National Institute of Allergy and Infectious Diseases, NIH

Volume 1

Frontiers in Research

Infectious Disease

Vassil St. Georgiev

For other titles published in the series, go towww.springer.com / humanaclick on the series disciplineclick on the heading Seriesclick on the name of the series

National Institute of Allergy and Infectious Diseases, NIH

Volume 1

Frontiers in Research

Edited by

Vassil St. Georgiev, PhDKarl A. Western, MDJohn J. McGowan, PhDNational Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

EditorsVassil St. Georgiev, PhDKarl A. Western, MDJohn J. McGowan, PhDNational Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

Series EditorVassil St. GeorgievNational Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD

ISBN 978-1-934115-77-0 e-ISBN 978-1-59745-569-5DOI: 10.1007/978-1-59745-569-5

Library of Congress Control Number: 2007941162

2008 Humana Press, a part of Springer Science+Business Media, LLCAll rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, 999 Riverview Drive, Suite 208, Totowa, NJ 07512 USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden.The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein.

Cover illustration: Adapted from Chapter 30, Fig. 30.3, showing the sCD4-17b bifunctional protein, which in turn is based on the atomic structure reported in Kwong et al., Nature, 393:648659 (1998).

Printed on acid-free paper

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Dedication

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

RESEARCH IS NOT A SYSTEMATIC OCCUPATION BUT AN INTUITIVE ARTISTIC VOCATION

Albert Szent-Gyrgyi

Preface

For more than 50 years, as part of the National Institutes of Health, the mission of the National Institute of Allergy and Infec-tious Diseases (NIAID) has been to conduct and support basic and applied research to better understand, treat, and prevent infectious, immunologic, and allergic diseases with the ultimate goal of improving the health of individuals in the United States and around the world.

In recent years, NIAID has responded to new challenges including emerging and re-emerging infectious diseases, potential bioterrorism threats, and an increase in pediatric asthma prevalence. A cornerstone of NIAID-supported research also continues to be the discovery and improvement of vaccines focused on an array of infectious diseases with global public health impor-tance.

As part of its mission to foster biomedical discovery and to reduce the burden of human disease, NIH and NIAID in par-ticular, are committed to encouraging the accelerated translation of biomedical discoveries into effective clinical care and public health practice throughout the world. In pursuit of this goal and its disease-specific scientific objectives, NIAID seeks to broaden research opportunities and collaborations involving scientists and institutions outside the United States.

During 2006, special emphasis was given to fostering scientific collaboration between U.S. researchers and investigators in Central and Eastern Europe, the Baltic Region, Russia, Ukraine, and other newly independent states that were formerly part of the Soviet Union. Although the countries of Central and Eastern Europe have strong traditions in biomedical research, scien-tists from this region have been less successful than their Western European colleagues in competing for NIAID funding and in forming partnerships with U.S. scientists. To help address this situation, NIAID convened a research conference in Opatija, Croatia (June 2430, 2006) so that U.S. and European scientists could explore shared research interests with a focus on micro-biology and infectious diseases, HIV/AIDS, and basic and clinical immunology.

In the field of microbiology and infectious diseases, major presentations at the conference focused on recent research devel-opments in emerging and re-emerging infections (anthrax and other potential biological weapons, vector-borne infections, tuberculosis, and influenza). A number of presentations discussed ongoing research targeting the development of infectious disease prophylactics and therapeutics.

One of the most serious problems worldwide that confronts efforts to control and treat infectious diseases is the increasing resistance of some pathogens to the current armamentarium of drugs. Microorganisms belonging to all four classes of infectious agents (bacteria, viruses, parasites, and fungi) have developed resistance to previously effective chemotherapeutics, thereby becoming serious threats to individual well-being and international public health. One striking example of drug resistance is the emergence of extensively drug-resistant tuberculosis. Several conference presentations were therefore focused on drug resistance.

HIV/AIDS also remains a major infectious disease research priority and it was well addressed during the conference. Since the start of the HIV/AIDS pandemic in the early 1980s, nearly 20 million people worldwide have died of the disease. According to an estimate issued by the Joint United Nations Programme on HIV/AIDS (UNAIDS) by the end of 2003, about 38 million adults and children were living with HIV/AIDS and in many countries overall prevalence still is rising. Although much prog-ress has been made in the treatment of AIDS and in understanding effective strategies to prevent HIV transmission, research is urgently needed on vaccines, microbicides, therapeutic agents, behavioral prevention strategies, and the management of HIV-related co-morbidities.

NIAID-funded research in basic and clinical immunology has led to significant discoveries that have guided the effective treatment of a host of immunological conditions. For example, tolerance induction research has enabled the selective block-ing of inappropriate or destructive immune responses while leaving protective immune responses intact. Major presentations at

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the conference discussed various topics in immunomodulation, autoimmunity, infections and immunity, and vaccine develop-ment.

Finally, two sessions at the research conference were designed to inform participants about NIAIDs research funding mech-anisms and the NIH application process.

With more than 100 participants, the 2006 NIAID Research Conference in Croatia clearly demonstrated NIAIDs com-mitment to a cutting-edge scientific exchange to help generate more research cooperation. Following the meeting, numerous research collaborations have been explored and numerous joint research applications have been prepared and submitted.

NIAID is pleased to have supported this important and unusual meeting and it welcomes publication of the important sci-entific findings presented there. The future of science lies in cooperation across national borders. Therefore, it is particularly rewarding to see research partnerships grow between scientists from countries previously characterized by a lack of commu-nication and mutual understanding. With a strong research base, talented investigators in the United States and abroad, and the availability of powerful new research tools, NIAID will continue to support scientists in the forefront of basic and applied infectious and immune-mediated disease research.

Vassil St. GeorgievBethesda, MD

viii Preface

Acknowledgments

ix

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

preparation of this volume.

Contents

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

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xv

PART I INTRODUCTION

National Institute of Allergy and Infectious Diseases (NIAID): An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Karl A. Western

PART II MICROBIOLOGY AND INFECTIOUS DISEASES

Section 1 Emerging and Re-Emerging Infections

1 Biotools for Determining the Genetics of Susceptibility to Infectious Diseases and Expediting Research Translation into Effective Countermeasures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Malak Kotb, Robert W. Williams, Nourtan Fathey, Mohamed Nooh, Sarah Rowe, Rita Kansal, and Ramy Aziz

2 Spore Surface Components and Protective Immunity to Bacillus anthracis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Patricia Sylvestre, Ian Justin Glomski, Evelyne Couture-Tosi, Pierre Louis Goossens, and Michle Mock

3 New Candidate Anthrax Pathogenic Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 Serguei G. Popov

4 Ehrlichiae and Ehrlichioses: Pathogenesis and Vector Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 H. L. Stevenson, N. Ismail, and D. H. Walker

5 Multiple Locus Variable Number Tandem Repeat (VNTR) Analysis (MLVA) of Brucella spp. Identifies Species-Specific Markers and Insights into Phylogenetic Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . 47Lynn Y. Huynh, Matthew N. Van Ert, Ted Hadfield, William S. Probert, Bryan H. Bellaire, Michael Dobson, Robert J. Burgess, Robbin S. Weyant, Tanja Popovic, Shaylan Zanecki, David M. Wagner, and Paul Keim

6 Expression of the MtrC-MtrD-MtrE Efflux Pump in Neisseria gonorrhoeae and Bacterial Survival in the Presence of Antimicrobials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55William M. Shafer, Jason P. Folster, Douglas E. M. Warner, Paul J. T. Johnson, Jacqueline T. Balthazar, Nazia Kamal, and Ann E. Jerse

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Section 2 Tuberculosis

7 What can Mycobacteriophages Tell Us About Mycobacterium tuberculosis ? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 Graham F. Hatfull

