11th Anniversary Annual International Meeting of the

11th Anniversary Annual International Meeting of the Institute of Human Virology Foreword from Robert C. Gallo, Founder and Director Downloaded from https://journals.lww.com/jaids by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3ltH2fbMApE2B3uinzTGcdJYjuMHPD8xXpoxjq8VhNhhXWRE0QWgv/g== on 05/23/2018

For more than a decade, the Institute of Human Virology (IHV) at the University of Maryland School of Medicine has pursued a mission of combined research, treatment and prevention, in addition to a commitment and sense of compassion towards a broader community that is not limited to the Baltimore region. IHV’s work knows no artificially drawn national boundaries. We are active at laboratories and clinics, in the United States and abroad, via teamwork and in partnership with scientists, clinicians, governments, and patients, among others. Our measure of success continues to be an ability to take the results of neverending scientific research directly to the patient and in a manner that allows those advances to have an effective global impact. The Annual International Meeting of IHV has been a centerpiece of our efforts to share with and learn from the broader scientific and clinical communities. The Meeting is one of the world’s leading HIV/AIDS conferences, undoubtedly because of the unparalleled quality of presentations and the level of expertise possessed by presenters and participants. It is also because of the vision and dedication of individuals like Hilary Koprowski and Stanley Prusiner who were instrumental in organizing IHV’s inaugural meeting. I also want to acknowledge Jeff Meshulam of Profectus BioSciences, Inc., who has contributed so much to so many of our meetings through the years, as well as Beth Peterson, whose tireless efforts now reach fruition. 2008 marks an especially significant year as the anniversary of the finding of the HIV virus, while next year marks the 25th anniversary of the recognition of HIV as the cause of AIDS and the development of the first HIV blood test. These significant milestones give us much reason to reflect upon what we did right, what has gone wrong and where we need to go in the future. Just recently, the field of HIV research suffered a major setback which in my view has too often been minimized. Interim data from a large, expensive vaccine trial, the STEP trial, co-sponsored by the National Institute of Allergy and Infectious Disease (NIAID) and Merck, showed that the vaccine employing an adenovirus vector with HIV genes had failed. Not only did the vaccine offer no protection from HIV, it apparently increased the risk of infection in recipients who had previously been exposed to adenoviruses similar to the vaccine vector. While failures are an unavoidable reality of grand scientific endeavors, the fallout from the STEP trial presents an opportunity to re-evaluate the entire HIV vaccine development process. In celebrating our 11th anniversary, we are honored to present the IHV Lifetime Achievement Award for Scientific Contributions to Dr. Isaac P. Witz of Tel Aviv University. We will also be presenting an unprecedented two IHV Lifetime Achievement Awards for Public Service to The Honorable Robert K. Gray, a Cabinet member in the Eisenhower Administration and former worldwide Chairman of Hill & Knowlton, and Mr. John Evans, co-founder of C-SPAN and an internationally recognized expert in the telecommunications industry. We will also pause to commemorate the life of a good friend, Dr. Bob Ting – a research scientist, entrepreneur and pioneer originating and helping to popularize the term ‘‘biotechnology.’’ Bob developed and produced the first FDAapproved diagnostic test kits for HIV antibody confirmation and was the founding president and original chief executive of Profectus Biosciences Inc. I am grateful to all who have chosen to join us this year and am confident that this Meeting will write another important chapter in the book of human retrovirology.

2008 International Meeting of the Institute of Human Virology

SEPTEMBER 11-13, 2008

Copyright © 2009 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.

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The Institute of Human Virology www.ihv.org Mission Statement The Institute of Human Virology (IHV) is a world-class center of excellence focusing on chronic viral diseases and virally linked cancers. IHV is dedicated to biomedical research leading to improved treatment and prevention of these diseases. Our unique structure connects cohesive, multidisciplinary research and clinical programs so that new treatments are streamlined from discovery to patients. IHV serves patients locally and the scientific community globally.

Robert C. Gallo Director Co-Director, Division of Basic Science and Vaccine Research

William A. Blattner Associate Director Director, Division of Epidemiology and Prevention

Robert R. Redfield Associate Director Director, Division of Clinical Care and Research

George Lewis Co-Director, Division of Basic Science and Vaccine Research

Joseph L. Bryant Director, Division of Animal Models

David Pauza Assistant Director

Dave Wilkins Chief Operating Officer

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Board of Advisors The Hon. Kathleen Kennedy-Townsend Chairperson Former Lieutenant Governor of Maryland Baltimore, Maryland

The Honorable Arthur Gajarsa United States Court of Appeals for the Federal Circuit Washington, DC

Her Royal Highness Princess Chulabhorn Mahidol Mahidol University Bangkok, Thailand

The Honorable Robert K. Gray

Timothy Moynahan

Chairman, Gray and Company II Miami Beach, Florida

Moynahan & Minella, LLC Waterbury, Connecticut

Assistant Secretary of Defense for Health Affairs Bethesda, MD

Stewart Greenebaum

Franco Nuschese

Greenebaum & Rose Associates, Inc. Baltimore, MD

Georgetown Entertainment Group Washington, DC

Fred Cannon

William Hall

Thomas Paese, Esquire

University College Dublin Dublin, Ireland

Buchanan Ingersoll and Rooney Harrisburg, Pennsylvania

William A. Haseltine

Chris YH Tan

Haseltine Foundation for Medical Sciences and the Arts Washington, DC

Asian Institute of Molecular and Cell Biology Vancouver, British Columbia

The Honorable Ernest F. Hollings

Lenny Wilkens

The Honorable Sue Bailey

Senior Vice President for Government Relations, BMI, Inc. New York, NY

Robert Charrow Greenberg-Traurig LLP Washington, DC

John P. Coale Attorney at Law Washington, DC

Medical University of South Carolina Charleston, SC

National Basketball Association Hall of Fame Coach and Player Medina, Washington

Science Journalist and Policy Consultant Washington, DC

Richard E. Hug

James Wyngaarden

HUG ENTERPRISES, INC. Baltimore, MD

The Honorable Elijah Cummings

Toshiaki Inoue

Former Director, National Institutes of Health Durham, North Carolina

Barbara J. Culliton

United States House of Representatives Baltimore, Maryland

Lynda M. Dee Attorney at Law, AIDS Activist Baltimore, Maryland

Sanyo E&E America Company Bensenville, IL

Ex-Officio Members

Mark Kaplan

Robert C. Gallo

University of Michigan Medical Center Ann Arbor, Michigan

Director, Institute of Human Virology

William E. Kirwan

The Honorable Nancy Kopp

Chancellor University System of Maryland

Founding Director, Project Inform San Francisco, California

State Treasurer, Maryland State Government Annapolis, Maryland

E. Albert Reece

The Honorable Sheila Dixon

Hilary Koprowski

Martin Delaney

Mayor of Baltimore Baltimore, Maryland

John Evans Evans Telecommunications Key West, Florida

Dean University of Maryland School of Medicine

Thomas Jefferson University Philadelphia, PA

Thomas Lynch Amarin Pharmaceutical, Ltd. Dublin, Ireland




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Scientific Advisory Board Hilary Koprowski, M.D.

William Hall, M.D., Ph.D.

Joseph Pagano, M.D.

Chair Thomas Jefferson University Philadelphia, Pennsylvania

University College Dublin Dublin, Ireland

University of North Carolina School of Medicine Chapel Hill, North Carolina

Edward A. Berger, Ph.D. National Institutes of Health Bethesda, Maryland

University of Michigan Medical Center Ann Arbor, Michigan

Farley Cleghorn, M.D.

Michel Klein, Ph.D.

Constella Group Washington, DC

Canadian Network for Vaccines and Immunotherapeutics Toronto, Canada

Myron S. Cohen, M.D. University of North Carolina School of Medicine Chapel Hill, North Carolina

Mark Kaplan, M.D., FACP

Myron Levine, M.D., D.T.P.H.

Max Essex, D.V.M., Ph.D.

University of Maryland School of Medicine Baltimore, Maryland

Harvard School of Public Health Cambridge, Massachusetts

Erling C. J. Norrby, M.D., Ph.D.

Warner Greene, M.D., Ph.D. Gladstone Institute of Virology and Immunology San Francisco, California

Kathleen Squires, M.D. Thomas Jefferson University Philadelphia, Pennsylvania

Mario Stevenson, Ph.D. University of Massachusetts Medical School Worcester, Massachusetts

Sten Vermund, M.D., Ph.D. Vanderbilt University Institute for Global Health Nashville, Tennessee

The Royal Swedish Academy of Sciences Stockholm, Sweden

Communications and Press Policy 11th Annual International Meeting of the Institute of Human Virology To enhance the exchange of information and communication among attendees of the Institute of Human Virology Annual International Meeting, the following must be adhered to by all participants: All comments at sessions are off-the-record and are not for attribution. No coverage, reporting or publication of scientific data or presentations at the Institute of Human Virology Annual

Meeting is permitted without the consent of the presenter(s) and the program organizers. One-on-one interviews with scientists and media may be arranged by contacting Nora Grannell, Director of Public

Relations and Marketing, Institute of Human Virology, [email protected] or (410) 706-1954.

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Special Acknowledgements The University of Maryland School of Medicine Institute of Human Virology expresses its gratitude to the corporations, foundations, and US government institutions whose support makes possible the 11th Annual International Meeting of the Institute of Human Virology.

Benefactor Fogarty International Center The Bill & Melinda Gates Foundation

Underwriter Office of AIDS Research National Institutes of Health

Supporters Gilead Sciences, Inc. National Cancer Institute OrthoClinicalDiagnostics, Inc. Tibotec, Inc.

Contributors Advanced Bioscience Laboratories, Inc. Bristol-Myers Squibb LabNow, Inc. Partec Essential Healthcare Sanofi-Aventis Sanyo Wyeth Profectus Biosciences, Inc




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11th Anniversary Annual International Meeting of the Institute of Human Virology

ONLINE ABSTRACTS TITLE INDEX To navigate to an abstract, simply click on the title of the abstract. Abstracts will be available online at www.jaids.com

Thursday, September 11, 2008 HIV Entry 100

3D structure of native HIV-1 gp120 trimers and mechanisms of cellular entry Sriram Subramaniam, National Cancer Institute, Bethesda, MD

101

Chemokine Receptor CCR5 Mediates Resistance to West Nile Virus Infection in Mouse and Man Carole Bewley, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD

102

Visualization of HIV-1 entry pathways in cells Gregory B. Melikian, Institute of Human Virology, University of Maryland School of Medicine

103

Tracking Dynamic HIV-1 Escape from CCR5 Antagonist Therapy in vivo by Deep Sequencing Athe Tsibris, Massachusetts General Hospital, Boston, MA

104

A V3-independent pathway to resistance to small molecule CCR5 inhibitors Kleio Anastasopolou, Weill Cornell Medical College, New York, NY

105

sCD4-17b, an Engineered Bifunctional HIV-1 Neutralizing Protein: Potent Cross Clade Activity and Potential Microbicide Use Laurel Lagenaur, National Institute of Allergy and Infectious Diseases, Bethesda, MD

HIV Infection Early Events 106

Early events in vaginal HIV transmission Ronald Veazey, Tulane University

107

Escape from Neutralizing Antibody in Early Subtype C HIV-1 Infection Cynthia A. Derdeyn, Emory University Vaccine Center, Altanta, GA

108

Indentification of Full-Length Transmitted HIV-1 Genomes Revelas Extraordinary Dynamics and Precise Molecular Pathways of Early Virus Diversification, Adaptation and Immune Evasion George Shaw, University of Alabama at Birmingham School of Medicine

109

Complexity of the Transmitted Virus and Compartmentalization In Its Seminal Source Ronald Swanstrom, Center for AIDS Research University of North Carolina Lineberger Cancer Center *Abstracts appear as provided by authors.

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110

HIV permeation efficiency in human cervicovaginal mucus Justin Hanes, Johns Hopkins University, Baltimore, MD

111

Reconstruction and Characterization of a Human Endogenous Retrovirus with the Original Sequence at the Time of Integration Reinhardt Kurth, Robert Koch-Institute, Berlin, Germany

112

hBD2 inhibits HIV via CCR6, a receptor expressed on memory, Dendritic and Th17 cells Alfredo Garzino-Demo, Instittute of Human Virology, University of Maryland School of Medicine

113

The Anti-Herpetic Drug Acyclovir Supresses HIV-1 in Herpesvirus-Infected Human Tissues After Conversion into a Nucleoside Reverse Transcriptase Inhibitor Andrea Lisco, National Institute of Child Health and Human Development, Bethesda, MD

114

NK Cells Contribute to the Constitution of HIV Reservoirs in Dendritic Cells, Involvement of HMGB-1 Marie-Lise Gougeon, Institut Pasteur, Paris, France

115

HIV Vpu Complexes with bTrCP to Direct the Degradation of the Virus Release Inhibitor BST-2 (Tetherin) Janet Douglas, Oregon Health and Science University

116

Molecular Studies of HIV-1 Rev Function Lili Gu, Centre for Research in Infectious Diseases, Dublin, Ireland

117

CA-Dependent HIV-1 Nuclear Import Relies on Transportin-SR2 Vineet KewalRamani, National Cancer Institute, Frederick, MD

118

Self-Inactivation of HIV by its own RT/Rnase H Karin Moellling, University of Zurich

119

HIV-1 Envelope gp120 Indicues a Stop Signal and Virological Synapse Formation in Non-infected CD4+ T Cells Gaia Vasiliver-Shamis, New York University School of Medicine

120

Promoter-Targeted shRNA Driven by Retroviral Vector Achieves Long-term Repression of HIV-1 Replication Makoto Yagamashi, University of Tokyo Institute of Medical Science

121

Plasmid DNA- and live viral vector-based vaccine approaches for the treatment and prevention of HIV Infection John Eldridge, Profectus Biosciences, Inc.

122

Differential CD4 T cell subset depletion at mucosal sites in HIV infection Daniel Douek, National Institute of Allergy and Infectious Diseases, Bethesda, MD

123

AIDS Restriction Genes-Genome Wide Association Study Stephen O’Brien, National Cancer Institute, Frederick, MD

124

NK cells: good guys or bad guys? Galit Alter, Massachusetts General Hospital, Boston, MA

Progression/Anti-Progression Factors




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125

Immunogenetic polymorphisms and their effects on AIDS progression Xiaojiang Gao, National Cancer Institute, Frederick, MD

126

Host genetic determinants of response to HIV-1: A genome-wide perspective Abstract not available at time of printing David B. Goldstein, Duke Institute for Genome Sciences and Policy

127

HTLV-1 Infection of WE17/10 CD4+ Cell Line Leads to Progressive Alteration of Ca2+ Influx that Eventually Results in Loss of CD7 Expression and Activation of an Antiapoptopic Pathway Involving AKT and BAD Which Paves the Way for Malignant Transformation Bassan Badram, University of Brussels, Belgium

128

Human Domain Antibodies Against HIV-1 as Exceptionally Potent Cross-Reactive Neutralizers Weizao Chen, National Cancer Institute, Frederick, MD

129

Human Endogenous Retrovirus-K (HML-2) in the Plasma of People with Lymphoma and Breast Cancer Rafael Contreras-Galindo, University of Michigan Medical Center, Ann Arbor, Michigan

130

Inhibition of HIV-1 Release by Cell Permeable Peptides Sarah Daniels, National Institutes of Health, Bethesda, MD

131

The gp41-derived immunosuppressive (isu) peptide of HIV-1 modulates cytokine release and gene expression in human immune cells Joachim Denner, Robert Koch Institute, Berlin, Germany

132

Argotom-VAX: A Proposed Investigational HIV DNA Vaccine Composed of Selected Sequences of Clade B Gag, Pol, Nef, Tat, Vif, and Env Linked to Lysosomal Membrane Associated Protein (LAMP) William Hearl, Immunomic Therapeutics, Inc., Gaithersburg, MD

133

O-linked N-Acetylglucosaminylation Represses HIV-1 Replication and Sp-1 Mediated Trans- Activation of the HIV-1 LTR Ramona Jochmann, University of Erlangen, Germany

134

Cell-cycle Dependent HIV-1 Killing is Mediated Through the Viral Protease Kyeongeun Lee, National Cancer Institute, Frederick, MD

135

Dramatic Enhancement of Uptake and Trafficking of HIV-1 Tat Protein via Modulation of Endocytosis Pathways Guan-Han Li, Johns Hopkins University School of Medicine, Baltimore, MD

136

Increased IL-15 Production is Associated with Higher Infection of Memory CD4 T Cells During Acute SIV Infection Joseph Mattapallil, Uniformed Services University of the Health Sciences, Bethesda, MD

137

Presence and Role of HLA-C in HIV-1 Infection Andrea Matucci, University of Verona, Italy

138

Prevalence of Human Papillomavirus (HPV) Infection among HIV-Positive Women in the Pre- HAART and HAART Era in a Nigeria Clinic Olanrewaju Onigbogi, University College Hospital, Ibadan, Nigeria

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139

HIV-2 Capsids Distinguishing High and Low Virus Load Patients in a West African Community Cohort Clayton Onyango, MRC - Fajara Laboratories, Banjul, Gambia

140

A Retrospective Study of HIV-Exposed Infacts/Children in Kitwe, Zambia Gilbert Siame, Zambia Rural Healthcare Outreach Services(ZARHOS), Kitwe, Zambia

141

Type C Coping and Alexithymia are Associated Differentially with Specific Immune Mechanisms (Interleukin-6 and Beta-Chemokine Production) Linked to HIV Progression Lydia Temoshok, Institute of Human Virology, Baltimore, MD

142

HIV Frequently Elicits Mucosl and Plasma Env-Specific IgA with a Rapid Initial Decline in Acute Infection Nicole Yates, Duke University Medical Center, Durham, NC

143

Broad Spectrum Neutralizing Antibodies Against HIV-1 Elicited by Immunizing with Fusion Complexes Donato Zipeto, University of Verona, Italy

Friday, September 12, 2008 144

In vivo and ex-vivo proteomics for target discovery in cancer Guiliano Elia, University College Dublin, Ireland

145

The V1/V2 Loop Region of Simian-Human Immunodeficiency Virus Envelope gp120 is the Major Determinant of the Strain Specificity of the Neutralizing Antibody Response Ronald C. Desrosiers, Harvard University, Cambridge, MA

146

a4b7: A Newly Discovered Receptor for HIV Anthony Fauci, M.D., National Institute of Allergy and Infectious Diseases, Bethesda, MD

147

Hepatitis C and cancer Michael Houghton, Epiphany Biosciences, San Francisco, CA

148

Towards a genetics of cancer resistance George Klein, Karolinska Institutet, Stockholm, Sweden - In Honor of Lifetime Achievement Awardee Isaac Witz

149

The Role of the Environment in EBV-Associated Lymphoid Malignancies Eva Klein, Karolinska Institutet, Stockholm, Sweden

150

HIV and cancer: current trends and insights Eric Engels, National Cancer Institute, Bethesda, MD

151

MicroRNAs and the biology of KSHV infection Don Ganem, University of California, San Francisco

152

HIV-Associated Lymphomas. Focus on unusual lymphomas occurring specifically in HIV-infected patients Antonino Carbone, Istituto Nazionale Tumori, Milano, Italy

HIV Malignancies




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Controlling Replication of Human Retroviruses 153

Insight into the molecular mechanisms of CIITA-mediated inhibition of HIV-1 and HTLV replication Roberto Accolla, University of Insubria, Varese, Italy

154

Apobec3: A Modern Twist to A Classic Retroviral Mystery Warner Greene, Gladstone Institute of Virology and Immunology, University of California San Francisco

155

Tetherin, An Interferon-alpha Induced Inhibitor of Retrovirus Release that is Antagonized by Vpu Paul Bienasz, Aaron Diamond AIDS Research Center, New York, NY

156

Humanized Mouse Models for the In-Vivo Analysis of HIV Infection Victor Garcia-Martinez, Southwestern Medical School, Dallas, TX

157

Vpr as a mediator of proteasomal degradation and other functions Carlos de Noronha, Albany Medical College, Albany, NY

158

The SET complex acts as a barrier to autointegration of HIV-1 Judy Lieberman, Immune Disease Institute, Harvard Medical College

159

Finding Host Proteins Required for HIV Replication Abraham Brass, Massachusetts General Hospital, Boston, MA

160

Polarization of M1 and M2 macrophages and HIV infection Guido Poli, San Raffaele Scientific Institute, Milan, Italy

161

Peptide stabilization of gp120 confirmation: a novel vaccine candidate Jonathan Gershoni, Tel Aviv University, Israel

Saturday, September 13, 2008 Clinical HIV Session 162

Transmission Networks of Drug Resistance Acquired in Primary/Early Stage HIV Infection Mark Wainberg, McGill AIDS Centre, Lady Davis Institute, Jewish General Hospital

163

HIV Persistence in Patients on HAART: Re-evaluating Prospects for Eradication John Bartlett, Center for Civilian Biodefense Strategies, Johns Hopkins University Bloomberg School of Public Health

164

An update on raltegravir (Isentress) Charles F. Farthing, Merck

165

Evaluation of Efficacy and Immune Recovery of Optimized Background Therapy (OBT) Plus Maraviroc (MVC) vs Placebo (PBO) in Treatment Experienced (TE) Patients with only R5 HIV-1, Combined Analysis of MOTIVATE 1 and 2 Randall Tressler, Pfizer

166

HIV Therapeutics in Resource Limited Settings – Status and Future Directions Anthony Amoroso, Institute of Human Virology, University of Maryland School of Medicine

167

Update on Gilead Sciences Anti-HIV Development Programs Tomas Cihlar, Gilead Sciences

168

Rapamycin enhances the anti-HIV activity of CCR5 antagonist Vicriviroc Olga Latinovic, Institute of Human Virology

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Vaccines, Immune Response and Innate Immunity 169

Why do so few Antibodies Neutralize HIV-1? Tests of the ‘‘Tolerance Hypothesis’’ Garnett Kelsoe, Department of Immunology, Duke University

170

Getting the Right Immune Response to HIV: Evaluation of Protective Immune Responses After Vaccination of Rhesus Macaques George Pavlakis, National Cancer Institute, Frederick, MD

171

Induction of Cross-clade Neutralizing Antibodies in Rabbits Using a DNA Prime/Protein Boost Immunization Regimen Susan Zolla-Pazner, New York University Medical Center

172

Prospects for an AIDS Vaccine: Can Effector-Memory T Cell Responses Contribute? Louis T. Picker, Oregon Health and Science University

173

Human defensins - Small in Size, but Big in Functionality Wuyuan Lu, Institute of Human Virology, University of Maryland School of Medicine

174

The C5 region of gp120; a therapeutic vaccine target? Angus Dalgleish, St. George’s University of London

175

Mechanisms by which Synergistic Combinations of TLR Ligands Enhance T-Cell Responses to Vaccines Jay Berzofsky, National Cancer Institute, Bethesda, MD

176

Vector-based Vaccines for Cancer Therapy Jeffrey Schlom, National Cancer Institute, Bethesda, MD

177

Recruitment of High Avidity Antigen-Specific T Cells in Pancreatic Cancer Patients Elizabeth M. Jaffee, M.D., Sidney Kimmel Cancer Center at Johns Hopkins

178

Development of Preventive HIV Vaccines in the US Military HIV Research Program Nelson Michael, United States Military HIV Research Program, Rockville, MD

179

Rational Vaccine Design and the Development of an AIDS Vaccine Gary Nabel, Vaccine Research Center, National Institutes of Health, Bethesda, MD

180

Continuing Clinical Trials for HIV Vaccine Research Glenda Gray, Perinatal HIV Research Unit, University of the Witwatersrand

181

HIV-specific immune responses in healthy volunteers immunized with a multigene, multiclade HIV-1 DNA vaccine and boosted with HIV-1 MVA in Sweden and Tanzania Abstract not available at time of printing Gunnel Biberfeld, Karolinska Institutet, Stockholm, Sweden

182

A Novel Model for In Vivo SIV Neutralization Philip Johnson, Children’s Hospital of Philadelphia

183

Morphogenomic immune responsiveness to preventive/therapeutic vaccines Luigi Buonaguro, Istituto Nazionale Tumori ‘‘Fond. G. Pascale,’’ Naples, Italy

184

Development of the broadly neutralizing human antibody m9 for anti-HIV prophylactics Antony Dimitrov, Profectus Biosciences, Inc.

Preventative Vaccines




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POSTER SESSION INDEX 185

Interaction between a domain in the fusion peptide proximal region (FPPR) of gp41 and the epitope domain in the membrane proximal external region (MPER) increases binding of 2F5 to its epitope Uwe Fiebig, Magdalena Eschricht, Mirco Schmolke, Reinhard Kurth, Joachim Denner, Robert Koch- Institute

186

Immunosuppressive human endogenous retrovirus K (HERV-K) is expressed in human villous and extravillous cytotrophoblasts Ulrike Ka¨mmerer1, Ariane Germeyer2, Michaela Kapp1, Sven Stengel3, Kristina Bu¨scher3, Reinhard Kurth3, and Joachim Denner3; 1University Women’s Hospital, Wuerzburg, Germany; 2University of Heidelberg, Dept. of Gynecological Endorinology and Reproductive Medicine, Heidelberg, Germany; and 3Robert Koch Institute, Berlin, Germany

187

Autoresistance to X4 HIV Infection by soluble suppressor factors secreted from CD4+ T cells F. Cocchi, A. DeVico, A. Garzino Demo and R. C. Gallo; Institute of Human Virology, University of aryland School of Medicine

188

Targeted delivery of antiHIV siRNAs to T cells in vivo Premlata Shankar, M.D., Department of Biomedical Sciences, Texas Tech University Health Sciences enter, Paul L. Foster School of Medicine

189

Defective HIV-1 Proviral Genomes in Natural Viral Suppressors L. M. Eyzaguirre, M. M. Sajadi, R. R. Redfield, W. A. Blattner and J. K. Carr; Institute of Human Virology, University of Maryland School of Medicine

190

The Immune Response to HIV: Friend or Foe M. Karen Newell1, Elizabeth Connick2, Evan Newell3, Haig Keledjian3, Monica Ord3, Robert Berliner3, Joshua Cabrera1, Richard Tobin1, Cassie Pleasant1 and Lisa Villalobos-Menuey1; 1University of Colorado, Colorado Springs; 2University of Colorado Health Sciences Center; and 3Viral Genetics, Inc., Asuza, California

191

Anti-FasL treatment preserves SIV-specific memory and slows progression to AIDS in rhesus macaques Bhawna Poonia, Maria S. Salvato, C. David Pauza, Institute of Human Virology

192

Significant Relationship between INNO-LIAä HIV I/II Positivity Bands and the Immunological Status of HIV Patients Fernanda Leite, Fa´tima Oliveira and Luciana Pinho; Clinical Haematology Dept. - Hospital Geral Santo Antonio

193

The HIV-Positive Inpatient: Psychosocial Risks and Adherence Implications Rebecca L. Wald, Stephen J. Synowski and Lydia R. Temoshok, Institute of Human Virology and Department of Medicine, University of Maryland School of Medicine

193a

Psychosocial Contributors to Antiretroviral Adherence: Stability and Change Rebecca L. Wald, Stephen J. Synowski and Lydia R. Temoshok; Institute of Human Virology and Department of Medicine, University of Maryland School of Medicine

194

Systolic Blood Pressure recovery following mental stress predicts immune dysregulation in persons with HIV Stephen J. Synowski Ph.D., Institute of Human Virology, University of Maryland School of Medicine

195

Stimulation of reverse cholesterol transport potently suppresses HIV-1 replication Michael Bukrinsky, The George Washington University

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196

Monocyte-Dependent and –Independent Modulatory Effects of Vitamin D3 on X4 and R5 HIV-1 Replication in IL-2 Stimulated PBMC Massimo Alfano1, Chiara Rizzi1, Giuseppe Penna2, Luciano Adorini2 and Guido Poli1,3; 1San Raffaele Scientific Institute; 2BioXell SpA; and 3Vita-Salute San Raffaele University, School of Medicine

197

Naturally Occurring C-terminally Truncated Isoform of STAT5 (STAT5D) Is a Transcriptional Repressor of HIV-1 Expression. Characterization of the Platinum-Based STAT Inhibitor CPA-7 Giulia Della Chiara1, Andrea Crotti1, Heidi Kay2 and Guido Poli1 ; 1AIDS Immunopathogenesis Unit - San Raffaele Scientific Institute, Milano, Italy; and 2College of Public Health, University of South Florida, Tampa, Florida

198

Modulation of CCR5 density with low doses of the transplant drug Rapamycin sensitizes Vicriviroc-resistant R5 HIV-1 Alonso Heredia, Institute of Human Virology, Baltimore, MD

199

Risk factors for Virologic Failure and Adverse Reactions among Patients on Triple Antiretroviral therapy Adedayo Adeyemi, Oluseyi Adesola and Oluyemisi Olaogun, Healthmatch International, Lagos, Nigeria

200

Design, Synthesis, Anti-HIV and Cytotoxicity of Novel Heterocyclic Compounds Periyasamy Selvam, Amrita School of Pharmacy, Elmakkara, Kerala, India

200a

Inhibition of HIV replication and integrase activity by isatin derivatives Periyasamy Selvam, Amrita School of Pharmacy, Elmakkara, Kerala, India

201

Pathogenesis induced acidosis and hpercalceamia evident in HIV/AIDS disease Abdulrazak Hamza Yahaya, HIV/Immunology Laboratory, Pathology Department Murtala Muhammad Specialist Hospital, Kano, Nigeria

202

Retinopathy and enteropathy in HIV patients, a hypothetical concept beyond Virus and CD4 levels- a study from Varanasi, North India Dr. V. Satya Suresh Attili, Prof. Shyam Sundra, Prof. V. P. Singh and Prof. A. K. Gulati, Yashoda Hospitals, Somajiguda, Hyderabad

203

Immunosuppression Level In HIV-1 –Infected Patients Doesn’t Affect the Serological Diagnosis of Hepatitis C Virus Fernanda Leite, Luciana Pinho; Clinical Haematology Dept- Hospital Geral Santo Antonio- Porto- Portugal

204

Serum albumin: Could it be an inexpensive and simple marker of immunosupression in HIV-infected individuals? Muthu Sundaram, YRG Centre for AIDS Research and Education (YRG CARE), Voluntary Health Services Campus, Chennai, India

205

Increased IFN-alpha expression in circulating plasmacytoid dendritic cells of HIV-1 infected patients despite selective loss Clara Lehmann, M.D.1,3, Dirk Taubert, Ph.D.2, Jill M. Harper, Ph.D.3, Norma Jung, M.D.1, Pia Hartmann, M.D.1, Gerd Fa¨tkenheuer, M.D.1 and Fabio Romerio, PhD.3; 1First Department of Internal Medicine, University of Cologne, Cologne, Germany; 2Department of Pharmacology, University of Cologne, Cologne, Germany; and 3Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD

206

HIV-Induced Alterations in Vg2Vd2 T-cell Phenotype and Function Impact Mechanisms for Tumor Immunity in AIDS Jean-Saville Cummings, Institute of Human Virology, Baltimore, MD




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207

Mechanisms regulating Vg2Vd2 tumor cell cytotoxicity: Roles for CD56 and T cell receptor Kun Luo, Institute of Human Virology University of Maryland, Baltimore, MD

208

Altered cord blood gaT cell repertoire in Nigeria: possible impacts of environmental factors on neonatal immunity C. Cairo, Institute Of Human Virology, Baltimore, MD

209

TNF-a is an autocrine factor for Va2Va2 T cell Haishan Li, Institute of Human Virology, Baltimore, MD

210

Peripheral blood mononuclear cells activated by HIV-VLPs in correlation with HIV-1 seropositivity status L. Buonaguro1, M. L. Tornesello1, R. C. Gallo2, F. M. Marincola3, G. K. Lewis2 and F. M. Buonaguro1; 1Lab. of Viral Oncogenesis and Immunotherapies and AIDS Reference Ceneter, Istituto Nazionale Tumori ‘‘Fond. G. Pascale’’, Naples - Italy; 2Institute of Human Virology, Univ. of Maryland School of Medicine, Baltimore, MD, USA; and 3 Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, NIH, Bethesda

211

Potent Orally Bioavailable HIV-1 Fusion Inhibitors Alter Env Conformation and Expose Conserved Neutralization Epitopes C. Finnegan, V. Dettmer, M. Bramah-Lawani, T. Nitz, P. Bullock, I. Burimski, M. Reddick, C. Matallana, C. Beaubien, D. Stanley, J. Pettitt, G. Allaway and K. Salzwedel; Panacos Pharmaceuticals, Gaithersburg, MD

212

In vivo alteration of humoral responses to HIV-1 envelope gp120 by antibodies to the CD4-binding site of gp120 Maria Luisa Visciano, New York, NY

213

Dendritic cell-specific delivery of siRNA targeting SOCS1 enhances HIV-gag-specific CD8 T cell response Sandesh Subramanya, Texas Tech University-Health Sciences Center, Department of Biomedical Sciences, El Paso, TX

214

Increased expression of Suppressor of Cytokine Signaling-1 (SOCS-1) by HIV-1 transgenic rats: A mechanism for dysregulated T helper-1 responses William Reid, Institute of Human Virology, Baltimore, MD

215

The A1 subunit of Cholera toxin as an adjuvant for HIV DNA vaccines Kenneth Bagley, Baltimore, MD

216

Genetics of Fc Gamma Receptors IIa and IIIa in Tanzania Gustavo H. Kijak, Pharm.D., Ph.D.; U.S. Military HIV Research Program, Rockville, MD

217

HIV Knowledge and Willingness to Participate in New Preventive Technologies (NPT) Trials among a Nigerian Refugee Population O. Akinyemi, Department of Community Medicine, University College Hospital, Ibadan, Nigeria

218

Knowledge, attitudes and practices (KAP) of sexually active men towards circumcision as a preventive measure against HIV infection in Kitwe district, Zambia Sthembile Ndopu and Gilbert Siame, Zambia Rural Healthcare Outreach Services (ZARHOS), Kitwe, Zambia

219

Preventing HIV infection in developing countries: focus on gender and age as the determinants of the spread Abdulrazak Hamza Yahaya, HIV/Immunology Laboratory, Murtala Mohammed Specialists Hospital Kano, Nigeria

16




220

The Translational Laboratory Shared Services Mariola Sadowska, Ph.D., Colette Burgess, Latreece Nance and Joseph Bryant, DVM, TLSS, UMGCC, Baltimore, MD

221

Humoral Immunity against Conserved Epitopes of HIV-1 Envelope Protein Archived in Memory B cells in Natural Viral Suppressors: Discordance with Plasma Antibodies Yongjun Guan1, Mohammad Sajadi1, Anthony L. DeVico1, Christine Obriecht1, Robin Flinko1, Karla Godfrey1, Timothy Fouts2, Ranajit Pal3, Robert Redfield1, Robert Gallo1 and George K. Lewis1; 1Divisions of Basic Sciences and Vaccine and Clinical Sciences, Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD; 2Perfectus BioSciences, TechCenter at University of Maryland Baltimore County, Baltimore, MD; and 3Advanced BioScience Laboratories, Kensington, MD

222

Antibody 2G12 recognizes a glycopeptide epitope on HIV-1 gp120 envelope glycoprotein Wei Huang, George K. Lewis and Lai-Xi Wang; Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD

223

An animal model for non-hodgkin’s lymphoma of the central nervous system (NHL-CNS) J. Bryant, H. Tran, M. Sadowska, E. Ateh and Y. Lunardi-Iskandar, Baltimore, MD

224

Persistent virological benefit in SIV-infected macaques upon therapeutic vaccination with DNA vectors by in vivo constant-current electroporation Barbara K. Felber1, A. Valentin2, A. von Gegerfelt2, M. Rosati2, V. Pate2, G. Miteloudis2, C. Alicea1, C. Bergamaschi2, R. Jalah1, A. Kha3, R. Draghia-Akli3 and G.N. Pavlakis2; 1Human Retrovirus Pathogenesis Section, 2Human Retrovirus Section, NCI, Frederick, MD; and 3VGX Pharmaceuticals, Inc., The Woodlands, TX

225

New Viral and Tuberculosis Therapeutics for the 21st Century Roger J. Pomerantz, M.D., Johnson & Johnson Corporation

226

Glycosylation of gp41 of Simian Immunodeficiency Virus Shields Epitopes That Can Be Targets for Neutralizing Antibodies Eloı´sa Yuste, Jacqueline Bixby, Jeffrey Lifson, Shuji Sato and Ronald Desrosiers; Harvard Medical School

227

Opportunity for Scale –Up; Mobile X-ray Technology- Strengthening TB Diagnosis in HIV+ve Patients; ACTION Experience in Zaria, rural Northern Nigeria U. I. Gebi, B. Musa, N. Alfred, A. Abimiku, P. Dakum, W. Blattner, E. Meshak, O. Obasanya, M. Gidado and A. Clement; Institute of Human Virology, Nigeria

228

Psychological distress as a risk factor for non-adherence to Highly active anti-retroviral therapy Etheldreda Nakimuli-Mpungu, Johns Hopkins School of Public Health

229

Baseline renal insufficiency and risk of death among HIV-infected adults on antiretroviral therapy in Lusaka, Zambia L. B. Mulenga1, G. Kruse1, S. Lakhi2, R. A. Cantrell1,3, S. E. Reid1,3, I. Zulu4,5, E. M. Stringer1,3, Z. Krishnasami3, A. Mwinga4, M. S. Saag3, J. S. A. Stringer1,3, and B. H. Chi1,3; 1Center for Infectious Disease Research, Lusaka, Zambia; 2University Teaching Hospital, Lusaka, Zambia; 3University of Alabama at Birmingham, AL; 4CDC, Lusaka, Zambia; and 5University of Zambia School of Medicine, Lusaka, Zambia

231

Lopinavir/ritonavir-based Second Line Antiretroviral Treatment in children at National Pediatric Hospital, Phnom Penh, Cambodia S. Sam, V. Ung1, C. Huot1, C. Courpotin2, Eric Nerrienet3, David Pugatch4, Kenneth Mayer4 and Y. M. Chhour5 ; 1Child Health Improvement Clinic, National Pediatric Hospital, Phnom Penh, Cambodia; 2French Red Cross, Phnom Penh, Cambodia; 3Pasteur Institute, Phnom Penh, Cambodia; 4Brown University, Miriam Hospital, Providence, RI, USA; and 5 National Pediatric Hospital, Phnom Penh, Cambodia

FOGARTY SCHOLARSHIP ABSTRACTS




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232

Explosive Expansion of HIVand associated risk factors among Male and Hijra Sex Workers in Sindh, Pakistan Arshad Altaf, HIV/AIDS Surveillance Project, Sindh AIDS Control Programme

233

Enhancement of HIV-1 Replication in Human Primary Cells by Macrophage Migration Inhibitory Factor in Nigerian Africans Dr. Busari, Olusegun Adesola, HIV Study Group, Federal Medical Centre

234

Neutralization efficiency and presence of anti-V3 antibodies in plasma of HIV-1 infected Northern Indians Alok K. Choudhary1, Subhashree Dutta1, Naveet Wig2, A Biswas. 2, Raiees Andrabi1, Rajesh Kalra1, Rama Bhasin3, Susan Zolla Pazner4, Suman Laal4, and Kalpana Luthra1; 1Department of Biochemistry; 2Department of Medicine; 3Blood Bank, CN Centre, All India Institute of Medical Sciences, New Delhi, India; and 4Veterans Affairs Medical Center, NYU School of Medicine New York, NY

235

Quality ART Scale Up Through Regionalization of Laboratory Services In Nigeria C. Ezeaku, J. Farley, P. Dakum, T. Croxton, N. Constantin2, A. Abimiku and W. Blattner; Institute of Human Virology Nigeria

236

T-Cell Receptor (TCR) Activation Mediates Efficient and Sustained HIV Transcriptional Elongation and Initiation through Multiple Signal Pathways Joseph F. Hokello; Department of Molecular Biology and Microbiology, CWRU School of Medicine

237

N-linked glycans on HIV-1 gp120 are critical determinants for the recognition of CD4 helper T cell epitopes Hualin Li, VA Medical Center, New York, NY

238

Comparative Evaluation of the Performance of Abbott m2000rt Real-Time HIV-1 Assay for Measurement of HIV-1 Plasma Viral Load M. Vidya1, S. Saravanan1, Kartik K. Venkatesh2, P. Balakrishnan1, N. Kumarasamy1, K. G. Murugavel1, Sunil S. Solomon1, Suniti Solomon1 and Kenneth H. Mayer2 ; 1YRG Centre for AIDS Research and Education, Voluntary Health Services, Taramani, Chennai, India; and 2Brown University, RI, USA

239

Barriers to timely initiation of antiretroviral therapy among HIV infected children admitted to Mulago Hospital Paediatric wards Dr. Eleanor Namusoke, Joint Clinical Research Centre, Kampala, Uganda

240

Patient Retention in a University Hospital-based ART program in Uganda Ouma Joseph

241

High Incidence Cohort of IDUs Infected with HIV with Low Genetics Diversity for HIV Vaccine Efficacy Trials Sergey Verevochkin, St. Petersburg, Russia

242

Mycobacterium avium KatG protein (MAV_2753): a putative candidate for the serodiagnosis of MAC disease Kapil Gupta1, Ajay Wanchu2, Romica Latawa1, S. Laal3, G.K. Khuller and Indu Verma1; 1Department of Biochemistry; 2Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, INDIA; and 3Department of Pathology, NYU Langone Medical Center, New York, USA

243

The Use of HIVQUAL as a Quality Improvement Tool for a Large Scale HIV/AIDS Public Health Program – THE ACTION PROJECT, IHV Nigeria U. Yakubu, U. Gebi, M. Babamaiyaki, I. Okoye, K. Falayajo, P. Dakum, J. Farley, W. Blattner, M. Etiebet, A. Zoakah and M. Charurat; Institute of Human Virology, Abuja, Nigeria

18




244

Mannose Binding Lectin and its variants: susceptibility to HIV-1 and Schistosoma haematobium infection in a rural Zimbabwean community Rutendo B. L. Zinyama-Gutsire, National Institute of Health Research Ministry of Health and Child Welfare Zimbabwe

245

Outcome of early infant diagnosis in exposed infants in Northern Nigeria O. D. Adegoke, Z. Basir, J. Jumare, S. Sani, R. Enzama, S. Peters, A. Abimiku, W. A. Blattner; Institute of Human Virology, Nigeria

246

Interface of public health implementation and research – Challenges and Prospects – IHVN/Action Project Experience O. Akinwande, Patrick Dakum, J. Farley, U. Gebi, C. Adebamowo, A. Abimiku, M. Charurat, W. Blattner; Institute of Human Virology, Nigeria

247

The effect of chronic alcohol exposure in HIV/AIDS patients on antiretroviral drugs (Triomune30 – AZT/3TC/NVP) in Uganda Dr. Godfrey Sande Bbosa; Supervisors: Professor J. Ogwal-Okeng; Professor W. W. Anokbonggo and Associate Professor B. D. Kyegombe; Department of Pharmacology and Therapeutics, Faculty of Medicine, Makerere University, Kampala, Uganda

248

Telemedicine in Peru: training physicians responsible for the administration of highly active antiretroviral therapy (HAART) in a developing country Katiuska Castillo1, Rau´l Gutie´rrez1,3, Leslie Soto1, Alfonso Silva-Santisteban1, David Iglesias1, Alberto GuerraGarcá1, Carlos Kiyan2, Carlos Seas1, Juan Echevarrá1, Ciro Maguinã1 and Eduardo Gotuzzo1; 1Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru; 2Facultad de Medicina, Universidad Peruana Cayetano Heredia, Lima, Peru; and 3Fogarty-NIH International Training and Research Program in HIV/AIDS and TB, University of Miami, Miller School of Medicine

249

Efficacy of a Behavioral Intervention to Reduce HIV Risk among Female Sex Workers in Urumchi, China Lin Han, Division of Policy and Information, NCAIDS, Beijing, China

250

Establishment of new Jurkat cell line Stably expressing HIV-1-Tat Mohamed Ali Jarboui, William W. Hall and Virginie W. Gautier; Centre for Research in Infectious Diseases, University College of Dublin, Dublin, Ireland

251

Migration, Pastoralists, HIV Infection and Access to Care: The Nomadic Fulani of Northern Nigeria J. Jumare, A. G. Habib, U. Gebi, A. Zoakah, P. Dakum, J. Farley and W. A. Blattner; Institute of Human Virology, Nigeria

252

Acceptance of and adherence to anti-retroviral therapy in Tanzania: the influence of lipodystrophy and of traditional medicine Sajida J. Kimambo

253

Changes of HIV risk behaviors of heroin drug users treated in methadone maintenance treatment clinics in Guizhou province, China Enwu Liu

254

Factors associated with development of Opportunistic Infections among patients on ART in Uganda S. Muhumuza, J. Ouma, F. Semitala, E. Mbabazi and M. Kamya; Mulago-Mbarara Teaching Hospital’s Joint AIDS Program (MJAP), Kampala, Uganda

