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35th Annual Symposium on Nonhuman Primate Models for AIDS
August 22-25, 2017 Overture Center for the Arts Madison, Wisconsin USA Hosted by the Wisconsin National Primate Research Center
This conference is supported (in part) by the Office of The Director, National Institutes of Health under Award Number R13OD024607 through PA16‐294 from the Office of Research Infrastructure Programs (ORIP). The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services, National Institutes of Health; nor does mention by trade names, commercial practices, or organizations imply endorsement by the U.S. Government.
Session 3 Virus‐host interactions and immune responses (abstracts 300‐310) Session 4 Genomics: Host and microbiome (abstracts 400‐410)
Friday, August 25 Program
Session 5 ‘SIV tools’ for other pathogens (abstracts 500‐510)
Oral Abstracts (listed by session)
Poster Abstracts (listed by poster board number)
All Author Index
List of Registrants (and their contact)
Things to do, places to see
Co‐Chair Welcome Dear friends and colleagues: Welcome to Madison and to the 35th Annual Symposium on Nonhuman Primate Models for AIDS. Thanks to the hard work of our scientific committee, we have assembled a program of talks and poster presentations that promises to be very exciting and informative. We hope this meeting will provide you a chance to meet and exchange ideas with a wide range of investigators, in keeping with the high standards set by previous Symposia. We are particularly pleased to be able to provide scholarship support to an outstanding group of junior investigators, who will be presenting their work here. We also hope that you will have an opportunity to enjoy some of the amenities Madison has to offer in the summer, including our beautiful lakes, great places to eat and drink, and of course cheese curds and beer (if they are your thing). We are particularly grateful to Leah Leighty and her staff at UW–Madison CALS Conference Services for their outstanding support in organizing this conference. We wish everyone a productive and enjoyable stay in Madison!
Thomas Friedrich and Shelby O’Connor
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Organizing Committee Dr. Shelby O’Connor Co‐Chair Associate Professor UW‐Madison
Dr. Thomas Friedrich Co‐Chair Associate Professor UW‐Madison
Dr. Jon Levine Professor and Director Wisconsin National Primate Research Center
Dr. David O’Connor Professor and Associate Director Wisconsin National Primate Research Center
Scientific Program Committee Dr. Ann Chahroudi Assistant Professor Emory University
Dr. Elizabeth Connick Professor University of Arizona
Dr. David Evans Professor Wisconsin National Primate Research Center
Dr. JoAnne Flynn Professor University of Pittsburgh
Dr. Thomas Friedrich Associate Professor UW‐Madison
Dr. Ann Hessell Assistant Professor Oregon Health and Science University
Dr. David O’Connor Professor UW‐Madison
Dr. Shelby O’Connor Associate Professor UW‐Madison
Dr. Sallie Permar Professor Duke University
Dr. Matthew Reynolds Assistant Professor UW‐Madison
Dr. Nathan Sherer Associate Professor UW‐Madison
Dr. Vaiva Vezys Associate Professor University of Minnesota
Dr. Ryan Westergaard Assistant Professor UW‐Madison
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Sponsor Acknowledgement The Organizing Committee gratefully acknowledges the generous support of the meeting sponsors. Many of the sponsors are in attendance. We strongly encourage you to take a moment to inquire about how their products could help you.