8 Clinical Mycobacterium tuberculosis Strains Differ in their Intracellular Growth in Human Macrophages . . . . . . . . 77 Sue A. Theus, M. Donald Cave, and Kathleen D. Eisenach

9 Mechanisms of Latent Tuberculosis: Dormancy and Resuscitation of Mycobacterium tuberculosis . . . . . . . . . . . . . 83 Galina Mukamolova, Elena Salina, and Arseny Kaprelyants

10 Separating Latent and Acute Disease in the Diagnosis of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 T. Mark Doherty

11 Mutant Selection Window Hypothesis: A Framework for Anti-mutant Dosing of Antimicrobial Agents . . . . . . . . . . 101 Karl Drlica and Xilin Zhao

Section 3 Avian Influenza

12 The NIAID Influenza Genome Sequencing Project . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109Lone Simonsen, Gayle Bernabe, Karen Lacourciere, Robert J. Taylor, and Maria Y. Giovanni

13 Lessons from the 1918 Spanish Flu Epidemic in Iceland . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 Magns Gottfredsson

14 Control of Notifiable Avian Influenza Infections in Poultry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123 Ilaria Capua and Stefano Marangon

15 Understanding the Complex Pathobiology of High Pathogenicity Avian Influenza Viruses in Birds . . . . . . . . . . . . . 131 David E. Swayne

Section 4 Prophylactics and Therapeutics for Infectious Diseases

16 Development of Prophylactics and Therapeutics Against the Smallpox and Monkeypox Biothreat Agents . . . . . . . . 145 Mark Buller, Lauren Handley, and Scott Parker

17 The Hierarchic Informational Technology for QSAR Investigations: Molecular Design of Antiviral Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163V. E. Kuzmin, A. G. Artemenko, E. N. Muratov, L. N. Ognichenko, A. I. Hromov, A. V. Liahovskij, and P. G. Polischuk

18 Antivirals for Influenza: Novel Agents and Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 William A Fischer, II and Frederick Hayden

19 Anti-Infectious Actions of the Proteolysis Inhibitor -Aminocaproic Acid (-ACA) . . . . . . . . . . . . . . . . . . . . . . . . . 193 V. P. Lozitsky

20 A New Highly Potent Antienteroviral Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199Lubomira Nikolaeva-Glomb, Stefan Philipov, and Angel S. Galabov

Section 5 Russian Perspectives in Emerging and Re-Emerging and Infections Research

21 Reduction and Possible Mechanisms of Evolution of the Bacterial Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205 George B. Smirnov

22 Interaction of Yersinia pestis Virulence Factors with IL-1R/TLR Recognition System . . . . . . . . . . . . . . . . . . . . . . . . 215Vyacheslav M. Abramov, Valentin S. Khlebnikov, Anatoly M. Vasiliev, Igor V. Kosarev, Raisa N. Vasilenko, Nataly L. Kulikova, Vladimir L. Motin, George B. Smirnov, Valentin I. Evstigneev, Nicolay N. Karkischenko, Vladimir N. Uversky, and Robert R. Brubaker

xii Contents

23 IS481-Induced Variability of Bordetella pertussis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227Ludmila N. Sinyashina, Alisa Yu. Medkova, Evgeniy G. Semin, Alexander V. Chestkov, Yuriy D. Tsygankov, and Gennagiy I. Karataev

24 Microarray Immunophosphorescence Technology for the Detection of Infectious Pathogens . . . . . . . . . . . . . . . . . . 233 Nikolay S. Osin and Vera G. Pomelova

25 Development of Immunodiagnostic Kits and Vaccines for Bacterial Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 Valentina A. Feodorova and Onega V. Ulianova

Section 6 Perspectives in Emerging and Re-Emerging InfectionsResearch in Central Asia and Caucasus

26 Research in Emerging and Re-Emerging Diseases in Central Asia and the Caucasus: Contributions by the the National Institute of Allergy and Infectious Diseases and the National Institutes of Health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251Katherine T. Herz

27 Disease Surveillance in Georgia: Benefits of International Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253Lela Bakanidze, Paata Imnadze, Shota Tsanava, and Nikoloz Tsertsvadze

28 Epidemiology (Including Molecular Epidemiology) of HIV, Hepatitis B and C in Georgia: Experience From U.S.Georgian Collaboration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 Tengiz Tsertsvadze

29 The National Tuberculosis Program in the Country of Georgia: An Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263Archil Salakaia, Veriko Mirtskhulava, Shalva Gamtsemlidze, Marina Janjgava, Rusudan Aspindzelashvili, and Ucha Nanava

PART III HUMAN IMMUNODEFICIENCY VIRUS AND AIDS

30 Virus Receptor Wars: Entry Molecules Used for and Against Viruses Associated with AIDS . . . . . . . . . . . . . . . . . . . 271 Edward A. Berger

31 HIV Latency and Reactivation: The Early Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 Guido Poli

32 HIV-1 Sequence Diversity as a Window Into HIV-1 Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289 Milloni Patel, Gretja Schnell, and Ronald Swanstrom

33 Human Monoclonal Antibodies Against HIV and Emerging Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 299 Dimiter S. Dimitrov

34 Biological Basis and Clinical Significance of HIV Resistance to Antiviral Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Mark A. Wainberg and Susan Schader

35 NIAID HIV/AIDS Prevention Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319 David N. Burns and Roberta Black

36 Epidemiological Surveillance of HIV and AIDS in Lithuania . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327 Saulius Caplinskas

PART IV IMMUNOLOGY AND VACCINES

Section 1 Immunomodulation

37 TACI, Isotype Switching, CVID, and IgAD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 Emanuela Castigli and Raif S. Geha

38 A Tapestry of Immunotherapeutic Fusion Proteins: From Signal Conversion to Auto-stimulation . . . . . . . . . . . . . . . 349 Mark L. Tykocinski, Jui-Han Huang, Matthew C. Weber, and Michal Dranitzki-Elhalel

Contents xiii

39 A Role for Complement System in Mobilization and Homing of Hematopoietic Stem/Progenitor Cells . . . . . . . . . . 357M. Z. Ratajczak, R. Reca, M. Wysoczynski, M. Kucia, and J. Ratajczak

40 Post-translational Processing of Human Interferon- Produced in Escherichia coli and Approaches for Its Prevention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365Maya Boyanova, Roumyana Mironova, Toshimitsu Niwa, and Ivan G. Ivanov

Section 2 Autoimmunity

41 B-cell dysfunctions in Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 Moncef Zouali

42 A Model System for Studying Mechanisms of B-cell Transformation in Systemic Autoimmunity . . . . . . . . . . . . . . 385Wendy F. Davidson, Partha Mukhopadhyay, Mark S. Williams, Zohreh Naghashfar, Jeff X. Zhou, and Herbert C. Morse, III

43 Breach and Restoration of B-Cell Tolerance in Human Systemic Lupus Erythematosus (SLE) . . . . . . . . . . . . . . . . . 397 Iaki Sanz, R. John Looney, and J. H. Anolik

Section 3 Infection and Immunity

44 Dendritic Cells: Biological and Pathological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 409Jacques Banchereau, John Connolly, Tiziana Di Pucchio, Carson Harrod, Eynav Klechevsky, A. Karolina Palucka, Virginia Pascual, and Hideki Ueno

45 Immunomic and Bioinformatics Analysis of Host Immunity in the Vaccinia Virus and Influenza A Systems . . . . . . 429Magdalini Moutaftsi, Bjoern Peters, Valerie Pasquetto, Carla Oseroff, John Sidney, Huynh Hoa-Bui, Howard Grey, and Alessandro Sette

46 Immunoreactions to Hantaviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 435 Alemka Markotic and Connie Schmaljohn

47 Innate Immunity to Mouse Cytomegalovirus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 445Djurdjica Cekinovic, Irena Slavuljica, Tihana Lenac, Astrid Krmpotic, Bojan Polic, and Stipan Jonjic