255

HIV Related TB: Prospects and Challenges in a High Burden Area; A Case Study of Isoniazid Prophylaxis (IPT) Utilization B. M. Musa, U. Gebi, K. Falayajo, P. Dakum, J. Farley and W. Blattner; Institute of Human Virology, Nigeria




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256

Dyslipidemia in HIV-1 infected individuals following generic efavirenz based HAART in a resource constrained setting Dr. Sundaram Muthu, YRG CARE, Voluntary Health Services, Taramani, Chennai, India

257

Does toxicity to fixed dose stavudine, lamivudine and nevirapine regimen affect virologic suppression among HIV infected adults at the Infectious Diseases Institute, Makerere University? Fred C. Semitala1, Harriet Mayanja- Kizza1, Andrew Kambugu2, Agnes K. Kiragga2, Barbara Castelnuovo2, Robert Kalyesubula1, Walter Schlech3, Robert Colebunders4, Keith McAdam2 and Moses R. Kamya1; 1Department of Medicine Makerere University, Kampala, Uganda; 2Infectious Diseases Institute, Makerere University, Kampala, Uganda; 3Dalhousie University Halifax, Canada; and 4Department of Clinical Sciences, Institute of Tropical Medicine Antwerp, Belgium

258

Intra patient evolution of Human Immunodeficiency Virus Type 1 protease and reverse transcriptase in antiretroviral naı¨ve patients Uma Shanmugasundaram1, Kumarasamy Nagalingeswaran1, Kailapuri G. Murugavel1, Saravanan Shanmugam1, Vidya Madhavan1, Sunil S. Solomon1, Suniti Solomon1, Kenneth H. Mayer2 and Balakrishnan Pachamuthu1; 1 Y. R. Gaitonde Centre for AIDS Research and Education, Chennai, India; and 2Department of Medicine, Brown University/Miriam Hospital, Providence, RI, USA

259

Quality of life among end-of-life former commercial plasma donors infected with HIV and the care model in rural Henan, China Yu Sheng, Beijing P. R. China

260

Scaling Up PMTCT Access Using a Hub and Spoke Model in Nigeria, Sub-Sahara Africa Dr. Akinmurele Timothy, Institute of Human Virology Nigeria

261

Impact of Community- and Home-Based Care on HIV/AIDS Patients and Their Families Emily Hauwa Umaru, Institute of Human Virology, Nigeria

262

Prevalence of Norovirus infections in children from Iquitos, Peru Daniel Velasquez Portocarrero, Jr. Enrique Barron, PERU

263

Adherence to highly active antiretroviral treatment and related factors in drug users with HIV/AIDS Wang Honghong, School of Nursing, Central South University, Changsha, China

264

Immunologic recovery among HIV infected children on first line antiretroviral therapy in Kano, Nigeria Bashir M. Zubayr

20




Author Index to Abstracts A Accolla Adegoke Adeyemi Akinmurele Akinwande Akinyemi Altaf Alter Amoroso Anastasopolou Attili

153 245 199 260 246 217 232 124 166 104 202 127 215 163 247 175 101 181 155 159 223 183 210 195 233

C Cairo Carbone Castillo Chen Choudhary Cihlar Cocchi Contreras-Galindo Cummings

208 152 248 128 234 167 187 129 206

D Dalgleish Daniels de Noronha Della Chiara Denner Denner Denner Derdeyn Desrosiers Desrosiers

184 122 115

E Eldridge Elia Engels Eyzaguirre Ezeaku

121 144 150 189 235

F

B Badram Bagley Bartlett Bbosa Berzofsky Bewley Biberfeld Bienasz Brass Bryant Buonaguro Buonaguro Burkrinsky Busari

Dimitrov Douek Douglas

174 130 157 197 131 185 186 107 145 226


Farthing Fauci Felber Finnegan

164 146 224 211

G Ganem Gao Garcia-Martinez Garzino-Demo Gebi Gershoni Goldstein Gougeon Gray Greene Gu Guan Gupta

151 125 156 112 227 161 126 114 180 154 116 221 242

H Han Hanes Hearl Heredia Hokello Honghong Houghton Huang

249 110 132 198 236 263 147 222

J Jaffee Jarboui Jochmann Johnson Joseph Jumare

177 250 133 182 240 251



21

K Kelsoe KewalRamani Kijak Kimambo Klein Klein Kurth

R 169 117 216 252 148 149 111

L Lagenaur Latinovic Lee Lehmann Leite Leite Li Li Li, H. Lieberman Lisco Liu Lu Luo

105 168 134 205 192 203 209 135 237 158 113 253 173 207

M Mattapallil Matucci Melikian Michael Moelling Muhumuza Mulenga Musa Muthu

136 137 102 178 118 254 229 255 256

N Nabel Nakimuli-Mpungu Namusoke Ndopu Newell

179 228 239 218 190

Reid

S Sadowska Sam Schlom Selvam Selvam Semitala Shankar Shanmugasundaram Shaw Sheng Siame Subramaniam Subramanya Sundaram Swanstrom Synowski

123 138 139

P Pavlakis Picker Poli Poli Pomerantz Poonia Portocarrero

22

170 172 160 196 225 191 262

220 231 176 200 200a 257 188 258 108 259 140 100 213 204 109 194

T Temoshok Tressler Tsibris

141 165 103

U Umaru

261

V Vasiliver-Shamis Veazey Verevochkin Vidya Visciano

119 106 241 238 212

W Wainberg Wald Wald

O O’Brien Onigbogi Onyango

214

162 193 193a

Y Yagamashi Yahaya Yahaya Yakubu Yates

120 201 219 243 142

Z Zinyama-Gutsire Zipeto Zolla-Pazner Zubayr


244 143 171 264



100

3D structure of native HIV-1 gp120 trimers and mechanisms of cellular entry

Sriram Subramaniam Center for Cancer Research, NCI, NIH, Bethesda, MD 20892 We are using electron tomography and related methods in 3D electron microscopy to analyze the structure of HIV-1 in the purified state, and at various stages of maturation in infected T-lymphocytes, macrophages and mature dendritic cells. Using cryo-electron tomography combined with 3D image classification and averaging, we have recently obtained the three-dimensional structures, at resolutions ˚ , of the trimeric envelope glycoprotein (Env) displayed on native HIV-1 in the unliganded state, of ;20 A in complex with the broadly neutralizing antibody b12 and in a ternary complex with CD4 and the 17b antibody. By fitting the known crystal structures of the monomeric gp120 core in the b12- and CD4/17b-bound conformations into the density maps derived by electron tomography, we have derived molecular models for the native HIV-1 gp120 trimer in unliganded and CD4-bound states. CD4 binding results in a major reorganization of the Env trimer, causing an outward rotation and displacement of each gp120 monomer. This is coupled with a rearrangement of the gp41 region along the central axis of the trimer, leading to closer contact between the viral and target cell membranes. Our findings identify the structure and conformational changes of trimeric HIV-1 gp120 relevant to antibody neutralization and attachment to target cells, and are likely to provide important clues to vaccine design.




23

101

Chemokine Receptor CCR5 Mediates Resistance to West Nile Virus Infection in Mouse and Man

Carole A. Bewley,1 Son N. Lam,1 Chih-Chin Huang,2 Priyamvada Acharya,2 Min Tang,2 Richard Wyatt,2 and Peter D. Kwong2 1

Laboratory of Bioorganic Chemistry, NIDDK, NIH; 2Vaccine Research Center, NIAID, NIH

The CCR5 co-receptor binds to CD4-activated HIV-1 gp120 surface envelope glycoprotein and facilitates HIV-1 entry into cells. Key portions of CCR5 involved in gp120 binding include its N-terminus and second extra cellular loop (ECL2). The N-terminus of CCR5 contains multiple tyrosine-sulfate (TyrSO4) residues that are necessary for binding gp120, as do several antibodies that react with the coreceptor-binding site on gp120. We have used a combination of NMR spectroscopy, X-ray crystallography and molecular docking to solve the structure of the CCR5 N-terminus and the tyrosine-sulfated antibody, 412d, in complex with gp120 and CD4. These analyses have revealed a highly conserved Tyr-SO4-specific binding site on gp120 for which HIV-1 Entry inhibitors may be developed, and provide the structural basis for post-translational mimicry between the immune system and an HIV co-receptor. In ongoing studies we are using NMR and biochemical techniques to probe the interactions between CCR5’s ECL-2 and gp120; results from these studies will also be discussed.

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102

Visualization of HIV-1 entry pathways in cells

Gregory Melikian Institute of Human Virology, 725 W. Lombard St., Baltimore, MD 21201 Viruses whose fusion proteins are activated by interactions with cellular receptors at neutral pH are generally thought to fuse directly to a plasma membrane. However, recent evidence suggests that these viruses may undergo receptor-mediated, pH-independent fusion with endosomes. To elucidate the HIV-1 entry pathways, we have developed an imaging assay capable of differentiating between surface fusion (SF) and endosomal fusion (EF). Pseudoviruses bearing the pH-independent HIV-1 Env or the Avian Sarcoma and Leukosis Virus (ASLV) Env, which has been shown to induce fusion in both receptor- and low pH-dependent manner, were generated. Viral membrane and content redistribution during fusion was visualized by co-labeled viruses with a core marker (Gag-GFP) and a red lipophilic dye (DiD). The Gag-GFP cleavage by viral protease generated a smaller GFP-tagged fragment that was readily released from virions upon their permeabilization. Double-labeled viruses were adhered to target cells expressing CD4 and coreceptors in the cold, and fusion was triggered by raising the temperature and monitored by a laser scanning confocal microscopy. Fluorescent viruses undergoing SF are expected to loose their lipid marker (hemifusion) and content marker (full fusion) due to dilution of these probes in the plasma membrane and the cytosol, respectively. By contrast, full fusion with an endosome should be manifested in disappearance of a content marker, but not a lipid dye. This strategy was validated by comparing the outcomes of ASLV fusion in endosomes (the natural pathway) with fusion at the cell surface induced by acidic pH. Imaging HIV-cell fusion (at neutral pH) detected both SF and EF events. Notably, HIV and ASLV fusion with a plasma membrane was incomplete, resulting in lipid, but not viral content transfer within the limited time frame of imaging experiments. In contrast, endosomal fusion events were associated with the viral content release into the cytosol. These results indicate that HIV-1 can fuse both directly with a plasma membrane and with endosomal compartments. It remains to be established which of these alternative entry pathways is the one that leads to productive infection.




25

103

Tracking Dynamic HIV-1 Escape from CCR5 Antagonist Therapy in vivo by Deep Sequencing

Athe M. N. Tsibris 65 Landsdowne St, Room 435, Cambridge, MA 02139 High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects who failed CCR5 antagonist (vicriviroc [VCV]) therapy. All subjects were receiving VCV at the time of VF. Three time points were analyzed for each subject: study entry (wk 0), an intermediate time point on study drug, and VF. HIV-1 RNA was extracted from plasma and subjectspecific primer sets were used to reverse transcribe and amplify plasma V3 loop-coding regions of env. V3 amplicons were then submitted in a blinded fashion for deep sequencing and custom analysis. Between 25,000–140,000 single genome sequences were obtained per subject per time point. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or emergence of VCV resistance, were present as minor variants at 0.8–2.8% of baseline samples. Deep sequencing provided a detailed view of both extreme shifts in population frequencies toward these forms and the rapid evolutionary impact of VCV selection. This degree of V3 loop sequence diversity has implications for vaccine design and the optimal use of HIV-1 CCR5 antagonists.

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104

A V3-independent pathway to resistance to small molecule CCR5 inhibitors

Dr. Kleio Anastasopoulou Weill Cornell Medical College, Dept. of Microbiology, 1300 York Ave, Rm: W-801, New York, NY 10065 CCR5-targeted entry inhibitors have now entered clinical practice for the treatment of HIV-1 infection. As with all antiretroviral drugs, the development of resistance should be anticipated and understood. The genetic pathways to resistance to CCR5 inhibitors described to date have almost invariably involved changes in the V3 region of the HIV-1 gp120 surface glycoprotein, irrespective of the identity of the parental virus, of whether resistance developed in vivo or in vitro, and of the method used to generate resistant variants. An exception is that no V3 changes were found in a variant (D1/85.16) of the R5 subtype B primary isolate CC1/85 that was selected for resistance to vicriviroc (VVC) during prolonged culture in PBMC (Marozsan et al. Virology 2005; 338: 182–199). The resistant virus was found to be stable during 19 passages of culture in the absence of the drug, so there are no obvious fitness costs associated with the development of VVC resistance. Moreover, the phenotype of D1/85.16 is generally similar to those of other variants with critical resistance sequence changes in V3. Thus the resistant variant has adapted to use the inhibitor-CCR5 complex, as well as the free co-receptor. To locate the non-V3 resistance mutations, a VVC-resistant clone was generated from the D1/85.16 isolate, and then used to produce chimeric viruses and site-directed mutants for further analysis. The critical determinants of VVC resistance have now been shown to lie within a specific region of the gp41 transmembrane glycoprotein. We are now investigating whether these amino acid changes are necessary and sufficient for complete resistance, as well as trying to define how they function. However, it is clear that although V3 changes are the dominant genetic pathway to CCR5 inhibitor resistance, there is a V3- (and gp120-) independent pathway that can create a similar phenotype. Understanding how this pathway operates may assist in further defining structurefunction relationships within the trimeric HIV-1 Env complex.




27

105

sCD4-17b, an Engineered Bifunctional HIV-1 Neutralizing Protein: Potent Cross Clade Activity and Potential Microbicide Use

Laurel A. Lagenaur,1,2 Vadim A. Villarroel,1 and Edward A. Berger1 1

NIAID, NIH, Bethesda, MD 20892, 2Osel, Inc., 4008 Burton Dr., Santa Clara, CA 95054, USA

We previously described sCD4-17b, an engineered single chain bifunctional protein that neutralizes HIV-1 infection (Dey et al., J. Virol., 2003). sCD4-17b contains the first two domains of CD4 attached by a flexible linker to a 17b mAb scFv, whose epitope overlaps the CD4i bridging sheet involved in coreceptor binding. Based on the crystal structure of a gp120 core complexed to two-domain sCD4 and the 17b Fab (Kwong et al., Nature, 1998), we originally anticipated that a 35 aa linker length would ˚ distance required for simultaneous binding of the sCD4 and 17b moieties, thus readily span the 56 A neutralizing free virions. Linkers too short to enable simultaneous binding were predicted to be much less active, perhaps equivalent to a mixture of unlinked sCD4 and 17b scFv. We expressed sCD4-17b constructs containing linkers of various lengths, as well as free sCD4 and 17b scFv. The purified proteins were tested against HIV-1 pseudotypes containing genetically diverse Envs in the TZM-bl single-round neutralization assay. sCD4-17b constructs with linkers of 35 or 40 aa displayed equivalently potent neutralization activities; as expected, a construct with a linker of only 5 aa was significantly less potent, although it was more effective than equimolar concentrations of unlinked sCD4 plus 17b (Perhaps reflecting cross-binding of the sCD4 and 17b moieties to different gp120 subunits, between different spikes on either the same or different virions). Unlinked sCD4 plus 17b scFv had minimal activities over the same concentration ranges. sCD4-17b with the 40 aa linker was tested against pseudotypes with Envs from genetically diverse HIV-1 isolates. Amongst Envs from standardized clade B and C panels, as well as several from clades D, E, and F, neutralization was observed in every case; the potencies were consistently high (IC50 2 – 40 nm, corresponding to 0.1 – 2 mg/mL). Amongst Envs from early infection subtype A isolates previously shown to be resistant to most broadly neutralizing mAbs (Blish et al., AIDS 2007), nearly all were neutralized by sCD4-17b albeit with higher IC50 values (40–200 nM, corresponding to 2–10 mg/mL). Neutralization of ‘‘live’’ virus via spreading infection in PBMC’s is currently being examined. The combined breadth and potency of sCD4-17b exceeds that reported for any anti-HIV mAbs. These findings suggest its potential use as a microbicide to prevent sexual transmission.

28




106

Early events in vaginal HIV transmission

Ronald S. Veazey1 and Thomas Hope2 1

Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, 2Northwestern University, Chicago IL

Worldwide, the vast majority of HIV-1 cases occur through heterosexual transmission. Although the initial events involved in vaginal transmission are uncertain, studies suggest that dendritic cells in the vaginal epithelium may be involved in trapping viral particles on the surface, and transporting them to susceptible memory CD4+ T lymphocytes in the mucosa. However, studies show that the vaginal epithelium do not express known receptors for HIV attachment including CD4, CCR5, or DC-SIGN. Although dendritic cells expressing CD1a are present in the epithelium, and CD4+CCR5+ memory cells are abundant in the underlying lamina propria, the mechanisms involved in transport of HIV across the epithelium to the underlying target cells remains debated. Using a photoactivateable HIV, we are currently examining the earliest events in vaginal HIV transmission in rhesus macaques in vivo, and in human vaginal/cervical explants ex vivo. These studies have shown that HIV passively penetrates through layers of the squamous epithelium of the vagina, and thus gains access to intraepithelial antigen presenting cells. Breaks or thinning of the epithelium increase the level of penetration, and cervical mucus provides a protective barrier that traps most particles in the lumen. Although virus is frequently observed to penetrate the vaginal epithelium, in experimental macaques, virus is rarely found in the endocervix or upper reproductive tract. Combined, these data suggest that the vaginal mucosa is the major site of HIV entry, and is likely the major site of viral transmission in the female reproductive tract.




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107

Escape from Neutralizing Antibody in Early Subtype C HIV-1 Infection

Rong Rong,1 Bing Li,1 Rebecca M. Lynch,1 Joseph Mulenga,2 Susan Allen,1 Eric Hunter,1 Jerry L. Blackwell,1 and Cynthia A. Derdeyn1 1

Emory University, Atlanta, GA, USA; 2Zambia Blood Transfusion Service, Lusaka, Zambia

HIV-1 subtype C viruses circulate predominantly in India and sub-Saharan Africa, accounting for most global infections. We previously demonstrated that 9 out of 11 subtype C infected seroconvertors followed in a Zambian cohort rapidly developed high titer autologous neutralizing antibody (Nab) against the infecting virus. Here we characterized the Nab sensitivity of individual envelope (Env) glycoproteins that evolved during early infection in four of these seroconvertors. Env genes were PCR amplified from longitudinal patient PBMC DNA and plasma samples using single genome analysis, cloned into an expression vector, and screened for biological function using a pseudovirus assay. Neutralization sensitivity of each Env pseudovirus was evaluated against autologous plasma from longitudinal time points to identify variants that had escaped from contemporaneous Nab. The molecular mechanism of Nab escape was then investigated in detail in two subjects by replacing Env domains of the initial, Nab sensitive virus with those derived from 6 to 10 escape variants from the same patient. The monophyletic founder virus in the newly infected subjects was rapidly replaced by a succession of variants that were resistant to contemporaneous Nab but were subsequently neutralized by a de novo antibody response. In one patient, the virus used multiple escape mechanisms, even at a single time point. One of these escape pathways required sequence changes in both gp120 and the gp41 ectodomain, while another involved only the V3-V5 region of gp120. The V1V2 domain, however, did not contribute to neutralization escape in this subject. In contrast, in the second patient, the V1V2 domain played a major role in resistance in every Env analyzed. The changes in V1V2 conferred early and complete resistance against a monoclonal antibody isolated from the PBMC of this patient four years later, but changes outside of V1V2 were required to escape from the polyclonal antibody response. Our data demonstrate that subtype C viruses use multiple mechanisms to escape from potent autologous Nab, even within a single patient. These findings highlight the need to define and understand potential escape mechanisms in strategies to induce protective Nab.

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Identification of Full-length Transmitted HIV-1 Genomes Reveals Extraordinary Dynamics and Precise Molecular Pathways of Early Virus Diversification, Adaptation and Immune Evasion

George M. Shaw, MD, PhD University of Alabama at Birmingham, 816 KAUL Bldg, 720 20th St. South, Birmingham, AL 35294-0024 Background: Identification of complete (9kb) genomes of HIV-1 viruses responsible for productive clinical infection could be instrumental in elucidating viral properties and biological events responsible for virus transmission and in characterizating subsequent virus evolution across the proteome. Methods: Recently, we developed a mathematical model of viral sequence evolution in acute HIV1 infection and an empirical dataset of 3449 complete HIV-1 subtype B env sequences derived by single genome amplication (SGA)-direct amplicon sequencing that allowed us to infer the exact nucleotide sequences of full-length env genes of transmitted and early founder viruses in 98 of 102 consecutively studied patients (Keele et al., PNAS 2008). Here, we applied the same experimental strategy to the identification of full-length HIV-1 genomes in 12 subjects with acute or early subtype B or C infection and characterized their early evolution. Results: Prior to antibody seroconversion, viruses generally exhibited a Poisson distribution of mutations and star-like phylogeny, which coalesced to inferred transmitted/early founder consensus sequences at or near the estimated time of virus transmission. 11 of 12 subjects were productively infected by a single virus (or virally-infected cell) and one was infected by two viruses. In four subjects, we performed sequential SGA analyses of complete viral genomes over the first year of infection and mapped each autologous CTL epitope recognized and escaped. We also mapped neutralizing antibody (Nab) recognition and escape in Env. Immune recognition and escape occurred substantially earlier for CTLs (;4 wks post-infection) than for Nabs (;12 wks post-infection). Within a two week period beginning at peak viremia, virtually the entire body’s population of productively infected cells was replaced by cells infected with viruses containing CTL escape mutations that were confirmed phenotypically. Complete replacement of transmitted wildtype virus by Nab escape virus occurred between 8 and 12 weeks post-infection. Conclusions: Identification of transmitted/founder full-length genomes allows for a comprehensive and dynamic assessment of immune recognition and escape in HIV-1 infected humans, including individuals immunized with candidate HIV-1 vaccines who experience breakthrough infections. We are currently conducting similar studies in monkeys infected intrarectally by SIVmac251 or SIVsmE660 to better define this animal model system and its relationship to humans infected mucosally by HIV-1.




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Complexity of the Transmitted Virus and Compartmentalization In Its Seminal Source

Ronald Swanstrom UNC Center For AIDS Research The transmission event has become a focal point for understanding the earliest steps of infection with HIV-1. Most transmission events occur during unprotected sex. Thus there is special interest in the virus that crosses the mucosal surface and also the source of virus in genital secretions, as these two sites define the bottleneck that occurs during transmission. We have employed the single genome amplification strategy to examine the env gene in the transmitted virus and also in the blood and semen of chronically infected men, all in the context of subtype C infection. A comparison of sequences from transmitted viruses to those found in chronic infection suggests differences in the proteins encoded by these env genes. While most infections are initiated with the transmission of a single variant, approximately 20% of infections are the result of infection with two or more variants. Multiple variants were found in both men and women, and the frequency of the presence of multiple variants is inconsistent with these representing independent transmission events of low probability. Thus transmission rates must be transiently high or the transmission of multiple variants must represent linked events. Comparison of virus in blood plasma and seminal plasma of chronically infected men shows frequent clonal amplification of virus in the seminal tract, changing the composition of the viral population from that seen in the blood. In some cases there is more extensive compartmentalization, indicative of sustained independent replication in the seminal tract. These results show the presence of genetic differences in both the donor and the recipient that affect the nature of the transmitted virus. This work represents a large collaborative effort within the CHAVI.

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HIV permeation efficiency in human cervicovaginal mucus

Justin Hanes Johns Hopkins University, 3400 N. Charles St., MD 221, Baltimore, MD 21218 HIV is transmitted sexually with unusually low efficiency (typically ,1 per ;1000 coital acts). To reliably infect a primate model for HIV, ;10,000-fold more virus must be delivered vaginally than intravenously. However, the vaginal mechanisms that help protect against HIV are poorly understood. Here we report that human cervicovaginal mucus (CVM), obtained from donors with normal lactobacillus-dominated vaginal flora, efficiently traps HIV, causing it to diffuse more than 1000-fold slower than it does in water. Lactobacilli acidify CVM to pH ;4 by continuously producing lactic acid. At this acidic pH we find that lactic acid, but not HCl, abolishes the negative surface charge on HIV. In contrast, in CVM neutralized to pH 6–7, as occurs when semen temporarily neutralizes the vagina, HIV maintains its native surface charge and diffuses rapidly, only 15-fold slower than in water. Thus, methods that can maintain the native CVM acidity during coitus may contribute to both vaginal and penile protection by trapping HIV before it can reach target cells. Our results reveal that CVM likely plays an important but currently unappreciated role in decreasing the rate of HIV sexual transmission.




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Reconstitution and Characterization of a Human Endogenous Retrovirus with the Original Sequence at the Time of Integration

Reinhard Kurth, Nadine Beimforde, Kirsten Hanke, and Norbert Bannert Robert Koch-Institute, Nordufer 20, D-13353 Berlin, Germany Human endogenous retroviruses (HERVs) are remnants of infectious exogenous retroviruses that invaded the germ line of our ancestors. These elements have since been transmitted vertically from generation to generation acquiring postinsertional mutations. In contrast to several other species, no infectious endogenous retroviruses have so far been identified in the human genome, although there is evidence for integration of active and replication competent HERV-K(HML-2) proviruses in our very recent evolutionary history and a still ongoing particle production in some tissues has been documented. The presence of multiple proviruses in the chromosomes and the expression of functional viral proteins seems to have various pathogenic and in some instances also beneficial implications for the host. To facilitate molecular and functional studies of HERV elements, its pathogenic potential and the human factors restricting their proliferation, we have cloned HERV-K113, one of the youngest and most complete of the HERV-K(HML-2) proviruses. Subsequently, we identified nonsynonymous postinsertional mutations in the HERV-K113 sequence by aligning it with other human specific HERV-K elements. These randomly acquired mutations were reverted by site-directed mutagenesis, reconstituting a provirus encoding proteins which the virus produced at the time of its integration. The properties of the viral proteins and of the generated viral particles as well as several aspects of its lifecycle will be discussed in the light of recent findings obtained by others using HERV-K consensus sequences.

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hBD2 inhibits HIV via CCR6, a receptor expressed on memory, Dendritic and Th17 cells

Mark K. Lafferty, Lingling Sun, Wuyuan Lu, and Alfredo Garzino-Demo Institute of Human Virology and Dept. of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore MD 21201 Human beta defensins (hBD) are antimicrobial peptides that are produced by epithelial cells, and are components of innate immunity in mucosae. hBD2 and 3 inhibit HIV infection. Based on the properties of these cationic peptides, which inhibit infectious agents by disrupting their negativelycharged membranes or to glycan moieties on their glycoproteins we expected that hBD2 would exert its HIV-suppressive activity by inactivating HIV, binding to its envelope. While we observed some degree of virus inactivation, we found that hBD2 inhibits HIV also after virus entry. Since hBD2 binds to, and triggers signaling via the chemokine receptor CCR6, we tested the hypothesis that CCR6 mediates the antiviral activity of hBD2. Three lines of evidence support this hypothesis. First, MIP-3a (CCL20), the chemokine ligand for CCR6, induces antiviral activity. Second, hBD2 loses efficacy when tested in CCR6- cells, both in single cycle and spreading infectivity assays. Third, Bordetella Pertussis toxin, an inhibitor of chemokine receptor –mediated intracellular signaling, abrogates the antiviral activity of hBD2 and MIP-3a. Our mechanistic studies show that HIV inhibition occurs at an early stage, prior to completion of reverse transcription. In summary, this is a novel antiviral activity mediated by a chemokine receptor. CCR6 is expressed in cells that are highly relevant to HIV infection, mucosally and in the periphery: memory CCR5+ T lymphocytes, dendritic cells and, based on our data, on LPS-activated macrophages. Interestingly, CCR6 is one of the markers for the Th17 subset of T lymphocytes. Published reports have shown that CCR6+ and Th17 cells are specifically lost in the course of progressive HIV disease or in primate models of AIDS. Thus, a mechanism that protects CCR6 cells from infection may yield novel preventive or therapeutic approaches to HIV infection.




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The anti-herpetic drug acyclovir suppresses HIV-1 in herpesvirus-infected human tissues after conversion into a nucleoside reverse transcriptase inhibitor

Andrea Lisco Section of Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, 10 Center Drive, Bldg.10, Room 9D58, 20892, Bethesda, MD Background: Ever-new, safe, efficient and inexpensive strategies against HIV are needed because of the increase of HIV-1 epidemics and the emergence of drug-resistant viruses. Such strategies have been developed against HSV, which can be efficiently treated with acyclovir (ACV). The anti-herpetic specificity of ACV is primarily based on the unique ability of HHV-encoded kinases to phosphorylate ACV to its monophosphate derivative, which is subsequently converted into the antivirally active ACVtriphosphate (TP). Consistently with its highly restricted anti-herpetic activity, ACV is not used as a direct HIV-1 inhibitor. Methods: We measured the effect of ACV-TP on HIV-1 reverse transcriptase (RT) activity in a cellfree system using a gel-based assay. Various HIV-1-infected human tissues were treated ex vivo with ACV and newly synthesized ACV-based prodrugs containing a masked phosphate group. We monitored viral replication in tissues and cell lines using real-time PCR and ELISA. Results: We demonstrate the direct inhibitory effect of ACV-TP, but not of ACV itself, on isolated HIV-1 RT. The kinetics of incorporation of ACV-TP by HIV-1 RT, evidence of its phosphorolytic excision, and the kinetics of formation and dissolution of a dead–end-complex with HIV-1 RT demonstrate that ACV-TP acts as a nucleoside RT inhibitor. Consequently, ACV suppresses HIV-1 replication in various human tissues and in T-cell line cultures carrying various HHVs that are capable of phosphorylating ACV. Furthermore, we demonstrate that monophosphorylated ACV-based prodrugs, which bypass the requirement for HHV-mediated phosphorylation, are able to inhibit HIV-1 replication irrespective of the presence of HHVs. Conclusions: In human tissues coinfected with various herpesviruses, ACV is activated into an HIV RT inhibitor that suppresses HIV-1. This newly discovered mechanism of HIV suppression by ACV may help to explain the results of several recent ACV trials in HSV-2/HIV-1-coinfected patients. The discovery of the antiretroviral activity of ACV-monophosphate prodrugs can promote the development of a new class of HIV-1 RT inhibitors.

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NK cells contribute to the constitution of HIV reservoirs in dendritic cells

Dr. Marie-Lise GOUGEON Institut Pasteur, 28 rue du Dr. Roux, Paris 75015, France Early stages of viral infections are associated with local recruitment and activation of effectors of innate immunity, i.e. NK cells and DCs. DCs are essential for both antigen-presentation and activation of na¨ve CD4+ T cells, and further Th1 polarization. DCs also constitute early targets for HIV. NK-DC crosstalk is important for DC homeostasis and maturation, but its contribution to susceptibility of DCs to infection, and to DC-dependent Th1 polarisation is unknown. We addressed this question in the present study. We report that HIV-infection of immature DC did not induce their maturation, as evaluated by the coexpression of CD86 and HLA-DR, while their coculture with activated NK cells did. NK-induced maturation of DC was inhibited by polyclonal antibodies specific for HMGB1 molecule or by glycyrrhizin, suggesting an important role of the pro-inflammatory cytokine HMGB1 in this process. Interestingly, activated NK were also able to polarize the production of IL-12 by DC that in turn induced a T helper 1-like immune response when cocultured with naive CD4+ T cells. In contrast, infected DCs were no more susceptible to NK-dependent IL-12 polarization, and thus no more able to induce a Th1 response. In addition, NK-dependent DC maturation was associated with an increased production of HIV-1 p24 and an increased expression of proviral DNA by DC. Altogether, our results show that activated NK cells 1- induce the maturation of DC through an HMGB-1-dependent mechanism; 2- trigger IL-12 production by DC and further Th1 polarization; 3- increase HIV production and the constitution of viral reservoirs in infected DC; 4- facilitate HIV-1 transmission from DC to T cells.




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HIV Vpu complexes with bTrCP to direct the degradation of the virus release inhibitor BST-2 (Tetherin)

Janet L. Douglas, Kasinath Viswanathan, Matthew N. McCarroll, Jean K. Gustin, Klaus Fruh, and Ashlee V. Moses Oregon Health & Science University-VGTI, 505 NW 185th Ave, Beaverton, OR 97006 The primary roles attributed to the HIV-1 Vpu protein are the targeted downregulation of the viral receptor CD4 and the enhancement of virion release. With regards to CD4 degradation, Vpu has been shown to act as an adaptor linking CD4 with the ubiquitin/proteasome machinery via interaction with the F-box protein bTrCP. This interaction was previously thought to be dispensable for Vpu’s virion release function. In an attempt to identify other cellular bTrCP-dependent Vpu targets, we performed quantitative proteomics on the plasma membrane fraction of Hela cells expressing either a wildtype Vpu, or a Vpu double point mutant (S52N/S56N) that no longer binds to bTrCP. We identified one cellular protein, BST-2 (CD317), that was significantly downregulated by wildtype Vpu, but not the mutant. Recently, BST-2 has been identified as the IFN-inducible cellular factor Tetherin, which limits HIV virion release in the absence of Vpu. We address here the mechanism of Vpu-mediated BST-2 downregulation. To verify the biological relevance of this phenotype, we show that in T-cells infected with HIV-1, BST-2 downregulation does indeed occur and is Vpu dependent. Interestingly, HIV-2, which does not encode Vpu, did not downregulate BST-2. Because our screen was designed to identify those proteins downregulated by Vpu in a bTrCP-dependent manner, we next sought to determine if Vpu targets BST-2 for degradation. We show via immunofluorescence and co-immunoprecipitations that both wildtype and mutant Vpu are able to interact with BST-2, but only wildtype Vpu results in BST-2 degradation. Further support for bTrCP’s role was provided by showing that the dominant negative mutant DF-bTrCP, which can no longer interact with the SCF ubiquitin ligase complex, blocks Vpu’s ability to downregulate BST-2. In addition, treatment of cells with either the proteasome inhibitor MG132 or the lysosome acidification inhibitor concanamycin A reduces BST-2 downregulation by wildtype Vpu. Taken together, these data support the hypothesis that, similar to its role in CD4 degradation, Vpu acts as an adaptor molecule linking BST-2 to the cellular ubiquitination machinery via bTrCP. This interaction then leads to the degradation of BST-2, which prevents viral tethering and therefore results in enhanced virion release.

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Molecular studies of HIV-1 Rev function

L. Gu, V. W. Gautier, N. Sheehy, T. Tsuji, H. Hayakawa, and W. W. Hall Centre for Research in Infectious Diseases (CRID), University College Dublin, Dublin 4, Ireland The HIV-1 regulatory protein Rev is critical for viral replication and modulates nuclear export of partially spliced and unspliced viral transcripts. Its function requires Rev to be actively imported and exported from the nucleus via nuclear pore complexes, a process mediated by recognition of its Nuclear Localisation Signal (NLS) domain by importin-b and its Nuclear Export Signal domain by CRM-1. Here we report that the human I-mfa domain-containing protein, HIC, modulates Rev nuclear localisation and function by physically interacting with its NLS domain, impairing nuclear import by rendering the NLS inaccessible to importin-b. Co-immunoprecipitation studies and GST- Pulldown assays demonstrated that HIV-1 Rev physically interacts with HIC via its NLS domain. Ectopic expression of HIC resulted in the cytoplasmic redistribution of Rev with a concomitant reduction in its nuclear accumulation. This correlated with a decrease in Rev activity as shown by the reporter gene assay. In vivo co-localisation studies in the presence of Leptomycin B suggested that HIC inhibits Rev nuclear import instead of promoting its nuclear export. This was subsequently confirmed by in vitro nuclear import assays, where competitive amount of recombinant 63His-HIC specifically inhibited Rev nuclear import by Importin-b. Functionally, the cytoplasmic sequestration of Rev may represent a novel mechanism for the control of Rev function and ultimately the regulation of HIV-1 replication, and may provide a new target for therapeutic intervention.




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CA-Dependent HIV-1 Nuclear Import Relies on Transportin-SR2

Kyeongeun Lee, Thomas D. Martin, Zandrea Ambrose, Alok Mulky, and Vineet N. KewalRamani HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702 We have previously shown that C-terminally truncated forms of cleavage and polyadenylation factor 6 (CPSF6), an SR family protein, interfere with infection by HIV-1 and SIV, but not MLV. HIV-1 entry and reverse transcription are not impaired in the presence of antiviral CPSF6, but nuclear forms of the vDNA are diminished. Strikingly, growth arrest of cells expressing antiviral CPSF6 intensifies the restriction of HIV-1 to over two orders of magnitude. A single amino acid change in HIV-1 CA, N74D, overcomes the restriction by antiviral CPSF6. While N74D HIV-1 can efficiently infect transformed cell lines and primary T cells in the presence or absence of antiviral CPSF6, this mutant virus is blocked at an early stage in the infection of macrophages. These data suggested that antiviral CPSF6 interfered with a nuclear entry pathway relevant to HIV-1 infection of primary target cells. To better understand how antiviral CPSF6 prevents HIV-1 nuclear entry, we examined whether a recently described HIV-1 dependency factor, TNPO3, identified in a genome-wide siRNA screen by Elledge and colleagues was relevant to infection by wild-type (WT) but not N74D HIV-1. Whereas siRNA knockdown of endogenous CPSF6 or its binding factor CPSF5 did not impair HIV-1 infection, knockdown of the transportin-SR2 spliced isoform of TNPO3 specifically restricted WT but not N74D HIV-1. Notably, growth-arrest of cells knocked down for transportin-SR2 intensified the block to WT HIV-1 infection, phenocopying the restriction by antiviral CSPF6. These data reinforce a role for transportin-SR2, a non-‘‘NLS’’ karyopherin, in the early replication of HIV-1 and suggest that its function may be particularly relevant for HIV-1 infection of nondividing cells. Our findings provide the first genetic evidence that HIV-1 CA regulates interactions with nuclear pore associated transport factors.

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118

Self-inactivation of HIV by its own RT/RNase H

K. Moelling, A. Matskevich, L. Wittmer-Elzaouk, J. Heinrich, J.-S. Jung,T. Kwok, and S. Mathur Section Viruses and Cancer, University of Zurich, Switzerland We have designed a method to inactivate HIV infectivity in the extracellular environment by activating the virion-associated Ribonuclease H. This enzyme has not been targeted for antiretroviral therapy. The effect is mediated by a short hairpin-loop DNA targeted to the highly conserved polypurine tract, the PPT (1–5). Treatment of HIV-infected serum isolated from patients and African strains showed about 100fold reduction of infectivity in 33% of the cases. A recombinant retrovirus was applied to the mouse vagina and showed a statistically significant reduction of viral RNA by prophylactic or therapeutic regimens of DNA application. Previously we showed in a retroviral mouse model that the viral load in the blood was reduced, disease progression delayed and infection prevented (6). We compared the effects of the hairpin-loop-structured DNA to antisense DNA and siRNA and find clear differences. The mechanism of uptake is independent of TLR9 and not yet understood. (1, 2) Jendis et al., AIDS Res. Hum. Retroviruses (1996, 98); (3) Moelling et al., FEBS Letters (2006); (4) Matskevich et al., AIDS Res. Hum. Retroviruses (2006); (5) Moelling et al. CSH Symp. 71 (2006); (6) Matzen et al. Nature Biotech. 23, 663 (2007).




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HIV-1 envelope gp120 induces a stop signal and virological synapse formation in non-infected CD4+ T cells

Gaia Vasiliver-Shamis, 1 Michael Cho, 2 Michael L. Dustin, 1 and Catarina E. Hioe3 1

Skirball Institute for Biomolecular Medicine, New York University School of Medicine; 2Case Western Reserve University School of Medicine; 3VA New York Harbor Health Care System and New York University School of Medicine HIV-1-infected T cells form a virological synapse (VS) with non-infected CD4+ T cells in order to efficiently transfer HIV-1 virions from cell to cell. The virological synapse is a specialized cellular junction that is similar in some respects to the immunological synapse involved in T cell activation and effector functions mediated by the T cell antigen receptor. The immunological synapse stops T cell migration to allow sustained interaction between T cell and antigen-presenting cells. In this study we have asked whether HIV-1 envelope gp120 presented on a surface to mimic an HIV-1 infected T cell also delivers a stop signal and if this is sufficient to induce a virological synapse. Using transmigration assay we demonstrate that HIV-1 gp120-presenting surfaces arrested the migration of primary activated CD4+ T cells that occurs spontaneously in the presence of ICAM-1. The VS dynamics were evaluated using real-time fluorescence imaging of non-infected CD4+ T cells on a glass-supported planar bilayer containing HIV-1 gp120 and ICAM-1. The data reveal for the first time high-resolution enface images of VS that is characterized by segregated supramolecular structures with a central cluster of HIV-1 gp120 surrounded by a ring of ICAM-1. Interestingly, unlike the immunological synapse that can be stable for .1 hour, the VS was formed transiently with initiation of T cell migration within 30 minutes. Both the stop signal and VS formation required HIV-1 gp120 interaction with CD4 but not the chemokine receptor. Thus, HIV-1 gp120-presenting surfaces induce a transient stop signal and supramolecular segregation in non-infected CD4+ T cells by a CD4-dependent manner. The HIV-1 gp120-induced transient arrest and VS assembly may constitute an efficient mechanism for virus transfer from infected cells to target cells without incurring cell-cell fusion that terminates the virus life cycle.

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Promoter-targeted shRNA driven by retroviral vector achieves long-term repression of HIV-1 replication

Makoto Yamagishi, Takaomi Ishida, Ariko Miyake, Kazuo Suzuki, and Toshiki Watanabe Laboratory of Tumor Cell Biology, Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minatoku, Tokyo 108-8639, Japan Regulation of gene expression by siRNA is a new therapeutic approach to a variety of viruses and related diseases. In this conference, we will report an improved procedure using an shRNA targeted to HIV-1 promoter region, which induces transcriptional gene silencing (TGS). Recently, some studies demonstrated that promoter-targeted siRNA induces TGS in mammalian cells. However, the mechanism of TGS remains unclear. When we use siRNA as a therapeutic tool against viruses, for example HIV-1, siRNA-mediated mRNA degradation can transiently suppress HIV-1. However, rapid emergence of ÔsiRNA escape mutantsÕ has been observed in vitro, since HIV-1 can quickly adapt to environmental pressures. In this study, we investigated the efficiency of inhibition and the mechanisms of TGS induced by shRNA-directed TGS of HIV-1 provirus integrated in Tcell. For this purpose, we prepared shRNAs homologous to HIV-1 U3 region and constructed retroviral expression vectors. Using these vectors, we established shRNA-expressing CD4 (+) T-cell lines. We measured the levels of HIV-1 replication and found that an shRNA targeting NF-kB binding site within LTR inhibited HIV-1 gene expression and its replication for unexpectedly long periods (more than 3 months) without escape mutants. Nuclear run-on assays revealed direct inhibition of transcription of HIV-1 genes, indicating that TGS was induced by this shRNA. Moreover, the shRNA inhibited reactivation of HIV-1 in latently infected cell lines. To elucidate the mechanism of TGS against HIV-1 promoter, we studied reactivation of HIV-1 replication by RT-PCR in cells where HIV-1 replication is suppressed by the shRNA, using HDAC inhibitor, TSA, or DNA demethylation agent, 5-AzaC. Furthermore, chromatin immunoprecipitation assays demonstrated induction of H3K27 trimetylation and subsequent facultative heterochromatin formation of shRNA-targeted promoter. These data suggested that shRNA targeted to HIV-1 promoter region induced TGS in an epigenetics-dependent manner, resulting in longterm suppression of HIV-1 replication.




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Plasmid DNA- and live viral vector-based vaccine approaches for the treatment and prevention of HIV infection

John H. Eldridge, PhD Profectus Biosciences, Inc. Almost two decades ago, it was first demonstrated that the direct injection of a plasmid DNA vaccine encoding a foreign antigen resulted in plasmid uptake, protein expression, and the induction of an antigen-specific cellular and humoral immune response. In the interim, the ability of DNA vaccine-elicited immune responses to protect against viral and bacterial infections, parasites, cancers, and autoimmune diseases has been well-documented in numerous animal models. Phase I human clinical trials have shown that experimental DNA vaccines are safe and well tolerated, however, these preliminary studies indicate that measures must be taken to improve vaccine immunogenicity. One particularly promising approach to improve the immunogenicity of DNA vaccines is through the codelivery of cytokine expression plasmids as genetic adjuvants. Studies in a variety of animal models clearly demonstrate that plasmid DNA encoded immunomodulatory cytokines (IL-12, IL-15) not only alter the magnitude and direction of the DNA vaccine-elicited immune response, but can also improve vaccine efficacy. Another approach to improve the immunogenicity of DNA vaccines is through the optimization of in vivo pDNA delivery. Recently, in vivo electroporation, i.e. the application of short electrical pulses, has been shown to enhance gene delivery and dramatically improve the induction of vaccine antigen-specific cell-mediated and humoral immune responses. Recombinant vesicular stomatitis virus (rVSV) vectors expressing HIV/SIV antigens elicit robust immune responses in rhesus macaques, significantly reduced viral load and alter disease pathogenesis following challenge with the simian-human immunodeficiency virus SHIV89.6P. However, when tested in a stringent non-human primate neurovirulence model, the lab-adapted rVSV vector appeared to be inadequately attenuated for use in humans. To address this concern, a variety of strategies were employed to produce more attenuated vectors, and these further attenuated vectors displayed markedly reduced neurovirulence in murine, ferret and non-human primate models, while retaining levels of immunogenicity equivalent to the more virulent rVSV lab-adapted vector. As a result of these efforts, a further attenuated replication competent rVSV clinical trial candidate with an appropriate safety profile that retains the ability to propagate to high levels in vitro and elicit robust immune responses in vivo has been identified. Collectively, improved pDNA expression vectors, co-delivery of plasmid-based immunomodulators and improved DNA delivery when used in combination with replication competent viral vectors in heterologus prime-boost vaccination regimens have the potential to bring the field closer to the design and development of an efficacious DNA vaccine for the prevention and/or treatment of HIV-1 infection.

