. 39. Ganta RR, Cheng C, Miller EC, McGuire BL, Peddireddi L, Sirigireddy KR, Chapes SK (2006) Differential clearance and immune responses to tick cell vs. macrophage culture-derived Ehrlichia chaffeensis in mice. Infect Immun 75:135–145. 40. Munz C, Steinman RM, Fujii S (2005) Dendritic cell maturation by innate lymphocytes: coordinated stimulation of innate and adaptive immunity. J Exp Med 202:203–207. 41. Mattner J, Debord KL, Ismail N, Goff RD, Cantu C III, Zhou D, Saint-Mezard P, Wang V, Gao Y, Yin N, Hoebe K, Schneewind O, Walker D, Beutler B, Teyton L, Savage PB, Bendelac A (2005) Exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature 434:525–529. 42. Dumler JS, Brouqui P, Aronson J, Taylor JP, Walker DH (1991) Identification of Ehrlichia in human tissue. N Engl J Med 325:1109–1110. 43. Walker DH, Dumler JS (1997) Human monocytic and granulocytic ehrlichioses. Discovery and diagnosis of emerging tickborne infections and the critical role of the pathologist. Arch Pathol Lab Med 121:785–791. 44. Dierberg KL, Dumler JS (2006) Lymph node hemophagocytosis in rickettsial diseases: a pathogenetic role for CD8 T lymphocytes in human monocytic ehrlichiosis (HME)? BMC Infect Dis 6:121. 45. Sehdev AE, Dumler JS (2003) Hepatic pathology in human monocytic ehrlichiosis. Ehrlichia chaffeensis infection. Am J Clin Pathol 119:859–865. 46. Ganta RR, Cheng C, Wilkerson MJ, Chapes SK (2004) Delayed clearance of Ehrlichia chaffeensis infection in CD4+ T-cell knockout mice. Infect Immun 72:159–167. 47. Feng HM, Walker DH (2004) Mechanisms of immunity to Ehrlichia muris: a model of monocytotropic ehrlichiosis. Infect Immun 72:966–971. 48. Ravyn MD, Korenberg EI, Oeding JA, Kovalevskii YV, Johnson RC (1999) Monocytic Ehrlichia in Ixodes persulcatus ticks from Perm, Russia. Lancet 353:722–723. 49. Vorobyeva NN, Korenberg EI, Grigoryan YV (2002) Diagnostics of tick-borne diseases in the endemic region of Russia. Wien Klin Wochenschr 114:610–612. 50. Shibata S, Kawahara M, Rikihisa Y, Fujita H, Watanabe Y, Suto C, Ito T (2000) New Ehrlichia species closely related to Ehrlichia chaffeensis isolated from Ixodes ovatus ticks in Japan. J Clin Microbiol 38:1331–1338. 51. Sotomayor E, Popov V, Feng H-M, Walker DH, Olano JP (2001) Animal model of fatal human monocytotropic ehrlichiosis. Am J Pathol 158:757–769. 52. Ismail N, Soong L, McBride JW, Valbuena G, Olano JP, Feng HM, Walker DH (2004) Overproduction of TNF-α by CD8+ type 1


53.

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60.

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62.

63. 64.

65.

66.

67.

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cells and down-regulation of IFN-γ production by CD4+ Th1 cells contribute to toxic shock-like syndrome in an animal model of fatal monocytotropic ehrlichiosis. J Immunol 172:1786–1800. Stevenson HL, Jordan JM, Peerwani Z, Wang HQ, Walker DH, Ismail N (2006) An intradermal environment promotes a protective type-1 response against lethal systemic monocytotropic ehrlichial infection. Infect Immun 74:4856–4864. Bitsaktsis C, Winslow G (2006) Fatal recall responses mediated by CD8 T cells during intracellular bacterial challenge infection. J Immunol 177:4644–4651. Ismail N, Stevenson HL, Walker DH (2006) Role of tumor necrosis factor alpha (TNF-alpha) and interleukin-10 in the pathogenesis of severe murine monocytotropic ehrlichiosis: increased resistance of TNF receptor p55- and p75-deficient mice to fatal ehrlichial infection. Infect Immun 74:1846–1856. Bitsaktsis C, Huntington J, Winslow GM (2004) Production of IFN-γ by CD4 T cells is essential for resolving ehrlichia infection. J Immunol 172:6894–6901. Li JS, Yager E, Reilly M, Freeman C, Reddy GR, Reilly AA, Chu FK, Winslow GM (2001) Outer membrane protein-specific monoclonal antibodies protect SCID mice from fatal infection by the obligate intracellular bacterial pathogen Ehrlichia chaffeensis. J Immunol 166:1855–1862. Li JS, Winslow GM (2003) Survival, replication, and antibody susceptibility of Ehrlichia chaffeensis outside of host cells. Infect Immun 71:4229–4237. Winslow GM, Yager E, Shilo K, Volk E, Reilly A, Chu FK (2000) Antibody-mediated elimination of the obligate intracellular bacterial pathogen Ehrlichia chaffeensis during active infection. Infect Immun 68:2187–2195. Yager E, Bitsaktsis C, Nandi B, McBride JW, Winslow G (2005) Essential role for humoral immunity during Ehrlichia infection in immunocompetent mice. Infect Immun 73:8009–8016. Estrada-Pena A, Jongejan F (1999) Ticks feeding on humans: a review of records on human-biting Ixodoidea with special reference to pathogen transmission. Exp Appl Acarol 23:685–715. Parola P, Paddock CD, Raoult D (2005) Tick-borne rickettsiosis around the world: emerging diseases challenging old concepts. Clin Microbiol Rev 18:719–756. Parola P, Davoust B, Raoult D (2005) Tick- and flea-borne rickettsial emerging zoonoses. Vet Res 36:469–492. Kramer VL, Randolph MP, Hui LT, Irwin WE, Gutierrez AG, Vugia DJ (1999) Detection of the agents of human ehrlichioses in ixodid ticks from California. Am J Trop Med Hyg 60:62–65. Alekseev AN, Dubinina HV, Semenov AV, Bolshakov CV (2001) Evidence of ehrlichiosis agents found in ticks (Acari: Ixodidae) collected from migratory birds. J Med Entomol 38:471–474. Cao WC, Gao YM, Zhang PH, Zhang XT, Dai QH, Dumler JS, Fang LQ, Yang H (2000) Identification of Ehrlichia chaffeensis by nested PCR in ticks from Southern China. J Clin Microbiol 38:2778–2780. Kocan AA, Ewing SA, Stallknecht D, Murphy GL, Little S, Whitworth LC, Barker RW (2000) Attempted transmission of Ehrlichia chaffeensis among white-tailed deer by Amblyomma maculatum. J Wildl Dis 36:592–594. Steiert JG, Gilfoy F (2002) Infection rates of Amblyomma americanum and Dermacentor variabilis by Ehrlichia chaffeensis and Ehrlichia ewingii in southwest Missouri. Vect Born Zoon Dis 2:53–60. Popov VL, Chen S-M, Feng HM, Walker DH (1995) Ultrasturctural variation of cultured Ehrlichia chaffeensis. J Med Microbiol 43:411-421.

Chapter 34 Biological Basis and Clinical Significance of HIV Resistance to Antiviral Drugs Mark A. Wainberg and Susan Schader

34.1

Introduction

HIV-1 drug resistance has emerged as a major factor that limits the effectiveness of antiviral drugs in treatment regimens. Many studies have shown that the development and transmission of drug-resistant (DR) HIV-1 is largely a consequence of incompletely suppressive antiretroviral regimens; HIV-1 drug resistance can significantly diminish the effectiveness and duration of benefit associated with combination therapy for the treatment of HIV/AIDS (1–6). Resistance-conferring mutations in both the HIV-1 reverse transcriptase (RT) and protease (PR) genes may precede the initiation of therapy due to both spontaneous mutagenesis and the spread of resistant viruses by sexual and other means. However, it is also generally believed that multiple drug mutations to any single or combination of antiretrovirals (ARVs) are required in order to produce clinical resistance to most ARVs and that these are in fact selected following residual viral replication in the presence of incompletely suppressive drug regimens (7–9). In the case of the PR inhibitors (PIs; refs. 10–12), and most nucleoside analog RT inhibitors (NRTIs), the development of progressive high-level phenotypic drug resistance follows the accumulation of primary resistance-conferring mutations in each of the HIV-1 PR and RT genes (13–15). Non-nucleoside RT inhibitors (NNRTIs) have low genetic barriers for the development of drug resistance and, frequently, a single primary drug resistance mutation to any one NNRTI may be sufficient to confer high-level phenotypic drug resistance to this entire class of ARVs (16, 17). Furthermore, differences have also been reported in regard to the development and evolution of ARV drug resistance between subtype B HIV-1 and several group M non-B subtypes. Non-B subtypes, e.g, subtype C HIV-1 variants, are known to possess naturally occurring polymorphisms at several From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

RT and PR codons that are implicated in drug resistance (18, 19). In some studies, the presence of these polymorphisms did not significantly reduce susceptibility to ARVs in phenotypic resistance assays or limit the effectiveness of an initial ARV therapy (ART) regimen for a period of up to 18 months (18–20). However, it has also been suggested that polymorphisms at resistance positions may sometimes facilitate selection of novel pathways leading to drug resistance, especially with incompletely suppressive ARV regimens (18). This, in turn, may have important clinical implications with respect to choice of effective ART. This warrants increased genotypic surveillance on a worldwide basis, as the prevalence of non-B HIV-1 infection is increasing rapidly. As illustrated in Figures 34.1-34.3, it has been possible to select numerous drug resistance mutations for all licensed ARVs and investigational agents such as the HIV-1 entry inhibitors that are currently undergoing final stages of clinical testing (5, 6, 21). In view of the hypervariability of HIV-1 and limitation of existing ARV combinations to completely suppress viral replication, it is essential that new anti-HIV drug discovery initiatives focus on the identification of new therapeutic targets and the development of ARV agents with more robust genetic barriers and a broader spectrum of activity against DR HIV-1 variants.

34.2 Generation of HIV-1 Drug Resistance Resistance mutations to ARVs may arise spontaneously as a result of the error-prone replication of HIV-1 and, in addition, are selected both in vitro and in vivo by pharmacological pressure (22–24). The high rate of spontaneous mutation in HIV-1 has been largely attributed to the absence of a 3’->5’exonuclease proof-reading mechanism. Sequence analyses of HIV-1 DNA have detected several types of mutations including base substitutions, additions and deletions (22). The frequency of spontaneous mutation for HIV-1 varies considerably as a result 309

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of differences among viral strains studied in vitro (25). Overall mutation rates for wild-type (WT) laboratory strains of HIV-1 have been reported to range from 97 X 10–4 to 200 X10–4 per nucleotide for the HXB2 clonal variant of HIV-1 to as high as 800 X 10–4 per nucleotide for the HIV-1 NY5 strain (22, 25). In addition to the low fidelity of DNA synthesis by HIV1 RT, other interdependent factors that affect rates of HIV mutagenesis include RT processivity, viral replication capac-

ity, viral pool size, and availability of target cells for infection (26–29). It follows that an alteration in any or combination of these factors might influence the development of HIV drug resistance. There is also data showing that thymidine analog mutations (TAMs) in RT can significantly increase the likelihood of further mutant HIV-1 distributions and evolution of drug resistance; furthermore, this can happen in the presence or absence of concomitant NRTIs (30, 31).

34. HIV Resistance to Antiviral Drugs

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Chart of common PR mutations associated with HIV drug resistance IDV

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Figure 34.3. The letter designation at the top of a box refers to the amino acid that is present in the wild-type sequence of PR. The letters at the bottom refer to substitutions associated with drug resistance. Sometimes, several different amino acid changes at the same codon can conferresistance, drug as for example both I and L in the case of position 46 (See Color Plates).

34.3

Inhibitors of RT

The RT enzyme is encoded by the HIV pol gene and is responsible for the transcription of double-stranded proviral DNA from viral genomic RNA. Two categories of drugs have been developed to block RT; these are NRTIs that act to arrest DNA chain elongation by acting as competitive inhibitors of RT and NNRTIs that act as non-competitive antagonists of enzyme activity by binding to the catalytic site of RT. NRTIs are administered to patients as precursor compounds that are phosphorylated to their active triphosphate form by cellular enzymes. These compounds lack a 3’ hydroxyl group necessary for elongation of viral DNA. These analogs can compete effectively with normal deoxynucleotide triphosphate (dNTP) substrates for binding to RT and incorporation into viral DNA (32, 33). NNRTI antiviral activity is incompletely understood but is known to involve the binding of these non-competitive inhibitors to a hydrophobic pocket close to the catalytic site of RT (34, 35). NNRTI inhibition reduces the catalytic rate of polymerization without affecting nucleotide binding or nucleotideinduced conformational change (36). NNRTIs are particularly active at template positions at which the RT enzyme naturally pauses. NNRTIs do not seem to influence the competition between dideoxynucleotide triphosphates (ddNTPs) and the

naturally occurring dNTPs for insertion into the growing proviral DNA chain (37). Both types of RT inhibitors have been shown to successfully diminish plasma viral burden in HIV-1 infected subjects. However, monotherapy with all drugs has led to drug resistance. Patients who receive combinations of three or more drugs are less likely to develop resistance, since these “cocktails” can suppress viral replication with much greater efficiency than single drugs or two drugs in combination. Although mutagenesis is less likely to happen in this circumstance, it can still occur, and the emergence of DR breakthrough viruses has been demonstrated in patients receiving highly active ART (HAART; refs. 38 and 39). Furthermore, the persistence of reservoirs of latently infected cells represents another major impediment to currently applied anti-HIV chemotherapy (40). Replication of HIV might resume once therapy is stopped or interrupted and, therefore, eradication of a latent reservoir of 105 cells might take as long as 60 years, a goal that is not practical with currently available drugs and technology (40, 41). Resistance to 3TC ((-)-2’, 3’-dideoxy-3’-thiacytidine, lamivudine) develops quickly whereas resistance to other NRTIs commonly appears only after about six months of therapy. Phenotypic resistance is detected by comparing the IC50 (or drug concentration capable of blocking viral replication by 50%) of pretreatment viral isolates with those obtained after

