Streptococcus - A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References

STREPTOCOCCUS A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R E FERENCES

J AMES N. P ARKER , M.D. AND P HILIP M. P ARKER , P H .D., E DITORS

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ICON Health Publications ICON Group International, Inc. 4370 La Jolla Village Drive, 4th Floor San Diego, CA 92122 USA Copyright 2004 by ICON Group International, Inc. Copyright 2004 by ICON Group International, Inc. All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America. Last digit indicates print number: 10 9 8 7 6 4 5 3 2 1

Publisher, Health Care: Philip Parker, Ph.D. Editor(s): James Parker, M.D., Philip Parker, Ph.D. Publisher's note: The ideas, procedures, and suggestions contained in this book are not intended for the diagnosis or treatment of a health problem. As new medical or scientific information becomes available from academic and clinical research, recommended treatments and drug therapies may undergo changes. The authors, editors, and publisher have attempted to make the information in this book up to date and accurate in accord with accepted standards at the time of publication. The authors, editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of this book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised to always check product information (package inserts) for changes and new information regarding dosage and contraindications before prescribing any drug or pharmacological product. Caution is especially urged when using new or infrequently ordered drugs, herbal remedies, vitamins and supplements, alternative therapies, complementary therapies and medicines, and integrative medical treatments. Cataloging-in-Publication Data Parker, James N., 1961Parker, Philip M., 1960Streptococcus: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References / James N. Parker and Philip M. Parker, editors p. cm. Includes bibliographical references, glossary, and index. ISBN: 0-597-84639-1 1. Streptococcus-Popular works. I. Title.

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Disclaimer This publication is not intended to be used for the diagnosis or treatment of a health problem. It is sold with the understanding that the publisher, editors, and authors are not engaging in the rendering of medical, psychological, financial, legal, or other professional services. References to any entity, product, service, or source of information that may be contained in this publication should not be considered an endorsement, either direct or implied, by the publisher, editors, or authors. ICON Group International, Inc., the editors, and the authors are not responsible for the content of any Web pages or publications referenced in this publication.

Copyright Notice If a physician wishes to copy limited passages from this book for patient use, this right is automatically granted without written permission from ICON Group International, Inc. (ICON Group). However, all of ICON Group publications have copyrights. With exception to the above, copying our publications in whole or in part, for whatever reason, is a violation of copyright laws and can lead to penalties and fines. Should you want to copy tables, graphs, or other materials, please contact us to request permission (E-mail: [email protected]). ICON Group often grants permission for very limited reproduction of our publications for internal use, press releases, and academic research. Such reproduction requires confirmed permission from ICON Group International, Inc. The disclaimer above must accompany all reproductions, in whole or in part, of this book.

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Acknowledgements The collective knowledge generated from academic and applied research summarized in various references has been critical in the creation of this book which is best viewed as a comprehensive compilation and collection of information prepared by various official agencies which produce publications on streptococcus. Books in this series draw from various agencies and institutions associated with the United States Department of Health and Human Services, and in particular, the Office of the Secretary of Health and Human Services (OS), the Administration for Children and Families (ACF), the Administration on Aging (AOA), the Agency for Healthcare Research and Quality (AHRQ), the Agency for Toxic Substances and Disease Registry (ATSDR), the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), the Healthcare Financing Administration (HCFA), the Health Resources and Services Administration (HRSA), the Indian Health Service (IHS), the institutions of the National Institutes of Health (NIH), the Program Support Center (PSC), and the Substance Abuse and Mental Health Services Administration (SAMHSA). In addition to these sources, information gathered from the National Library of Medicine, the United States Patent Office, the European Union, and their related organizations has been invaluable in the creation of this book. Some of the work represented was financially supported by the Research and Development Committee at INSEAD. This support is gratefully acknowledged. Finally, special thanks are owed to Tiffany Freeman for her excellent editorial support.

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About the Editors James N. Parker, M.D. Dr. James N. Parker received his Bachelor of Science degree in Psychobiology from the University of California, Riverside and his M.D. from the University of California, San Diego. In addition to authoring numerous research publications, he has lectured at various academic institutions. Dr. Parker is the medical editor for health books by ICON Health Publications. Philip M. Parker, Ph.D. Philip M. Parker is the Eli Lilly Chair Professor of Innovation, Business and Society at INSEAD (Fontainebleau, France and Singapore). Dr. Parker has also been Professor at the University of California, San Diego and has taught courses at Harvard University, the Hong Kong University of Science and Technology, the Massachusetts Institute of Technology, Stanford University, and UCLA. Dr. Parker is the associate editor for ICON Health Publications.

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About ICON Health Publications To discover more about ICON Health Publications, simply check with your preferred online booksellers, including Barnes&Noble.com and Amazon.com which currently carry all of our titles. Or, feel free to contact us directly for bulk purchases or institutional discounts: ICON Group International, Inc. 4370 La Jolla Village Drive, Fourth Floor San Diego, CA 92122 USA Fax: 858-546-4341 Web site: www.icongrouponline.com/health

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Table of Contents FORWARD .......................................................................................................................................... 1 CHAPTER 1. STUDIES ON STREPTOCOCCUS ....................................................................................... 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Streptococcus ................................................................................ 5 E-Journals: PubMed Central ....................................................................................................... 61 The National Library of Medicine: PubMed ................................................................................ 94 CHAPTER 2. NUTRITION AND STREPTOCOCCUS ........................................................................... 143 Overview.................................................................................................................................... 143 Finding Nutrition Studies on Streptococcus ............................................................................. 143 Federal Resources on Nutrition ................................................................................................. 146 Additional Web Resources ......................................................................................................... 147 CHAPTER 3. ALTERNATIVE MEDICINE AND STREPTOCOCCUS..................................................... 149 Overview.................................................................................................................................... 149 National Center for Complementary and Alternative Medicine................................................ 149 Additional Web Resources ......................................................................................................... 165 General References ..................................................................................................................... 167 CHAPTER 4. DISSERTATIONS ON STREPTOCOCCUS....................................................................... 169 Overview.................................................................................................................................... 169 Dissertations on Streptococcus .................................................................................................. 169 Keeping Current ........................................................................................................................ 171 CHAPTER 5. PATENTS ON STREPTOCOCCUS ................................................................................. 173 Overview.................................................................................................................................... 173 Patents on Streptococcus ........................................................................................................... 173 Patent Applications on Streptococcus........................................................................................ 201 Keeping Current ........................................................................................................................ 231 CHAPTER 6. BOOKS ON STREPTOCOCCUS ..................................................................................... 233 Overview.................................................................................................................................... 233 Book Summaries: Federal Agencies............................................................................................ 233 Book Summaries: Online Booksellers......................................................................................... 235 Chapters on Streptococcus ......................................................................................................... 236 CHAPTER 7. PERIODICALS AND NEWS ON STREPTOCOCCUS ....................................................... 241 Overview.................................................................................................................................... 241 News Services and Press Releases.............................................................................................. 241 Newsletter Articles .................................................................................................................... 243 Academic Periodicals covering Streptococcus............................................................................ 244 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 247 Overview.................................................................................................................................... 247 NIH Guidelines.......................................................................................................................... 247 NIH Databases........................................................................................................................... 249 Other Commercial Databases..................................................................................................... 251 The Genome Project and Streptococcus ..................................................................................... 251 APPENDIX B. PATIENT RESOURCES ............................................................................................... 255 Overview.................................................................................................................................... 255 Patient Guideline Sources.......................................................................................................... 255 Finding Associations.................................................................................................................. 259 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 261 Overview.................................................................................................................................... 261 Preparation................................................................................................................................. 261 Finding a Local Medical Library................................................................................................ 261 Medical Libraries in the U.S. and Canada ................................................................................. 261

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ONLINE GLOSSARIES................................................................................................................ 267 Online Dictionary Directories ................................................................................................... 267 STREPTOCOCCUS DICTIONARY ........................................................................................... 269 INDEX .............................................................................................................................................. 351

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FORWARD In March 2001, the National Institutes of Health issued the following warning: "The number of Web sites offering health-related resources grows every day. Many sites provide valuable information, while others may have information that is unreliable or misleading."1 Furthermore, because of the rapid increase in Internet-based information, many hours can be wasted searching, selecting, and printing. Since only the smallest fraction of information dealing with streptococcus is indexed in search engines, such as www.google.com or others, a non-systematic approach to Internet research can be not only time consuming, but also incomplete. This book was created for medical professionals, students, and members of the general public who want to know as much as possible about streptococcus, using the most advanced research tools available and spending the least amount of time doing so. In addition to offering a structured and comprehensive bibliography, the pages that follow will tell you where and how to find reliable information covering virtually all topics related to streptococcus, from the essentials to the most advanced areas of research. Public, academic, government, and peer-reviewed research studies are emphasized. Various abstracts are reproduced to give you some of the latest official information available to date on streptococcus. Abundant guidance is given on how to obtain free-of-charge primary research results via the Internet. While this book focuses on the field of medicine, when some sources provide access to non-medical information relating to streptococcus, these are noted in the text. E-book and electronic versions of this book are fully interactive with each of the Internet sites mentioned (clicking on a hyperlink automatically opens your browser to the site indicated). If you are using the hard copy version of this book, you can access a cited Web site by typing the provided Web address directly into your Internet browser. You may find it useful to refer to synonyms or related terms when accessing these Internet databases. NOTE: At the time of publication, the Web addresses were functional. However, some links may fail due to URL address changes, which is a common occurrence on the Internet. For readers unfamiliar with the Internet, detailed instructions are offered on how to access electronic resources. For readers unfamiliar with medical terminology, a comprehensive glossary is provided. For readers without access to Internet resources, a directory of medical libraries, that have or can locate references cited here, is given. We hope these resources will prove useful to the widest possible audience seeking information on streptococcus. The Editors

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From the NIH, National Cancer Institute (NCI): http://www.cancer.gov/cancerinfo/ten-things-to-know.

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CHAPTER 1. STUDIES ON STREPTOCOCCUS Overview In this chapter, we will show you how to locate peer-reviewed references and studies on streptococcus.

The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and streptococcus, you will need to use the advanced search options. First, go to http://chid.nih.gov/index.html. From there, select the “Detailed Search” option (or go directly to that page with the following hyperlink: http://chid.nih.gov/detail/detail.html). The trick in extracting studies is found in the drop boxes at the bottom of the search page where “You may refine your search by.” Select the dates and language you prefer, and the format option “Journal Article.” At the top of the search form, select the number of records you would like to see (we recommend 100) and check the box to display “whole records.” We recommend that you type “streptococcus” (or synonyms) into the “For these words:” box. Consider using the option “anywhere in record” to make your search as broad as possible. If you want to limit the search to only a particular field, such as the title of the journal, then select this option in the “Search in these fields” drop box. The following is what you can expect from this type of search: •

Streptococcus Milleri: A Cause of Pyogenic Liver Abscess Source: Journal of the National Medical Association. 93(7-8): 276-277. July-August 2001. Contact: Available from National Medical Association. 1012 Tenth Street, NW, Washington, DC 20001. (202) 347-1895, ext. 267. Website: www.NMAnet.org. Summary: Anemia, leukocytosis (high levels of white blood cells in the blood), elevated abnormal liver function enzymes, hypoalbuminemia (low levels of protein in the blood), fever, and right upper quadrand abdominal pain are common signs and symptoms of liver abscesses. Mortality is high: 100 percent without treatment, and 50 to 65 percent with medical treatment. The bacteria Streptococcus milleri has been found to be associated with liver abscesses significantly more frequently than any other streptococci. S. milleri is also a common cause of liver abscess in patients with Crohn's disease. This

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article reports a case of S. milleri in a 47 year old patient with a history of hypertension, alcohol abuse, and tobacco smoking. The authors stress that increased awareness of S. milleri has come from better isolation of Streptococci species. The clinical importance of this awareness is that S. milleri is resistant to metronidazole. Therefore, patients with liver abscesses who receive metronidazole may not respond if S. milleri is the infecting organism. Effective antibiotics include ampicillin, erythromycin, clindamycin, and the cephalosporins. 1 figure. 1 table. 3 references. •

Prognosis of Streptococcus Pneumoniae-Induced Hemolytic Uremic Syndrome Source: Pediatric Nephrology. 16(4): 362-365. April 2001. Contact: Available from Springer-Verlag. Service Center Secaucus, 44 Hartz Way, Secaucus, NJ 07094. (201) 348-4033. Summary: Streptococcus pneumoniae induced hemolytic uremic syndrome (HUS) is known to be a severe acute disease leading to death in one third of cases, but data regarding the long term followup are lacking. This article reports on a series of 11 patients with HUS induced by the bacteria Streptococcus pneumoniae; all patients had meningitis and pneumonia. Among 9 patients with a severe acute infectious disease, 3 died from meningitis (infection of the meninges, or outer membranes of the brain) and 1 from neurological sequelae after a partial recovery of renal (kidney) function. The mean duration of dialysis was 32 days in patients with acute renal failure (ARF) who survived the acute infectious period. Cortical necrosis (tissue death) was documented in five of six kidney specimens. Among the 7 surviving patients, 5 developed end stage renal disease (ESRD) between 4 and 17 years later. The authors note that S. pneumoniae induced HUS is a rare disease, and they recommend the use of a registry of patients, in order to follow prospectively a larger number of patients. 1 table. 21 references.

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Acute Otitis Media: Management and Surveillance in an Era of Pneumococcal Resistance-A Report from the Drug-Resistant Streptococcus Pneumoniae Therapeutic Working Group Source: Pediatric Infectious Disease Journal. 18(1): 1-9. January 1999. Contact: Available from Lippincott Williams and Wilkins. P.O. Box 350, Hagerstown, MD 21740-0350. Website: www.lww.com. Summary: This article summarizes the report of a therapeutic working group that was convened to provide consensus recommendations for the management of acute otitis media (AOM) and the surveillance of drug resistant Streptococcus pneumoniae (DRSP). Five areas were addressed, considering published and unpublished data from the scientific literature and from the experts present. Oral amoxicillin should remain the first line antimicrobial agent for treating AOM. In view of the increasing prevalence of DRSP, the safety of amoxicillin at higher than standard dosages and evidence that higher dosages of amoxicillin can achieve effective middle ear fluid concentrations, an increase in the dosage used for empiric treatment from 40 to 45 mg per kg per day to 80 to 90 mg per kg per day is recommended. For patients with clinically defined treatment failure after 3 days of therapy, useful alternative agents include oral amoxicillin clavulanate, cefuroxime axetil and intramuscular ceftriaxone. Many of the 13 other FDA approved otitis media drugs lack good evidence for efficacy against DRSP. Currently local surveillance data for pneumococcal resistance that are relevant for the clinical management of AOM are not available from most areas in the U.S. Recommendations to improve surveillance include establishing criteria for setting susceptibility breakpoints for clinically appropriate antimicrobials to ensure relevance for treating AOM, testing

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middle ear fluid or nasal swab isolates in addition to sterile site isolates and testing of drugs that are useful in treating AOM. The authors stress that these recommendations are intended to provide a framework for appropriate clinical and public health responses to this problem. 4 tables. 59 references. (AA-M). •

Streptococcus Mutans, Early Childhood Caries and New Opportunities Source: JADA. Journal of the American Dental Association. 130(12): 1787-1792. December 1999. Summary: This commentary article, written by the director of the National Institute of Dental and Craniofacial Research, discusses the prevalence of early childhood caries (cavities) and the role of Streptococcus mutans (a caries promoting bacteria). The author begins by reviewing epidemiological data that shows dental caries are at epidemic proportions among lower income children. The author contends that the condition of early childhood caries (ECC) results from a complication combination of factors, including human and microbial genetics, microbial ecology, and the complexities of pathogen, host, and environment interactions. One section describes the S. mutans bacteria in some detail, then discusses other factors related to caries susceptibility, including bottle habits, early dietary habits, and malnutrition. The author then turns to prevention strategies, including antimicrobial therapy, the oral application of genetically engineered antibodies, and fluoridation. Research studies continue to investigate the associations among strains of S. mutans, parent-child transmission, and susceptibility to ECC. The author reminds readers that educational interventions, combined with regular preventive care, can go a long way in helping to prevent ECC. One sidebar offers three resources through which readers can obtain additional information. 1 table. 25 references.

Federally Funded Research on Streptococcus The U.S. Government supports a variety of research studies relating to streptococcus. These studies are tracked by the Office of Extramural Research at the National Institutes of Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other institutions. Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen. You will have the option to perform targeted searches by various criteria, including geography, date, and topics related to streptococcus. For most of the studies, the agencies reporting into CRISP provide summaries or abstracts. As opposed to clinical trial research using patients, many federally funded studies use animals or simulated models to explore streptococcus. The following is typical of the type of information found when searching the CRISP database for streptococcus:

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Healthcare projects are funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Healthcare Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH).