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Differential CD4 T cell subset depletion at mucosal sites in HIV infection

Daniel C. Douek, MD, PhD National Institutes of Health, Vaccine Research Center, 40 Convent Dr., MSC 3022, Bethesda, MD 20892 Acute HIV infection is characterized by massive loss of CD4 T cells from the gastrointestinal (GI) tract. Th17 cells are critical in the defense against microbes, particularly at mucosal surfaces. We analyzed Th17 cells in the blood, GI tract and broncheoalveolar lavage (BAL) of HIV-infected and uninfected humans, and SIV-infected and uninfected sooty mangabeys. We found that (i) human Th17 cells are specific for extracellular bacterial and fungal antigens, but not common viral antigens; (ii) Th17 cells are infected by HIV in vivo, but not preferentially so; (iii) CD4 T cells in blood of HIV-infected individuals are skewed away from a Th17 phenotype towards a Th1 phenotype with cellular maturation; (iv) there is significant loss of Th17 cells in the GI tract of HIV-infected individuals (v) Th17 cells are not preferentially lost from the BAL of HIV-infected individuals and (vi) SIV-infected sooty mangabeys maintain healthy frequencies of Th17 cells in the blood and GI tract. These observations further elucidate the immunodeficiency of HIV disease and may provide a mechanistic basis for the mucosal barrier breakdown that characterizes HIV infection. Finally, these data may help account for the non-progressive nature of non-pathogenic SIV infection in sooty mangabeys.




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AIDS Restriction Genes-Genome Wide Association Study

Stephen J. O’Brien Laboratory of Genomic Diversity, NCI The screening of common genetic polymorphisms among candidate genes for AIDS pathology in HIV exposed cohort populations has led to the description of twenty AIDS Restriction Genes (ARGs), variants that affect susceptibility to HIV infection or to AIDS progression. All of these ARGs validated by our group have been discovered using the candidate gene approach. We have recently undertaken a genome wide association analysis (GWAS) for some 6500 study participants registered in longitudinal AIDS cohort populations using he Affymetrix 6.0 genotyping array which screens .900,000 human SNPs and as many features for copy number variation ( CNV) . The genetic associations were analyzed using both single monotypic AIDS association as well as multivariate hypothesis analyses using non-independent genetic association tests as internal replication . My presentation will demonstrate the gene discovery approaches and the results of a GWAS screen for undiscovered ARGs that were revealed by this GWAS.

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NK cells: friends or foes?

Galit Alter, 1 Daniel Kavanagh, 1 Suzannah Rihn, 1 Maureen Martin, 2 Mary Carrington,2 and Marcus Altfeld1 1

Partners AIDS Research Center and Infectious Disease Unit, Massachusetts General Hospital and Division of AIDS, Harvard Medical School Boston, MA; 2Basic Research Program, SAIC-Frederick, Inc., Laboratory of Genomic Diversity, Frederick, MD Decline of peak viremia during acute HIV-1 infection occurs prior to the development of vigorous adaptive immunity and the level of decline correlates inversely with rate of AIDS progression, implicating a potential role for the innate immune response in determining disease outcome. Natural Killer (NK) cells are critical both in the early control of various viral infections but also play a critical role in inducing a vigorous antiviral adaptive immune response. The combined expression of the activating NK cell receptor KIR3DS1 and its presumed ligand HLA-B Bw4-80I has been associated with slow progression to AIDS, however it is uncertain whether subjects with this genotype exhibit qualitatively different patterns of NK cell expansions and contractions during acute infection that may contribute to differences in patterning of the adaptive immune response. This presentation will highlight our recent findings demonstrating that NK cells play a role in both promoting pathogenic events as well as protecting against disease progression in the presence of protective KIR/HLA genotypes in HIV infection.




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Immunogenetic polymorphisms and their effects on AIDS progression

Xiaojiang Gao Cancer and Inflammation Program, SAIC-Frederick, National Cancer Institute, Frederick, MD, USA For the past 10 years we have been investigating the differential effects of genetic diversity on AIDS pathogenesis and HIV infection. In particular, we are interested in two highly polymorphic immunogenetic systems, namely human leukocyte antigen (HLA) and killer immunoglobulin-like receptor (KIR). The allelic polymorphism of HLA genes and chromosomal organization of KIR genes were characterized in multiple AIDS cohorts to examine their effects on AIDS progression and HIV-1 infection. HLA zygosity and three HLA-B alleles show a consistent association with AIDS: HLA-B*35Px shows a susceptible effect associated with a more rapid progression to AIDS, whereas HLA-B*27, B*57 and HLA heterozygosity show protective effects associated with a slower disease progression. Most immunogenetic effects are detected at the stage of CD4 T-cell depletion, but the B27 protection is most significant in the later stages of AIDS-defining illness. The same three HLA alleles are also associated with different risks for HIV transmission: hemophiliac patients with B*35Px were more likely to transmit HIV-1 to their female partners whereas patients with B*27 and B*57 were less likely to transmit the virus. The protective HLA alleles also correlate with a lower level of HIV viral loads compared to the susceptible alleles. Therefore, one mechanism to explain the observed HLA associations with AIDS is their influence on viral load control. The newly described KIR system is an important component of the innate immune mechanism. These polymorphic receptors regulate NK killing of abnormal cells through recognition of class I HLA expression. Therefore KIR may be involved in many of the diseases for which an HLA influence has been identified. Genetic epistasis between two functionally-relevant immunogenetic systems was observed: the combination of KIR3DS1 and its putative ligand HLA-Bw480I show a significant protection for AIDS progression, but either alone has no effect. Our results demonstrate that genetic polymorphisms of both acquired and innate immune systems influence AIDS pathogenesis and the disease progression. This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

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David Goldstein Abstracts not available at time of printing




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HTLV-I infection of WE17/10 CD4+ cell line leads to progressive alteration of Ca2+ influx that eventually results in loss of CD7 expression and activation of an antiapoptotic pathway involving AKT and BAD which paves the way for malignant transformation

H. Akl, B. Badran, G. Dobirta, N. E. Zein, F. Bex, C. Sotiriou, K. E. WillardGallo, A. Burny, and P. Martiat Laboratory of Experimental Hematology, Jules Bordet Institute, University of Brussels (ULB), Brussels, Belgium Adult T-cell leukemia/lymphoma (ATLL) is a malignancy slowly emerging from human T-cell leukemia virus type 1 (HTLV-I)-infected mature CD45 T-cells. To characterize the molecular modifications induced by HTLV-I infection, we compared HTLVI-infected WE17/10 cells with control cells, using micro-arrays. Many calcium-related genes were progressively downmodulated over a period of 2 years. Infected cells acquired a profound decrease of intracellular calcium levels in response to ionomycin, timely correlated with decreased CD7 expression. Focusing on apoptosisrelated genes and their relationship with CD7, we observed an underexpression of most antiapoptotic genes. Western blotting revealed increasing Akt and Bad phosphorylation, timely correlated with CD7 loss. This was shown to be phosphatidylinositol 3-kinase (PI3K)-dependent. Activation of PI3K/Akt induced resistance to the apoptotic effect of interleukin-2 deprivation. We thus propose the following model: HTLV-I infection induces a progressive decrease in CD3 genes expression, which eventually abrogates CD3 expression; loss of CD3 is known to perturb calcium transport. This perturbation correlates with loss of CD7 expression and induction of Akt and Bad phosphorylation via activation of PI3K. The activation of the Akt/Bad pathway generates a progressive resistance to apoptosis, at a time HTLV-I genes expression is silenced, thus avoiding immune surveillance. This could be a major event in the process of the malignant transformation into ATLL.

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Human domain antibodies against HIV-1 as exceptionally potent cross-reactive neutralizers

Weizao Chen, PhD CCR Nanobiology Program, NCI-Frederick, Bldg. 469, Rm. 144, Miller Drive, Frederick, MD 21702 HIV-1 has evolved to escape neutralization by antibodies generated by the immune system but not by antibody fragments of smaller size that could be able to gain access to the highly guarded conserved structures on the envelope glycoprotein (Env). Such small fragments targeting sterically restricted regions on the Env could exhibit neutralization activity superior to larger antibodies as has been demonstrated for Fab and scFv X5 which are significantly more potent than the full-size antibody (IgG1 X5). We have hypothesized that further decreasing the size of the antibody fragments to the smallest independently folded fragments, the antibody domains (about 10-fold smaller than an IgG, but maintaining high binding affinity) could lead to exceptionally potent and broadly cross-reactive neutralizers. To identify such fragments, we constructed a large (size 2.5 3 1010) highly diversified library of human antibody variable domains (domain antibodies, dAbs), and used it for selection of binders to conserved Env structures by panning sequentially against Envs from two different isolates, one of which was complexed with CD4. The highest affinity binder, m36, neutralized all tested HIV-1 primary isolates from clades A, B, C, and D with an activity on average higher than that of C34, a peptide similar to the fusion inhibitor T20 which is in clinical use, and that of m9 which is an improved derivative of scFv X5. Increasing the size by joining m36 to Fc with hinge regions of varying length or to Ch3 domains diminished its neutralizing activity likely due to the sterically restricted nature of its epitope. M36 is the first representative of a novel class of potent and broadly crossreactive HIV-1 inhibitors based on human dAbs. It has potential as a candidate therapeutic, and as an agent for exploration of the highly protected conserved Env structures with implications for the design of small molecule inhibitors and elucidation of the mechanisms of virus entry into cells. Key words: drug; domain antibody; entry; epitope; HIV-1; neutralization; steric restriction; therapeutic




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Human Endogenous Retrovirus-K (HML-2) Elements in the Plasma of People with Lymphoma and Breast Cancer

Rafael Contreras-Galindo University of Michigan, Department of Internal Medicine, Division of Infectious Diseases, 1150 West Me Medical Center Dr., 5240 MSRBIII, Ann Arbor, MI 48108 Actively replicating endogenous retroviruses entered the human genome millions of years ago and became a stable part of the inherited genetic material. They subsequently acquired multiple mutations, leading to the assumption that these viruses no longer replicate. However, certain human tumor cell lines have been shown to release endogenous retroviral particles. Here we show that RNA from the human endogenous retrovirus HERV-K (HML-2), a relatively recent entrant into the human genome, can be found in very high titers in the plasma of patients with lymphomas and breast cancer as measured by either RT-PCR or Nucleic Acid Sequence Based Amplification (NASBA). Further, these titers drop dramatically with cancer treatment. We also demonstrate the presence of reverse transcriptase and viral RNA in plasma fractions that contain both immature and correctly-processed HERV-K (HML-2) Gag and Envelope proteins. Finally, we show the presence of HERV-K (HML-2) virus-like particles in the plasma of lymphoma patients using immunoelectron microscopy. Taken together, these findings demonstrate that elements of the endogenous retrovirus HERV-K (HML-2) can be found in the blood of modern-day humans with certain cancers.

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Inhibition of HIV-1 Release by Cell Permeable Peptides

Sarah I. Daniels, David A. Davis, and Robert Yarchoan NIH, 9000 Rockville Pike, Bldg 10, Rm. 6N106, Bethesda, MD 20892-1868 Despite the effective use of highly active antiretroviral therapy to treat HIV infection viral, resistance occurs over time and therefore new strategies to treat HIV infection re necessary. In the process of developing peptide dimerization inhibitors of HIV-1 protease (Davis et al., Antiviral Research, 2006, 72, 88), we found that certain peptides, originally designed as dimerization inhibitors, were able to inhibit HIV-1 release. The peptide that was most effective at blocking dimerization, P27, revealed a block in virus release of HIV-1 as measured by p24 in the media of treated H9 cells with a corresponding increase in processed virus within the cells as evaluated by Western blot. P27 and analogs also blocked the release of virus from transfected 293T cells. In the transfected 293T cells, the virus did not accumulate significantly within the cells, but appeared to be degraded. The IC50 of blockage of wt HIV-1 release from 293T cells by P27 was approximately 1.0 mM, and no significant toxicity was seen at that concentration. P27 was designed to bind to the HIV protease dimer interface. The potency of various analogs of P27 on inhibition of protease activity and dimerization did not correlate to their potency on blocking virus release. Some peptides that were very weak at inhibiting dimerization were still quite effective inhibitors of virus release. Also, different plasmid constructs transfected in 293T cells showed that the inhibition of HIV-1 release was independent of the effects on HIV protease. Moreover, peptides could inhibit the release of viral-like particles from cells transfected with plasmid encoding only Gag. Characterization of P27 revealed that at least three of the four domains are required to block the release of wild type and protease mutant virus. Further studies will be required to understand the precise mechanism of action of these peptides and provide further insights into the pathways important for viral release.




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The gp41-derived immunosuppressive (isu) peptide of HIV-1 modulates cytokine release and gene expression in human immune cells

Magdalena Eschricht, Michael Lauck, Levent Akyu¨ z, Rayk Behrendt, Reinhard Kurth, and Joachim Denner Robert Koch Institute, D-13353 Berlin, Germany The transmembrane envelope protein gp41 of HIV-1 and a synthetic peptide corresponding to a highly conserved domain of gp41, the so-called immunosuppressive (isu) domain, have been shown to inhibit mitogen-triggered stimulation and to modulate cytokine production of human immune cells. The expression of cytokines such as IL-10, IL-6, IL-8, RANTES, MCP-1, MCP-2, TNF-alpha, MIP-1alpha, MIP1beta, MIP-3 increased upon exposure to gp41 or the corresponding isu-peptide. In contrast, the expression of IL-2 and MIG (CXCL-9) decreased. The modulation of cytokine expression induced by the isu-peptide is similar to the changes in cytokine expression in HIV-1 infected individuals. To analyse changes in gene expression in peripheral blood mononuclear cells (PBMCs) from healthy donors when incubated with the isu-peptide, a microarray analysis (AB1700, 29098 human genes) was performed. The microarray analysis confirmed the cytokine data and showed elevated expression of more than 400 genes, among them some of the above mentioned cytokines, MMP-1 (matrix metallopeptidase 1), TREM-1 (triggering receptor expressed on myeloid cells 1) and others. By real time PCR studies overexpression of these genes was confirmed and kinetics of the changes in gene expression were analysed. The changes in gene expression were also shown at the protein level using ELISA and Western blot analysis. In addition, preliminary data showed an elevated expression of FoxP3 and an increase in the number of CD4+CD25+FoxP3+ regulatory T cells. These data indicate that gp41 and the corresponding isu-peptide of HIV-1 modulate cytokine release and gene expression in normal human PBMCs.

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Argotom-VAX: A Proposed Investigational HIV DNA Vaccine Composed Of Selected Sequences Of Clade B Gag, Pol, Nef, Tat, Vif, And Env Linked To Lysosomal Membrane Associated Protein (LAMP)

William G. Hearl, PhD and J. Thomas August, MD (if available) Immunomic Therapeutics, Inc., 9290 Gaither Road, Gaithersburg, MD 20877 HIV infection results in a progressive loss of CD4+ cells and the immune response of the host. The aim of immune intervention in the chronic stage is to restore immunological competence of infected individuals, reduce viral load, and retard CD4 cell loss. It has been shown by extensive animal and human studies that the immunogenicity of the protein antigen can be enhanced by targeting the antigen as a lysosome associated membrane protein (LAMP) chimera to the major histocompatibility complex type 2 (MHC II) processing compartment of professional antigen presenting cells for enhanced presentation to CD4 cells. Each of the three plasmids of the vaccine product will contain three distinct domains of LAMP: (1) the highly glycosylated luminal domain with an N-terminal endoplasmic reticulum translocation signal; (2) the transmembrane domain, and (3) the cytoplasmic domain at the C-terminal. The antigen sequences are inserted between the luminal and transmembrane domains. Studies of HIV-1 DNA immunogen formulations have described an HIV-1 p55Gag DNA construct with strong, broad and poly-functional cellular and humoral immune responses in immunized mice when the Gag sequence was incorporated into the complete LAMP cDNA sequence. The LAMP/Gag chimera with the complete LAMP protein co-localized with the MHC II of transfected cells and elicited strong cellular and humoral responses in immunized mice compared to DNA-encoding native Gag, with a 4-to 5-fold increase in CD8+ T-cell response and antibody titers of .100,000. The proposed investigational vaccine, Argotom-VAX, is composed of three closed, circular supercoiled DNA plasmids containing the 8L-pCMV vector with three characterized nucleotide sequences that encode HIV antigens: LAMP/gag, LAMP/pol, and LAMP/NTVE. The complete LAMP protein will be used in each chimeric construct, designed for optimum protein expression and targeting to the major histocompatibility II (MHC II) compartment of selected sequences of HIV-1 Clade B proteins, p55 gag, pol, and a fusion of Nef-Tat-Vif-Env (gp41).




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O-linked N-Acetylglucosaminylation Represses HIV-1 Replication and Sp1-Mediated Trans-Activation of the HIV-1-LTR

Ramona Jochmann, Mathias Thurau, Elisabeth Naschberger, Susan Jung, Elisabeth Kremmer, and Michael Stu˜rzl Division of Molecular and Experimental Surgery, Department of Surgery, University of ErlangenNuremberg, Schwabachanlage 10, 91054 Erlangen, Germany The gene expression and replication of the human immunodeficiency virus-1 (HIV-1) is regulated by the promoter/enhancer located in the U3 region of the proviral 5’-long terminal repeat (LTR). Binding of the cellular transcription factor Sp1 to specific regulatory sites in the 5’-LTR is a key event in the replication cycle of HIV-1. O-linked N-acetylglucosaminylation (OGlcNAcylation) is a metabolismassociated highly dynamic and inducible posttranslational modification found on cytoplasmic and nuclear proteins in eukaryotes, catalyzed by O-GlcNAc transferase (OGT) and reversed by OGlcNAc hexosaminidase (O-GlcNAcase). Since the activity of Sp1 has been described to be regulated by O-GlcNAcylation, we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the OGlcNAcylation- enhancing agent glucosamine (GlcN) repressed viral replication in a dose dependent manner, and that this effect is due to a decreased activity of the LTR promoter. Overexpression of OGT specifically and dose dependently inhibited the activation of the LTR promoter containing the Sp1binding sites and strongly augmented the O-GlcNAcylation of Sp1. Knockdown of Sp1 by short hairpin RNA blocked OGT repression of the HIV-1-LTR promoter, and depletion of OGT by RNA interference completely restored the promoter activity. In contrast to the LTR promoter, OGT had no inhibitory effect on the Sp1-dependent control promoter EF1-alpha. Finally, gel shift assays demonstrated that O-GlcNAcylation does not alter the DNA-binding affinity of Sp1. From this study, we conclude that OGlcNAcylation of Sp1 represses trans-activation of the HIV-1-LTR promoter. OGT does not act as a general repressor of Sp1-triggered gene expression, but selectively inhibits the Sp1-mediated HIV-1LTR promoter activity. Thus, modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation, and may link viral replication to the metabolic state of the host cell.

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Cell-cycle dependent HIV-1 killing is mediated through the viral protease

Kyeongeun Lee, Taichiro Takemura, Yujing Xue, Alok Mulky, and Vineet N. KewalRamani HIV Drug Resistance Program, National Cancer Institute, Frederick, MD 21702, USA HIV-1 infection in vivo is characterized by the profound, cytopathic depletion of CD4+ helper T cells. Different mechanisms have been proposed to underlie HIV-1 cell killing. High multiplicity HIV-1 infection is thought to kill cells via toxic levels of unintegrated vDNA or lethal integration events. Killing by low multiplicity HIV-1 infection is thought to require the accumulation of toxic gene products. Using an HIV-1 vector that lacks several cytotoxic genes (vif, vpr, env) and can be stably transduced, we observed killing of human cells at infection frequencies as low as one hit per cell. Interestingly, slowly dividing cells were less rapidly killed after infection. Consistent with this observation, cells growth arrested with anti-proliferative drugs were not killed by HIV-1 vector infection. However removal of drugs and relief of the cell-cycle block rapidly led to death in the infected cells. HIV-1 vector expression was found to be required for the killing. A comparison of cells stably or acutely infected with the HIV-1 vector revealed higher viral protein expression during acute infection and more efficient proteolysis of Gag. Treatment of cells with HIV-1 PR inhibitors (PIs) alleviated killing during acute infection but did not reduce overall viral protein expression. PIs also protected initially growth-arrested HIV-1 vector infected cells after relief of the cell-cycle block. By contrast, HIV-1 vectors encoding drug-resistant PR killed dividing cells in the presence of PIs. Collectively, our data indicate that HIV-1 PR expressed from a single provirus is a cytotoxic hazard to proliferating cells.




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Dramatic enhancement of uptake and trafficking of HIV-1 Tat protein via modulation of endocytosis pathways

Guan-Han Li, Wenxue Li, Yan Huang, and Avindra Nath Johns Hopkins School of Medicine, Dept of Neurology, 600 N Wolfe St, Baltimore, MD 21287 HIV-1 Tat protein can be secreted from infected cells and taken up by other target cells. Tat uptake and trafficking has been widely investigated. This property is also exploited for drug delivery. We initially confirmed that Tat mainly entered Hela cells by clathrin-dependent endocytosis and was degraded in lysosomes, whereas chloroquine could inhibit the degradation and enhanced its trafficking to the nucleus. Interestingly, a similar effect was noted in presence of chlorpromazine (CPZ). Most likely, the uptake of Tat was shifted to caveolar endocytosis after the clathrin pathway was blocked by CPZ. However, the LTR transactivation of Tat could be enhanced by at least 50 fold when a liposome reagent was introduced into the Tat delivery system. Further evidence showed that liposome reagents could switch the Tat uptake from clathrin-dependent endocytosis to lipid raft pathway and bypass the degradation in lysosomes. This was supported by both pharmacological assays using inhibitors of caveolae-mediated endocytosis (MbCD, etc.) or microtubules (cytochalasin D & lutrunculin A) and co-localization using Tat101venus and immunstaining with anti-clathrin1, anticaveolin1, anti-EEA1, anti-Lamp1 or anti-Rab7. Subsequently, chemical molecules carrying ‘‘+’’ or ‘‘2’’ charges were tested for blocking the Tat uptake facilitated with the liposome reagent, including macromolecules (e.g., heparin, polybrene) and small molecules (e.g., spermine). Both ‘‘+’’ and ‘‘-’’ charged molecules could inhibit the uptake of Tat, but the ‘‘+’’ molecules actually attached to the membrane carrying ‘‘–’’ charges and the ‘‘–’’ molecules bound to the Tat-liposome complex with ‘‘+’’ charges. However, the Tat-liposome complex was probably formed by hydrophobic bonds linking liposome’s lipid to Tat hydrophobic domain because both Tat and the liposome reagent carried ‘‘+’’charges. A peptide containing partial hydrophobic domain of Tat (CFITKGLGISYGRKK) could competitively inhibit the uptake of Tat-liposome complex. In conclusion, the liposome reagent could facilitate Tat protein to traverse the membrane by changing endocytosis pathways and significantly enhance its delivery to the nucleus in a lysosome-independent manner. These observations have important implications for elucidating the mechanism of Tat uptake and trafficking and exploring drug delivery. Key Words: HIV-1 Tat; liposome; uptake

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Increased IL-15 production is associated with higher infection of memory CD4 T cells during acute SIV infection

Joseph Mattapallil F. W. Herbert School of Medicine, USUHS, 4301 Jones Bridge Road, Bethesda, MD 20814 Acute SIV infection is characterized by explosive infection of memory CD4 T cells in peripheral and mucosal tissues. Interestingly, relatively few memory CD4 T cells in either the mucosa or periphery of SIVinoculated rhesus macaques are infected until as late as day 7–8 after challenge. However, by day 10 pi most of the memory CD4 T cells in both tissues are infected and carry viral DNA. The rapid pace with which infection expands within 2–3 days to encompass virtually the entire memory CD4 Tcell compartment suggests significant alterations in the susceptibility of memory CD4 T cells to infection during this period. The mechanism(s) underlying this increased permissiveness for infection is not known. Here we show that IL-15 secretion significantly correlates with the upregulation of CD4 expression on memory CD4 T cells leading to their increased permissiveness to SIV infection. Activation and proliferation of memory CD8, but not memory CD4 T cells, preceded the amplification of viral infection that significantly correlated with IL-15 production. Though memory CD4 T cells did not express normal activation markers they displayed a significant upregulation in the density of CD4 expression between day 7 and 10 pi that correlated with increased infection of these cells. Culture of purified populations of CD4 T cells with IL-15 and/or SIV upregulated the expression of CD4 on these cells, and was accompanied by a significant increase in the level of infection in these sorted cells. Our results demonstrate that IL-15 contributes to the increased susceptibility of memory CD4 T cells to SIV during the early phase of acute SIV infection.




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Presence and role of HLA-C in HIV-1 infection

Andrea Matucci,1 Paola Rossolillo,1 Marco Turci,1 Pierpaolo Racchiolli,1 Antonio G. Siccardi,2 Alberto Beretta,2 and Donato Zipeto1 1

Section of Biology and Genetics, University of Verona, Italy and 2San Raffaele Scientific Institute, Milan, Italy

Background: A whole genome association study reported a SNP at -35Kb from the HLA-C gene strongly associated to HIV-1 viral set point and HLAC expression level (Fellay J. et al., 2007). HLA-C is not down-modulated by HIV-1 Nef and can be specifically incorporated in viral membrane enhancing infectivity and resistance to neutralizing antibodies (Cosma A. et al., 1999). We investigated the role of HLA-C in modulating HIV-1 infectivity using cell fusion and pseudovirus infection models and the interaction between HLA-C and Env at membrane level and in purified fusion complexes. Methods: Human cell lines expressing different HIV-1 gp120/gp41 were specifically silenced for HLA-C expression. Cells or pseudoviral particles were used for fusion and single-cycle infection analysis with TZM-bl cells. Fusion efficiency and viral infectivity were compared. Fusion complexes from fusing cells were purified and the molecular proximity of HLA-C and Env was analyzed by using BRET2. Results: The absence of HLA-C significantly decreased the fusion efficiency of cells expressing different R5 and X4 tropic gp120/gp41s. Similarly, pseudovirus infectivity was significantly reduced if they were produced on HLA-C silenced 293T cells. The X4 tropic NDK Env was insensitive to HLA-C at higher infectious doses. VSV-G pseudovirus used as control was not sensitive to HLA-C presence. HLAC could be detected associated to gp120 in cells taken before fusion, albeit a stronger association was evident during fusion process with target cells. Conclusions: The co-expression of HLA-C with X4/R5 HIV-1 Env increases the fusogenicity of cell lines and the infectivity of pseudotyped viruses. This data point to a specific interaction between HLAC and gp120 on the cell surface membrane. HLA-C and gp120 increase their association during fusion complex formation. This interaction can stabilize Env trimers, increasing the kinetic of conformational changes or the exposure of the receptor and/or coreceptor binding site favoring membrane fusion. Inhibiting the HLA-C/Env interaction might be of great importance in reducing virus infectivity and become the rationale for the development of new inhibitors of HIV-1 entry.

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Prevalence of Human Papillomavirus (HPV) Infection among HIV-positive women in the Pre-HAART and HAART era in a Nigerian Clinic

Dr. Olanrewaju Onigbogi Department of Community Medicine, University College Hospital, Ibadan Objectives: The prevalence of HIV infection has been on the increase in Nigeria in recent times. HIV-positive patients frequently have anogenital malignancies due to HPV. HAART was introduced in Anti-retroviral (ARV) centers in Nigeria in the year 2002. The aim of the study is to determine trends in incidence of anogenital malignancies among HIV-positive women undergoing treatment in the clinic in the pre-HAART and HAART era. Methods: A retrospective review of 541 case notes of HIV-positive female patients from January 1999 to December 2004 were analyzed by utilizing an on-going observational database at the ARV center. Rate ratios, comparing incidence rates (number of malignancies per 1000 person years) were calculated. Results Obtained: Twenty-four (4.43%) of the patients had one form of anogenital manifestation of HPV or the other. The incidence rate for HPV rose from 2.28 in the pre-HAART era to 6.40 in the HAART era (Rate ratio = 3.15; 95% confidence interval (CI) = 1.31 – 7.44; P = 0.0002). Conclusions: There has been a significant rise in the incidence of HPV since the introduction of HAART. This may be due to the longer survival of HIV-infected patients, surpassing the latency period for the anogenital malignancies. Care providers should be more vigilant for HIV-associated malignancies as patients live longer in this part of the world.




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HIV-2 Capsids Distinguishing High and Low Virus Load Patients in a West African Community Cohort

Clayton Onyango, 1 Aleksandra Leligdowicz, 1,2 Masaru Yokoyama, 4 Hironori Sato,4 Haihan Song,3 Emi Nakayama,3 Tatsuo Shioda,3 Assan Jaye,1 Hilton Whittle,1 Sarah Rowland-Jones,1,2 and Matthew Cotten1 1

MRC Laboratories, The Gambia; 2WIMM, John Radcliffe Hospital, Oxford, OX3 9DS, United Kingdom; Department of Viral Infections, Research Institute for Microbial Diseases, Osaka University, Japan; and 4 Laboratory of Viral Genomics, Center for Pathogen Genomics, National Institute of Infectious Diseases, Tokyo, Japan 3

HIV-2 causes AIDS similar to HIV-1; however, most HIV-2 infected patients show no disease and have low plasma virus load (VL). A detailed sequence analysis of HIV-2 p26 capsid (P26) variation in a community cohort was undertaken with the primary objective of detecting p26 variation that correlated with virus load. Activities: Sensitive methods were developed to generate virus sequences from high and low virus load subjects, providing information from both healthy and progressing HIV-2+ patients. Results: Three amino acid polymorphisms in p26 were identified that correlated significantly with VL. Proline residues at the three sites (PPP) were found more frequently in lower VL subjects while p26 with non-proline residues at all three sites (non-PPP) were isolated more frequently from high VL subjects. Structural modeling of these capsid variants revealed an increase in capsid dimer binding energies associated with the high VL (non-PPP) variants. In vitro virus replication supported the conclusion that these residues influence TRIM5a interaction, with PPP variants having increased susceptibility to TRIM5a restriction. The cellular immune responses to HIV-2 antigens in PPP patients were significantly higher than in non-PPP patients, indicating that capsid phenotype may also influence antigen presentation. Lessons: These results demonstrate that HIV-2 replication in vivo can be linked to p26 variation and provide a new basis for understanding HIV-2 pathogenesis.

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Risk factors associated with a high chance of mother to child transmission (MTCT) of HIV: a retrospective study of HIV-exposed infants/children in Kitwe, Zambia

Gilbert Siame Zambia Rural Healthcare Outreach Services (ZARHOS), Kitwe, Zambia C/O Theresa Chalwe, Jambo Flats No.11, Jambo Drvie, Parklands, Kitwe, Zambia Background: In the absence of intervention to curb transmission to children, studies suggest that the risk of transmission from an HIV infected mother to her child is 25–35%. In Zambia, about 112 new HIV infections in babies occur per day, of the 520,000 babies born annually. Objective: To identify risk factors that make a mother more likely to transmit HIV to her baby, and come up with recommendations to reduce MTCT of HIV and improve infant survival. Method: This was a retrospective study in which 100 case records of HIV exposed babies were reviewed. Polymerase Chain Reaction (PCR) to detect HIV DNA had been done in all cases. Results: From the 100 cases reviewed,13% of the babies were HIV positive. Of these positive babies, 69% were still b/feeding beyond 9 months. 54% were b/fed exclusively, while 15% were having mixed feeding. 62% of the mothers to these positive babies did not get any intervention to prevent MTCT(PMTCT). 23% of their mothers got SD NVP whereas 15% had AZT+SD NVP.As for the babies, 31% received SD NVP, while 7% got AZT+SD NVP. 85% of the babies’ mothers experienced an obstetric event with bleeding, with 31% of the positive babies asphyxiated. There was h/o untreated STI in 15% of the mothers, with more than half of the mothers (54%) having booked late in pregnancy. 31% of the mothers had CD4 counts ,200 cells/ml around time of conception, with 0% having done viral loads. Conclusion: 1) Vertical transmission (MTCT) is still the overwhelming source of HIV infection in babies, with low maternal immunity/poor clinical status, prolonged b/feeding, obstetric trauma, playing a major contributory role. 2) Women should be encouraged to do viral loads/CD4 counts prior to conception, and avoid pregnancy if viral loads .50 000 copies/ml or CD4,200 cells/mL. 3) Need to avail women in developing countries with appropriate and affordable alternatives to b/feeding. 4) Skilled health workers should attend to HIV positive pregnant women in labour to minimize adverse obstetric events such as trauma, hemorrhage, birth asphyxia, stillbirth. 5) PMTCT programs should be part of a broader health care strategy; they need to be holistic, requiring an expanded and strengthened prenatal, delivery and postnatal care programme. 6) Policy makers should address the cultural, legal and economic factors that make girls and women especially vulnerable to HIV infection.




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Type C Coping and Alexithymia are Associated Differentially with Specific Immune Mechanisms (Interleukin-6 and Beta-Chemokine Production) Linked to HIV Progression

Lydia R. Temoshok,1 Rebecca L. Wald,1 Stephen J. Synowski,1 Alfredo GarzinoDemo,1 and James Wiley2 1

Institute of Human Virology, University of Maryland School of Medicine, Baltimore MD, 2Public Research Institute, San Francisco State University, San Francisco CA

We hypothesized that b-chemokines MIP-1a/b which inhibit HIV entry into CD4+ T lymphocytes via the CCR5 coreceptor, and/or proinflammatory cytokines (Particularly IL-6) which activate HIV replication, may be mechanisms underlying our previously reported finding that higher Type C coping predicted HIV progression. The present study examined baseline relationships between Type C coping (which allows unrecognized, unexpressed emotions to remain as chronic stressors) and the adjacent personality construct of alexithymia (emotion processing/regulation deficits) and these immune mechanisms linked to HIV progression. Participants were 200 HIV+ outpatients (92% African-American, 53% male, mean age 45). Type C was assessed by the Vignette Similarity Rating Method; alexithymia by the Toronto Alexithymia Scale. In vitro production of IL-6 and MIP-1a/b was measured in response to Candida, PHA, and the HIV core protein p24. A Stimulation Index was defined as antigen-stimulated chemokine/IL-6 production compared to unstimulated controls. In regression analyses with independent variables Type C and the total alexithymia score, controlled for age, CD4+ count, antiretroviral medications, and adherence, higher Type C coping was significantly associated with IL-6 production stimulated by Candida (b =.207, P , 01), PHA (b = .182, P , 02), and p24 (b = .138, P = .075). Regression analysis with the same IVs revealed a significant negative association for MIP-1a; and alexithymia (b = 2.196, P = .008). To summarize: higher Type C coping is associated differentially with higher IL-6 production, which amplifies HIV replication; while higher alexithymia is associated with lower production of the anti-HIV chemokine MIP-1a, suggesting a role for psychoneuroimmunological processes in HIV progression.

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HIV Frequently Elicits Mucosal and Plasma EnvSpecific IgA With a Rapid Initial Decline In Acute Infection

Nicole L. Yates, Judith Lucas, Rob Parks, Vicki C. Ashley, Glenn Overman, David C. Montefiori, Kent J. Weinhold, Robin Shattock, Myron Cohen, Barton F. Haynes, and Georgia D. Tomaras, the CHAVI 001 Team Duke University Medical Center, MSRBII, Research Drive, Durham, NC 27710 An optimal HIV vaccine would elicit protective immune responses, preferably neutralizing antibodies, at mucosal surfaces in order to prevent infection. One key to eliciting vaccine-induced antibody responses at mucosal surfaces is to understand what antibodies are present at these sites soon after transmission during acute HIV infection (AHI). However, past attempts to detect HIV-specific IgA in mucosal specimens have been largely unsuccessful. The goal of this project is to characterize the binding antibody reponses elicited during AHI in plasma and mucosal (semen and cervicovaginal lavage (CVL)) samples compared to chronic infection with respect to antibody isotype, specificity, and timing in the CHAVI 001 acute infection cohort. To determine the presence of binding antibodies to HIV envelope proteins (gp140 consensus oligomers and recombinant gp41) and p55 gag protein, a standardized colorimetric ELISA as well as a customized Luminex assay were performed. Luminex is a bead-based flow cytometric assay that we customized to create a highly sensitive multiplexed assay for determining HIV-specific isotypes in plasma and mucosal specimens. Our results are the first to demonstrate the presence of HIV-specific IgA in approximately 88% of mucosal samples obtained during AHI, whereas approximately 33% of samples from chronically infected patients were positive for HIV-specific IgA. Each of the AHI patients had env-specific mucosal IgA, some of which mapped to the immunodominant region of gp41. High levels of HIV-specific IgG were also observed in the acute and chronic mucosal samples while HIV-specific IgM was undetectable. Furthermore, HIV env-specific plasma and mucosal IgA levels rapidly declined over time. In addition, in approximately 40% of AHI patients, systemic levels of p55 gag-specific IgA rose over the course of the infection despite the decrease in env-specific IgA. The rapid decline in HIV env-specific IgA suggests mucosal B cells may lose the capacity for env-specific IgA production during the course of AHI. Furthermore, the concurrent rise in systemic gag-specific IgA levels over time may be a reflection of HIV env-mediated differential regulation of IgA production in activated mucosal B cells. Future work includes functional studies to determine the role of HIV-specific IgA in AHI versus chronic infection as well as more epitope mapping to determine the specificity of isotypes in mucosal samples. These results have important implications in our understanding of how HIV infection regulates mucosal B cell responses and the barriers that need to be overcome by an HIV vaccine.




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Broad Spectrum Neutralizing Antibodies Against HIV-1 Elicited by Immunizing with Fusion Complexes

P. Rossolillo,1 A. Matucci,1 P. Racchiolli,1 L. Mainetti,2 S. Dispinseri,1 G. Scarlatti,2 and D. Zipeto1 1

University of Verona, Italy; 2DIBIT-HSR, Milan, Italy

Background: The development of neutralizing antibodies against HIV-1 is of pivotal importance for the development of an AIDS vaccine. We immunized mice with fusion complexes and elicited antibodies with neutralizing activity against heterologous HIV-1 isolates (Zipeto et al., Microb Infect 2006). Methods: We prepared murine hybridomas from mice whose sera showed the highest neutralizing activity. Their supernatants were tested for reactivity against cells expressing CD4 and CCR5, gp120/41, and the gp120/CD4 complex formed by capture ELISA. Hybridoma antibodies with no reactivity against HIV-1 receptors were selected. IgGs were purified and tested for their neutralizing activity (1–5 mg/mL) using both the TZM-bl neutralization assay (Li M. et al, J Virol 2005) and pNL43.Luc.R-E- based pseudoviruses on U87R5 cells with the standard group B Env panel. Specificity was tested against the VSV-G envelope protein. The neutralizing activity of selected antibodies was confirmed using the PBMC-based neutralization assay (Polonis VR et al, Virol 2008). Results: We screened 150 different hybridoma groups; 8% showed a neutralizing activity higher than 40% and 1 between 50 and 80% against the different pseudoviruses tested, similar or higher than the Tri-mAb positive control. Cells from this hybridoma group were cloned; 7% showed a neutralizing activity higher than 40% and 2% higher than 50%. Among the latter as many as 44% showed a neutralizing activity higher than 75% against the different pseudoviruses tested. Conclusions: Hybridomas produced from mice immunized with fusion complexes secrete antibodies neutralizing a panel of HIV-1 clade B Envs. We are screening monoclonal antibodies for their broad spectrum neutralizing activity against HIV-1. The isolation of such monoclonal antibodies will be of great interest for the development of an AIDS vaccine.

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In vivo and ex-vivo proteomics for target discovery in cancer

Giuliano Elia University College Dublin Conway Institute, Belfield, Dublin 4, Ireland There is an urgent need for more selective anticancer drugs. Targeted delivery of bioactive molecules to the tumor environment by means of binders (such as recombinant human antibodies and their fragments) specific to tumor-associated markers is a promising strategy towards this goal. This approach crucially relies on the availability of good quality, tumor-specific antigens and suitable high affinity ligands. Target proteins located around tumor blood vessels and in the stroma appear to be particularly suited for targeted anticancer strategies in view of their abundance, stability and accessibility by intravenously administered biopharmaceuticals. We have established ‘‘2D peptide mapping’’ as a new method for selective labeling, separation, relative quantification and identification of membrane proteins. This method is based on biotinylation of cell surface proteins, purification on streptavidin columns, elution, digestion with trypsin and peptide separation through reverse-phase HPLC. MALDI mass spectrometric profiles of each fraction can be displayed in two-dimensional maps that allow the immediate comparison of related samples and quantification of significant differences. We have applied this method to an in vivo proteomic approach for discovery of tissue specific targets accessible from circulation. We performed terminal perfusion of tumor-bearing rodents with a biotin derivative, which cannot diffuse through biological membranes. Comparative proteomic analysis by 2D peptide mapping revealed candidate targets differentially expressed in normal organs vs. experimental tumors. Finally, we have performed the ex vivo perfusion and biotinylation of whole, surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using 2D peptide mapping, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the antigens identified are indeed preferentially expressed in tumors.




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The V1/V2 Loop Region of Simian-Human Immunodeficiency Virus Envelope gp120 is the Major Determinant of the Strain Specificity of the Neutralizing Antibody Response

Melissa E. Laird, Tatsuhiko Igarashi, Malcolm A. Martin, and Ronald C. Desrosiers Harvard Medical School Plasma samples from individuals infected with human immunodeficiency virus type 1 (HIV-1) are known to be highly strain-specific in their ability to neutralize HIV-1 infectivity. Such plasma samples exhibit significant neutralizing activity against autologous HIV-1 isolates but typically exhibit little or no activity against heterologous strains. Monkeys infected with the simian-human immunodeficiency virus (SHIV) clone DH12 generated antibodies that neutralized SHIV DH12 but not SHIV KB9. Conversely, antibodies from monkeys infected with the SHIV clone KB9 neutralized SHIV KB9 but not SHIV DH12. To investigate the role of the variable loops of the HIV-1 envelope glycoprotein gp120 in determining this strain specificity, variable loops 1 and 2 (V1/V2), 3 (V3) or 4 (V4) were exchanged individually or in combination between SHIV DH12 and SHIV KB9. Despite the fact that both parental viruses exhibited significant infectivity and good replication in the cell lines examined, three of the ten variable loop chimeras exhibited such poor infectivity that they could not be used further for neutralization assays. These results indicate that a variable loop that is functional in the context of one particular envelope background will not necessarily function within another. The remaining seven replication-competent chimeras allowed for unambiguous assignment of the sequences principally responsible for the strain specificity of the neutralizing activity present in SHIV-positive plasma. Exchange of the V1/V2 loop sequences conferred a dominant loss of sensitivity to neutralization by autologous plasma and gain of sensitivity to neutralization by heterologous plasma. Substitution of V3 or V4 had little or no effect on the sensitivity to neutralization. These data demonstrate that the V1/V2 region of HIV-1 gp120 is principally responsible for the strain specificity of the neutralizing antibody response in monkeys infected with these prototypic SHIVs.

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146 a4b7: A Newly Discovered Receptor for HIV Anthony S. Fauci, MD National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD Infection with HIV-1 results in the dissemination of virus to gut-associated lymphoid tissue (GALT). Subsequently, HIV mediates massive depletion of gut CD4+ T cells, which contributes greatly to HIV-induced immune dysfunction. The migration to and retention of lymphocytes in GALT is mediated by integrin a4b7. We have demonstrated that the HIV envelope protein gp120 binds to an activated form of a4b7. This interaction is mediated by a tripeptide in the V2 loop of gp120, a tripeptide that mimics structures presented by the natural ligands (MadCAM, VCAM-1, and fibronectin) of a4b7. Engagement by gp120 of a4b7 on CD4+ T cells results in rapid activation of LFA-1, the major integrin involved in the establishment of virological synapses, which facilitate efficient cell-to-cell spread of HIV. These findings may have important implications in the further delineation of the pathogenic mechanisms of HIV infection.