*Jointly supported by ORIP and OAR
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Young Investigator Scholarships With generous financial support from the National Institutes of Health, jointly supported by ORIP and OAR, the Scientific Committee is delighted to provide a number of full scholarships for promising young scientists to attend this conference. The recipients of the Young Investigator Scholarships are listed below. Dr. Ben Burwitz – Oregon Health & Science University Dr. Yoshi Fukazawa – Oregon Health & Science University Adam Kleinman – University of Pittsburgh Dr. Danijela Maric – Northwestern University Paul Munson – University of Washington Dr. Megan O'Connor – Washington National Primate Research Center Dr. Jeffrey Schneider – Northwestern University Spandan Shah – Beth Israel Deaconess Medical Center Lisa Smith – Texas Biomedical Research Institute Dr. Helen Wu – Oregon Health & Science University Outstanding Poster Awards Kawthar Machmach – California National Primate Research Center Naofumi Takahashi – Tulane National Primate Research Center
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Schedule at a Glance Tuesday, August 22, 2017 17:00 – 22:00
KEYNOTE ADDRESS ‐ Deborah Persaud, PhD, Johns Hopkins Promenade Hall 99 FROM THE “MISSISSIPPI BABY” TO THE NONHUMAN PRIMATE MODEL: CONTRIBUTIONS TO UNDERSTANDING HIV PERSISTENCE AS A STEP TOWARD CURE Welcome Reception
Scientific Session 1 Interventions: Therapeutic vaccines and functional cures
Chairs: Douglas Nixon and Vaiva Vezys 8:45 – 9:15 100 HIV: WHERE DID IT COME FROM AND WHERE IS IT GOING? Douglas Nixon1 1 George Washington University 9:15 – 9:30 101 ANTI‐CD20 ANTIBODY MEDIATED B CELL DEPLETION ENHANCES VIRAL CONTROL IN SIV‐ INFECTED RHESUS MACAQUES Yoshi Fukazawa1,2, Richard Lum1,2, Jin Young Bae1,2, Alden Ho1,2, Scott Hansen1,2, Jeseph Clock1,2, Bryan Randall1,2, Haesun Park1,2, Alfred Legasse1,2, Michael Axthelm1,2, Jeffrey Lifson3, Afam Okoye1,2, Louis Picker1,2 1 Vaccine and Gene Therapy Institute, Oregon Health & Science University, 2Oregon National Primate Research Center, Oregon Health & Science University, 3AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory 9:30 – 9:45 102 FULLY MHC‐MATCHED ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION IN SIV‐INFECTED, CART‐SUPPRESSED MAURITIAN CYNOMOLGUS MACAQUES Dr. Helen L Wu1, Dr. Benjamin J Burwitz1, Dr. Shaheed A Abdulhaqq1, Christine Shriver‐Munsch2, Tonya Swanson2, Alfred W Legasse2, Katherine B Hammond1, Jason S Reed1, Mina Northrup1, Dr. Stephanie L Junell4, Dr. Gabriela M Webb1, Dr. Justin M Greene1, Dr. Benjamin N Bimber2, Dr. Wolfram Laub4, Dr. Paul Kievit2, Dr. Rhonda MacAllister2, Dr. Michael K Axthelm2, Dr. Rebecca Ducore2, Dr. Anne Lewis2, Dr. Lois Colgin2, Dr. Theodore R Hobbs2, Dr. Lauren D Martin2, Dr. Charles R Thomas4, Dr. Angela Panoskaltsis‐Mortari5, Dr. Gabrielle Meyers3, Dr. Jeffrey J Stanton2, Dr. Richard T Maziarz3, Dr. Jonah B Sacha1,2 1 Vaccine and Gene Therapy Institute, Oregon Health & Science University, 2Oregon