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Project Title: AGE ASSOCIATED CHANGES IN B LYMPHOCYTE FUNCTION Principal Investigator & Institution: Bondada, Subbarao A.; Professor; Microbiology Immunology, and Molecular Genetics; University of Kentucky 109 Kinkead Hall Lexington, Ky 40506 Timing: Fiscal Year 2002; Project Start 01-AUG-1986; Project End 31-JUL-2006 Summary: (provided by applicant): Immune responses to pneumococcal polysaccharide antigens are important for protection against pneumococcal infections but are absent in neonates and are reduced in the aged. Using Pnu-Imune vaccine that is made up of 23 serotypes of pneumococcal polysaccharides and TNP-Ficoll, two type 2 thymus independent (TI)-2 antigens, we have shown that neonates and the aged have an accessory cell deficiency in addition to their B-cell defects. This deficiency is overcome by supplementation with accessory cell derived cytokines, IL-1 and IL-6. The neonatal macrophages make less IL-l and IL-12 in response to TI stimuli such as TNP-Ficoll and lipopolysaccharide. This application is directed at testing the molecular basis of accessory cell defects leading to B-cell unresponsiveness to polysaccharide antigens in the neonates and the aged. It is hypothesized that toll-like receptor (TLR) expression and/or TLR signaling pathways may be defective in neonatal macrophages. Also, B-cell macrophage interactions required for TI responses may involve the macrophage derived cytokine BLyS and its B-cell receptors, TACI and BCMA, which may be dysfunctional in the neonate. Towards this goal five specific aims are proposed. 1. To determine if the defect in neonatal and the aged macrophages is limited to IL-1 and IL-12 or is a general property of macrophages in the neonate and the aged. 2. To test the hypothesis that the molecular nature of the defect in neonatal accessory cells that contributes to TI-2 unresponsiveness is in TLR expression or in the TLR signaling pathway. 3. To determine the role of dendritic cells in neonatal and aged unresponsiveness to polysaccharide antigens. 4. To determine if the restoration of neonatal B-cell responses to polysaccharides is due to their ability to signal neonatal B-cells/accessory cells via TLR signaling pathway. 5. To determine the role of monocyte-derived TNF like molecule, BLyS and its receptor (TACI), in B-cell-macrophage interactions and to determine if neonates or aged have defects in this ligand receptor system. Results from these studies should in future allow us to design and test agents that can augment accessory cell function and in turn to develop more effective polysaccharide vaccines for the neonate and the aged. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ALCOHOL, APOPTOSIS AND LUNG OPPORTUNISTIC INFECTION Principal Investigator & Institution: D'souza, Nympha B.; Medicine; University of Kentucky 109 Kinkead Hall Lexington, Ky 40506 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2008 Summary: (provided by applicant): The long-term objective of this proposal is to elucidate the mechanisms by which chronic alcohol (EtOH) ingestion predisposes the host to pulmonary infections. In spite of years of extensive research, the precise mechanisms whereby pulmonary infections develop in alcoholics are not fully elucidated. Therefore, novel approaches need to be undertaken to further our understanding of how these infections develop. Based on the gaps in our understanding of why alcoholics have a greater propensity to acquire lung infections, and the recently discovered pivotal role for apoptosis in health and diseases, the project proposes to test three hypotheses: 1) EtOH alters basal rate of lung apoptosis. The primary targets of this EtOH effect are the alveolar macrophages (AM) and during an infection, also infiltrating

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cells; 2) EtOH alters apoptosis of lung immune cells by modulating the amount and interaction of apoptotic protein adapters; and 3) The EtOH-induced apoptotic death of lung cells overwhelms the inherent clearance mechanisms in the lung. As a result, the uncleared dead cells undergo secondary, necrosis leading to persistent lung inflammation. Accordingly, the proposal has four specific aims: 1) to determine the temporal relationship between EtOH-altered rate of lung apoptosis and severity of infection; 2) to determine the lung cell types undergoing apoptosis following EtOH ingestion and infection; 3) to determine the mechanism(s) underlying EtOH effects on lung immune cell apoptosis and 4) to determine the temporal relationship between EtOH-induced alterations in the kinetics of lung apoptosis and the ability of AM to remove apoptotic cells. To achieve these goals, mice will be fed EtOH for various periods and infected with Streptococcus pneumoniae (SP). These mice will be used to assess the apoptotic and pathologic state of the lung. To evaluate the potential deleterious effects of EtOH enhanced lung cell apoptosis, the lung propensity, to clear an SP infection, will be evaluated in presence and absence of apoptosis inhibitors. By applying biochemical, molecular and cell biology techniques, the steps of apoptotic signaling pathway where EtOH acts will be established. Finally, the project will determine if there is a correlation between EtOH-induced apoptosis, decreased phagocytosis, and increased inflammation. The project will provide essential and novel information to: 1) broaden our understanding of how EtOH increases the hosts' susceptibility to pulmonary infections and 2) pave the way to developing therapeutic interventions to control pulmonary infections. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ALTERNATIVE APPROACHES FOR E FAECALIS INFECTIONS Principal Investigator & Institution: Murray, Barbara E.; Professor of Medicine; Internal Medicine; University of Texas Hlth Sci Ctr Houston Box 20036 Houston, Tx 77225 Timing: Fiscal Year 2002; Project Start 01-AUG-2000; Project End 31-MAY-2005 Summary: (Adapted from the Applicant's Abstract): Enterococci are the 3rd most common cause of endocarditis, behind streptococci and staphylococci, and the 2nd or 3rd most common cause of hospital acquired infections, with Enterococcus faecalis being the predominant species isolated. Antimicrobial resistance likely facilitates the establishment of enterococci in nosocomial infections and certainly makes it more difficult to successfully treat patients, particularly those with endocarditis. The central hypothesis of this project is that by better understanding enterococci, new therapeutic or preventative modalities can be developed. Work during a previously funded grant identified and characterized a number of antigen encoding genes; a polysaccharide gene cluster (epa) that appears to influence virulence in mice; different adherence phenotypes, and a gene, ace, that appears to be involved in adherence; and a gene locus with homology to the accessory gene regulator (agr) locus of staphylococci that is involved in expression of an E. faecalis gelatinase and a serine protease that also influence virulence in mice. In this application, the investigators propose (1) to verify that the E. faecalis agr-like locus regulates gelE and sprE and determine if all are important for virulence; to investigate the distribution of these genes among E. faecalis; and to determine how the enterococcal agr-like locus is regulated and if it, like the staphylococcal agr, regulates other genes. They also plan (2) to test the hypothesis that Ace (a newly described adhesin for collagen of enterococci) is the cause of the adherence we have reported and is important for virulence; to explore the regulation of Ace production; and to determine the distribution and effect of variations in ace, if Ace elicits an antibody response in humans (using recombinant Ace and patient sera)

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infected by E. faecalis and if antibody made during infection, or antibody to recombinant Ace, is protective. In their third specific aim, they plan (3) to establish if the polysaccharide gene cluster is the cause of a recently described mucoid phenotype, to study its regulation, and to further test its contribution to adherence to foreign material, virulence and protection. They will also explore a system for constructing non-polar deletion mutants using counter-selection based on their prior work with the E. faecalis pyr genes, and to explore additional assays that would help to avoid lethality models. The investigators hope that results from this work will provide solid leads in the quest for methods to prevent, control, or combat E. faecalis infections. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ANALYSIS OF MGA PROTEIN FROM THE STREPTOCOCCUS PYOGENES Principal Investigator & Institution: Mciver, Kevin S.; Assistant Professor; Microbiology; University of Texas Sw Med Ctr/Dallas Dallas, Tx 753909105 Timing: Fiscal Year 2002; Project Start 30-SEP-2001; Project End 31-MAY-2005 Summary: (provided by applicant): The group A streptococcus (Streptococcus pyogenes, GAS) is a bacterial pathogen of enormous medical importance to humans, causing a variety of disease syndromes that range in severity from minor to lifethreatening. Mga is a DNA-binding protein of GAS that activates the transcription of several key virulence genes in response to changing environmental conditions, likely through interactions with other regulatory components in the cell. The Mga regulon encodes products essential for the survival of GAS in the host, including the antiphagocytic M protein, M-like immunoglobulin binding proteins, the secreted inhibitor of complement, a collagen-like protein, and a C5a peptidase. Thus, Mga provides an excellent model system to study global regulatory networks involved in GAS pathogenesis and how they may interact. However, we currently know very little about Mga, including what domains of the protein are critical for its function and how environmental signals control the Mga regulon. The specific aims of this project are: (1) To identify domains of Mga involved in DNA-binding and characterize their role in targeting specific promoters; (2) To determine a consensus Mga binding element for each of the known promoter sites through identification of specific Mga/nucleotide interactions; (3) To investigate whether domains of Mga interact directly with other bacterial components to transduce environmental signals (i.e., is Mga a two-component response regulator?); (4) To identify additional factors required for the environmental regulation of the Mga regulon and assess their role in global virulence regulation. An attractive feature of this proposal is our ability to study Mga both as a purified protein in vitro and as a native molecule in its GAS background. As such, we will be able to thoroughly address key questions of GAS pathogenesis associated closely with the environmental regulation of the Mga regulon. The overall objectives of this proposal are (A) to undertake a structure/function analysis of Mga and determine the mechanisms by which this key GAS virulence regulator contributes to streptococcal disease, and (B) improve our general understanding of regulatory pathways that broadly control virulence in these gram-positive pathogens. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ANTIBODY RESPONSE TO PNEUMOCOCCAL CARRIAGE IN HUMANS Principal Investigator & Institution: Mccool, Tera L.; Microbiology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104

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Timing: Fiscal Year 2003; Project Start 01-JUN-2003; Project End 31-MAY-2006 Summary: (provided by applicant): An analysis of the immune response to pneumococcal proteins expressed during carriage in humans may reveal potential protein vaccine candidates. Half of the subjects in an experimental model of human carriage became colonized for 27-122 days; the other subjects were naturally resistant to colonization by the type 23F pneumococcus. IgG antibodies that recognized a 22 kD band (subsequently identified as the truncated N-terminus of pneumococcal surface protein A (PspA)) appeared after inoculation with pneumococcus in colonized subjects (6 of 7), while in all uncolonized subjects (7 of 7), PspA-specific antibodies were present prior to inoculation, suggesting a correlation between presence of PspA-specific IgG and resistance to colonization. The human serum and secretory antibody response to PspA was further elucidated by ELISA. This proposal examines the potential for PspA as a vaccine candidate against pneumococcal colonization and utilizes rational vaccine design based on the susceptibility to carriage in humans to identify novel vaccine candidates as well as to evaluate previously described vaccine antigens. In addition, the conservation of PspA expression in clinical isolates of pneumococcus will be examined to determine the effectiveness of a protein vaccine based soley on PspA. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ANTI-INFECTIVES WITH MULTI-DOMAIN RIBOSOMAL BINDING Principal Investigator & Institution: Katz, Leonard; Vice Present of Biological Sciences; Kosan Biosciences 3832 Bay Center Pl Hayward, Ca 94545 Timing: Fiscal Year 2002; Project Start 15-JUN-2001; Project End 31-MAY-2003 Summary: Resistance to macrolide antibiotics has increased at alarming rates in recent years, driving the need to develop new and more effective antibiotics. The long term objective of this proposal is to develop a novel 16-membered macrolide antibiotic that is active against erythromycin-resistant Streptococcus pneumoniae and other Gram positive pathogens and which can be produced at reasonable cost. The proposed compound is designed to exhibit its potency through the novel mechanism of synergistic multi-domain ribosomal binding. Consequently, the compound should not induce macrolide resistance and evade all known efflux mechanisms that confer macrolide resistance. Phase I is a proof of principle project to produce a small series of derivatives of a 16- membered macrolide that is a readily available fermentation product, and determine whether the derivatives bind to domain II of the ribosomes and exhibit increased potency against macrolide-resistant strains. Phase II Specific Aims will be to optimize the derivatives to achieve oral bioavailability. Lead compounds will be examined in vitro and in animals for efficacy, toxicity and pharmacokinetics with the intent of advancing one or more to clinical development. PROPOSED COMMERCIAL APPLICATION: Clinical development candidates could be commericialized as antiinfective agents only after approval by the appropriate regulatory authorities. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: B7 COSTIMULATION RESPONSE TO EXTRACELLULAR BACTERIA Principal Investigator & Institution: Snapper, Clifford M.; Professor; Henry M. Jackson Fdn for the Adv Mil/Med Rockville, Md 20852 Timing: Fiscal Year 2002; Project Start 01-JUL-2000; Project End 31-MAR-2005 Summary: (Adapted from the Applicant's Abstract): Infections with extracellular, polysaccharide (PS)-encapsulated, bacteria represent a major source of morbidity and mortality in the U.S. Increasing antibiotic resistance to these agents, makes their control

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by immunotherapeutic means more compelling. Induction of PS- and protein-specific Ig play major roles in immunity to these bacteria. Preliminary results, using a model grampositive extracellular bacterium, Streptococcus pneumoniae (strain R36A), indicate that both PS- and protein-specific humoral responses to R36A are T cell-dependent and B7 ligand-dependent. Modulating B7 ligand interactions has therapeutic potential for modifying the ongoing immune response and for vaccine development, yet few studies have examined the role of these interactions during the T cell-dependent immune response to bacterial pathogens. This proposal will examine the role of B7 interactions during primary and memory Ig isotype responses to the PS and protein components of R36A. CD28 and CTLA-4 function will be examined using genetically deficient mice and blocking antibodies with the working hypothesis that these molecules differentially regulate the progression of the anti-PS and anti-protein response and that they may be useful targets for modifying this in vivo immune response, both at the initiation stage and subsequent to immunization. The individual roles of B7-1 and B7-2 will also be examined, and the specific APCs that provide B7-1 vs. B7-2-mediated costimulation will be identified using adoptive transfer experiments in genetically deficient mice. These experiments will provide insight into the mechanism of why B7-1 vs. B7-2 blockade differentially influences the R36A response. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BACTERIAL CONJUGATE VACCINES Principal Investigator & Institution: Madhi, Shabir; Univ of Witwaterstrand Johannesburg, Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-MAY-2007 Summary: Evaluation of quantitative and qualitative antibody responses to Streptococcus pneumoniae and Haemophilus influenzae type b conjugate vaccines The vertical transmission of HIV-1 infection from mother to infant can be reduced by at least two-thirds using simple perinatal anti-retroviral (ARV) drug regimens. Nevertheless, at least 10-12% of children born to HIV-1 infected mothers will still be HIV-1 infected. The management of these children through anti-retroviral treatment (ART) and prophylaxis against common opportunistic pathogens has been successful in developed countries. Implementation of such an intervention in resource-constrained countries will only be possible through novel strategies. Maj or causes of morbidity and mortality in HIV-1 infected children are infection by Pneumocystis carinii and bacteria, specifically Streptococcus pneumoniae and Haemophilus influenzae type b (Hib). Prophylaxis against these pathogens includes co-trirnoxazole and the new conjugate vaccines. This project will allow for an evaluation of the effectiveness of cotrimoxazole prophylaxis in HIV-1 infected children and its impact on selecting for antibiotic-resistant bacteria. Furthermore, the impact of ART on the immunogenicity of the conjugated pneumococcal and Hib vaccines will be evaluated in 250 HIV-1 infected children receiving ART and 125 who are not receiving ART at the time of their primary series of vaccines. These children will also be compared to HIV uninfected infants, born to seronegative (n = 125) and seropositive (n = 125) mothers. Both quantitative and qualitative antibody responses to these vaccines will be evaluated. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: BACTERIAL GENE EXPRESSION IN MODEL ORAL BIOFILMS Principal Investigator & Institution: Jacques, Nicholas A.; Principal Research Scientist; Institute of Dental Research 2 Chalmers St Sydney,

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Timing: Fiscal Year 2001; Project Start 20-SEP-1999; Project End 31-JUL-2004 Summary: It is now recognised that the transition of microbial species from a free-living, suspended or planktonic state to a component of an adherent community involves fundamental behavioural change. Both dental caries and periodontal diseases are essentially dependent on the polymicrobial plaque that develops with increasing complexity after the initial colonization of salivary components that coat the tooth surface. While there has been extensive analysis of the growth of dental plaque organisms as mixed communities in the planktonic state, to date there is little knowledge of interactions between adherent bacteria. The proposed studies will employ two powerful and complementary new technologies to monitor changes in gene expression occurring during initial colonisation and maturation of a model biofilm. Recognition of the factorial expansion of complexity in polymicrobial systems limits detailed analysis to two key microorganisms, Streptococcus gordonii as a major early coloniser of the acquired salivary pellicle and Streptococcus mutans which is strongly implicated as a major pathogen initiating dental caries. Both organisms can be genetically manipulated. The technique of In Biofilm Expression Technology (IBET) will enable comprehensive analysis of new gene expression patterns during biofilm formation on saliva-coated hydroxyapatite while Proteome analysis will facilitate recognition of altered protein profiles, particularly at the cell surface. Structural and time course analysis of altered gene expression patterns in situ will be enhanced by Confocal Laser Scanning Microscopy. Regulatory mechanisms relating to surface adhesins, the influence of extracellular sugar polymers, the response to unfavourable environments including acidic conditions and to the activity of key enzyme activities will be a focus. These parameters are highly pertinent and are the subject of study at the Institute. This program of research will provide essential data necessary for a therapeutic strategy aimed at the establishment and maintenance of a non-pathogenic dental plaque. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BACTERIAL VACCINE ANTIGEN DISCOVERY Principal Investigator & Institution: Paoletti, Lawrence C.; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-MAR-2007 Summary: (provided by applicant): New directions in bacterial vaccine discovery may arise from studies of host-microbe interactions, particularly through the use of a newly described technology: the dynamic in vitro attachment and invasion system (DIVAS). DIVAS was developed to study bacterial attachment and invasion with cells held at specific and controlled conditions of growth, metabolism, and nutrient levels. Results from experiments performed with DIVAS and group B Streptococcus (GBS) type III strains substantiated earlier findings that capsular polysaccharide is not critical for invasion of respiratory epithelial cells. Moreover, GBS invaded these cells only when held at a fast as opposed to a relatively slower rate of growth and they expressed several proteins solely under growth conditions conducive for invasion. In this proposal, we seek to test the hypothesis that GBS proteins involved with invasion of eukaryotic cells are new and important targets of protective immunity. GBS is a major cause of neonatal sepsis and meningitis, and is increasingly prevalent among non-pregnant adults and the elderly with underlying illnesses. Preclinical and clinical trials have been successfully performed with protein conjugate vaccines prepared with GBS polysaccharides. GBS proteins with virulence properties have been described and some with vaccine potential have been tested preclinically. In this proposal, we will use DIVAS to identify