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Hepatitis C and cancer

Michael Houghton, PhD Epiphany Biosciences Inc., San Francisco The hepatitis C virus (HCV) is a positive-stranded RNA virus classified within the Flaviviridae family. It infects 3-4 MM individuals in the USA alone and an estimated 170 MM worldwide. Around 75% of acute infections develop into life-long infections which are associated with varying levels and types of liver and other diseases. While the majority of infected individuals only have mild forms of hepatitis, some can develop chronic lobular hepatitis that then proceeds sequentially through chronic active hepatitis, liver fibrosis, liver cirrhosis and primary hepatocellular carcinoma. The latter is always preceded by cirrhosis suggesting that the continually-inflamed liver may itself be a direct cause of tumorigenesis .However, certain HCV gene products may also play a role since the nucleocapsid and nonstructural proteins 3 & 4 have been shown to induce tumorigenesis when overexpressed in various cellular and animal models. HCV-associated extrahepatic diseases include mixed cryoglobulinemia, membranoproliferative glomerulonephritis, porphyria cutanea tarda and there is some association with non-Hodgkin’s B cell lymphoma. The latter may be mediated by the engagement of the HCV receptor, CD81 by envelope glycoprotein 2 within circulating HCV virions. Many co-factors of HCV-associated disease are known including gender (with the incidence of hepatocellular carcinoma being lower in females), alcohol use, duration of infection and co-infection with other hepatotropic viruses as well as co-infection with HIV. Individuals co-infected with HIV & HCV often develop severe liver disease. Vaccine strategies to prevent the development of persistent infection are now looking promising. Vaccines eliciting cross-neutralising antibodies or cross-reacting cellular immune responses in nonhuman primates are able to prevent chronic infection following experimental challenge with heterologous viral strains thus mirroring the correlates of protection in humans that are able to spontaneously eradicate acute HCV infection. Standard-of-care therapy comprises pegylated alpha-interferon along with ribavirin, a mixture that is thought to have direct antiviral and immunomodulatory activity. Patients with chronic hepatitis and even liver cirrhosis can be normalized by this therapy with virus eradication, although 12 months of treatment are required for the common genotype 1 infections and even then, less than 50% of patients are permanently cured. However, progress is occurring through the use of more specific antivirals that inhibit the viral serine protease, the viral replicase and other targets like nonstructural protein 5a that plays an essential role in virion production. Therefore, we can anticipate a combination therapy to emerge within the next decade that will cure the large majority of HCV patients and a vaccine that could prevent the chronic sequelae of the estimated 25,000 new infections still occurring annually in the USA alone.

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Towards a genetics of cancer resistance

George Klein Microbiology and Tumor Biology Center (MTC), Karolinska Institute, Stockholm, Sweden Approximately two of three humans never develop cancer. Not even the majority of heavy smokers get cancer. Are there cancer resistant genotypes? Mice can be readily selected for low cancer incidence by selective breeding. Feral-derived mice, e.g. M.spretus are highly cancer resistant. Among known cancer protection mechanisms, DNA repair, the stringency of epigenetic imprinting, apoptotic propensity and immune response to virus associated tumors are influenced by genetic variation. Contactual inhibition of tumor by adjacent normal cells may reflect a form of microenvironmental control that could be responsible for preventing the cancerous progression of initiated cells, the growth of disseminated and dormant tumor cells, and the progression on incipient neoplastic foci. This mechanism has not been studied for genetic variation. A high throughput microwell system has been developed in our laboratory that may be suitable for such studies. In the field of somatic cell genetics, our studies on monochromosomal microcell hybrids have revealed the existence of ‘‘asymmetric’’ tumor suppressor genes that can inhibit tumor growth in vivo but do not influence in vitro growth. This study may open the way towards the exploration of cellular receptors for contactual control.




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The role of the microenvironment in EBV associated lymphoid malignancies

Eva Klein Department of Microbiology, Tumor and Cell Biology, Karolinska Institute, 17177 Stockholm Sweden Depending on the differentiation and maturation of EBV-carrying cells, virally encoded proteins are expressed in various combinations. These proteins determine the fate of the viral genome harbouring cells. Virus transformed B lymphocytes - lymphoblastoid cell lines- LCL- express six virally encoded nuclear and three surface localised proteins. This phenotype is encountered only in B lymphocytes and it is associated with cell proliferation. It is usually referred to as Type III EBV expression or growth transformation program. Such cells are readily recognized by the immune response. Their proliferation therefore is inhibited in healthy individuals. In immunosupressed patients this inhibition is impaired and EBV associated haematological malignancies may arise. The presence of the EBV genome in lymphocytes with a more restricted viral protein expression, like the type II pattern that lacks the nuclear protein EBNA-2, does not induce proliferation. However it may affect the behaviour of the cell within the dynamics of the immune system. Such cells may avoid apoptosis, they may induce an enrichment of inflammatory cells and/ or may respond to intercellular contacts or to cytokines with proliferation. This may lead to malignancy. There is evidence for such mechanisms in EBV associated Hodgkin´s lymphoma and nasal NK lymphoma.

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HIV and cancer: current trends and insights

Eric A. Engels National Cancer Institute, 6120 Executive Blvd, Room 7076, Rockville, MD 20852 HIV-infected individuals have an elevated risk for cancer. This high risk partly arises from HIVinduced immunosuppression, and partly from a high prevalence of cancer risk factors such as tobacco use and infections with oncogenic viruses. HAART, widely available since 1996, can result in substantial improvements in immune status and has led to declines in AIDS incidence and mortality. Monitoring of cancer trends in HIV-infected persons in the U.S. has revealed population-level declines in the incidence of Kaposi sarcoma and non-Hodgkin lymphoma, to some extent attributable to introduction of HAART in 1996. Of interest, Hodgkin lymphoma incidence has risen during the HAART era. Hodgkin lymphoma risk is related to CD4 count in a non-linear manner, declining at the lowest CD4 counts, and the rise in Hodgkin lymphoma incidence may reflect effects of HAART (i.e., a shift in immune function to a mid-range with highest Hodgkin lymphoma risk). Among cancers caused by human papillomavirus, cervical cancer incidence has remained steady, but incidence of anal cancer has increased over time. Lung cancer is the most common non-AIDS-defining cancer in HIV-infected persons. This reflects very high smoking rates, but lung cancer incidence appears greater than can be attributed to smoking alone. The high risk of lung cancer in HIV-infected persons may be due to repeated lung infections or chronic pulmonary inflammation, in concert with tobacco use. Incidence of non-melanoma skin cancers is also elevated. Data on basal cell and squamous cell skin carcinomas are limited, but risk is strongly increased for Merkel cell carcinoma and sebaceous carcinoma, two rare types of skin cancer. A novel polyomavirus was recently discovered in Merkel cell carcinoma. Trends in cancer incidence in HIV-infected individuals reflect the effects of HAART as well as other factors. Studies of cancers that arise at increased frequency in this population may reveal novel biological insights.




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MicroRNAs and the biology of KSHV infection

Don Ganem HHMI, UCSF San Francisco, CA 94143 Pre-miRNAs as part of its latency program; these are processed to yield 17 mature viral miRNAs. The functions of most of these miRNAs are unknown. To address this issue, we have developed an expression- based screen for candidate cellular mRNA targets of each of these miRNAs. Here, we report the results for one such miRNA, miR-K5. Following ectopic expression of individual miRNAs, expression profiling was carried out, looking for mRNAs whose abundance declines in the presence of the miRNA; in addition, we transfected latently infected cells with antisense antagonists of each miRNA, then looked for transcripts whose level rose in response to miRNA inhibition. Transcripts passing both sets of tests were then examined for seed sequence homologies in their 3’UTR. For miRK5, the leading candidate to emerge was the mRNA encoding the protein BCLAF1. This was validated as a target by extensive formal genetic tests. Additional testing revealed that BCLAF1 was also targeted by at least 2 other KSHV miRNAs, pointing to an important role in the life cycle. Inhibition of BCLAF1 by siRNA phenocopied the expression of KSHV miRNAs, and resulted in enhanced spontaneoud lytic reactivation. We conclude that KSHV miRNA-mediated modulation of BCLAF1 promotes the reversibility of latent infection, thereby preventing latency from becoming a dead-end pathway from which virus cannot escape.

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HIV-ASSOCIATED LYMPHOMAS — Focus on unusual lymphomas occurring specifically in HIV-infected patients

Antonino Carbone Fondazione IRCCS Istituto Nazionale Tumori – Via G. Venezian, 1 – 20133 Milano, Italy Lymphomas that develop in HIV-positive patients are predominantly aggressive B-cell lymphomas. These disorders include the same lymphomas that develop sporadically in the absence of HIV infection and those seen much more often in the setting of HIV infection. The most common HIV-associated lymphomas are Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) (also involving the central nervous-system-CNS). Classic Hodgkin lymphoma (HL) is also increased in the setting of HIV. Lymphomas occurring specifically in HIV positive patients include primary effusion lymphoma (PEL) and its solid variants, plasmablastic lymphoma of the oral cavity type and lymphoma associated with HHV8-related multicentric Castleman disease. These lymphomas together with BL and immunoblastic lymphoma subtypes with plasmacytoid differentiation carry EBV infection and display a phenotype related to plasma cells. EBV is identified in the neoplastic cells of approximately 40% of HIV-associated lymphomas, but the detection of EBV varies considerably with the site of presentation and histological subtype. EBV infection occurs in 80-100% of primary CNS lymphomas and PELs, 80% of DLBCLs with immunoblasticplasmacytoid features, and 30-50% of BL-plasmacytoid. Nearly all HL cases in the setting of HIV infection are associated with EBV. KSHV/HHV8 is specifically associated with PEL, which usually occurs in the late stages of disease, in the setting of profound immunosuppression. The current knowledge about HIV-associated lymphomas can be summarized in the next essential points: 1) lymphomas specifically occurring in patients with HIV infection are closely linked to other viral diseases; 2) most of these lymphomas exhibit plasmablastic differentiation; and 3) among HIV-associated lymphomas the incidence of classic HL is increasing in the setting of improved immunity.




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Insight into the molecular mechanism of CIITAmediated inhibition of HIV-1 and HTLV transactivators

Giovanna Tosi, 1 Chiara Orlandi,1,4 Luisa Bozzo, 1 Elisabetta Pilotti,2 Claudio Casoli,3 and Roberto S. Accolla1 1

Dept. of Clinical and Biological Sciences, University of Insubria, Varese, Italy; 2Dept. of Clinical Medicine, Nephrology and Health Sciences, University of Parma, Parma, Italy; 3Dept. of Clinical Sciences L. Sacco, Infectious Diseases and Immunopathology Section, University of Milan, Milan, Italy; and 4PhD, Program in Experimental Medicine and Oncology, University of Insubria, Varese, Italy We previously showed that CIITA, the master regulator of MHC-II genes transcription, acts as a potent inhibitor of HIV and HTLV-2 replication by suppressing the activity of their respective viral transactivators Tat and Tax-2. Here we show that CIITA inhibits also the activity of Tax-1 the transcriptional activator of HTLV-1, the aetiological agent of Adult T cell Leukemia (ATL). Interestingly, both Tax-2 and Tax-1 are inhibited by the same region of CIITA that we have now restricted to 60 aa at the N-term of the molecule. This region is distinct from the one inhibiting the HIV-1 Tat transactivator. Thus, our hypothesis is that Tax-1 and Tax-2 transactivators are inhibited through a common molecular mechanism which is different from the one inhibiting HIV-1 replication. Our previous results have shown that another transcription factor, NF-Y, which cooperates with CIITA in the activation of MHC-II genes transcription, inhibits Tax-2 activity when over expressed in cells. A similar effect is observed also with Tax-1 and we are assessing whether NF-Y is involved in CIITA-mediated inhibition of Tax-1 and Tax2. We show that Tax-1 interacts with NF-YB subunit and report on a new interaction between CIITA and both Tax-1 and Tax-2 in vivo. Studies are also in progress to determine whether the suppressive effect of CIITA on Tax-1 correlates with the inhibition of HLTV-1 replication as well. These findings reveal that CIITA, beside its well known role in adaptive immunity has an important function in innate immunity, counteracting retrovirus replication and spreading.

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Apobec3: A Modern Twist to A Classic Retroviral Mystery

Mario L. Santiago,1 Mauricio Montano,1 Robert Benitez,1 Ronald J. Messer,3 Wes Yonemoto,1 Bruce Chesebro,3 Kim J. Hasenkrug,3 and Warner C. Greene1,2 1

Gladstone Institute of Virology and Immunology, San Francisco, CA 94158; 2Department of Medicine, University of California, San Francisco, CA 94143-1230; and 3Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, Hamilton, MT 59840 Recovery from Friend Virus 3 (Rfv3) corresponds to a single autosomal gene encoding a resistance trait that promotes the survival of mice infected with the Friend Virus complex (FV). Rfv3 acts by promoting the production anti-FV neutralizing antibodies and by controlling FV viremia. Rfv3 was discovered 30 years ago but its genetic identity has remained unknown. We now demonstrate that Rfv3 is encoded by the Apobec3 gene. Apobec3 maps to the same region of chromosome 15 as Rfv3. Of note, the Apobec3 family of deoxycytidine deaminases are known to exert inhibitory activity against many retroviruses including HIV. Genetic inactivation of Apobec3 converts Rfv3-resistant mice to a susceptible phenotype characterized by diminished neutralizing antibody titers and increased viremia. Furthermore, while Rfv3 susceptible mice contain an Apobec3 gene and produce Apobec3 mRNA, we find that Apobec3 is naturally disabled in Rfv3-susceptible mice by aberrant mRNA splicing resulting in the removal of exon 2 sequences. This newly discovered link between Apobec3 and neutralizing antibody responses highlights an Apobec3-dependent mechanism of host protection that might extend to HIV. We speculate that the ability of HIV Vif to induce degradation of Apobec3G and 3F during acute infection could contribute to the poor neutralizing antibody response observed HIV infection.




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Tetherin, an interferon-alpha induced inhibitor of retrovirus release that is antagonized by Vpu

Paul D. Bieniasz Aaron Diamond AIDS Research Center and Laboratory of Retrovirology, The Rockefeller University, New York, NY 10016 The HIV-1 Vpu protein overcomes the action of an interferon-regulated antiviral restriction factor, termed tetherin, which causes the retention of virions on infected cell surfaces. Using comparative gene expression analysis and deductive constraints, we identified tetherin as a membrane protein with an unusual topology consisting of a N-terminal cytoplasmic tail, a transmembrane domain, an ectodomain predicted to form a coiled-coil, and anchored at the C-terminus by a putative GPI-linkage. Tetherin expression is necessary and sufficient to restrict the release of Vpu-defective HIV-1 from human cells. Tetherin induces dramatic accumulations of mature HIV-1 virions on the cell surfaces and, following endocytosis, in late endosomes. To understand mechanisms by which tetherin might function, we examined its ability to inhibit the release of a variety of virions or virus like particles (VLPs) derived from retroviruses that share little sequence homology. Notably, tetherin appears capable of restricting the release of every retrovirus that we have tested. Additionally, tetherin inhibited the release of VLPs assembled by expression of the Ebola virus matrix protein, suggesting that its activity against enveloped viral particles might be very broad indeed, and may simply involve crosslinking of lipid bilayers. Immunofluorescence and electron microscopy studies indicate that tetherin localizes to nascent tethered virions, consistent with the notion that it is a physical component of the tethers that retain virions on cell surfaces. We have cloned orthologs of tetherin from rhesus macaque, African green monkey and mouse. These non-human tetherins are inhibit HIV-1 release, but in contrast to the human protein, are not antagonized by Vpu. Determinants of Vpu sensitivity in tetherin reside in the transmembrane domain and these findings suggest that HIV-1 Vpu has specifically evolved to antagonize the human tetherin protein. Notably, Vpu only modestly affects tetherin expression levels, but abolishes tetherin’s to localize with nascent virions, suggesting that Vpu functions by sequestering tetherin from sites of virus particle assembly.

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Humanized Mouse Models for The In Vivo Analysis of HIV Infection

J. Victor Garcia-Martinez University of Texas Southwestern Medical Center, at Dallas HIV is predominantly transmitted by unprotected sexual contact. Currently, women worldwide account for more than half of the estimated 11,000 newly acquired infections every day with a majority of those transmissions occurring via the vaginal route. However, of the estimated 341,524 male adults and adolescents living with HIV/AIDS in the US, 61% had been exposed through male-to-male sexual contact. If untreated, the outcome of HIV infection is a systemic depletion of CD4+ T cells leading to immunodeficiency, opportunistic infection or neoplasm resulting almost invariably in death. ART blunts virus replication resulting in dramatic decreases in the levels of plasma viremia to undetectable levels by standard clinical assays. This drop in plasma viremia is accompanied by increases in the level of peripheral CD4+ T cells that remains relatively constant for the duration of ART. However, despite this apparent disappearance of virus from peripheral blood, infected cells remain and therapy interruption usually results in viral rebound to levels that are similar to those observed pre-treatment. In order to advance our overall understanding of HIV and AIDS we developed and implemented a novel small animal model where human stem cells are used to reconstitute the hematopoietic system of immunodeficient mice. In these humanized mice (designated BLT to represent the fact they are generated from a bone marrow transplant of mice previously implanted with a piece of autologous human fetal liver and thymic tissue) there is systemic reconstitution with human lymphoid cells in all hematopoietic and non-hematopoietic tissues tested. These humanized mice are susceptible to infection with HIV-1 administered intravenously, intrarectally or intravaginally. Infection results in plasma antigenimia, development of anti-HIV specific human antibodies, progressive depletion of human CD4+ cells from the peripheral blood as well as systemic CD4 depletion especially in the gut associated lymphoid tissue (GALT). This model presents unique opportunities to evaluate novel therapeutic interventions, to advance our understanding of HIV pathogenesis and to evaluate novel approaches aimed at virus eradication.




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Vpr as a mediator of proteasomal degradation and other functions

Carlos de Noronha Albany Medical College MC151/MS216, 47 New Scotland Avenue, Albany, New York 12208 Vpr is a 15 kDa HIV1 accessory protein that promotes infection of terminally differentiated macrophages and blocks the cell cycle of dividing cells in the G2 phase. Interestingly, HIV2 and SIVmac/sm encode two Vpr-like proteins, Vpx and Vpr that perform these functions separately. Our goal is to discover the mechanisms of Vpr action and their role in HIV pathogenesis by identifying the cellular protein partners of Vpr. Work from our lab and others revealed that Vpr from HIV1 or 2 blocks cell cycle progression by engaging a cellular ubiquitin ligase complex. The complex, characterized by inclusion of the proteins DCAF1, DDB1 and cullin4A must be functional for Vpr-triggered G2 arrest. We hypothesize that a protein required for cell cycle progression is recruited to the complex by Vpr, ubiquitinated, and thereby blocked, redistributed or degraded. Subsequent work from other labs demonstrated that both SIV mac/sm and HIV2 Vpx also act through the same ubiquitin ligase complex to overcome an innate block to macrophage infection that interferes with reverse transcription. Our work to identify targets of Vpr-mediated ubiquitination revealed that HIV1 Vpr and HIV2 Vpx both engage rpS3, a protein that is a ribosomal component, an endonuclease and a subunit of NF-kB complexes that act on specific promoters. Previous work showing that Vpr can both stimulate and block NF-kB action lead us to hypothesize that Vpr modulates specific NF-kB function through its interaction with rpS3. We found that expression of HIV1 Vpr and HIV2 Vpx but not HIV2 Vpr inhibits NFkB reporter activity in response to TNF-a and reconfirmed that siRNA-mediated rpS3 depletion has a similar effect. We are currently working to determine whether Vpr blocks NF-kB function by modulating free rpS3 levels, whether the block is specific to the subset of genes that are regulated by the rpS3-containing NF-kB complexes and whether activity of the DCAF1/DDB1/Cullin4A ubiquitin ligase complex is required for this Vpr function.

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The SET complex acts as a barrier to autointegration of HIV-1

Nan Yan,1 Peter Cherepanov,2 Janet E. Daigle,3 Alan,3 and Judy Lieberman1 1

Immune Disease Institute; 2Dana Farber Cancer Institute, Harvard Medical School; and 3Division of Medicine, Imperial College London Retroviruses and retrotransposons are vulnerable to a suicidal pathway known as autointegration, which occurs when the 3’-ends of the reverse transcript are activated by integrase and then attack sites within the viral DNA. Retroelements have diverse strategies for suppressing autointegration, but how HIV-1 protects itself from autointegration is not well understood. We found that the SET complex, an endoplasmic reticulum-associated complex that contains the base excision repair (BER) endonuclease APE1 and is mobilized to the nucleus in response to oxidative stress, acts as a barrier to autointegration. Antibodies to SET complex proteins capture HIV-1 DNA in the cytoplasm. Knocking down any of the SET complex proteins, as well as upstream and downstream BER enzymes, increases autointegration and decreases chromosomal integration. The SET complex likely acts to inhibit autointegration via its presumed role in BER since enhancing uracil misincorporation by culturing cells in the presence of added dUTP (or BrdU) or by infection with viruses encapsulating the cytidine deaminase APOBEC3G decreases autointegration in a SET complex-dependent manner.




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Finding Host Proteins Required for HIV Replication

Abraham Brass Massachusetts General Hospital HIV-1 exploits multiple host proteins during infection. Each host protein that HIV relies on is a potential viral weakness, that once discovered represents a therapeutic target, both for prophylaxis and treatment. We performed a large-scale siRNA screen to identify host factors required by HIV, and identified more than 200 new, and 38 previously known, HIV-dependency factors (HDFs). These proteins participate in a broad array of cellular functions and implicate new pathways in the viral lifecycle. Further analysis revealed previously unknown roles for retrograde Golgi transport proteins (Rab6 and Vps53) in viral entry, a karyopherin (TNPO3) in viral integration, and the Mediator complex (Med28) in viral transcription. This effort begins to illustrate the power with which RNA interference and forward genetics can be used to expose the dependencies of human pathogens such as HIV, and in so doing identify potential targets for therapy.

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Polarization of M1 and M2 macrophages and HIV infection

Guido Poli DIBIT, Via Olgettina n. 58, 20132, Milano, Italy Permissiveness of macrophages to productive HIV-1 infection is modulated by various cytokines. Here, we demonstrate that stimulation of primary human monocyte-derived macrophages (MDM) with pro- (M1) and anti- (M2) inflammatory cytokines, i.e. TNF-a plus IFN-g vs. IL-4, generates two clearly distinct populations of macrophages that differ in their ability to support CCR5-dependent (R5) productive HIV-1 infection. Viral suppression was more profound but less sustained in M1- compared to M2-MDM. M1 polarization was associated with a marked down-regulation of CD4, increased secretion of CCR5-binding chemokines (i.e. MIP-a/CCL3, MIP-1a/CCL4, RANTES/CCL5) and .90% decrease in HIV-1 DNA accumulation, suggesting inhibition of virion entry. Despite reduced virus production in M2-MDM, CD4 was only moderately downregulated, there was no upregulation of CCR5-binding chemokines and the early HIV-1 DNA levels were unaffected in respect to control cells. However, M2-MDM expressed very high levels of MDC/CCL22 that we have previously linked to a predominantly post-entry inhibition of HIV replication in MDM (M. Cota et al., PNAS 97: 9162, 2000). Polarization also resulted in a delayed, reciprocal down-regulation of the release of M1-related chemokines and cytokines in M2-MDM and vice versa. Most changes were fully reversible 7 days after removal of the polarizing stimulus with the exception of IP-10/CXCL10 and MDC/CCL22, which remained elevated in M1- and M2-MDM, respectively. In both M1- and M2-MDM, reversion to a nonpolarized state was associated with a renewed capacity to support productive infection, suggesting a mechanism for which infected macrophage may cycle between latent and productive infection.




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Peptide stabilization of gp120 conformation: a novel vaccine candidate

Jonathan M. Gershoni, Anna Roitburd-Berman, and Gal Dela Department of Cell Research and Immunology, Tel Aviv University – Israel Binding of CD4 locks gp120 into a unique conformation, enabling chemokine-receptor recognition necessary for viral entry. Stabilized gp120 in the CD4 induced conformation (CD4i) has been generated via a single chain CD4-gp120 recombinant molecule which has been demonstrated to be a preferred vaccine modality in macaques (DeVico et al 2007 PNAS 104:17477). These results indicate that CD4-gp120 complex reveals epitopes able to elicit neutralizing antibodies; however, this probably is at the expense of occlusion of the CD4 binding-site (BS) epitopes on gp120 - which are also regarded as highly desirable vaccine targets. We have isolated peptides that bind gp120 and in doing so induce the CD4i conformation - as demonstrated by binding of defining mAbs such as 17b, 19e and CG10. Moreover, the peptide-gp120 complex continues to bind CD4, illustrating that this site remains accessible. Furthermore, the peptide-gp120 complex efficiently binds a panel of CD4-BS defining mAbs, such as b12, b6 and M14 thus illustrating that the CD4i conformation is compatible with simultaneous presentation of the CD4-BS epitopes. We therefore propose that the peptide-gp120 complex represents a novel vaccine candidate that benefits from ‘‘both worlds’’; a CD4i stabilized conformation that maintains accessible neutralizing epitopes associated with the CD4-BS of gp120.

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Transmission Networks of Drug Resistance Acquired in Primary/Early Stage HIV Infection

Brenner BG, Roger M, Moisi D, Oliveira M, Hardy I, Turgel R, Charest H, Routy J-P, and Wainberg MA, the Montreal PHI Cohort HIV Prevention Study Groups Jewish General Hospital – McGill University AIDS Centre, 3755 Cote Ste-Catherine Road, Montreal, Quebec H3T 1E2 Objectives: Population-based sequencing of primary/recent HIV infections (PHI) can provide a framework for understanding transmission dynamics of local epidemics. In Quebec, half of PHI represent clustered transmission events. This study ascertained the cumulative implications of clustering on onward transmission of drug resistance. Methods: HIV-1 pol sequence datasets were available for all genotyped PHI (,6 months postseroconversion (n = 848 subtype B infections, 1997–2007). Phylogenetic analysis established clustered transmission events, based on maximum likelihood topologies having high bootstrap values (.98%) and short genetic distances. The distributions of resistance to nucleoside and non-nucleoside RT inhibitors (NRTIs and NNRTIs) and protease inhibitors (PIs) in unique and clustered transmissions were ascertained. Results: Episodic clustering was observed in half of recent/early stage infections from 1997–2008. Overall, 29% and 28% of new infections segregated into small (,5 PHI/cluster, n = 242/848) and large transmission chains ($5 PHI/cluster, n = 239/848), averaging 2.8 6 0.1 PHI/cluster and 10.3 6 1.0 PHI/cluster, respectively. The transmission of nucleoside analogue mutations and 215 resistant variants (T215C/D/I/F/N/S/Y) declined with clustering (7.9% vs. 3.4% vs. 1.2% and 5.8% vs. 1.7% vs. 1.1% for unique, small and large clustered transmissions, respectively). In contrast, clustering was associated with the increased transmission of viruses harbouring resistance to NNRTIs (6.6% vs. 6.0% vs. 15.5%, respectively). Conclusions: Clustering in early/PHI stage infection differentially affects transmission of drug resistance to different drug classes. Public health, prevention and diagnostic strategies, targeting PHI, afford a unique opportunity to curb the spread of transmitted drug resistance.




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The State of the ART in HIV Therapeutics

John G. Bartlett 1830 East Monument St., Rm. 447, Baltimore, MD Presentation based on guidelines from U.S. and Europe (which are nearly identical). When to start: 2 NRTIs: TDF/FTC, ABC/3TC, ?AZT/3TC 3d drug: EFV, LPV/r, ATV/r, FPV/r, SQV/r When to change: Toxicity, convenience or virologic failure (defined as VL. 50 c/mL 3 2) What to change to: Toxcitiy - single drug substitution Viral failure - based on resistance testing Major issues: 1. Should HIV screening be ‘‘routine’’ for all? 2. Should everyone be treated except long-term progressors? 3. Does ART play an important role in prevention? 4. When will point-of-care diagnostics be available in clinics for patients?

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An update on raltegravir (Isentress)

Charles Farthing, MD U.S. Dir of Scientific Affairs for Merck & Co Inc., 770 Sumneytown Pike, WP97A-279, PO Box 4, West Point, PA 19486-0004 Raltegravir, the first in class integrase inhibitor from Merck, will be presented with an update of phase 2 and 3 clinical trials, and an update on PK, drug – drug interaction data, and resistance data, obtained to date. 96 week phase two data in na¨ve patients shows a sustained virological suppression of viral load with TNF+FTC+RAL similar to the control arm of TNF+FTC+EFV with all four doses of RAL studied – 100 mg, 200 mg, 400 mg and 600 mg bid. Drug related clinical adverse events with RAL at 48%, were less then with EFV - 71%. RAL had a neutral effect on serum lipids. Suppression of viral load with RAL was somewhat more rapid initially with RAL than with EFV although the relevance of this finding is unknown. 48 week data in two phase three studies with RAL and OBR (BENCHMRK) show sustained virological suppression to ,50 copies/mL in 62% of patients versus 33% with OBR alone. Side effects for patients treated with OBR+RAL were similar as for those treated with OBR alone suggesting RAL added little to toxicity. PK with RAL is highly variable and no relationship between blood levels and results has so far been determined. RAL is metabolized by glucuronidation by the enzyme UGT1A1. Studies to date show no significant drug level alterations with different UGT1A1 polymorphisms - and few concerning drug drug interaction with drugs that induce or inhibit UGT1A1. Tipranavir lowers RAL levels by 55% and rifampin lowers levels by 61%. Phase 3 data suggests tipranavir’s effect is not significant – no data yet exists for use with rifampin. Resistance to RAL usually follows one of two pathways with primary mutations at either position Q148 or N155, which are usually rapidly followed by the development of secondary mutations which increase resistance and return fitness to the virus.




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Evaluation of Efficacy and Immune Recovery of Optimized Background Therapy (OBT) Plus Maraviroc (MVC) vs Placebo (PBO) in Treatment Experienced (TE) Patients with only R5 HIV-1, Combined Analysis of MOTIVATE 1 and 2

R. Tressler,1 J. Goodrich,2 N. Rajicic,1 H. Valdez,1 and H. Mayer2 1

Pfizer, Inc., New York; 2Pfizer, Inc., New London

Intro: In studies with MVC a numeric increase in CD4 cell count has been observed in the groups receiving MVC vs Placebo or efavirenz. The clinical parameters associated with and clinical significance of the increase in CD4 cell count are unknown. This post hoc analysis of the Wk 48 data combined MOTIVATE 1 and 2 data explores efficacy of MVC by degree of immune deficiency at BL, the clinical relevance of and predictors of the CD4 cell count increase observed in the MVC vs PBO arms. Methods: Descriptive statistics of antiviral efficacy (ITT), time to virologic failure, change in CD4 cell count (LOCF) and time to a Category C event were computed for all patients who received at least one dose of randomized study drug. A multivariate analysis of BL demographic and disease characteristics was conducted to determine parameters of changes in CD4 count at Wk 48. Results: There were no differences at BL among randomized treatment arms in CD4 cell count, viral load, age, gender, race and number of new drugs used in OBT. At all clinically relevant CD4 cell count strata a greater proportion of patients receiving MVC achieved HIV RNA ,50 copies/mL at Wk 48 as compared to the PBO arm. Conclusions: Efficacy of MVC was observed in all CD4 strata and CD4 cell increases were greater in patients who received MVC vs PBO independent of viral load change or achieving a viral load ,50 copies/mL. Predictors of CD4 count increase, other than MVC use, were similar to what has previously been reported. The higher proportion of patients achieving CD4 cell count .200 cells/mm3 on MVC, and the fact that on treatment CD4+ cells (but not treatment arm) were associated with lower risk of Category C events, suggests a clinical relevance to the CD4 cell count increases on MVC observed in the MOTIVATE studies.

(Continued on next page)

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Evaluation of Efficacy and Immune Recovery of Optimized Background Therapy (OBT) Plus Maraviroc (MVC) vs Placebo (PBO) in Treatment Experienced (TE) Patients with only R5 HIV-ombined Analysis of MOTIVATE 1 and 2 (continued) Placebo (n = 209*)

MVC QD (n = 414*)

MVC BID (n = 426)

HIV RNA ,400 copies/mL

22.5%

51.7%

56.1%

HIV RNA ,50 copies/mL

16.7%

43.2%

45.5%

+24

+92

+103

Median change in CD4 cell count at Wk 48 for pts who achieved HIV RNA ,50 copies/mL at least once (cells/mm3)

+96 (n = 68)

+125 (n = 251)

+126 (n = 267)

Median change in CD4 cell count at Wk 48 for pt who never achieved HIV RNA ,50 copies/mL (cells/mm3)

+10 (n = 140)

+43 (n = 162)

+59 (n = 159)

32 (n = 118)

43 (n = 235)

47 (n = 250)

Median change in CD4 cell count at Wk 48 (cells/mm3)

Percent of patients with CD4 ,200 at BL who achieved .200 at Wk 48 Percent of patients who experienced a Category C event through Wk 48 (unadjusted for exposure)

7.7%

7.0%

5.2%

*One patient in the PBO and MVC QD arm were missing BL CD4 data, therefore CD4 cell counts are based on 208 pts in the PBO and 413 pts in the MVC QD groups. (Time to occurrence of a Category C event was statistically significantly longer for MVC (QD or BID) vs PBO arms. Higher on-treatment CD4 cell counts were independently associated with a decreased risk of a Category C event. The discontinuation rate and adverse event profile was similar in the MVC and PBO arms. BL CD4 cell count, BL viral load, change in viral load at Wk 48, age and MVC use were independent predictors of CD4 cell increases.




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HIV Therapeutics in Resource Limited Settings – Status and Future Directions

Anthony Amoroso Institute of Human Virology, 725 West Lombard Street, Baltimore, MD 21201 In 2003 there was an estimated 50,000 patients on antiretroviral therapy in sub-Sahara Africa. Today there is an estimated 2 million. Concerns regarding efficacy and durability of antiretroviral therapy remain. In addition the potential for race/gender specific drug toxicity profiles in the targeted populations exist. This session will review data on long term durability of first line regimens used in large scale-up treatment programs to include toxicity and efficacy.

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Update on Gilead Sciences Anti-HIV Development Programs

Tomas Cihlar, PhD Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, CA 94404 Gilead Sciences has two current development programs for anti-HIV small molecule inhibitors. Elvitegravir (EVG, GS-9137) is an HIV-1 integrase inhibitor (INI) which has completed a phase 2 doseranging study (Study GS-US-183-0105). Patients were randomized to 20 mg, 50 mg or 125 mg QD doses of ritonavir-boosted EVG (EVG/r) plus optimized background therapy (OBT) comprised of NRTIs +/2 T-20. Study 0105 patients were highly experienced with a median of 3 TAMs and 11 PI-R mutations at baseline. Patients receiving EVG 125 mg had rapid viral load declines (mean decline in HIV-1 RNA of 2.1 log10 copies/mL at week 24); those with $1 active drug(s) in their background therapy had durable responses. Among patients with virologic failure on EVG/r 125 mg, the most common integrase mutations detected were E92Q, E138K, Q148R/K/H and N155H (observed in 11/28, 39%), S147G (9/28, 32%) and T66I/A/K (5/28, 18%), some of which have been selected by raltegravir (MK-0518) in vivo. RC declined from a median of 108% at baseline to 54% at virologic failure. EVG is currently entering phase 3 studies. In another program, a novel nucleotide analog, phosphonomethoxy-2’-fluoro-2’, 3’-dideoxydidehydroadenosine (GS-9148), was selected as an NRTI with an improved pharmacological and resistance profile. The prodrug of GS-9148 (GS-9131) exhibits potent antiretroviral activity both in primary cells and T-cell lines (EC50 = 25-200 nM), low cytotoxicity (CC50 . 50 iM) in multiple cell types, and shows no effect on mitochondrial DNA or lactate production in HepG2 cells. In phenotypic assays, the activity of GS-9148 was not affected by the K65R, L74V, or M184V mutations in RT (EC50 fold change ,1). Viruses with 4 or more thymidine analog mutations showed 0.74 to 2.0-fold change in the susceptibility, a shift that was smaller than that of any other tested NRTI. Passage of HIV-1 in the presence of GS-9148 selected for a primary K70E mutation and two other RT mutations that together conferred ,3-fold resistance to GS-9148. Oral administration of GS-9131 in dogs resulted in high and persistent levels of GS-9148 diphosphate in PBMCs and lymph nodes. Overall, GS-9131 exhibits a favorable in vitro virological and in vivo pharmacological profile and is entering phase I trials for once daily dosing in NRTI-experienced patients.




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Rapamycin enhances the antiviral activity of CCR5 antagonist Vicriviroc

Olga Latinovic Institute of Human Virology School of Medicine, University of Maryland, 725 West Lombard Street, Baltimore, MD 21201 The CCR5 chemokine receptor plays a crucial role in human immunodeficiency virus type I (HIV-1) infection. We previously demonstrated that reduction of CCR5 density with low doses of the transplant drug Rapamycin (RAPA) potentiates the antiviral activity of HIV-1 fusion inhibitor Enfuvirtide (T-20) against R5 strains of HIV-1 [Heredia et al, AAC, 2007]. In the current study, we show that RAPA enhances the potent antiviral activity of the CCR5 antagonist, Vicriviroc (VCV), which is currently being evaluated in Phase III clinical trials. The goal of this investigation is to identify most effective combination of antiviral drugs with the lowest level of side effects. Drug interaction based studies show that the RAPA/VCV combination has significant antiviral synergy (combination indexes of 0.1–0.04) in both spreading-cycle and single-cycle infection of lymphocytes, as well as in a cell-cell fusion assay. The drug, RAPA, enhances VCV antiviral activity against both B and non-B clade isolates, potently suppressing clade G viruses with reduced sensitivities to VCV and to Maraviroc, a second CCR5 antagonist. An existing synergy between RAPA and VCV translated into Dose Reduction Indices (DRI) results in 8–41 fold reduction for RAPA and 19– 658 fold reduction for VCV. In addition, we found that the RAPA induced reduction of CCR5 density in lymphocytes increased sensitivity to VCV in VCV-resistant strains, inhibiting virus production by ;90%. Moreover, by using cell lines and donor lymphocytes expressing a range of CCR5 densities, we further demonstrate the importance of CCR5 density to VCV activity. These research results direct to the future avenue of enhancing the potency of CCR5 antagonists, reducing potential for toxicity, potentially controlling emerging VCV-resistant variants.

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Why do so few Antibodies Neutralize HIV-1? Tests of the ‘‘Tolerance Hypothesis’’

Garnett Kelsoe Department of Immunology, Duke University, Durham, North Carolina 27710 USA The emergence of serum antibody responses only after early cellular immune responses have suppressed HIV replication has led to doubts regarding the importance of humoral immunity in controlling HIV infections. Nonetheless, rare, broadly cross-reactive antibodies are capable of neutralizing multiple isolates of HIV-1 in vitro, and when passively administered to monkeys, prevent experimental infection by SHIV. Why are such antibodies rarely produced by HIV-infected patients? Recently, several of these rare, HIV neutralizing antibodies were shown to react with self-antigens leading to the possibility that effective HIV humoral responses are constrained by the tolerization of HIV-reactive B cells that also recognize self-antigens. We have begun to test the hypothesis that B cells that recognize phylogenetically conserved, neutralizing epitopes of the HIV-1 gp-41 membrane proximal external region (MPER) are present in early, developmentally immature B cells but are tolerized and lost during their subsequent development/maturation. Our studies indicate that murine B cells specific for the 2F5 and 4E10 MPER epitopes are present at significant frequencies in the immature and transitional B-cell compartments of bone marrow but are undetectable in peripheral Bcell pools including phenotypically identical transitional B cells. Perhaps for this reason, immunization of normal mice with MPER peptides containing the 2F5 and 4E10 epitopes peptides results in little antibody production and poor germinal center responses. In contrast, reconstitution of RAG1 deficient mice with B lymphocytes that develop outside of the tolerizing environment of the bone marrow is permissive both for robust autoantibody responses, the production of germinal centers, and greatly increased levels of IgG antibody to MPER peptide epitopes.




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Getting the Right Immune Response to HIV: Evaluation of Protective Immune Responses After Vaccination of Rhesus Macaques

George N. Pavlakis,1 Antonio Valentin,1 Margherita Rosati,1 Agneta von Gegerfelt,1 Vainav Patel,1 Cristina Bergamaschi,1 Brunda Ganeru,1 Osamu Usami,1 Viraj Kulkarni,2 Rashmi Jalah,2 Candido Alicea,2 and Barbara K. Felber2 1

Human Retrovirus Section; and 2Human Retrovirus Pathogenesis Section, NCI, Frederick

Rhesus macaques infected by SIV develop a disease remarkably similar to human AIDS and provide the best animal model for AIDS vaccine evaluation. We have used this model to study immune responses after vaccination and challenge with pathogenic heterologous stocks of SIV. Our experience includes DNA vaccination either alone or in combination with recombinant virus or protein boost, and also vaccination with attenuated non-pathogenic (or weakly pathogenic) SIV molecular clones. Analysis of immune responses from several vaccination/challenge experiments show the development of protective immune responses that prevent high viremia in the challenged macaques. The correlates of protection remain poorly defined. It is anticipated that more detailed analysis of immune responses will provide a better understanding of the nature of protective immune response. The methodology of DNA vaccination has advanced rapidly. In the past it produced meager results in primates and humans, whereas at present DNA vaccination with optimized vectors and delivery is shown to efficiently produce high and durable cellular and humoral immune responses. These responses are disseminated to mucosa and protect the animals from high viremia after challenge. The best protection in the SIV macaque model has been achieved by the use of non-pathogenic, attenuated SIV clones. Such animals are able to fully suppress the pathogenic challenge virus for many years and protect the animals from disease development. Comparison of immune responses of animals protected after different vaccine regimens suggest that the quality, timing and location of immune responses are critical. Better assays to evaluate immune responses are essential.

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Induction of Cross-clade Neutralizing Antibodies in Rabbits Using a DNA Prime/Protein Boost Immunization Regimen

S. Zolla-Pazner,1,2 S. Cohen,2 C. Krachmarov,3 A. Pinter,3 S. Wang,4 and S. Lu4 1

Veterans Affairs Medical Center, New York, NY 10010; 2Dept. of Pathology, NYU School of Medicine, New York, NY 10016; 3Public Health Research Institute, Newark, NJ 07103; and 4University of Massachusetts Medical School, Worcester, MA 01605 Previous studies showed that an immunization regimen that focuses the immune response on the structurally conserved V3 epitope of gp120 can result in the induction of cross-clade neutralizing antibodies (Abs). Experiments were undertaken to optimize the breadth and strength of this response by immunizing rabbits with three doses of gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by protein boosts of a V3-gp70 fusion protein carrying the V3 sequence from a clade B virus with the GPGR motif (V3B-FP), a V3A-FP with a clade A V3 loop with the GPGQ motif, and/or a V3C-FP (GPGQ motif). Neutralization was demonstrated against viruses pseudotyped with the SF162 envelope in which V3 was replaced with consensus V3 loops from subtypes A1, CRF02_AG, B, C, CRF01_AE, F or H. .50% neutralization was also achieved at titers ranging from 1:10–1:559 against 14 of 15 primary isolates from the four subtypes tested (A, CRF02_AG, B and C). The polyclonal Abs in the immune rabbit sera were able to neutralize viruses that none of 10 human anti-V3 mAbs could neutralize. Thus, cross-clade neutralizing Abs were induced using a DNA prime/protein boost regimen designed to focus the immune response on a neutralizing epitope, The broadest neutralizing responses were generated when using a gp120 DNA prime derived from a clade C virus carrying a V3 loop with the GPGQ motif.




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Prospects for an AIDS Vaccine: Can Effector-Memory T Cell Responses Contribute?

L. Picker Vaccine and Gene Therapy Institute and the Oregon National Primate Research Center at Oregon Health & Science University, Beaverton, OR Memory T cells, both CD4+ and CD8+, can be described in terms of their differentiation along the so-called ‘‘central-memory’’ (TCM) to ‘‘effector-memory’’ (TEM) axis. TCM recirculate among secondary lymphoid tissues and exhibit the capacity to expand and under-go further differentiation to TEM or active effectors upon antigenic stimulation. In contrast, TEM populations are based primarily in nonlymphoid tissues, have less proliferative potential upon re-stimulation than TCM, but can manifest potent immediate effector function. TEM would therefore provide an immediate, but limited, response to microbial invasion of non-lymphoid tissues, whereas TCM would potentially provide a much larger, but delayed, response. Prime-boost vaccines or vaccines with non-persistent vectors typically yield TCM predominant responses that are dependent on post-challenge expansion for effective effector generation. The limited efficacy of these approaches to date might be due to a kinetic mismatch between this delayed production of T cell effectors and the rapid replication and diversification of HIV/SIV after transmission. As TEM are pre-positioned and ‘‘armed’’ at sites of initial HIV/SIV replication, vaccines that elicit and maintain these populations might have an advantage in mediating anti-HIV/SIV protection early, in the window of opportunity prior to massive systemic viral replication. To test this hypothesis, we have developed CMV-based vectors that are able to prime and maintain strong SIVspecific TEM responses in rhesus macaques. In keeping with our hypothesis, these SIV-specific TEM responses endow resistance to the acquisition of pathogenic SIV infection delivered by a mucosal route, but do not provide substantial protection when progressive systemic infection does occur. These data suggest that vaccines capable of generating and maintaining HIV-specific TEM might be able to decrease the incidence of HIV infection after sexual exposure.