National Primate Research Center, Oregon Health & Science University, 3Division of Hematology and Medical Oncology, Oregon Health & Science University, 4Division of Medical Physics, Oregon Health & Science University, 5Division of Blood and Marrow Transplantation, University of Minnesota 9:45 – 10:00 103 THE IL‐15 SUPERAGONIST ALT‐803 DECREASES PLASMA VIRAL LOADS IN SIV INFECTED RHESUS MACAQUES IN THE ABSENCE OF ANTIRETROVIRAL THERAPY Dr. Amy Ellis1, Alexis Balgeman1, Katie Zarbock2, Gabrielle Barry2, Andrea Weiler2, Thomas Friedrich3, Jeffrey Miller4, Timothy Schacker5, Ashley Haase6, Emily Jeng7, Jack Egan7, Hing Wong7, Eva Rakasz2, Shelby O'Connor1 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, 3Department of Pathobiological Sciences, University of Wisconsin‐Madison, 4Division of Hematology, Oncology, and Medicine, University of Minnesota, 5 Department of Medicine, University of Minnesota, 6Department of Microbiology, School of Public Health, University of Minnesota, 7Altor Bioscience Corporation
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10:00 – 10:15
10:15 – 10:30
10:30 – 11:00
104 THE HUMAN IL‐15 SUPERAGONIST COMPLEX ALT‐803 ACTIVATES NK AND MEMORY T CELLS, REACTIVATES LATENT SIV, AND DRIVES SIV‐SPECIFIC CD8+ T CELLS INTO B CELL FOLLICLES Gabriela Webb1,2, Katherine Hammond1,2, Shengbin Li8, Gwantwa Mwakalundwa8, Justin Greene1,2, Jason Reed1,2, Jeffrey Stanton2, Alfred Legasse1, Byung Park1, Michael Axthelm1, Emily Jeng3, Hing Wong3, James Whitney4,5, Brad Jones6, Douglas Nixon7, Pamela Skinner8, Jonah Sacha1,2 1 Oregon Health & Science University/Vaccine And Gene Therapy Institute, 2Division of Pathobiology and Immunology, Oregon National Primate Research Center, Oregon Health & Science University, 3 Altor BioScience Corporation, 4Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, 5Ragon Institute of MGH, MIT, and Harvard, 6Department of Microbiology Immunology and Tropical Medicine, 7School of Medicine & Health Sciences, The George Washington University, 8Department of Veterinary and Biomedical Sciences, University of Minnesota 105 NEXT GENERATION GENE PROTECTION AND RESERVOIR TARGETING APPROACHES FOR HIV CURE Christopher Peterson1,2, Claire Deleage3, Anjie Zhen4, Andreas Reik5, Michael C. Holmes5, Scott Kitchen4, Jacob D. Estes3, Hans‐Peter Kiem1,2 1 Fred Hutchinson Cancer Research Center, 2University of Washington, 3AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., 4 UCLA, 5Sangamo Therapeutics Coffee Break
106 THERAPEUTIC VACCINES FOR HIV Dr. Vaiva Vezys1 1 University of Minnesota 107 CONSERVED ELEMENTS (CE) DNA VACCINATION INDUCES CE RESPONSES IN SIV INFECTED, CART TREATED MACAQUES Paul Munson1,2, Hillary Tunggal1,2, Nika Hajari1,2, Megan O'Connor1,2, Debra Bratt2, James T. Fuller1,2, Drew May2, Solomon Wangari2, Brian Agricola2, Jeremy Smedley2, Xintao Hu3, Barbara K. Felber3, George N. Pavlakis4, James I. Mullins1, Deborah Heydenburg Fuller1,2 1 Department of Microbiology, University of Washington , 2University of Washington National Primate Research