Section 4 Vaccines

48 Research and Development of Chimeric Flavivirus Vaccines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459 Simon Delagrave and Farshad Guirakhoo

49 Correlates of Immunity Elicited by Live Yersinia pestis Vaccine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473Vivian L. Braciale, Michael Nash, Namita Sinha, Irina V. Zudina, and Vladimir L. Motin

PART V BUILDING A SUSTAINABLE PERSONAL RESEARCH PORTFOLIO

50 Strategies for a Competitive Research Career . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483Hortencia Hornbeak and Peter R. Jackson

51 Selecting the Appropriate Funding Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487Priti Mehrotra, Hortencia Hornbeak, Peter R. Jackson, and Eugene Baizman

52 Preparing and Submitting a Competitive Grant Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497Peter R. Jackson and Hortencia Hornbeak

53 Identifying Research Resources and Funding Opportunities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507Eugene Baizman, Hortencia Hornbeak, Peter R. Jackson, and Priti Mehrotra

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

xiv Contents

Contributors

Vyacheslav M. Abramov Department of Biochemistry of Immu-nity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia

J. H. Anolik Department of Medicine, Division of Clinical Immu-nology and Rheumatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

A. G. Artemenko A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Rusudan Aspindzelashvili National Center for Tuberculosis and Lung Diseases / National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia

Ramy Aziz The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA

Eugene Baizman Scientific Review Program, Division of Extra-mural Activities, National Institute of Allergy and Infectious Dis-eases, National Institutes of Health, Bethesda, MD, USA

Lela Bakanidze National Center for Disease Control and Medi-cal Statistics of Georgia, Tbilisi, Republic of Georgia

Jacqueline T. Balthazar Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA

Jacques Banchereau Baylor Institute for Immunology Research, Dallas, TX, USA

Bryan H. Bellaire Louisiana State University Health Science Center, Shreveport, LA, USA

Edward A. Berger Laboratory of Viral Diseases, National Insti-tute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Gayle Bernabe Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Roberta Black Prevention Sciences Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Maya Boyanova Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Vivian L. Braciale Department of Microbiology and Immunol-ogy, University of Texas Medical Branch, Galveston, TX, USA

Robert R. Brubaker Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, USA

Mark Buller Department of Molecular Microbiology and Immu-nology, Saint Louis University Health Sciences Center, St. Louis, MO, USA

Robert J. Burgess Armed Forces Institute of Pathology, Wash-ington, DC, USA

David N. Burns Prevention Sciences Branch, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Saulius Caplinskas Lithuanian AIDS Center, Mykolas Romeris University, Vilnius, Lithuania

Ilaria Capua OIE/FAO Reference Laboratory for Newcastle Dis-ease and Avian Influenza, Istituto Zooprofilattico Sperimentale delle Venezie, Legnaro, Padova, Italy

Emanuela Castigli Division of Immunology, Childrens Hospi-tal, Boston, MA, USA

M. Donald Cave Neurobiology and Developmental Science, University of Arkansas for Medical Sciences and Central Arkan-sas Veterans Healthcare System, Little Rock, AR, USA

Djurdjica Cekinovic Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia

Alexander V. Chestkov State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia

Evelyne Couture-tosi Unit Toxines et Pathognie Bactri-ennes, Institut Pasteur, Paris, France

Wendy F. Davidson Marlene and Stewart Greenebaum Cancer Center and Department of Microbiology and Immunology, and the Center for Vascular and Inflammatory Diseases, BioPark Building 1, University of Maryland, Baltimore, MD, USA

Simon Delagrave Acambis Inc., Cambridge, MA, USA Tiziana Di Pucchio Baylor Institute for Immunology Research,

Dallas, TX, USA Dimiter S. Dimitrov Protein Interactions Group, Center for Can-

cer Research Nanobiology Program, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

Michael Dobson Armed Forces Institute of Pathology, Washing-ton, DC, USA

T. Mark Doherty Statens Serum Institut, Department of Infec-tious Disease Immunology, Copenhagen, Denmark

Michal Dranitzki-Elhalel Hadassah Medical Center, Ein Kerem, Israel

Karl Drlica Public Health Research Institute, Newark, NJ, USA Kathleen D. Eisenach Departments of Pathology, Microbiology

and Immunology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas, USA

Valentin I. Evstigneev Department of Biochemistry, Immunity, and Biodefense, Institute of Immunological Engineering, Lyubu-chany, Russia

Nourtan Fathey The MidSouth Center for Biodefense and Secu-rity at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA

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Valentina A. Feodorova Scientific and Research Department, Saratov State University, Saratov Russia, Russia

William A Fischer, II Johns Hopkins Hospital, Baltimore, MD; Global Influenza Program, World Health Organization, Geneva, Switzerland; and University of Virginia, Charlottesville, VA, USA

Jason P. Folster Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA

Angel S. Galabov Department of Virology, The Stephan Ange-loff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Shalva Gamtsemlidze National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia

Raif S. Geha Division of Immunology, Childrens Hospital, Bos-ton, MA, USA

Vassil St. Georgiev Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Maria Y. Giovanni Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Magns Gottfredsson Department of Medicine, Landspitali University Hospital and University of Iceland School of Medicine, Reykjavik, Iceland

Ian Justin Glomski Unit Toxines et Pathognie Bactriennes, Institut Pasteur, Paris, France

Pierre Louis Goossens Unit Toxines et Pathognie Bactriennes, Institut Pasteur, Paris, France

Howard Grey Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Farshad Guirakhoo Senior Director, External Research & Development, Global Research and R&D, Sanofi Pasteur Acam-bis Inc., Cambridge, MA, USA

Ted Hadfield Armed Forces Institute of Pathology, Washington, DC, USA

Lauren Handley Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, MO, USA

Carson Harrod Baylor Institute for Immunology Research, Dallas, TX, USA

Graham F. Hatfull Department of Biological Sciences, Univer-sity of Pittsburgh, Pittsburgh, PA, USA

Frederick Hayden J ohns Hopkins Hospital, Baltimore, MD; Global Influenza Program, World Health Organization, Geneva, Switzerland and University of Virginia, Charlottesville, VA, USA

Katherine T. Herz Office of Global Research, National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA

Huynh Hoa-bui Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Hortencia Hornbeak Scientific Review Program, Division of Extramural Activities, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

A. I. Hromov A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Jui-han Huang Department of Pathology and Laboratory Medi-cine, University of Pennsylvania, Philadelphia, PA, USA

Lynn Y. Huynh Department of Biological Sciences, Northern Ari-zona University, Flagstaff, AZ, USA

Paata Imnadze National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia

Ivan G. Ivanov Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Peter R. Jackson Scientific Review Program, Division of Extra-mural Activities, National Institute of Allergy and Infectious Dis-eases, National Institutes of Health, Bethesda, MD, USA

Marina Janjgava National Center for Tuberculosis and Lung Dis-eases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia

Ann E. Jerse Veterans Affairs Medical Center, Decatur; and Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA

Paul J. T. Johnson Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA, USA

Stipan Jonjic Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia

Nazia Kamal Department of Microbiology and Immunology , Emory University School of Medicine, Atlanta, GA, USA

Rita Kansal The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN

Arseny Kaprelyants Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia

Gennagiy I. Karataev Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia

Nicolay N. Karkischenko Scientific Center of Biomedical Tech-nologies RAMS, Russia

Paul Keim Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA

Valentin S. Khlebnikov Department of Biochemistry, Immunity and Biodefense, Institute of Immunological Engineering, Lyubu-chany, Russia

Eynav Klechevsky Baylor Institute for Immunology Research, Dallas, TX, USA, TechnionIsrael Institute of Technology, Tech-nion City, Haifa, Israel

Igor V. Kosarev Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia

Malak Kotb The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA

Astrid Krmpotic Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia

M. Kucia Stem Cell Biology Program, University of Louisville, Louisville, KY, USA

Nataly L. Kulikova Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubu-chany, Russia