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therapy. Thus, higher IC50 values obtained after several months of treatment reflect a loss in viral susceptibility to ARV agents. Selective polymerase chain reaction (PCR) analysis of the RT genome confirms that the number of mutations associated with drug resistance increases concomitantly with increases in IC50 values. Mutations associated with drug resistance have been reported in response to the use of any single NRTI or NNRTI (42). However, not all drugs elicit the same mutagenic response; sensitivity and resistance patterns must be considered on an individual drug basis. For example, patients on 3TC monotherapy may develop high-level, i.e. 1000-fold resistance within weeks, whereas six months or more are often required in order for sensitivity to ZDV to drop by 50-100-fold. In contrast, HIV may appear to remain reasonably sensitive, even after prolonged monotherapy, to four of the other commonly used nucleoside analogs: ddI (didanosine), ddC (zalcitabine), d4T (stavudine) and ABC (abacavir). In the case of ZDV, increases in IC50 below threefold are regarded as non-significant, while 10-50 fold increases usually represent partial resistance, and increases above 50-fold denote high-level resistance. Patient resistance to nucleoside analogs can often develop independently of the dose of drug that is administered. Tissue culture data have shown that HIV-1 resistance can be easily demonstrated against each of NRTIs, NNRTIs and PIs, by gradually increasing the concentration of compound in the tissue culture medium (43, 44). Cell lines are especially useful in this regard, since HIV replication occurs very efficiently in such hosts. Tissue culture selection provides an effective pre-clinical means of studying HIV mutagenesis, especially since the same resistance-conferring mutations that arise in cell culture also appear clinically. Owing to the high turnover and mutation rate of HIV-1, the retroviral quasispecies will also include defective virions and singly mutated DR variants that are present prior to commencement of therapy. Multiply mutated variants appear later, because it requires time to accumulate multiple mutations within a single viral genome, and are not commonly found in the retroviral pool of untreated patients. An exception to this involves cases of new infection with DR viruses transmitted from extensively treated individuals. Patients with advanced infection have a higher viral load and a broader range of quasispecies than newly-infected individuals. Such patients are often immunosuppressed and may also have diminished ability to immunologically control viral replication, possibly leading to more rapid development of drug resistance. Site-directed mutagenesis has shown that a variety of RT mutations encode HIV resistance to both NRTIs and NNRTIs. Crystallographic and biochemical data have demonstrated that mutations conferring resistance to NNRTIs are found in the peptide residues that make contact with these compounds within their binding pocket (34, 35). Resistance-encoding mutations to NRTIs are found in different regions of the RT enzyme, probably due to the complexity of nucleoside incorporation, which involves


several distinct steps. These mutations can decrease RT susceptibility to nucleoside analogs. A summary of primary RT mutations has been published elsewhere (42). It has also been shown that a family of insertion and deletion mutations between codons 67 and 70 can cause resistance to a variety of NRTIs including ZDV, 3TC, ddI, ddC, and d4T. Usually, these insertion mutations confer multidrug resistance (MDR) when present in a ZDV-resistant background. Another less frequently observed resistance mutation, K65R, has been shown to be associated with prior treatment with ABC-containing regimens and results in reduced antiviral susceptibility to both ABC and the acyclic RT nucleotide analog tenofovir (TDF). Hence, resistance to these ARV agents can develop via genetic pathways involving either the TAMs or K65R as hallmark drug resistance mutations (45). In recent years, the proportion of genotyped clinical samples containing K65R has increased from less than 1 to almost 4%, reflecting the increased use of TDF in treatment regimens. Diminished sensitivity to NNRTIs appears quickly both in culture selection protocols and in patients (34, 37). NNRTIs share a common binding site, and mutations that encode NNRTI resistance are located within the binding pocket that makes drug contact (34, 35, 37, 44, 46–49). This explains the finding that extensive cross-resistance is observed among all currently approved NNRTIs (49, 50). A substitution at codon 181 (tyrosine to cysteine; Y181C) is a common mutation that encodes cross-resistance among many NNRTIs (46, 51, 52). Replacement of Y181 by a serine or histidine also conferred HIV resistance to NNRTIs (53). A mutation at amino acid 236 (proline to leucine; P236L), conferring resistance to a particular class of NNRTIs that include delavirdine, can also diminish resistance to nevirapine and other NNRTIs, particularly if a Y181C mutation is also present in the same virus (54). Other important substitutions are Y188C and Y188H that can also confer NNRTI resistance. Another drug resistance mutation, namely K103N (lysine to asparagine), is commonly observed and is responsible for reduced susceptibility to all approved NNRTIs (46, 51, 52). Substitution of K103N results in alteration of interactions between NNRTIs and RT. The K103N mutation shows synergy with Y181C in regard to resistance to NNRTIs, unlike antagonistic interactions involving Y181C and P236L (55). Resistance to NNRTIs is also observed in cell-free enzyme assays (51, 53, 56–58). Both Y181I and Y188L mediate decreased sensitivity to NNRTIs without affecting either substrate recognition or catalytic efficiency, supporting the idea that resistance to NNRTIs is attributable to diminished ability of these drugs to be bound by RT.

34.4

PR Inhibitors

DR viruses have been observed in the case of all PR inhibitors (PIs) developed to date (11, 12, 59). In addition, some strains of HIV have displayed cross-resistance to a variety of


PIs after either clinical use or in vitro drug exposure (11, 12, 59). In general, the patterns of mutations observed with PIs are more complex than those observed with RT antagonists. First, a greater number of mutations within the PR gene are involved. This involves greater variability, as well, in temporal patterns of appearance of different mutations and the manner in which different combinations of mutations can give rise to phenotypic resistance. These data suggest that the PR enzyme can adapt more easily than RT to pressures exerted by antiviral drugs. At least 40 mutations in PR have been identified as responsible for resistance to PIs (11, 12, 59). Certain of the mutations within the PR gene are more important than others and can confer resistance, virtually on their own, to at least certain PIs (11, 12, 59). One mutation, in particular, D30N, is probably unique to nelfinavir, a potent HIV PR inhibitor. However, a variety of other mutations may confer cross-resistance among multiple drugs within the PI family. In addition, wide arrays of secondary mutations have been observed, that, when combined with primary mutations, can cause increased levels of resistance to occur. On the other hand, the presence of certain of these secondary mutations on their own may not lead to drug resistance, and, in this context, some of these amino acid changes should be considered to represent naturally-occurring polymorphisms. In addition, it should be noted that resistance to PIs can also result from mutations within the substrates of the PR enzyme, i.e. the gag and gag-pol precursor proteins of HIV. A variety of studies have now shown that mutations at cleavage sites within these substrates can be responsible for drug resistance, both in tissue culture as well as in treated patients. However, the full clinical significance of cleavage site mutations in regard to PR resistance is not yet understood.

34.5 ARV Drug Resistance in Non-B Subtypes of HIV-1 Group M Genotypic divergence of pol gene sequences between different HIV-1 subtypes is only beginning to be investigated, although the RT and PR enzymes are the main targets of anti-retroviral therapy (60–64). Group O and HIV-2 viruses carry natural polymorphisms Y181C and Y181I that confer intrinsic resistance to NNRTIs (65–67). Subtype F isolates, showing 11% nucleotide sequence variation from subtype B and group M viruses, have also been reported to have reduced sensitivity to some NNRTIs while retaining susceptibility to others such as nevirapine and delavirdine, NRTIs and PIs (68, 69). In contrast, the drug sensitivity of subtype C isolates from treatment-naive patients in Zimbabwe was reported to be similar to that of subtype B isolates (69, 70). Recent studies conducted with Ethiopian subtype C clinical isolates showed natural resistance to NNRTIs in one case and resistance to ZDV in another, due to natural polymorphisms at positions G190A and K70R, respectively (71). Another study reported no differences in drug susceptibility among subtypes A, B,

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C, and E; subtype D viruses showed reduced susceptibility due to rapid growth kinetics (72). High prevalence (i.e. 94%) of a valine polymorphism (GTG) at position 106 in RT from subtype C HIV-1 clinical isolates has also been reported (73). In tissue culture experiments, selection of subtype C with efavirenz (EFV) was associated with development of high-level (i.e. 100-1000 fold) phenotypic resistance to all NNRTIs. This was a consequence of a V106M mutation that arose in place of the V106A substitution that is more commonly seen with subtype B viruses (73). This V106M mutation conferred broad cross-resistance to all currently approved NNRTIs and was selected on the basis of differential codon usage at position 106 in RT, due to redundancy in the genetic code. Genotypic diversity and drug resistance may be particularly relevant in establishing treatment strategies against African and Asian strains. First, since many antiviral drugs have been designed based on sequences of subtype B RT and PR enzymes, and drug resistance profiles, if not responses, may be different for non-B viral strains. Second, drug resistance may develop more rapidly in resource-poor countries if only sub-optimal therapeutic regimens are available. Global phenotypic and genotypic screening of non-B subtypes is warranted so as not to jeopardize the outcome of recently introduced ARV treatments (74).

34.6 Transmission of HIV Drug-Resistance As stated, HAART, including drugs that inhibit the RT and PR enzymes of HIV-1, has resulted in declining morbidity and mortality (75). The failure to completely suppress viral replication allows for the development of genotypic changes in HIV-1 that confer resistance to each of the three major classes of ARV drugs (76–78). Cumulative data indicate that single DR variants can be transmitted to approximately 10 to 15% of newly infected persons in western countries in which ARVs have been available for many years, with transmission of dual and triple-class MDR observed in 3–5% of cases (79–82). There is concern that the transmission of MDR viruses in primary HIV-1 infection (PHI) may limit future therapeutic options. Treatment failure has been observed in several individuals harboring MDR infections (82–84). Some reports have shown an impaired fitness of transmitted MDR variants compared with WT infections acquired in PHI (85), and the mutations that were transmitted in such patients persisted in the absence of treatment (85). This persistence differs from the rapid outgrowth of WT viruses in established infections upon treatment interruption, due to the selective growth advantage and fitness of WT variants (85–87). Taken together, these findings suggest that archival WT viruses may not exist in MDR infections transmitted during PHI. Several reports have also documented cases of intersubtype superinfection (A/E and B) in recently infected (RI) intravenous drug users (IDUs; refs. 88 and 89). Other

314

studies have failed to confirm superinfection following intravenous drug exposure, suggesting that superinfection is a relatively rare event (90, 91). Several subsequent reports demonstrated superinfection in subtype B infections. In one case, a WT superinfection arose following a primary MDR infection (92, 93). It is important to assess the virological consequences of transmission of DR variants in primary infection, as well as the time to disappearance of resistant virus in those patients not initially treated. Genotypic analysis indicates that a single dominant HIV-1 species can persist for more than 2 years in circulating plasma and peripheral blood mononuclear cells, regardless of route of transmission. Resistant and MDR infections can persist for 2 to 7 years following PHI. Superinfection with a second MDR strain in a patient originally infected with a MDR strain from an identified source partner has also been described (85). Despite a rapid decline in plasma viremia suggestive of an effective immune response, this patient was susceptible to a second infection which occurred concomitant with a dramatic rise in viral load. Five other subtype B superinfections have been described, as well as three intersubtype A/E and B superinfections (85, 88, 89, 92–95). Six of the seven superinfections described have occurred in the first year following initial infection. Many have attributed superinfection to co-infection during primary infection. Two longitudinal studies involving IDU populations (n = 37 in both studies) indicated that superinfection is a rare phenomenon that was not observed during 1 to 12 years of follow-up spanning 215 and 1072 total years of exposure (92, 93). However, it is not known whether any patients were recruited within the first year of HIV-1 exposure in these studies. In the case of the MDR infections cited previously, identification of the source partner of infection argues against co-infection (95). Findings of HIV-1 superinfection are a matter of concern insofar as such results challenge the assumption that immune responses can protect against re-infection. Of course, the impaired viral fitness of the initial MDR infection described above may be a factor in permitting superinfection. The initial MDR strain showed a 13-fold impaired replicative capacity from a WT variant strain from the isolated source partner following a treatment interruption. Fitness considerations may also have been important in a WT superinfection of an initial MDR infection and cases of subtype B superinfection following A/E infections that elicited low-level viremia (88, 89). In newly infected individuals, multi-mutated viruses conferring MDR may represent a new determinant of virological outcome. Persistence of MDR in the absence of treatment raises serious issues regarding HIV-1 management. For RI MDR patients, drug resistance analysis and viral fitness may provide useful information in regard to ultimate therapeutic strategies.


It is interesting to note that the presence of the M184V mutation in RT, associated with high-level resistance to 3TC, seems to have been associated with the persistence of low viral load. In two PHI cases, rebounds to a high level of plasma viremia occurred only at times when the M184V mutation in RT could no longer be detected. A third PHI patient maintained low plasma viremia over 5 years, and his virus also contained the M184V mutation throughout this time. In an additional individual, high viral loads were present at times after primary infection in spite of the M184V mutation, but virus could only be isolated from this individual in coculture experiments after loss of the M184V mutation (85). These data are consistent with previous findings on loss of fitness conferred by the M184V mutation in RT, alongside multiple other pleiotropic effects, including diminished processivity, diminished rates of nucleotide excision, and diminished rates of initiation of reverse transcription (96–98). Other studies suggest that despite reduced ARV susceptibility, MDR infections may be of some immunological and virological benefit due to the impaired replicative capacity of MDR variants (86, 99–101). Moreover, in all cases, RT assays and competitive fitness assays showed MDR viruses to have compromised replicative capacity. The absence of genotypic changes in these viruses over time further supports the concept of expansion of predominant MDR quasispecies during primary infection. Recombination events can also occur in this period. It is also important to point out that the replication fitness of a given virus vs its transmission fitness may represent two very different concepts. ART, by reducing HIV-1 replication, has been shown not only to impact significantly on morbidity and mortality but also to reduce the spread of HIV-1 (102, 103). Treatment effectiveness is hampered by the development of DR strains, leading inexorably to virological failure (76). The transmissibility of DR strains is not fully understood and may differ from that of WT strains for at least two reasons: first, the relative fitness of DR strains compared with WT in the absence of therapy and second, the degree to which partially active therapies can reduce viral load in persons harbouring resistant viruses (80, 104). As a consequence of widespread use of ART in North America, the transmission of DR strains in RI individuals has increased from 3.8% in 1996 to 14% in 2000. Such an increase of primary DR is of public health concern since a clear association between DR and early treatment failure has been reported (82). However, several groups in Europe and Australia have reported a recent stable or decreasing trend in DR transmission for RT and/or PIs (105, 106), and have attributed this decline to the widespread use of suppressive triple ARV regimens since 1996. This presupposes that transmission of DR variants may have earlier been more common due to the widespread use of suboptimal biotherapy or even monotherapy regimes prior to 1996 and the likelihood that these suboptimal regimens may have selected for drug resistance mutations with very high frequency (106).


34.7

Conclusion

The accumulation of specific resistance-conferring mutations is associated with the development of phenotypic resistance to anti-HIV drugs which can significantly diminish the effectiveness and longevity of ART. Cross-resistance among drugs of the same class also occurs frequently and is most problematic with NNRTIs due to their lower genetic barrier for rapid selection of drug resistance compared to other classes of ARVs. There is now also data indicating that cross-resistance amongst the NRTIs may in fact be more widespread than was initially thought (107). Furthermore, the emergence of new drug resistance mutations is helping to establish new mutant distributions with additional pathways for developing crossresistance to ARVs (108). These new patterns of cross-resistance together with increasing transmission of MDR HIV-1 variants are problematic and seriously limit the number of effective treatment options that are now available for longterm management of HIV-infection. Additional strategies, in addition to new drug discovery programs, are urgently required to help curb the development of DR HIV-1. One possible approach that merits further consideration is based on the maintenance of specific fitnessattenuating drug mutations in therapeutic regimens for HIV1 infection (108, 109). The M184V substitution in RT has been extensively studied in this regard because of its ability to impair viral replication capacity while limiting the development of subsequent drug resistance mutations in HIV-1 RT, e.g. TAMs and the Q151M multi-drug complex resistance mutation associated with use of AZT and d4T (110, 111). Of course, restricted evolution of drug resistance in these circumstances may also result from other alterations of RT function by M184V (96). One recent study has shown that viruses containing the M184V mutation may be transmitted less frequently than viruses containing other mutations associated with drug resistance (98), perhaps because M184V compromises viral replicative capacity. Further work on these and other topics is needed to improve our understanding of HIV drug resistance in the context of clinical relevance, successful antiviral chemotherapy, and likelihood of transmission of resistant strains (112).