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Human defensins – small in size but big in functionality

Wei Gang, 1 Erik de Leeuw, 1 Guozhang Zou, 1 Mohsen Rajabi, 1 Marzena Pazgier,1,2 Weirong Yuan,1 Jing Li,1 Jacek Lubkowski,2 and Wuyuan Lu1 1

Laboratory of Synthetic Protein Chemistry, Division of Basic Science and Vaccine Research, Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201, USA; 2Macromolecular Assembly Structure and Cell Signaling Section, NCI, National Institutes of Health, Frederick, MD 21702, USA Defensins are a family of small cationic peptides expressed primarily in leukocytes and epithelial cells. Lehrer and colleagues originally discovered, two decades ago, the prototype alpha-defensins – human neutrophil peptides or HNPs – as antibiotic peptides important for phagocytosis. Recent studies have unveiled a great variety of new functions of these antimicrobial molecules in many biological processes. However, the molecular basis for the functional promiscuity of defensins remains poorly understood. Using an extensive mutational analysis aided by structural biology, we have systematically investigated the structural determinants for HNP1 as a bactericidal agent and a potent inhibitor of anthrax lethal factor. As a result, a clear picture of how human defensins work at the molecular level is beginning to emerge.




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The C5 region of gp120: A therapeutic vaccine target?

Angus Dalgleish, and Martin Cadogan SGUL, tooting, London SW17 ORE, UK HIV infection does not always lead to disease as demonstrated by very long term non progressors and chimpanzees. The difference between progressors and non progressors is the degree of non specific pan activation of the immune system which is absent in the latter. The cause of this genetically restricted pan activation has yet to be confirmed. However, recognized causes such as hypervariability and superantigen stimulation are not relevant to HIV. Another potential cause is allogeneic stimulation . HIV has several regions of homology to HLA class 1 and 11. We have reported that C5 is structurally very similar in that it and no other region bind specific class 1 and class 2 peptides. Moreover, when the region is itself presented as a peptide in context of self HLA it is recognized as an alloepitope . As such it would be closest to a self peptide in HLA-27 and most pronounced as an alloepitope in HLA-8. This is consistent with HLA-27 being associated with slow progression and HLA-8 with fast progression. Furthermore, chimps have very restricted HLA types similar to HLA-27/57. Several large studies have reported the presence of anti-bodies to C5 in non progressors but these observations have been discounted as the antibodies are non neutralizing. Here, we propose that if these antibodies are preventing the C5 region from inducing the activation that drives AIDS, then a therapeutic vaccine aimed at inducing this response following immune normalization with HAART may allow some patients to convert to non progressors and no longer be dependant on HAART therapy. As this region is so highly conserved amongst all viable isolates it may well be a good candidate for a prophylactic vaccine as sterilising immunity is clearly not necessary in order to prevent progression to AIDS. Therapeutic non-neutralising vaccines need not be limited to HIV!

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Mechanisms by which synergistic combinations of TLR ligands enhance T cell responses to vaccines

Qing Zhu, 1 Igor M. Belyakov, 1 Dennis Klinman, 2 and Jay A. Berzofsky1 1

Vaccine Branch and 2Laboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, MD 20892 USA Ligands for Toll-Like Receptors (TLRs) are usually microbial products that are naturally encountered in combinations. Thus, we asked whether synergies occurred among them, and what the mechanisms of those synergies might be, and how we could exploit that for enhancing vaccineinduced T cell responses. We found several synergies between pairs of TLR ligands that used different intracellular signal transduction pathways. In each case, the synergy depended on dendritic cell (DC) activation either in vitro or in vivo to induce an enhanced T cell response. Dendritic cells were similarly activated both in vitro and in vivo, and when treated with the synergistic combinations of TLR ligands in vitro, the DCs could be used in vivo as a vaccine to induce an enhanced immune response. The increased responses correlated with dendritic cell production of cytokines like IL-12, that depended on the MyD88 pathway, but not with increased costimulatory molecules, that depended only on the TRIF pathway. However, the TRIF pathway was found to amplify the cytokine production through the MyD88 pathway in a unidirectional crosstalk, accounting for the synergy. The synergistic combinations activate CD4+ and CD8+ T cells independently. Taking advantage of these synergistic combinations of TLR ligands could allow the design of more effective vaccine compositions and adjuvants and potentially overcome deficiencies in DC maturation or lack of CD4+ help in cancer or HIV infection.




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Vector-based Vaccines for Cancer Therapy

Jeffrey Schlom, James W. Hodge, Kwong-Yok Tsang, Claudia Palena, John W. Greiner, Ravi A. Madan, and James L. Gulley Laboratory of Tumor Immunology and Biology, Center for Cancer Research, NCI, NIH Our program has been involved in the design and development of recombinant vector based vaccines for the therapy of human carcinomas. Emphasis has been based on the development of a pox virus-based regimen, i.e., recombinant vaccinia prime vaccination followed by multiple booster vaccinations with recombinant fowlpox vectors. Clinical trials have demonstrated that one can administer multiple boosts of recombinant fowlpox without host neutralizing activity. Vaccines have now been developed which contain transgenes for three human T-cell costimulatory molecules (TRICOM) and transgenes for either PSA for prostate cancer vaccines, or CEA-MUC-1 for use in the therapy of most carcinomas. These vaccines have demonstrated evidence of antigen-specific T-cell responses, the initiation of antigen cascade, objective clinical responses, drops in serum markers, delays in time to progression, and most importantly, evidence of increase in patient survival. In two separate clinical trials, evidence of increased survival has been observed in prostate cancer patients with hormone refractory disease and with metastatic disease, respectively. Preclinical studies have clearly demonstrated that vaccine-induced T-cell immunity can act synergistically with radiation therapy, and certain chemotherapeutic agents by altering the phenotype of tumor cells to make them more susceptible to T-cell–mediated attack. Evidence is also emerging that the dynamic process of T-cell–mediated immunity initiated by vaccine therapy can have a positive influence on subsequent therapies. REFERENCE 1. Schlom, J, P.M. Arlen, J.L. Gulley. 2007. Cancer vaccines: moving beyond current paradigms. Clin. Cancer Res. 13:3776–3782.

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Recruitment of high avidity antigen-specific T cells in pancreatic cancer patients

Elizabeth M. Jaffee, MD Sidney Kimmel Cancer Center at Johns Hopkins, 4M07 CRB I, 1650 Orleans St., Baltimore, MD 21231 T cell responses are often observed in cancer patients who have been treated with antigenspecific vaccines. However, the majority of responses are often weak and ineffective at controlling tumor growth. In some instances, this may be due to an ineffective vaccine approach. However, in many cases mechanisms of T cell tolerance or ‘‘immune checkpoints’’ to specific tumor antigens are at play. Understanding these mechanisms in the context of tumor antigens is critical for the development of interventions that can reverse the tolerant state or modify ‘‘immune checkpoints’’ and allow these T cells to more effectively respond to tumors. We have described the existence of immune tolerance in the HER-2/neu transgenic (neu-N) mouse model of breast cancer and used these mice to understand the mechanisms that suppress high avidity antigen-specific CD8+ T cells. We previously reported that treatment of neu-N mice with immune modulating doses of Cyclophosphamide (Cy) prior to vaccination resulted in tumor protection in a proportion of mice. More recently we reported that CD8+ T cells specific for the immunodominant neu epitope, RNEU420-429, were identified only in Cy plus vaccine treated neu-N mice that rejected tumor challenge, but not in neu-N mice given vaccine only. Furthermore, high avidity RNEU420-429-specific CD8+ T cells were also identified in vaccine treated mice that were first depleted of CD25+ T regulatory cells (Tregs). Thus, the mechanism by which immune modulating doses of Cy augments vaccine induced high avidity RNEU-specific CD8+ T cell responses is by inhibiting and/or deleting CD4+CD25+ Tregs. ‘‘Proof of principle’’ clinical trials have confirmed that vaccines given with immune modulating Cy can recruit higher avidity T cells that correlate with improved clinical outcomes. The antigen-specific T cell responses associated with these clinical trials will be discussed.



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Development of Preventive HIV Vaccines in the US Military HIV Research Program

Nelson L. Michael, MD, PhD Approaches to develop a preventive HIV-1 vaccine have been challenged by the failure of protein subunit and some non-replicating vector immunizations to reduce HIV-1 acquisition risk or postinfection viremia in breakthrough infections. Recent trials of adenovirus type 5 vectored HIV-1 vaccines have raised concerns that some vaccines under certain circumstances may increase HIV-1 acquisition risk. Long-standing challenges to develop vaccines that produce functional B-cell responses, effective T-cell responses, mucosal immunity, and that work across the wide range of HIV-1 sequence variation have not been overcome. The HIV vaccine development field has undergone much introspection resulting in greater emphasis on vaccine discovery research, relevant small animal and non-human primate models, and new approaches to vaccine delivery. The US Military HIV Research Program has major efforts that address this new paradigm. Clinical data will be presented from novel MVA vectored vaccine phase I trials and planned experiments with heterologous vector approaches will be described. Finally, an update on the progress of the phase III trial of ALVAC-gp120 protein in Thailand will be provided.

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Rational Vaccine Design and the Development of an AIDS Vaccine

Gary J. Nabel, MD, PhD Vaccine Research Center, NIAID, National Institutes of Health, Bethesda, MD 20892-3005, USA Advances in our basic understanding of the immune system have provided the tools to make more effective vaccines—rationally designed vaccines developed with a scientific understanding of the mechanisms of microbial pathogenesis and immunity. Current HIV vaccine candidates target multiple internal and external gene products. Next generation vaccines will need to enhance the immunogenicity of the envelope (Env), with the goal of improving the breadth of the neutralizing antibody response while concurrently stimulating cell-mediated immunity. Our recent efforts have centered on the use of genetic and structural biological information to improve both Env and Gag immunogen design. The genetic approach has focused on the conserved regions of Gag and Env to improve CD8 cell breadth. To target such conserved domains, site-specific mutagenesis has been employed to generate and analyze mosaic, variant, and chimeric Env and Gag proteins. In other studies, the VRC and collaborators have determined the structure of the broadly neutralizing monoclonal antibody, b12, as well as its corresponding HIV-1 epitope. This information is being used to design epitope mimics that can induce additional insight into the immune response. The status of the rational immunogen design and the development of vaccines that elicit broadly neutralizing antibody responses will be reviewed.



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Continuing Clinical Trials for HIV Vaccine Research

Glenda Gray PHRU, Chris Hani Baragwanath Hospital, Old Potch Road, Diepkloof, Soweto, Johannesburg There is a pressing need to continue developing novel biological platforms that will interrupt HIV transmission as is evident from the explosive epidemic seen in South Africa. South Africa, with an estimated 5.7 million infected with HIV, where 90% of all new infections amongst 15–24 year olds occur in girls and women, is in urgent need for a biomedical intervention that is not coital dependent. HIV Vaccine related research presents many challenges to scientists in the post ‘‘STEP’’ era. These challenges include the continued need for discovery, design and manufacturing of candidate vaccines as well designing clinical trials that address specific immunological and virological questions that advance our understanding of HIV and impact on immunogen design and development. Continuing clinical research that addresses genetic diversity of HIV-1, host immune responses to HIV-1, anti-vector immunity, and genetic susceptibility to HIV-1 are critical. The nature of protective CTL remains unidentified and can only be revealed by doing further clinical testing. The successful execution of this will require mechanisms for continued global financing and commitment to basic science clinical trials.

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Gunnel Biberfeld Abstracts not available at time of printing



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A Novel Model for In Vivo SIV Neutralization

Phil Johnson Children’s Hospital of Philadelphia, 1216B ARC, 3615 Civic Center Blvd, Philadelphia, PA 19104 The holy grail of HIV vaccine development is an immunogen that elicits antibodies which broadly neutralize field strains of the virus. While tremendous insights have been gained into the structure and function of the HIV envelope glycoprotein, precious little progress has been made in designing such immunogens. In fact, it remains formally possible that with current technologies, engagement of the adaptive immune system will not lead to an effective HIV vaccine. Considering this possibility, we have taken a markedly different approach to the generation of serum neutralizing activity against HIV that we have dubbed ‘‘reverse immunization.’’ In this model, we pre-select the antibody or antibody-like molecule of interest (i.e, one that broadly neutralizes) then synthetically derive the representative gene and place it into the context of an adeno-associated virus (AAV) gene transfer vector. When injected intramuscularly into a vaccinee, the AAV vector genome directs in vivo production of the gene product that leads to serum neutralizing activity against HIV. Recently, we have refined this approach for the SIV model system, and can now provide in a single dose, sterilizing immunity to vaccinated monkeys against a virulent SIV challenge virus. Thus, reverse immunization holds significant promise as a ‘‘designer’’ approach to an effective HIV vaccine.

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Morphogenomic immune responsiveness to preventive/therapeutic vaccines

A. Monaco,1 M. Sabatino,1 Z. Pos,1 D. F. Stroncek,1 E. Wang,1 R. C. Gallo,2 G. K. Lewis,2 M. L. Tornesello,3 F. M. Buonaguro,3 F. M. Marincola,1 and L. Buonaguro3 1

Immunogenetics Section, Dept Transf Medicine, Clinical Center, NIH, Bethesda; 2Inst. Human Virology, Univ. of Maryland School of Medicine, Baltimore, MD, USA; 3Lab. of Viral Oncogenesis and Immunotherapies & AIDS Reference Center, Istituto Nazionale Tumori ‘‘Fond. G. Pascale’’, Naples – Italy

We have previously reported that the transcriptional profile of VLPstimulated MDDCs shows the up-regulation of classic markers of MDDC activation. In particular, VLP-stimulation induced the activation of genes associated with antigen presentation and MDDCs migration. Subsequently, we have shown that a comparable transcriptional profiling can be obtained using PBMCs without further in vitro maturation in MDDCs. Fresh human peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation and plated in 6-well plates at a concentration of 1 3 107/well. PBMCs were pulsed with 6 mg/mL of HIV-VLPs. After 16 hrs, the cells were harvested, washed, total RNA was isolated and RNA quality/quantity was estimated. Amplified antisense RNA (aRNA) was obtained from total RNA via two round of in vitro transcription. Testreference sample pairs were mixed and co-hybridized to a custom-made 37K oligo-based microarray platform encompassing the whole human genome. In the present study we show that VLPs induce ex vivo transcriptional activation comparable to that observed in in vitro matured MDDCs and the transcriptional patterns induced ex vivo by VLPs in PBMCs from normal donors equated the patterns observed ex vivo in unstimulated PBMC from HIV in patients. Moreover the baseline activation of immune genes observed in HIV-infected patients is further enhanced by ex vivo stimulation by VLPs resulting in a transcriptional activation superior to that observed in PBMC obtained from normal non HIV-infected volunteers. In summary, this study may provide a road map for the study of HIV-infected patients based on HIVspecific signatures, their heterogeneity among patients, their response to vaccine strategies. Future studies should take into account this findings for the discovery of potentially correlative patterns associated with the natural history of the disease and/or it response to treatment.



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Development of the broadly neutralizing human antibody m9 for anti-HIV prophylactics

Antony S. Dimitrov, Wenlei Zhang, Ilia Prado, and Timothy Fouts Profectus BioSciences, Inc., 1450 South Rolling Road, Baltimore, MD 21227 ScFv m9 is a human antibody fragment that exhibits potent and broad anti-HIV activity. Identified by using phage display, scFv-m9 targets a highly conserved CD4-induced (CD4i) epitope on gp120 and potently neutralizes Groups M (clades A-G) and N HIV-1 in PBMC/primary isolate-based and cell line/pseudovirus-based assays most likely by interfering with envelope engagement to the coreceptor. This monoclonal antibody fragment neutralized more than 80% of the tested primary isolates and showed superior potency and breadth to b12, 2F5, 4E10, and 2G12. In addition, scFv-m9 lacks reactivity to self antigens such as cardiolipin that plagued the development of 2F5 and 4E10. While effective as a scFv or a Fab, the potency of m9-IgG is significantly less suggesting that the target CD4i epitope has a size restriction. To exploit scFv m9 or Fab m9 as clinical products, we must extend their pharmacokinetic half-life in-vivo. Binding to albumin via albumin binding peptides has been used as a strategy to dramatically improve the pharmacokinetics of antibody fragments without significant increase in their size. We have engineered two variants of scFv m9, called m9hc and m9sa, in which two albumin binding peptides independently were fused to the C-terminus of the scFv m9. The new constructs have been expressed in E-coli and tested for binding to gp120-CD4 complex and for neutralization of HIV-1. Albumin binding peptide scFv m9 variants preserved the binding and neutralizing properties of the original scFv m9. If pharmacokinetic characteristics of m9sa and m9hc constructs are appropriate for once a day injection, these human antibody fragments will be developed as clinical candidates for anti-HIV prophylactics in incidences of accidental exposure, mother-to-child transmission; or salvage treatment therapy.

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Interaction between a domain in the fusion peptide proximal region (FPPR) of gp41 and the epitope domain in the membrane proximal external region (MPER) increases binding of 2F5 to its epitope

Uwe Fiebig, Magdalena Eschricht, Mirco Schmolke, Reinhard Kurth, and Joachim Denner Robert Koch-Institute, Nordufer 20, D-13353 Berlin, Germany Neutralizing antibodies such as 2F5 and 4E10 have been isolated from HIV-1 infected patients. They recognize epitopes in the MPER of gp41 and neutralize a broad range of HIV strains. Previously we reported induction of neutralising antibodies against the porcine endogenous retrovirus (PERV), the feline leukaemia virus (FeLV) and the Koala retrovirus (KoRV) by immunization with the ectodomain of their transmembrane envelope (TM) proteins p15E. Antisera obtained in rats, goats and cats recognized two epitopes: One, designated E1, was located in the FPPR and the other was located in the MPER of p15E (E2). E2 corresponded to the 2F5/4E10 epitope in gp41. Despite the evolutionary distance between HIV on one hand and PERV, FeLV and KoRV on the other, limited sequence homology exists (F/YEGWFN in the case of gammaretroviruses and NWFNIT, the 4E10 epitope, in the case of HIV-1, identical amino acids underlined). Immunising cats with p15E of FeLV resulted in protection from FeLV infection in vivo. Using ELISA, epitope mapping and surface plasmon resonance analysis we also identified an E1 domain in the FPPR of gp41 of HIV and showed that the interaction of peptides containing E1 significantly increased the binding of 2F5 to peptides containing the E2 (2F5/4E10 epitope) domain. In addition, neutralisation of HIV-1 by 2F5 was inhibited more effectively with both gp41-derived peptides E1 and E2 than with peptide E2 alone. Since E1 peptides increased the interaction of 2F5 with peptides containing its E2/ELDKWA epitope, it is most likely that both domains are required to induce neutralizing antibodies (WO 2005/021574, WO 2007/107597). This strategy may be used to generate a vaccine inducing broadly neutralising antibodies to prevent or curtail HIV infection.



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Immunosuppressive human endogenous retrovirus K (HERV-K) is expressed in human villous and extravillous cytotrophoblasts

Ulrike Ka¨mmerer,1 Ariane Germeyer,2 Michaela Kapp,1 Sven Stengel,3 Kristina Bu¨scher,3 Reinhard Kurth,3 and Joachim Denner3 1

University Women’s Hospital, Wuerzburg, Germany, 2University of Heidelberg, Dept. of Gynecological Endorinology and Reproductive Medicine, Heidelberg, Germany, and 3Robert Koch Institute, Berlin, Germany Overexpression of different human endogenous retroviruses (HERVs) has been shown in stem cell tumors, melanomas, human embryonic stem cells as well as in the placenta. The envelope proteins of HERV-W (also known as syncytin 1) and HERV-FRD (syncytin 2) were shown to be involved in cell fusion allowing to generate the syncytiotrophoblast in the human placenta. One of them, syncytin 2, was shown to be immunosuppressive, the immunosuppressive activity was associated with its immunosuppressive (isu) domain. Here we report the expression of another HERV, HERV-K, which is characterised by open reading frames for all viral genes. Using real time PCR, immunohistochemistry and Western blot analysis, expression of HERV-K was studied in normal placental tissue of different gestational ages. The transmembrane envelope (TM) protein of HERV-K was found exclusively in villous and extravillous trophoblast cells, sparing syncytiotrophoblast and other cells. The expression increased up to the second trimester and later decreased. This is the first report showing expression of the TM protein of HERV-K in normal human placental tissue with an exclusive expression in the trophoblast, suggesting a potential involvement of HERV-K in placentogenesis and pregnancy. In parallel the immunosuppressive properties of the TM protein and the corresponding isu peptide were demonstrated. Therefore expression of the immunosuppressive TM protein of HERV-K may contribute to the protection of the human embryo.

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Autoresistance to X4 HIV Infection by soluble suppressor factors secreted from CD4+ T cells

F. Cocchi, A. DeVico, A. Garzino Demo, and R. C. Gallo Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street, Baltimore, MD 21201 Control of persistent viral infection relies particularly on cell-mediated immunity comprised of CD4+ and CD8+ T cells. There is accumulating evidence on the relevance of soluble factor(s) secreted by CD8+ and CD4+ T cells in controlling HIV replication in vivo. While the a chemokines RANTES, MIP1a and MIP1-b collectively account for the suppression of R5 viruses, yet information is lacking on the identity of the molecules involved in the suppression of X4 viruses. Proteins that inhibit the replication of X4 HIV isolates were purified from the conditioned media (CM) of immortalized CD8+ and CD4+ T cell lines from HIV+ long-term non-progressors subjects (LTNPs) and identified as the a chemokines macrophage-derived chemokine (MDC), thymus and activation-regulated chemokine (TARC) and I309. These chemokines are secreted primarily by CD4+ T cells but also by CD8+T cells. CD4+ T cells of asymptomatic HIV+ individuals secreted significantly higher levels of MDC and TARC compared with subjects who progressed to AIDS. Recombinant human MDC, TARC and I309 induced a dose dependent inhibition of X4 viruses. A cocktail of neutralizing antibodies against MDC, TARC and I309 abrogated the inhibition of the replication of X4 viruses mediated by the endogenous chemokines in a dose dependent manner in PBMC and CD8-depleted PBMC cells acutely infected in vitro. These studies demonstrate that MDC, TARC and I309 represent a major component of the soluble anti-X4 activity of T cells, suggesting that the mechanism whereby CD8+ and CD4+ T cells contribute to the control of HIV-1 replication may relate to the secretion of these molecules.



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Targeted delivery of antiHIV siRNAs to T cells in vivo

Premlata Shankar, MD Department of Biomedical Sciences, Texas Tech University Health Sciences Center, Paul L. Foster School of Medicine, 5001, El Paso Drive, El Paso, Texas 79905 RNA interference is a promising therapeutic alternative for HIV infection, provided the barrier of effective delivery to T cells can be overcome. We developed a method for targeted delivery of siRNA to T cells that relies on a single chain antibody to the pan T cell antigen, CD7 (scFvCD7). The scFV was conjugated to the positively charged oligo-9-arginine (9R) peptide (scFvCD7/9R) to enable nucleic acid binding. NOD/SCID IL2rgc-/- mice that are receptive human cell engraftment and support systemic HIV-1 infection were used as a preclinical model to test the delivery strategy. The chimeric scFvCD7-9R reagent efficiently delivered siRNA and silenced CD4 gene expression in T lymphocytes in the Hu/PBL model, where human cell repopulation is by activation and expansion of adoptively transferred peripheral blood lymphocytes (PBLs). In animals reconstituted with PBLs from normal donors, weekly treatment with scFvCD7-9R/siRNA complexes targeting a combination of viral genes and the host coreceptor molecule CCR5, contained viral replication and maintained CD4 T cell numbers after exogenous challenge with HIV-1. Similar results were also observed with endogenous virus in animals reconstituted with HIV+ PBLs to mimic established infection. In contrast untreated control mice displayed a marked reduction in CD4 T cell numbers with significantly higher serum p24 antigen levels. scFvCD7-9R was also able to deliver siRNA to na¨ve/resting T cells in the Hu/HSC model where the adaptive immune system was reconstituted from engrafted cord blood CD34+ hematopoietic stem cells. In Hu/HSC mice, infected with HIVBaL siRNA treatment could control viral replication and CD4 T cell loss for up to 7 weeks. Thus, we have demonstrated the feasibility of T cell targeted delivery of siRNA for potential therapeutic applications in HIV infection, using SCID gamma chain ko mice repopulated with human cells as a novel preclinical model.

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Defective HIV-1 Proviral Genomes in Natural Viral Suppressors

L. M. Eyzaguirre, M. M. Sajadi, R. R. Redfield, W. A. Blattner, and J. K. Carr Institute of Human Virology, University of Maryland School of Medicine, 725 West Lombard Street Room S 524 Baltimore, MD 21201 Background: A very small proportion of people infected with HIV-1, referred to as Natural Viral Suppressors (NVS), are able to suppress viral replication to undetectable levels without treatment. One possible mechanism for this phenotype is inactivation of the virus at the onset of the infection such that most viral genomes are defective. Methods: Twenty-nine NVS patients who have been identified from inner city Baltimore have longitudinal data confirming this phenotype over many years. A group of 18 controls was also selected (HIV-infected individuals with undetectable viral loads on HAART treatment and those with high viral loads off of treatment). Nearly full-length proviral genomes were amplified from PBMC DNA and sequenced; all genes were analyzed for open reading frames, and hypermutation was assessed by HyperMut software. Results: The NVS patients were 100% African American, 63% male and 63% injecting drug users. The controls were similar: 84.6% African American and 69.3% male. Ten of the 29 NVS patients had proviral genomes that were defective (34.5%): 8 due to G-to-A hypermutation and 2 with frame shift mutations in either pol or env. Among the 18 controls there were 2 defective genomes (11.1%), both due to hypermutation. Of note, both of the hypermutated strains were from the 4 controls that had been on treatment. Most of the hypermutated sites were in a GG context, rather than GA, implicating APOBEC3G as the enzyme responsible. The mean pairwise genetic distance among the NVS patients over the entire genome was 5.05% (+/2 1.7%) compared to 6.63% (+/2 1.0%) among the controls. Conclusions: A study of 29 NVS patients revealed that 34.5% had defective proviral genomes compared to 11.1% of the controls. The NVS patients also had less genetic diversity than the controls. Both of these observations suggest that one of the mechanisms responsible for the NVS phenotype may be inactivation of the virus early after transmission, leading to the prevention of wide-scale replication and reduced genetic diversity.



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The Immune Response to HIV: Friend or Foe

M. Karen Newell,1 Elizabeth Connick,2 Evan Newell,3 Haig Keledjian,3 Monica Ord, 3 Robert Berliner, 3 Joshua Cabrera, 1 Richard Tobin, 1 Cassie Pleasant,1 and Lisa Villalobos-Menuey1 1

University of Colorado, Colorado Springs; 2University of Colorado Health Sciences Center; 3Viral Genetics, Inc., Asuza, California

Our work aims to investigate a newly discovered link between inflammatory events that characterize early stages of HIV infection and a link to an infected person’s MHC alleles. Hypothesis: We propose that it is the inflammation characteristic of an innate immune response that is responsible for the loss of CD4+ T cells in HIV. Experiments and Results: We base this proposition on five observations: (1) There is evidence that, in the absence of functional T regulatory cells (Tregs), the impact of polyclonal B cell activation is chronic inflammation and the activation of abT cells*; ab T cells have been shown to kill CD4 T cells during HIV infection**; (2) It has been shown that as T reg numbers decline, there is a concomitant rise in viral load in the HIV-infected patient*** (3) We have discovered that the B cells from an HIV infected lymph node have high levels of an important self peptide on their surfaces that indicates the lymph node B cells have been non-specifically activated. (4) Our data suggests peptide loading of nonspecifically-activated B cells with targeted peptides can stimulate the activity of Tregs in an MHC alleledependent manner; and (5) Treatment with a mixture of targeted peptides, reduces viral load in approximately 25 to 30% of patients treated, a result potentially explained by differences in MHC between patients. Conclusions: The consequences of unraveling the link between inflammation, cellular activation, and MHC may result in understanding a mechanism that may lead to development of targeted peptides that can redirect the immune response to, and interfere with, the inflammation-dependent pathology of HIV. Future Directions: Our exciting findings suggest that, with the proper predictions, we can synthesize targeted peptides to treat people with all different types of MHC alleles providing an inexpensive and potentially efficacious biological therapy for HIV.

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Anti-FasL treatment preserves SIV-specific memory and slows progression to AIDS in rhesus macaques

Bhawna Poonia, Maria S. Salvato, and C. David Pauza Institute of Human Virology, 725 W Lombard Street, Baltimore, MD Previously we reported an increased survival advantage in SIV-infected rhesus macaques after injection with a FasL-blocking monoclonal antibody (Salvato et al., Clin Dev Immunol. 2007: 93462). In the present study we analyzed the effect of such FasL blocking on immune parameters and disease progression in a cohort of SIV-infected rhesus macaques. Upon infection, various immune parameters in the blood developed highly significant differences between animals given anti-FasL RNOK203 antibody and those given an isotype control antibody. The anti-FasL treated animals had a clear preservation of central memory CD4 lymphocytes starting from acute infection through at least set point viremia. The total memory CD4 lymphocytes in these animals were also significantly higher throughout the 25-week study period. Anti-FasL treatment resulted in significantly higher percentages of memory CD8 lymphocytes in peripheral blood. Besides these changes in the memory lymphocyte compartment, the treated animals had a higher response to infection as evidenced by increased turnover of CD4 and CD8 lymphocytes expressing Ki67. Importantly, the antigen-specific cellular immune responses were significantly higher at 4, 6 and 17 weeks after infection in anti-FasL treated animals. Preservation of memory lymphocytes and cellular immunity are considered the main correlates of protection in SIV infection. Our study demonstrates that FasL is a major contributor towards depletion of anti-SIV immunity and that blocking this pathway is a beneficial strategy that should be considered for vaccine formulations.



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Significant Relationship between INNO-LIAä HIV I/II Positivity Bands and the Immunological Status of HIV Patients

Fernanda Leite, Fa´tima Oliveira, and Luciana Pinho Clinical Haematology Dept. -Hospital Geral Santo Antonio- 4050 Porto-Portugal Objective: the aim of our work was to study the relation between the INNO-LIAä HIV I/II (ILH) positivity band patterns and the immunological status of HIV patients. Methods: We looked for HIV patients whose ILH was performed between June 2007 and June 2008. The ILH bands were classified after the grade of positivity of the different antibodies: antisgp120, anti-gp41, anti-p31, anti-p24, anti-p17, anti-sgp105 and anti-gp36. We looked for age, gender, n° total leucocytes, n° total lymphocytes, n° of CD3+, CD4+, CD8+, CD19+, NK cells and HIV viral load. We looked also for HCV, HBV and HAV status. We studied 247 individuals, 65 were female (26,3%) and 182 male (73,7%) with ages between 15 and 76 years old with mean age of 40. Five patients were HIV-2 patients. Data statistical analysis was performed with SPSS program and P value ,0.05 was regarded significant. Results: Sgp120 positivity was significantly related with the decreasing lymphocyte number (P = .046) and in the linear regression equation the HAV independent variable showed the highest predictive value for sgp120 positivity (P = .031). Gp41 was significantly related with HIV viral load (P = .040) and with HCV co-infection (P = .005); HCV and HAV were independent variables with predictive value for gp41 positivity (P = .001). P31 was associated significantly with the decreasing n° of leucocytes (P = .043) and CD19+ cells (P = .011); HIV viral load had the highest predictive value for p31 positivity. The positivity of p24 band was associated with increasing number of CD4+ (P = .0.000) and CD19+ cells (P = .000) and decreasing CD8+ cells (P = .001). There wasn’t any significant correlation between p17, sgp105 nor gp36 and immunological parameters. Women showed higher CD4+ cells (P = .048) and p24 positivity (P = .010), being the gender the independent variable with the highest predictive value for the p24 positivity (P = .007). Men had higher HIV viral load (P = .019) and higher incidence of HCV co-infection (P = .021). CD4 cells decreased with age (P = .015). NK cells showed a decrease till 60 and increased afterwards (P = .000). HCV incidence was higher till 49 (P = .000), being 38, 8% of the population HCV co-infected. HBV carrier status was presented in 5, 8% and 40, 2% of the population had HBV resolved infection. HAV IgG was presented in 85, 8% of the individuals. CD4+ cells was significantly associated with HIV viral load (P = .000) and CD19+ cells (P = .000).

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Significant Relationship between INNO-IAä HIV I/II Positivity Bands and the Immunological Status of HIV Patients (Continued)

Discussion and Conclusions: In spite of the limitations of a cross-sectional study, we found a significant relation between INNO-LIAä HIV I/II positivity bands and immunological parameters in HIV infected patients. Anti-sgp120, anti-gp41 and anti-p31positivity are associated with a more immunodepressed status and a higher HIV viral replication. Concerning p24 antibody: is there a protective role for women? Is there a role for anti-p24 in vaccine issue? HCV co-infection and past HAV infection are associated with poorer immunological parameters. This issue highlights the importance of the chronic immune activation as the major factor for AIDS progression. The correlation found between CD4+ cells, CD19+cells and HIV viral load highlights the importance of the study of B cells in the pathogenesis and therapy for HIV infection.



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The HIV-Positive Inpatient: Psychosocial Risks and Adherence Implications

Rebecca L. Wald, Stephen J. Synowski, and Lydia R. Temoshok Institute of Human Virology and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA Prior research on adherence to anti-retroviral therapy (ART) has focused on correlates of adherence among outpatients. Little is known about adherence among hospital inpatients with HIV, or about the prevalence of psychosocial factors associated with nonadherence in inpatient populations, despite the fact that their poorer health status typically denotes a critical need for ART initiation and adherence. 200 HIV-positive inpatients and 200 outpatients (52% male, 95% African-American) completed a structured interview assessing nonadherence to ART and psychosocial variables associated in previous research with nonadherence. Inpatients reported missing significantly more doses of ART in the week prior to hospitalization than did outpatients (t = 5.67, P , 001). Inpatients had poorer scores on measures of multiple psychosocial predictors of nonadherence: social instability (t = 23.42, P = 001), depressive symptoms (t = 23.61, P , 001), other psychiatric symptoms (t = 2.95, P = 003), recent heroin or cocaine use (x2 = 25.2, P , 001) and cognitive dysfunction (t = 22.76, P = 006), and reported life stressors of greater severity (t = 3.99, P , 001). Inpatients were significantly less likely to have a regular HIV doctor (x2 = 37.2, P , 001) or to have seen an HIV specialist in the 6 months prior to hospitalization (x2 = 74.37, P , 001); those in care reported lower satisfaction with their provider (t = 217.34, P , 001). In summary, chronic and severe psychosocial problems found in prior research to be associated with nonadherence are widespread among HIV-positive inpatients. Inpatients also display a substantial lack of engagement in HIV care. Adherence studies conducted with outpatients do not adequately reflect the difficulties of this population. Most inpatients will require intensive psychosocial interventions in order to achieve ART success.

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Psychosocial Contributors to Antiretroviral Adherence: Stability and Change

Rebecca L. Wald, Stephen J. Synowski, and Lydia R. Temoshok Institute of Human Virology and Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA Adherence to anti-retroviral therapy (ART) is critical to HIV treatment success, yet many fail to achieve the .95% adherence necessary for viral suppression. This study examined the stability of psychosocial predictors of adherence. 127 HIV+ adults (92% African-American, 51% female, mean age 44.5), enrolled in a longitudinal study of HIV progression and receiving ART, completed a structured interview assessing nonadherence and related psychosocial variables at baseline and at 6- and 12-month follow up. The Hardiness Scale (HS), Perceived Stress Scale (PSS), and a coping measure (Vignette Similarity Rating Method; VSRM) were also completed. Patients were classified as adherent if they took .95% of ART doses in the past week. Adherence was stable from baseline to 6-month (O2 = 15.86, P , 01) and 12-month (O2 = 8.31, P , 01) follow-up. At each point, different psychosocial problems were associated with concurrent nonadherence: at baseline, depression (t = 2.13, P = 04), psychiatric symptoms (t = 2.23, P = 03), and interviewer-rated stress severity (t = 2.63, P = 01); at 6 months, stress severity (t = 2.36, P = 02) and hopelessness (VSRM) (t = 2 1.88, P = 07); and at 12 months social support (t = 22.33, P = 02), unsupportive social interactions (t = 2.073, P = 042), alcohol use (t = 22.86, P , 01), stress severity (t = 1.875, P = 06), and PSS scores (t = 1.863, P = 06). Being adherent at follow-up was associated with baseline coping variables: higher scores for optimism (t = 2.86, P , 01); adaptive coping (VSRM) (t = 22.07, P = 042); and the Commitment (t = 22.51, P = 02) and Control (t = 22.15, P = 04) subscales of the HS, which respectively assess a sense of purpose and a sense of autonomy. Nonadherence in this sample was stable over time, but the associated acute psychosocial problems differed at each time point. The stability of adherence appears to be driven by more stable, trait-like coping patterns.



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Systolic Blood Pressure recovery following mental stress predicts immune dysregulation in persons with HIV

Stephen J. Synowski, PhD 725 West Lombard Street, Baltimore, Maryland 21201 Cardiovascular reactivity (CVR) to, and prolonged recovery from, mental stress have been associated with impaired immune function. In 128 HIV-infected patients enrolled in a longitudinal study of potential mechanisms of HIV progression, baseline analyses revealed that exaggerated CVR and a poorer recovery were associated with decreased production of the HIV-inhibiting betachemokines MIP-1a/b (which block the HIV co-receptor CCR5 and thus, HIV entry into CD4+ T-cells). We hypothesized that these relations would be part of a chronic pattern of psychophysiologic dysregulation. At 6-month follow-up, participants were 95 HIV+ adults (92% African-American, 51% female, mean age 44.5). At baseline, systolic and diastolic blood pressure (SBP, DBP) and heart rate (HR) were monitored during emotion-provoking anger recall and role play tasks; each task was preceded by 10 minutes of rest and followed by 5 minutes of recovery. At 6 month follow-up, in vitro production of MIP-1a/b was assessed as stimulated by the core HIV protein p24. Supernatants were collected on days 3 and 6, and assays performed by ELISA. Poorer recovery to resting SBP levels following role play (at baseline) predicted decreased 6-month production of MIP-1a (b = 20.21, P , 044, R = 0.74) and MIP-1b (b = 20.26, P , 016, R = 0.63). Additionally, the relations between SBP reactivity (b = 20.18, P , 093, R = 0.67) and poorer recovery (b = 20.19, P , 079, R = 0.64) following anger recall and decreased MIP-1b production approached statistical significance. In sum, prolonged SBP recovery from mental stress at baseline significantly predicted decreased HIV-specific immune response at 6 month follow-up. These findings are consistent with previous results obtained at baseline, and suggest that a chronic pattern of psychophysiological dysregulation may be associated with suppression of anti-HIV b-chemokine production, and thus, may be implicated in HIV progression.

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Stimulation of reverse cholesterol transport potently suppresses HIV-1 replication

Michael Bukrinsky The George Washington University, 2300 Eye St. NW, Washington, DC 20037 Cholesterol plays an important role in HIV life cycle, and infectivity of cholesterol-depleted HIV virions is significantly impaired. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits activity of the cholesterol transporter ABCA1 and impairs cholesterol efflux from infected macrophages. However, the role of this pathway in HIV infection of CD4+ T cells remained unclear. In this study, we investigated the effect on HIV replication of an LXR agonist, TO-901317, which is a potent inducer of ABCA1 expression and of reverse cholesterol transport. Treatment with TO-901317 restored cholesterol efflux from HIV-infected macrophages and CD4+ T lymphocytes, and potently suppressed HIV-1 replication in both cell types. The drug also inhibited HIV-1 replication in ex vivo cultured lymphoid tissue and in humanized Rag2/2gc2/2 mice. Cholesterol analysis demonstrated a significant enrichment with cholesterol of membrane rafts in cells infected with wild-type, but not Nef-deficient, HIV-1. Stimulation with LXR agonist reduced raft cholesterol in both infected and uninfected cells. The amount of virion-associated cholesterol correlated with raft cholesterol: Nefdeficient virus had less cholesterol than the wild-type virus, and cholesterol in both types of virions was reduced by LXR agonist. Importantly, infectivity of virions closely matched the amount of virionassociated cholesterol. These results demonstrate an extreme dependence of HIV infectivity on activity of reverse cholesterol transport and describe a novel approach to reduce HIV virion cholesterol. They suggest that drugs targeting LXR-ABCA1 pathway, in addition to their anti-atherogenic activity, are potential anti-HIV agents working through a novel mechanism of action. Such drugs may provide a double benefit to HIV-infected patients by reducing HIV replication and preventing development of atherosclerosis associated with HIV infection.



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Monocyte-Dependent and –Independent Modulatory Effects of Vitamin D3 on X4 and R5 HIV-1 Replication in IL-2 Stimulated PBMC

Massimo Alfano,1 Chiara Rizzi,1 Giuseppe Penna,2 Luciano Adorini,2 and Guido Poli1,3 1

San Raffaele Scientific Institute; 2BioXell SpA; 3Vita-Salute San Raffaele University, School of Medicine, Via Olgettina n. 58, 20132 Milan, Italy

Background: Vitamin D3 (calcitriol) is a well known mononuclear phagocyte differentiating agent as well as a potent modulator of the immune system. We investigated the potential role of Vit D3 on the replication of R5 and X4 HIV-1 strains in primary PBMC stimulated with IL-2 in the absence of PHA, a model system in which virus replication is dependent upon monocyte-T cell interaction and release of endogenous cytokines. Methods: IL-2 PBMC were incubated with Vit D3 or vitamin D receptor (VDR) agonists like calcitriol analog BXL219 and infected with laboratory adapted or primary X4 and R5 HIV-1 strains. Virus production and cytokines levels were measured in culture supernatants, chemotaxis and the levels of CD4, CCR5 and CXCR4 were determined by cytofluorimetric analysis. Results: VDR agonists enhanced laboratory-adapted and primary R5 and X4 HIV-1 replication in IL2 PBMC. Unlike R5 HIV-1, X4 virus spreading was initially inhibited by VDR agonists in the first 2-3 weeks of infection, as determined by virion-associated RT activity in cell culture supernatants. Neither the levels of CD4, CCR5 or CXCR4 expression nor the chemotactic response to their ligands CCL4 and CXCL12 were affected by calcitriol. Neither R5 nor X4 HIV-1 infection affected significantly the production of pro-inflammatory cytokines (TNF-a, IL-1b, IFN-g and IL-6), IL-1ra and chemokines (CCL3, CCL4, CCL5, CXCL8 and CCL11) by IL-2 PBMC. Calcitriol significantly inhibited the production of TNF-a, IFN-g and CCL5, but not of the other tested molecules, in both infected and uninfected cells. Of interest, X4 infection did not modify calcitriol effect on TNF-a and IFN-g whereas it abrogated the inhibitory effect induced by calcitriol on CCL5 expression. Stimulation of IL-2 PBMC with calcitriol enhanced the differentiation of monocytes into adherent macrophages. The replication of both R5 and, remarkably, X4 HIV-1 was greatly enhanced in adherent IL-2 PBMC (mostly monocyte/macrophages) upon stimulation with VDR agonists whereas non-adherent lymphocytes showed patterns of virus replication superimposable to those of unfractionated IL-2 PBMC. Conclusions: VDR agonists predominantly enhance HIV replication in both lymphocytes and, particularly, monocyte/macrophages regardless of which chemokine entry co-receptor is used in PBMC stimulated with IL-2 suggesting caution in their clinical use in HIV-infected individuals.

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Naturally Occurring C-terminally Truncated Isoform of STAT5 (STAT5D) Is a Transcriptional Repressor of HIV-1 Expression. Characterization of the PlatinumBased STAT Inhibitor CPA-7

Giulia Della Chiara,1 Andrea Crotti,1 Heidi Kay,2 and Guido Poli1 1

AIDS Immunopathogenesis Unit - San Raffaele Scientific Institute, Milano, Italy; 2College of Public Health, University of South Florida, Tampa, Florida Signal transducers and activator of transcription (STAT) proteins, namely STAT1 and STAT5D, are frequently constitutively activated in the PBMC of most of HIV-1+ individuals. We previously described that STAT5D is also the only STAT5 isoform detectable in the chronically HIV-1 infected promonocytic cell line U1 characterized by a constitutive state of viral latency and inducibility of virus expression by PMA or several cytokines. We have recently reported that STAT5D can act as inhibitor of HIV-1 transcription in GM-CSF-stimulated U1 cells and IL-2 stimulated PBMC of HIV+ individuals due to its binding to target DNA sequence in the provirus LTR causing an impaired recruitment of RNA Pol II (A. Crotti et al., Blood, 2007). GM-CSF also triggered the late activation of an ERK/AP-1 dependent pathway inducing HIV-1 expression in U1 cells. Selective inhibition of this pathway turned off, while inhibitors of STAT5 enhanced, viral expression in GM-CSF stimulated U1 cells. We are currently investigating a new platinum-based compound, termed CPA-7, reported to interfere with STAT3 in several solid tumor cell lines by inducing apoptosis. Our preliminary results demonstrate that CPA-7 increased HIV-1 expression in GM-CSF stimulated U1 cells. Further investigation will clarify the role of this compound in the context of STAT-induced HIV-1 expression and will suggest a possible therapeutic use of CPA-7 in the context of HIV reactivation from latently infected cells.