Center, 3Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute , 4Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute 108 COMPARATIVE ASSESSMENT OF THREE LATENT SIV RESERVOIR REACTIVATION STRATEGIES IN ELITE CONTROLLER RHESUS MACAQUES Adam Kleinman1, Dr. Ranjit Sivanandham1, Benjamin Policicchio1, Dr. Egidio Brocca‐Cofano1, Kevin Raehtz1, Tianyu He1, Dr. Cui Ling Xu1, Dr. Paola Sette1, Kathryn Martin1, Ellen Penn1, Dr. Ivona Pandrea1, Dr. Cristian Apetrei1 1 University Of Pittsburgh 109 SIV‐SPECIFIC RNA‐GUIDED CAS9 NUCLEASES AND PAIRED NICKASES INHIBIT SIV REPLICATION THROUGH PROVIRAL GENOME EDITING Lisa Smith1, Vida Hodara2, Laura Parodi2, Zhao Lai1, Yi Zou1, Luis Giavedoni2 1 University of Texas Health San Antonio, 2Texas Biomedical Research Institute
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12:00 – 12:15
12:15 – 16:00 12:15 ‐ 14:00
110 ADOPTIVE T CELL IMMUNOTHERAPY USING CMV‐SPECIFIC T CELLS GENETICALLY MODIFIED WITH ΑHIV‐CAR VECTORS Chengxiang Wu1, Shan Yu1, Agnus Lo2, Hui Li3, Gautam Sahu4, Preston Marx1, Dorothee von Laer5, Gail Skowron4, George Shaw3, Amitinder Kaur1, Richard Junghans2, Stephen E. Braun1 1 TNPRC, 2Tufts University Medical School, 3University of Pennsylvania, 4Roger Williams Medical Center, 5Medizinische Universität Innsbruck Poster set up
Chairs: Ann Chahroudi and Matt Reynolds 14:00 – 14:30 200 OVERVIEW OF VACCINE APPROACHES IN ADULT AND INFANT MACAQUES Ann Chahroudi1 1 Emory University 14:30 – 14:45 201 CROSS‐SPECIES CMV VACCINATION REVEALS VIRAL DETERMINANTS FOR INDUCTION OF NON‐CLASSICAL MHC‐E‐RESTRICTED T CELLS Dr. Justin Greene1, Dr. Daniel Malouli1, Dr. Scott Hansen1, Dr. Travis Whitmer1, Abigail Ventura1, Roxanne Gilbride1, Colette Hughes1, Jason Reed1, Dr. Helen Wu1, Luke Uebelhoer2, Jennie Womack1, Matthew McArdle1, Junwei Gao1, Alfred Legasse1, Dr. Michael Axthelm1, Dr. Louis Picker1, Klaus Fruh1, Jonah Sacha1 1 Oregon Health & Science University/Vaccine And Gene Therapy Institute, 2Oregon Health & Science University/ Department of Pediatrics 14:45 – 15:00 202 A SINGLE V2 MONOCLONAL ANTIBODY REDUCES LYMPHOID TISSUE VIREMIA AND PARTIALLY PROTECTS MACAQUES AFTER REPEATED SHIV CHALLENGES Dr. AJ Hessell1, DC Malherbe1, SM McBurney1, S Pandey1, T Cheever1, P Barnette1, WF Sutton1, S Zolla‐Pazner2, NL Haigwood1 1 Oregon Health & Science University, Oregon National Primate Research Center, 2Icahn Mt. Sinai School of Medicine 15:00 – 15:15 203 AN ORAL PRIME/ BOOST PEDIATRIC VACCINE STRATEGY FOR THE PREVENTION OF HIV TRANSMISSION BY BREAST‐FEEDING Alan Curtis1, Bonnie Phillips1, Neelima Choudhary1, Ryan Tuck1, Koen Van Rompay2, Pamela Kozlowski3, Rama Amara4, Kristina De Paris1 1 University Of North Carolina At Chapel Hill, 2California National Primate Research Center, 3 Louisiana State University, 4Emory University 15:15 –15:30 204 A NOVEL SHIV EXPRESSING THE B41 HIV‐1 ENV: IMPLICATIONS FOR HIV VACCINE DESIGN AND TESTING Jessica Smith1, Hui Li1, Wenge Ding1, Shuyi Wang, Alexander Murphy, Maho Okumura, Beatrice H. Hahn1, George M. Shaw1 1 Perelman School of Medicine 15:30 – 15:45 205 DRUG DISTRIBUTION AT SHIV INFECTION SITES IN THE MACAQUE FEMALE REPRODUCTIVE TRACT Dr. Katarina Halavaty1, Dr. Adina K. Ott1, Dr. Danijela Maric1, Dr. Jonathan T. Su1, Edgar Matias1, Dr. Lara Pereira2, Dr. James M. Smith3, Dr. Patrick F. Kiser1, Dr. Thomas J. Hope1 1 Northwestern University, 2Lifesource Biomedical LLC, 3Centers for Disease Control and Prevention