V. E. Kuzmin A .V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Karen Lacourciere Division of Microbiology and Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Tihana Lenac Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia

A. V. Liahovskij A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

xvi Contributors

R. John Looney Department of Medicine, Division of Clinical Immunology and Rheumatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

V. P. Lozitsky Ukrainian I.I. Mechnikov Research Anti-Plague Institute, Odessa, Ukraine

Stefano Marangon OIE/FAO Reference Laboratory for New-castle Disease and Avian Influenza, Istituto Zooprofilattico Speri-mentale delle Venezie, Legnaro, Padova, Italy

Alemka Markotic University Hospital of Infectious Diseases, Zagreb, Croatia

John J. McGowan National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Alisa Yu. Medkova Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia

Priti Mehrotra Scientific Review Program, Division of Extra-mural Activities, National Institute of Allergy and Infectious Dis-eases, National Institutes of Health, Bethesda, MD, USA

Roumyana Mironova Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Veriko Mirtskhulava National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia and Emory University, Atlanta, GA, USA

Michle Mock Unit Toxines et Pathognie Bactriennes, Institut Pasteur, Paris, France

Herbert C. Morse, III Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA

Vladimir L. Motin Departments of Pathology/Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA

Magdalini Moutaftsi Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Galina Mukamolova Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia

Partha Mukhopadhyay Marlene and Stewart Greenebaum Can-cer Center and Center for Vascular and Inflammatory Diseases, University of Maryland, Baltimore, MD, USA

E. N. Muratov A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Ucha Nanava National Center for Tuberculosis and Lung Dis-eases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia

Michael Nash Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA

Toshimitsu Niwa Department of Clinical Preventive Medicine, Nagoya University School of Medicine, Nagoya, Japan

Lubomira Nikolaeva-glomb Department of Virology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria

Mohamed Nooh T he MidSouth Center for Biodefense and Secu-rity at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA

Zohreh Naghashfar Laboratory of Immunopathology, National Institute of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA

L. N. Ognichenko A .V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Carla Oseroff Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Nikolay S. Osin Department of Biological Microanalysis, State Research Center, R&D Institute of Biological Engineering, Moscow, Russia

A. Karolina Palucka Baylor Institute for Immunology Research, Dallas, TX, USA

Scott Parker Department of Molecular Microbiology and Immu-nology, Saint Louis University Health Sciences Center, St. Louis, MO, USA

Virginia Pascual Baylor Institute for Immunology Research, Dallas, TX, USA

Valerie Pasquetto Division of Vaccine Discovery, La Jolla Insti-tute for Allergy and Immunology, La Jolla, CA, USA

Milloni Patel Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Bjoern Peters Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Stefan Philipov Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria

Guido Poli AIDS Immunopathogenesis Unit, San Raffaele Scien-tific Institute, Milano, Italy

Bojan Polic Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia

P. G. Polischuk A.V. Bogatsky Physico-Chemical Institute of the National Academy of Sciences of Ukraine, Odessa, Ukraine

Vera G. Pomelova Laboratory of Molecular Diagnostics, Depart-ment of Biological Microanalysis, State Research Center, R&D Institute of Biological Engineering, Moscow, Russia

Serguei G. Popov National Center for Biodefense and Infectious Disease, George Mason University, Manassas, VA, USA

Tanja Popovic United States Centers for Disease Control and Pre-vention, Atlanta, GA, USA

William S. Probert California State Department of Health Ser-vices, Microbial Diseases Laboratory, CA, USA

J. Ratajczak Stem Cell Biology Program, University of Louis-ville, Louisville, KY, USA

M. Z. Ratajczak Stem Cell Biology Program, University of Lou-isville, Louisville, KY, USA

Sarah Rowe The MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis, TN, USA

Archil Salakaia National Center for Tuberculosis and Lung Diseases/National Tuberculosis Program (NCTBLD/NTP), Tbilisi, Republic of Georgia

Elena Salina Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow, Russia

Iaki Sanz Department of Medicine, Division of Clinical Immu-nology and Rheumatology. University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

Susan Schader McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Mon-treal, Quebec, Canada

Connie Schmaljohn U.S. Army Medical Research Institute of Infectious Diseases, Frederick, MD, USA

Gretja Schnell Department of Microbiology and Immunology, UNC Center For AIDS Research, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

Contributors xvii

Evgeniy G. Semin Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia

Alessandro Sette Division of Vaccine Discovery, La Jolla Insti-tute for Allergy and Immunology, La Jolla, CA, USA

John Sidney Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA, USA

Lone Simonsen Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Namita Sinha Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA

Ludmila N. Sinyashina Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia

Irena Slavuljica Department of Histology and Embryology, Fac-ulty of Medicine, University of Rijeka, Rijeka, Croatia

George B. Smirnov The Gamaleya Institute of Epidemiology and Microbiology, Moscow, Russia

William M. Shafer Department of Microbiology and Immunol-ogy and Laboratories of Microbial Pathogenesis, Emory Univer-sity School of Medicine, Atlanta, GA, USA

H. L. Stevenson Department of Pathology and Center for Bio-defense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA

Ronald Swanstrom Department of Microbiology and Immunol-ogy, UNC Center For AIDS Research, University of North Caro-lina at Chapel Hill, Chapel Hill, NC, USA

David E. Swayne Southeast Poultry Research Laboratory, Agri-cultural Research Service, U.S. Department of Agriculture, Athens, GA, USA

Patricia Sylvestre Unit Toxines et Pathognie Bactriennes, Institut Pasteur, Paris, France

Robert J. Taylor Office of the Director, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Sue A. Theus Department of Pathology, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA

Shota Tsanava National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia

Nikoloz Tsertsvadze National Center for Disease Control and Medical Statistics of Georgia, Tbilisi, Republic of Georgia

Tengiz Tsertsvadze Infectious Diseases, AIDS, and Clinical Immunology Research Center, Tbilisi, Republic of Georgia

Yuriy D. Tsygankov State Research Institute of Genetics and Selection of Industrial Microorganisms, Moscow, Russia

Mark Tykocinski Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

Hideki Ueno Baylor Institute for Immunology Research, Dallas, TX, USA

Onega V. Ulianova Scientific and Research Department, Saratov State University, Saratov, Russia

Vladimir N. Uversky Department of Biochemistry of Immu-nity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia

Matthew N. Van Ert Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA

Anatoly M. Vasiliev Department of Biochemistry of Immu-nity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia

Raisa N. Vasilenko Department of Biochemistry of Immunity and Biodefense, Institute of Immunological Engineering, Lyubuchany, Russia

David M. Wagner Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA

Mark A. Wainberg McGill University AIDS Centre, Lady Davis Institute for Medical Research, Jewish General Hospital, Mon-treal, Quebec, Canada

Matthew C. Weber Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA

D. H. Walker Department of Pathology and Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch, Galveston, TX, USA

Douglas E. Warner Veterans Affairs Medical Center, Decatur; and Department of Microbiology and Immunology, USA

Karl A. Western Office of Global Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Robbin S. Weyant United States Centers for Disease Control and Prevention, Atlanta, GA, USA

Mark S. Williams Department of Microbiology and Immunol-ogy, University of Maryland School of Medicine, and Center for Vascular and Inflammatory Diseases, University of Maryland, Baltimore, MD, USA

Robert W. Williams T he MidSouth Center for Biodefense and Security at the University of Tennessee Health Sciences Center and the VA Medical Center, Memphis TN, USA

M. Wysoczynski Stem Cell Biology Program, University of Louisville, Louisville, KY, USA

M. Shaylan Zanecki Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ, USA

Xilin Zhao Public Health Research Institute, Newark, NJ, USA Jeff X. Zhou Laboratory of Immunopathology, National Institute

of Allergy and Infectious Disease, National Institutes of Health, Rockville, MD, USA

Moncef Zouali Inserm, Paris, University of Paris, France Irina V. Zudina Department of Pathology, University of Texas

Medical Branch, Galveston, TX, USA

xviii Contributors

3

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

The National Institute of Allergy and Infectious Diseases (NIAID) of the U.S. National Institutes of Health (NIH) is within the U.S. Department of Health and Human Services (DHHS; Figure 1). The NIH is the DHHS agency responsible for biomedical research and research training. In the U.S. fed-eral system, health is considered primarily a local and state responsibility, with the federal government providing support and assistance as required. Biomedical research, however, is viewed as a federal responsibility. For that reason, the NIH size and budget have resulted in its becoming the largest of the DHHS agencies.