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Modulation of CCR5 density with low doses of the transplant drug Rapamycin sensitizes Vicrivirocresistant R5 HIV-1

Alonso Heredia Institute of Human Virology, Rm N549, 725 W. Lombard Street, Baltimore, MD CCR5 antagonist (CA) are a new class of anti-HIV drugs. With a novel mechanism of action, they offer additional treatment options to patients, especially those infected with drug-resistant HIV-1. Maraviroc is the first approved CA. Vicriviroc (VCV), another CA, is undergoing evaluation in Phase III clinical trials. We show that reduction of CCR5 density (CCR5 receptors/cell) with low doses of the transplant drug Rapamycin (RAPA) synergistically enhances VCV antiviral activity (see Abstracts by Latinovic et al.). We have also evaluated the antiviral activities of RAPA and RAPA/VCV combination against D1/85.16 and CC101.19, two VCV-resistant R5 HIV-1 strains that use CCR5 but can also use VCVbound CCR5. RAPA inhibited these viruses by up to 60%, in agreement with their dependence on CCR5 for infection. Importantly, RAPA partially sensitized these viruses to VCV, with the RAPA/VCV combination inhibiting viral production in lymphocytes by ;90%. We further demonstrate that resistance to VCV in IL-2 primed lymphocytes depends on donor CCR5 density, with lower densities partially inhibiting virus replication. We confirmed the effect of CCR5 density on VCV resistance in pseudovirus infection of cell lines with same CD4 but varying CCR5 densities. Titration of VCV against VCV-resistant pseudovirus yielded incomplete inhibition curves, with inhibition plateau heights inversely correlated to CCR5 density. In T-20 time-of-addition experiments, VCV-resistant pseudovirus infection kinetics were considerably slower in VCV-bound than in CCR5 free usage. These results indicate that VCV-resistant HIV uses VCV-bound CCR5 less efficiently than free CCR5 and suggest that the affinity of the VCV-resistant envelope is lower for bound CCR5 than for free CCR5. An alternative explanation to differences in affinity is that the energy requirements for induction of conformational changes in gp41 following CCR5 binding are higher in use of VCV-bound CCR5 than in use of free CCR5. Together, these results suggest CCR5 modulation with low doses of RAPA as a potential approach to prevent and control resistance to VCV in patients.

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Risk factors for Virologic Failure and Adverse Reactions among Patients on Triple Antiretroviral therapy

Adedayo Adeyemi, Oluseyi Adesola, and Oluyemisi Olaogun Healthmatch International, Lagos, Nigeria Introduction: Adverse effects from antiretroviral (ARV) drugs can result in poor compliance and which may results in treatment failure. Recognition and management of adverse effects are important issues in improving quality of lives of HIV positive patients. This study examined the prevalence of adverse effects and the risk factors associated with adverse effects and virologic failure. Methods: Retrospective chart review of 146 naive patients on antiretroviral therapy between January 2007 and December 2007 was done in Lagos Nigeria. The following information was obtained: time of first diagnosis, demographical information, missing clinic, CD4 count, weight, tuberculosis/opportunistic infections, viral loads (at least one in 6 months after starting ARV), and partner/family notification. Results: Female 93 (64%), male 53 (36%); mean age: 33.4 years; 234 person-years of follow-up with a mean of follow-up of 1.6 6 0.7 years; 126 (86%) had undetectable viral load at 6 months of ARV; mean weight change from 56 kg to 62 kg; and 53 (36%) experienced adverse effects. Adverse effects were peripheral neuropathy 35 (24%); gynecomasia 6 (4%) lactic acidosis 10 (7%); diarrhea 18 (12%); nausea and vomiting 4 (3%); hepatitis 6 (4%); Stevens-Johnson syndrome 3 (2%); rash 20 (14%); insomnia 12 (8%); lipodystrophy/lipoatrophy 9 (6%). CD4 count and Viral load at initiation were 2 significant predictors of virologic failure, with CD4 , 100 cells/mm3 (OR 1.6, 95% CI 1.2 to 1.9) and viral load at initiation $100,000 copies/mL (OR 2.1, 95% CI 1.5–2.7). For the adverse effects: patients with weight ,60 kg (OR: 3.2, 95% CI 1.9 to 4.2) and with age .40 years OR 2.4 (1.2 to 3.9) are more likely to experience adverse effects. Mean change of regimen was 13.5 6 6.2 months. Conclusion: Despite increasing access to ARV, good drug toxicity monitoring system should be put in place so that life threatening adverse effect is adequately screened for and proper managed. However, care should be taken with D4T associated peripheral neuropathy. More surveillance studies on HIV drug toxicity are needed to enhance safety and effectiveness of the therapy in Nigeria.



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Design, Synthesis, Anti-HIV and Cytotoxicity of Novel Heterocyclic Compounds

Periyasamy Selvam Amrita School of Pharmacy, Elmakkara, P.O. Kochi, 682026, Kerala, India AIDS is a fatal pathogenic diseases caused by retrovirus HIV. Recently much attention has been devoted for searching of effective chemotherapeutic agents for combat HIV/AIDS. Present work is to design and synthesis of novel heterocyclic compounds form Indole, Benzoxazine, Quinoxaline, Quinazolinone, Flouroquinolone, Phthalimide, benztriazole and benzimidazole lead molecules and characterized by spectral analysis. Synthesized compounds were screened for in vitro antiviral activity against HIV-1 & 2 in MT-4 cells. Cytotoxicity is also investigated in uninfected MT-4 cells by MTT assay. From the results of anti-HIV activity, 2,3-diphenylquinoxaline and 3-sulphonamido-quinazolinones inhibits and sulphadimidinylmethyl benzimidazole replication of HIV-1 and 2. Benztriazole and Benzimidazole derivatives displayed marked cytostatic activity in MT-4 cells. Details of Design, Synthesis, anti-HIV activity and cytotoxicity will be presented.

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Inhibition of HIV replication and integrase activity by isatin derivatives

Periyasamy Selvam Amrita School of Pharmacy, Elmakkara, P.O. Kochi, 682026, Kerala, India We evaluated the broadspectrum antiretroviral activity of some novel isatin (SPIII) and its derivatives using different strains of HIV-1, HIV-2 and SIV. Several SPIII derivatives were found to inhibit HIV-1 (IIIB and NL4.3) replication in MT-4 cells at EC50 values ranging from 3.6 to 49.6 mg/mL. No cytotoxicity was observed at concentrations up to 125 mg/mL in mock-infected MT-4 cells. The compounds were evaluated for their inhibitory effects against a variety of mutant HIV strains. The SPIII derivatives showed low level resistance (up to 11-fold) to NL4.3 virus strains resistant to agents that block virus entry, i.e. NL4.3 strains resistant to the binding inhibitor dextran sulphate or the CXCR4 antagonist AMD3100. Moreover, the SPIII-5Br and 5Cl derivatives showed some low level crossresistance to an integrase inhibitor resistant virus. We observed no cross-resistance against the fusion inhibitor, nucleoside reverse transcriptase, nonnucleoside reverse transcriptase or protease inhibitor resistant HIV strains. The SPIII compounds were found to inhibit the human immunodeficiency virus type 1 integrase enzymatic activity, HIV-1 integrase binding to target DNA and adsorption of HIV-1 to MT-4 cells, all at IC50 values in the mg/mL range. We conclude that it would be interesting to synthesize and evaluate more derivatives of SPIII to identify more selective congeners. These compounds would enable us to determine the structural requirements for inhibition of entry or integration steps in the replication cycle of HIV.



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Pathogenesis induced acidosis and hpercalceamia evident in HIV/AIDS disease

Abdulrazak Hamza Yahaya HIV/Immunology Laboratory, Pathology Department Murtala Muhammad Specialist Hospital, Kano, Nigeria Background: Metabolism is essential to functioning of cellular systems. HIV destroys the host immune system and induced metabolic changes that interfere with their normal functions. It is imperative to control the induced changes caused by the viral disease while rehabilitating the host immune system through therapeutic care. Methods: Serum electrolytes and calcium were studied among eighty volunteers, 50% HIV infected and 50% were uninfected. Volunteers’ bio data were obtained using structured questionnaires and blood samples were collected and tested for HIV antibodies and electrolytes data were analyzed by SPSS v 12.0. Results: Study observed marked variation in mean values of electrolytes between HIV infected and uninfected volunteers. Respective serum concentration of Calcium, bicarbonate and potassium recorded in infected and uninfected groups were 35.9 6 16.8 mg/dL, 18.6 6 3.8 mMol/L, 4.45 6 1.0 mMol/L and 10.4 6 1.0 mg/dL, 29.7 6 1.0 mMol/L, 3.9 6 0.9 mMol/L. HIV pathogenesis induce changes in host system, most interesting are those related to immune coordination and metabolism; observed alteration in calcium and bicarbonate in HIV/AIDS patients studied are significant at P , 0.01. Conclusions: Great deal of care is required for HIV/AIDS patients to have a quality life. Finding from our biochemical evaluation results showed variation in serum bicarbonate and calcium level among infected cohorts as compared with uninfected cohorts, thus there is need for correcting these abnormalities in HIV/AIDS disease. Normal hosts’ metabolisms required for correct cellular processes and safety from drug toxicity can be altered by electrolytes imbalance. Key Words: alteration; electrolytes; HIV

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Retinopathy and enteropathy in HIV patients, a hypothetical concept beyond Virus and CD4 levelsa study from Varanasi, North India

Dr. V. Satya Suresh Attili, Prof, Shyam Sundra, Prof, V. P. Singh, Prof, and Prof, A. K. Gulati Room No 20, Yashoda Hospitals, Somajiguda, Rajbhavan Road, Hyderabad-500082 Background: Based on our previous observations which were published (Asian journal of ophthalmology, 2006. July p341-48 and BMC Infectious Diseases 2006, 6:39) we thought of re evaluating the results of the hospital based cohort to propose possible hypothesis and evaluate it in prospective manner. Aim: To evaluate the effect of local immunity on development of HIV retinopathy and enteropathy in a hospital based cohort at Institute of Medical Sciences, Banaras Hindu university, Varanasi, India. Methods: All the patients with HIV retinopathy or enteropathy (diagnosis of which was established by ruling out all other pathologies like opportunistic infections, malignancies in cases of enteropathy and infections, malignancies and other confounders like hypertension, diabetes in retinopathy cases) were enrolled and equal number of controls, who had similar symptoms during the same time was recruited for comparison. The baseline characters, CD4 levels, duration of symptoms, number of episodes and other possible confounders were compared in the groups. Results: The multivariate analysis for the retinopathy suggested that the local inflammation & immunity are most important risk factors as significant number of patients have near normal circulating CD4 counts. All retinopathy patients had some form of local infection (which indirectly indicated a decreased local immunity) which further supports our hypothesis. Similarly in patients with enteropathy, we found that around 78% of chronic and 45% of acute diarrheal episodes were experienced by patients with CD4 counts of .200 cells/mL, indicating a regional immunosuppression. Similar observations were made earlier (GUT. 1995;37:524–29) where authors observed that, loss of CD4 cells in intestinal mucosa of the patients with diarrhea, were more pronounced than peripheral CD4 levels and their relation is quite variable. Conclusions: The local immunity, an important factor to prevent retinopathy as well as enteropathy may therefore be variable and therefore they can be observed even in patients with good immunity (i.e. peripheral CD4 . 200). However, it would be premature to draw definitive conclusions beyond this, as the present study is retrospective one and number of patients analyzed was relatively small.



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Immunosuppression Level In HIV-1 –Infected Patients Doesn’t Affect the Serological Diagnosis of Hepatitis C Virus

Fernanda Leite, and Luciana Pinho Clinical Haematology Dept- Hospital Geral Santo Antonio- Porto- Portugal Objective: The aim of our work was to study immunological variables in RIBAÒHCV 3.0 results in HIV-1/HCV Co-Infected Patients (CIP). Methods: we retrospectively looked for HIV-1/HCV CIP with RIBAÒHCV 3.0 Indeterminate (Group 1) and RIBAÒHCV 3.0 Positive (Group 2), between 1/2002 and 1/2007. We looked for age, gender, n° total leucocytes, n° total lymphocytes, n° of CD3+, CD4+, CD8+, CD19+, NK cells and HIV-1 viral load. We studied 87 individuals, 41-Group 1 (47, 1%) and 46- Group 2 (52, 9%); 17 were female (19, 5%) and 70 male (80, 5%) with ages between 1 and 69 years old with mean age in the subgroup 30–39 years. Two individuals were excluded from the study because data wasn’t coincident in time. Data statistical analysis was performed with SPSS program and P value , 0.05 was regarded significant. Results: The 2 groups didn’t show differences statistically significant considering age. In Group 1 there wasn’t a significant relation between the HIV-1 viral load and CD4 cells in contrast with Group 2 that showed an inverse relation between these two variables (P = .040). In Group 2 the n° of CD4+ cells was lower than Group 1 (P = 0.49) with mean CD4+ cells in total population of 407/il. We found a significant positive relation between CD19+ cells and CD4+ cells in all population (Group 1- p. = .006; Group 2-p. =.003). We didn’t find significant differences in the parameters studied in the two Groups; except when CD4 were inferior to 50 cells/il (n = 7), Group 1 showed lower CD3+ cells than Group 2 (P = .029). Conclusions and Discussion: We didn’t find significant differences between the two Groups considering the immunological variables and HIV-1 viral load. The number of CD4+ cells didn’t influence RIBA HCV indeterminate results, in spite of only 7 individuals had less than 50 T CD4+ cells. We hypothesize there’s a higher decrease in circulating memory B cell population in the HIV-1/HCV coinfected RIBA indeterminate Group since very early in the infection occurs a preferential depletion of splenic marginal zone-like peripheral blood CD27+B220- memory cells in HIV-1 individuals after M. Morrow et al. (2006) not related with CD4 cells nor viral load what may contribute to the impaired immunity response against pathogenic antigens. The positive significant relation found between CD19+ and CD4+ cells highlight the importance of the study of B cells in the pathogenesis and treatment of HIV disease.

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Serum albumin: Could it be an inexpensive and simple marker of immunosupression in HIV-infected individuals?

Muthu Sundaram YRG Centre for AIDS Research and Education (YRG CARE), Voluntary Health Services Campus, Chennai 600 113, India Background and Aims: Several studies have suggested that albumin could serve as a useful marker of HIV disease progression in resource-limited settings. We identified the relationship between serum albumin and absolute CD4+ cell count in subjects attending a HIV referral centre in Chennai, Southern India. Methods: In a cross-sectional study, 200 HIV-1 infected individuals were studied. The absolute CD4+ cell counts were determined using Guava Personal Cell Analyser (Guava Technologies Inc., Hayward, CA, USA). Serum albumin was estimated by Bromo Cresol Green method (BCG) using Olympus AU 400 clinical chemistry autoanalyzer (Olympus Optical, Japan). Results: We observed that the correlation co-efficient of the serum albumin assay as compared with the absolute CD4+ cell count was 0.90 (CI:058–0.92; P = 0.0002). Subjects that had absolute CD4+ cell count ,200 cells/mL had a better correlation (R = 0.83, 95% CI:0.72–0.92; P = 0.001) than those with 200–350 cells/mL (R = 0.82, 95% CI:0.72–0.93; P = 0.001) and .350 cells/mL (R = 0.71, 95% CI:0.52–0.80; P = 0.106). The sensitivity and specificity between serum albumin and absolute CD4+ cell ,200 cells/mL were 97% and 44% respectively. Conclusions: Serum albumin was strongly associated with CD4+ cell count. However, this warrants further investigation on their possible application as a useful marker of HIV disease progression and monitoring of treatment outcome in resource-limited settings.



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Increased IFN-alpha expression in circulating plasmacytoid dendritic cells of HIV-1 infected patients despite selective loss

Clara Lehmann, MD,1,3 Dirk Taubert, PhD,2 Jill M. Harper, PhD,3 Norma Jung, MD,1 Pia Hartmann, MD,1 Gerd Fa¨tkenheuer, MD,1 and Fabio Romerio, PhD3 1

First Department of Internal Medicine, University of Cologne, Cologne, Germany; 2Department of Pharmacology, University of Cologne, Cologne, Germany; and 3Institute of Human Virology, University of Maryland School of Medicine, Baltimore, MD

Background: The role of plasmacytoid dendritic cells (PDC) and interferon alpha (IFNg) in HIV-1 infection is still unclear. On one hand, HIV-1 disease is associated with a progressive decline of pDC, which display reduced ability to produce IFNg after in vitro challenge. On the other hand, high IFNg serum levels in HIV-1 infected individuals have been proposed to promote immune hyper-activation and disease progression. Further, it remains unknown whether distinct IFNg subtypes are expressed with disease progression. Methods: We sought to determine whether disappearance of pDC in HIV-1 disease is due to homing in lymphoid tissues. We also studied IFNg and MxA expression as well as the profile expression levels of twelve known IFNg subtypes in unstimulated pDC, and correlated these results with selected clinical and laboratory parameters in PBMC from normal donors and HIV-1 patients at CDC stage A and stage C of the disease. Results: PDC decline markedly in peripheral blood of HIV-1 patients progressing to disease, but at the same time express much higher levels of IFNg and MxA compared to control individuals. On the other hand, steady pDC counts in lymph nodes of HIV-1 patients are observed, which correlated directly with CD4 cell counts and inversely with viral load. However, no correlation between IFNg and MxA expression levels, CD4 counts, and viral load were found. The analysis of IFNga subtype expression pattern demonstrated high IFNg2 and 6 mRNA levels in both patient groups. Especially, IFNg2 was upregulated .60-fold in stage A and .400-fold in stage C patients compared to controls, which correlated with declining CD4 counts. The subtypes IFNg1/13, 8, 14, 16, 17 and 21 were upregulated in stage C but not stage A patients. The other IFNg subtypes did not change among the three groups. Conclusions: Circulating pDC decline sharply in the course of HIV-1 disease, but express high levels of IFNg. Furthermore, distinct IFNg subtypes are sequentially activated during HIV-1 infection, which may be predictive of disease progression.

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HIV-Induced Alterations in Vg2Vd2 T-cell Phenotype and Function Impact Mechanisms for Tumor Immunity in AIDS

Jean-Saville Cummings IHV- Room s507A, 725 W. Lombard St, Baltimore, MD 21201 Human Vg2Vd2 T-cells represent 3-10% of CD3+ lymphocytes and are known to function in innate tumor surveillance. Several studies have demonstrated that HIV infection causes rapid and lasting defects in the population of Vg2+ cells, specifically describing a reduction in the overall frequency of circulating Vg2Jg1.2-Vd2 T-cells. In an effort to further describe the impact of HIV on these cells, we examined PBMC samples from HIV+ patients receiving HAART who had displayed prolonged viral control and CD4 counts above 300 cells/mm3. Among HIV+ individuals we observed lower frequencies of CD27-/CD45RA- Vg2Vd2 cells when compared to uninfected controls. Vg2+ T-cells from HIV+ donors had lower expression of granzyme B and displayed reduced cytotoxicity against Daudi cell targets after in vitro stimulation. Additionally, the frequency of tumor reactive CD56+ Vg2 effectors was reduced among HIV+ individuals when compared to controls. Similar reductions in CD56+ frequency were observed among CD3- NK cells and CD3+Vg2- aa T cells within the HIV patient group. There was increased expression of Fas receptor (CD95) on Vg2 cells from HIV+ PBMC, which may be a mechanism for depletion of Vg2 cells during disease. In addition to the well-characterized defects in Vg2 repertoire and functional responses to phosphoantigen, the proportion of CD27-/CD45RAVg2Vd2 T-cells after IPP stimulation was reduced sharply in HIV+ donors versus controls. Similarly, the CD56+ frequency of Vg2+ cells generated after PBMC stimulation in vitro was also reduced among HIV+ individuals. None of the differences observed among HIV+ donors were related to CD4 count or vRNA burden. Thus, HIV infection has multiple impacts on the circulating Vg2Vd2 T-cell population that combine to reduce the potential effector activity in terms of tumor cytotoxicity. Changes in Vg2Vd2 T-cells, along with concomitant effects on NK and NKT cells that also contribute to tumor surveillance, may be important factors for elevating the risk of malignancy during AIDS.



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Mechanisms regulating Vg2Vd2 tumor cell cytotoxicity: Roles for CD56 and T cell receptor

Kun Luo Institute of Human Virology University of Maryland, 725W Lombard Street, Baltimore, MD 21201 Human Vg2Vd2 T lymphocytes respond to stimulation with non-peptidic phosphoantigens plus IL-2 and become cytotoxic for a variety of human tumor cell lines. Recently, we reported that CD56 expression marked a subset of activated Vg2Vd2 T cells that were most potent in cytotoxicity assays (Alexander, et al., Clin. Cancer Res. 14: 4232, 2008). These observations raised important question about tumor cell recognition and cytotoxicity byVg2Vd2 T cells. We found that high level CD56 expression was induced when CD56- cells were stimulated with IL-2. Cell surface CD56 expression reflected a substantial accumulation of CD56 mRNA due to elevated levels of the RunX1p48 transcription factor. Vg2 chain repertoire studies revealed that CD56 expression was not related to TCR specificity or strength of response to phosphantigen, but rather reflected a pre-existing property of individual Vg2Vd2 T cells that included higher cytokine expression, resistance to Fas- or TNF- mediated cell death and down-regulation of several cell death-related genes. CD56 cross-linking on CD56+ Vg2Vd2 T cells without TCR ligation was sufficient to drive production of the cytokines IFN-g or TNF-a and to increase IPP-induced proliferation of Vg2Vd2 T cells. Thus, CD56 and the lack of CD56+ Vg2Vd2 cells in patients with HIV, is consistent with reduced timor immunity in this disease. The absent of specific Vg2Vd2 cells and the inability to expression CD56 after stimulation, are key defects related to HIV infection, that might lead to the increased risk to malignant disease.

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Altered cord blood ga T cell repertoire in Nigeria: possible impacts of environmental factors on neonatal immunity

C. Cairo Institute Of Human Virology, 725 W Lombard St, Baltimore, MD 21201 Infectious diseases during pregnancy can impact the development of fetal immunity, resulting in reduced neonatal resistance to infection and decreased responses to pediatric vaccines. In tropical and subtropical countries, high rates of infectious diseases including malaria will change the landscape for HIV vertical transmission and for future pediatric vaccine programs. Here we show initial studies on fetal immunity for Nigeria, where P. falciparum, Mycobacterium tuberculosis and HIV infection are prevalent. We focus on aa T cells because they respond to all three pathogens and provide a marker for fetal immune system capacity. Responses to Bacille Calmette-Guerin (BCG), malaria and HIV are dictated by the Vg2Vd2 T cell receptor. We compared cord blood gd T cells from deliveries to HIV- mothers in Jos (Nigeria), or in Rome (Italy) and we noted substantial differences in the Vg2 repertoire. In specimens from Jos, we observed a specific absence of Vg2 chains, in particular the Vg2-Ja 1.2 subset, that is most responsive to pathogen stimulation and that we expect will respond to BCG. Differences in the levels of aa T cells able to recognize pathogens or vaccines might be due to host genetics, prior pathogen exposure, or other environmental factors including nutrition. At present, we cannot discriminate these possibilities. The unique feature of aa T cells, that they react to an array of pathogens, suggests that lower levels due to any the of possible causes predispose infants to vertically (HIV) and horizontally (malaria and tuberculosis) transmitted pathogens and may reduce responses to pediatric vaccines.



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TNF-a is an autocrine factor for Va2Va2 T cell

Haishan Li Institute of Human Virology, 725 W Lombard Street, Baltimore, MD 21201 Human Vg2Vd2 T cells recognize phosphoantigen and play important roles in host defense and immunoregulation. For example, the Va2Ja1.2Va2+ subset responds to multiple pathogens including mycobacteria and malaria but is extensively and specifically depleted in HIV disease. The T cell receptor is required for Vg2Vd2 T cell responses to phosphoantigens which are mostly isoprenoid intermediates of cholesterol synthesis, but less is known about soluble or cell-associated costimulatory molecules that might regulate effector responses. In the present study, we focused on the impact of TNF-g on Vg2Vd2 T cell function. The Vg2Vd2 T cell responses to antigen stimulation, including activation, proliferation, cytokine production and tumor cell cytotoxicity, were impaired significantly when TNF-g or its receptor was blocked. Since Vg2Vd2 T cells are also the major source of TNF-g in IPP-stimulated PBMC, this is an autocrine regulatory mechanism that may act to amplify Vg2Vd2 T cell responses and further increase cytokine production. Impaired proliferation in the TNF-g or TNF receptor blocking agents can be rescued by a TLR2 agonist, Pam3Cys that also acts as a costimulator. Our studies demonstrate for the first time that TNF-g plays a critical role in regulating Vg2Vd2 T cell immune responses and provide new insight into their potential roles in infectious disease.

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Peripheral blood mononuclear cells activated by HIV-VLPs in correlation with HIV-1 seropositivity status

L. Buonaguro,1 M. L. Tornesello,1 R. C. Gallo,2 F. M. Marincola,3 G. K. Lewis,2 and F. M. Buonaguro1 1

Lab. of Viral Oncogenesis and Immunotherapies and AIDS Reference Ceneter, Istituto Nazionale Tumori ‘‘Fond. G. Pascale’’, Naples - Italy; 2Institute of Human Virology, Univ. of Maryland School of Medicine, Baltimore, MD, USA; 3Immunogenetics Section, Department of Transfusion Medicine, Clinical Center, NIH, Bethesda We have recently shown that HIV-1 Pr55gag Virus-Like Particles (HIVVLPs), produced in a baculovirus expression system and presenting a gp120 molecule from an Ugandan HIV-1 isolate of the clade A (HIV-VLPAs), induce maturation and activation of monocyte-derived dendritic cells (MDDCs) with a production of Th1- and Th2-specific cytokines. Furthermore, HIV-VLP-loaded MDDCs are able to induce a primary and secondary response in autologous human CD4+ T cells, in an ex vivo immunization assay. Fresh human peripheral blood mononuclear cells were isolated by Ficoll-Hypaque density gradient centrifugation and plated in 6well plates at a concentration of 1 3 107/well. PBMCs were pulsed with 6_g/mL of HIV-VLPs. After 16 hrs, the cells were harvested, washed, and stained for phenotypic analysis by flow cytometry. The cellular supernatants were collected for quantification of cytokine production by ELISA. In the present study we show that similar data can be obtained directly on fresh peripheral blood mononuclear cells (PBMCs) and the HIV-1 seropositivity status, with either low or high viremia, does not significantly impair the immune activation status and the responsiveness of circulating monocyte CD14+ cell populations to an immunogenic stimulus. The established Th2 polarization in both HIV seropositive groups is efficiently boosted by HIV-VLP induction and does not switch into a Th1 pattern, strongly suggesting that specific Th1 adjuvants would be required for a therapeutic effectiveness in HIV-1 infected subjects. These results indicate the possibility of screening PBMCs for donor susceptibility to an immunogen treatment, which would greatly simplify the identification of ‘‘responsive’’ vaccinees as well as the understanding of eventual failures in individuals enrolled in clinical trials.



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Potent Orally Bioavailable HIV-1 Fusion Inhibitors Alter Env Conformation and Expose Conserved Neutralization Epitopes

C. Finnegan, V. Dettmer, M. Bramah-Lawani, T. Nitz, P. Bullock, I. Burimski, M. Reddick, C. Matallana, C. Beaubien, D. Stanley, J. Pettitt, G. Allaway, and K. Salzwedel Panacos Pharmaceuticals, Gaithersburg, MD, USA, 20877 Fusion inhibitors are a relatively new class of antiretroviral drugs that block conformational changes in the HIV envelope glycoprotein (Env) critical for virus-cell fusion. Proof-of-concept for this therapeutic approach is provided by enfuvirtide, a peptide that inhibits terminal conformational changes in gp41. The identification of non-peptide, orally bioavailable fusion inhibitors may provide valuable new therapeutic options for people living with HIV/AIDS. Using a cell-based assay for inhibition of critical Env conformational changes we identified three structurally distinct series of drug-like small molecule fusion inhibitors (median MW ;470; cLogP ;4.3). The most potent compounds inhibited HIV-1 (but not HIV-2 or SIV) infection with an IC50 of approximately 5 nM and retained activity against X4 or R5 primary isolates from clades A, B, C, D, F, G, H, and group O. These compounds exhibited oral bioavailability of up to 30% in animal models. Unlike enfuvirtide, these compounds bind to Env prior to CD4 binding and stabilize gp120-gp41 association. Binding of these compounds unmasks conserved neutralization epitopes on Env. The compounds specifically block receptor-induced conformational changes that occur after exposure of the gp120 coreceptor binding site, but before exposure of the gp41 intermediate to which enfuvirtide binds. In vitro experiments indicate that these fusion inhibitors act synergistically with neutralizing antibodies, thereby enhancing their inhibitory potential. In vitro resistance determinants map to conserved regions in both gp120 and gp41. We have discovered a novel class of potent, orally bioavailable HIV-1 fusion inhibitors with a unique mechanism of action and resistance profile. Optimized lead compounds are currently in pre-clinical drug development. These compounds may also have promising applications in Env structural studies.

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In vivo alteration of humoral responses to HIV-1 envelope gp120 by antibodies to the CD4-binding site of gp120

Maria Luisa Visciano 423 East 23rd Street New York, NY, 10010 USA Background: The binding of antibodies (Abs) to the CD4-binding site (CD4bs) of gp120 has been shown to induce gp120 to undergo conformational changes that exposes and/or shields specific epitopes on this glycoprotein. Here we study alteration in the antigenicity and immunogenicity of gp120 when complexed with human monoclonal antibodies (mAbs) specific for the CD4 binding site of gp120. Material and Methods: ELISA was used to assess changes in the antigenicity of gp120 when complexed with anti-CD4bs mAbs or with other anti-gp120 mAbs. To evaluate the immunogenicity of gp120/mAb complexes, BALB/c mice were inoculated with gp120/mAb complexes or with uncomplexed gp120 in RIBI adjuvant. The levels and specificities of serum anti-gp120 Ab responses induced in mice immunized with gp120/anti-CD4bs mAb complexes were compared to those in mice immunized with uncomplexed gp120 or other gp120/mAb complexes. Virus-neutralizing activities of the sera were also tested against HIV-1 isolates in the TZM-bl neutralization assay. Results: The data demonstrate that the binding of anti-CD4bs mAb to gp120 specifically enhanced the reactivity of mAbs to the N-terminal C1 region and the neutralizing epitopes in the V3 loop. Importantly, immunization with gp120/anti-CD4bs mAb complexes consistently elicited higher levels of serum anti-gp120 Abs directed especially to the V3 region. Sera from mice immunized with gp120/anti-CD4bs mAb complexes also exhibited much more potent virus-neutralizing activity than sera of mice receiving uncomplexed gp120 or other gp120/mAb complexes, although the\ neutralizing activity was observed primarily against the homologous HIV-1 strain.



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Dendritic cell-specific delivery of siRNA targeting SOCS1 enhances HIV-gag-specific CD8 T cell response

Sandesh Subramanya Texas Tech University-Health Sciences Center, Department of Biomedical Sciences, 4800 Alberta Avenue, El Paso, TX 79905 Dendritic cells (DC) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNAi-mediated silencing of negative immuno-regulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. To accomplish this, we developed a novel method to deliver siRNA specifically to DCs. A DC-targeting-12-mer peptide when fused to nonamer D-arginine residues (9dR) was able to bind and transduce siRNA into human monocyte-derived DC (MDDC), but not into monocytes or primary T cells. Peptide-mediated delivery of siRNA targeting the suppressor of cytokine signaling-1 (SOCS-1) molecule resulted in efficient gene silencing in MDDC and enhanced their cytokine responses to LPS stimulation. The modified DCs were able to elicit a stronger T cell proliferation in a mixed lymphocyte reaction as compared to mock or irrelevant siRNA-treated DCs. SOCS-1 silenced DCs were also found to enhance primary in vitro response of na¨ve CD8 T cells from healthy A2 (+) donors to the well- characterized Melan-A /MART-1 A2-restricted epitope as well as the HIV-gag specific A2-SLYNTVATL epitope. Finally, in comparison to irrelevant siRNA, in vitro stimulation with autologous SOCS-1 siRNA-transduced DCs resulted in significant increase in HIV-gag specific proliferation and IFN-gamma production of CD8 T cells from seropositive subjects. Collectively, these results demonstrate the feasibility of using a peptide-based approach for targeted delivery of siRNA to DCs. Silencing SOCS-1 expression in DCs enhanced primary and secondary HIV-specific CD8 T cell responses, suggesting that the approach can be exploited for enhancing immunogenicity of DCs for potential immunotherapeutic applications.

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Increased expression of Suppressor of Cytokine Signaling-1 (SOCS-1) by HIV-1 transgenic rats: A mechanism for dysregulated T helper-1 responses

William Reid Institute of Human Virology; 725 W. Lombard Street, Baltimore, MD Antigen-specific T cell proliferation, maintenance of Th1 responses, and dendritic cell (DC) functions are compromised in HIV-1 infected individuals. These abnormalities include increased production of the immunosuppressive cytokine IL-10 and decreased IL-12 by DC, as well as decreased IFN-g production by effector/memory CD4+ T cells. To better understand these immune abnormalities and the extent to which they are independent of chronic antigenic stimulation, we developed an HIV-1 transgenic (Tg) rat, which has defects in Th1 cytokine production and responses and in generation/maintenance of effector/memory phenotype CD4+ T cell subsets. We now report that the Tg rats show defects in both innate and adaptive immune responses. Tg DCs induce elevated levels of SOCS-1 mRNA and secrete decreased IL-12p40 and elevated levels of IL-10 following TLR-4 stimulation by LPS. This leads to further induction of SOCS-1 by IL-10 and decreased IFN-g-mediated induction of interferon response factor (IRF)-1 and IL-12Rb1 expression in CD4+ T cells and to decreased IL-12-induction of IFN-g production by Th1 polarized T cells. IL-12p40 production can be restored by siRNA knockdown of SOCS-1 expression. We also show that SOCS-1 is elevated in CD4+ T cells from HIV-1 infected progressors (900-1 3 106 copies of HIV-1 RNA/mL), and is correlated with defective induction of IRF-1 following IFN-g stimulation, compared with healthy controls and HIV-1 natural viral suppressor (NVS) patients. These results suggest a link between high levels of SOCS-1, defects in innate immunity as determined by LPS induction of IL-12p40 in DCs, and dysregulation in adaptive Th1 immune responses that may be reflected in the loss of Th1 immune competence observed with AIDS patients.



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The A1 subunit of Cholera toxin as an adjuvant for HIV DNA vaccines

Kenneth Bagley 1450 South Rolling Road, Room 4011, Baltimore, MD 21227 Vaccination using plasmid DNA remains a promising means to develop effective prophylactic and therapeutic vaccines against HIV. Unfortunately, their poor immunogenicity in primates demands the use of genetic adjuvants and/or improved delivery techniques. Our approach is to exploit adjuvants, such as the A1 domain of cholera toxin (CTA1), that directly activate dendritic cells. Using SIV gag and HIV gp120 as model antigens in mouse studies, we found that the adjuvanticity of CTA1 is modulated significantly by dose, route (i.m. vs Gene Gun vs electroporation), frequency (1, 2, 3 doses), and type of antigen administered. When delivered via Gene Gun in Cynomolgus macaques, a CTA1 adjuvanted DNA prime significantly enhanced (.10-fold) for a SIV gag humoral response after boosting with a recombinant SIV gag protein. We continue to evaluate CTA1 with additional immunogens to determine if the adjuvanticity diminishes with each subsequent use.

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Genetics of Fc Gamma Receptors IIa and IIIa in Tanzania

Gustavo H. Kijak, PharmD, PhD U.S. Military HIV Research Program, 1600 East Gude Drive, Rockville, MD 20850, USA Background: Non-neutralizing Abs (NNAbs) can combat viral infections by mediating immune complex clearance and Ab-dependent cell cytotoxicity (ADCC) by interacting with FcGamma receptors (FcGRs). In some cases, though, NNAbs can enhance viral infection. Genetic variants of FcGRIIa and FcGRIIIa are important due to their different binding preference and affinity for IgG subclasses, and were shown to influence levels of NNAbs elicited in a vaccine trial with rgp120 (Forthal, 2007). Few reports, and mostly from Caucasian and Asian populations, showed that polymorphic FcGRs exhibit different frequencies in diverse ethnicities. Due to the potential influence of FcGR variants for vaccines, the aim of this study was to define their distribution in East Africa, where HIV vaccine trials are underway or planned. Methods: 174 HIV seronegative consented adults from a cohort development study conducted in Mbeya, Tanzania, were studied. After DNA extraction from PBMCs, polymorphisms at FcGRIIa (131H/R) and FcGRIIIa (158V/F), were typed using real-time PCR (ABI, and newly-developed test). Genetic analyses were conducted using Pypop and Arlequin software packages. Results: Allele frequencies of variants FcGRIIa131H and FcGRIIIa158V were 0.475 and 0.326, respectively. Homozygosity for high-affinity alleles, genotypes putatively associated with enhancement of HIV infection, was frequent (131HH = 25.8%; 158VV = 12.3%; and 131HH:158VV = 4.4%). Individuals homozygous for low-affinity variants, genotypes previously associated with higher benefit from ADCC, were abundant (131RR = 30.8%; 158FF = 47.1%; and 131RR:158FF = 16.4%). Both loci were in Hardy-Weinberg equilibrium and appeared to segregate independently. Conclusion: This is an initial description of the distribution of variants of FcGRIIa and FcGRIIIa in East Africa. The high representation of genotypes having the potential for beneficial, and for detrimental effects on vaccination, makes this a preferred setting to define the impact of variation of FcGRs on vaccine efficacy, and the levels, breath and IgG subclass of neutralizing Abs and NNAbs elicited by vaccination.



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HIV Knowledge and Willingness to Participate in New Preventive Technologies (NPT) Trials among a Nigerian Refugee Population

O. Akinyemi Department of Community Medicine, University College Hospital, Ibadan, Nigeria Objectives: Refugees in Nigeria unlike refugees in some other countries are not restricted to camps but rather interact freely with the general population. It is known that refugees act as a reservoir of sexually transmitted infections and HIV/AIDS within any population because of their vulnerable nature. This study was designed to assess their knowledge about HIV/AIDS and their willingness to participate in NPT trials. Methods: The subjects were recruited by simple random technique utilizing a list of camp inhabitants. The study was conducted utilizing standardized questionnaires with 37 structured questions administered by trained assistants. Two hundred and fifty two questionnaires administered randomly were analyzed using SPSS version 11. Results: The larger percentage (60.7%) of the respondents comprised of Liberians. SierraLeoneans constituted 30.7% of respondents. Other nationalities represented were Cameroonians, Congolese, Ghanaians, Ivorians and Sudanese. One hundred and twenty nine of the 252 respondents (51.2%) were females. The mean age was 27.7 years (SD 6 10.1years). Majority of the respondents (88.1%) had at least some secondary school education while 48.8% had lived in the refugee camp for 5 or more years prior to the survey. Most respondents with at least a secondary school education believed that HIV/AIDS can be cured using local herbs (P = 0.021). Twenty-three (69.7%) of those who had used an illicit drug in the past one month were willing to subject themselves to clinical trials for HIV vaccine (P = 0.06). Eighty-five respondents (45%) with less than secondary school education had never heard of microbicides compared with those (55%) who had a tertiary education or its equivalent (P = 0.04). Conclusion: More still needs to be done in educating this vulnerable group about HIV and also increasing awareness about NPT if we are to reduce the spread of HIV/AIDS among the refugees.

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Knowledge, attitudes and practices (KAP) of sexually active men towards circumcision as a preventive measure against HIV infection in Kitwe district, Zambia

Sthembile Ndopu, and Gilbert Siame Zambia Rural Healthcare Outreach Services (ZARHOS), Kitwe, Zambia Background: Male circumcision has attracted a lot of attention since studies in Africa showed that it can reduce HIV transmission by a significant amount(50–60%). In Zambia many people are now imploring policy makers to embark on a countrywide drive to sensitize and circumcise sexually active men. Even if one NGO have already embarked on this programme in partnership with the University Teaching Hospital, there is as yet no national policy in place. Objective: To find out the knowledge, attitudes and practices men have on circumcision as a preventive measure against HIV infection. Method: This was a primary survey of 115 sexually active men in Kitwe district, Zambia, who were interviewed using a questionnaire. These men were picked at random, but they had to be at least 20 yrs and older. Results: Majority of respondents were aged 30–40 years (44%), were married (52%) and had a high school education (37%). More than half reported having multiple sexual partners (59%). A large number of men knew what circumcision is (94%). 71% of the men were not circumcised compared to 29% who were circumcised. Of those who were not circumcised 57% were willing to be circumcised. Three main reasons for seeking circumcision were:HIV prevention (60%), cultural reasons (29%) and hygiene (11%). About two thirds (64%) of those who knew what circumcision is also said that circumcision reduces the chances of HIV infection, with almost three quarters (72%) of these men believing that there is no need to wear a condom when one is circumcised. The source of information on circumcision was mainly from the mass media (37%), health care provider (19%) and friends (13%). Conclusion: 1) Most men have heard that circumcision helps reduce chances of contracting HIV infection. 2) A good number of men are willing to be circumcised for the purpose of protecting themselves against HIV infection. 3) There is need to break the myth that when one is circumcised they cannot contract HIV and therefore no need of using condom. If unchecked, it is likely to lead to rampant spread of HIV. 4) More men must be encouraged to undergo circumcision, but it must be safe and performed by a skilled health worker. 5) There is potential for a greater synergy in reduction of HIV infection rate if circumcision is combined with increased use of other preventive interventions such as condom and microbicide.



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Preventing HIV infection in developing countries: focus on gender and age as the determinants of the spread

Abdulrazak Hamza Yahaya HIV/Immunology Laboratory, Murtala Mohammed Specialists Hospital Kano, Nigeria Background: HIV Prevention remains an important goal of public health services. HIV infects mainly population groups at their prime reproductive and productive age; visual consequences of HIV/AIDS in communities are poverty resulting from cost of care, mortality and morbidity among productive age groups. Methods: In this retrospective study for the presence of HIV antibodies two thousand asymptomatic volunteers 55% males and 45% females were screened from 2002 to 2005 using a rapid screening, double ELISA confirmation and structured questionnaires. Data generated was analyzed by SPSS v 14.0 using ANOVA and LSD comparison Results: One hundred and seventy two subjects (8.6%) were confirmed positive for HIV, 99.4% were infected with HIV-1 and 0.60% HIV-2. Respective Males’ to females’ infection ratio is 55.2% to 44.8%. At age group #44 years and $45 years 88.9% and 11.1% of the total infection were recorded; 3.90% and 0.85% among males and 3.75% and 0.10 % among females respectively. HIV infection spread and age are related (OR = 32.18; P , 0.001). Lost of human resource to HIV/AIDS exceeds that recorded from any single infectious disaster, HIV infection risk among the volunteers studied is significantly associated with their age and sex at P , 0.01. Conclusions: Strategizing HIV/AIDS prevention campaign by gender and age should be prioritized as a means of salvaging communities’ consequences and health crisis. Impact of HIV/AIDS disease on families can be reduced through care provision, diagnosis, treatment, and support in ways acceptable to at risk populations and communities.