206 ALLOIMMUNIZATION OF MAURITIAN CYNOMOLGUS MACAQUES WITH ALLOGENEIC CELLS FAILS TO PROTECT AGAINST REPEATED, LIMITING‐DOSE SIV CHALLENGE Dr. Matt Reynolds1 1 UW‐Madison 207 TRACKING FLUOROPHORE‐CONJUGATED VRC01 FOLLOWING IV INJECTION IN THE RHESUS MACAQUE REVEALS THAT TISSUE DISTRIBUTION IS SLOW AND CAN TAKE APPROXIMATELY 1 WEEK TO ACHIEVE STEADY STATE. Dr. Jeffrey R. Schneider1, Dr. Ann M. Carias1, Dr. Amarendra Pegu2, Dr. Arangassery R. Bastian1, Dr. Gianguido C. Cianci1, Dr. Patrick F. Kiser1, Dr. Ronald S. Veazey3, Dr. John R. Mascola2, Dr. Thomas Hope1 1 Northwestern University, 2Vaccine Research Center, 3Tulane National Primate Research Center 208 DNA AND PROTEIN CO‐DELIVERY VACCINES USING TLR‐4‐BASED ADJUVANTS INDUCE POTENT IMMUNE RESPONSES ABLE TO DELAY HETEROLOGOUS SIV ACQUISITION Barbara Felber1, Shakti Singh1, Antonio Valentin1, Margherita Rosati1, Eric Ramierz1, Rami Doueiri1, Xintao Hu1, Jenifer Bear1, Vanessa Hirsch2, Kate Broderick3, Niranjan Sardesai3, Steven Reed4, Hung Trinh5, Mangala Rao5, David Montefiori6, Guido Ferrari6, Xiaoying Shen6, Georgia Tomaras6, George Pavlakis1 1 National Cancer Institute At Frederick, 2NIAID, 3Inovio Pharmaceuticals Inc., 4IDRI, 5MHRP, 6Duke University Medical Center 209 IMPACT OF A TLR‐5 LIGAND AS ADUVANT ON IMMUNOGENICITY AND EFFICACY OF A RHCMV‐SIV VACCINE Dr. Ellen Sparger1, Dr. William Chang1, Dr. Jesse Deere1, Mr. Hung Kieu1, Dr. Diego Castillo1, Dr. Shelley Blozis1, Dr. Jeffrey Lifson2, Dr. Xiaoying Shen3, Dr. Georgia Tomaras3, Dr. Barbara Shacklett1, Dr. Peter Barry1 1 University Of California Davis, 2Frederick National Laboratory, 3Duke Human Vaccine Institute 210 DISTINCT REPLICATION PATTERNS AND NEUTRALIZING ANTIBODY RESPONSES IN RHESUS MACAQUES INFECTED BY SHIVS BEARING 15 DIFFERENT PRIMARY OR TRANSMITTED/FOUNDER HIV‐1 ENVS Dr. Hui Li1, Dr. Fang‐Hua Lee1, Mr. Ryan Roark1, Ms. Jessica Smith1, Ms. Maho Okumura1, Mrs. Shuyi Wang1, Mrs. Wenge Ding1, Dr. Beatrice Hahn1, Dr. George Shaw1 1 University Of Pennsylvania
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17:30 – 20:00 Poster Session
17:30 – 20:00 Reception Promenade Lobby Posters are listed by poster board number. All poster will be on display in the Promenade Lobby. 