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physiological conditions conducive for bacterial attachment/invasion of eukaryotic cells using GBS as a model pathogen. We will isolate and identify GBS membrane proteins expressed solely under invasive conditions (Sp. Aim 1). GBS mutants lacking genes for these proteins will be constructed and tested for invasiveness in DIVAS and virulence in mice (Sp. Aim 1). Several of these proteins will be purified directly from GBS, or cloned and recombinantly expressed, and tested as vaccine candidates in mice (Sp. Aim 2). Findings from these studies utilizing this unique approach to vaccine antigen discovery could be directly applied to other bacterial pathogens. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: BINDING ENDOCARDITIS

PROTEINS

AND

ORAL

STREPTOCOCCAL

Principal Investigator & Institution: Holt, Robert G.; Associate Professor; Meharry Medical College 1005-D B Todd Blvd Nashville, Tn 37208 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2007 Summary: Infective endocarditis is the most common serious and life-threatening cardiac infection in the United States. The number of cases is expected to increase because of the increasing numbers of intravenous drug users and elderly individuals with underlying valvular degenerative changes. Both groups are at risk for the development of the disease. Over 50% of all cases of infective endocarditis are caused by oral streptococci of the mitis group and the mutans group of streptococci, where historically many members were called viridans streptococci. The generally nonnivasive Streptococcus mutans is a member of the dental caries-causing mutans group streptococci and is the most frequent species of the mutans streptococci isolated from the oral cavity of humans. The long term goal of this study is to broaden the understanding of the pathogenesis of infective endocarditis caused by oral streptococci. We hypothesize that mutans streptococcal cells have specific receptors that bind extracellular matrix molecules and fibrinogen and this interaction functions to facilitate colonization of heart tissue in the pathogenesis of endocarditis caused by mutans streptococci. The goal of this project is to identify and characterize the bacterial cell surface molecules that are involved in the adherence of heart tissue by mutans streptococci. Binding studies in our laboratory have indicated that S. mutans cells have the ability to bind to the human extracellular matrix molecules, fibronectin, laminin, and collagen type I, and the plasma protein, fibrinogen. Also, we have determined that an isogenic antigen I/II (spaF)-deficient mutant strain of S. mutans has a reduced ability to bind fibronectin, collagen and fibrinogen which suggests that antigen I/II contributes to the interaction of S. mutans cells with these molecules. The specific aims of this study are: 1) to clone and characterize genes encoding proteins that mediate the interaction of S. mutans cells with extracellular matrix molecules (fibronectin, taminin and collagen) and fibrinogen; 2) to construct mutant S. mutans strains having defects in their ability to bind extmcellular matrix molecules and fibrinogen; 3) to evaluate using a rat model of experimental endocarditis the role of binding of S. mutans cells to extracellular matrix molecules and fibrinogen in the pathogenesis of infective endocarditis; and 4) to examine the virulence of an antigen I/II(P 1) mutant strain using the rat model of endocarditis. The results of these studies should identify candidate antigens for the production of protective immunity against infective endocarditis caused by oral streptococci as well as provide information on virulence mechanisms that function during infective endocarditis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: BIOMARKER OF NEUROTOXICITY IN MENINGITIS Principal Investigator & Institution: Mulchahey, James J.; Assistant Professor; Phase 2 Discovery, Inc. 3130 Highland Ave, 3Rd Fl Cincinnati, Oh 45219 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2003 Summary: (provided by applicant): The objective of this Phase I application is to develop a sensitive biomarker for quantifying neurotoxicity and neuroprotectant efficacy in meningitis. Previous research documents that brain injury caused by meningitis affects multiple brain areas with a heterogeneous distribution. We have previously shown that the cytoskeletal protein MAP-tau is cleavedin brain during axonal degeneration. We developed a sensitive ELISA that specifically quantifies this biomarker of neuronal degeneration, cleaved-tau (C-tau). Our preliminary studies demonstrate that levels of C-tau are increased over 300-fold in an animal model of group B streptococcus meningitis (GBM). We will use the well-documented neurotoxicity of GBM to validate the C-tau as a biomarker of neurotoxicity and a measure of neuroprotectant efficacy. Our Specific Aims are: Specific Aim 1: Determine whether GBM results in a time-dependent elevation in brain, plasma and CSF concentrations of C-tau. Specific Aim 2: Determine it C-tau levels in brain, CSF and plasma correlate with traditional measures of neuronal damage in GBM. Specific Aim 3: Determine whether perihperal tissues such as liver, kidney or spleen are potential sources of C-tau measured in GBM. Specific Aim 4: Determine whether a neuroprotectant intervention known to be effective in GBM has predictable effects on Ctau levels. PROPOSED COMMERCIAL APPLICATIONS: The C-tau ELISA may be used to quantify the severity of brain injury in meningitis and to quantify the effects of neuroprotectant interventions in basic science, preclinical and clinical research settings. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CARIES PREVENTION IN ALASKAN NATIVE INFANTS Principal Investigator & Institution: Grossman, David; University of Washington Grant & Contract Services Seattle, Wa 98105 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2003 Summary: Alaska Native children are disproportionately affected by early childhood caries, compared to all U.S. children. Dental care needs for adults and children in rural Alaska far exceed the acute care and prevention resources available. As a result, there is a high level of dental morbidity present among adults that likely contributes to early transmission of mutants streptococci (MS) from adult caregivers to infants in the household. Furthermore, the cultural practice of per- mastication of solid food for infant feeding amplifies the transmission of oral secretions from adult to child. The prevention of early MS acquisition and subsequent caries in infants and toddlers required efforts starting at birth. Since Alaska Natives are a rural population at high risk for caries, interruption of vertical transmission of MS using a combination of improved oral hygiene practice, and topical antimicrobials and bacteriostatic agents may be an ideal prevention strategy for childhood caries. Chlorhexidine and xylitol are two agents that have been shown to reduce dental decay and MS counts. The specific aim of this proposal is to conduct a community based, randomized blinded trial to determine if the serial use of chlorhexidine and xylitol in 250 mothers will reduce the vertical transmission of caries between Alaska Native mothers and infants. The YukonKuskokwim (YK) Delta of southwestern Alaska is the site of the study. We hypothesize that a two week period of twice-daily chlorhexidine mouthwash use prior to deliver followed by a subsequent two year period of maternal xylitol gum use, will lead to a

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significant reduction in the age-specific prevalence of early childhood caries at 12 and 24 months of age among the offspring of mothers in the intervention group, compared to control group mothers. We also hypothesize that, compared to controls, mothers and children in the intervention group will have significant reductions in oral MS counts at each follow-up interval. If proven successful, this intervention could have a significant impact on the prevalence of caries among young Alaska Native children and other population groups at high risk for childhood caries. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CD-18 DEPENDENT/INDEPENDENT WBC RESPONSES IN THE LUNG Principal Investigator & Institution: Doerschuk, Claire M.; Professor of Pediatrics and Pathology; Pediatrics; Case Western Reserve University 10900 Euclid Ave Cleveland, Oh 44106 Timing: Fiscal Year 2003; Project Start 01-AUG-1994; Project End 31-MAY-2008 Summary: (provided by applicant): Neutrophil emigration into the lungs can occur through at least two different pathways depending upon the stimulus, one that requires the CD11/CD18 adhesion complex, and one that does not. Our studies provide evidence that mice deficient in the NF-kappaB p65 (Rel A) subunit, mice deficient in both TNF R1 and IL-1R1, or mice with blockade of ICAM-1 have defects in E. coli-induced CD18dependent emigration. In contrast, mice deficient in the leukocyte non-receptor Src tyrosine kinases Lyn, Fgr, and Hck, in the small GTPase Rac2, or in interferon-(IFN-g) have defects in S. pneumoniae-induced CD18-independent but not E. coli-induced CD18-dependent emigration. Moreover, exogenous IFN-( switches CD18-dependent to CD18-independent emigration, whereas genetic deficiency of IFN-( switches CD18independent to CD18-dependent emigration. Studies comparing gene expression during these bacterial pneumonias also provided many new ideas. Our goal is, to understand the mechanisms, through which CD18-dependent and CD18-independent adhesion pathways are elicited and function; and to identify ways of modulating the acute inflammatory process to benefit the host. Our working hypothesis is that neutrophil emigration occurs through CD11/CD18-dependent pathways when early stages of host defense result in nuclear translocation of NF-kappaB, production of TNF-alpha and IL-1, and increased expression of ICAM-1 on pulmonary capillary endothelial cells, while CD11/CD18- independent mechanisms are selected when IFN-( is produced and the leukocyte Src kinases Lyn, Fgr, and Hck and the small GTPase Rac2 are activated. The proposed Aims will test this hypothesis and examine the role of each of these required molecules in the mechanisms of neutrophil emigration. Aim 1 will determine the role of NF-(B -mediated gene transcription and the function of TNF-alpha and IL-1 in CD18dependent and -independent neutrophil emigration. Aim 2 will determine the role of IFN-g in CD18- independent emigration. Aim 3 will determine the role of Lyn, Fgr, and Hck and of Rac2, and the functional relationships between these molecules and IFN-g in neutrophil recruitment and function. Aim 4 will determine the functional role of molecules identified by gene microarray technology to be differentially expressed in S. pneumoniae but not E. coli pneumonia. These studies will help to elucidate the molecular mechanisms of neutrophil recruitment and identify potential targets for therapy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CHARACTERIZATION OF IRON TRANSPORT IN GBS Principal Investigator & Institution: Clancy, Kathryn A.; Children's Hospital and Reg Medical Ctr Box 5371, 4800 Sand Point Way Ne, Ms 6D-1 Seattle, Wa 98105 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2005 Summary: (provided by applicant): Group B streptococcus is the predominant etiological agent of neonatal bacteremia, sepsis and meningitis, and has been correlated with preterm membrane rupture and premature birth. While a limited number of GBS virulence determinants have been identified, the contribution of iron and iron acquisition to the pathogenesis of GBS infection is unknown. All pathogens require iron and a correlation between bacterial virulence and iron acquisition has been established. However, as the concentration of available iron in the human host (10 [-18]M) is well below that required by bacteria (10 [-8] M), successful pathogens express specific systems to acquire sufficient concentrations of intracellular iron. One such system involved in siderophore-mediated iron uptake, is the focus of this application. Siderophores are high-affinity iron chelators secreted from the bacterial cell to scavenge iron from host iron-binding proteins. Specialized uptake systems transport the siderophore-iron complex across the bacterial membrane. We have identified a putative siderophore-mediated iron transport system, the fhu operon, in GBS. The operon is comprised of four genes, fhuC, fhuD, fhuB, and fhuG, encoding a putative ATPhydrolysis protein, siderophore (ferrichrome)- iron receptor protein, and two permeases, respectively. In this application, the role of the fhu operon in iron acquisition by GBS will be examined. Basic information on the requirement of GBS for iron, the ability of GBS to utilize siderophores as an iron source, and whether the organism secretes siderophores to acquire iron will first be established. The biochemical characterization of an isogenic mutant strain deficient for fhu will examine the role of this operon in GBS siderophore-iron transport. In order to define the siderophore specificity of the fhu operon, the construction and biochemical characterization of isogenic mutant strains deficient for each gene is proposed. In addition, heterologous expression studies will provide further evidence for the role of the fhu-encoded proteins in siderophore-iron transport. These studies will provide a basic understanding of the molecular mechanism of iron acquisition in GBS and will allow us to design appropriate in vivo studies to determine the role of iron transport in GBS pathogenesis. The potential of identifying novel therapeutic targets to prevent or treat infections is also proposed. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CHEMISTRY AND IMMUNOLOGY OF STREPTOCOCCAL M PROTEINS Principal Investigator & Institution: Dale, James B.; Professor; Medicine; University of Tennessee Health Sci Ctr Memphis, Tn 38163 Timing: Fiscal Year 2002; Project Start 01-JUN-1996; Project End 31-MAY-2006 Summary: (provided by the applicant): The overall goal of this project is to develop a safe and broadly effective vaccine that will prevent group A streptococcal infections. Previous studies have shown that the surface M proteins, which are the major protective antigens, contain tissue-crossreactive epitopes as well as protective epitopes. The serotype-specific protective epitopes may be separated from potentially harmful autoimmune epitopes by using limited N-terminal peptides of M proteins. The protective M protein fragments representing multiple serotypes of group A streptococci may then be combined to form a multivalent vaccine. The specific aims of this proposal

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are: 1) To identify the primary structures of M proteins or other surface proteins that contain opsonic (protective) epitopes from serotypes of group A streptococci that are epidemiologically important and, therefore, necessary vaccine components, 2) To construct recombinant, multivalent vaccines that evoke optimal opsonic antibody responses in laboratory animals against 26 different serotypes of group A streptococci, 3) To test immune rabbit sera evoked by multivalent vaccines for opsonic and bactericidal antibodies against clinical isolates of group A streptococci collected from children with pharyngitis in 10 geographic sites in the U.S., 4) To develop strategies of intranasal delivery of multivalent M protein-based vaccines that result in secretory and systemic immune responses, and 5) To directly compare the protective immunogenicity of multivalent M protein-based vaccines delivered to mice via either the intramuscular or intranasal routes. In our preliminary studies, we have identified six epidemiologically important serotypes of group A streptococci that are not opsonized by antisera against the N-terminal M protein peptides. We propose a series of experiments to determine the covalent structures of the M proteins, M-like proteins, or other surface proteins that contain opsonic epitopes so that these M serotypes may be included in multivalent vaccines. We will construct a 26-valent vaccine composed of 4 different recombinant, hybrid proteins. The individual hybrid proteins will be tested for protective and tissuecrossreactive immunogenicity after intramuscular injection of rabbits. Because mucosal delivery of streptococcal vaccines may have both immunological and practical advantages over parenteral delivery, we will assess different strategies of intranasal delivery and then directly compare the protective efficacy of i.n. vs i.m. vaccines in mice. The studies should provide the detailed information needed to develop a safe and effective multivalent vaccine that could prevent the majority of streptococcal infections in North America and Western Europe. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CLINICAL CENTER IN COPD Principal Investigator & Institution: Bailey, William C.; Professor and Director; Medicine; University of Alabama at Birmingham Uab Station Birmingham, Al 35294 Timing: Fiscal Year 2003; Project Start 15-AUG-2003; Project End 31-JUL-2008 Summary: (provided by applicant): Chronic Obstructive Pulmonary Disease (COPD) is a serious public health problem responsible for more than 500, 000 hospitalizations, 100,000 deaths, and $15 billion in direct costs of medical care in the United States each year. We propose to form a collaborative COPD CRN Clinical Center at the University of Alabama at Birmingham (UAB) and the Birmingham Veterans Affairs Medical Center (BVAMC). The UAB/BVAMC Clinical Center will be headed by, Drs. William C. Bailey and J. Allen D. Cooper. The proposed investigators and their research staff have extensive experience in recruitment and retention for clinical trials, as well as study design and implementation. The collaborative effort between UAB and the BVAMC will allow us to quickly and efficiently recruit large numbers of COPD patients for clinical research studies. In addition, the UAB/BVAMC COPD Clinical Center will have access to a large network of experts at the University of Alabama at Birmingham who can assist in the design and implementation of clinical trials. The UAB/BVAMC Clinical Center proposes two studies aimed at enhancing treatment for moderate-to severe COPD. The first study will examine measures of inflammation and responsiveness to bronchodilator challenge, responsiveness to systemic steroid treatment, and responsiveness to inhaled steroid treatment. This study may provide a new method for identifying subgroups of COPD patients who are most likely to respond to inhaled corticosteroids. The second project will examine the causes of poor responsiveness to the