The NIH consists of 27 institutes and centers, 24 of which carry out and fund biomedical research and three that support the NIH biomedical research endeavor (Figure 2). Each insti-tute consists of two major components: the extramural and the intramural. Intramural programs consist of NIH scientists working in NIH government laboratories. Intramural research constitutes of about 10 to 20% of each institutes research effort and budget. Intramural researchers select scientists to come to their laboratories for research training and conduct international research using the funding available to their lab-oratory. The extramural program of each institute is approxi-mately 80 to 90% of its total funding and operates through both unsolicited and solicited research applications for grants, collaborative agreements, and contracts. Applications are sub-mitted to the NIH Center for Scientific Review, which assigns each application to the appropriate initial review group for sci-entific peer review and to an institute according to the scien-tific content of the application and the research mission of the institute. NIH is unique among national biomedical research agencies in that nearly one-half of the intramural scientists are not U.S. citizens and that foreign scientists are eligible to apply directly or as a partner in extramural awards.

NIAID is similar in its organization to other NIH institutes in that it has three intramural divisions and five extramural divi-sions (Figure 3). The Division of Intramural Research heav-ily emphasizes basic biomedical research, while the Vaccine Research Centers mission includes the discovery and early development of vaccine products. The Division of Clinical Research was established in 2006 to set up domestic and inter-national sites to carry out human subject studies on new or improved diagnostic tests, drugs, vaccines and other preven-tion products. The Division of Microbiology and Infectious Diseases is responsible for all infectious and parasitic diseases except for the human acquired immunodeficiency syndrome (AIDS). The Division of AIDS is responsible for AIDS and related conditions. The Division of Allergy, Immunology, and Transplantation is concerned with the human immune system. The Division of Extramural Activities provides support to the other three extramural divisions through NIAID-organized initial review groups, grant and contract management, and award databases.

The NIAID mission is to understand, treat, and ultimately prevent infectious, immunological, and allergic diseases that affect or threaten U.S. populations and hundreds of millions of people worldwide. The major areas of NIAID investigation currently are (in alphabetical order): AIDS; acute respiratory infections, including influenza; antimicrobial drug resistance, asthma and allergic diseases; civilian biodefense; emerging infectious diseases; enteric infections; genetics, transplanta-tion, and immune tolerance; immune disorders; malaria and other tropical diseases; sexually transmitted diseases; tuber-culosis, and vaccine development and evaluation.

The evolution of the NIAID budget is summarized in Figure 4. Prior to the recognition of AIDS, NIAID was the seventh larg-est NIH Institute. As a result of its research responsibilities in infectious diseases and immunology, funding for AIDS and AIDS-related research rose to become one-half of the NIAID budget. Subsequent to the anthrax attacks in 2001, NIAID was given lead responsibility for the U.S. Civilian Biodefense Research Initiative. At the present time, NIAID is the second

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

4 K. A. Western

largest institute after the National Cancer Institute. NIAID research funding is approximately one-third AIDS, one-third civilian biodefense, and one-third non-AIDS/non-biodefense.

Following a Congressional mandate to double the NIH budget in the 1990s, the NIH budget has been flat for the past several years, resulting in overall inflation-adjusted negative growth. During this period, NIAID funding for international research has maintained a slow and steady growth (Figure 5) so that international research now accounts for 10% of the total NIAID budget. This remarkable sustainability is due to the globaliza-tion of health problems, the relevance of health conditions glob-ally to domestic U.S. health problems, humanitarian objectives, and the economic development, political stability, and increas-ing investment in international health on the part of key interna-tional partners such as Brazil, China, and India. This sustained interest and growth in international research is not seen across NIH. One major factor that fuels NIAIDs global research activ-ities is that our mission in infectious diseases necessitates that we partner with countries that have heavier burdens of disease and/or different risk factors in the development of clinical sites and the evaluation of new or improved diagnostic tests, treat-ment modalities, or prevention products.

NIAID operates under five guiding principles in Global Health Research. First, every effort is made to target collab-orative research efforts to the needs of the partner country or region. Second, it strives to develop collaborative relationships that begin with collaboration in basic research and discovery so that intellectual property can be shared and proceed through product development, the design of human subject studies, and the conduct of rigorous clinical trials that generate data resulting in approval of the product by regulatory agencies. Third, to achieve multidisciplinary research collaboration, research capacity must be built and sustained in the host coun-try. Fourth, NIAID strives to stimulate scientific collaboration and global multi-sector partnerships. Finally, NIAID interna-tional collaboration must develop training, communication, and outreach programs.

NIAID uses six approaches to support its international research. The first is through the NIAID intramural research divisions for pre- and postdoctoral research training. This research training frequently results in sustained collabora-tion once the visiting scientists have returned to their home countries. Intramural collaboration is limited by the resources available in each laboratory but has the advantages of being

U.S. Department of Health and Human Services

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Figure 1. (See Color Plates).

NIAID: An Overview 5

decentralized and scientifically driven, and it provides the opportunity to establish long-term collaboration with the NIAID laboratory and other researchers who have trained there. Because about 50% of NIH intramural scientists are from outside the United States and only 10% of intramural scientists become tenured, the intramural research training experience provides an opportunity to become part of a global network linking trainees and their home institutions with NIAID-tenured scientists, U.S. scientists who take academic or private sector appointments or join other U.S. agencies, and foreign scientists who return home to continue their research careers.

Foreign investigators are encouraged to partner with U.S. extramural investigators in the submission of investigator-initiated research applications or in response to solicited program announcements (PAs) and requests for applications (RFAs). This is how NIAID supports the bulk of its interna-tional research. If the collaboration is between U.S. scientists

and scientists in another industrialized country, there may be no NIAID funding involved. On the other hand, if the collabo-rating overseas scientist is from a middle- or lower-income country and/or does not have his or her own funding, NIAID will provide the U.S. investigator with research funds to sup-port the overseas component.

NIH is unique among national domestic research agen-cies in that foreign investigators are eligible to apply directly for investigator-initiated research awards. Foreign scientists and institutions are also eligible to apply for most solicited grant and collaborative agreement solicitations. There are no international set-aside funds, and foreign investigators must compete against experienced U.S. investigators. All unsolic-ited foreign applications with a competitive score must also be approved by the National Allergic and Infectious Diseases Council before funding. Because of the intense competi-tion and grantsmanship required, NIAID does not encourage foreign investigators to apply directly unless their ideas are

Office of the Director

National Instituteon Alcohol Abuseand Alcoholism

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Figure 2. (See Color Plates).

6 K. A. Western

truly novel and the investigator has considerable experience preparing NIAID grant applications. NIH is obligated to fol-low U.S. contracting laws, so that foreign institutions can be funded in response to requests for proposals only if there is a prior determination that there is no viable U.S. source, or the foreign application is clearly superior to responses from U.S. institutions.

NIAID also participates in a number of bilateral programs with foreign governments and institutions. These agreements may be developed at the Presidential, State Department, DHHS, or NIH levels in science and technology, health, or biomedical research. In the majority of cases, these agreements have no NIAID funding associated with them and collaborative activities must be undertaken with resources currently at hand in intramu-ral laboratories or using extramural funding mechanisms.