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The Translational Laboratory Shared Services

Mariola Sadowska, PhD, Colette Burgess, Latreece Nance, and Joseph Bryant, DVM TLSS, UMGCC, 655 West Baltimore St. Rm. BRB9-039, Baltimore, MD 21201 The Translational Laboratory Shared Services (TLSS) established at the University of Maryland Marlene and Stewart Greenebaum Cancer Center (UMGCC) facilitates the preclinical studies (drug screening and in vivo animal models) and clinical development of novel, anticancer agents for patients with skin cancer such as melanoma, breast cancer, lung cancer, multiple myeloma, leukemia and lymphoma. The goals of TLSS are (1) to make preclinical in vitro and in vivo model expertise available to UMGCC members and facilitate translation of novel therapeutic concepts; (2) to add value to advanced preclinical developments of pharmaceutical companies and create a pipeline of new drugs for UMGCC’s Phase I and II clinicians; and (3) to provide UMGCC clinicians with the means to perform early clinical trials and assessment of pharmacodynamic endpoints of molecularly targeted drugs. TLSS offers several in vitro assays: proliferation/cytotoxicity assays including MTT, XTT and WST-1 (mitochondrial function), SRB (total cellular protein), propidium iodide staining (total DNA content); proliferation/drug combination assays for single agents and the drug combination; tumor stem cell proliferation assays; in vivo nude mouse assays, including MTD and tissue collection, establishment of tumor xenograft mouse models, pharmacodynamic endpoints efficacy studies, establishment of cell lines from tumors. TLSS offers immunohistochemistry or immunofluorescence, Western blotting, Real time PCR, development of stably transfected cell lines, proteomic and genomic analyses before and after drug treatment. The clinical services include patient sensitivity testing; collection and processing of clinical materials (blood, bone marrow, tissue); development of Standard Operation Procedures (SOPs) and clinical trial protocol. Currently, several clinical Phase I and II trials, with a translational research initiative component, are ongoing. Some of them are sponsored by the National Cancer Institute (NCI). TLSS also provides these services to small biotechnology companies. Future plans for TLSS include services for researchers studying the HIV-1-related tumors such as Kaposi’s sarcoma and B lymphoma.



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Humoral Immunity against Conserved Epitopes of HIV-1 Envelope Protein Archived in Memory B cells in Natural Viral Suppressors: Discordance with Plasma Antibodies

Yongjun Guan,1 Mohammad Sajadi,1 Anthony L. DeVico,1 Christine Obriecht,1 Robin Flinko,1 Karla Godfrey,1 Timothy Fouts,2 Ranajit Pal,3 Robert Redfield,1 Robert Gallo,1 and George K. Lewis1 1

Divisions of Basic Sciences and Vaccine and Clinical Sciences, Institute of Human Virology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201; 2Perfectus BioSciences, TechCenter at University of Maryland Baltimore County,1450 South Rolling Road, Baltimore, MD 21227; and 3Advanced BioScience Laboratories, 5510 Nicholson Lane, Kensington, MD 20895 Long-lived memory B cells (BMem) are an archive of past antibody (Ab) responses that persists for much of the host lifespan. By contrast, circulating antibodies typically decline to low or undetectable levels once the immunogen is cleared. This raises the possibility that the BMem pool might provide a more accurate picture of discrete antibody specificities that arise and decline during the course of an antibody response. This could be especially important for correlating antibody specificity with protective immunity in infectious diseases such as AIDS. By studying Ab and BMem of HIV-1 infected individuals who naturally suppress HIV-1 without therapy (NVS), we found high frequencies of BMem specific for CD4 induced (CD4i) or CD4 binding site (CD4bs) epitopes of gp120 that were discordant with contemporaneous plasma antibody responses to these epitopes. These data suggest that plasma Ab responses can underestimate the breadth of humoral immunity and that analyses of BMem should be included in studies correlating antibody specificity with protective immunity to HIV-1. In addition, a novel strategy to rapid selectively clone human monoclonal antibody (mAb) from BMem was developed and 25 novel mAbs of CD4i or CD4bs were obtained.

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Antibody 2G12 recognizes a glycopeptide epitope on HIV-1 gp120 envelope glycoprotein

Wei Huang, George K. Lewis, and Lai-Xi Wang Institute of Human Virology, University of Maryland School of Medicine, 725 W. Lombard Street, Baltimore, MD 21201 Identification of novel neutralizing epitope on HIV-1 envelope glycoproteins is an essential step toward developing an effective HIV-1 vaccine. Human antibody 2G12 is one of the few broadly neutralizing antibodies capable of neutralizing a range of HIV-1 primary isolates. Previous studies have suggested that 2G12 recognizes a carbohydrate antigen involving several N-glycans that form a novel oligosaccharide cluster. The two N-glycans at the base of the V3 loop, N295 and N332, seem to be essential as mutation of any of them will diminish gp120’s binding to 2G12. The role of the V3 domain is still unclear although mutational studied implicated that V3 might not be involved in 2G12 recognition. Using a chemoenzymatic approach, we have synthesized the full-size V3 domain glycopeptide, in which the two N-glycans at N295 and N332 were assembled in the context of the V3 domain. Our 2G12-binding results implicate that the V3 polypeptide not only provided a scaffold to hold the carbohydrate cluster, but was also involved in the interaction with 2G12. Thus, we propose that a unique HIV-1 V3 glycopeptide constitutes the epitope for antibody 2G12. A molecular modeling study with 2G12 and the synthetic antigen supports this notion. This discovery provides insights into the mechanism of 2G12-mediated recognition and neutralization, which will facilitate the design of a glycopeptide-based HIV-1 vaccine.



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An animal model for non-hodgkin’s lymphoma of the central nervous system (NHL-CNS)

J. Bryant, H. Tran, M. Sadowska, E. Ateh, and Y. Lunardi-Iskandar 725 W. Lombard Street, Room S110, Baltimore, MD 21201 Primary CNS lymphoma (PCNSL) is a rare form of extranodal non-hodgkin’s lymphoma, that occurs in the brain, leptomeninges, spinal cord, or eyes, and typically remains confined to the CNS. HIV is a primary factor responsible for the recent increase in the incidence of PCNSL. In fact, persons infected with HIV have a 3,600 fold increased risk of developing PCNSL compared with the general population. Because PCNSL is heterogenous in its clinical behavior, considerable attention has been focused on the identification of clinically relevant prognostic biomarkers. Patients with PCNSL typically present with neurological symptoms compared to systemic NHL-CNS. This lymphoma is a lethal disease and development of therapies for the treatment is highly desired, however, preclinical studies for PCNSL is hindered by the lack of a relevant animal model. We describe here in this paper the observation and development of a small animal model of PCNSL that occurs naturally in a HIV-1 transgenic mouse model carrying the HIV-1 genes. In this model, clinically the mice are presented with neurological signs, ranging from wobbling to fatal paralysis in the hind limbs. On necropsy there were no signs of any gross lesion suggesting it was not systemic spread and appears to have originated in the CNS. Cultures of the blood and spleen were grown in culture and passed back into HIV-1 TG mice and wild type mice. In 15 days all the animals developed the disease identical to what we observed in the natural occurring disease in the transgenic line. After two months none of the wild type animals developed disease, suggesting that the HIV-1 genes play a role in the development of this model. Phenotyping of the tumor in this model reveal the following monotypic surface immunoglobulin or Bcell associated antigens CD40+{25–32%}, CD28+{35–42%}, CD19+[59–86%], IgS+[47–62%], Igk+[23– 35%], Igx+[49–55%], CD23+[66–72%], CD25+[7–14%] and lack of T cell associated antigens in B-CFC cells. This is similar to the pan-B-cell markers seen in human PCNSL. To our knowledge this is the first report of NHL-CNS like disease in a mouse model that carries HIV-1 genes. Ultimately this model can be used to study the pathogenesis of this disease and be used as a model for preclinical studies for therapy.

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Persistent virological benefit in SIV-infected macaques upon therapeutic vaccination with DNA vectors by in vivo constant-current electroporation

Barbara K. Felber,1 A. Valentin,2 A. von Gegerfelt,2 M. Rosati,2 V. Pate,2 G. Miteloudis,2 C. Alicea,1 C. Bergamaschi,2 R. Jalah,1 A. Kha,3 R. DraghiaAkli,3 and G. N. Pavlakis2 1

Human Retrovirus Pathogenesis Section; 2Human ?Retrovirus Section, NCI, Frederick, MD 21702; and 3 VGX Pharmaceuticals, Inc., The Woodlands, TX 77381

Background: We have previously shown that SIV-infected macaques immunized during ART by intramuscular injection of optimized plasmid DNA mount an immune response able to reduce viremia in both prophylactic and therapeutic interventions against SIV infection. Here, we tested the more efficient in vivo constant-current electroporation as DNA delivery method. Methods: Rhesus macaques chronically infected with SIVmac251 were treated with combination antiretroviral therapy (ART). During this time, the animals were vaccinated by in vivo constant-current electroporation with optimized forms of DNA vectors expressing the majority of SIV proteins and then released from ART. The animals were monitored for rebounding virus and changes in SIV specific immune responses during and after termination of therapy. Cellular immune responses were monitored in peptide-stimulated PBMC by multiparametric flow cytometry combining cell surface and intracellular cytokine staining. Results: DNA vaccination by in vivo electroporation was very effective in inducing recall immune responses. These responses were between 5-10-fold higher that those previously observed by IM injection. Animals treated with a 2nd cycle of ART and therapeutic vaccination showed additional persistent virological benefit (.1 log10), as measured by decreased viremia after release from ART. Control of viremia was associated with a very strong induction of SIV-specific responses, and was characterized by the production of IFN-g, IL22, and TNFa either alone or in combination as double or triple cytokine positive multifunctional T cells. A significant induction of CD4+ T cell responses, mainly targeting Gag, Nef, and Pol, as well as of CD8+ T cells, mainly targeting Env, was found in both T cells with central memory and effector memory markers. Conclusions: In vivo electroporation of optimized forms of DNA vaccines during ART induced immune responses able to persistently decrease viremia after ART release. DNA vaccination has many advantages, including the possibility to administer the vaccine several times, and was shown to induce potent, efficacious, and long-lasting recall immune responses. Therefore, these data support the concept of adding DNA therapeutic vaccination to the HAART regimen to boost the HIV-specific immune responses.



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New Viral and Tuberculosis Therapeutics for the 21st Century

Roger J. Pomerantz, MD, FACP President, Tibotec and Global Therapeutic Area Head of Virology, Johnson & Johnson Corporation There remains high medical need in various area of virology and drug treatment of resistant tuberculosis. Tibotec has worked for the last decade in developing novel anti-retroviral drugs including Prezista and Intelence to combat resistant HIV-1 infections, and treat patients with simple and effective regimens during initial na¨ve treatment (eg. TMC278). We are now also looking at important cotravelers with HIV including Hepatitis C virus with the use of new protease inhibitors (VX-950 and TMC435). Finally, a novel anti-tuberculosis compound TMC207 using a new mechanism of action, ATP synthase inhibition, is being developed initially for MDR tuberculosis. As such, this portfolio of compounds will be critical in treating these viral and mycobacterial pandemics in both the developed and developing world.

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Glycosylation of gp41 of Simian Immunodeficiency Virus Shields Epitopes That Can Be Targets for Neutralizing Antibodies

Eloı´sa Yuste, Jacqueline Bixby, Jeffrey Lifson, Shuji Sato, and Ronald Desrosiers Harvard Medical School Human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) possess three closely-spaced, highly-conserved sites for N-linked carbohydrate attachment in the external domain of the transmembrane protein gp41. We infected rhesus monkeys with a variant of cloned simian immunodeficiency virus strain 239 (SIV239) lacking the second and third sites or with a variant strain lacking all three of SIV239’s glycosylation sites in gp41. For each mutation, asparagine (N) in the canonical N-X-S/T recognition sequence for carbohydrate attachment was changed to the structurally similar glutamine such that two nucleotide changes would be required for reversion of the mutated codon. By 16 weeks, experimentally infected monkeys made antibodies that neutralized the mutant viruses to high titer. Such antibodies were not observed in monkeys infected with the parental virus. Thus, new specificities were revealed as a result of the carbohydrate attachment mutations and antibodies of these specificities had neutralizing activity. Unlike monkeys infected with the parental virus, monkeys infected with the mutant viruses made antibodies that reacted with peptides corresponding to the sequences in this region. Furthermore, there was strong selective pressure for the emergence of variant sequences in this region during the course of infection. By analyzing the neutralization profiles of sequence variants, we were able to define three mutations (Q625R, K631N and Q634H) in the region of the glycosylation site mutations that conferred resistance to neutralization by plasma from the monkeys infected with mutant virus. Based on the reactivity of antibodies to peptides in this region and the co-localization of neutralization escape mutations, we conclude that Nlinked carbohydrates in the ectodomain of the transmembrane protein shield underlying epitopes that would otherwise be the direct targets of neutralizing antibodies.



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Opportunity for Scale –Up; Mobile X-ray Technology- Strengthening TB Diagnosis in HIV+ve Patients; ACTION Experience in Zaria, rural Northern Nigeria

U. L. Gebi, B. Musa, N. Alfred, A. Abimiku, P. Dakum, W. Blattner, E. Meshak, O. Obasanya, M. Gidado, and A. Clement Institute of Human Virology, Nigeria Background: HIV is the most powerful risk factor for the reactivation of latent TB infection to active disease. Currently, the tuberculosis case detection rate is put at 27% which is a far cry from the expected 70% CDR. Presently, it is estimated that 27% of Nigerians infected with HIV have Tuberculosis, a significant proportion of which are smear negative. Currently, the only diagnostic tool available for TB diagnosis is sputum AFB if there is sputum production with a sensitivity of about 50% and CXRay, which is rarely available at rural centers that see a large proportion of co-infected patients. IHVN has piloted successfully a mobile easy to deploy x-ray machine at the National TB Training which has remarkably improved diagnosis of TB in smear negative clients. Results: As at end of June, 2008 1,385 patients attending the NTBLTC in rural Zaria are in care for HIV, with 1,045 patients on ARVs, of which 233 have received co-treatment for TB at this center. Following the inception and traing in the use of the mobile MiniXray machine in this facility in February, 2008, a total of 208 patients have been diagnosed with TB with 56 (26.92%) being HIV+. Of this, 27 were sputum smear positive (12.98%) and an almost equal proportion of co-infected were sputum smear negative 29 (13.94%) for TB. The presence of this new easily usable digital X-ray at this rural site increased the proportion of TB diagnosis in HIV+ patients to 51.79% and access to treatment in line with the national guidelines for TB treatment, thus protecting these patients from certain death if they were not put on treatment. Conclusion: The successful roll out of this robust technology shows increased potential particularly in rapid TB diagnosis in community settings amongst high risk groups. This could be colinked in mobile HCT facilities screening for one of the ‘‘captains of the ship’’ of death in people living with HIV.

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Psychological distress as a risk factor for nonadherence to Highly active anti-retroviral\therapy

Etheldreda Nakimuli-Mpungu, MBChB, MMED, (Psych) Johns Hopkins School of Public Health Background: Adherence to highly active anti-retroviral therapy (HAART) is critical to the success of HIV/AIDS treatment. Non-adherence to antiretroviral therapy leads to incomplete viral suppression and the emergence of resistant virus. Poor mental health is one of the factors associated with nonadherence. Various psychiatric problems have been described among HIV positive adults in Uganda and yet there are virtually no HIV treatment centers in Uganda which provide mental health services for HIV positive individuals. Objective: To determine the factors associated with non-adherence to HAART among individuals receiving HIV care at the Butabika HIV clinic. Method: 92 adults receiving care for HIV and using HAART therapy at the Butabika Hospital HIV Out patient clinic were enrolled in the study. Participants were administered a brief structured psychiatric instrument (SRQ) that screened for psychological distress during the previous month and adherence to HAART. Sociodemographic and clinical factors associated with non-adherence to HAART were examined in multivariate logistic regression analyses. Results: 30% of the sample reported being non adherent to HAART at some point in time since initiating this therapy. Psychological distress in the previous month and living in isolation were significantly associated with non-adherence to HAART. Having a severe mental disorder was not significantly associated with non-adherence to HAART. Conclusion: Psychological distress and living in isolation may interfere with adherence to HAART. Regular mental health checks may lead to early identification and alleviation of these problems and thus prevent further complications.



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Baseline renal insufficiency and risk of death among HIV-infected adults on antiretroviral therapy in Lusaka, Zambia

L. B. Mulenga,1 G. Kruse,1 S. Lakhi,2 R. A. Cantrell1,1,3 S. E. Reid,1,3 I. Zulu,4,5 E. M. Stringer,1,3 Z. Krishnasami,3 A. Mwinga,4 M. S. Saag,3 J. S. A. Stringer,1,3 and B. H. Chi1,3 1

Center for Infectious Disease Research, Lusaka, Zambia; 2University Teaching Hospital, Lusaka, Zambia; 3University of Alabama at Birmingham, AL; 4CDC, Lusaka, Zambia; 5University of Zambia School of Medicine, Lusaka, Zambia Objective: To examine the association between baseline renal insufficiency and mortality among adults initiating antiretroviral therapy (ART) in an urban African setting. Design: Open cohort evaluation. Methods: We examined mortality according to baseline renal function among adults initiating ART in Lusaka, Zambia. Renal function was assessed by the Cockcroft-Gault method, the Modification of Diet in Renal Disease (MDRD) equation, and serum creatinine. Results: From April 2004 to September 2007, 25,779 individuals started ART with an available creatinine measurement at baseline. When creatinine clearance was calculated by the Cockcroft-Gault method, 8, 456 (33.5%) had renal insufficiency: 73.5% were mild (60–89 mL/min), 23.4% moderate (30– 59 mL/min), and 3.1% severe (,30 mL/min). Risk for mortality at or before 90 days was elevated for those with mildly (adjusted hazard ratio [AHR] = 1.7; 95% CI = 1.5–1.9), moderately (AHR = 2.3; 95% CI = 2.0–2.7), and severely (AHR = 4.1; 95% CI = 3.1–5.5) reduced creatinine clearance. Mild (AHR = 1.4; 95% CI =1.2–1.6), moderate (AHR = 1.9; 95% CI = 1.5–2.3), and severe (AHR = 3.6; 95% CI = 2.4–5.5) insufficiency were also associated with increased mortality after 90 days, when compared to those with normal renal function. Trends were similar when renal function was estimated with MDRD or serum creatinine. Conclusions: Renal insufficiency at time of ART initiation was prevalent and associated with increased mortality risk among adults in this population. These results have particular relevance for settings like Zambia, where tenofovir – a drug with known nephrotoxicity – has been adopted as part of first-line therapy. This emphasizes the need for resource-appropriate screening algorithms for renal disease, both as part of ART eligibility and pre-treatment assessment.

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Lopinavir/ritonavir-based Second Line Antiretroviral Treatment in children at National Pediatric Hospital, Phnom Penh, Cambodia

S. Sam,1 V. Ung,1 C. Huot,1 C. Courpotin,2 Eric Nerrienet,3 David Pugatch,4 Kenneth Mayer,4 and Y. M. Chhour5 1

Child Health Improvement Clinic, National Pediatric ?Hospital, Phnom Penh, Cambodia; 2French Red Cross, Phnom Penh, Cambodia; 3Pasteur Institute, Phnom Penh, Cambodia; 4Brown University, Miriam Hospital, Providence, RI, USA; and 5National Pediatric Hospital, Phnom Penh, Cambodia

Background and Objectives: Cambodia has scaling up a large national ART program using 1st line therapy (d4T or AZT+3TC+NVP or EFV). As March 31st 2008, 2689 HIV infected children were on HAART in Cambodia. 1059 children were registered (664 on HAART) in Child Health Improvement Clinic (CHIC), an HIV outpatient clinic set up by the National Pediatric Hospital (NPH), with French Red Cross technical support. We evaluated the current pediatric cases with 2nd line regimen. Methods: Data and medical records from a retrospective cohort followed at CHIC to 31st March 2008 were analyzed. Patients meeting the Cambodian National Guidelines for the Use of Pediatric ART for treatment failure were evaluated. Treatment failure was confirmed based on clinical and immunological failure and/or virological failure. Plasma viral load has been assessed by HIV RNA real time PCR using 2nd generation ANRS Kit. Genotypic resistance results were done at Institute Pasteur according to ANRS algorithm (v.sep.07). Results: 29/612 patient (4.7%), 31% female, switched to 2nd line were enrolled in this study. Median age was 10.4 years (4.3–17.1). Median duration in the 1st line was 1.9 years (0.6–6.3). Median of CD4 percentage when switching to 2nd line was 5.0% and VL was 5.1Log (4.0–6.3) with +/2 clinical failure. At switch, 22 patients were tested for HIV drug resistance for RT gene. The results revealed that 95.4% (21/22) children were resistance to NVP/EFV, 77.3% to AZT/d4T, 54.5% to ABC/ddI, and 9.1% to TD. Median time in the 2nd line was 0.9 years (0.2–2.5). 18 of 29 patients (62.1%) receive standard 2nd line regimen (ABC/ddI/LPV/r), 5 (17.2%) with 3TC/TDF/LPV/r) and 2 (6.9%) with 3TC/AZT/LPV/r). Median CD4 gain on 2nd line regimen were 11.5% (1–31%) at M6 (n = 22); 15% (0–27) at M12 (n = 11); 15.5% (10–26) at M18 (n = 6); 18% (16–25) at M24 (n = 3); and 17% at M30 (n = 1). Patients who achieved undetectable VL (VL,2.4Log) at M2 were 62.5% (n = 24); 84.2% at M6 (n = 19); 80% at M12 (n = 10), 75% at M18 (n = 4) and 100% at M24 (n = 2). Conclusion: We report here the first immunological and virological results of children on 2nd line of ART in Cambodian. This data confirm a strong efficacy of LPV/r-based 2nd line regimen and provide important information for appropriate and effective HAART in children.



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Explosive Expansion of HIV and associated risk factors among Male and Hijra Sex Workers in Sindh, Pakistan

Arshad Altaf HIV/AIDS Surveillance Project, Sindh AIDS Control ?Programme, PAKISTAN Background: Pakistan has progressed from nascent to concentrated level of the HIV epidemic. Among IDUs the HIV prevalence is 16.5% in the most industrialized province of Sindh (Pakistan) which has a population of over 60 million. Male and hijra (transvestite) sex workers are emerging as the second highest risk group. With present estimate of almost 16,000 MSWs in Sindh (considered a conservative estimate) their transmission dynamics with other risk group suggest that this group along with IDUs could kick start the epidemic in general population. Methods: We investigated specific demographic and behavioral characteristics of male (MSW) and hijra sex workers (HSW) in four cities of Sindh Province i.e Larkana, Karachi, Sukkur and Hyderabad as part of second generation surveillance (SGS). A cross sectional study integrated behavioral and biological survey (IBBS) was conducted between August 2006 and January 2007. A total of 1610 MSW/HSWs (800 MSWs and 810 HSWs) were recruited. All interviews were conducted in the field maintaining privacy and confidentialities. Respondent driven sampling was used to recruit MSWs and cluster sampling to include HSWs. Results: Mapping results estimate 7650 MSWs and 7300 HSWs in selected cities of Sindh. Average age of MSWs was 21 years and HSWs 27.3 years. The reported paid clients in past one month for MSWs were 33.5 and HSW 48.8. Condom use in last sexual encounter was reported to be 26.7% among MSWs and 20.4% among HSWs. Cumulative sero prevalence of for four cities among MSWs was 20/800 (2.5%) (95% CI: 1.42–3.58) and among HSWs 38/810 (4.7%) (95% CI: 3.23–6.15). HIV prevalence was higher at each city level. 13.1% also reported having sex with IDUs. Conclusion: The preventive measures taken in this relatively neglected group are going to be far more effective and would have much better impact than the preventive work among IDUs population.

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Enhancement of HIV-1 Replication in Human Primary Cells By Macrophage Migration Inhibitory Factor in Nigerian Africans

Dr. Busari Olusegun Adesola, HIV Study Group, Federal Medical Centre, Ido-Ekiti, Nigeria Introduction: Cytokines play a critical role in the pathogenesis of HIV-1 infection. However, the involvement of macrophage migration inhibitory factor (MIF) in HIV-1 infection has not been documented. The objective was to analyse if MIF modulates HIV-1 replication in monocyte-derived macrophages (MDM) and phytohaemaglutinin-activated peripheral blood mononuclear cells (PBMC). Methods and Materials: PBMC was obtained from 12 healthy donors by density gradient centrifugation and MDM by plastic adherence of PBMC. These cells were infected with HIV-1 and latter exposed to human MIF or anti-MIF antibodies. Flow cytometry was used to analysed cellular expression of CD4, and ELISA to measure levels of MIF and p24 Ag in cell culture supernatants. Results: The study shows that HIV-1 infected PBMC induced an increased MIF secretion in 8 (66.7%) of donors. MIF also increased viral replication in PBMC (P = 0.002). In PBMC treated with antiMIF antibodies, HIV-1 replication was inhibited by 64% (P = 0.0001). MIF did not enhance cellular expression of CD4 cells suggesting that MIF-induced enhanced HIV-1 replication was not as a result of increased expression of HIV-1 receptors. Conclusion: The study concluded that MIF enhanced HIV-1 replication in peripheral human cells. There is need for further studies to unravel the mechanisms of MIF-enhanced HIV-1 replication and to develop an MIF-inhibitory concept that can result into potential antiretroviral compounds.



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Neutralization efficiency and presence of anti-V3 antibodies in plasma of HIV-1 infected Northern Indians

Alok K. Choudhary,1 Subhashree Dutta,1 Naveet Wig,2 A. Biswas,2 Raiees Andrabi,1 Rajesh Kalra,1 Rama Bhasin,3 Susan Zolla Pazner,4 Suman Laal,4 and Kalpana Luthra1 1

Department of Biochemistry; 2Department of Medicine; 3Blood Bank, CN Centre, All India Institute of Medical Sciences, New Delhi, India; and 4Veterans Affairs Medical Center, NYU School of Medicine New York, NY

Background: Clade C viruses are primarily responsible for the HIV pandemic. Antibodies which bind HIV and effectively neutralize the virus are needed to reduce the viral load. These antibodies are targeted to different regions of the viral envelope such as the membrane proximal external region of gp41, the CD4 binding on gp120, complex glycans on gp 120, the CD4 induced epitopes in and around the gp120 bridging sheet and the V3 loop of gp120. The present day novel immunization approach is directed at designing an ‘‘immunofocussing vaccine’’. In order to enhance the quality and/or quantity of neutralizing antibodies, the V3 loop of the gp120 has been focused. Anti V3 antibodies have been shown to efficiently neutralize primary isolates of HIV. Methods: In order to characterize the immune response elicited by HIV-1 infected patients visiting AIIMS, New Delhi, we detected the presence of anti V3 antibodies in their plasma and determined the neutralizing efficiency. Neutralizing activity of the patient plasma at different dilutions was measured using the TZM assay against a primary isolate raised. Presence of anti V3 antibody in the plasma of these patients was detected using consensus C V3 peptide ELISA. Results: Out of the 29 plasma samples, 17 showed .90% neutralization, 2 showed neutralization between 70–80%, 6 showed ,50% neutralization, while 4 plasma samples revealed increased infection in comparison to the virus control. All the plasma samples were found positive for V3 antibodies in comparative amounts based on the A405 obtained. Conclusions: Our study suggests the presence of immunogenic epitopes which are more exposed and therefore responsible for eliciting neutralizing antibodies, in majority of the patients. Besides V3, there may be other immunogenic determinants responsible for efficient neutralization of HIV and this need to be characterized. These sera are being tested further to check for neutralization of different primary isolates raised in the laboratory to determine if they exhibit a broad cross-clade neutralization of different viruses.

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Quality ART Scale Up Through Regionalization of Laboratory Services In Nigeria

C. Ezeaku, J. Farley, P. Dakum, T. Croxton, N. Constantine, A. Abimiku, and W. Blattner Institute of Human Virology, Nigeria Issues: Setting up of standard lab infrastructure and provision of free laboratory tests by PEPFAR has improved health care delivery system and services to PLWHAs in Nigeria. We here describe the challenges and successes of the tier laboratory infrastructure established by the Institute of Human Virology - University of Maryland PEPFAR funded AIDS Care and Treatment in Nigeria (ACTION) program to support the diagnosis, care and treatment of HIV infected Nigerians. Description: The ACTION program currently treats over 80,000 and cares for another 150,000 HIVinfected Nigerians; also, about 300,000 are screened. To support this, the laboratory infrastructure expanded from 12 to a regionalized tier system with 22 tertiary, 25 secondary and 12 primary laboratories all linked with appropriate oversight and referral system. Choice of laboratory equipment was based on ease of use, in country vendor/service provider, ease of purchase of reagents, and overall manufacturer’s support. Implementation included: 1) Decentralization of laboratory field services into regions with central oversight 2) Detailed site assessments/renovations 3) Step wise site activation in HIV Rapid Testing, Pregnancy Testing, Manual CD4 Count, HbAg screening, CyFlow SL (tertiary) or Counter (secondary) for CD4, Sysmex KX 21N for Hematology, Vitros V250 (tertiary) DT 60 II (tert/sec) or Reflotron (sec/prim) for Chemistry assays. 4) Monitoring and mentoring of site personnel through an aggressive QA/QC program. 5) Hub and spoke linkage between comprehensive and satellite sites. Lessons learned: Tier lab network with adequate mentoring and referral led to a successful lab expansion to 59 (22 tertiary, 25 secondary and 12 primary) sites performing HIV Serology, CD4, Pregnancy, Hematology, Chemistry, Hepatitis B, Cryptococcus, and TB tests. Also, 57 HCT/PMTCT sites performing HIV Serology Tests. Improved proficiency at sites from 40–60% to 80–95%. Recommendations: Regionalization and tier lab system is critical for scale up. Defined roles for lab personnel creates a good network to support rapid scale up. QA/QC program involving site monitoring is critical for sustained high quality lab results.



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T-Cell Receptor (TCR) Activation Mediates Efficient and Sustained HIV Transcriptional Elongation and Initiation through Multiple Signal Pathways

Joseph F. Hokello Department of Molecular Biology and Microbiology, CWRU School of Medicine The persistence of HIV in a latent pool during intensive drug therapy remains the single greatest obstacle to successful HIV eradication. T-cell receptor (TCR) activation induces multiple signal transduction pathways resulting in activation of several factors previously reported to regulate HIV transcription such as NF-kB, NF-AT and AP-1. We analyzed the contribution of each of these transcription factors in the reactivation of latent HIV using Jurkat T-cell clones harboring single latent HIV proviruses. Jurkat T-cells were activated through the TCR by co-treatment with anti-CD3 and CD28 antibodies. The kinetics of proviral reactivation following mobilization of NF-kB by treatment with TNFa which induces NF-kB as the major transcription factor is characterized by an initial 4 hr lag phase where elongation is restricted until new Tat is synthesized. In contrast, TCR activation induces efficient and sustained HIV transcriptional elongation of latent proviruses. At initial time points, RNAP II is recruited to the HIV LTR in parallel to NF-kB induction. However, during latter stages of activation when AP-1 is induced, both NF-kB and AP-1 co-assemble on the promoter. Treatment of cells with MAPK inhibitor PD98059 blocked AP-1 mobilization and recruitment to the promoter and these result in a remarkable loss of RNAP II elongation as demonstrated by strong reduction in RNAP II levels present downstream of the promoter. Studies using cyclosporine and LTR mutants demonstrate that NF-AT does not play a major role in activating HIV transcriptional elongation or initiation when NF-kB is present. However, in the absence of NF-kB, a combination of NF-AT and AP-1 can stimulate transcriptional initiation. In addition to regulation of elongation by AP-1, we have found that P-TEFb is ‘‘pre-activated’’ by a novel pathway following TCR activation. During the first hour after TCR activation, there is enhanced P-TEFb recruitment and RNAP II processivity which can be blocked by ERK inhibitor U0126. We conclude that HIV transcription is a highly dynamic process which is tightly regulated by multiple and complimentary signal pathways during TCR activation.

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N-linked glycans on HIV-1 gp120 are critical determinants for the recognition of CD4 helper T cell epitopes

Hualin Li VA Medical Center, New York, NY Background: The heavy glycosylation of HIV-1 envelope gp120 shields this important antigen from recognition by neutralizing antibodies and cytolytic CD8 T cells. However, very little work has been done to understand the influence of glycosylation on the generation of MHC class II-restricted gp120 helper epitopes and their recognition by the CD4 T cells. Methods: Three glycans in the C4 region of gp120 (linked to N406, N448 and N463) and three glycans in the C2 region (linked to N197, N230 and N234) were removed individually or in combination by N-to-Q substitutions. These glycans were selected because they flank Ôhot-spotsÕ where many of the helper gp120 epitopes are located. The wild type and mutant gp120 proteins were produced in CHO cells and tested for recognition by CD4 T cell lines specific for different gp120 epitopes. The effect of the N-linked glycan removal on proteolytic processing of gp120 was evaluated by trypsin digestion and mass spectrometry (MS) analysis. Results: The mutant proteins lacking the N448- or N230-glycans did not effectively stimulate CD4 T cells specific for the nearby epitopes, although the same mutants were recognized well by CD4 T cells specific for more distant epitopes. Notably, both of these glycans were not part of the helper epitopes and were located 2 amino acids downstream from the core epitopes. Data from trypsin digestion and MS analyses demonstrated that the N448-glycan removal impeded the proteolytic cleavage of the nearby C4 region, without affecting more distant sites. In contrast, the N230-glycan had no discernible effects on the trypsin digestion of the neighboring C2 region, but the exact enzymatic processing necessary for the generation of the C2 epitopes remains unclear. Conclusions: The presence or absence of a single N-glycan on HIV-1 gp120 can dramatically alter CD4 T cell recognition of this antigen. While the mechanisms by which the N-glycans affect CD4 T cell recognition of gp120 are not yet fully understood, the glycans may dictate the local conformations of the nearby gp120 regions and therefore influence the way CD4 helper epitopes are processed and generated. Hence, N-linked glycans are critical determinants that can profoundly influence CD4 T cell recognition of HIV-1 gp120.



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Comparative Evaluation of the Performance of Abbott m2000rt Real-Time HIV-1 Assay for Measurement of HIV-1 Plasma Viral Load

M. Vidya,1 S. Saravanan,1 S. Kartik,1 K. Venkatesh,2 P. Balakrishnan,1 N. Kumarasamy,1 K. G. Murugavel,1 Sunil S. Solomon,1 Suniti Solomon,1 and Kenneth H. Mayer2 1

YRG Centre for AIDS Research and Education, Voluntary Health Services, Taramani, Chennai, India; Brown University, RI, USA

2

Background: Measurement of HIV-1 plasma viral load has become a corner stone in the clinical management particularly in the resource-limited settings. The objective of the present study is to evaluate the performance of recently introduced ABBOTT Real Time HIV-1 PCR assay (ABBOTT m2000rt) in comparison with the standard, COBAS Roche HIV-1 Monitor version 1.5 assay. Materials and Method: A total of 50 stored (270°C) HIV-1 positive plasma samples with known viral copies/mL {,400 not detected (n = 10); ,400 detected (n = 10); .400 – 5000 (n = 10); .5000 – 50000 (n = 10) and .50000 – 500000 (n = 10)} tested using the Roche HIV-1 Monitor ver. 1.5 assay and 20 HIV-1 low-risk seronegative (SN) samples were randomly used for the validation. Plasma RNA was extracted and concentrated using ABBOTT manual specimen preparation, which uses magnetic particle technology to capture nucleic acid extracted from 600 uL of plasma (detection range of 40– 100000 copies/mL). Plasma HIV-1 RNA levels were log10 transformed before being subjected to statistical analysis. Averages were statistically compared using the paired Z test. Linear regression and Bland-Altman were done to assess the agreement between these two assays. Results: The good correlation and agreement between ABBOTT Real Time and Roche was observed. Although the difference in viral load determinations was positively skewed in favor of the Roche assay, the difference in each PVL category, between the assays was within 0.5 log and the results were well correlated [R = +0.89 (P , 0.05)]. Bland-Altman analysis found 95% of the samples within clinically acceptable limits of agreement (21.001 to 0.52 log copies/mL). The sensitivity and specificity of ABBOTT Real Time PCR detecting the viral copies of more than 1000/mL is 100% and .87%, respectively. The specificity of 100% by ABBOTT Real Time PCR with the testing of SN for HIV-1/2 plasma samples was observed. Conclusion: The new ABBOTT RealTime PCR plasma viral load assay is sensitive and meets the requirements for a wide dynamic range and providing higher throughput reliable quantification of HIV-1 RNA in plasma specimens.

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Barriers to timely initiation of antiretroviral therapy among HIV infected children admitted to Mulago Hospital Paediatric wards

Dr. Eleanor Namusoke Joint Clinical Research Centre, Kampala, Uganda Background: The median survival of HIV infected children in the absence of treatment is 2 years; thus early initiation of ART is desirable. In Uganda, ART is now being provided free of charge. However, some children still do not access the treatment. No study has been done in our setting to establish what could be the possible barriers to timely initiation of ART. Objective: To determine the proportion of children eligible to start antiretroviral therapy who have not yet started by 4 weeks after discharge, and the factors that hinder access to timely initiation of antiretroviral therapy in these children. Methods: A cross sectional study was conducted on the paediatric wards at Mulago Hospital and the Paediatric Infectious Disease Clinic (PIDC). 113 newly diagnosed HIV infected children in WHO clinical stage 3 and 4, aged 3 months to 12 years were recruited. Questionnaires to assess factors affecting timely initiation of ART were administered at recruitment and at 4 weeks after discharge. Home visits were also done. Qualitative data was collected using focus group discussions, and indepth interviews. Results: The proportion of children who were eligible to start antiretroviral therapy but had not yet started by 4 weeks after discharge was 97.3% (110/113). Incomplete adherence counselling, default and death were the major barriers that prevented initiation of ART. Conclusion: 97.3% of the children had not started ART by 4 weeks. Caregiver factors were the main barriers that prevented initiation of ART in these children.



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Patient Retention in a University Hospital-based ART program in Uganda

Ouma Joseph Faculty of Medicine, Makerere University, Kampala, Uganda Background: Access to antiretroviral therapy (ART) in resource limited settings has increased over the past five years. ART programs report good adherence but these data are usually generated from patients who are retained in care. Documentation on long term follow up of patients on ART is limited. We evaluated the two-year retention of patients receiving ART from 12 clinics (8 in urban and 4 in rural settings) supported by Mulago-Mbarara Teaching Hospitals’ Joint AIDS program (MJAP). MJAP provides ART to over 14,000 patients. Patients are initiated on ART if CD4+ counts ,200 cells/mm3 or WHO stage III or IV. The preferred first line ART regimen is Nevirapine and Combivir containing. Patients are given monthly appointments for ART refills, clinical evaluation and counselling. Treatment supporters are encouraged. Telephone calls and/or home visits are used to track patients who miss appointments. Methods: We retrospectively analyzed data for 6,067 ART na¨ve adult (18+ years) patients initiated on ART between November 2005 and November 2007. We assessed predictors for retention on ART using Cox proportional hazard model. Results: The median CD4+ count at ART initiation was 132 cells/mm3 (IQR: 56,193). Total attrition was 15% for urban based sites and 7% for rural based sites. Death rate was 5.4% overall (5.7% urban and 4.9% rural). Overall transfer out was 5.5% (6.5% urban and 2.3% rural), and lost to follow up 2.2% (2.8% urban and 0.2% rural). Estimated attrition rate was 30.5 per 100 person years over 2.4 years of follow up. Nevirapine based ART regimen, Female sex, weight .50 kilograms, CD4+ counts .200cells/mm3, and rural clinics were associated with lower loss to follow-up, P-value ,0.001. Conclusion: Retention was high and comparable to other ART programs in Africa. Loss to followup is higher in urban sites, possibly related to mobility especially of male patients. Enhanced patient tracking and support may be necessary for patients accessing ART services in the urban areas and those with baseline CD4 count below 200 cells/mm.

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High Incidence Cohort of IDUs Infected with HIV with Low Genetics Diversity for HIV Vaccine Efficacy Trials

Sergey Verevochkin St. Petersburg, Russia Background: In the previous study (NIH HPTN 033) HIV incidence was determined in the cohort of injection drug users (IDU) from St. Petersburg, Russia. Between March and December 2002, 898 IDU were screened for HIV and 270 (30%) were HIV positive. 520 uninfected IDU were enrolled in the cohort study. The retention rate at the 12-month follow-up period was 80%. HIV-1 incidence rate was 4.5 per 100/PY. Similar incidence (4.8%) was obtained using STARHS detuned method. Methods: There are two current studies at the Biomedical Center among high-risk groups for HIV infection. The cross-sectional study (SATH-CAP) used the respondent-driven methodology to recruit DUs, their sex partners, and sex partners of sex partners through chain referral. Sera from HIV positive study participants were tested with Calypte HIV-1 Incidence EIA. Incidence was calculated in accordance with CDC consensus formula (comes with manual). Interview data were used for confirmation of infection date. The Randomized Controlled Trial of Russia IDU Peer Network HIV Prevention Intervention includes follow-up at 6 and 12 month. Incidence data based on seroconvertions during follow-up visits were compared with Calypte assay and interview data. Results: During 2005–2006 years 419 from 909 IDU patients were found positive for HIV-1 antibodies in EIA and confirmed by WB (46,1%). 72 of them were negative on Calypte assay. Interview showed that 15 patients were found positive for HIV-1 antibodies at least 6 months before Calypte test. In addition, follow-up sera from 6 patients were negative on Calypte assay at least 6 month after first Calypte test. So, only 51 specimens were classified as recent infections, which gives 21, 2 per 100 p-y. The incidence data from the cohort study performed during the same period showed 19.6 per 100 p-y. Conclusion: These two studies show that incidence of HIV infection among IDU has substantially increased during the last several years. Different approaches (the cohort study and laboratory testing for incidence cases) produced close results of 19.6 and 21.2 percent incidence. This is the highest published incidence of HIV infection to our knowledge. The current situation of high HIV incidence rates suggests the need for implementation of intense prevention strategies. In the context of a homogeneous infecting HIV strain the situation is also ideal for mounting efficacy trials of HIV vaccines among IDUs in St. Petersburg.



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Mycobacterium avium KatG protein (MAV_2753): a putative candidate for the serodiagnosis of MAC disease

Kapil Gupta,1 Ajay Wanchu,2 Romica Latawa,1 S. Laal,3 G. K. Khuller,1 and Indu Verma1 1

Department of Biochemistry; 2Department of Internal Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, INDIA; 3Department of Pathology, NYU Langone Medical Center, New York, USA Disseminated Mycobacterium avium complex (MAC) infection is a severe complication of advanced HIV disease. Recent studies suggest that Mycobacterium avium, a member of M. avium complex (MAC) is responsible for about 5–7% of the mycobacterial infections in HIV+ patients in India. Currently available diagnostic methods for differentiation of M. tuberculosis (M. tb) and MAC infections require culture of bacteria, which is not routinely available in India and is time-consuming and complex. A simple and rapid diagnostic assay that can identify M. avium disease and can discriminate between M. tb and M avium is urgently required. Present study was aimed to identify a M. avium secretory protein as a candidate serodiagnostic marker for MAC bacteremia in HIV patients. Based on a combination of biochemical and immunological approaches, we have identified the ;81 kDa M. avium KatG protein to be immunodominant antigen that is recognized by antibodies in 90% of HIVinfected patients who were confirmed to have disseminated MAC infection on the basis of blood culture. In contrast, no antibodies to this protein were detected in sera from TB patients, healthy subjects, or HIV-infected subjects. These data suggest that the M. avium KatG protein can be useful in designing a simple and specific serodiagnostic assay to rapidly diagnose the bacteremia due to M. avium in AIDS patients. Such a diagnostic test could be of great help to clinicians in determining the therapeutic strategies for HIV-infected patients suffering from mycobacterial diseases.