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pneumococcal vaccine with the long term goal of using this information to reduce pneumococcal infections and related exacerbations in COPD patients. The UAB/BVAMC Clinical Center also proposes a Clinical Research Skills Training Core. Dr. J. Allen D. Cooper, the Director for fellowship training in Pulmonary and Critical Care Medicine at UAB, will head this core. Trainees will be funded for a total of two years and will be required to enroll in one of the UAB K30 Clinical Research Curriculum Development Award components (either the Clinical Research Training Program or the Master's of Science in Public Health in Clinical Research). Trainees will also receive funding for a pilot project, which will be developed and conducted under the supervision of the Training Core Investigators. These Investigators have expertise in COPD, Clinical Research and Study Design, Behavioral Science, Epidemiology, and Statistics. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CLONING ANALYSIS OF S. MUTANS PUTATIVE COLLAGENASE Principal Investigator & Institution: Dao, My Lien L.; Biology; University of South Florida 4202 E Fowler Ave Tampa, Fl 33620 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-MAY-2005 Summary: Dental root decay is prevalent among older individuals as their gum recesses exposing Dental root surface to attack by cariogenic bacteria. In a study involving 449 subjects of an age range of 79-101 years, 96% had coronal decay experiences, and 64% had root caries experience with 23% of the group having untreated root caries (ADA News Releases, 2000). Streptococcus mutans, an etiologic agent in the development of coronal caries, has also been implicated in Dental root decay; data in support of this implication include the finding of S. mutans in Dental root section, its ability to bind collagen, and to degrade FALGPA, a known synthetic peptide substrate for collagenase. Bacterial collagenases are considered as virulence factors as they facilitate the invasion and destruction of host tissues by the pathogens. It is not yet known whether S. mutans produces a true collagenase enzyme. Considering the increase in incidence of Dental root caries as the population lives longer, the long-term goal of the current study is to develop effective and safe methods to control this disease, and improve the nutrition and quality of life of the population at risk. In order to determine whether the collagenolytic enzyme in S. mutans is a good candidate antigen for vaccine development, the Specific Aim of the current research is to learn more about the S. mutans enzyme in order to explore this avenue. A putative S. mutans collagenase gene has been obtained previously, and sequence analysis showed a high homology with the 35-kDa collagenase of various clinical isolates of Porphyromonas gingivalis, a bacterium causing periodontitis. The plan is to clone the 1.2 kbp putative collagenase coding sequence into an expression plasmid under the control of a strong promoter in order to obtain the corresponding protein, which in turn will be isolated and characterized by biochemical methods. Antibody will be prepared against the S. mutans enzyme and tested for the ability to block collagen binding and/or collagen degrading properties. The data obtained will be compared with other known bacterial collagenases. This information is essential in determining future directions for research on the role of S. mutans in Dental root caries, and other diseases that may involve collagen binding and collagen degrading activity such as periodontitis and endocarditis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: COMPLEMENT AS AN ADJUVANT IN POLYSACCHARIDE IMMUNITY Principal Investigator & Institution: Test, Samuel T.; Children's Hospital & Res Ctr at Oakland Research Center at Oakland Oakland, Ca 946091809 Timing: Fiscal Year 2002; Project Start 01-APR-2001; Project End 31-MAR-2004 Summary: (Adapted from Applicant's Abstract) The complement system plays a critical role in the antibody response to both thymus-dependent and thymus-independent antigens. Recently, the C3d fragment of complement C3 has been shown to have a profound adjuvant effect when coupled to a model T-dependent protein antigen and used to immunize mice. However, the utility of this approach for enhancing the humoral immune response to either T-independent or clinically relevant antigens has not been addressed. This project will test the hypothesis that C3d conjugated to the capsular polysaccharides of Streptococcus pneumoniae, clinically important Tindependent type 2 antigens, has an adjuvant effect similar to that seen for T-dependent antigens. Preliminary data show that conjugation of C3d to pneumococcal serotype 14 capsular polysaccharide results in a significant enhancement of the murine antibody response to this polysaccharide. The ultimate goal of these studies is to gain an understanding of the immune response to C3d-polysaccharide conjugates with proteinpolysaccharide conjugates in pneumococcal vaccines. The aims of this research are as follows. First to determine the effects of C3d conjugation on the humoral immune response to pneumococcal capsular polysaccharides as compared with the response to polysaccharides coupled to ovalbumin, a T-dependent protein carrier. The specific questions that will be addressed as part of this aim as follows: 1) Are there qualitative differences in the antibodies produced in response to immunization with the different types of polysaccharide conjugate? 2) What is the role of the complement system in the humoral immune response to the different conjugates? 3) What is the role of T lymphocytes in the antibody response to C3d-polysaccharide versus ovalbuminpolysaccharide conjugates? 4) Is the presence of a spleen necessary for mice to mount an effective humoral immune response to polysaccharide conjugates? 5) Within the spleen of immunized mice, how does the presence of C3d or ovalbumin coupled to pneumococcal polysaccharides influence the identity and distribution of the cells involved in the immune response? The second aim is to determine whether the adjuvant effect of C3d is a general phenomenon with respect to pneumococcal capsular polysaccharides. Conjugates of mouse C3d and capsular polysaccharide from serotypes 14, 6B and 23F S. pneumoniae will be prepared and evaluated in different strains of mice. These experiments should provide new insights into the immune response to conjugate vaccines and facilitate future efforts to improve vaccine efficacy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CONDITIONAL B INTEGRIN BLOCKADE, PMN TRAFFICKING Principal Investigator & Institution: Malik, Asrar B.; Distinguished Professor and Head; Pharmacology; University of Illinois at Chicago 1737 West Polk Street Chicago, Il 60612 Timing: Fiscal Year 2002; Project Start 10-JUL-2000; Project End 31-MAY-2005 Summary: Acute lung injury is characterized by polymorphonuclear leukocyte (PMN) accumulation in the pulmonary microcirculation and transalveolar PMNmigration, and resultant injury of the microvessel-alveolar barriers. The mechanisms of migration of PMN across both pulmonary microvessel endothelial and alveolar epithelial barriers and the role of transalveolar PMN migration in the pathogenesis of acute lung injury remain unclear. In the proposed studies, we will direct the expression of the selective

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anti-adhesive beta2 CD11/CD18 integrin binding protein, Neutrophil Inhibitory Factor (NIF), in a site- and inflammation- specific manner. NIF will be expressed in either pulmonary microvascular endothelium or type II alveolar epithelial cells using inducible cell-specific promoters. Constructs of NIF cDNA driven by the inducible endothelial cell-or type II alveolar epithelial cell-specific promoters (i.e., E-selectin and surfactant protein C promoters, respectively) will be introduced in mice to express NIF at endothelial and epithelial sites. Mice will be challenged with TNFalpha, or with gram negative or gram positive bacteria, E. coli or S. pneumoniae, administered intraperitioneally or directly into airway to simulate systemic or lung-localized infection. We will determine the effects of site- specific PMN beta2 integrin blockade in either microcirculation or alveolar space on the migration of PMN across microvessel- alveolar epithelial barriers and in the injury of these barriers. We will address the role of beta2 integrins in directing PMN traffic across the pulmonary microvessel endothelial and alveolar epithelial barriers and mechanism of injury of these barriers using a strategy based on cell-selective and inducible expression of beta2 integrin antagonist. In separate studies, we will determine using gene knockout mouse models the contributions of Lselectin (another PMN adhesion molecule involved in PMN migration) as well as ICAM-1 (a PMN beta2 integrin counter- receptor in endothelial and alveolar epithelial cells) and E- selectin (an endothelial cell-specific adhesion molecule) in the mechanisms of alveolar PMN migration and injury of microvessel and alveolar epithelial barriers. With the completion of these studies, new information will be obtained on the role of adhesion molecules in the mechanisms of PMN migration into the airspace and novel strategies will be developed to prevent acute lung injury based on the conditional and site-specific expression of a beta2 integrin antagonist. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CONSEQUENCES OF GROUP B STREPTOCOCCAL PROPHYLAXIS Principal Investigator & Institution: Sinha, Anushua; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 30-SEP-2000; Project End 31-MAY-2005 Summary: (adapted from applicant's abstract): This application for a K23 award combines coursework, a research plan, and personal mentoring to train Anushua Sinha M.D., M.P.H. for independence as a clinical investigator. Dr. Sinha is boarded in Internal Medicine, Pediatrics, and Infectious Diseases. She currently holds an individual NRSA from NIAID to develop and use automated medical record linkage systems to assess the epidemiology of infections during the first 30 days of life. Dr. Richard Platt (Harvard Medical School) and Dr. Milton Weinstein (Harvard School of Public Health) will serve as research mentors. The research builds on Dr. Sinha's NIAID-funded work to investigate the impact of maternal antibiotic prophylaxis against neonatal group B streptococcus on non-GBS infectious outcomes of both infants and mothers. The adoption in the mid-199Os of prophylaxis during labor for approximately 25% of women greatly reduced the occurrence of neonatal GBS disease. However, little is known about its effects on other antibiotic susceptible and resistant infections of mothers and infants, or more generally on the emergence of antimicrobial resistance. It is important to elucidate these issues, and to ascertain patient utilities, to guide decisions about whether and how to use a GBS vaccine that is currently being developed at the investigator's institution (The Streptococcal Initiative, PI: D. Kasper, NO1-AI-75326). The study thus has 5 specific aims: Assess the impact of GBS prophylaxis on 1) neonatal nonGBS infections; 2) maternal non-GBS outcomes, 3) antibiotic resistant infections among prophylaxis recipients, and among newborns and postpartum women generally; and 4)

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Streptococcus

resource utilization. Aim 5 assesses the cost effectiveness of antibiotic prophylaxis compared to immunization. Dr. Sinha will address these issues using direct patient interviews together with data from an existing cohort of 15,532 deliveries from 10/1/90 to 3/31/98 for which she has linked extensive, automated inpatient and outpatient records. Case-control and cohort methods will be used to address aims 14, and decision analysis for aim 5. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CPG DNA ADJUVANTS AND VACCINES FOR ENCAPSULATED BACTERIA Principal Investigator & Institution: Harding, Clifford V.; Professor; Pathology; Case Western Reserve University 10900 Euclid Ave Cleveland, Oh 44106 Timing: Fiscal Year 2002; Project Start 29-SEP-2000; Project End 31-MAY-2005 Summary: (Adapted from the Applicant's Abstract): CpG oligodeoxynucleotides (ODN) have immunomodulatory effects that may be useful for many future vaccine applications. The goal of this proposal is to understand how CpG ODN alter antigen processing and presentation of peptides to T cells. The project will also investigate how CpG ODN alter humoral immunity to polysaccharide Ags, as induced by immunization with either unconjugated PS or PS-protein conjugate vaccines. Aim 1: To determine the effect of CpG ODN on the ability of Ag presenting cells to process Ag and stimulate T cell responses to protein Ags. It is hypothesized that CpG ODN enhance Ag processing by dendritic cells and B cells. Investigators will determine the effects of CpG ODN on the ability of these cells to process and present exogenous protein Ags, including CRM 197, the carrier protein for glycoconjugate vaccines studied in Aims 2 and 3. Mechanisms for these effects will be explored, including the influence of CpG ODN on factors such as MHC-II synthesis and expression, half-life of peptide:MHC-II complexes, and expression of Ag processing components. Aim 2: To explore the adjuvant effects of CpG ODN on responses to PS and peptide epitopes of glycoconjugate vaccines, primarily using an experimental vaccine for Streptococcus pneumoniae. It is suggested that CpG ODN will enhance Ab responses to PS epitopes of glycoconjugate vaccines and alter the Ab isotypes that are elicited (e.g., to induce IgG2a and IgG3 responses in mice). The mechanisms of these effects will be determined including the roles of cytokines and T cells. Aim 3: Experiments will test whether CpG ODN can act as effective adjuvants in concert with vaccines containing only unconjugated PS immunogen to enhance PSspecific IgM and IgG1 responses and induce PS-specific Ab of other isotypes. Mechanisms of these effects will be determined (e.g., roles of T cells and cytokines). Understanding the modulation of Ag presenting cells by CpG ODN would increase our understanding of the basic mechanisms of adjuvant function for CpG ODN. The ability of CpG ODN to enhance humoral immunity to PS Ags would allow the development of improved vaccines for encapsulated bacteria. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: DEGP PROTEINASE INHIBITORS: NOVEL ANTI-INFECTIVES Principal Investigator & Institution: Hruby, Dennis E.; Chief Scientific Officer; Siga Technologies, Inc. 420 Lexington Ave, Ste 620 New York, Ny 10170 Timing: Fiscal Year 2002; Project Start 01-AUG-2000; Project End 31-MAY-2004 Summary: (provided by applicant): The DegP (HtrA) protease is essential for virulence in several Gram-negative pathogens: S. typhimurium, B. melitensis, Y. enterocolitica and P. aeruginosa. The phenotype of a degP knockout in Escherichia coli, temperature and

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oxidative sensitivity, suggests a role in virulence. The DegP protease is a multifunctional protein essential for the removal of misfolded and aggregated proteins in the periplasm of Gram-negative bacteria. We have identified the major pilin subunit of the Pap pilus, PapA, as a native DegP substrate and demonstrate vigorous proteolysis of this substrate in vitro. The DegP cleavage site in PapA was mapped and an in vitro cleavage assay suitable for HTS was developed. Hits that arise from the HTS will be passed through a series of secondary screening assays and in vivo models to identify bioavailable inhibitors of DegP that display good selectivity. We recently identified DegP homologues in S. pyogenes and S. aureus and demonstrated that a degP knockout in S. pyogenes has reduced virulence. We propose to validate S. aureus DegP as a virulence factor and develop this target for screening. There is ample precedent for the efficacy of protease inhibitors as therapeutic agents. Moreover, nonessential virulence targets may be less prone to resistance development. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: EFFECTS OF NATURAL AGENTS WITH FLUORIDE ON CARIES Principal Investigator & Institution: Koo, Hyun; Eastman Dentistry; University of Rochester Orpa - Rc Box 270140 Rochester, Ny 14627 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-MAY-2005 Summary: (provided by applicant): Apigenin and tt-farnesol, were identified as potentially novel natural anti-caries agents (Koo et al., 2002b; c). Both agents exhibited cariostatic properties in rats without significant effects on the animals' oral flora. Apigenin is a potent inhibitor of glucosyltransferases (GTFs), both in solution and on a surface (60-95% inhibition at 1.33mM), and without effect on bacterial growth, ttFarnesol, in contrast, affects the growth rate and metabolism of mutans streptococci biofilms by disrupting the membrane function. The aim of the proposed study is to evaluate the effects of the combination of apigenin, tt-farnesol and fluoride on the formation and composition of mutans streptococci biofilms in vitro, and on caries development in rats. Fluoride is a clinically proven anti-caries agent; its main effect is to interfere physicochemically with caries development. However, fluoride has antibacterial activity, and in addition, may interfere with GTF production (Bowen and Hewitt, 1974). Our hypothesis is that the association of the natural agents may enhance the anti-caries effect of fluoride by synergistically diminishing the virulence factors of mutans streptococci involved in the pathogenesis of dental caries. The rationale for this study is that the combination of the therapeutic agents will reduce the formation and virulence of cariogenic biofilms by (a) inhibiting the synthesis of glucans, e.g., inhibition of the activity and production of GTFs; Co) reducing the acid tolerance, e.g., inhibition of F-ATPase; (c) reducing the acidogenicity, e.g., enhancing the access of fluoride into cells by increasing the bacterial membrane permeability. We will test our hypothesis using a series of experiments as follows: I) In vitro- By determining the effects of the combination of agents on (1) formation, (2) viability, (3) pH, and (4) polysaccharide and inorganic composition of mutans streptococci biofilms. Streptococcus mutans biofllms formed on hydroxyapatite disks will be used in this part of the investigation. The polysaccharide composition will be determined by a series of colorimetric assays, liquid scintillation counting and gas chromatography/mass spectrometry. The concentrations of fluoride will be determined using a fluoride-selective electrode, calcium will be analyzed by atomic absorption spectrophotometry and phosphorus will be determined colorimetrically. The pH of the biofilms will be measured using a Beetrode pH electrode. II) In vivo - By evaluating the ability of combination(s) of agents (selected from in vitro analyses) to reduce dental caries, and to affect the pH and the composition of plaque