NIH intramural scientists are encouraged to collaborate with counterparts at other U.S. government agencies such as the Centers for Disease Control and Prevention, the Food and

Drug Administration, and the U.S. Army or Navy. U.S. Gov-ernment scientists, however, may not compete for NIH extra-mural research funds. When there is mutual interest, however, NIH may negotiate interagency agreements with these and other agencies such as the State Department or the U.S. Agency for International Development that serve as a contrac-tual mechanism to transfer funds and resources between the participating agencies.

Finally, NIAID collaborates with multilateral agencies such as the World Health Organization (WHO), the Pan American Health Organization, and the Joint United Nations Program on HIV/AIDS through consultation, serving on advisory boards, and participation in technical meetings. NIAID has provided targeted funding to the WHO/World Bank/UNDP Special Program for Research and Training in Tropical Dis-ease Research. NIAID also has a Congressional mandate to provide funding to the Global Fund to Combat AIDS, Tuber-culosis, and Malaria.

Office of the Director

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Figure 3. (See Color Plates).

NIAID: An Overview 7

Figure 5. (See Color Plates).

Figure 4. (See Color Plates).

Figure 6. (See Color Plates).

8 K. A. Western

The NIAID strategy to respond globally to new or emerg-ing infectious diseases and scientific opportunity has been first to encourage the intramural research community to turn their talent and attention to the new or underserved research area. The second step is to encourage extramural investiga-tors working in relevant research areas to submit supplemen-tal research proposals. The third step is to alert the more general scientific community about NIAIDs research priori-ties and interests in the area through notices, PAs, and RFAs in the NIH Guide for Grants and Contracts. Foreign investi-gators are ordinarily eligible to partner with U.S. applicants and, if they prefer, apply directly for NIAID funding. The result of these solicitations is to increase the research, the research training base, and eventually the pool of investiga-tors in the targeted area. NIAID fulfills the need for directed activities in support of research in the targeted area through contracts to build the infrastructure and to provide research reagents and repositories.

After a critical mass of individual extramural awards has been reached, NIAID usually puts out an RFA to establish multidisciplinary centers of excellence in the field. These centers of excellence provide further opportunities for research training of U.S. and foreign scientists. The centers of excellence are usually encouraged to engage in interna-tional research and/or carry out research training through the center award and/or independent research and research train-ing awards. Examples of NIAID centers of excellence pro-grams include the Sexually Transmitted Disease Research Centers, the Tropical Disease Research Units, the Centers for AIDS Research, the Tuberculosis Research Unit, the

Regional Centers for Emerging Infectious Diseases, and the recently announced Centers for Influenza Research and Sur-veillance.

Once the domestic centers of excellence are established, the next phase is the establishment of special programs to link the domestic network to international partners. RFAs are published to solicit applications for collaboration with one or more foreign partners. This is the time when the NIH Fogarty International Center solicits applications from U.S. institutions for international research training in the targeted area. Examples of linkage programs include the International Collaboration in Infectious Disease Research Program, the HIV Vaccine Trials Network, HIV Prevention Trials Net-work, the NIAID International Centers of Excellence, and the International Emerging Infectious Disease Research and Training Program.

The third phase is reached when the linkage programs are mature and international partners have developed the capacity to carry out and account for their own research. NIAID develops solicitations open to foreign institutions to apply directly to NIAID in the targeted area. Examples of mechanisms to support foreign researchers include the Trop-ical Medicine Research Centers, the Multilateral Initiative on Malaria, the Comprehensive International Program for Research on AIDS, and the International Research in Infec-tious Diseases Program.

Further information on NIAID and NIH international grants and funding opportunities may be found at http://grants1.nih.gov/grants; http:// www.niaid.nih.gov/ncn/; and http://www.niaid.nih.gov/ncn/grants/int/default.htm.

http://grants1.nih.gov/grantshttp://grants1.nih.gov/grantshttp:// www.niaid.nih.gov/ncn/http://www.niaid.nih.gov/ncn/grants/int/default.htmhttp://www.niaid.nih.gov/ncn/grants/int/default.htm

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

20.1 Introduction

The enteroviruses are widely spread viruses associated with diverse clinical syndromes and diseases, ranging in severity from minor febrile disorders to severe and potentially life-threatening conditions. They may affect various organs and systems: the central nervous system, the respiratory system, the skin, the heart, the pancreas, and the eye. The enteroviruses are the most common etiological agent of viral meningitis. They may also cause encephalitis. In addition, these viruses can cause summer colds, herpangina, pleurodynia, hemor-rhagic conjunctivitis, uveitis, and chronic fatigue syndrome. They are implicated in cardiac infections such as myocarditis and pericarditis that both in some cases may lead to dilated cardiomyopathy, where the singular option of recovery is the heart transplantation. Enteroviruses have also been implicated to play a role in the development of juvenile-onset (type 1) insulin-dependent diabetes mellitus (1) .

Antienteroviral therapy until now has had certain limita-tions. To date, there is no enterovirus-specific drug available for clinical use. Indeed, a great number of enterovirus inhibi-tors have been described so far, but only a few of them have shown effectiveness in vivo and none has been approved for clinical use yet. Thus, etiological therapy remains elusive, and there is a clear need for continued development of new and effective inhibitors of enteroviral replication.

20.2 Oxoglaucine

In a pilot study performed by our research group, a series of aporphinoid alkaloids were isolated from Glaucium flavum Crantz (yellow horn poppy) or obtained synthetically have been tested in vitro for their antiviral activity against viruses

belonging to several taxonomic groups, including picorna-, orthomyxo-, paramyxo-, and herpesviruses. One of the com-pounds, oxoglaucine, has manifested a well-pronounced inhibitory effect against the replication of poliovirus type 1 (Mahoney), of the Picornaviridae family. The antiviral effects in the preliminary screening tests have been evaluated by the semi-quantitative agar-diffusion test (2) .

Oxoglaucine is isolated from the epigean parts of Glaucium flavum Crantz (3) and it can also be obtained synthetically from the main plant alkaloid glaucine (4) .

The cytotoxic effect of oxoglaucine has been tested in two experimental procedures. The first was a microscopic evalu-ation of the effect of different concentrations of oxoglaucine on the morphology of the cell monolayer resulting in deter-mination of the maximal tolerated (nontoxic) concentration (MTC). The second involved tracing the growth curve of the cell culture in the presence of different concentrations of the compound followed by determination of the concentration that reduces the number of viable cells by 50%, the cell growth inhibitory concentration 50 (CGIC

50 ). The concentration of

oxoglaucine, which produces no visible cytotoxic effect on monolayer FL cells, the MTC, is 6.4 g/mL, and the concen-tration reducing their growth by 50% (CGIC

50 ) is 4 g/mL

( Figure 20.1 ). The antienteroviral spectrum of oxoglaucine has been

tested against 16 enterovirusespoliovirus type 1 (Mahoney), poliovirus type 1 (LSc-2ab), and a series of viruses belong-ing to human enterovirus B. The following enteroviruses have been included in the test: poliovirus type 1 (PV-1), coxsacki-evirus (CV)-A9, the six coxsackie B viruses (CV-B1, CV-B2, CV-B3, CV-B4, CV-B5, and CV-B6), and echovirus (EV)-2, EV-4, EV-6, EV-9, EV-13, EV-15, and EV-19. The endpoint dilution method in the multi-cycle cytopathic effect (CPE)-inhibition setup in FL cells and the plaque-inhibition test has been used for determining the antiviral effect.