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317 71. Loemba H, Brenner B, Parniak MA, et al (2002) Genetic divergence of human immunodeficiency virus type 1 Ethiopian clade C reverse transcriptase (RT) and rapid development of resistance against nonnucleoside inhibitors of RT. Antimicrob Agents Chemother 46:2087–2094. 72. Palmer S, Alaeus A, Albert J, Cox S (1998) Drug susceptibility of subtypes A, B,C,D, and E human immunodeficiency virus type 1 primary isolates. AIDS Res Hum Retroviruses 14:157–162. 73. Brenner B, Turner D, Oliveira M, et al (2003) A V106M mutation in HIV-1 clade C viruses exposed to efavirenz confers crossresistance to non-nucleoside reverse transcriptase inhibitors. AIDS 17:F1–F5. 74. Petrella M, Brenner B, Loemba H, Wainberg MA (2001) HIV drug resistance and implications for the introduction of antiretroviral therapy in resource-poor countries. Drug Resist Updat 4:339–346. 75. Palella FJ Jr, Delaney KM, Moorman AC, et al (1998) Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 338:853–860. 76. Wainberg MA, Friedland G (1998) Public health implications of antiretroviral therapy and HIV drug resistance. JAMA 279:1977–1983. 77. Hirsch MS, Brun-Vezinet F, Clotet B, et al (2003) Antiretroviral drug resistance testing in adults infected with human immunodeficiency virus type 1: 2003 recommendations of an International AIDS Society-USA Panel. Clin Infect Dis 37:113–128. 78. D’ Aquila RT, Schapiro JM, Brun-Vezinet F, et al (2003) Drug resistance mutations in HIV-1. Top HIV Med 11:92–96. 79. Salomon H, Wainberg MA, Brenner B, et al (2000) Prevalence of HIV-1 resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or injecting drug use. Investigators of the Quebec Primary Infection Study. AIDS 14:F17–F23. 80. Yerly S, Kaiser L, Race E, Bru JP, Clavel F, Perrin L (1999) Transmission of antiretroviral-drug-resistant HIV-1 variants. Lancet 354:729–733. 81. Boden D, Hurley A, Zhang L, et al (1999) HIV-1 drug resistance in newly infected individuals. JAMA 282:1135–1141. 82. Little SJ, Holte S, Routy JP, et al (2002) Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med 347:385–394. 83. Hecht FM, Grant RM, Petropoulos CJ, et al (1998) Sexual transmission of an HIV-1 variant resistant to multiple reverse-transcriptase and protease inhibitors. N Engl J Med 339:307–311. 84. Gandhi RT, Wurcel A, Rosenberg ES, et al (2003) Progressive reversion of human immunodeficiency virus type 1 resistance mutations in vivo after transmission of a multiply drug-resistant virus. Clin Infect Dis 37:1693–1698. 85. Brenner BG, Routy JP, Petrella M, et al (2002) Persistence and fitness of multidrug-resistant human immunodeficiency virus type 1 acquired in primary infection. J Virol 76:1753–1761. 86. Verhofstede C, Wanzeele FV, Van Der Gucht B, De Cabooter N, Plum J (1999) Interruption of reverse transcriptase inhibitors or a switch from reverse transcriptase to protease inhibitors resulted in a fast reappearance of virus strains with a reverse transcriptase inhibitor-sensitive genotype. AIDS 13:2541–2546. 87. Devereux HL, Youle M, Johnson MA, Loveday C (1999) Rapid decline in detectability of HIV-1 drug resistance mutations after stopping therapy. AIDS 13:F123–F127.

318 88. Jost S, Bernard M-C, Kaiser L, et al (2002) A Patient with HIV-1 superinfection. N Engl J Med 347:731–736. 89. Ramos A, Hu DJ, Nguyen L, et al (2002) Intersubtype human immunodeficiency virus type 1 superinfection following seroconversion to primary infection in two injection drug users. J Virol 76:7444–7452. 90. Gonzales MJ, Delwart E, Rhee SY, et al (2003) Lack of detectable human immunodeficiency virus type 1 superinfection during 1072 person-years of observation. J Infect Dis 188:397–405. 91. Tsui R, Herring BL, Barbour JD, et al (2004) Human immunodeficiency virus type 1 superinfection was not detected following 215 years of injection drug user exposure. J Virol 78:94–103. 92. Altfeld M, Allen TM, Yu XG, et al (2002) HIV-1 superinfection despite broad CD8+ T-cell responses containing replication of the primary virus. Nature 420:434–439. 93. Koelsch KK, Smith DM, Little SJ, et al (2003) Clade B HIV1 superinfection with wild-type virus after primary infection with drug-resistant clade B virus. AIDS 17:F11–F16. 94. Smith DM, Wong JK, Hightower GK, et al (2004) Incidence of HIV superinfection following primary infection. JAMA 292:1177–1178. 95. Allen TM, Altfeld M (2003) HIV-1 superinfection. J Allergy Clin Immunol 112:829. 96. Petrella M, Wainberg MA (2002) Might the M184V substitution in HIV-1 RT confer clinical benefit. AIDS Rev 4:224–232. 97. Turner D, Brenner B, Routy JP, et al (2004) Diminished representation of HIV-1 variants containing select drug resistanceconferring mutations in primary HIV-1 infection. J Acquir Immune Defic Syndr 37:1627–1631. 98. Wainberg MA, Hsu M, Gu Z, Borkow G, Parniak MA (1996) Effectiveness of 3TC in HIV clinical trials may be due in part to the M184V substitution in 3TC-resistant HIV-1 reverse transcriptase. AIDS 10:S3–S10. 99. Baxter JD, Mayers DL, Wentworth DN, et al (2000) A randomized study of antiretroviral management based on plasma genotypic antiretroviral resistance testing in patients failing therapy. CPCRA 046 Study Team for the Terry Beirn Community Programs for Clinical Research on AIDS. AIDS 14:F83–F93. 100. Colgrove RC, Pitt J, Chung PH, Welles SL, Japour AJ (1998) Selective vertical transmission of HIV-1 antiretroviral resistance mutations. AIDS 12:2281–2288.

M. A. Wainberg and S. Schader 101. Dickover RE, Garratty EM, Plaeger S, Bryson YJ (2001) Perinatal transmissionmajor, of minor, and multiple maternal human immunodeficiency virus type 1 variants in utero and intrapartum. J Virol 75:2194–2203. 102. Quinn TC, Wawer MJ, Sewankambo N, et al (2000) Viral load and heterosexual transmission of human immunodeficiency virus type 1. Rakai Project Study Group. N Engl J Med 342:921–929. 103. Yerly S, Vora S, Rizzardi P, et al (2001) Acute HIV infection: impact on the spread of HIV and transmission of drug resistance. AIDS 15:2287–2292. 104. Phillips A (2001) Will the drugs still work? Transmission of resistant HIV. Nat Med 7:993–994. 105. Chaix ML, Descamps D, Harzic M, et al (2003) Stable prevalence of genotypic drug resistance mutations but increase in non-B virus among patients with primary HIV-1 infection in France. AIDS 17:2635–2643. 106. Ammaranond P, Cunningham P, Oelrichs R, et al (2003) No increase in protease resistance and a decrease in reverse transcriptase resistance mutations in primary HIV-1 infection: 1992–2001.AIDS 17:264–267. 107. Kuritzkes D (2002) Drug resistance. Navigating resistance pathways. AIDS Read 12:395–400, 407. 108. Nijhuis M, Deeks S, Boucher C (2001) Implications of antiretroviral resistance on viral fitness. Curr Opin Infect Dis 14:23–28. 109. Brenner BG, Turner D, Wainberg MA (2002) HIV-1 drug resistance: can we overcome. Expert Opin Biol Ther 2:751–761. 110. Ait-Khaled M, Rakik A, Griffin P, et al (2002) Mutations in HIV-1 reverse transcriptase during therapy with abacavir, lamivudine and zidovudine in HIV-1-infected adults with no prior antiretroviral therapy. Antivir Ther 7:43–51. 111. Ait-Khaled M, Stone C, Amphlett G, et al (2002) M184V is associated with a low incidence of thymidine analogue mutations and low phenotypic resistance to zidovudine and stavudine. AIDS 16:1686–1689. 112. Daar ES, Richman DD (2005) Confronting the emergence of drug-resistant HIV type 1: impact of antiretroviral therapy on individual and population resistance. AIDS Res Hum Retroviruses 21:343–357.

Chapter 36 Epidemiological Surveillance of HIV and AIDS in Lithuania Saulius Caplinskas

36.1

Introduction

Epidemiological surveillance of HIV and AIDS is regulated by Law on Prevention and Control of Human Communicable Diseases of the Republic of Lithuania and its modifications (1). The Law provides for basics on management of human communicable diseases prevention and control, controversy elimination and harm restitution, responsibility of violation of judicial acts on issues of communicable disease prevention and control; rights and duties of natural person’s and body’s in the field of communicable disease prevention and control; specialities of budgeting of communicable diseases prevention and control, and compensation of their costs. In September 1999, the government of the Republic of Lithuania established the state registries of communicable diseases and their agents (1, 2) and the functions of chief manager were delegated to the Center of Communicable Diseases Prevention and Control. All cases of communicable diseases are reported to the State Registries of communicable diseases and communicable diseases agents. Personal healthcare institutions report to the territorial public health institutions on all individual cases of HIV and AIDS, and summarized data from the public health institutions are submitted to the registry. The Government of the Republic of Lithuania, the Ministry of Health and subordinated institutions are in charge of the management of communicable disease prevention and control. District governors are in charge of the management of communicable diseases prevention and control on district level according to legal regulations, and the mayors on the municipal level according to their competence. Governmental supervision of implementation of the communicable diseases prevention and control is delegated to the Ministry of Health, and institutions subordinated to the Ministry of Health, the Chief Epidemiologist of the Republic of Lithuania, district doctors and district From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

chief epidemiologists; municipal doctors are in charge of program implementation. The functions of government services, chief epidemiologists, district and municipal doctors in the field of prevention and control of communicable diseases management and supervision of the program implementation are regulated by present law and other legal regulations (Figure 36.1). The Lithuanian AIDS Center is in charge of the epidemiological surveillance of STI, HIV, AIDS on national level, evaluation of epidemiological situation, prognosis, conclusions, proposals, methodical government of personal and territorial public health case institutions on issues of epidemiological STI, HIV, and AIDS surveillance.

36.2

HIV Epidemiological Situation

Although Lithuania, with a mainly Catholic population of around 3.4 million (3), still appears to be a low prevalence country for HIV, it is situated in a sub-region bordered by neighbors worse affected than most European countries. Notably, one close neighbor, Estonia, had a similarly low prevalence state of HIV for many years, until the turn of the millennium, when suddenly it found itself to have the second highest prevalence of HIV infection in Europe due to an outbreak that spread rapidly in their intravenous drug users (IDUs) (3). Similarly worryingly high rates are found in Latvia, Belarus, Ukraine, Kaliningrad, and the rest of the Russian Federation.

36.3

General Overview

The first HIV-positive person in Lithuania was reported in 1988. From January, 1988 to January 1, 2006 a cumulative total of 1100 (29.41 per 100,000 population; refs. 4 and 5) HIV infections were registered in Lithuania, of these 96 have developed AIDS and 99 have died. However, the actual number of HIV-infected people may be higher than this. For example, international agencies estimated that there were 3,300 people 327

328

S. Caplinskas

Ministry of Health Governmental Public Health Care Service under the Ministry of Health

District administraion

Health Information Centre

EuroHIV

Statistical data

Centre of Communicable Diseases Prevention and Control Analysis and prognosis of epidemiological situation

Centre of Extremal Health Situation

Lithuanian AIDS Centre (Dept. .of Epidemiological Surveillance)

Laboratory of the Lithuanian

Test result Territorial Public Health Care Institutions

Personal Health Care Institutions Lithuanian AIDS Centre

Figure 36.1. Data flows.

Figure 36.2. HIV/AIDS registered per year cases in Lithuania (January 1, 2006) (See Color Plates).

living with HIV in Lithuania at the end of 2005 (Figure 36.2; ref. 6). HIV cases were identified in the majority of the Lithuanian districts. The highest prevalence is reported in Klaipeda (on the Baltic Sea): 28.9% of all HIV cases in the country (Figure 36.3). By the end 2005 the highest HIV prevalence in 100,000 population was reported in Klaipeda: 154.28/10000, the

capital Vilnius ranked only sixth by HIV prevalence with 24.93 cases per 100,000. There were 49 HIV-positive foreigners registered, including 14 Russian citizens, 16 Latvians, four from Belarus, two from Estonia and one HIV case from France, Denmark, Spain, Poland, Ukraine, Uzbekistan, Thailand, Vietnam; and five unknown cases (7).

36. Epidemiological Surveillance of HIV and AIDS in Lithuania 35

HIV prevalence

329

HIV incidence

30

26,13

22,78

25

29,4

19,6

20 15

11,44

9,72

10 5

1,45

0

0,33

1996

3,82 2,33 1,47 0,87 1997

1998

7,63

5,72

1999

2000

3,93

3,18

2,07

1,86

1,87

3,51

2001

2002

2003

2004

2005

Figure 36.3. HIV prevalence and incidence per 100,000 population (1996–2004) in Lithuania (See Color Plates).

450 400 350 300 250

299

200

26

34

2 12 10 80

4 5 15 4 55

31

37

20 04

15 12 2 3 45

20 02

19

20 01

6 3 4 4 7 12 22 29 29 19

99 20 00

98

97

19

96

92 19 93

9

3 17 1 8 5 5 15 2417 23

1 11 7 12

1 10

11

2

19

19

1 4

19

1

19 95

1 7

19 94

1

19 91

19 89

19 88

0 1

19 90

50

11 4 10 49

20 03

100

20 05

150

HIV outbreak in Alytus Corrective Institution Lithuanian AIDS center(outpatien depatment) detoxication, rehabilitation and methadone needle exchange cabinet (include AIDS centre needle exchange) Pre-Trial Wards health care institucion

Figure 36.4. HIV infection cases by place of diagnosis. Source: Lithuanian AIDS Center, 2006 (See Color Plates).

seksual

Intravenous drug use

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

1994

1993

1992

1991

1990

1989

1988

100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

unknow

Figure 36.5. HIV mode of transmission by year in Lithuania (%) (See Color Plates).