22

Streptococcus

using our rat model. The outcome of this study may lead to new and effective therapeutic combinations interventions to prevent dental caries, and possibly other plaque related diseases by using novel compounds in association with a well-known anti-caries agent. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: EFFICACY OF DNA VERSUS PROTEIN VACCINE Principal Investigator & Institution: Dao, My Lien.; Associate Professor; Biology; University of South Florida 4202 E Fowler Ave Tampa, Fl 33620 Timing: Fiscal Year 2003; Project Start 15-JUL-2003; Project End 31-MAY-2005 Summary: (provided by applicant): Dental caries is an infectious disease caused by Streptococcus mutans. Despite fluoridation and improved Dental hygiene in industrialized countries, worldwide Dental cades is still a significant public health problem affecting 50-90 percent of the population 0Nodd Health Organization). In the United Sates of America, 59 percent of first graders have at least one cavity, one third of the adult population does not see a Dentist annually, and 24 percent of the elderly experience caries-associated tooth loss (Oral Health America). Animal studies demonstrated that this disease was preventable by immunization with so mutans antigens. Gene cloning technology was applied to produce recombinant bacteria, protein and peptide vaccines for active immunization, and antibody for passive immunization. Various levels of protection were achieved with these vaccines. Considering that the population at risk is mostly from low socioeconomic groups (Surgeon General's report, U.S. DHHS, 2000), it is desirable to find means to lower the costs of vaccine production for mass immunization. One possibility is to directly immunize with a plasmid DNA (cDNA) containing the gene(s) of interest and obtain expression of the target protein(s) in the host. Thus, expensive protein isolation from recombinant clones is avoided. The Long Term Goal of the current study is to prepare an efficacious, safe and economical Dental Cades vaccine. The Specific Aim for the proposed period is to explore the prospect of a DNA vaccine against S. mutans with special emphasis on comparing the efficacy, duration and costs with those of corresponding Protein Vaccine. As models, the S. mutans antigen A (AgA), a recognized candidate vaccine antigen, and its precursor the wall-associated protein A (WapA), a factor involved in colonization and buildup of Dental plaque, will be used. The work proposed is supported by the availability of the recombinant clones needed for the production of WapA and AgA protein vaccines, and wapA-pDNA and agApDNAvaccines, and by the expression of WapA and AgA in mammalian cells transfected with these pDNA constructs. The results obtained will determine the feasibility and cost-effectiveness of genetic immunization against Dental caries, and the work performed will serve as a model for vaccine research against other infectious agents invading the body through mucosal surfaces. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ELDERLY POLYSACCHARIDE

IMMUNE

RESPONSE

TO

PNEUMOCOCAL

Principal Investigator & Institution: Westerink, M a J.; Medicine; Medical College of Ohio at Toledo Research & Grants Admin. Toledo, Oh 436145804 Timing: Fiscal Year 2002; Project Start 01-APR-2000; Project End 31-MAR-2005 Summary: Infection is one of the leading causes of morbidity and mortality in the elderly. Streptococcus pneumoniae is the organism most commonly isolated from

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elderly patients with pneumonia. Increased susceptibility to infections that occur in the elderly has been attributed to deteriorating health, decreased pulmonary function and a functional decline of the immune system. The immune system is unique in that it may be manipulated to achieve a desirable response. Studies in aged mice demonstrate both quantitative and qualitative changes in the immune response to T-independent type 2 (TI-2) antigens. Reports indicate age related loss of affinity, fine specificity and protective immunity are associated with a molecular shift in V gene usage and changes in cytokine profile. Studies of the in vivo immune response in elderly have been limited to vaccine efficacy studies and quantitative analysis of the magnitude and duration of the post-vaccination antibody response. The results of these studies suggest that despite adequate quantitative immune response the elderly show decreased vaccine efficacy. Current knowledge concerning the aging immune response to TI-2 antigens is mostly based on animal models and may not be applicable to humans. Human studies are fragmented and address quantitative and qualitative immune response as separate issues. We propose to study and characterize the immune response to S. pneumoniae capsular polysaccharide in the elderly. We will focus on both quantitative and qualitative changes in the immune response on molecular and functional levels. The quantitative immune response, isotype and IgG subclass, will be correlated with opsonophagocytic activity. We hypothesize that the discrepancy between the quantitative and qualitative immune response in the elderly results from altered V region sequence. We will characterize the immunoglobulin gene usage pattern and V-DJ joint diversity of the antibody response to pneumococcal polysaccharides (PPS) of serotypes 4 and 14 in elderly and young adults. This will be accomplished by gene family specific ELISA and by isolating single responding cells and determining the sequence of the V chains. Second, we propose to evaluate the influence of soluble regulatory factors on the aging immune response. The reconstituted SCID mouse model will be used to study the aging human B cell response to PPS 4 and 14 in a controlled cytokine environment allowing us to differentiate altered response intrinsic to the B cells versus altered responses secondary to environmental factors such as cytokines. The results of these studies will form the essential baseline for the rational development of vaccine and adjuvant strategies for the elderly. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: EXPERIMENTAL MODEL PREMATURITY

FOR CHORIOAMNIONITIS

AND

Principal Investigator & Institution: Gravett, Michael G.; Chief; None; Oregon Health & Science University Portland, or 972393098 Timing: Fiscal Year 2002; Project Start 01-SEP-1997; Project End 31-MAY-2006 Summary: Prematurity is the leading cause of neonatal morbidity and mortality in the United States. Intrauterine infections are an important, and potentially treatable cause of prematurity, and are associated with increased risk of neonatal white matter lesions of the brain and cerebral palsy. However, the mechanisms by which infection leads to prematurity and/or cerebral palsy remain speculative and treatment strategies untested largely because humans cannot be longitudinally studied following infection. We propose to use chronically instrumented pregnant rhesus monkeys at 120-130 day gestation with experimental intrauterine infection, as previously described (Gravett et al, Am J Obstet and Gynecol; 171:1660-1667,1994) to study the temporal and quantitative relationships among infection, cytokines, prostaglandins, steroid hormones, cytokine antagonists, preterm labor, and neonatal white matter lesions of the brain in order to develop effective interventional strategies. After postoperative stabilization in a tether,

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Streptococcus

we will; (1) inoculate Group B Streptococci (GBS) into the amniotic fluid to establish intrauterine infection and preterm labor. Uterine contractility will be continuously monitored and periodic samples of amniotic fluid and maternal and fetal blood (1-4 cc) will be obtained for assays of eicosanoids, steroid hormones, cytokines, matrix metalloproteinases and for microbial studies; (2) utilize antibiotics with and without potent inhibitors of proinflammatory cytokine production (dexamethasone,IL-10) o prostaglandin production (indomethacin) to ascertain the most effective intervention to down-regulate the cytokine/prostaglandin cascade and associated uterine activity; (3) infuse proinflammatory cytokine IL-1beta into the amniotic cavity through indwelling catheters in the absence of infection. Prior to infusion of IL-1beta in the absence of infection, specific novel proinflammatory cytokine inhibitors (IL-1ra and sTNF-R1 PEG) will be used to identify other potentially useful immunomodulators. Samples of the decidua, fetal membranes, tissues, and brain will be obtained at cesarean section for microbiologic, histopathologic studies, immunohistochemistry for cytokines, localization and quantitation of mRNA for cytokines and PGHS-2. Fetal brain will be examined for increased apoptosis associated with white matter lesions. Leukocytes in amniotic fluid and tracheal aspirates will be assessed by flow cytometry Postpartum, the mother will be treated with appropriate antibiotics to eradicate the GBS from the genital tract and returned to the colony. These studies will clarify the pathophysiology of infection-associated preterm labor and will suggest effective interventional strategies. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: FUNCTIONAL DIFFERENTIATION IN B LYMPHOCYTES Principal Investigator & Institution: Rittenberg, Marvin B.; Professor; Molecular Microbiology and Immunology; Oregon Health & Science University Portland, or 972393098 Timing: Fiscal Year 2002; Project Start 01-JUL-1988; Project End 31-MAR-2004 Summary: (Adapted from the Investigator's abstract): The ability of somatic mutation to modify the course of a humoral immune response is well documented. However, the focus has been almost exclusively on the ability of this process to improve the functional characteristics of representative antibodies; the harmful effects have not been well characterized. Yet in terms of cell numbers, all evidence suggests that B-cell wastage caused by harmful somatic mutations probably far exceeds the number of cells whose antibodies are improved through mutation. The purpose of this project is to gain quantitative insight into the contribution of mutation to B-cell wastage and secondly to exploit the well-known power of harmful mutations, to illuminate function. The investigators have previously made and characterized in vitro the binding of a large number of mutants of the T15 antibody to the hapten, phosphocholine (PC). The hypothesis is that mutant Abs displaying defective Ag binding or secretion in vitro would lead to apoptosis and B-cell wastage if they were to occur in vivo. This hypothesis will be tested in three ways: 1) by examining the ability of mutant antibodies to recognize PC which is displayed in different structural contexts on the surfaces of the pathogenic organisms, Streptococcus pneumoniae, Ascaris suum and Trichinella spiralis as well as Proteus morganii; 2) by testing the ability of mutant antibodies to transmit antigen-induced signals to transfected B lymphoma cells, and 3) by examining apoptotic GC B-cells for mutations in the VH1 gene of T15 shown to be harmful in vitro. These studies bear on B-cell wastage and homeostasis and the causes of apoptosis in germinal centers where recent evidence has suggested some lymphoid tumors such as Hodgkin's disease may originate. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: GENETIC ANALYSIS OF ORAL STREPTOCOCCAL BIOFILM FORMATION Principal Investigator & Institution: Ganeshkumar, Nadarajah; Associate Professor; Molecular and Cell Biology; Boston University Medical Campus 715 Albany St, 560 Boston, Ma 02118 Timing: Fiscal Year 2002; Project Start 01-APR-2000; Project End 31-MAR-2004 Summary: Initial colonization of tooth surfaces by oral viridans streptococci, including Streptococcus gordonii, leads to the eventual formation of biofilms called dental plaque. The most common disease of man, caries and periodontal disease, result from imbalances in the oral microflora, which allow pathogenic species to dominate. Initial plaque formation is characterized by the adhesion of planktonic cells of bacteria such as streptococci to tooth surfaces via specific salivary proteins of the acquired pellicle. Subsequent growth of these initial colonizers and other bacteria on the abiotic surface leads to the formation of dental plaque. Studies have extensively characterized of the initial binding of the planktonic bacteria to saliva-coated/hydroxyapatite surfaces, but the prerequisite signals that trigger the transition from a planktonic to a sessile mode of life and the subsequent accumulation of dental biofilms are poorly understood. It is hypothesized that novel genes are required for initial dental biofilm formation, and identification of such genes and characterization of their expression will be crucial for the development of novel methods of dental plaque control. A simple, but effective method of microbial accumulation on polystyrene surfaces will be used in this study to characterize isolation of biofilm-defective mutants of S. gordonii using Tn916 transposan mutagenesis, (2) characterization of biofilm-defective mutants, and (3) cloning and genetic analyses of biofilm genes. These studies of biofilm formation in streptococci will provide valuable information on the initial stages of dental plaque formation. Understanding the mechanisms involved in biofilm formation will be crucial for the development of novel therapeutic strategies to modify the composition of dental biofilm flora towards that found in health. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: GENETIC ANALYSIS OF S. PYOGENES VIRULENCE FACTORS Principal Investigator & Institution: Scott, June R.; Professor; Microbiology and Immunology; Emory University 1784 North Decatur Road Atlanta, Ga 30322 Timing: Fiscal Year 2003; Project Start 01-JUN-1984; Project End 31-JAN-2008 Summary: Streptococcus pyogenes, the group A streptococcus (GAS) is an important human pathogen causing frequent self-limiting diseases which may lead to serious sequelae. In addition, the GAS seems to be "reemerging" as a cause of life-threatening invasive disease. Because of the great diversity of syndromes produced by many strains of GAS, we wish to improve our understanding of the pathogenesis of this organism by focusing on the regulation of expression of its genes, which presumably occurs on interaction with the human host to determine disease outcome. This proposal focuses on the two major global regulators that alter expression of many GAS proteins, including virulence factors. The regulation of expression of Mga (Aim 1) and CovR/S (CsrR/S) (Aim 3) will be studied. The genes controlled by Mga and CovR/S and the mechanisms by which these regulatory proteins control expression of these genes will also be investigated (Aims 2 and 4). We hope these analyses will improve our understanding at the molecular level of the interactions of GAS with its human host and may identify new targets for development of therapies and vaccines. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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•

Streptococcus

Project Title: GENETIC FUNCTIONS OF AN ENTEROCOCCAL R FACTOR Principal Investigator & Institution: Dunny, Gary M.; Professor; Microbiology; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070 Timing: Fiscal Year 2002; Project Start 01-SEP-1992; Project End 31-MAR-2005 Summary: (Verbatim from Applicant's Abstract): The enterococci have become prevalent as causes of nosocomial infections. The high incidence of resistance of these organisms to the most efficacious antibiotics (e.g., vancomycin) causes major problems in treating enterococcal infections, and the tremendous reservoir of enterococcal resistance determinants serves as a vector for the spread of these genes to other, more pathogenic bacterial genera. This research is focused on dissection of a mechanism of horizontal genetic transfer of the antibiotic resistance plasmid pCF10 in Enterococcus faecalis. The most novel feature of this transfer system is that the pCF10-containing donor cell perceives the presence of potential recipients in its vicinity by sensing a small peptide signal (a sex pheromone called cCF10) excreted by the recipients. The donor cell only expresses genes required for plasmid transfer when exogenous cCF10 is detected in the growth medium. Both the donor cells response to exogenous pheromone, and the negative control system that prevents expression of transfer functions in the absence of exogenous pheromone, are complex processes that have been studied in detail. Enterococci produce a variety of peptide pheromones, consisting of hydrophobic peptides 7-8 amino acids in length. Different families of plasmids each encode a highly specific response to a single cognate pheromone. When a single cell carrying multiple pheromone plasmids is exposed to one pheromone, only the corresponding plasmid is transferred, even though the sensing systems are quite similar for all the plasmids examined to date. In the next funding period, the focus of the experiments will be on the molecular and genetic basis for the specificity of the pheromone response. The specific aims are: 1) Determine the molecular basis for specificity of pheromone cCF10 interactions with PrgZ (the extracellular pCF10-encoded pheromone binding protein), and with PrgX (the putative intracellular receptor for cCF10 believed to comprise the molecular switch involved in the intracellular phase of pheromone induction. 2) Determine the molecular basis for specific abolition of endogenous cCF10 activity in pCF10-containing donor cells by PrgY (a pCF10-encoded membrane protein), and by iCF10 (a plasmid-encoded peptide inhibitor of cCF10). 3) Determine the molecular basis for the activity and specificity of a novel regulatory RNA, Qa in blocking expression of conjugation in uninduced cells via its interaction with PrgX and with the Qs RNA encoded in the positive control region of pCF10. 4) Begin an experimental analysis of the genetic and molecular basis for specificity of the downstream steps in pheromone induction. These steps include post-transcriptional activation of transfer gene expression, and conjugative DNA processing. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: FIMBRIAE

GLYCOSYLATION

&

BIOGENESIS

OF

STREPTOCOCCAL

Principal Investigator & Institution: Wu, Hui; Microbiol & Molecular Genetics; University of Vermont & St Agric College 340 Waterman Building Burlington, Vt 05405 Timing: Fiscal Year 2003; Project Start 04-JUN-2003; Project End 31-MAY-2008 Summary: (provided by applicant): Dr. Wu's isolation and characterization of the Fap1 molecule, a glycosylated fimbrial structural subunit of Streptococcus parasanguis, was a significant breakthrough in understanding the basic for fimbrial biosynthesis and adhesion in Gram-positive bacteria. Fap1-like molecules and genes involved in Fapl

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glycosylation are not only highly conserved across oral streptococci but are also present in Streptococcus pneumoniae and in the staophlococci. Dr. Wu's current research on the glycoslylation of Fap1 is extremely exciting. It is likely that a universal glycosylation machinery is present in Gram-positive bacteria and the findings from these studies would be applicable to other organisms. Recently, he identified a gene cluster that is required for the glycosylation of Fap1. He is currently in the process of determining the biosynthetic pathway by which Fap1 is glycosylated using molecular genetic, structural biology, and biochemical approaches. With the support of a K22 award, Dr. Wu will take formal courses in mass spectrometry and bioinformatics and receive hands-on training in Mass spectrometry analysis of the carbohydrate structure and biochemical analysis of key enzymes involved in protein glycosylation. The support of a K22 will provide Dr. Wu with new skills and knowledge in carbohydrate biochemistry that will position him to pursue new avenues in the study of glycosylation of streptococcql adhesions. Dr. Wu is in a unique position to determine the mechanisms of prokaryotic glycosylation and Gram-positive fimbrial biogenesis as the explict genetic tools have been generated in Dr. Fives-Taylor's laboratory over the last 20 years. Dr. Wu's strength in molecular genetics coupled with the training in carbohydrate biochemistry specifically in mass spectrometry analysis and enzymology will enhance his career development in this exciting research area. The results of this proposal will help define the biosynthetic pathway for Fap1 glycosylation. New genes in the pathway may endow microbes with new effector functions or aid in the evasion of host defense, strategies that are important components of disease-causing capabilities of a number of bacterial pathogens. Therefore, elucidation of Fap1 glycosylation may provide a framework for understanding the role of glycoslyation in bacterial pathogenesis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GROUP B. STREPTOCOCCI AND TOLL-LIKE RECEPTORS Principal Investigator & Institution: Golenbock, Douglas T.; Chief, Division of Infectious Diseases &; Medicine; Univ of Massachusetts Med Sch Worcester Office of Research Funding Worcester, Ma 01655 Timing: Fiscal Year 2002; Project Start 15-AUG-2002; Project End 31-JUL-2007 Summary: (provided by applicant): Group B streptococci (GBS) are the leading cause of neonatal sepsis, the third most frequent cause of bacterial meningitis and an increasingly important cause of bacteremia and sepsis in adults in the United States today. Preliminary studies have identified a novel proinflammatory component of GBS, which we have designated GBS-factor (GBS-F). Based on experiments in mice with targeted genetic deletions in Toll-like receptor (TLR) expression and on experiments with engineered cell lines, we have determined that responses to GBS-F require expression of CD14, TLRs 2 and 6, and the Toll-adapter protein, MyD88. Activation of this receptor complex by GBS-F initiates important signaling events such as the activation of NF-kB, the phosphorylation of MAP kinases, the formation of proinflammatory cytokines, and the intracellular production of the toxic oxidant peroxynitrate. In contrast, although other components of GBS appear to engage TLRs, the exact identity of contributing TLRs is entirely unknown. The overall goal of this proposal is to identify and define components of GBS, focusing first on GBS-F, and their cognate Toll-like receptors (TLRs). We propose to characterize the structure of GBS-F and assess its function in vitro and in vivo. Furthermore, we will determine if TLR2, and related downstream signal transduction molecules, mediate a variety of important innate immune responses to GBS, including leukocyte chemotaxis and the intracellular killing of bacteria. Finally, we intend to determine what other TLRs, and associated