Oxoglaucine reveals a marked inhibitory effect on all of the tested viruses. Oxoglaucine concentrations that reduce virus titer by 1, 1.67, and 2 lg as compared to that of virus control

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

200 L. Nikolaeva-Glomb et al.

(with no oxoglaucine in the maintenance medium) have been determined in the CPE-inhibition test and results are shown on Table 20.1 . CV-B4 and CV-B5, followed by CV-B3, CV-A9, and EV-9 were the most susceptible to the antiviral effect

of oxoglaucine. On the opposite end, the highest dose of oxo-glaucine was required to inhibit the replication of CV-B1. The antiviral effects of oxoglaucine against the replication of CVs and the EVs included in the investigation are presented on Figure 20.2 and Figure 20.3 , respectively.

On the basis of the results presented in Table 20.1 and the cytotoxicity parameters of oxoglaucine, the selectivity index (SI) has been calculated as the ratio of MTC and the inhibitory concentration 50 (IC

50 ). The results are shown on Table 20.2 .

As expected, oxoglaucine exerts the highest selectivity against CV-B4, followed by EV-9, EV-13, and EV-19 as well as by CV-B3 and CV-A9.

In general, oxoglaucine reveals a broad-spectrum antientero-viral activity accompanied by high selectivity. This conclusion is supported by the results obtained in the plaque-inhibition test, which is considered the gold standard in experimental in vitro antienteroviral chemotherapy. The plaque-inhibition test has been carried out for poliovirus type 1 (Mahoney), poliovirus type 1 (LSc-2ab), and the six coxsackie B viruses. The concentration that reduces the number of plaques by 50% relative to the control with no inhibitor present in the agar overlay (IC

50 ) was determined and SI calculated. Results are

presented in Table 20.3 . From the tested viruses, CV-B4 is again the most sensitive one to the antiviral effect of oxoglaucine demonstrating SI approximating 400.

0.1 1 10 1000

50

100

num

ber

of v

iabl

e ce

lls a

s %

of t

he u

ntre

ated

con

trol

concentration (g/ml)

Figure 20.1. Effect of oxoglaucine on the growth of Fl cells. Cells are seeded in a growth medium containing various concentrations of the compound. After formation of cell monolayer in the untreated control (no compound in the growth medium), cells are trypsinized and viable cells counted. The number of viable cells in each sample is compared with the number of viable cells in the control and is presented as percent of the untreated control.

Figure 20.2. Antiviral effect of oxoglaucine against the replication of CV-A9, CV-B1, CV-B3, CV-B4, and CV-B5 in FL cells. Mono-layer FL cells in 96-well plates are inoculated with 0.1 mL virus suspension containing 100 000, 10 000, 1000, 100, 10, and 1 CCID

50

(or 320 000, 32 000, 3 200, 320, 32, and 3 CCID 50

). After an hour for virus adsorption, the excessive virus is discarded and cells are inoculated with 0.2 mL of maintenance medium containing 0.5 lg concentrations of the tested compound. The antiviral effect is scored according to the appearance of the cytopathic effect on the 48th hour p.i., virus titer in the presence or absence of the compound is deter-mined and the defference of titers ( lg) of the untreated virus control and the oxoglaucine-treated samples is calculated.

0.001 0.01 0.1 1

0

1

2

3

4

5

6

lg

concentration(g/ml)

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

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

Virus type

IC (g/mL)

lg = 1 lg = 1.67 lg = 2

PV-1 (LSc-2ab) 0.0007 0.018 0.33 PV-1 (Mahoney) n.d. n.d. n.d. EV-2 0.01 0.2 EV-4 0.03 0.12 0.17 EV-6 0.003 0.06 EV-9 0.04 0.06 0.08 EV-13 0.03 0.09 0.1 EV-15 0.14 0.2 0.25 EV-19 0.03 0.12 0.15 CV-B1 0.17 0.25 0.30 CV-B2 n.d. n.d. n.d. CV-B3 0.04 0.07 0.1 CV-B4 0.01 0.03 0.04 CV-B5 0.04 0.06 CV-B6 n.d. n.d. n.d. CV-A9 0.04 0.12 0.15

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

20. Antienteroviral Compound 201

Table 20.2. Selectivity of oxoglaucine determined accord-ing to the CPE-inhibition test.

Virus type

SI (MTC/IC)

lg = 1 lg = 1.67 lg = 2

PV-1 (LSc-2ab) 9 142 355 19 PV-1 (Mahoney) n.a. n.a. n.a. EV-2 640 32 EV-4 213 53 38 EV-6 2 133 107 EV-9 160 107 80 EV-13 213 71 64 EV-15 45 32 26 EV-19 213 53 43 CV-B1 38 26 21 CV-B2 n.a. n.a. n.a. CV-B3 160 91 64 CV-B4 640 213 160 CV-B5 160 107 CV-B6 n.a. n.a. n.a. CV-A9 160 53 43

n.a., not applicable

Table 20.3. Antienteroviral effect of oxoglaucine deter-mined in the plaque-inhibition test.

Virus type

IC 50

(g/mL) SI (MTC/

IC 50

)

PV-1 (LSc-2ab) 0.15 42 PV-1 (Mahoney) 0.041 156 CV-B1 0.03 213 CV-B2 0.017 376 CV-B3 0.038 168 CV-B4 0.017 376 CV-B5 0.02 320 CV-B6 0.042 152

Figure 20.3. Antiviral effect of oxoglaucine against the replication of EV-2, EV-4, EV-6, EV-9, EV-13, EV-15, and EV-19 in FL cells.

0.001 0.01 0.1 10

1

2

3

4

5

6

7

8

9

lg

concentration (g/ml)

EV-2

EV-4

EV-6

EV-9

EV-13

EV-15

EV-19

0 1 2 3 4 5 6 7 82

3

4

5

6

7

8

9

infe

ctio

us v

irus

titer

(C

CID

50/m

l)

hours post infection

VC

0h

1h

2h

3h

4h

5h

6h

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

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

Table 20.4. Direct virucidal effect of oxoglaucine.

TC

Virus titer lg CCID50

/mL

0 min

15 min

30 min

60 min

6 h

24 h

4C Oxoglaucine control

7.5 8.0

7.5 8.0

8.0 8.0

8.0 8.5

8.5 8.5

Room tem- perature

Oxoglaucine control

8.0

7.5 8.5

7.5 8.0

8.0 7.5

8.5 8.0

8.5 8.0

37C Oxoglaucine control

8.0 7.5

8.5 8.5

8.5 8.0

8.0 8.0

8.5 8.5

Experiments have been carried out on undiluted poliovirus type 1 (LSc-2ab; 10 8.25 CCID

50 /mL) by the virucidal quantitative suspension test in the pres-

ence of 5 g/mL and virus samples have been titrated by the endpoint dilution method in FL cells

202 L. Nikolaeva-Glomb et al.

Initial studies on the specific mode of action of oxoglau-cine have been carried out in the timing-of-addition study in the one-step replication cycle of poliovirus type 1 (LSc-2ab) at high multiplicity of infection. The study reveals that the susceptible period to the antiviral effect of oxoglaucine is the latent and lag phase of the virus replication cycle ( Fig 20.4 ).

The following conclusions may be drawn out from the results obtained so far: (i) oxogalaucine possesses a strong antiviral effect in vitro against a broad spectrum of enetero-viruses, (ii) a high selectivity ratio is observed for all tested viruses exceeding 100 in most cases, and (iii) the latent and the lag phase of the virus replication cycle is the susceptible period to the effect of oxoglaucine.

References

1. Galabov AS, Angelova A (2006) Antiviral agents in the prevention and treatment of virus-induced diabetes. Anti-Infective Agents in Medicinal Chemistry 5: 293 - 307 .

2. Galabov AS, Nikolaeva L, Philipov S (1995) Aporphinoid alka-loid glaucinone: a selective inhibitor of poliovirus replication. Antivir Res 26: A347 .

3. Kuzmanov BA, Philipov SA, Deligiozova-Gegova IB (1992) Comparative photochemical and chemosystematic research of pop-ulations of Glaucinum flavum Crantz in Bulgaria. Fitologia 52-57 .