The HIV epidemic in Lithuania appears to have passed through two phases. The first phase runs from the mid-1980s to the year 2002 (7). This was a quiet phase with only a few endemic infections, mainly due to contacts abroad (8). The second phase started when the official statistics jumped from 328 registered HIV cases at the end of 2001 to 845 HIV cases at the end of 2003. This was due to an outbreak of HIV infection

transmitted through the use of contaminated needles used in the Alytus Correctional Facility (CF; Figure 36.4). Until 1997, these infections were transmitted either heterosexually or homosexually: in 1989–1993 the virus spread among men having sex with men (MSM); in 1993–1996 heterosexual HIV transmission prevailed, especially in seafarers who have been infected in African countries. Starting from 1997, HIV

330

S. Caplinskas 100%

50%

heteroseksual

homoseksual

Intravenous drug use

2005

2004

2003

2002

2001

2000

1999

1998

1997

1996

1995

1994

1993

1992

1991

1990

1989

1988

0%

unknow

Figure 36.6. Tendencies HIV of transmission mode in Lithuania (See Color Plates).

has been mainly spread through blood in the IDU population. The annual share of HIV infection acquired through injection equipment reached 70–77%, except of 2002—the year of an HIV outbreak in Alytus CF. In 2002, the mode of HIV transmission through drug injection bounced up to 95% (9). Correspondingly, the proportion of new diagnosed heterosexual case has steadily increased from 9.1% in 2001 to 17.7% in 2004 and to 16.6% in 2005 (Figure 36.5). The number and proportion of new cases reported among MSM remains low (2004: four cases; 2005: three cases) and stable (2.5–2.9%; Figure 36.6). Until 2006, in Lithuania IDUs clearly dominate: 866 (78.7%) people were infected via illicit drug injection, 114 (10.4%) heterosexually, 70 (6.4%) homosexually; the transmission route remains unknown in 50 cases (10). HIV has been mostly reported in the 25–29 age group (25.8%) and 30–34 age group (18.4%), while 76.8% of the total cases were identified in the 20–39 age group, which is continually replenished with newly infected IDUs (Table 36.1). The youngest patient in Lithuania was 15 years old and the oldest was 68 years old at the time of the HIV diagnosis. Average age according to the mode of transmission also differs; 37 years of age for those who have been infected via sexual intercourse and 30 years of age for

Table 36.1. HIV infection cases by age in Lithuania, December 31, 2005 Age

Males

Females

IDU

Total

15–19

41

9

42

50

20–24

159

31

166

190

25–29

254

30

240

284

30–39

327

44

307

371

40–49

128

12

88

140

50–59

32

3

10

35

60 and older

9

3

—

12

Unknown

16

2

13

18

infection through contaminated drug injecting equipment (Table 36.1). The annual rate of female HIV cases has continuously increased, thus leading to a decrease in the male/female ratio: in 2002 it was 12/1; in 2003, 7/1; in 2004, 5/1; and in 2005, 3/1 (Figure 36.7) (11). The majority of the reported females are at their reproductive age, at average 31 years. The majority of them were infected through intravenous drug use (67.9%). The number of female HIV cases occurring heterosexually has annually increased. This fact is seriously increasing the threat of further HIV spread in general population. So far, there are no cases reported of mother-to-child HIV transmission, nor HIV infection in children younger than 15 years of age. Until 2006, a total of 11 pregnant women with HIV infection have been reported. Based on 100,000 population, the prevalence of HIV infection has been on a steady increase. Thus, HIV prevalence in 1996 was 1.45 for 100,000 of population (Table 36.2) and 29.4 in 2005 (which is the lowest in the Baltic Sea region). The HIV incidence has also gradually increased. In 1996, the HIV incidence for 100,000 population was 0.33, and in 2005 it was 3.51 (Table 36.3) (ref. 10, 12). A primary study of the HIV-1 molecular typification in Lithuania proved as predominating the subtype B virus. Thus, HIV-1 subtype B, which is the most prevalent in Western Europe, was also the most common subtype in all three Baltic countries (Lithuania, Latvia, and Estonia) and Russia, and was linked to homosexual transmission. However, after the HIV outbreak in the Alytus CF in 2002, the HIV-1 subtype A has been prevailing (13).

36.4

AIDS Cases

The first case of AIDS diagnosed in Lithuania was a man in 1988. In 1999, 11 years later, the first woman in Lithuania was diagnosed with AIDS. The overall AIDS incidence rate

36. Epidemiological Surveillance of HIV and AIDS in Lithuania

331

450

moteris

400

vyrai

350 300 250 200 150 100 50 0 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Figure 36.7. Dynamics of HIV spread by gender (See Color Plates).

Table 36.2. HIV prevalence rate in Lithuania and neighbor states (per 100,000 population) 1985

1986

1987

1988

1989

1990

1991

1992

1993

Estonia

0

Latvia

0

Lithuania

0

0

0.06

0.19

0.51

0.51

0.58

0

0.04

0.04

0

0.22

0.11

0.04

0

0

0

0.03

0.03

0.21

0.03

Belarus

0

0

0.2

0.11

0.12

0.14

Poland

0.03

0.02

0.08

0.16

1.36

0

0

0.02

0.03

0.18

Russian Federation

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

0.33

0.8

0.74

0.55

0.19

0.27

0.28

1.29

0.62

0.7

0.85

27.99

107.07

66.07

63.3

56.7

46.02

1.01

6.61

9.94

19.25

33.54

22.66

17.5

14.1

0.13

0.11

0.24

0.3

12.93

0.32

0.84

1.4

1.78

1.76

1.95

10.87

3.18

3.93

0.12

0.2

0.1

0.05

3.51

0.08

9.9

6.35

5.4

4.02

5.17

5.7

9.05

7.08

7.91

7.76

2.12

1.46

1.25

1

0.07

0.06

0.06

0.07

1.1

1.4

1.43

1.5

1.65

1.36

1.63

1.46

1.49

1.58

1.72

1.7

0.11

0.14

1.03

2.96

2.76

13.64

40.66

61.01

35.15

27.64

23.62

—

Table 36.3. HIV incidence rate in Lithuania and neighbor states (per 100,000 population) 1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

1995

1996

1997

1998

1999

2000

2001

2002

2003

2004

2005

Estonia

0

0

0

0.06

0.19

0.51

0.51

0.58

0.33

0.8

0.74

0.55

0.62

0.7

0.85

27.99

107.07

66.07

63.3

56.7

46.02

Latvia

0

0

0.04

0.04

0

0.22

0.11

0.04

0.19

0.27

0.28

1.29

1.01

6.61

9.94

19.25

33.54

22.66

17.5

14.1

12.93

Lithuania

0

0

0

0.03

0.03

0.21

0.03

0.13

0.11

0.24

0.3

0.32

0.84

1.4

1.78

1.76

1.95

10.87

3.18

3.93

3.51

Belarus

0

0

0.2

0.11

0.12

0.14

0.12

0.2

0.1

0.05

0.08

9.9

6.35

5.4

4.02

5.17

5.7

9.05

7.08

7.91

7.76

Poland

0.03

0.02

0.08

0.16

1.36

2.12

1.46

1.25

1

1.1

1.4

1.43

1.5

1.65

1.36

1.63

1.46

1.49

1.58

1.72

1.7

0

0

0.02

0.03

0.18

0.07

0.06

0.06

0.07

0.11

0.14

1.03

2.96

2.76

13.64

40.66

61.01

35.15

27.64

23.62

—

2001

2002

2003

2004

2005

Russian Federation

Table 36.4. AIDS incidence rate in Lithuania and neighbor states (per 100,000 population) 1985

1986

1987

1988

1989

1990

1991

1992

1993

1994

Estonia

0

0

0

0

0

Latvia

0

0

0

0

0

Lithuania

0

0

0

0.03

0

0.03

0.03

Belarus

0

0

0

0

0

0

0.04

Poland

0

0.002

0.01

0.01

0.07

0.06

0.12

Russian Federation

0

0

0.002

0.003

0.02

0.03

0.02

1995

1996

0

0

0.13

0.07

0.07

0.04

0.04

0.12

0.03 00.04 0.09 0.02

0.07

0.2

0.48

0.21

0.28

0.14

0.22

0.15

0.37

0.74

1.92

2.22

0.08

0.12

0.2

0.16

0.49

0.74

0.95

1.66

2.3

2.44

3.33

3.11

0

0.05

0.03

0.13

0.08

0.24

0.16

0.19

0.27

0.26

0.26

0.61

0.29

0.02

0.02

0.03

0

0.02

0.04

0.05

0

0.06

0.21

0.37

0.85

1.38

0.18

0.26

0.3

0.29

0.32

0.33

0.35

0.31

0.34

0.32

0.33

0.44

0.49

0.01

0.02

0.03

0.03

0.05

0.04

0.03

0.01

-

-

-

-

-

has been increasing slowly during the past 12 years (from 0.03 in 1990 to 0.29 in 2005), but still remains quite low in comparison to the rates in the neighboring countries (Table 36.4).

1997

1998

1999

2000

From the introduction of the HIV registry, AIDS as the advanced HIV stage was diagnosed in 96 persons (Table 36.5). There were 41 deaths of AIDS.

332

S. Caplinskas

Table 36.5. AIDS cases in Lithuania, December 31, 2005 AIDS cases Number of AIDS diagnosed Number of deaths

Males

Females

IDU

Total

84

12

17

96

9

12

54

99

From AIDS

40

1

4

41

Others reasons

47

11

50

58

Table 36.6. HIV/TB co-infection in Lithuania, December 31, 2005 1992 1993 1994 1999 2000 2001 2002 2003 2004 2005 Total HIV/TB

1

1

1

2

3

3

2

5

8

7

33

Of all registered AIDS cases, 56% are MSM, 24% AIDS cases were caused by heterosexual contacts, and only 12% might be attributed to IDU; in 8% of the AIDS cases, the transmission was unknown. In 3.8% of the cases, AIDS was diagnosed at the time of HIV/AIDS diagnosis (14).

36.5

HIV/TB Co-infection in Lithuania

The first case of active TB in person with HIV was identified in 1992. Up to 2006, the number of HIV/TB co-infection reached 33 (Table 36.6). This means that about 3% of all HIV cases are also TB-infected. The prevalence of active tuberculosis in people with HIV is 10 times higher than in the general population (4, 5).

36.6

HIV Outbreak in Alytus CF

The regulations in the Lithuanian prison system on HIV testing in the inmates provides for testing: · · · ·

in 3 months upon entry to the pre-trial house or CF 3 months prior to release from CF once a year in convicts according to epidemiological or medical indications (Figure 36.8)

Until 2002, there were no cases of HIV in prison inmates reported. With the continuous increase in the number of HIV cases in Lithuania, chances of incarcerating people with HIV also increased. In some cases prison inmates upon entry into the penitentiary system have already been included into outpatient care system of the Lithuanian AIDS Center. However, about 30 to 40% of HIV cases used to be identified only on entry to pre-trial wards. The first person with HIV was imprisoned in 1992. The mode of transmission in this case was sexual intercourse. The second person with HIV has entered the prison in 1996. The spread of HIV infection among IDUs had

Figure 36.8. HIV/AIDS in Lithuanian prison settings (See Color Plates).

a significant impact on the increase of HIV cases in penitentiaries. By the end 2001, 118 cases of HIV infection were newly identified in or already served their sentence in prison; by the end of 2002, there were 8 cases more (Figure 36.9) (15).

36.6.1 State Mental Health Center, Lithuanian AIDS Center, and Prison Department Data, 2005 In May 2002, the diagnosis of HIV infection in a person recently released from prison, necessitated a follow up study of the possible contacts of the former inmate. This study led to the discovery of the HIV outbreak in the Alytus CF (Figure 36.9) (16).

36.6.2

Prison Department Data, 2005

From May to June, 2005, 2,000 prisoners were tested for HIV infection in the Alytus CF and 207 new HIV cases were identified. During July-August of the same year, 1,813 tests were done repeatedly and 77 positive persons were identified. 17 of them were new prisoners, 60 become positive, with existing high probability to be infected after first wave of testing performed. In September-October, 1,481 HIV tests were done and only 15 HIV-positive persons were identified. The majority of the inmates with HIV infection admitted that a possible mode of their infection could be intravenous drug use at their home and needle/syringe sharing. Only 13 of those tested were revealed to have a CD4 cell count less than 500 cell/mm3. Serological markers of HCV infection were found in 252 (98%) of 257 HIV-infected persons, 22 (7%) have had HBV surface antigen. Due to the active national HIV epidemiological surveillance system, the HIV outbreak in the Alytus CF was practically identified at the very first stage of its development. The latter was confirmed by laboratory testing. The rapid spread of HIV in the Alytus CF was successfully confined due to all preventive measures taken, from administrative-organizational to technical-educational. The measures taken to fight HIV in prisons are

333

180

375

160

145,1

140

77,4

77,5

135,2 13,3%

95,3

100 80

126,3

117,2

120 83,3

156,8

14,4% 212

11,3%

20% 18% 18,1% 16% 14%

15,6%

12%

229

10% 8%

8,8%

60

6,6%

7,5%

128

6%

40

4%

20 0 1997

1998

22

32

33

1999

2000

2001

2% 0% 2002

2003

2004

2005

2006

Percent of drug users in prison settings

Dinamisc of drug and toxic substances addiction (100 000 population)


Prevalence of HIV infection in penitentiaries (abs. Nu)

Chronology of HIV and measures taken in prisons of Lithuania

2003 May - HIV/AIDS Couseling Center established in Central Prison Hospital 2003 − Local Zone cancelled 2002 − HIV outbreak in Alytus 1998 - opening of

1997 − several persons with the Local Zone HIV positive. Problem of their accomodation during their term of imprisonment 1992 − the first HIV infected person

Figure 36.9. Drug and toxic substances addiction, percentage of drug users in prison settings and prevalence of HIV infection in penitentiaries (See Color Plates).

effective at the moment. But total prevention of drug use in penal establishments is hard to achieve; therefore, the World Health Organization (WHO) has recommended that the prison system should follow strict procedures to stop the transmission of blood-borne infections, including improving the accessibility of disinfectants; establishing of drug-free zones; and providing addiction treatment. Screening in other penitentiaries in the same year did not reveal any new HIV infection cases. Although HIV testing in other correctional facilities did not reveal any new HIV infection cases among the inmates, the HIV and drug use prevention was reinforced in all Lithuanian penitentiary institutions. (Figure 36.10)

Thanks to the joint multi-sectorial actions, the outbreak in Alytus prison was stopped: in 2003 there were 15 new cases reported, two cases in 2004, and four cases in 2005 (Figure 36.11) (16).

36.7 HIV Transmission Through Sexual Contacts According to WHO estimations (2003), there might have been 7,000 to 11,000 IDUs, 17,000 to 44,000 MSM, 5,000 to 8,000 sex workers (SWs), and 11,400 (327 per 1,000,000) prisoners in Lithuania.