28

Streptococcus

signal transduction molecules, are involved in GBS recognition and response. The data learned from these studies should help in the development of rational therapeutic strategies to interfere with the deleterious hyperinflammation triggered by GBS and similar microbial organisms. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: IMMUNE RESPONSE TO THE GROUP A STREPTOCOCCAL CAPSULE Principal Investigator & Institution: Wessels, Michael R.; Professor; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 01-JUL-1991; Project End 31-MAY-2005 Summary: Description (Adapted from applicant's abstract): A global increase in invasive group A Streptococcus (GAS) disease (bacteremia, necrotizing fasciitis, and streptococcal toxic shock syndrome) that began in the 1980's has continued through the present decade and has focused attention on investigation of mechanisms of GAS pathogenesis. During the previous funding period, work in their laboratory and others further documented the central role of the hyaluronic acid capsular polysaccharide in GAS virulence in experimental models of local and systemic infection. These studies showed that the capsule interferes with the phagocytic killing, prevents internalization of GAS by epithelial cells, modulates adherence mediated by other GAS surface molecules, and acts as a ligand for attachment of GAS to CD44 on pharyngeal keratinocytes. Despite these other advances, it remains undefined how the capsule or other virulence determinants control the processes of tissue invasion and persistent colonization in the host. During the next funding period, their objectives are to define the role of the hyaluronic acid capsule in invasion of GAS from an epithelial surface to deep tissue, to characterize the effects of capsule on intracellular trafficking of GAS in epithelial keratinocytes, and to determine how regulation of capsule expression in vivo contributes to pathogenesis of GAS infection. The proposed experiments will make use of primary keratinocyte cultures and a model system simulating intact human skin in conjunction with confocal fluorescence microscopy in order to study the tissue and cell biology of GAS translocation through human skin, the phenomenon of persistence within cells, and the regulation of capsule expression at various phases of the infection process. Results of these studies will elucidate the basic pathogenic mechanisms involved in GAS disease and may suggest strategies for intervention. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: IMMUNE RESPONSES TO PNEUMOCOCCAL ANTIGENS IN CHILDREN Principal Investigator & Institution: Kobrynski, Lisa J.; Pediatrics; Emory University 1784 North Decatur Road Atlanta, Ga 30322 Timing: Fiscal Year 2002; Project Start 01-AUG-2000; Project End 31-JUL-2004 Summary: (Adapted from applicant's description): The objective of this proposal is to provide a period of mentored, multi-disciplinary training in clinical and laboratory research in pediatric immunology. CD 1 has recently been implicated in the antigen presentation of certain bacterial lipo-polysaccharides in a non-MHC dependent fashion. The applicant's data suggest that there is a role for CD1 restricted T cells in immune responses to pneumococcal polysaccharides and that a lack of these cells results in the inability to produce pneumococcal specific IgG. The proposed project will characterize these cells, delineate their role in polysaccharide antibody responses and ascertain

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whether patients in at-risk populations have alterations in this T cell subset. Identification of the precise role of these cells and the nature of the defect in susceptible hosts has the potential to lead to novel therapeutic regimes. This work has potentially broader applications in the immune responses to other polysaccharide antigens of common infectious agents. In addition, the applicant will pursue further training in study design and research methods, including didactic courses at the Rollins School of Public Health and the Graduate Division of Biological and Biomedical Sciences, at Emory. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: IMMUNITY TO PNEUMOCOCCAL SURFACE PROTEIN A AND C Principal Investigator & Institution: Briles, David E.; Professor; Microbiology; University of Alabama at Birmingham Uab Station Birmingham, Al 35294 Timing: Fiscal Year 2002; Project Start 01-AUG-1984; Project End 31-OCT-2002 Summary: (Adapted from the applicant's abstract): The proposal is aimed at continuing Dr. Briles' research efforts in the study of pneumococcal surface antigens. The application focuses on the first cell-associated protein described, PspA, as well as a second newly discovered protein, PspC. These two proteins may serve as vaccines themselves or as protein carriers for capsular polysaccharide-protein conjugates. PspA is present on all pneumococcal strains and can elicit protective immunity against sepsis and nasopharyngeal carriage in mice. PspC is related to PspA, but larger in size, and shows virtual identity with PspA in its C-terminal half. The proposed studies will determine whether PspC is a virulence factor and whether it can elicit protection. The relative roles of PspA and PspC in virulence and carriage in nonimmune animals will be examined. In addition, the relative roles of immunity against PspA and PspC in carriage, sepsis, and spread of pneumococci from the nasopharynx will be explored. The ability of human antibody to these molecules to protect mice from infection will be evaluated. Cross-reactive regions between the PspA and PspC proteins will be identified as well as the regions of each molecule most useful as a vaccine. Immunity to PspA and PspC will be evaluated to determine whether it involves opsonization, blocks virulence functions, or acts by other mechanisms. The data obtained will assist with the development of correlates of protective immunity for PspA and PspC that can be applied to vaccine development. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: IMMUNOCHEMISTRY POLYSACCHARIDES

OF

GROUP

B

STREPTOCOCCAL

Principal Investigator & Institution: Kasper, Dennis L.; Associate Director; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2001; Project Start 01-SEP-1985; Project End 31-JUL-2004 Summary: The major approach to the prevention of infection by encapsulated bacteria has been the use of vaccines consisting primarily of the polysaccharide capsules of these bacteria. The group B Streptococcus (GBS) is the most serious pathogen for the neonatal age group. Antibody to the capsular polysaccharides of GBS has been shown to be protective. Attempts to induce protective immunity to GBS utilizing polysaccharide capsules have met with only modest success due to the overall poor immunogenicity of these polysaccharide antigens in humans. The most effective approach to overcoming the problem of poor immunogenicity of polysaccharides has been covalent coupling to carrier proteins to produce conjugate vaccines, which have significantly enhanced

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immunogenicity. While it is clear that induction of T cell help is one important aspect of the enhanced immunogenicity of these conjugates, another factor, which we believe to be important, is the effect of conjugation on the polysaccharide epitope presentation and expression. Our studies of GBS polysaccharides have defined a unique system that allows the determination of how specific physicochemical characteristics, such as antigen conformation, affect the host recognition of polysaccharide antigens. Antibodies directed against these polysaccharides appear to recognize a conformational epitope that is fully expressed in higher-molecular-weight forms of the polysaccharide. Although a conjugate vaccine is likely to enhance the immune response by eliciting T cell help, conjugate designs that stabilize the conformational epitope may also increase the immune response by enhancing expression of this epitope. In this proposal we present a program to rigorously define model conjugate vaccines that allow the differentiation of the effects of conjugation in modifying polysaccharide epitope expression as distinct from the T cell dependent effects. Conjugation may modify particular structural features of the polysaccharide making it more immunogenic by changing the interaction of the polysaccharide and immune system. There are five specific aims in this proposal. In Specific Aims 1 and 2, oligosaccharides of differing length will be conjugated either at the reducing end or at multiple sites along the chain to determine what effect conformational stability has on the chain-length dependent epitope. The effect of single versus multisite coupling on T cell dependence will be studied. In Specific Aim 3,the effect on T cell dependence of the degree of cross-linking between the full length polysaccharide and the carrier protein molecules in a conjugate vaccine will be studied. In Specific Aims 4 and 5, specific chemical parameters which define conformation, NMR spectroscopy, and X-ray crystallography will define precisely how conformationally dependent polysaccharide epitopes interact directly with antibody. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: IMMUNOPROTEOMICS Principal Investigator & Institution: Boyle, Michael D.; Professor; Biology; Juniata College 1700 Moore St Huntingdon, Pa 16652 Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-MAY-2005 Summary: (provided by applicant): The proposed studies are designed to evaluate new methods using a mass spectral readout to provide sensitive, detection of selected proteins, as well as methods to monitor post-translational modification events of targeted antigens. The proposed assay involves an antigen capture step mediated by immobilized antibody (immuno) and an analytical step involving mass spectral analysis of bound antigen (proteomics). The goal of the project is to develop rapid sensitive methods of antigen capture from complex mixtures of unrelated proteins in a maimer that permits the subsequent precise molecular weight determination of the bound antigen using time of flight mass spectrometry. The ability to distinguish subtle variation in the size of a targeted antigen will allow analysis of post-translational modification events for any targeted antigen to be achieved. In addition, the ability to obtain semi-quantitative data based on the area under a specific molecular weight peak on the mass spectral read-out will be critically evaluated. The proof of concept studies will focus on a number of properties of the secreted streptococcal cysteine protease SpeB which is known to post-translationally modify the surface anti-phagocytic M protein, and degrade the secreted bacterial plasminogen activator SK. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: INHALED PARTICLES AND HOST DEFENSE IN THE PRIMED LUNG Principal Investigator & Institution: Kobzik, Lester; Associate Professor; Environmental Health; Harvard University (Sch of Public Hlth) Public Health Campus Boston, Ma 02115 Timing: Fiscal Year 2003; Project Start 07-JUL-2003; Project End 31-MAY-2008 Summary: (provided by applicant): The Problem: Hospital admissions for pneumonia are increased by elevated air particle levels. The mechanism(s) underlying particle effects on lung infection are unknown, but may reflect increased incidence of infection, increased severity of infection, or both. Hypothesis: The pathogenesis of the pneumococcal pneumonia (the most common variety and the disease we will study) suggests three possible mechanisms for particle effects: enhancement of lung cell 'receptors' used by bacteria for initial adhesion, damage to antimicrobial function of host cells (AMs and PMNs), and exaggerated inflammation in established infection leading to worse signs and symptoms. Hence, the central thesis of this research is that oxidant components of air particles mediate 1) dysfunction of host defenses against infection (incidence) and 2) increased inflammation in extant pneumonia (severity). Experimental Plan: Aim 1 will measure expression and function of pneumococcal 'receptors' (e.g., PAF receptor) used by pneumococcal for initial adhesion after exposure to concentrated ambient particles (CAPs) or control particles. Aim 2 will determine effects of air particles on pulmonary inflammation before and after onset of pneumococcal pneumonia. The hypothesis to be tested is that particles cause enhanced release of cytokine mediators by primed AMs, leading to increased inflammation and ultimately oxidant damage to both AM and PMN In vivo and in vitro studies will measure release of pro-inflammatory cytokines, cell influx and viability and severity of pneumonic inflammation. Aim 3 will test the hypothesis that particle exposure inhibits bacterial clearance via oxidantdependent damage of anti-microbial functions of AMs and PMNs. Component analysis will be performed using a panel of CAPs samples to provide links of particle constituents (e.g., metals, organics, endotoxin) with biologic effects. Rotated factor analysis will be used to correlate source types with CAPs toxicity. Specific intracellular oxidant pathways will be identified by measurement of oxidant production, intracellular levels of antioxidants, and the effect of a panel of anti-oxidants and other inhibitors. Significance: This research is relevant to the public health question of how inhaled particles cause pulmonary health effects and to the pathophysiology of lung host defense against environmental agents. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: INTERGENERIC SIGNALING MOLECULE OF STREPTOCOCCUS CRISTATUS Principal Investigator & Institution: Xie, Hua; Associate Professor; Meharry Medical College 1005-D B Todd Blvd Nashville, Tn 37208 Timing: Fiscal Year 2003; Project Start 01-AUG-2003; Project End 31-JUL-2007 Summary: Dental plaque (biofilm) has been implicated as a primary causative agent of adult periodontal disease. The key event leading to initiation of the disease is the transition from commensal dental biofilm to pathogenic biofilm. It is well known that the process of the transition is involved in the colonization of several specific periodontal pathogens such as Porphyromonas gingivalis. Our long-range goal is to understand events and factors leading to the transformation of healthy plaque to pathogenic plaque and to change the course of development of periodonpathogenic

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biofilm by preventing attachment of P. gingivalis. In our ongoing studies, we have identified several environmental factors that can influence expression of fimA gene, a virulence gene encoding a major protein unit (fimbrillin) of fimbriea. One of the striking findings is that the presence of Streptococcus cristatus molecule(s) could significantly repress fimA expression in P. gingivalis at the transcriptional level. As a result, S. cristatus could inhibit the formation of P. gingivalis biofilm in vitro. In this grant proposal, we will put our focus on characterization of S. ctristatus signaling molecule, biochemically and genetically. The hypothesis for this proposal is that S. cristatus plays an important role in impeding P. gingivalis' colonization on dental biofilm through intergenric signaling systems. To test this hypothesis, we will start with identification and purification the signaling molecule(s) of S. cristatus. The signaling molecule will be characterized in the terms of functional and genetic structures. We will also attempt to understand regulation of the signaling gene expression in oral biofilm. Therefore, the signaling gene of S. cristatus will be cloned. The promoter region of the gene will be fused with the reporter gene such as chloramphenical acetyltransferase gene, and level of the gene expression will be determined by measuring enzymatic activity. Finally the role of this molecule in the formation of pathogenic oral biofiim will be investigated. Studies will be initiated to determine the distribution of the signaling molecule in the dental plaques from healthy subjects and pedodontitis patients. Our ultimate goal is to convert the knowledge gained from these laboratory studies to practical technology that may be used to reprogram development of the dental biofilm and to reduce the incidence of adult periodontitis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: INTRARTICULAR SLPI THERAPY FOR RHEUMATOID ARTHRITIS Principal Investigator & Institution: Labhasetwar, Vinod D.; Associate Professor; Pharmaceutical Sciences; University of Nebraska Medical Center Omaha, Ne 681987835 Timing: Fiscal Year 2002; Project Start 28-SEP-2001; Project End 31-JUL-2003 Summary: (provided by applicant): Rheumatoid arthritis (RA) is a chronic inflammatory disease, characterized by the loss of joint structure and function, resulting in significant pain and morbidity. In this proposal, we plan to investigate a new therapeutic strategy for the treatment of RA. In normal joints, the cartilage matrix turnover is maintained due to a balance between the activities of proteases and protease inhibitors. This balance is lost in arthritic joints, resulting in a greater protease activity, leading to cartilage and bone degeneration. Secretory leukocyte protease inhibitor (SLPI) has been identified as an endogenous potent protease inhibitor that maintains the critical balance against the proteases in the joint. Since SLPI is not produced by the joint tissue and the arthritic joints loose their ability to sequester SLP1 from the blood, it is hypothesized that intra articular administration of SLPI in a sustained release formulation would be effective in reinstating the balance between the proteases and protease inhibitors, and in inhibiting the progression of the disease. SLPI is also considered to be involved in protecting the cartilage growth factor (Link N) in the joint from protease-mediated degradation. The link N promotes the synthesis of proteoglycan and collagen, which are required for maintaining normal cartilage composition in the joint. Therefore, localized SLPI therapy could also lead to regeneration of the cartilage matrix and restoration of joint functions. Therefore, the objective of the proposed studies is to determine the efficacy of sustained intra articular delivery of SLPI using an injectable thermo reversible (TR) gel system in RA. The specific aims of the research program are: (1) To formulate a sustained release TR gel system for SLP1 using biodegradable and biocompatible Polyethylene oxide-Poly (L-Lactide)-Polyethylene oxide copolymer, and to evaluate the gel for sustained protein

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release properties, (2) To investigate the kinetics of intra articularly injected SLPI-gel system to provide localized and sustained delivery of the protein, and to determine the therapeutic efficacy of the gel to inhibit the progression of the disease in a rat streptococcal. cell wall-induced model of inflammatory erosive arthritis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: LETHAL PNEUMOCOCCUS