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The Use of HIVQUAL as a Quality Improvement Tool for a Large Scale HIV/AIDS Public Health Program – THE ACTION PROJECT, IHV Nigeria

U. Yakubu, U. Gebi, M. Babamaiyaki, I. Okoye, K. Falayajo, P. Dakum, J. Farley, W. Blattner, W. Etiebet, A. Zoakah, and M. Charurat Institute of Human Virology, Abuja, Nigeria

Background: With 67, 543 patients currently in care, the ACTION project is a comprehensive HIV/AIDS care and treatment program sponsored by The Institute of Human Virology, University of Maryland School of Medicine since March, 2005. Initial efforts at rapid scale-up in order to address unmet demands for HIV/AIDS services within the country have been successful and the organization has decidedly shifted its focus from acquiring more patients to improving the quality of clinical care and other services that are being delivered to our patients who are already in care. Design/Methods: The ACTION project provides comprehensive HIV/AIDS care at 40 sites. Random sampling was used to select 23 sites spread across the six geopolitical zones of the country for a Quality Assessment Exercise spanning 2 weeks. The methodology employed the use of HIVQUAL indicators to assess the level of clinical care. A total of 5 tools were administered to assess the overall quality of care. These included a random chart review, administered questionnaires, focus group discussions, and site observation charts. For the random chart review a case list of active patients (i.e. any patient who has had at least one clinical visit in the last 6 months) was generated by the clinic. Sample size for each clinic was determined by active caseload. A randomized sample of patients was generated from the case list using the HIVQUAL sampling table. Core indicator abstraction which included Continuity of care, CD4 count monitoring, TB screening and assessment, Cotrimoxazole preventive therapy, Weight monitoring, Prevention education, appropriate ARV therapy and Adherence assessment were extracted from medical records and charts. Data was entered into the software and reports were generated automatically. A special element of this exercise was the specific auditing of Ôkidney function monitoringÕ by scheduled creatinine assessments of patients who had been newly initiated on TDF–based regimens with at least 12 months of follow-up after ART initiation. Questionnaire administration using the second tool involved ÔInterviewsÕ with the ARV focal person, the Matron of the clinic, one junior medical officer and one nursing officer in order to obtain their subjective impressions of the quality of key clinical activities that

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The Use of HIVQUAL as a Quality Improvement Tool for a Large Scale HIV/AIDS Public Health Program – THE ACTION PROJECT, IHV Nigeria (continued)

were occurring in their clinics. The third tool involved the conduction of Focused Group discussions with separate focus groups including Physicians, Nurses, M & E officers, Record staff and the cleaning and messenger staff. The fourth instrument was the employment of a ÔQuality of care Observation toolÕ which assessed the more general aspects of clinic flow and clinic conditions including patient waiting time, sitting space, physician to patient ratio on clinic day and activity coordination. The ready availability of relevant guidelines, SOP’s, cleanliness of the clinic environs, use of a team system and the presence of functional basic clinic equipment was also incorporated into this observation tool. Results: The results showed that compliance with protocol for laboratory testing was highest at baseline with CD4 counts, Full blood counts and liver function tests being ordered for virtually all of the patients. TB symptom screening was also high at baseline (85%). Ordering of follow up investigations however depreciated progressively with time, falling to about 60% at 6 months when all the sites are averaged together, and to about 50% at 12 months. Creatinine assay request was over 98% for most sites at baseline but dropped sharply to less than 40% across the board at 6 months and to less than 10% at 12 months. Patients being placed on Cotrimoxazole prophylaxis when their CD4 count was below 350 was about 80% while the correct and complete filling of clinical evaluation forms had a compliance of 45% across all sites. Weight measurement, which is carried out by nurses in the clinic, was the quality indicator with the highest level of compliance, consistently showing .98% documentation for all sites, across the entire study period. This particular finding of high compliance suggests that this capacity could be exploited in graduated task shifting processes which will relieve other overburdened staff, such as doctors prescribing ARV’s. Challenges and Lessons Learned: Feedback meetings were carried out with all the site project staff to share study findings and solicit their opinions. The most important and consistent obstacle to provision of quality service was Manpower. Critical lab investigation benches such as CD4 count and chemistry were manned by only one staff. If such a staff was indisposed or resigned there was immediate interruption of service which was a common occurrence. Doctor to patient ratio on clinic day was also suboptimal in most sites with one doctor commonly attending to more than fifty patients. Task shifting to the nurses who achieved a high compliance on weight monitoring should be considered as a result of the findings of this exercise. In conclusion it can be said that the HIVQUAL approach to quality assessment can serve as a very useful tool for quality monitoring and quality improvement for large-scale public health programs such as IHVN’s ACTION PROJECT.

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Mannose Binding Lectin and its variants: susceptibility to HIV-1 and Schistosoma haematobium infection in a rural Zimbabwean community

Rutendo B. L. Zinyama-Gutsire National Institute of Health Research Ministry of Health and Child Welfare Zimbabwe Introduction: The last decade has seen an emerging interest in Mannose Binding Lectin (MBL) due to its role in innate immunity. Low serum concentrations of MBL are associated with increased susceptibility to infections. We investigated the role of MBL in HIV-1 and Schistosoma haematobium coinfected and uninfected individuals. Objective: To determine levels of MBL in plasma of HIV-1 and Schistosoma haematobium coinfected and uninfected individuals in a rural Zimbabwean community and to test whether MBL concentrations or its genetic variants are associated with increased risk of HIV-1 infection and death. Methods: Through a cross-sectional survey, during which HIV-1 and schistosomiasis screening were done, a cohort of 379 participants was established. Outcome measures consisted of HIV-1 and schistosomiasis status and levels of MBL in plasma at baseline. Non-parametric statistical methods were used to assess the relationship of MBL levels or its genetic variants, with HIV-1 and S. haematobium infections. Results: A total of 379 adults formed the established follow up cohort comprising of 74 (19, 5%) men and 305 (80, 5%) women. Mean age was 33.25, range 17 to 62 years. There was no difference in the mean MBL plasma concentration of HIV-1 positive and HIV-1 negative participants (P = 0.066) but there was a significant difference in the mean MBL plasma concentration of S. haematobium positive and negative participants (P = 0.0173). There was a significant difference in the mean MBL plasma concentration between HIV-1 and S. haematobium coinfected and uninfected individuals (P = 0.087). The distribution of the three genotypes (A/A, A/O and O/O did not differ between the HIV-1 infected and the healthy controls and neither between those S. haematobium infected and uninfected. When HIV-1 S. haematobium infected participants were analysed separately, there was a significant difference in MBL levels between the three genotypes. In HIV-1 positive participants, there was no difference in rate of death between those with normal MBL compared to those with variants, log rank test 0.274. Conclusions: The results of our study show that HIV-1 infection had no influence on MBL plasma concentrations but that there were significantly higher levels of MBL in S. haematobium infected than those uninfected. The distribution of the three MBL genotypes (A/A, A/O and O/O did not differ between the infected and the healthy controls. We conclude that MBL homozygosity nor heterozygosity is not associated with increased risk of HIV-1 infection in this Zimbabwean cohort but there was increased production of MBL in S. haematobium infection. Among those HIV-1 positive, those with variant MBL were not at increased rate of death.



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Outcome of early infant diagnosis in exposed infants in Northern Nigeria

O. D. Adegoke, Z. Basir, J. Jumare, S. Sani, R. Enzama, S. Peters, A. Abimiku, and W. A. Blattner Institute of Human Virology, Nigeria Background: The rate of vertical transmission of HIV-1 in the Northern Nigeria was 35%. This prompted the intervention of PMTCT and Early Infant Diagnosis program in Northern Nigeria. Aim Evaluating the outcome of early infant diagnosis of HIV in exposed infants in Northern Nigeria and the rate of mother to child transmission of HIV in the region. Method: A total of 1236 HIV exposed infants from the Northern Nigeria, (Kano, Kaduna, Katsina, Jigawa, and Sokoto states of Nigeria) who had Dry Blood Spot (DBS) HIV-1 DNA Polymerase Chain Reaction test were enrolled for the study. The age of enrollment was between 6 weeks and 18 months with mean age of 7 months. Roche HIV 1 DNA version 1.5 method was used for analysis. Result: Total number of women who had PMTCT was 1,036 (83.8%), 182 (14.7%) had no PMTCT and 18 (1.5%) women had unknown PMTCT status. 124 (10%) children were HV-1 positive and 1112 (90%) were HIV-1 negative. 109 (87.9%) of those whose mothers had PMTCT were HIV-1 positive and those whose mother did not have PMTCT had 15 (12.1)% HIV-1 positive results. 520 (42%) children were not breast fed NBF and 495 (95.6%) of this children were HIV-1 DNA PCR negative and 25 (4.4%) children were HIV-1 DNA PCR positive. 437 (84%) of the NBF had PMTCT(PMTCT +) while 70 (16%) had no PMTCT (PMTCT2). 345 ceased breast feeding (BF Ceased) before 6weeks and 18 months and after their babies were tested for HIV. 285 (82.8%) children from this BF ceased mothers were tested HIV-1 DNA PCR negative and 60 (17.2%) was HIV-1 DNA PCR positive. Conclusion: Through the PMTCT and EID interventions, HIV-1 prevalence rate among the children of exposed mothers was dropped from 35% to 10% in Northern Nigeria. Attitude of women to absolute breast feeding in the Northern Nigeria have been changed for the benefit of the children. Through the EID program, children’s status are known and early ARV intervention is instituted. These result in decline mortality rate and more and more children are living meaningful lives in this part of the world.

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Interface of Public Health Implementation and Research – Challenges and Prospects – IHVN/ Action Project Experience

O. Akinwande, Patrick Dakum, J. Farley, U. Gebi, C. Adebamowo, A. Abimiku, M. Charurat, and W. Blattner Institute of Human Virology, Nigeria Background: The first case of HIV was described in Nigeria in 1986 with a characterization of the predominant subtype as AG recombinant and since then the prevalence has risen from an ANC Sentinel survey of 1.8 in 1991 peaking to 5.8 in 2001 and declining in 2005 to 4.4. With a population of 140m Nigeria has the second highest burden of HIV globally after South Africa. There are an estimated 5 million Nigerians currently living with the virus. In 2002 the Nigeria Government with assistance from IHV and other Partners commenced a pilot ART program that had a total of 10,000 people on therapy by 2004. Public Health Implementation: With PEPFAR Funding in 2004, the IHV commenced implementing the ACTION Project which currently supports 52 Health Facilities ranging from primary to tertiary (Teaching Hospital) sites. As at Jun 2008, ACTION Project has 57399 person receiving ART out of which 3711 are children below 15 years and 1310 are pregnant women. Laboratory and Data Management capacities have been built. Research: IHVN Research department currently coordinate research efforts focusing on assisting collaborators in site coordination of research staff, laboratory assays including sample transfers, data management and coordination of an Investigators working group. With Fogarty Funding, IHVN supported the training of 38 persons from 8 hospitals within its network on Ethical Issues related to research. IHVN has also facilitated the establishment of credible CITI certified IRBs in most of its collaborating sites. Challenges including lack of trained research assistants, committed and dedicated research staff, CITI certification for a number of site IRBs and power are issues that are being addressed. Conclusions: With about 5 internationally certified researches ongoing, Several accepted abstracts in international conferences and meetings and a number of proposals in final stages, ACTION presents a model for the interface of international public Health implementation and research and an opportunity for collaborators interested in international research both basic science and public health to provide evidence based answers to emerging public health questions in PEPFAR and Global Fund Implementation.



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The effect of chronic alcohol exposure in HIV/AIDS patients on antiretroviral drugs (Triomune30 – AZT/ 3TC/NVP) in Uganda

Dr. Godfrey Sande Bbosa, J. Ogwal-Okeng, W. W. Anokbonggo, and B. D. Kyegombe Department of Pharmacology and Therapeutics, Faculty of Medicine, Makerere University, Kampala, Uganda Alcohol is a major business worldwide and Uganda is ranked among the top alcohol consumers globally. Alcohol consumption is known to be a serious risk factor in the HIV transmission due to its effect on the CNS where it interferes with the judgment and decision making processes in the brain thus making people to engage in unsafe sex. Alcohol may also interfere with the drug metabolizing enzymes in the body like the ARVs hence leading to therapeutic failures if enzymes are induced or toxicity if the enzymes are inhibited. It may also affect the adherence of the HIV/AIDS patients on ARVS by forgetting to take their drugs on time. However, many of these patients are observed to consume alcohol despite being told to stop the habit but with no scientific data on alcohol, HIV and ARVs interaction. The longitudinal cohort study (still on-going PhD work) is being conducted to determine the effect of the chronic alcohol exposure in HIV/AIDS patients on triomune-30 in Uganda. This study is in the final stages of completion and is aimed at determining the triomune-30 drug plasma concentrations (Pharmacokinetics), the viral load, CD4 count, CBC and the LFT parameters in these patients exposed to chronic alcohol (therapeutic response).The outcome of this study will enlighten researchers on the alcohol, HIV and ARVs interaction which will be important in the policy formulation and guiding the clinicians on how to use these drugs instead of just switching drugs because of the therapeutic failure or toxicity like lactic acidosis associated with triomune-30 as observed in various ART clinics in the country that might lead to increased resistance to these drugs.

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Telemedicine in Peru: training physicians responsible for the administration of highly active antiretroviral therapy (HAART) in a developing country

Katiuska Castillo,1 Rau´ Gutie´rrez,1,3 Leslie Soto,1 Alfonso Silva-Santisteban,1 David Iglesias,1 Alberto Guerra-Garcıá,1 Carlos Kiyan,2 Carlos Seas,1 Juan Echevarrıá,1 Ciro Maguinã,1 and Eduardo Gotuzzo1 1

Instituto de Medicina Tropical Alexander von Humboldt, Universidad Peruana Cayetano Heredia, Lima, Peru; 2Facultad de Medicina, Universidad Peruana Cayetano Heredia, Lima, Peru; 3Fogarty-NIH International Training and Research Program in HIV/AIDS and TB University of Miami, Miller School of Medicine Issues: With support of the Global Fund, free HAART was implemented in Peruvian public hospitals and health centres in 2004. This effort included the conformation and training of multidisciplinary teams (Physicians, nurses, psychologists, social workers) responsible for the care of people living with HIV/AIDS throughout the country. Physicians working in remote regions would administer HAART for the first time and had to be thoroughly trained with limited resources. Description: The Telemedicine Unit of the Alexander von Humboldt Institute of Tropical Medicine developed a long-distance course on ‘‘Use of antiretrovirals’’ using an electronic platform as ‘‘classroom’’ Participants could access internet in their local hospitals or in public cabins which are widely distributed throughout Peru. The course took place from April–December 2005, covering 12 modules each lasting 2 weeks. Topics included adverse effects, antiretroviral schemes, resistance, vertical transmission, ethics, among others. (see www.upch.edu.pe/tropicales/telemedicinatarga). Each module consisted of a pre and post test, presentation, discussion forums, clinical cases and summaries of scientific articles. Lessons Learned: Sixty-eight physicians from 21 of the 24 Peruvian Regions started and 81% (n = 55) successfully completed the course. While 73% (n = 41) were already administering HAART, the others were waiting to be programmed by the Ministry of Health. Forty-nine physicians participated in an evaluation at the end of the course. They all pointed out the usefulness of the course in their daily practice; 98% (n = 48) classified the course as good/very good and 88% (n = 43) stated that the operational system was easy/very easy to use. Recommendations: After the successful implementation of a long-distance course about use of antiretrovirals, this course will be repeated for a broader target group of Peruvian physicians administrating HAART. Telemedicine has shown to be a powerful and practical tool for continuous training of health care personnel throughout the country and for the reinforcement of quality care to people living with HIV/AIDS.



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Efficacy of a Behavioral Intervention to Reduce HIV Risk among Female Sex Workers in Urumchi, China

Lin Han Division of Policy and Information, NCAIDS, Beijing, China Objectives: To assess the effectiveness of the behavioural intervention among FSWs in minority area of China. Methods: An experimental study using convenience sampling was conducted in Urumchi, capital of Xinjiang Uygur Autonomous Region. Participants attended a series of three biweekly 60-minute sessions and a three-month follow-up. The intervention program was culturally specific, addressing HIV-related knowledge, safer sexual practices, which consisting of education session, discussion session, counseling session and condom distribution. Results: A total of 229 brothel-based FSWs were recruited and 164 of the original participants were interviewed at the three months follow-up. As for the HIV transmission mode, the variables of kissing, sharing food, toilet, shaking hands and mosquitoes were statistically significant before and after the intervention (P , 0.001). Exact test found FSWs had more positive attitudes on health care, work ability of PLWHA and their own responsibility on AIDs prevention (P , 0.05) in follow-up survey. Consistent condom use and condom use during last sexual encounter with primary and other partners last month were significantly different before and after the intervention (P , 0.05). Conclusions: The intervention program consisting of culturally specific education, discussion and counselling sessions can be effective in changing self-reported, high-risk sexual behaviours with primary and other partners.

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Establishment of new Jurkat cell line Stably expressing HIV-1-Tat

Mohamed Ali Jarboui, William W. Hall, and Virginie W. Gautier Centre for Research in Infectious Diseases, University College of Dublin, Dublin, Ireland The trans-activator Tat protein is a critical regulatory factor for HIV-1 replication. We have established a new Jurkat-Tat cell line, which constitutively expresses HIV-1 Tat. Using retroviral gene delivery, we infected Jurkat cells with a VSV-G pseudo-typed virus. The virus was produced using Pheonix-GP cell line co-transfected with a VSV-G plasmid and the pCeMM-NTAPTat(GS)-Gw plasmid. HIV-1-Tat was cloned downstream a Tandem affinity purification (TAP) tag and upstream an IRES region, followed by a GFP coding sequence. We confirmed the activity of the NTAPTat protein using luciferase LTR reporter gene assay. The GFP expression allowed us to monitor the integration and the expression efficiency of the trans-gene and to isolate a heterogeneous population using FACS cell sorting. We enriched our population up to 90 % GFP expressing cells, representing different integration events. The level of expression of HIV-1-Tat was confirmed using Western-Blot analysis. Interestingly, even after high passages number (35), our Jurkat-Tat cells continue to stably express GFP and Tat. Furthermore, after preliminary observations, the expression of Tat does not alter the viability of the cells when compared to Jurkat cells even at very high density cell culture. The advantage of these Jurkat-Tat cells over several existing Jurkat clones is that Tat is expressed without being fused to GFP, meanwhile the trans-gene integration and expression can be monitored using the GFP expression level. Moreover, Tat is linked to a TAP tag which allows more efficient purification of Tat. Ultimately, the Jurkat-Tat cells represent a good model to study the molecular pathogenesis of HIV-1-Tat.



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Migration, Pastoralists, HIV Infection and Access to Care: The Nomadic Fulani of Northern Nigeria

J. Jumare,1 A. G. Habib,3 U. Gebi,1 A. Zoakah,1 P. Dakum,1 J. Farley,2 and W. A. Blattner2 Institute of Human Virology Nigeria Background: The burden of HIV infection among the nomadic Fulani of northern Nigeria is unknown. Migration — a way of life for this population — is known to increase the rate of HIV transmission and may limit individuals’ access to treatment and care. Many of Africa’s other traditional, pastoral societies are similarly affected. Objective: This paper explores cultural practices and factors among the Fulani that may influence HIV transmission, vulnerability to infection, sustainability and challenges to treatment access, as well as avenues and models for outreach services. Method: An extensive literature search with cross-referencing was done, and relevant publications on similar themes were reviewed. Three cases of Fulani nomads with HIV are presented to illustrate the challenge of providing a care continuum as well as to demonstrate successes when appropriate HIV interventions are employed. Findings: Community mobility limits opportunities for counseling, testing and diagnosis, as well as HIV-related care access and maintenance. Consanguinity and certain cultural practices among the Fulani have clear amplification potential for HIV transmission. Treatment support through the use of coaches and life partners improves adherence to antiretroviral therapy (ART). Existing programs for nomads afford opportunities for absorption and integration of HIV services. Recommendations: Nomadic communities should be provided with basic HIV-related services, including risk-reduction education and methods, counseling and testing, ART, medication adherence counseling, access to laboratory tests and health monitoring. These services should be taken to nomadic communities using novel approaches such as mobile units, extension services, case management, directly observed care, and treatment supporters linked to neighboring health facilities in a hub-and-spoke model. Stronger collaborations are recommended between programs for nomads and HIV services, and also between veterinary and public health services. Community participation and leadership should be encouraged to ensure the sustainability of HIV-related care delivery. Conclusion: More research is needed on the epidemiology and sociology of HIV infection and the best ways to provide services to hard-to-reach nomadic populations. They may well constitute HIV infection sanctuaries and should not be ignored.

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Acceptance of and adherence to anti-retroviral therapy in Tanzania: the influence of lipodystrophy and of traditional medicine

Sajida J. Kimambo Objectives: To assess the influence of lipodystrophy and of traditional medicine use on acceptance of, and adherence to, anti-retroviral therapy (ART). Design: Cross sectional study of HIV-infected subjects enrolled in a tuberculosis vaccine trial in Tanzania. Methods: ART eligible subjects who had either accepted (ARTpos) or refused ART (ARTneg) were interviewed and examined to identify subjective and objective features of lipodystrophy and to assess use of traditional medicine (TM). Results: 52 ARTpos and 53 ARTneg subjects had median CD4 counts of 296/mm3 and 160/mm3 respectively. Subjective features of lipodystrophy were noted by 39 (75%) ARTpos and 11 (21%) of ARTneg subjects (P = 0.0001). Median waist to hip ratios in males were .98 in ARTpos and .92 in ARTneg subjects (P = 0.04); in females 0.90 vs 0.83 (P = 0.004). Concern about lipodystrophy was reported to affect acceptance of ART by 19 (36%) of ARTneg subjects (P = 0.0001) and adherence to ART among 3 (6%) of ARTpos subjects. Traditional medicine was used by approximately 50% of subjects in both groups and affected ART acceptance in 11 (21%) of ARTneg subjects and ART adherence in 7 (13%) of ARTpos subjects. Conclusion: Subjective and objective features of lipodystrophy are more common among ARTpos than ARTneg subjects in Tanzania and appear to have a substantial effect on acceptance of ART, but not on adherence to ART. Traditional medicine is common regardless of ART status, and appears to have a modest effect on both acceptance of and adherence to ART.



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Changes of HIV risk behaviors of heroin drug users treated in methadone maintenance treatment clinics in Guizhou province, China

Enwu Liu Aims: To evaluate the HIV risk behaviors among heroin drug users who were treated in eight methadone maintenance treatment clinics in Guizhou province, China. Design and Methods: The study used a prospective cohort study design. Patients who had been treated in the methadone clinic no more than two and half months were recruited into the cohort beginning at June, 2006 and followed up until June, 2007. Face to face interviews were conducted to collect baseline and follow up information on HIV risk behaviors. McNemar tests were used to test the changes of HIV risk behaviors at baseline and follow-up. Results: 1003 patients were interviewed at baseline and 469 patients were interviewed both at baseline and follow-up for HIV risk behaviors. Comparing the 469 patients at baseline and follow up the study found that needle sharing behavior in the last 30 days decreased from 1.3% to 0.2% (P , 0.001); 5.5% patients had multiple sex partners in the last 30 days at baseline and 3.4% at follow up (P = 0.334); 6.4% had casual sex with casual sex partners or strangers in the last 30 days at baseline and 5.4% at follow up (P = 0. 030); when had casual sex in the last 30 days, condom use decreased from 39.1% at baseline to 19.6% at follow up (P = 0.039). Discussion and Conclusions: Methadone maintenance treatment was effective for decreasing needle sharing behaviors among heroin drug users. However, there was no significant decrease on HIV risk sexual behaviors among heroin drug users treated in the methadone maintenance clinics. Condom use with casual sex partners even decreased after patients treated in methadone clinics. Methadone clinics should let patients know that methadone itself will not prevent HIV transmission.

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Factors associated with development of Opportunistic Infections among patients on ART in Uganda

S. Muhumuza, J. Ouma, F. Semitala, E. Mbabazi, and M. Kamya Mulago-Mbarara Teaching Hospital’s Joint AIDS Program (MJAP), Kampala, Uganda Introduction: The introduction of Anti Retroviral Therapy (ART) has dramatically reduced the incidence of Opportunistic Infections (OIs) among HIV positive people who have received ART; however, potent ART does not replace the need for antimicrobial prophylaxis and treatment of OIs among patients with severe immune suppression. Background: Mulago-Mbarara Teaching Hospital’s Joint AIDS Program (MJAP) established under the Faculty of Medicine, Makerere University in 2004 using a grant awarded by PEPFAR provides ART to patients in 12 clinics basing on the eligibility criteria of CD4+ cell count ,200/mm3. By far, a total of 15,984 patients have been enrolled on ART. Methodology: Retrospective cohort analysis on adult patients initiated on ART between November 2005 and November 2007. The socio demographics, clinical and laboratory characteristics of patients that developed opportunistic infections were studied. Results: Data of 4,878 patients on ART was analyzed. 3,247 (67.2%) were females. Mean age 34.1 years (SD 8.5) and Mean weight 53.5 kg (SD 10.1). Prevalence of OIs 329 (6.7%); Oral candidiasis 106 (32%), Vaginal candidiasis 80 (24%), Kaposi’s sarcoma 40 (12%), Herpes simplex 28 (9%), Oesophageal candidiasis 25 (8%), Tuberculosis 15 (5%), others 35 (11%). WHO stage 3 &4, median CD4 count ,90/ mm3, not being married and having no formal education were significantly associated with development of OIs among patients on ART (P , 0.001). Sex, age, body weight, ART regimen and Karnofsky score were not associated with development of OIs. Conclusion: The risk of OIs is higher among patients with a low CD4 count, high WHO clinical stage, no formal education and in those that are not married.



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HIV Related TB: Prospects and Challenges in a High Burden Area, A Case Study of Isoniazid Prophylaxis (IPT) Utilization

B. M. Musa, U. Gebi, K. Falayajo, P. Dakum, J. Farley, and W. Blattner Institute of Human Virology, Nigeria Background: The complex relationship between tuberculosis (TB) and HIV does not only have an impact on the natural history of either diseases but also influence the design and implementation of programs intended to deal with the needs of people affected by the diseases. Interventions to control HIV-related TB comprise measures against both TB and HIV. However, in most settings, strategies to accomplish this are only beginning to reach the sites, where their impact will be made and the expectation of improving the outcome of both diseases realized. This study looked at the impact of TB/HIV collaborative activities; its prospect and challenges. Method: Data record of HIV+ patients accessing care were analyzed for information on TB prevention and treatment; data were also extracted from the TB registers maintained and kept at the DOTS center for tuberculosis treatment and HIV counseling and testing. Result: Of the 60 Sites under UMD/ACTION project 23 (38.3%) have access to TB/HIV services. Of the of 286,747 HIV+ patients; 18043 (6.3 %) were screened for TB; where 714 (4 %) had active TB. 1040 (6 %) of those patient in whom active TB has been ruled out were commenced on IPT. Of the 286747 that are under care in the program 2443 (0.85 %) had HCT in DOTS clinics. Conclusion: TB/HIV collaborative activity is feasible while trying to rapidly scale up HIV care and treatment. There is also a modest achievement in treating TB/HIV co-infection and there is the goodwill of clients to count upon while providing improved diagnostic capabilities and better access to care and treatment. In spite of the effort made in expanding access to TB/HIV care a lot of work has to be done to increase the uptake of IPT and the screening of HIV + patients for TB, as well as strengthening the linkage between DOTS clinics and ART points of care.

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Dyslipidemia in HIV-1 infected individuals following generic efavirenz based HAART in a resource constrained setting

Dr. Sundaram Muthu YRG CARE, Voluntary Health Services, Taramani, Chennai, India Background&Aims: Very little is known about the alterations of lipid profile among HIV-1 infected individuals initiating antiretroviral therapy containing efavirenz based regimen in resource limited settings. The study was aimed at evaluating the changes of lipid profile among HIV infected patients with and without HAART in Southern India. Methods: In a prospective study, 44 HIV-1 infected individuals (18 ART na¨ve and 26 on HAART) were enrolled for this study at YRG Centre for AIDS Research and Education in Southern India. Lipids and lipoproteins were estimated at baseline, 6 and 12 months using an Olympus AU 400 autoanalyzer. Plasma HIV RNA concentrations were determined by the Cobas Amplicor (Roche molecular Systems, NJ, USA) and Absolute CD4 T Lymphocytes count was determined using the Guava Personal Cell Analyser (Guava Technologies, Inc., USA). Baseline characteristics are described using mean and standard deviation and for non-normal data, median and interquartile range has been used. The Wilcoxon Signed rank test was used to compare the change in lipid levels from baseline to 12 months. A P value less than 0.05 were considered statistically significant. All analysis was done using SPSS v 13.0. Results: Of 44 HIV-1 infected individuals, 65% were males in the treated group and were 61% in the untreated group. At baseline, HIV infected individuals with and without HAART had a mean age of 32 and 30 years, BMI of 16.86 and 20.06 kg/m2 respectively. The median CD4 and Viral load in HIV treated and untreated patients were 197 cells/mL (IQR: 125–279), 146000 copies/mL (IQR: 38300– 214000), and 492 cells/mL (IQR: 327–595), 9670 copies/mL. All patients were initiated on efavirenz based HAART. From baseline to 12 months, patients on HAART had a significant increase in total cholesterol (134 to 170 mg/dL, P , 0.01), LDL-c (80 to 120 mg/dL, P , 0.05), Triglycerides (124 to 162 mg/dL, P , 0.01) and VLDL-c (25 to 32 mg/dL, P , 0.01) when compared to the untreated patients. HDLc showed significant increase from baseline to 6 months (32 to 51 mg/dL), but there was a significant drop at 12 months (51 to 44 mg/dL) in patients on HAART. Conclusions: Significant lipid and lipoprotein alterations are seen after 48 weeks of HAART. Serum lipid profile should be monitored annually to avoid morbidity among patients on generic HAART.



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Does toxicity to fixed dose stavudine, lamivudine and nevirapine regimen affect virologic suppression among HIV infected adults at the Infectious Diseases Institute, Makerere University?

Fred C. Semitala, 1 Harriet Mayanja- Kizza, 1 Andrew Kambugu, 2 Agnes K. Kiragga, 2 Barbara Castelnuovo, 2 Robert Kalyesubula, 1 Walter Schlech,3 Robert Colebunders,4 Keith McAdam,2 and Moses R. Kamya1 1

Department of Medicine Makerere University, Kampala, Uganda; 2Infectious Diseases Institute, Makerere University, Kampala, Uganda; 3Dalhousie University Halifax, Canada; 4Department of Clinical Sciences, Institute of Tropical ?Medicine Antwerp, Belgium

Introduction: Use of fixed dose combination of stavudine, lamivudine and nevirapine (Triomune) for the treatment of HIV/AIDS is wide spread in Resource Limited Settings. However, Triomune has been associated with toxicities which might affect the virologic outcome. The extent of the toxicities and their effects are not well studied in Uganda. Objectives: To establish factors associated with selected Triomune related toxicities and the effect of toxicities on virologic outcome during the first year of treatment among HIV-1 infected adults at the IDI. Methodology: Retrospective cohort study using the database and records from a cohort of patients started on Triomune between April 2004 and April 2005. Univariate and multivariate analysis was used to compare demographic, clinical and laboratory characteristics of patients who developed toxicity with those who did not. Results: 388 patients were included in this study; 73% were female. Median age was 37 (IQR = 32– 44) years, median BMI 20 kg/m2 (IQR = 18–22) and 92% were WHO stage 3 or 4, median CD4 count was 91 (IQR = 14–158) cells/mm3 and median viral load 5.4log10 [(IQR = 5.1–5.7)] copies /mL. 143 (37%) patients developed at least one toxicity during the first year;. 122 (31%) peripheral neuropathy (PN), 16(4%) skin rash, 11 (3%) liver injury and 6 (2%) hyperlactaemia. Baseline factors associated with toxicity were: over weight OR 5.69 (95% CI, 0.021–1.00), haemoglobin ,11.5g/dL [OR 1.92 (95% CI: 1.25–2.96)] and being started on 40 mg of stavudine compared to 30 mg [OR 1.96 (95% CI: 1.24–3.49)]. CD4 count .200 cells/mm [OR 2.39 (95% CI 1.18–4.84)] and viral load .5 log10 copies OR 1.82 (95% CI, 1.01–3.26), were associated with PN, while baseline CD4 count ,100 cells/mL [OR 3.27 (95% CI 1.60– 6.68)] with liver injury. Of the 306, patients with complete data at 12 months, 86.6% had virologic suppression. By intention to treat, only 56.6% had viral suppression at 12 months and this was statistically significant OR 5.43 (95% CI 3.09–9.63). Conclusions: There was a high incidence of toxicity especially PN and toxicity was associated with virologic failure. Over weight, low haemoglobin and receiving 40mg of stavudine were risk factors for Triomune toxicity. 184




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Intra patient evolution of Human Immunodeficiency Virus Type 1 protease and reverse transcriptase in antiretroviral naı¨ve patients

Uma Shanmugasundaram,1 Ku marasamy Nagalingeswaran,1 Kailapuri G. Murugavel,1 Saravanan Shanmugam,1 Vidya Madhavan,1 Sunil S. Solomon, 1 Suniti Solomon, 1 Kenneth H. Mayer, 2 and Balakrishnan Pachamuthu1 1

Y.R.Gaitonde Centre for AIDS Research and Education, Chennai, India; 2Department of Medicine, Brown University/Miriam Hospital, Providence, RI, USA

Objective: Intra patient HIV-1 evolution reflects the strong selection pressure driving viral escape from cytotoxic T-lymphocyte (CTL) recognition. We studied the intra host variation of HIV-1 protease (PR) and reverse transcriptase (RT) sequences from the antiretroviral na¨ve (ART) patients. Methods: Peripheral blood samples were collected longitudinally between 10–12 months from 18 HIV-1 infected ART na¨ve patients. HIV-1 RNA was isolated using Qiagen extraction kit. RT (n = 18) and PR (n = 10) gene was successfully amplified using nested PCR and direct sequencing was performed. Intrapatient nucleotide and amino acid distance of RT and PR based on pairwise comparison was performed using MEGA v3.1. The ratio of frequencies of synonymous mutations per nonsynonymous site (ds/dn) was implemented using Synscan (www.hivdb.stanford.edu). Mann Whitney U Test applied to compare the ds/dn ratio between PR and RT gene. Results: All the sequences were HIV-1 subtype C. The median intrapatient nucleotide distance of PR and RT measured based on pairwise comparison was 0.01 and 0.00 respectively. The majority of nucleotide changes in the PR and RT gene involved synonymous substitutions, resulting in a ratio of ds/dn .1 and the ratio was significantly lower (P = 0.001) in the PR sequences compared to RT indicating that the former is less conserved than the latter. The amino acids under selection located away from the active sites and some of which were under the predefined CTL epitope were observed at frequencies of above 50%. Conclusion: Negative selection observed in HIV-1 PR and RT gene indicates the absence of host’s selective forces in the absence of antiretroviral therapy and the fixation of an escape variant in the population could result in the loss of the epitope as a potential target to the immune system.



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Quality of life among end-of-life former commercial plasma donors infected with HIV and the care model in rural Henan, China

Yu Sheng Beijing P. R. China Objective: To investigate the quality of life of rural, terminally ill former plasma donors, and to discuss the trial hospice care provided by the Henan provincial government. Method: A descriptive cross-sectional design was used. The 100 terminally ill AIDS participants in this study were selected from twelve villages in Weishi county, Zhenping county and Tanghe County in Henan province using convenience sampling. The Would Health Organization Quality of Life for HIV (WHOQOL-HIV) BREF (WHO QOL-HIV-BREF Chinese version) was used to measure the quality of life of the patients. The Memorial University of Newfoundland Scale of Happiness (MUNSH) was used to measure the patients’ emotional feelings. Qualitative interviews and a focus group discussion were used to investigate the hospice care model. Results: Patients quality of the life score was 12.37 6 1.46 which is classified as moderate. The patients scored higher in spirit/religion/self-faith a dimension and psychological area while in independence dimension, social relationship dimension and environment dimension the scores are relatively low but are all in the moderate level too. The average score of the patients’ feelings of welfare is 20.91, which is also in the moderate level. The result which interviewed with village doctors indicated that there was a lower stigma for the AIDS patients in that area because the most patients were infected by plasma donation. On the other hand, they were depended on a very strong local social system which consisted of village doctors, nurses and patient’s assistants to provide the care for the end of life AIDS patients. Conclusions: There are a modirate quality of life and feelings of welfare for end-of-life AIDS patients in Henan. This care model which was base on local cultural social system is effective and available for the end of life AIDS patients in Henan. It’s necessary to develop the nurses’ function and involve them into the team for the HIV/AIDS treatment and prevention.

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Scaling Up PMTCT Access Using a Hub and Spoke Model in Nigeria, Sub-Sahara Africa

Dr. Akinmurele Timothy Institute of Human Virology, Nigeria Issues: PMTCT services in Nigeria were initially limited to comprehensive ART centers, limiting access. Extending PMTCT to small secondary hospitals and primary health centers necessitates access to CD4 count and clinical staging for HIV+ women, linkage to an ART center for women requiring HAART for their own health care, and quality site monitoring. Description: PMTCT networks were developed regionally, with small secondary hospital and primary health center ‘‘Spoke’’ sites linked to a larger hospital ‘‘Hub’’ sites. For HIV+ women at ‘‘Spoke’’ sites, blood was drawn for CD4 count and transported to the ‘‘Hub’’ site laboratory. Those HIV+ women at ‘‘Spoke’’ sites requiring HAART for their own health care were provided transportation to the ‘‘Hub’’ site, while all other HIV+ women were offered short course ARV prophylaxis at the ‘‘Spoke’’ site. A network coordinator from the ‘‘Hub’’ site provided training and site monitoring and organized monthly network coordination meetings. Results and Lessons Learned: Networks consisting of 3 ’Hub" sites and 13 ‘‘Spoke’’ sites providing services in 2007 were evaluated. At ‘‘Hub’’ sites, 6,882 new ANC clients were served, with 74% counseled, HIV tested and receiving results. At ‘‘Spoke’’ sites, 33,119 new ANC clients were served, with 87% counseled, tested and receiving results. 583 HIV+ pregnant women were identified/referred and received services at the ‘‘Hub’’ sites, while 536 were identified and received services at the ‘‘Spoke’’ sites. Women were less likely to return for delivery at the ‘‘Spoke’’ sites (48.5%) than ‘‘Hub’’ sites (61.2%) Next Steps: The network model provided a fourfold increase in women accessing PMTCT services. Use of a regional coordinator ensures quality of care and linkage and addresses implementation/ coordination challenges. Intensive interventions to reduce loss to follow up of HIV+ women are even more critical as PMTCT services are provided at the primary health center level.



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Impact of Community- and Home-Based Care on HIV/AIDS Patients and Their Families

Emily Hauwa Umaru Institute of Human Virology, Nigeria Background: In efforts to mitigate HIV pandemic, the federal government of Nigeria collaborated with PEPFAR implementers in selected health facilities around the federation to enhance services provided to HIV infected and affected clients. IHVN-ACTION program through its multidisciplinary team is building capacity for provision of comprehensive HIV treatment, care and support. This includes HCT, Lab investigations, treatment/ adherence support, palliative care (care & support and home based care). In addition Home based care services were commenced to enhance continuum of care for PLHAs and infected children. Formal and informal care givers receive training in home care and other community services to promote, restore and maintain a person’s maximum level of comfort, function and health. Health professionals work in collaboration with volunteers to provide them with technical assistance in their communities while volunteers in turn refer clients to health facilities. Approach: Advocacy to policy and community leaders on HIV/AIDS is paramount to gain their support for a successful program implementation. Community leaders are sensitized and intimated on the benefits of the program and requested to nominate volunteers to support the program. In addition volunteers are drawn PLHA support groups who also serve as peer educators to encourage other PLWHAs. Health care providers (nurse coordinator and social workers) from the facility serve as a link to provide technical assistance to the volunteers. These volunteers undergo six days intensive training to equip them with knowledge and skills in HIV/AIDS care. Volunteers are trained to conduct various activities such as home visit to the client for: Palliative/basic nursing care, psychosocial, nutritional, spiritual and adherence support. They also assist with activities of daily living, provide HBC kits where necessary, educate relatives and friends on care for PLHA, identify care givers for OVCs, counsel and refer relatives and friends for HCT and psychosocial support. Networking, linkages and collaboration with other CBOs/NGOs are done to leverage client support for other services. They conduct ongoing community sensitization for HIV/AIDS prevention, stigma reduction and behavior change for positive living, follow up defaulters (loss to follow up/poor adherence) and refer clients for health services, mobilize communities for ongoing mobile HCT in collaboration with counselors/Lab teams. The HBC services had a humble beginning, with only a few clients as many were afraid they’ll be stigmatized by neighbors and other family members. This number has grown tremendously, services have continued to expand as volunteers now identify more patients in need of care through

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Impact of Community-and Home-ased Care on HIV/ AIDS Patients and Their Families? (continued)

aggressive community mobilization, serve as buddy for community adherence and refer sick persons to hospital for HCT etc. To enhance their impact the HBC nurses were trained by ACTION to do HIV counseling and testing in the community and at homes. The linkage between the community and the hospital has been strengthened through the services of the HBC team. The utilization of volunteers has made it possible for smooth transition and effective provision of palliative care from the facility into the community thus enhancing the proverbial adage that ‘‘human beings are their brothers’ keepers’’. The community involvement and support has been overwhelming to the surprise of both clients and care providers. They have mobilized additional resources e.g. funds and food items to assist clients. Radio programs and other modes of communication are used to create awareness about HIV and services for those infected and affected. Ongoing HIV/AIDS sensitization to communities and in the media is gradually changing the attitude of individuals. Family and community members are informed on HIV/AIDS and provide care for their loved ones which has tremendously reduced stigma, people can now talk about HIV, and opt for HCT, treatment and care. Challenges: Despite the effort from CHBC volunteers, some challenges abound, like need for increased funding support and transportation assistance especially to hard to reach areas as their client load grows. Conclusion: Despite challenges, the benefits of community and home based services have been encouraging to the program and service providers. Many volunteers verbalize how improving patient quality of life and ability for self care continue to keep them motivated. Many of the patients themselves return to work as volunteers and adherence supporters for others so as to share their lives with others.



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Prevalence of Norovirus infections in children from Iquitos, Peru

Daniel Velasquez Portocarrero Jr. Enrique Barron, PERU Background: Noroviruses are the most commonly identified cause of outbreaks and sporadic cases of acute Gastroenteritis. Norovirus is a group of non-enveloped RNA viruses that belong to the family Caliciviridae. A number of studies in both developed and developing countries have shown that Norovirus is a major enteric pathogen, (second only to rotavirus), causing acute diarrhea in children which can sometimes be fatal. Objective: To characterize the burden of diarrheal disease caused by Norovirus in children from Iquitos, Peru. Methods: Norovirus testing was done for genogroup I and genogroup II using sensitive and specific TaqMan Real-time Reverse Transcription-PCR in 338 fecal specimens from children in a periurban area near Iquitos, Peru. Results: Norovirus was present in 23.6% of 254 diarrheal samples as compared to 9.3% of 84 nondiarrheic controls (P = 0.007). GII was present in 14.8% of diarrheal stools tested and 5.3% of stools from children in the absence of diarrhea. GI was present in 8.7% of diarrheal stools and 4.0% of asymptomatic controls. The presence of GII, but not GI was associated with the presence of diarrhea (Fisher’s exact 0.03 and 0.22, respectively) although the power of the study to detect such a difference with this sample size was only 17%. We also found that the identification of Norovirus in stool samples was age-related and decreased 32% per year (95% CI: 15–45%). The characterization of the enteric disease caused by Norovirus was done by comparing the presence of clinical symptoms and signs. Norovirus disease detected by active surveillance is best characterized as acute uncomplicated watery diarrhea. There was no difference in maximum stool frequency reported in a 24 hour period during the diarrheal episode in Norovirus as compared with diarrhea from other causes (6.75 and 7.03 episodes/24 hrs, P = 0.58). There was no difference in the reported incidence of nausea, vomiting, or fever between Norovirus and all-cause diarrhea. Conclusions: This study demonstrates that Norovirus is an important enteric pathogen in this population. GII was more prevalent than GI, which is consistent with other studies of Norovirus in the world. It also documents for the first time the prevalence of Norovirus infections in Peru.

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Adherence to highly active antiretroviral treatment and related factors in drug users with HIV/AIDS

Wang Honghong School of Nursing, Central South University, Changsha, China Objectives: To explore adherence to antiretroviral treatment (ART) and related factors in drug users with HIV/AIDS. Methods: From July to September 2007, 111 HIV-infected drug users who received national free HAART were investigated in the HAART clinics in Hengyang, Yueyang, and Chenzhou districts of Hunan Province. A questionnaire of Community Programs for Clinical Research on AIDS (CPCRA ) Antiretroviral Medication Self-Report was administered to assess adherence to ART, and Zung Depression Scale and Adaptation Partnership Growth Affection Resolve Scale were used to assess patients’ depression and family function respectively. Results: The average level of adherence to ART was 83%. Among 111 patients, 28.8% of patients reported poor adherence and took medication less than 90%. There were 83.9% patients demonstrating depressive symptom and only 30.6% patients’ family had good function. Logistic regression analysis showed that the degree of depression and treatment time were significantly associated with adherence negatively, while family function and the time of being free from drug were positively associated with adherence. Conclusion: The level of adherence to ART is low in the drug users with HIV/AIDS. Comprehensive interventions are needed to improve adherence to ART, including managing depression, encouraging drug rehabilitation, improving family function, and evaluating adherence periodically.



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Immunologic recovery among HIV infected children on first line antiretroviral therapy in Kano, Nigeria

Bashir M. Zubayr, Background: HIV disease progression in infants and children is rapid compare to adults. This picture is gloomier in sub Sahara Africa due to the co-morbidities of malaria, diarrhea disease and malnutrition. Therefore, the goal of treatment and care of the HIV infected child is to promote health and prevent disease progression. This present study evaluates immunologic recovery after one year of antiretroviral therapy using CD 4 cells counts. Aims and Objectives: To determine the pattern of CD4 count rise after one year of Zodovudine base first line therapy in HIV infected children. Method: HIV infected children who were treatment na¨ve and commenced on Zodovudine based regimen were enrolled into the study. Age, weight, CD4 counts and Hematocrit prior to initiation of treatment were all recorded and entered into a proforma. These parameters were reassessed again at 6 and 12 months on treatment and recorded. Data collected was analyzed using SPSS version 12 and presented as means and standard deviation. Results: A total of 92 HIV infected children completed the study, of which 48 were males and 44 females. Mean age at initiation of therapy was 5.4years while mean baseline Cd4 counts was 688 cell/mm. mean CD4 counts at 12 months was 746 cells, gain in CD4 among subjects was found to be statistically significant. (P , 0.001) Conclusions: There is significant increase in the Cd4 count after one year of treatment but no significant change in weight and hematocrit.

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