334

S. Caplinskas 500

Total HIV cases in Lithuania

400

HIV cases discovered in prisons

300 200 100 0 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005

Figure 36.10. Registered HIV cases in prisons and total HIV number registered in Lithuania (See Color Plates).

207

new HIV cases in Alytus prison

77 15 2002 May-June

2002 July-August 2002 September-October

15 2003

2 2004

4 2005

Figure 36.11. HIV outbreak in Alytus prison (See Color Plates).

36.8

Homosexual Transmission

The first HIV-positive cases in Lithuania were diagnosed in homosexual men’s community in 1989. It was estimated that the virus entered this community in 1980-1983. There were seven HIV-positive persons registered in 1990. These cases were disclosed mostly by partner notification. In 1989–1993, the virus spread among MSM. Until 1997, the HIV transmission has been mostly through sexual contacts: 26 HIV cases by heterosexual transmission and 20 cases by homosexual transmission (Figure 36.6). Since 1996, the number and proportion of new cases reported among MSM remained low (2004, four cases; 2005, three cases) and stable (2.5–2.9%) (17). One major way of transmitting HIV is through sexual intercourse. In this regard, the Lithuanian national program laid plans for prevention activities with the most at-risk populations (MARPs): SWs, their clients, IDUs, MSM, seafarers, long-distance truck drivers, soldiers, and police officers. It has also planned for improving the availability of good-quality condoms to these high-risk groups, and youth and general public education. It was estimated by national experts during a WHO and The Joint United Nations Programme on HIV/AIDS (UNAIDS) workshop in 2003, that there might have been from 17,000 to 44,000 MSM in the country. This is still a marginalized group

that had remained relatively underground and far from being reached by any prevention activities. It is no surprise that again very few non-government organizations (NGOs) have shown any interest in working with this MARP. Also the mass media generally give a very negative picture relevant to MSM issues. In order to provide some prevention services, the LAC established a “Target Group” Health Consulting Room to provide services for MSM, and other at-risk groups. This provides health advice and counselling to MSM and even clinical services, including testing. All the attendees were tested for Hepatitis B, syphilis, gonorrhoea, HIV, hepatitis C, and Chlamydia infection. One case of each gonorrhoea and chlamydia infection, three cases of Candida infection, and one of hepatitis C were identified in 50 consultations with a dermato-venerologist. This outlet also runs a popular website where there is some discussion of safer sex, sexually-transmitted infections (STI), and prevention. The services were expanded to include anonymous STI clinic services with anonymous testing and treatment accessible to all patients (MSM, SW, and others). In 2005, in total 4,369 visits were registered (the majority [2686] attended a dermato-venerologist office while 1,683 attended a gynaecologist office). In a survey of 90 MSM in 2003, 61 declared that they have used a condom with their last partner (68%), while an internet questionnaire resulted in 117 out of 213 saying that they have used a condom with the last sex partner (55%).


Sentinel surveillance performed by the Lithuanian AIDS Center in MSM proved the following statistics: in 2003, of 242 MSM, two were HIV-positive (0.8%); in 2004, 79; and in 2005, also 79 MSM were tested for HIV and no cases of infection were found.

36.9

Heterosexual Transmission

The first HIV cases of heterosexual HIV transmission were identified in 1988. In 1993-1996, heterosexual HIV transmission prevailed, especially in seafarers who were infected in African countries. Until 1997, the HIV transmission through sexual contacts prevailed, with 26 cases of heterosexual HIV transmission reported (Figure 36.12) (18). Since then, the proportion of newly diagnosed heterosexual cases has steadily increased from 9.1% in 2001 to 17.7% in 2004 and 16.6% in 2005. It has been estimated by national experts during a WHO and UNAIDS workshop in 2003, that there might have been about 5,000 to 8,000 SWs (both male and female) in the country. In 2005, about 120 street SWs regularly visited the AIDS Center site of low threshold services. This facility offered testing to SWs and 111 voluntarily underwent HIV, syphilis, HBV, and HCV tests. No cases of HIV were found at that time, but 15 cases of gonorrhoea, five of chlamydeous infection, five of hepatitis C (all were IDUs), one case of hepatitis B, and one case of syphilis were identified. Some SWs also agreed to answer a questionnaire on their behavior. Sixty-five percent claimed that they have used a condom with their most recent client in 2003, while in 2004, 70% said that have used a condom with their most recent client. This model should be expanded to all the major cities, by supporting the establishment of new NGOs with similar scope of activities.

36.10

SWs

Sentinel surveillance of SWs in the Women Health Site at the Lithuanian AIDS Center provided the following statistics: in 2003 a total of 89 SW were tested; in 2004, 86; in 2005, 111; and no cases of HIV have been identified (Figure 36.13).

unknown 5%

IDU 12%

homo 38%

hetero 8% homo 5%

hetero 50%

Year 1988-1996 (n = 52)

IDU 82% Year 1996-2005 (n = 1048)

Figure 36.12. Distribution of newly diagnosed HIV cases by transmission group (See Color Plates).

335

96% 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%

75% 68%

0,4% 1999

2000

2002

2004

Figure 36.13. Low threshold center for street prostitutes in LAC. STI among focus group of Vilnius street prostitutes (n = 56). The Lithuanian AIDS Center data, 2005 (See Color Plates).

Percentage of IDUs prostitutes out in Vilnius streets: in 1998, 23%; in 2001, 65%; and in 2004, 78%. Prostitution trends have changed from prostitution ® drug use into drug use ® prostitution. So far, 19 HIV-positive cases were identified in SWs (the rate of heterosexually infected women has also increased significantly).

36.11

IDU

The first HIV-positive IDU in Lithuania were diagnosed in 1994. Two years later, four HIV-positive IDUs were reported in the Klaipeda harbor. Starting from 1997, HIV has been mainly spread through blood in the IDU population. In 1997, IDUs accounted for 70% of all HIV-positive registered cases (19). The Drug Control Department is working with the European Monitoring Center for Drugs and Drug Addiction to improve the current estimates of the size of the drug problem in Lithuania. Their main priorities are to prevent addiction, especially among children and young adults, as well as to: institute policies to decrease supply; provide healthcare; rehabilitation and social rehabilitation; and establish a strong IT database. Recent surveys have demonstrated that 80% of the 5,371 registered drug users use opiates, with most of these drug users using poppy straw, and 402 are on methadone. The estimated real number of IDUs is more than 7,000 IDU. However, additional funding is still needed to carry out a more scientific study of the true extent of the problem. A main area of concern is promoting safer drug injection behavior. The principles include: ensuring that there is a good distribution of harm-reduction activities, especially easily accessible clean needles and syringes and/or bleach for cleaning injection equipment; peer counselling; programs that teach persons from the IDU scene to counsel other drug users in their own environment and in their own language; drug substitution/methadone programs should be used to getting IDUs away from risky use of illegal substances and try to channel them into a controlled, curative program (rehabilitation). All these measures must be provided in a suitably supportive social and legal environment.

336

S. Caplinskas

The first needle and syringe exchange programs were initiated in 1997 in the capital city of Vilnius. In 1998, the LAC had opened a “low threshold” consulting office aiming at promoting counselling and similar services among IDUs, and reducing HIV transmission and other STIs among IDUs and their sexual partners. During 1998-2005, a total of 2,408 IDU clients of Low Threshold Site at the Lithuanian AIDS Center for drug users were surveyed, and 90 HIV cases identified (3.74%). The majority of site visitors are IDUs with HIV infection; therefore, data on HIV prevalence in this subgroup might be higher compared to the whole IDUs population in Vilnius. In 2004, this “low threshold” services unit registered 397 new visitors; of those, 325 (81.9%) were men, and 72 (18.1%) were women. The majority of clients (375 or 94.5%) are injecting drug users and family members or sexual partners of these individuals. The number of visits is increasing every year with 10,635 for syringe exchange registered in 2004 (compared to 8,271 visits in 2003). 27,428 syringes and 28,709 injection needles were provided and 33,570 syringes and 35,437 needles were collected in 2004. In 2005, 134 new clients and 5,848 visits were registered. 12,808 syringes and 15,275 needles were exchanged while 14,512 syringes and 17,204 needles were collected in all, and 1,550 condoms distributed. In 2005, the unit tested 345 visitors (134 were new clients) and found three HIV-positive individuals (2.2%). When tested for the hepatitis C virus, 94 were found to be positive out of 229 clients (94 out of 134 new, 70.1%). Sentinel surveillance of IDUs performed on outreach basis (mobile needle exchange site “Blue bus” of Vilnius Center for Treatment of Addictive Disorders) in Vilnius in 2005 resulted in iidentification of 22 (3%) HIV-positive cases (n = 681;Table 36.7). Comparison of risky behavior and knowledge survey in IDUs (%) The Lithuanian AIDS Center data, 2005.

36.12

trend toward “feminization” of the HIV epidemic in Lithuania would eventually have an influence on HIV mother-to child transmission rates. So far in Lithuania there have been 11 HIV-positive women observed during their pregnancy (have taken ARV) and all of the infants tested negatively.

36.13

Mother-to-Child Transmission

In Lithuania, there is an HIV test available to pregnant women, and antiretroviral therapy is available for free to pregnant women, women in child-birth and newborns. This

Conclusions

1. From the data available, it appears that Lithuania might be one of the countries with a significantly localized HIV epidemic, particularly confined among IDUs which is also affecting MSM and “bridging groups,” e.g., SWs and their clients, sex partners of IDUs, and prisoners, while the general population is likely to have had very little exposure to HIV so far. 2. By risk groups: in Lithuania, the HIV epidemic has developed similarly to other countries of Eastern and Central Europe: the first cases were diagnosed in the MSM community, then in IDUs, and, recently, infection has been more rapidly spreading among heterosexuals, though the prevailing transmission mode still is injection drug use in men. 3. HIV infection cases are more concentrated in the seaports and the capital Vilnius. 4. The low TB/HIV co-infection rate and the lack of MTC cases in Lithuania may have the characteristics of an early HIV epidemic. 5. The HIV epidemic peaked in Lithuania in 2002 and, until recently, there was a downward trend in incidence rates. However, in the last few years, the incidence rate has once again increased, fuelled by infections through the use of contaminated injecting equipment and in prisons, leading to the likelihood of an outbreak affecting wider population. 6. Treatment of drug addiction, harm reduction, re-integration of ex-prisoners into society is a main factor in HIV prevention on this stage of the HIV epidemic. There are signs of spill-over and spread of HIV from IDUs to other population groups. This indicates the possibility for further spread of HIV infection from high risk groups by bridging populations (IDUs sexual partners) into the general population.

Table 36.7. Comparison of risky behavior and knowledge survey in IDUs (%) Mean duration of drug use, in years Drugs injected exceptionally with new needle/syringe Knowledge on HIV risk because of injection equipment sharing Never have got drug treatment Use of other drugs while in MT Stable sexual partner in last 12 months Awareness of HIV test result Unsafe sex with commercial partner during recent intercourse Abstinence of commercial sex in last 12 months Underwent HIV test some when The Lithuanian AIDS Center data, 2005.

2000

2004

4.0 60 98.6 28.9 85.2 45.5 94.4 86.4 76.1 69

6.4 77.7 100 56.8 95.6 64.7 87.5 70 98.1 94.1


7. Though the male IDUs with HIV still prevail, feminization of the HIV epidemic is being presently observed in Lithuania. 8. Counselling and care of HIV-infected pregnant women should be expended. 9. The number of TB/HIV co-infection cases has increased in Lithuania, though it is still not a serious problem among patients living with HIV/AIDS. However, even wider and deeper cooperation between HIV and TB control and prevention programs must be stimulated, HIV/TB counselling, surveillance, and treatment should be expanded accordingly. 10. Second generation epidemiological surveillance should be intensified in the target groups and country regions.

References 1. Resolution of the Government of the Republic of Lithuania (1999) No. 1046, On establishment of the state registry of communicable diseases agents and approval of regulations. Official Gazette No. 81, Publ. No. 2401. 2. Resolution of the Government of the Republic of Lithuania (1999) No. 1047, On establishment of the state regsitry on communicable diseases and approval of regulations. Offical Gazette No 81, Publ. No. 2402. 3. HIV/AIDS epidemic in the Baltic countries in 1987-2005: comparative study / S. Caplinskas, A. Ferdats, I. Januskevica, T. Pertel // 7th Nordic-Baltic congress on infectious diseases “Current challenges and new opportunities”, Riga, September 18-20, 2006: poster. – P. 49. ”, , 22-26 2006: abstract. – 2006, . 10, 2, c. 51. 4. Statistical Department of Lithuania Website. Available at: www. std.lt. 5. Health Information Center of Lithuania Website. Available at: www.lsic.lt. 6. Ministry of Health, Lithuanian Health Information Center (2006) Health and health care institutions in 2005. Vilnius ISSN 392- 8155. 7. Caplinskas S (2004) Epidemiology of HIV/AIDS in Lithuania in 1988–2001: review of present situation and prognosis of HIV transmission trends. Medicina 40 tomas, Nr. 2. 8. Likatavicius G, Caplinskas S, Rakickiene J (2004) HIV/AIDS epidemiology in Lithuania. Acta medica Lithuanica. Supplement 6. 9. Trends by transmission category in HIV/AIDS in Lithuania (1988-2005) / Oksana Strujeva, Saulius Caplinskas // 7 th NordicBaltic congress on infectious diseases “Current challenges

337 and new opportunities”, Riga, September 18-20, 2006: poster. – P. 32-33. 10. Epidemiology of the human immuno deficiency virus (HIV) in Lithuania: 19 year surveillance result / Oksana Strujeva, Saulius Caplinskas // 4th IAS conference on HIV pathogenesis, treatment and prevention, Sydney, July 22-25, 2007: abstract. – Hannover, 2007. – P. 80. 11. Characteristics of HIV transmission in women in Lithuania / Oksana Strujeva, Vilma Uzdaviniene, Saulius Caplinskas // 7 th NordicBaltic congress on infectious diseases “Current challenges and new opportunities”, Riga, September 18-20, 2006: poster. – P. 33. 12. The epidemiology of HIV infection in Lithuania, 1988-2005 / S. Caplinskas, O. Strujeva, V. Uzdaviniene. – Diagr. – Lent. – Bibliogr.: 19 pavad. - Tas pats rus.: p. 19-25 // EpiNorth. – 2007, vol. 8, no. 2, p. 19-26. 13. Molecular epidemiology of HIV-1 in Eurasia / E. V. Karamov, Ivanovsky institute of virology, Moscow; V. V. Lukashov, Academic medical center, university of Amsterdam; V. F. Eremin, Research institute for epidemioplogy and microbiology, Minsk; S. Caplinskas, Lithuanian AIDS center; I.G. Sidorovich, Institute of immunology, Moscow // Status of HIV vaccine research: an exploratory workshop on perspectives and potential for vaccine development, St. Petersburg, June 1-2, 2007: abstract. – St. Petersburg, 2007, p. 14-15. 14. HIV/AIDS ir Lithuania / Saulius Caplinskas. – Diagr. - Lent. – Bibliogr.: 8 pavad. // HIV/AIDS in Russia and Eurasia / ed.by Judyth L. Twigg. – {New York, 2006]. – P.171-186. 15. Caplinskas S, Likatavicius G (2002) Recept sharp rise in registered HIV infections in Lithuania. Eurosurveillance Weekly 6:27/06/2002. 16. HIV outbreak in prison follow-up / Saulius Caplinskas, Oksana Strujeva, Irma Caplinskiene // 10th European AIDS conference / EACS, Dublin, Ireland, Nov 17-20, 2005: abstract. – [Dublin, 2005]. - P.152-153. 17. HIV transmission trends in Lithuania / S.Caplinskas, I.Caplinskiene, O.Strujeva // 6-th Nordic-Baltic Congress on Infectious Diseases In Cooperation with the Task Force on Communicable Disease Control in the Baltic Sea Region “Current Strategies for Prevention and Treatment of Infectious Diseases”, Palanga, Jun 3-6, 2004: poster. – [Vilnius, 2004]. – P.43. 18. HIV/AIDS epidemiology in Lithuania / G. Likatavi ius, V. Uzdaviniene, V. Lipnickiene, J. Rakickiene, S. Caplinskas // 14th international conference on the reduction of drug related harm “Strengthening partnerships for a safer future”, Chiangmai, Thailand, April 6—10, 2003: abstract. – [Chiangmai, 2003]. – P. 174. 19. Links between HIV and injection drug use (IDU) in Lithuania / Irma Caplinskiene // 10th European AIDS conference / EACS, Dublin, Ireland, Nov 17-20, 2005: abstract. – [Dublin, 2005]. - P.131.