SYNERGISM

BETWEEN

INFLUENZA

AND

Principal Investigator & Institution: Mccullers, Jonathan A.; Assistant Member; St. Jude Children's Research Hospital Memphis, Tn 381052794 Timing: Fiscal Year 2002; Project Start 01-APR-2001; Project End 31-MAR-2004 Summary: (provided by applicant): The K08 Award will provide an opportunity for the applicant to extend his virology training in the area of viral-bacterial interactions and to develop new expertise in pneumococcal pathogenesis in the setting of prior viral infection. These skills will enable the applicant to achieve his long term career goals by becoming a fully independent research scientist who can translate observations made at the bench into therapeutics and interventions at the bedside. Epidemiologic evidence suggests that there is a lethal synergism between influenza A virus and Streptococcus pneumonias accounting for excess mortality (average 20,000 influenza-related deaths per year in the US) during influenza epidemics. However, the pathogenic mechanisms underlying this interaction are poorly understood, and the lack of a suitable animal model of pneumonia following infection with both organisms has hampered study. The goal of the proposed research plan is to determine the role of receptor alterations engendered by influenza virus infection in the pathogenesis of pneumococcal pneumonia. A newly developed murine model of dual infection will be used to examine the relationships of timing and of infectious doses of influenza virus and pneumococcus to morbidity and mortality. Influenza viruses with different pathogenic features in the mouse will be utilized to determine how cytokine expression varies with different viruses. Expression of receptors permissive for pneumococcal adherence and invasion will be examined in the context of cytokine expression following influenza virus infection, and a correlation to development of pneumonia and in the murine model of dual infection will be made. Identification of specific pneumococcal proteins involved in this synergistic interaction will provide drug and vaccine targets for future intervention in human disease and death caused by pneumococcal superinfection following influenza. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: LUXS-MEDIATED QUORUM SENSING IN STREPTOCOCCUS MUTANS Principal Investigator & Institution: Wen, Zezhang; Oral Biology; University of Florida Gainesville, Fl 32611 Timing: Fiscal Year 2003; Project Start 15-JUN-2003; Project End 31-MAY-2005 Summary: (provided by applicant): Streptococcus mutans is recognized as the principal etiological agent of dental caries, the most prevalent infectious disease of humans. The ability to metabolize carbohydrates and generate acids, to survive acidic pH and other adverse conditions, and to adhere to and form tenacious biofilms on the tooth surface are believed to be critically associated with the cariogenicity of this human pathogen. Known for its high degree of acid tolerance (aciduricity) and its high capacity to produce acid (acidogenicity), S. mutans lives primarily on the tooth surface at high cell-

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Streptococcus

density in a high diversity ecosystem better known as dental plaque, the structure and composition of which is known to be largely influenced by such factors as the source and availability of nutrients, the pH in the oral cavity and by the ability of the biofilm organisms to adapt to the fluctuations in environmental conditions. Quorum sensing is a cell density--dependent regulatory mechanism that is known to be involved in regulation of a variety of physiologic processes and virulence in both Gram (+) and Gram (-) bacteria. We have recently generated evidence that the S. mutans possesses a gene encoding a functional homologue of the new family of autoinducer synthases (LuxS) that are responsible for production of autoinducers of the quorum sensing system 2, AI-2. This study is designed to yield novel information concerning LuxSmediated quorum sensing and virulence regulation in S. mutans, which will contribute to our understanding of the pathogenesis of this microorganism and the ecology of the oral flora. The Specific Aims of this proposed study are: 1) to investigate the role of luxS in acid tolerance by S. mutans. By using functional assays, reporter gene fusions, Northern hybridization, and proteomics, we will investigate acid tolerance and its regulation by luxS, and identify novel factors (proteins) that are involved in luxSregulated acid tolerance responses. 2). To use confocal laser scanning microscopy (CLSM) and mixed, known-species consortia to determine the impact of luxS of S. mutans on bacterial adherence by S. mutans and the inter- and intra-generic interactions between S. mutans and other oral bacteria in terms of biofilm initiation development and structure. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MATERNAL IMMUNIZATION TO PREVENT INFANT OTITIS MEDIA Principal Investigator & Institution: Ferrieri, Patricia; Professor; Pediatrics; University of Minnesota Twin Cities 200 Oak Street Se Minneapolis, Mn 554552070 Timing: Fiscal Year 2003; Project Start 01-APR-2003; Project End 31-MAR-2006 Summary: (provided by applicant): Vaccine strategies to prevent childhood otitis media (OM) are being sought due to its major public health impact and the troubling increase in antibiotic resistant bacteria. The pneumococcus bacteria is a prime target for vaccine prevention. Maternal immunization is a strategy designed to prevent early infant OM, since it is one of the greatest risk determinants for recurrent and chronic OM. A recently licensed 7-valent pneumococcal conjugate vaccine (PCV7) is now given routinely to all infants beginning at age 2 months. This vaccine is highly protective against invasive pneumococcal disease after 7 months of age, has modest (6-9%) protection against all OM episodes between 7 and 24 months, and a 57% reduction in vaccine-type pneumococcal AOM. But it does not significantly reduce OM before 7 months. Thus, there remains a potentially important role for maternal pneumococcal immunization to prevent pneumococcal AOM in the first 6 months of life. Thus, it is important to test the hypothesis that maternal pneumococcal immunization during pregnancy does not produce immune interference in the infant, impairing response to the infant vaccine, before proceeding with a maternal vaccine OM efficacy trial. This Phase I/II trial will enroll 154 pregnant women and follow subjects and their infants to age 13 months. Primary aims are: (1) to determine if infants of women immunized with 9-valent PCV (PCV9) and infants of control women who receive placebo during the third trimester of pregnancy have equivalent anti-capsular polysaccharide (PS) IgG antibody responses to PCV7 measured one month after the third vaccine injection at 6 months of age, and (2) to compare local and systemic adverse events among women immunized with PCV9 or placebo. We will also investigate the hypotheses that (1) maternal immunization does

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not interfere with the infant's antibody subclass and pneumococcal opsonic responses to the primary PCV7 vaccine series or to booster PCV7 immunization at 12 months, (2) pregnant women have a significant antibody response to PCV9 vaccine compared to placebo vaccine and increased antibody persists 13 months after delivery, (3) anti-PS IgG and secretory IgA antibodies are present in the milk of immunized lactating women, and (4) maternal immunization does not interfere with the infants' antibody response to H. influenzae type b conjugate and diphtheria toxoid vaccines. Transplacental anti-PS IgG antibody transfer, the natural decline of two anti-PS antibodies against PS antigens in the maternal but not the infant vaccine, and the natural production of one anti-PS antibody not in either vaccine will be measured. The trial has enrolled and randomized 87 subjects to date with continuous, steady subject accrual since November 2000. Results of this trial will have a profound impact on the broad field of maternal immunization to prevent early infant disease caused by a variety of infant bacterial and viral pathogens. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MCP--STRUCTURE, MICROBIAL INTERACTIONS AND FUNCTION Principal Investigator & Institution: Atkinson, John P.; Professor of Medicine & Molecular Biolog; Medicine; Washington University Lindell and Skinker Blvd St. Louis, Mo 63130 Timing: Fiscal Year 2002; Project Start 01-APR-1995; Project End 31-MAR-2005 Summary: (adapted from investigator's abstract): The complement system is ingeniously designed to prevent infections as well as to process immune complexes and damaged tissue. Strict control of its activation during innate and acquired humoral immune responses is critical to minimize damage to host tissue. Membrane cofactor protein (MCP; CD46) is widely expressed inhibitor of complement activation at the critical step of C3/C5 convertase generation. It serves as a cofactor for the serine protease factor I to cleave and thereby inactivate C3b and C4b that deposit on host tissue. Most cells and tissues express MCP as a family of four isoforms that differ in their O-glycosylation and cytoplasmic tails. MCP is a receptor for three human pathogens: measles virus, Streptococcus pyogenes and Neisseria. Attachment of the measles virus of Neisseria to MCP transmits signals for IL-12 down-regulation in monocytes or for calcium fluxes in epithelial cells, respectively. MCP has also been implicated in reproduction, in large part due to its dense expression on placental trophoblast and on the inner acrosome membrane of spermatozoa. Because of its potent complement regulator activity, MCP has been recombinantly produced for use as a soluble therapeutic agent and engineered into pigs whose organs are being employed for xenografting. In this grant application, we propose to continue our studies on the structure, microbial interactions and function of MCP. We postulated and later demonstrated during the prior grant period that each of four regularly expressed isoforms of MCP possesses functional advantages. In this renewal application we continue this focus while placing an increased emphasis on microbial connections and cell signaling. The active sites of MCP will be characterized by NMR spectroscopy and X-ray crystallography. We have recently demonstrated that in three cell types MCP is tyrosine phosphorylated on one of its two cytoplasmic tails. For this signaling event, we propose a systematic analysis of the site(s), responsible kinase(s), and related downstream events. Additionally, we will explore in depth the microbial interactions with MCP as they relate to binding sites, signaling events, and three-dimensional structure. We will characterize three strains of transgenic mice expressing human MCP. We anticipate these animals will be a valuable tissue source. We also propose a targeted disruption of the MCP mouse gene since MCP is expressed

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Streptococcus

predominantly on the inner acrosomal membrane of mouse spermatozoa. The fertility of these mice will be assessed and they will be crossed with other deficient mice strains to explore the role of MCP in reproduction. Lastly, the mechanism of action of MCP in situ will be analyzed by quantitative methods developed during the prior granting period. We will address such questions as the role of membrane versus fluid phase inhibitors and contribution of MCP versus decay accelerating factor. The specific aims of this proposal outline a broad-based approach to increase our understanding of complement regulation by one if its major inhibitory proteins. Additionally, these experiments will expand our knowledge of the fascinating interactions of this complement regulator with three common infectious diseases and its role in cell signaling events and reproduction. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MECHANISMS OF CELL INJURY IN BURN COMPLICATED BY SEPSIS Principal Investigator & Institution: Horton, Jureta W.; Professor; Surgery; University of Texas Sw Med Ctr/Dallas Dallas, Tx 753909105 Timing: Fiscal Year 2002; Project Start 01-AUG-1999; Project End 31-JUL-2003 Summary: (adapted from applicant's abstract): Despite aggressive fluid resuscitation and topical antimicrobial therapy after burn trauma, sepsis frequently results from the loss of dermis; thus sepsis and resultant multiorgan failure are a major cause of death in the burn unit. Studies from the PI's lab and others' have shown that burn trauma and sepsis independently alter cardiocirculatory performance, and recent studies suggest that myocardial abnormalities after burn, trauma or sepsis are related to intracellular accumulation of calcium with subsequent cellular injury and dysfunction. Although this field has grown rapidly, much is still unknown about the cellular mechanisms underlying cardiac dysfunction after either trauma or sepsis. The PI's group have focused their attention on a clinically relevant model of burn injury complicated by sepsis (intratracheal administration of S. pneumoniae administered 24 hours postburn) and have shown progressive cardiocirculatory dysfunction in this two-hit model. Specific Aim 1a will determine if burn/sepsis exacerbates the increased [Ca2+] and [Na2+] shown to occur after burn alone and will determine the contribution of altered Na+/Ca2+ to cardiac contractile dysfunction. Specific aim 1b will determine the contribution of transient cellular acidosis and altered H+/Na+ exchange to increased [Na2+], and whether increased [Na+] in turn promotes Na+/Ca2+ exchange in [Ca2+] overload. Specific Aim 2 will determine the contribution of burn/ sepsis-mediated alterations in SR Ca2+ handling (SR Ca2+ efflux, Ca2+-ATPase activity, SERCA, and SR Ca content) to cellular Ca2+ and cardiac contractile deficits and determine the contribution of burn/sepsis induced myofilament Ca2+ insensitivity to cardiac contractile dysfunction. Studies in Specific Aim 3 will examine the role of PKC activation in intracellular Na+/Ca2+ accumulation and cardiac contractile dysfunction in burn sepsis. Studies in Specific Aim 4 will determine the contribution of increased [Ca2+] and reactive oxygen species to apoptosis in burn/sepsis and further determine the contribution of apoptosis to burn/sepsis-induced ionic derangements as well as cardiac contractile dysfunction. Only by understanding the cellular events involved in the postburn inflammatory cascade can adequate prevention and treatment modalities be designed to improve outcome. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: MECHANISMS OF TANDEM REPEAT DELETION IN GROUP B STREP Principal Investigator & Institution: Puopolo, Karen M.; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 07-FEB-2002; Project End 31-JAN-2007 Summary: (Provided by Applicant): Group B Streptococcus (GBS): is a leading cause of serious infection in newborns and pregnant women, and in adults immunologically impaired by liver disease, diabetes and malignancy. GBS contain a family of immunogenic surface proteins that are characterized by the presence of long tandemlyrepeated elements, the prototype of which is the alpha C protein. Previous work has demonstrated that variation in the number of tandem repeats in the gene for the alpha C protein (bca), alters the antigenicity of the protein and the virulence of the strain in the presence of specific antibody. Tandem repeat deletion in the alpha C protein allows antigenic variation and may thus serve as a virulence mechanism in GBS. The molecular mechanism by which excision of tandem repeat units in bca is accomplished is unknown. Identification of the molecular factors involved in tandem repeat deletion of broader interest as tandem repeat sequences of DNA are found in both prokaryotic and eukaryotic genomes, and variation in these sequences is associated both with changes in bacterial virulence and the genesis of inherited human diseases. Previous work by the applicant has demonstrated that inactivation of recA, the principal gene involved in bacterial homologous recombination, does not affect tandem repeat deletion in GBS. The scientific goals of this project are to identify genes involved in tandem repeat deletion in GBS, and to determine the role of conserved nucleotide sequences within repeats in directing tandem repeat deletion. To meet these goals, a plasmid -based reporter system will be constructed to complement studies of chromosomal tandem repeat deletion. The training goal of this project is to prepare the applicant for a career in bacterial genetic research by providing didactic education and the opportunity to develop laboratory expertise in this rapidly evolving field. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: MOLECULAR DETERMINANTS OF HUMAN PNEUMOCOCCAL IMMUNITY Principal Investigator & Institution: Reason, Donald C.; Associate Scientist; Children's Hospital & Res Ctr at Oakland Research Center at Oakland Oakland, Ca 946091809 Timing: Fiscal Year 2002; Project Start 01-APR-2000; Project End 31-MAR-2005 Summary: Antibodies directed against the capsular polysaccharides of the pathogen Streptococcus pneumoniae protect humans against infection, and are elicited by vaccination with polysaccharide or polysaccharide conjugated to protein carriers. In the proposed study the variable region gene usage and junctional diversity of human antibodies specific for S. pneumoniae capsular serotypes 6B, 14, and 23F will be determined by repertoire cloning and sequence analysis. The influence of plain polysaccharide and polysaccharide-protein conjugate vaccine formulations on the expressed repertoire will be investigated, and the degree to which these thymusindependent and thymus-dependent forms of the vaccine induce somatic mutations and affinity maturation will be determined. The structural determinants of antipolysaccharide antibody affinity will be defined by sequence comparison, site directed mutagenesis, and molecular modeling. Sequence-defined Fab fragments will be expressed in vitro , their affinity and fine specificity determined, and the relationship between antibody affinity and protective efficacy established using an in vitro

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Streptococcus

opsonophagocytosis assay. Our overall hypothesis is that the quality of an oligoclonal antibody response, such as that seen in humans to bacterial capsular polysaccharides, is influenced to a greater degree by the affinities of the individual antibody binding domains than would be a polyclonal response. These differences in antibody affinity arise as a consequence of variable region gene usage, junctional diversity, and somatic mutation. The generation of affinity loss variants by somatic mutation could therefore leads a diminution of overall antibody quality. These studies will determine if antibodies to structurally distinct polysaccharides utilize the same or distinct variable region genes and the degree to which maturation of the response through somatic mutation determines overall affinity of the response. These studies will also determine if the same clonotypes occur in unrelated individuals, and if a single clonotype predominates the response to a given specificity. Defining the relationship between binding site affinity and antibody functional quality will provide better surrogate markers of protective immunity. Understanding the molecular mechanisms that shape the human antibody repertoire to pneumococcal polysaccharides may also suggest strategies that would facilitate the development of more efficacious vaccines. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MOLECULAR INTERACTIONS BETWEEN SPEA AND THE TCR Principal Investigator & Institution: Collins, Carleen M.; Professor; Microbiology; University of Washington Grant & Contract Services Seattle, Wa 98105 Timing: Fiscal Year 2002; Project Start 01-AUG-1998; Project End 31-JUL-2004 Summary: (Adapted from the applicant's abstract): In recent years in both this country and abroad, there has been a resurgence of acute, often life threatening infections due to Streptococcus pyogenes (group A streptococcus). Many patients experience symptoms mimicking this associated with staphylococcal toxic shock syndrome, and the designation streptococcal toxic shock syndrome (STSS) has been assigned to these invasive streptococcal infections. There is strong epidemiologic evidence implicating streptococcal pyrogenic exotoxin A (SpeA) in the pathogenesis of STSS. SpeA is a bacterial superantigen that is capable, in combination with class II major histocompatibility molecules, of activating a large fraction of T cells. The pathology of STSS and other bacterial superantigen mediated disease is believed to result from the massive and unregulated release of bioactive cytokines from the activated T cells. SpeA and other bacterial (and viral) proteins have been termed superantigens due to their unique mechanisms of interacting with the class II MHC expressing antigen-presenting cells and the T lymphocytes. Superantigens bind to class II MHC as intact molecules at sites distinct from the antigen-presenting groove. In addition to binding the class II MHC molecule, superantigens interact with the T cell receptor (TCR) in regions encoded by the V-gene segments. Each superantigen activates a specific set of V-beta chainencoding T cell, and thus is able to activate a much larger percentage of the T cell population than conventional peptide antigens. The goals of this grant proposal are to examine in detail the interaction between SpeA and the human TCR. The amino acid residues of SpeA needed for a productive TCR interaction will be defined. V-beta chains amino acids residues needed for a productive interaction with SpeA will be identified. These studies will include measurements of the affinities of the toxin- V-beta interactions. In addition, the X ray crystal structure of the toxin, mutant and allelic toxin forms, as well as the toxin completed with a human V-beta chain will be determined. These data will help characterize the interaction needed for activation of a T cell by the superantigen. In addition, the data obtained here are the first step in the design and development of compounds, such as TCR specific peptides, to interfere with the SpeA-