4. Philipov S, Ivanovska N, Nikolova P (1998) Glaucine ana-logues as inhibitors of mouse splenocyte activity. Die Pharmazie 53: 694 - 698 .

193

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

19.1 Introduction

For various proteins, proteolytic cleavage represents the universal mechanism of activation. The activation of prote-olysis plays an important role in the pathogenesis of many diseases. So, our supposition about antiviral activity of the proteolytic inhibitors (1) has been well founded.

Previous research data has made it possible to formulate the vicious circle concept of viral virulence. That is: the virus activates the proteolytic systems, which in turn assists in the development, generalization, and aggravation of the infec-tious process at the expense of influence on the etiologic and pathogenesis factors (see Scheme 19.1 ; refs. 1 and 2 ).

The inhibitors of proteolysis may prevent the forming of or destroy this vicious circle. The antiviral action of proteolytic inhibitors was discovered on all levels from subcellular to whole organism in both the experiment stage and the clinic. Further-more, the antiviral therapeutic and prophylactic action of the proteolytic inhibitors was demonstrated against a wide spectrum of RNA and DNA viruses, such as the influenza A and B, herpes, HIV, Newcastle disease viruses (NDVs), and the adenoviruses (3) . In this study, we present results on anti-infectious action of the proteolytic inhibitor -aminocaproic acid (-ACA).

19.2 Materials and Methods

1. -ACA manufactured by pharmaceutical company Zdorovya (Kharkiv, Ukraine) was used.

2. Patients: sick children with influenza and other ARVI, adult patients with genital herpetic infection.

3. Laboratory animals: inbred white mice, white rats.

4. Viruses: human influenza A viruses H1N1, H2N2, H3N2; avian influenza A viruses H5N3 and H7N3; influenza B virus; NDV; adenovirus; HSV-1; and HIV.

5. Tissue and cell cultures: tissue cultures of chorioallantoic membranes (CCM) of 12- to 14-day-old chicken embryos, cell culture Hep-2.

6. Bacterial agents of emerging and nosocomial infections: vac-cine strain 15 and virulent strain 29 of Francisella tularensis; strains of Vibrio cholerae : Vibrio cholerae cholerae strain 569; Vibrio cholerae El-Tor strains 754 and 878; Vibrio chol-erae non-01 strain 146/11; hospital isolates of Staphylococ-cus aureus , Pseudomonas aeruginosa , and Escherichia coli.

7. Anti-influenza and anti-NDV activities in vitro were stud-ied by inhibition of virus replication in tissue cultures CCM. CCM was infected with 1000 TID

50 (tissue infec-

tion dose) of virus. Samples contained -ACA were called experimental and those without inhibitor were named control. Control and experimental samples were tested on viral infective titers after incubation (24 hours, 37C for influenza A viruses and NDV, and 32C for influenza B viruses). At least five experiments were carried out of the investigation of each compound. Anti-influenza activity is expressed in log

10 TID

50 and reflected suppression of viral

replication in experimental samples to control. 8. Anti-influenza activity of -ACA in vivo was studied in

mouse models of models lethal and non-lethal experimen-tal influenza infections (2, 4) .

9. Antiherpetic action of -ACA was tested using cyto-morpho-logical method. Hep-2 cells were infected with HSV-1 strain US in dose 5 IFU/cell. The cells were incubated at 37C dur-ing 48 hours in maintenance medium that contained -ACA (experimental samples) or without its (control samples). Then, cells were fixed with 96% ethanol and stained with 0.01% acri-dine orange solution. The amount of infected cells with DNA-containing virus inclusion bodies was counted by fluorescent microscopy. Anti-HSV activity of compounds was estimated as the ratio of the percentage of infected cells in treated to percentage of infected cells in untreated cell cultures.

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

194 V. P. Lozitsky

19.3 Results and Discussion

Usually -ACA is used for hemostasis when fibrinolysis contributes to bleeding. -ACA is a low toxic drug. The intra-venous and oral LD

50 values of -ACA were 3.0 and 12.0 g/kg,

respectively, in the mouse and 3.2 and 16.4 g/kg, respectively, in the rat. An intravenous infusion dose of 2.3 g/kg was lethal in the dog.

-ACA prevented the enhancement of proteolysis during the interaction of virions with cell membranes and decreased the penetration of virions into sensitive cells. It brought down the proteolytic cleavage of the HA precursor to HA-1 and HA-2, and reduced the infectious virus harvest. High levels of -ACA anti-influenza efficacy were shown by us in vitro on the H1N1, H2N2, and H3N2 subtypes of influenza A human viruses, influenza B viruses (2, 4) , and on H5N3 and H7N3 subtypes of avian influenza viruses (5) .

The obtained results showed that while both H5N3 and H7N3 avian influenza viruses are sensitive to -ACA, the H5 subtype was more sensitive ( Figure 19.1 ).

Further results have shown that -ACA prevented enhance-ment of the alkaline proteases activity in lungs of mice infected with influenza virus in addition to exhibiting therapeutic and prophylactic effects (4 , 6 , 7) .

-ACA intensified the production of specific antibodies, increased cell immunity, prevented vessels permeability and hemorrhagic phenomena, and decreased the destruction of bronchial epithelium.

Mice treated by -ACA during infection were more pro-tected from re-infection with influenza virus (7) . -ACA, when used in the treatment of influenza, decreased the virus reproduction in lungs and also enhanced the humoral immune response ( Figure 19.2 ). The antibody titers on day 21 post-infection were significantly higher in the treated animals.

On day 30 after challenge with the homologous strain A/Hong Kong/1/68 (H3N2), the virus reproduced to low level in the lungs of untreated convalescent mice; however, no virus was detected in the lungs of mice (except one animal) that had been treated with -ACA during the primary infection ( Figure 19.3A ). A marked increase of the antibody level was found in such mice ( Figure 19.3B ).

Upon challenge with lethal doses of the virulent strain A/Leningrad/49/32 (H1N1), the protection was significantly higher among animals treated with -ACA during the primary infection with sublethal virus dose. We believe that the immu-nomodulatory action of -ACA may play an important role in the increased resistance to challenge exhibited by treatment.

The reproduction of influenza virus in the lungs was reduced in half 10 days after a single application of -ACA, and 4 weeks after 5-day prophylactic course (6) . This correlated with the ability of the proteolysis inhibitor to stimulate the early produc-tion of specific serum antibodies. The favorable effect of pro-phylactic administration of -ACA was especially significant in experimental lethal influenza ( Figure 19.4 ). A significant protection was observed from days 3 to day 14 post-infection.

The prophylactic effects produced by different types of anti-viral preparations, such as inactivated vaccine and -ACA, used separately or in combination in experimental lethal infection induced by influenza virus A/Leningrad/49/32 (H1N1) in mice were compared (8) . The quantitative evaluation of the anti-influenza effect was carried out by using the method of multi-factor analysis after the optimum second-order plan based on the mathematical theory of experiment. This made it possible to determine the best combination of the preparations and their doses to establish the time of the formation of reliable protection from influenza in mice. The results of study on combined use of inactivated vaccine and -ACA condition for prevention of lethal experimental influenza in mice are exhibited on Figure 19.5 .

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

19. Anti-Infectious Actions of Proteolysis Inhibitor -Aminocaproic Acid (-ACA) 195

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

0

0,5

1

1,5

2

2,5

3

3,5

4

4,5

- lo

g10

TID

50

1 2

control

30 mg/ml E-ACA

20 mg/ml E-ACA

15 mg/ml E-ACA

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

A

00,5

11,5

22,5

33,5

44,5

5

-lo

g10

TID

50

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Days of experiment

mice treated with E-ACA

control group

B

0

50

100

150

200

250

Days of experiment

Tit

res

of

anti

bo

die

s

control groupmice treated with E-ACA

5 7 10 14 21

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

A

0

0,2

0,4

0,6