Chapter 31 HIV Latency and Reactivation: The Early Years Guido Poli

31.1 HIV as a Retrovirus: A New Pathogenic Entity HIV is a short, 9.2-Kb retrovirus encoding a handful of structural (gag, pol, env) and non-structural genes. These are divided into “regulatory” (tat, rev) and “accessory” (nef, vif, vpu, vpr) genes to underscore the fact that the virus cannot efficiently replicate in vitro in the absence of the former, but can handle quite well the deletion of the latter (1). The discovery of HIV (2, 3) was shortly followed by the identification of its primary receptor, the CD4 molecule displayed on the very subset of T lymphocytes selectively and progressively depleted during AIDS (4). In addition, CD4 is expressed also on circulating monocytes and tissue macrophages later shown to be infected in vivo and infectable in vitro (5, 6). It took another 12 years, however, to completely unlock the secrets of how HIV infects T cells and macrophages with the discovery of the second receptor, or co-receptor, mandatory for the virus to interact with in order to enter these cells (7). This second receptor is a chemokine receptor, either CCR5 (8) or CXCR4 (7). The virus external glycoprotein gp120 Env must bind firstly to CD4 to undergo a conformational change enabling it to recognize the chemokine receptor. This sequential interaction frees the second Env glycoprotein, gp41, that, springing like a jackknife, inserts itself into the target cell membrane and promotes its fusion with the viral particle (virion) membrane. Once inside the cell, with the virion membrane and proteins dispersed in the plasma membrane, the virus is uncoated and initiates its most characteristic biochemical process: the transcription of its single-stranded RNA genome into an equivalent cDNA form utilizing a unique enzyme known as reverse transcriptase (RT). This process begins in the cytoplasm but is completed into the nucleus as a consequence of the interaction between the so-called pre-integration complex and specific From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

determinants of the nuclear membrane. Linear DNA genomes of the virus can integrate in hot spots of the host DNA (9) by effect of the viral enzyme integrase, while surplus circular DNA remains as a scar of recent infection. Because RT does not possess a proof reading capacity, this process spontaneously introduces random mutations into the viral genome, thus contributing to one of the greatest problems in the clinical management of HIV infection: its extraordinary molecular variability (10). Once integrated as a “provirus,” the virus DNA lives as long as the cell that has just been infected and no actual drug regimen is capable of affecting this condition (although integrase inhibitors are on the verge of being introduced into the clinical arena). From this moment on, the virus behaves as a multi-gene complex subjected to the rules of activation and inhibition of transcriptional and post-transcriptional processes typical of the infected cell, as described later in greater detail. Novel HIV RNA is synthesized by the host RNA polymerase II complex and is subjected to splicing into fully spliced (2Kb) and partially spliced (4.5-Kb) mRNA coding for Tat, Rev, and Nef and for Env, respectively. The Tat protein reenters the nucleus and binds to its target RNA sequence known as TAR, thus potentiating the process of viral transcription. Similarly, Rev binds to dispersed RRE sequences promoting the export of the partially spliced (4.5-Kb) and unspliced (9.2-Kb) RNA from the nucleus to the cytoplasm where they can be translated into novel viral proteins (10). A p55 Gag precursor polyprotein needs to be cleaved by the viral protease to generate novel infectious particles; protease inhibitors have represented indeed the breakthrough in the mid-1990s that, in together with different kinds of RT inhibitors, have generated the combination protocols known as highly active antiretroviral (HAART) therapy, a very important landmark in the partial conquer of HIV disease (Figure 31.1A). Assembly of new proteins and genomic HIV RNA at the inner side of the plasma membrane of the infected cell is the prerequisite for the production of a new virion progeny after sealing of the particles by the p6 Gag protein (10). 279

280

G. Poli

Figure 31.1. In vivo and in vitro HIV infection. (A) Typical course of natural HIV-1 infection in the absence of antiretroviral therapy. The three phases (acute infection, clinical latency, and AIDS) are associated with variations in the levels of virus replication that is partially (in some case effectively) controlled during the intermediate phase. Modified from ref. 62. (B; left panel) Schematic profile of in vitro cytopathic infection of CD4+ cells. Peak virus replication coincides with the peak of virus-induced cytopathic effect (variable according to the type of cell, type and amount of infectious virus and stimulatory conditions). After the peak, virus replication tends to fade away while cell proliferation regains pre-infection levels. These “surviving” cells frequently carry integrated proviruses in their genome; virus replication, unless stimulated by specific factors, is frequently latent or at low levels. (B; right panel) Limiting dilution cloning of cells surviving acute HIV infection has resulted in clonal cell lines such as U1 and ACH-2. Modified from ref. 63.

31.2 Surrogate Model Systems for Studying HIV Infection In Vitro All features briefly described previously have been learned on the basis of previous knowledge of the retroviral life cycle and, to some extent, from the study of infected cells of HIV+ individuals. However, the relatively easy possibility to infect a variety of human cell types, including both primary cells (such as mitogen-stimulated leukocytes or T-cell blasts and, later, monocyte-derived macrophages [MDMs]) and several cell

lines of T lymphocytic and myelomonocytic origin, has been of substantial help to grow significant amounts of HIV for sequencing and drug testing. In addition, it was noted earlier that not all the cells in culture died as a consequence of HIV infection (Figure 31.1B). Surviving cells, regaining viability and proliferative capacity, were frequently infected although not always productively. This immediately suggested that they could be used as surrogate models for studying the molecular mechanisms underlying viral latency as opposed to unchecked replication and to fish out relevant factors capable of converting a latently infected cell culture into a viral factory.

31. HIV Latency and Reactivation

Classical cell activators already known as inducers of viral replication, including mitogens such as phytohemagglutinin (PHA), phorbol esters (PMA), and demethylating agents, indicated that indeed HIV could be reactivated from bulk infected cultures entered into a quiescent phase and (Figure 31.2). In addition, the immortal nature of the cell lines used allowed a much more detailed analysis of virus–host cell interaction upon limiting dilution cell cloning. Clonal cell lines were generated and scrutinized for their pattern of either latent or

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active viral replication. Both “truly latent” (i.e., methylated), “restricted” (i.e., inducible by multiple factors), and “permissive” chronically infected cell lines were generated from lymphocytic and myelomonocytic tumor cells (11). In our Laboratory of Immunoregulation at the National Institute of Allergy and Infectious Diseases (NIAID), both directed by A.S. Fauci, under the guidance of Thomas M. Folks and in close collaboration with the Laboratory of Molecular Microbiology headed by Malcolm A. Martin, also at NIAID, two

Figure 31.2. U1 and ACH-2 cell lines as surrogate models for studying modulation of virus expression from integrated proviruses. (A) Conversion from latent to active virus production occurs after 24 to 72 hours in these cell lines as a function of the stimulus applied. Virus production does not cause a cytopathic effect unlike what observed in acutely infected cells. (B) Northern blot analysis and indirect immunofluorescence for expression of HIV proteins in the U1 cell line stimulated with PMA. The kinetics of HIV RNAs accumulation after cell stimulation resemble those observed during acute infection (starting with 2 Kb spliced messages to full length 9.2 Kb RNA). Spontaneous expression of HIV proteins occurs in a 1 to 2% of unstimulated cells, whereas cell stimulation results in a potent induction of expression in most cells at a given time point, as here observed after 24 hours of stimulation. Modified from ref. 64.

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cell lines became focus of general interest: the U1 cell line, derived from acutely infected U937 promonocytic cells, and the ACH-2 cell line, originated from similarly infected CEMderived A301 T lymphocytic cell line (12, 13). They were infected with the same laboratory-adapted viral strain known as HIV-1LAI/IIIB, using CXCR4 for infection (X4 virus) and represented an easy-to-handle model of relative (“restricted”) viral latency. U1 possessed two integrated, defective proviruses, while ACH-2 had only one that, provirus per cell, could generate an infectious progeny of passageable virus. Later, U1 was shown to be defective in the Tat/TAR axis (14, 15), contrary to early claims, while the ACH-2 provirus was shown to be defective but self-repairing during viral reactivation (16). Nonetheless, the exploitation of these two models, and of similar others generated independently (like the HL60-derived OM10.1 cell line), has provided a significant contribution to dissect out factors and modeling mechanisms regulating HIV replication and its latent phase.

31.3 Cytokines as Physiological Factors Controlling HIV Latency and Replication After the demonstration that integrated HIV provirus could be reactivated by either its own regulatory protein Tat (either endogenously synthesized or exogenously loaded as a protein) or cell-activating factors such as mitogens (PHA), cytokine-reach supernatants derived from either activated T cells or MDM (17) or PMA (known activators of protein kinase C, PKC, dependent pathways; Figure 31.2), the search for physiological modulators of virus replication begun. PMA and related stimuli were shown to induce the activation of a dormant cytoplasmic transcription factor originally thought to be restricted in its action to the regulation of Ig light chain synthesis: nuclear factor κB (NF-κB). Almost simultaneously a potent cytokine, tumor necrosis factor (TNF)α (cachectin, and its related molecule TNF-β/lymphotoxin) was shown to activate transcription of proviruses integrated in either T cells or macrophages by the same NF-κB-dependent pathway (18–20). Thus, the first molecular pathway through which an extracellular stimulus (such as a cytokine) could directly influence the expression of an integrated provirus through activation of a cellular transcription factor was demonstrated. Endogenously released TNF-α could activate HIV expression in U1 cells in an autocrine fashion, i.e. interacting with cell surface receptors, after PMA stimulation of either U1 or ACH-2 cells (21), as later demonstrated also in primary PBMC stimulated with IL-2 and in MDM as discussed further. Was the TNF–NF-κB axis the only regulatory pathway capable of influencing virus replication or was it simply the first example described out of several? Interleukin (IL)1 (either –α or –β, another fundamental pro-inflammatory cytokine) was shown to act like TNFs in terms of activation of NF-κB-dependent transcription in at least some macro-

G. Poli

phage infection models (19, 22). Another late inflammatory molecule, IL-6, crucial for inducing the synthesis of liver proteins collectively responsive for inducing the acute phase response, induced HIV expression in the monocytic U1 cell line and in primary MDM. However, after several attempts of demonstrating activation of NF-κB, we realized that IL-6 must have exploited a different modality of upregulating virus production. At the same time, we noticed that cells stimulated with IL-6 and TNF together showed a much greater induction of virus expression than cells stimulated with each cytokine separately (and even outscoring a simple additive effect of the two stimuli). Synergy was thus demonstrated between TNF and IL-6 (but also between IL-1 and IL-6, as later demonstrated; ref. 23). At the molecular level, still in the pre-polymerase chain reaction (PCR) era, we also noticed that IL-6 stimulation alone failed to induce detectable levels of HIV RNA (as assessed by Northern blotting) and yet it induced levels of virion production only two- to threefold lower than those induced in the same cells by TNF or PMA. Thus, we proposed that IL-6 stimulation alone was acting predominantly at one or more post-transcriptional levels (i.e., increasing the efficiency of translation of low levels viral RNAs) although it also synergized at the transcriptional level in the presence of TNF (23). Only in recent years it has been defined (24, 25) that IL-6 mostly, if not exclusively, activates HIV expression in U1 cells via activation of another family of transcription factors: Fos and Jun, forming AP-1 complexes capable of binding to target sequences present both in the HIV LTR and in an intragenic enhancer present in the gag gene (26, 27). AP-1 binding results in transcriptional activation of HIV expression, although, even by quantitative PCR, IL-6 stimulation of viral RNA synthesis is usually 10fold lower than that induced by TNF-α. After the demonstration that multiple cytokines could upregulate HIV expression in monocytic cells by activating different synergistic pathways, the next question was whether HIV-suppressive cytokines existed. It was already known that class I interferons (IFNs), IFN-α/-β, suppressed acute HIV infection of T cells and macrophages and that they inhibited virion production from chronically infected cell lines by acting on the very late step of budding and release of new progeny virions (28). But did cytokines distinct from IFNs capable of interfering with HIV replication exist? In 1986, Walker and Levy demonstrated the existence of “non-cytolytic soluble suppressor factor(s)” secreted by CD8+ T lymphocytes that inhibited virus replication during experiments of HIV isolation from PBMC of infected individuals (29). Depletion of CD8+ cells (including NK cells, later shown to be also effective in terms of suppression of virus replication) became a common procedure to increase the frequency of virus isolation, while the search for the inhibitory soluble factor(s) (also known as “CAF” for CD8-antiviral factor) remained elusive for at least 10 years, when HIV-inhibitory chemokines (RANTES, MIP1α, MIP-1β; ref. 30) and IL-16 (31, 32) were claimed to mediate such a biological activity.