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TCR interaction, and in turn prevent SpeA from acting as a superantigen. It is possible that components that can specifically block the superantigenic capabilities of SpeA might prove useful as treatments to ameliorate and possibly prevent STSS in an infected individual. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: MOLECULAR COLONIZATION

MECHANISMS

OF

PNEUMOCOCCAL

Principal Investigator & Institution: Sebert, Michael E.; Microbiology; University of Pennsylvania 3451 Walnut Street Philadelphia, Pa 19104 Timing: Fiscal Year 2002; Project Start 01-JUL-2002; Project End 31-MAR-2007 Summary: (provided by applicant): This application describes a 5-year program designed to provide training for Dr. Michael Sebert in the skills necessary to become an independent researcher in the field of microbial pathogenesis. Dr. Sebert is currently completing his fellowship training in pediatric infectious diseases. His present work in the laboratory of Dr. Jeffrey Weiser, his sponsor for this application, has provided preliminary exposure to the study of the molecular mechanisms of bacterial infection. The educational plan outlined herein consists of both a comprehensive didactic program intended to provide a solid foundation in molecular genetics, prokaryotic cell biology, immunology, and bacterial pathogenesis as well as a roadmap for the in-depth laboratory experience necessary for his maturation into a productive, independent investigator. Colonization of the upper respiratory tract by the major bacterial pathogen Streptococcus pneumoniae serves as the common first step in the pathogenesis of the wide range of disease processes caused by this organism. How S. pneumoniae senses and adapts to this environment is unknown. Genomic analyses of the pneumococcus have revealed over a dozen putative two-component signal transduction systems. PreliminaryInvestigations have revealed that one of these systems, CiaRH, is required for persistence of the organism in the infant rat nasopharynx and that a portion, but not all, of this failure to colonize effectively can be attributed to downregulation of a putative serine protease, HtrA. The hypothesis to be tested in the proposed research is that a set of genes is regulated by the CiaRH system that collectively account for the requirement of this system for effective carriage. The first specific aim toward this objective is the biochemical and molecular characterization of the HtrA protein in order to define its precise role in nasal carriage. The second specific aim of this project is the definition of the sequence elements upstream of htrA responsible for its regulation by CiaRH and the subsequent usage of this information to identify other genes under the control of CiaRH that contribute to colonization. Together these lines of investigation hold the potential to expand the understanding of the molecular basis of this critical step in the pathogenesis of S. pneumoniae. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: NOVEL BIFUNCTIONAL MOLECULES FOR INTRAORAL DRUG DELIVERY Principal Investigator & Institution: Periathamby, Antony R.; Sullivan Professor; Periodontics; Marquette University P.O. Box 1881 Milwaukee, Wi 532011881 Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-MAY-2004 Summary: The goal of this innovative research project is to develop simple bifunctional molecules for intraoral delivery of antimicrobial agents. The bifunctional hybrid molecules will each be composed of a carrier sequence possessing high affinity for tooth

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and pellicle surfaces, and a natural antimicrobial peptide. These two sequences will be linked to each other with a biodegradable bond. With this linkage, antimicrobial sequences inherently linked to the carrier will be released efficiently from the tooth surface and in saliva through the oral physiological and microbial environment for a controlled and sustained release of the antimicrobial agent, thereby providing a novel and efficient method for intraoral drug delivery. This research project involves: 1) Synthesis of hybrid molecules by rationally selecting carrier sequences from salivary statherin, and antimicrobial sequences from bactenecins and defensins; 2) Determination of toxicity of hybrid molecules to ensure that they are cytotoxic only to microbes; 3) Delineation of the effect of whole saliva on the stability of hybrid molecules; 4) Assessment of the adsorption and desorption characteristics of hybrid molecules onto hydroxyapatite surfaces; 5) Determination of the extent of adherence of Candida albicans, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Streptococcus mutans, Streptococcus gordonii and Streptococcus sanguis onto the hybrid adsorbed hydroxyapatite surfaces. This proposed research will identify new, safe, and affordable hybrid molecules for the prevention and treatment of plaquerelated oral diseases. The hybrid molecules can be directly used as a topical rinse, or irrigant or they may be applied professionally to sub-gingival areas. The oral physiological and microbial environment will naturally induce the dissociation and the release of the antimicrobial peptide from the tooth surface into the site of oral infection. The hybrid molecules will serve as an efficient local drug delivery system and eliminate the discomfort and retention problems associated with the existing local delivery devices. The hybrid molecules will have a high potential for clinical and commercial application as anti-plaque agents. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: NUCLEASE-RESISTANT OPSONIZATION

APTAMERS

FOR

ANTHRAX

Principal Investigator & Institution: Bruno, John G.; Operational Technologies Corporation Suite 230 San Antonio, Tx 78229 Timing: Fiscal Year 2004; Project Start 01-APR-2004; Project End 31-OCT-2004 Summary: (provided by investigator): The anti-phagocytic poly-D-glutamic acid (PDGA) capsule of Bacillus anthracis is a major virulence factor of inhalation anthrax. Although generation of antibodies to PDGA might be of value in opsonizing vegetative anthrax, such antibodies would require "humanization". A simpler and less expensive approach would be the development of nuclease-resistant ('shielded') DNA aptamers against PDGA with an Fc-TR or C3bR binding domain at the other end of a 'hybrid' aptamer. In Phase I, Operational Technologies (OpTech) in conjunction with the Biochemistry Department of the University of Texas at San Antonio (UTSA), proposes to develop 2'- amino pyrimidine modified shielded DNA aptamers against PDGA, murine Fc-yR, and C3bR by the SELEX process. OpTech will link the aptamers and compare phagocytosis of PDGA-conjugated microbeads by the RAW 264.7 macrophage-like cell line in the presence and absence of shielded aptamers and versus serum opsonized PDGA-conjugated microbeads. Since polyanionic PDGA appears to inhibit phagolysosome formation and killing of ingested bacteria, targeting PDGA with 2'amino-modified aptamers may serve to partially neutralize the polyanionic charge and enable bacterial killing by the respiratory burst. In Phase II, OpTech will clone and sequence all aptamers, then assess aptamer-opsonization and bacterial killing in RAW 264.7 cells with virulent anthrax at the Southwest Research Foundation. Since anthrax and some other infectious bacteria utilize capsules to evade phagocytosis, any

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inexpensive reagent that acts as an effective opsonin would be of tremendous value in the medical, veterinary or agricultural communities. If successful, OpTech will possess a pharmaceutical substance of great commercial value in the post-exposure treatment of anthrax. Further development of the concept for shielded aptamers against hyaluronic acid or other capsule materials, for example, would also lead to better treatments for many Streptococcus-related infections. Such shielded aptamers could even be used in inhalers to enhance alveolar macrophage phagocytosis. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: PANDAS AND STREP INFECTION: ARE THEY LINKED? Principal Investigator & Institution: Kurlan, Roger M.; Professor; Neurology; University of Rochester Orpa - Rc Box 270140 Rochester, Ny 14627 Timing: Fiscal Year 2002; Project Start 25-SEP-2001; Project End 31-JUL-2005 Summary: Sydenham's chorea is the only accepted immune-mediated central nervous system manifestation of group A beta hemolytic streptococcal (GABHS) infection. Several lines of evidence now suggest that there may be a spectrum of post-GABHS immune- mediated neurobehavioral sequelae termed "Pediatric Autoimmune Neuropsychiatric Disorders after Streptococcal Infection (PANDAS)". Tics, including Tourette's syndrome (TS), and obsessive-compulsive disorder (OCD) have been reported as the characteristic features of PANDAS. Proposed is a multicenter prospective case control cohort study involving 40 cases of PANDAS and 40 matched controls with TS and/or OCD but without evidence of PANDAS. All subjects will undergo intensive clinical and laboratory prospective observation for 24 months to determine whether antecedent GABHS infection is: 1) temporally associated with exacerbations of PANDAS, 2) specifically associated with exacerbations of tics and OCD, and 3) a specific trigger for exacerbations of PANDAS. All determinations of case/control status, GABHS infection and clinical exacerbation will be determined independently by blinded review in order to limit selection and clinical biases. Establishing a post-infectious etiology for PANDAS would dramatically change our understanding of the causes of TS and OCD, alter our therapeutic approach and may have a critical public health implication of preventing potentially fatal rheumatic cardiac sequelae in affected children. Disproving the PANDAS hypothesis would prevent the use of expensive and potentially dangerous therapies (e.g., plasma exchange, immune globulin, antibiotics) that have been proposed for PANDAS patients. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: PERA HELICASE AND REPLICATION OF DRUG RESISTANCE PLASMID Principal Investigator & Institution: Khan, Saleem A.; Professor; Molecular Genetics & Biochem; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260 Timing: Fiscal Year 2003; Project Start 15-APR-2003; Project End 31-MAR-2005 Summary: (provided by applicant): Plasmids that replicate by a rolling-circle (RC) mechanism are ubiquitous in Gram-positive bacteria and a vast majority of over 200 rolling-circle replicating (RCR) plasmids belong to four major families. Many RCR plasmids carry antibiotic resistance genes and evidence suggests that horizontal transfer of RCR plasmids is quite common. Many RCR plasmids also contain genes that are involved in plasmid mobilization and transfer. While some RCR plasmids are able to replicate stably only in their native hosts, many have a broad host range. RC replication involves synthesis of the leading strand that requires the PcrA helicase, single strand

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Streptococcus

DNA binding protein and DNA polymerase III. Lagging strand replication requires primer RNA synthesis by the host RNA polymerase, and subsequently replication is carried out by DNA Pol I and DNA Pol III. PcrA helicase is required for plasmid RC replication as well as survival of Gram-positive organisms. The goal of this R21 application is to test the hypothesis that interaction between plasmid initiator proteins and the PcrA helicases is critical for efficient plasmid replication and may determine narrow versus broad host range replication ofRCR plasmids. No studies have been performed dealing with this issue and the exploratory/development nature of the current application is consistent with the R21 format. We plan to utilize a few plasmids with a relatively narrow host range and a few with broad host range in our study. We will study the replication of these plasmids in S. aureus, B. subtilis, B. cereus and S. pneumoniae. RCR plasmids defective in replication in a particular host will be complemented by a cloned copy of their cognate pcrA gene. We will purify the PcrA helicases from the above Gram-positive organisms and study their interactions with the plasmid initiator proteins. We will also study the in vitro replication of the above plasmids in cell free extracts made from S. aureus in the presence and absence of the various PcrA helicases. These studies should reveal whether PcrA is critical in determining the efficient replication and host range of RCR plasmids. These studies may facilitate the development of antimicrobial drugs targeting the PcrA helicase and/or the initiators of RCR plasmids. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: PNEUMOCOCCAL EPIDEMIOLOGY

INFECTIONS

IN

SCD--PATHOLOGY

&

Principal Investigator & Institution: Tuomanen, Elaine I.; Chair, Professor; St. Jude Children's Research Hospital Memphis, Tn 381052794 Timing: Fiscal Year 2003; Project Start 01-JUL-2003; Project End 31-MAR-2008 Summary: Patients with sickle cell disease experience a 400 fold increased risk of severe pneumococcal infection. This risk is not shared by other encapsulated pathogens or by other anemias suggesting that there is a potentially causal relationship between severity of pneumococcal disease and the SS phenotype. This application seeks to investigate two aspects of pneumococcal infection in sickle cell disease. First we will take advantage of the presence in this SS Center of a program in pneumococcal pathogenesis and expertise in creating an SS transplant mouse model (Project 4). These two programs will cooperate to characterize which step in pneumococcal invasion differs between wild type and SS mice. The steps of colonization and invasion will be dissected at the molecular level and intervention with specific receptor antagonists will be tested. We will further determine the level of protection from pneurnococcal disease afforded by progressive hematologic correction by transplantation. Second we will build on a strong history of this Center's study of the colonization of SS patients with antibiotic resistant pneumococci. The introduction of the new seven-valent conjugate pneumococcal vaccine into the SS population has not been studied. We will determine its effect on nasopharyngeal carriage with specific reference to antibiotic susceptibility and shifts away from vaccine serotypes. Further we will measure the prevalence of a new property of antibiotic tolerance emerging in clinical isolates. Tolerance prevents antibiotic killing of pneumococci and may adversely affect the outcome of infection. Spread of this property may have implications for the efficacy of continued penicillin prophylaxis in this at risk population. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: PNEUMOLYSIN, INNATE AND ACQUIRED IMMUNITY TO PNEUMOCOCCI Principal Investigator & Institution: Malley, Richard; Assistant Professor; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2002; Project Start 15-APR-2002; Project End 31-MAR-2006 Summary: Streptococcus pneumoniae remains a major cause of morbidity and mortality worldwide. Two components of pneumococci are thought to be responsible for much of the deleterious inflammatory hot response, the thiol-activated secreted toxin pneumolysin and the peptidoglycan component of the ell wall. Both of these molecules have been shown to cause inflammatory changes in vitro studies in epithelial and neuronal cells, as well as activate monocytes and macrophages of the inactive immune system. Preliminary work in our laboratory supports the hypothesis that pneumolysin activates the innate arm of the immune system through a transmembrane receptor, Tolllike receptor 4 (TLR4), in synergy with peptidoglycan (a TLR2 ligand) and/or whole pneumococci. In addition, we have some preliminary data to suggest that the activation of the innate immune response by pneumolysin may greatly augment acquired immunity to pneumococcus as well. The scientific goals of this project are divided in two phases. In the first phase, the PI will study the TLR- dependent signaling pathway of pneumolysin, alone and in combination with peptidoglycan or whole pneumococci. Studies will be done to test the hypothesis that pneumolysin binds directly to TLR4. This phase will also include an intense didactic component, to complement the work in the laboratory. In the second phase, the PI will study the role of this pathway in the modulation of disease and acquired immunity to pneumococci. This phase of the project will test the hypothesis that TLR4-mediated cellular activation by pneumolysin plays an important role in the pathophysiology of pneumococcal disease. Pneumococci carrying mutations in the pneumolysin gene, with diminished or abrogated TLR4-signaling capability, will be studied both in vitro (in macrophages) and in vivo (in murine models of colonization and disease). The possible role of TLR4-mediated response to pneumolysin in the development of acquired immunity will be studied in the final aim of the proposal. The establishment of a link between the innate and the acquired arm of the immune response to pneumococcus may provide a better understanding of the mechanisms by which immunity to pneumococcus is acquired. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: PREVENTION OF PNEUMOCOCCAL DISEASE IN HIV INFECTION Principal Investigator & Institution: Mcellistrem, Mary C.; Medicine; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260 Timing: Fiscal Year 2002; Project Start 30-SEP-2000; Project End 31-MAY-2005 Summary: (adapted from the application s abstract): A K24 award would afford the candidate the opportunity to attain the following objectives: 1) Short-term Objectives: Determine the epidemiology of Streptococcus pneumoniae infections in HIV-infected patients and establish the optimum clinical conditions necessary to facilitate utilization of an innovative, tailored, immunogenic and safe vaccine. 2) Long-term Objectives: The candidate has become proficient at pulse field gel electrophoresis (PFGE) and computer assisted gel analysis. This award will allow her to increase her current technical ability while realizing her aspiration of performing clinical trials. She will obtain her MPH in Epidemiology at the University of Pittsburgh School of Medicine s Program in Clinical Effectiveness/Evaluation Sciences and the Graduate School of Public Health with a focus on epidemiological methods, design of clinical trials, data management and

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Streptococcus

analyses. The candidate will be able to synthesize her technical expertise in bacterial subtyping with her desire to prevent bacterial disease in high risk populations. S. pneumoniae disease is associated with more deaths than any other vaccine- preventable bacterial pathogen. While the 23-valent pneumococcal polysaccharide vaccine covers the majority of invasive serotypes, this vaccine has inferior immunogenicity in HIV-infected patients with CD4 lymphocyte counts