Our laboratory at the NIH investigated the hypothesis that anti-inflammatory cytokines could counteract the HIVinductive effects of pro-inflammatory cytokines also in terms of viral replication. The hypothesis turned to be correct, at least in part. Transforming growth factor (TGF)-β and, later, IL-10 (likely the most potent known anti-inflammatory cytokines) did inhibit and even suppressed HIV replication in U1 cells and MDM. TGF-β inhibited virus expression induced by stimulation of U1 cells with either PMA, IL-1, or IL6 (but not with TNF; ref. 33). If U1 cells were stimulated with PMA in the presence of anti-TNF-α neutralizing Ab (which reduced the levels of virus expression approximately of 50% by interfering with the endogenous TNF-α dependent loop of HIV expression) and TGF-β virus expression was virtually abolished. Finally, TGF-β inhibited HIV transcription as demonstrated by both Northern blotting and runon experiments (33). Thus, the existence of HIV-suppressive cytokines was indeed demonstrated; later on, retinoic acid, a well known differentiating agent, was shown to exert inhibitory effects on virus expression from monocytic cells similar to those of TGF-β (34). However, in primary MDM, TGF-β (as well as retinoic acid) demonstrated a bipolar behavior: it inhibited acute viral replication when given after infection, but, quite surprisingly, it exerted upregulatory effects when cells were prestimulated with TGF-β (34). Independent investigators had earlier described inductive effects of TGF-β on acute infection of primary MDM or activated PBMC (35– 37). Similarly, IL-10 could inhibit HIV replication in MDM (38); we confirmed this observation but observed that this occurred at fully biologically active concentrations capable of inhibiting the secretion of endogenous pro-inflammatory cytokines such as (TNF-α, and IL-6; (39). In contrast, when MDM were incubated with lower concentrations of IL-10 (not effective in suppressing cytokine secretion) enhancement rather than inhibition of virus replication was observed; in addition, IL-10 potentiated the HIV-inductive effects of these cytokines in U1 cells (although it did not activate virus expression per se; ref. 40). It was shortly demonstrated that IL-10 enhanced endogenous TNF-α dependent HIV expression in U1 cells (41, 42). IFN-γ also showed opposite effects on HIV replication, depending on the experimental conditions. Alone, it stimulated virus expression in U1 cells, but it inhibited virus production in combination with PMA and shut-off TNF-mediated HIV expression in these cells (43). However, an ultrastructural analysis showed unequivocally that IFN-γ was a per se inducer of virus production in U1 cells, but that it, in the presence of PMA, shifted the major site of virion assembly and release from the plasma membrane to intracytoplasmic vacuoles. IFN-γ actually potently synergized with TNF in terms of virus transcription, but it simultaneously cooperated in promoting cytokine-mediated cell death thus resulting in an abortive activation of virus production (43, 44). If U1 cells were co-stimulated with anti-Fas Ab and IFN-γ, no induction of virus was observed (since Fas engagement does not lead to NF-κB activation as

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in the case of TNF receptors) but the cells were synergistically killed by the two stimuli (44). Thus, bimodal cytokines, acting as either inducers or inhibitors of virus expression as a function of the experimental conditions were described. It was later realized that several cytokines and chemokines do exert opposite effects on viral entry (via direct engagement of chemokine co-receptors, as in the case of CCR5-binding chemokines, or through downregulation of CD4 and chemokine receptors, as shown for TNF and IFN-γ) and post-integration events in the HIV life cycle. In this regard, also chemokines such as IL-8, MCP-1, and CCR5-binding chemokines were shown to upregulate virus expression as in infected cells recently reviewed (45).

31.4 Cytokine-mediated Modulation of HIV Replication: From Cell Lines to Primary Cells Infected In Vitro or In Vivo The demonstration of a complex network of cytokines and related factors showing regulatory activities on virus expression in different cell lines made urgent understanding whether these phenomena were restricted to a bizarre set of randomly selected tumor cell lines or were they demonstrable in primary cells, including cells isolated from infected individuals. As already mentioned, there were already good evidence that both endogenous and exogenous cytokine stimulation modulated virus replication in primary MDM. The demonstration of their relevance for infected CD4+ T lymphocytes initially difficult because the standard protocol through which these primary cells were infected (i.e., via pre-activation with PHA and then incubation with IL-2) did not show evidence of cytokine-driven virus replication. Addition of specific anticytokine Ab did not result in meaningful and reproducible effects and the direct measurement of cytokine production after removal of PHA and change of its conditioned medium was essentially negative. However, we observed that stimulation of PBMC with IL-2 in the absence of preactivation with PHA resulted in a productive infection so-called T-cell- and macrophage-tropic viruses (46). Indeed, before the discovery of CXCR4 and CCR5 as entry co-receptors there were several, partially overlapping classification of primary isolates and laboratory-adapted viral strains, including their tropism for these two main target cells, coinciding quite well with that based on the capacity to infect and induce cytopathic effects in a reference cell line: the HTLV-1 transformed MT-2 T-cell line. Viruses scoring positive in the infection of this cell line were defined as syncytia-inducing (SI) while those testing negative were simply defined as non-SI (NSI) viruses (47, 48). It was later shown that MT-2 cells, like most cell lines, are exclusively positive for CXCR4 expression and, therefore, permit infection and replication of only those viruses displaying a gp120 Env utilizing this chemokine receptor; conversely, NSI viruses engage selectively CCR5.

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Figure 31.3. Multiple effects of IL-2 on HIV replication. (A) PBMC or lymph node mononuclear cells (LNMC) obtained from HIV-infected individuals were stimulated in vitro with either IL-2 or IL-12. In the presence of CD8+ cells, only IL-12 induced virus replication. Removal of CD8+ cells unleashed the upregulatory effects of IL-2 outscoring those of IL-12; the lower panels indicate RT activity, a much less sensitive assay than the p24 Gag assay indicated in the top panels. Modified from ref. 53. (B) A schematic model of multiple IL-2 mediated effects on HIV infected cells. HIV- Suppressive effects mediated by CD8+ cells are dominant over the inductive ones and are hypothetically proposed to contribute to the net gain in CD4+ T cells observed in individuals receiving intermittent IL-2 therapy (references in the text).


IL-2 stimulated PBMC secreted several cytokines already characterized as inducers of virus expression in cell lines (TNF-α, IL-1β, IFN-γ, and IL-6) and their peak level of production coincided or preceded that of virus replication. More importantly, their neutralization by Ab or IL-1 receptor antagonist resulted in a significant inhibition of virus replication. This was an important demonstration of two concepts: first, cytokines do act in an autocrine/paracrine fashion in terms of regulation of HIV replication in primary cells, as independently reported (49); second, IL-2 is not a direct inducer of virus replication but it is the induction of this pro-inflammatory cytokine cascade responsible for the virus-inductive effects triggered by this cytokine. This observation had relevance for the initial studies just published on the potential clinical use of IL-2 in HIV-infected individuals, showing expansion of CD4+ T lymphocytes without alterations of steady state viremia levels (but inducing only spikes of transient virus replication) (50, 51). After several successful phase II trials, intermittent IL-2 therapy is currently under phase III evaluation in thousands of patients worldwide (52). Even more convincingly, follow-up studies conducted with either peripheral or lymph node cells of infected individuals demonstrated that among other effects IL-2 potently activated CD8+ cells to exert their “CAF” activity on infected CD4+ cells in that removal of CD8+ cells unleashed the upregulatory effect of IL-2 on virus replication (53), as independently observed (Figure 31.3; ref. 54). After the demonstration of the existence of latent HIV reservoirs in resting memory CD4+ T cells, it was shown that their stimulation with a cocktail of pro-inflammatory cytokines, including IL-2, IL-6, and TNF-α (55), and later, IL-7 (56–59), resulted in the activation of virus replication.

31.5

Conclusions and Perspectives

Looking back at the early days when a number of basic paradigms were established may be helpful for the new challenges facing modern investigators dealing with the unsolved problems of the HIV/AIDS pandemic. Will IL-2 based therapy be successful in terms of AIDS-sparing, lifesaving events? How much of its activity is attributable to a direct effect on target cells and how much is the contribution of secondary cascades triggered by a pharmacological use (millions of units per day in cycles of 5 days every 4–8 weeks) of this cytokine? Will other cytokine join IL-2 in clinical trials involving HIV infected individuals? IL-7, IL-15, and GM-CSF, sharing with IL-2 the activation of the Janus kinase/signal transducer activator of transcription (JAK/STAT) pathway and, particularly, the activation of STAT5A and STAT5B (60, 61), have already demonstrated interesting evidence of in vivo activity. Will cytokines such as IL-2 be relevant in the design of strategies aimed at curtailing viral reservoirs or, at least, at controlling their capacity to ignite virus replication in conditions of absent or weak control by ARV therapy?

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In conclusion, the beauty of studying HIV infection in in vitro models, either from a molecular, virological, or immunological perspective, as here exemplified for the study of cytokine, is that most observations will bear relevance to clinical application, if rightly analyzed and interpreted. Acknowledgements. This chapter mostly relies on studies conducted during my stage in the Laboratory of Immunoregulation at NIAID, NIH, Bethesda, MD. I wish to express my sincere gratitude to Tony Fauci and to all my friends and collaborators of that time for what I learned in those years. Whatever I have accomplished thereafter is the consequence of attending a very good school! This study is supported by my grant from the V° National Program of Research Against AIDS of the Istituto Superiore di Sanità, Rome, Italy.

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54.

55.

56.

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58.

59.

60.

61.

62.

63. 64.

JM, Berrey MM, Corey L, Lane HC, Fauci AS (1999) Effect of interleukin-2 on the pool of latently infected, resting CD4+ T cells in HIV-1-infected patients receiving highly active antiretroviral therapy. Nat Med 5:651–655. Emery S, Abrams DI, Cooper DA, Darbyshire JH, Lane HC, Lundgren JD, Neaton JD (2002) The evaluation of subcutaneous proleukin (interleukin-2) in a randomized international trial: rationale, design, and methods of ESPRIT. Control Clin Trials 23:198–220. Kinter AL, Bende SM, Hardy EC, Jackson R, Fauci AS (1995) Interleukin 2 induces CD8+ T cell-mediated suppression of human immunodeficiency virus replication in CD4+ T cells and this effect overrides its ability to stimulate virus expression. Proc Natl Acad Sci USA 92:10,985–10,989. Barker E, Mackewicz CE, Levy JA (1995) Effects of TH1 and TH2 cytokines on CD8+ cell response against human immunodeficiency virus: implications for long-term survival. Proc Natl Acad Sci USA 92:11,135–11,139. Chun TW, Engel D, Mizell SB, Ehler LA, Fauci AS (1998) Induction of HIV-1 replication in latently infected CD4+ T cells using a combination of cytokines. J Exp Med 188: 83 – 91 . Uittenbogaart CH, Anisman DJ, Zack JA, Economides A, Schmid I, Hays EF (1994) Effects of cytokines on HIV-1 production by thymocytes. Thymus 23:155–175. Smithgall MD, Wong JG, Critchett KE, Haffar OK (1996) IL-7 up-regulates HIV-1 replication in naturally infected peripheral blood mononuclear cells. J Immunol 156:2324–2330. Ducrey-Rundquist O, Guyader M, Trono D (2002) Modalities of interleukin-7-induced human immunodeficiency virus permissiveness in quiescent T lymphocytes. J Virol 76:9103–9111. Wang FX, Xu Y, Sullivan J, Souder E, Argyris EG, Acheampong EA, Fisher J, Sierra M, Thomson MM, Najera R, Frank I, Kulkosky J, Pomerantz RJ, Nunnari G (2005) IL-7 is a potent and proviral strain-specific inducer of latent HIV-1 cellular reservoirs of infected individuals on virally suppressive HAART. J Clin Invest 115:128–137. Bovolenta C, Camorali L, Lorini AL, Ghezzi S, Vicenzi E, Lazzarin A, Poli G (1999) Constitutive activation of STATs upon in vivo human immunodeficiency virus infection. Blood 94:4202–4209. Crotti A, Lazzarin A, Bovolenta C, Poli G (2006) Negative regulation of HIV-1 expression by the natural isoform C-terminus truncated STAT5 (STAT5Delta). Retrovirology 3:S101. Pantaleo G, (1993) New concepts in the immunopathogenesis of human immunodeficiency virus infection. N Engl J Med 328:327–335. Vicenzi E, Poli G (2005) Curr Prot Immunol 12.3.1–12.3.17. Vicenzi E, et al (2007) Immunopathogenesis of HIV infection. In The Biology of Dendritic Cells: Role in the Pathogenesis and Immunity of HIV Infection (Gessani S, Belardelli F, ed.), Springer, New York.

Chapter 32 HIV-1 Sequence Diversity as a Window Into HIV-1 Biology Milloni Patel, Gretja Schnell, and Ronald Swanstrom

32.1

Overview

Sequence diversity represents a rich source of information about the selective pressures placed on a genetically dynamic organism such as HIV-1. Information can be gleaned by sampling infected subjects cross-sectionally, sampling individuals longitudinally, and comparing the virus present in different compartments within an individual. Patterns of sequence diversity and evolution can provide insights into the nature of the selective pressure and represent an intimate part of the response of the virus to changing virus–host interactions. This chapter discusses research trends in the study of newly transmitted HIV-1, the evolution of co-receptor use, compartmentalization of HIV-1 in the central nervous system (CNS), and differences between HIV-1 subtypes and the neutralizing antibody response. Deciphering the nature of the transmitted virus will contribute to an understanding of the selective pressures at the time of transmission and refine the target of vaccine development. Determining the ways in which the virus is compartmentalized will add to our understanding of virus–host interactions and viral pathogenesis. Part of understanding viral pathogenesis is the evolution of co-receptor use that is associated with advanced disease state. Finally, the natural immune response that prevents disease progression in some patients is partially due to an efficient neutralizing antibody response, and understanding why a particular antibody is broadly cross-reactive will help in designing immunogens that will generate these important neutralizing antibodies.

From: National Institute of Allergy and Infectious Diseases, NIH Volume 1, Frontiers in Research Edited by: Vassil St. Georgiev, Karl A. Western, and John J. McGowan © Humana Press, Totowa, NJ

32.2 Complexity of Newly Transmitted Virus The HIV-1 population within a chronically infected individual is heterogeneous and complex, and varies in sequence as much as 8% in the env gene (1, 2). However, transmission of virus from a chronically infected person is limited to one or a few variants from the entire viral population, resulting in a genetic bottleneck at the time of transmission (1, 3–6). To study the transmission of HIV-1 viral variants, we focused on cross-sectional examination of primary infection subjects. Primary infection is the earliest stage of HIV-1 infection and occurs in the absence of an adaptive immune response (5–8). The study of transmitted variants is complicated by the early diversification of the virus population. Approximately 8 to 11 weeks post-infection, simian immunodeficiency virus (SIV) diversification begins in response to host immune pressure (9). Thus, the study of transmitted virus requires the correct identification of subjects during primary infection to prevent a bias toward the presence of multiple variants as a result of this diversification. The method we used to examine the complexity of newly transmitted virus is the heteroduplex tracking assay (HTA). The HTA is a gel-based assay that separates viral variants based on sequence differences, and can resolve variants that comprise as little as 3% of the total viral population (1, 4, 5, 10, 11). The HTA has advantages over conventional cloning procedures in that the entire viral population can be sampled at one time, and reproducing the HTA pattern can validate the quality of sampling (5, 11–13). Studies with SIV have shown the transmission of multiple variants under experimental conditions (9). A study by Rybarczyk et al. detected multiple variants by HTA during primary infection in rhesus macaques inoculated with a genetically complex SIVsm strain. A comparison of intravenous versus intrarectal-challenged macaques showed no evidence for selection of specific variants at the mucosal membrane (9). In addition, the timing of SIVsm diversification was correlated with the neutralizing antibody titer (9). 289

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The potential for the transmission of multiple HIV-1 variants has been documented in previous studies (2, 3, 5, 14, 15). However, a link to specific viral or host factors is incompletely understood, and the reported frequency of the transmission of multiple variants varies widely. A study by Ritola et al. assessed HIV-1 viral diversity of the V1/V2 regions of env in a primary infection cohort using HTA. Examination of the V1/V2 region of env detected multiple variants in about half of the primary infection subjects (5). In contrast, a primary infection study by Delwart et al. detected only single HIV-1 variants in men infected via homosexual transmission (3). Studies by Long et al. have shown a correlation between the transmission of multiple variants and the sex of the infected individual, where more than half of women infected via heterosexual transmission had multiple HIV-1 variants and only single variants were found in men infected via heterosexual transmission (14). Because transmission of HIV-1 is a low probability event, we have suggested that multiple variants are present in infected cells, and the transmission of an infected cell could result in the transmission of multiple variants (5). More recently, we have developed evidence that the estimation by HTA of the number of variants transmitted can be an overestimate in this type of cross-sectional analysis. It is likely that multiple variants can be transmitted but not at as high a frequency as previously reported (Schnell, unpublished observation). Our results suggest that the initial diversification of virus in humans occurs earlier than we observed in macaques, suggesting that the higher viral loads in macaques may blunt the initial immune response. Also, this observation indicates how important it is to work with viruses collected as close to the time of transmission as possible to avoid the problem of early genetic diversification.

32.3 Compartmentalization and HIV-associated Dementia Compartmentalized HIV-1 variants have been identified in the CNS and the genitourinary tract of HIV-1 infected individuals (12, 13, 15–19). HIV-1 infection of the CNS occurs shortly after peripheral infection through the trafficking of infected monocytes across the blood–brain barrier (16, 20, 21). In a subset of infected individuals, HIV-1 infection of the CNS leads to compartmentalization and neurological complications, including the development of HIV-1-associated dementia (HAD) or minor cognitive motor disorder (MCMD; refs. 12, 20–23). Invasion of the CNS may provide HIV-1 an opportunity to establish an autonomously replicating viral population in the presence of therapy due to the poor CNS penetration of some antiretrovirals (ARVs; refs. 22, 24–26). Compartmentalized variants in the CNS are genetically distinct from peripheral virus, but the factors important for compartmentalization and the development of neurological disease are not currently known. The HTA is a sensitive assay

M. Patel et al.

that can be used to study HIV-1 population complexity and dynamics and identify compartmentalized variants within the population (4, 5, 10–13, 27). Examination of viral populations in the CNS frequently relies on studies of HIV-1 in cerebrospinal fluid (CSF), which is thought to contain virus originating from both the periphery and the CNS (19, 23, 28–30). We used HTA to distinguish between viral variants in the CSF that originated from CNS tissue and variants that are shared between peripheral blood and CSF. Viral variants can be considered compartmentalized by HTA if the variants are unique to the CSF, or if their relative abundance significantly differs between the periphery and the CSF (12, 13). Previous studies have found increased HIV-1 compartmentalization in the CNS of individuals with HAD or MCMD (12, 18, 31, 32). A study by Ritola et al. investigated the relationship of CNS compartmentalization and the severity of neurological disease by examining the V1/V2 and V3 regions of env in plasma and CSF from individuals with HAD, MCMD, or no neurological symptoms (12). HTA analysis showed at least some compartmentalized viral variants in the CSF of most subjects; thus, the simple detection of CSF-compartmentalized variants was not correlated with the level of neurological disease. However, the fraction of virus in the CSF that represented CNS-unique variants was present at a higher frequency in the CSF of HAD subjects. The molecular mechanisms that lead to the development of HAD are still unknown. Several studies have suggested that as disease advances, virus present in the CSF shifts to a higher percentage of CNS-derived virus (12, 18, 22, 28, 33). The immunological failure associated with advanced HIV-1 disease may result in increased viral replication in the CNS, leading to a higher fraction of CNS-derived virus in the CSF (12, 28, 33). Perivascular macrophages and microglia in the CNS can be productively infected by HIV-1, and previous studies have suggested that macrophage-tropic viruses are neurotropic (16, 21). In addition, Gonzalez et al. reported that a specific genotype of MCP-1, the MCP-1-2578G allele, was correlated with an increased risk of HAD (34). This specific genotype of MCP-1 was associated with increased inflammation and the ability to upregulate HIV-1 infection, which may increase the risk of developing HAD. The time at which CNS compartmentalization occurs during HIV-1 infection is also unknown. Determining whether compartmentalization initiates during the primary or chronic stages of HIV-1 infection could have major implications for ARV treatment regimens, including the timing of CNSpenetrating drugs (18). A study by Ritola et al. used HTA to examine whether HIV-1 compartmentalization occurs during primary infection using a cross-sectional comparison of the env V1/V2 variable regions for plasma, semen, and CSF (5). Virus was detected in plasma, semen, and CSF from acute primary infection subjects, indicating the rapid expansion of virus in these compartments. However, compartmentalization was not evident between the CSF or semen and the plasma compartments, suggesting that compartmentalization does not

32. HIV-1 Sequence Diversity as a Window Into HIV-1 Biology

occur during primary HIV-1 infection and must develop later in the course of infection.

32.4 Source of Compartmentalized Virus in the CNS HIV-1 invades the CNS shortly after initiating a peripheral infection (20, 21). Once HIV-1 has moved across the blood–brain barrier, it comes in contact with different cell types in the brain parenchyma, including perivascular macrophages, microglia, astrocytes, oligodendrocytes, and neurons (21). Perivascular macrophages and microglia are the only cell types in the CNS that are known to sustain productive HIV-1 infection, although astrocytes may be non-productively infected by HIV-1 (21, 35–37). Our laboratory is interested in characterizing the cellular sources of HIV-1 in the CNS, and determining the mechanisms HIV-1 uses to persist in the CNS. Previous studies examining the kinetics of HIV-1 decay have shown two phases of viral decay in the peripheral blood of subjects initiating highly active ARV therapy (HAART; refs 38–40). The first phase of decay is rapid and results from the death and clearance of short-lived infected CD4+ T cells and free virus (38–40). The second phase of viral decay is slower and of unknown source, but may be the result of clearance of long-lived infected cells like macrophages and/or resting CD4+ T cells (38–40). Several studies examining viral decay rates in the CSF of HIV-1-infected subjects reported similar viral decay kinetics in both CSF and plasma for most individuals, but some subjects showed a slower clearance of virus from the CSF compared to plasma (18, 22, 28, 33). Slower clearance of HIV-1 from the CSF has been associated with the presence of neurological disease (18, 22, 28, 33). A study by Eggers et al. found that HIV-1-infected individuals with severe neurological symptoms show much slower viral decay rates in the CSF compared to plasma after initiating HAART (22). An examination of asymptomatic (no neurological symptoms) subjects initiating HAART by Harrington et al. found that the majority of compartmentalized virus in the CSF is produced by short-lived cells (13). This study utilized the HTA to identify CNS-compartmentalized virus, then measured the viral decay rates for each detected variant. The HIV-1 population in the CSF of these asymptomatic subjects was found to be 11 to 85% compartmentalized (enriched and/or unique to the CSF), and the compartmentalized variants decayed rapidly once HAART was initiated for each subject (13). HIV-1 persistence in the CNS can be described by two pathways based on previous studies (18). The first pathway is based on the continuous trafficking of short-lived infected CD4+ T cells into the CNS. Virus in the CNS is constantly replenished from the periphery, resulting in similar HIV-1 populations in both the periphery and the CSF (12, 18). In this model, virus in both the CSF and plasma should decay rapidly upon the initiation of HAART (18). This type of CNS infec-

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tion has been described in previous studies involving asymptomatic HIV-1 infected subjects (12, 13, 18). The second pathway is based on HIV-1 infection of longlived perivascular macrophages and microglia in the CNS. Virus originates from local CNS tissue, resulting in large differences in HIV-1 populations in the CSF and periphery (12, 18). The second model predicts that virus in the CSF will have slower decay kinetics compared to the periphery due to the slower turnover time of macrophages and microglia (18, 22). Previous studies involving subjects with severe neurological disease that are initiating HAART have described strong compartmentalization between the CSF and plasma, and slower elimination of HIV-1 from the CSF (12, 18, 22, 28, 33). The connection between HIV-1-associated neurological disease and actual viral replication in the CNS has not been established, but is an area of active research.

32.5 Evolution of CCR5 Usage to CXCR4 Usage For reasons that are just beginning to be understood, virus that is most frequently transmitted by any route of infection uses CCR5 as a co-receptor (R5-tropic virus; refs. 41–44). It has been suggested that this is due to the uptake of R5-tropic virus by dendritic cells at mucosal surfaces where most transmission occurs (45, 46). This is further demonstrated in the population that contains a deletion in the CCR5 allele ( 32), where individuals who are homozygous for this allele are usually resistant to infection, and heterozygous individuals have delayed disease progression (47–49). The evolution of virus from CCR5 usage into CXCR4 usage (X4-tropic virus) occurs in 50% of patients infected with subtype B HIV-1, and CXCR4 usage is strongly associated with disease progression (50–53). It is not yet clear whether X4-tropic viruses cause a more rapid decline in T cells or whether X4-tropic viruses grow out when the host is experiencing a rapid decline in T cells due to loss of even modest control of the infection, although both possibilities have been considered (54, 55). Attempts to characterize the progression from R5- to X4-tropism have come up with varied results. The mutations associated with co-receptor switch were at first attributed solely to mutations in V3; however, subsequent data suggests that the story is more complicated (56–59). A study by Bagnarelli et al. suggests that the V3 loop is the only determinant of co-receptor usage in 76% of viruses (56). Another study used CCR5/CXCR4 chimeras to show that R5-tropic virus evolve to acquire broader use of CCR5 (accompanied by an increasing loss of sensitivity to CCR5 ligand RANTES), suggesting that ongoing evolution is not directed to specific V3 amino acids (57). The final question that arises in understanding co-receptor usage is why the switch occurs; unfortunately the answer is not clear. Affinity of gp120 is much higher to CCR5 (4–15 nM) than CXCR4 (200–500 nM) and although CCR5 is expressed

292

on many cell types (including activated memory T cells, natural killer T cells, monocytes, macrophages, immature dendritic cells), HIV-1 can only infect cells that co-express CD4 with the co-receptor (60–62). Therefore, R5-tropic virus can only infect 10% of CD4+ T cells, whereas X4-tropic virus can infect 85% of CD4+ T cells (such as naïve T cells; refs. 63, 64). This suggests that co-receptor usage can be driven by appropriate T-cell availability. The co-receptor switch may be important (even with an affinity loss) in increasing host range after depletion of CCR5+ T cells, which is now understood to take place during the acute phase of infection (65–67). However, if CD4+/CCR5+ T-cell depletion takes place so early, how and why does virus persist for so long in a limited host range? There are many intrinsic obstacles to co-receptor switching: X4-tropic virus has diminished replication compared to parent R5-tropic virus; evolutionary intermediates are more sensitive to both CCR5 and CXCR4 inhibitors than parent or final virus; non-random changes in amino acids and glycosylations are required to use CXCR4 as a co-receptor, and R5-tropic virus may release more virus/cell (68, 69). Further questions of the need to change co-receptor arise when we consider the 50% of patients that do not evolve to use CXCR4 (57, 70, 71). How are the viruses in these patients able to utilize CCR5 efficiently enough to cause disease progression? Finally, extended use of a CXCR4 inhibitor (AMD3100) shifted X4-tropic virus back to using CCR5, suggesting that there is a competition between the mixed populations of virus in vivo for the most efficient co-receptor that can be used (72). Current evidence points to a slow evolution to CXCR4 usage through mutations in V3, which may be enhanced or compensated by non-V3 mutations. There have been many studies on the most efficient method of predicting virus co-receptor phenotype using statistical approaches, many of which focus on the V3 region of Env. The original rule uses the net charge of the V3 as an indicator of co-receptor usage, since there is an increase in net V3 positive charge of CXCR4-using Envs (73, 74). The 11/25 rule, with a basic amino acid at position 11 and/or 25 (HXB 306 and 322, respectively), is also strongly associated with an X4-tropic phenotype (75–77). Furthermore, several studies have shown that positions 429, 440, 424, and a cluster of amino acids between 190 and 200 are all involved in the co-receptor switching (78, 79). Jensen et al. created a position-specific scoring matrix (PSSM) specific to subtype B V3 sequences to improve co-receptor prediction that is 84% sensitive and 96% specific (80). PSSM is used to detect non-random distributions of amino acids at adjacent sites associated with empirically determined groups of sequences. Often, PSSM uses background genetic variations as a baseline comparison (or null model) to facilitate comparison of the residues of a sequence fragment to those of a group of aligned sequences known to have the desired property. Using this principle, V3 sequences can be assigned a score; the higher the score, the more closely the sequence resembles those of known X4 sequences. Jensen et al. have also designed PSSM for subtype C V3 sequences to predict co-receptor use (81). These methods have been used extensively to predict

M. Patel et al.

co-receptor use, but biostatistical modeling is not 100% efficient, because they are not robust for evolutionary intermediates and their accuracy is limited by the volume of X4 sequences available to define different evolutionary pathways.

32.6 CCR5 and CXCR4 Usage Differences Between Subtype B and Subtype C HIV-1 Phylogenetic analysis shows that HIV-1 can be divided into three groups: M (main), N (new), and O (outlier). The M-group (99% of infections) is further subdivided into nine subtypes (A to K) and circulating recombinant forms in which each subtype has an env nucleotide divergence of 35% between subtypes, 20% divergence within subtypes, and up to 8% diversity within one person (1, 82–84). Subtype B is prevalent mostly in North America and Western Europe, and subtype C is the world’s most prevalent strain, found in sub-Saharan Africa, India, and Southeast Asia, accounting for half of the new infections worldwide. As described previously, subtype B virus can evolve from using CCR5 to using CXCR4 in 50% of patients; however, Ping et al. and others have determined that subtype C is less likely to use CXCR4 during disease progression (85–93). Thus, two major subtypes of HIV-1 have evolutionary pathways that are different from each other. The consensus V3 sequence differs in only five amino acid positions between subtypes B and C, suggesting that sequence changes affect structure in a way that limits subtype C HIV-1 from evolving to use CXCR4 efficiently. A recent study by Coetzer et al. suggests that 10% of subtype C isolates can evolve to use CXCR4 (strongly associated with CD4+ T cell count 1/12,500

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Drug Interactions in Infectious Diseases, 3rd Edition (Infectious Disease)

Evolution of Infectious Disease

Infectious Disease in the Aging: A Clinical Handbook (Infectious Disease)

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Case Studies in Infectious Disease

Infectious Disease Informatics and Biosurveillance

Serology of infectious disease syndromes

Mims' Pathogenesis of Infectious Disease

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Medical Management of Infectious Disease

Encyclopedia of Infectious Diseases

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