1 THE EFFECTS OF M. TUBERCULOSIS ON SIV EVOLUTION IN A MAURITIAN CYNOMOLGUS MACAQUE CO‐INFECTION MODEL Alexis Balgeman1, Amy Ellis‐Connell1, Katie Zarbock2, Jaffna Mathiaparanam1, Mark A. Rodgers3, Cassandra Updike3, Tonilynn Baranowski3, Charles A. Scanga3, Shelby O'Connor1 1 University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, 3University of Pittsburgh 2 NOVEL SHIVS BEARING HIV‐1 TRANSMITTED/FOUNDER ENVS FOR CURE RESEARCH: REPLICATION, CART SUPPRESSION, RESERVOIRS AND REBOUND Dr. Katharine Bar1, Anya Bauer1, Dr. Fang‐Hua Lee1, Dr. Hui Li1, Dr. George Shaw1 1 University Of Pennsylvania 3 SIMULTANEOUS EXPRESSION OF INTERFERON‐GAMMA AND INTERLEUKIN‐22 FROM INNATE LYMPHOID AND NATURAL KILLER CELLS IN THE COLON OF SIV‐INFECTED RHESUS MACAQUES Andrew Cogswell1, Moriah Castleman2, Stephanie Dillon2, Cara Wilson2, Dr Ed Barker1 1 Rush University Medical Center, 2University of Colorado 4 NOVEL FEATURES OF CRM1‐DEPENDENT HIV AND SIV RNA NUCLEAR EXPORT REVEALED USING LIVE CELL IMAGING Ryan Behrens1, Christina Higgins1, Nathan Sherer1 1 McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin ‐ Madison 5 NKTT320‐INDUCED ACTIVATION OF INVARIANT NATURAL KILLER T‐CELLS (INKTS) IN VIVO AND IMPLICATION FOR ITS USE IN MODULATING AIDS PATHOGENESIS Dr. Nell Bond1, Dr. Shan Yu1, Dr. Namita Rout1, Ms. Dollnovan Tran1, Mrs. Dawn Szeltner1, Dr. Robert Schaub2, Dr. Amitinder Kaur1 1 TNPRC, 2NKT Therapeutics 6 CHARACTERIZATION OF VACCINE‐INDUCED ANTIBODY RESPONSES Tysheena Charles1, Samantha Burton1, Lori Spicer1, S. Abigail Smith1, Tiffany Styles1, Pradeep Reddy1, Traci Legere1, Vijayakumar Velu1, Dr Rama Amara1, Dr Cynthia Derdeyn1 1 Emory University 7 EARLY CNS DAMAGE IN PEDIATRIC SIV INFECTION Heather Carryl1, Bonnie Phillips2, Koen Van Rompay3, Angela Amedee4, Mark Burke1, Kristina DeParis2 1 Howard University, 2UNC Chapel Hill, 3UC Davis‐ CNPRC, 4LSU 8 GUT MICROBIOTA IS ASSOCIATED WITH THE IMMUNE RESPONSE TO HIV‐1 ENVELOPE VACCINATION OF NEWBORN RHESUS MACAQUES
Amir Ardeshir1, Holly Heimsath2, Olaf Mueller2, Bonnie Phillips2, Joshua Eudailey2, Erika Kunz2, Genevieve Fouda2, Laura‐Leigh Rowlette2, Holly Dressman2, Kristina De Paris3, Koen Van Rompay1, Sallie Permar2 1
University of California Davis, 2Duke University, 3University of North Carolina
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9 HIGH‐THROUGHPUT GENERATION OF SIMIAN‐HUMAN IMMUNODEFICIENCY VIRUS AND SIMIAN TROPIC HIV REPLICATION‐COMPETENT MOLECULAR CLONES Dr. Debashis Dutta1, Samuel Johnson1, Alisha Dalal1, Martin J. Deymier2, Eric Hunter2, Siddappa N Byrareddy1 1 University of Nebraska Medical Center, 2Emory Vaccine Center, Emory University 10 IMPACT OF MATERNAL IM/IN HIV‐ENV IMMUNIZATION DURING PREGNANCY ON POSTNATAL HIV TRANSMISSION. Maria Dennis1, Josh Eudailey1, Morgan Parker1, Bonnie Philips2, Genevieve Fouda1, Koen Van Rompay3, Kristina DeParis2, Sallie Permar1 1 Duke Human Vaccine Institute, 2UNC, 3UC Davis 11 NIAID REAGENT RESOURCE SUPPORT CONTRACT FOR AIDS VACCINE DEVELOPMENT Dr. Valerie Fremont1, Dr. Rosemarie Mason2, Michael Fisher1, Dr. Mario Roederer2, Dr. Ronald L. Brown1 1 Quality Biological, 2Vaccine Research Center, NIAID, NIH 12 BI‐FUNCTIONAL ENTRY INHIBITORS SENSITIZE MACAQUE‐TROPIC HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 (HIV‐1MT) TO ANTIBODIES GENERATED IN HIV‐1MT‐ INFECTED MACAQUES Dr. Shigeyoshi Harada1, Yuta Hikichi1, Dr. Yohei Seki2, Dr. Akatsuki Saito2, Dr. Takeshi Yoshida2, Dr. Hirotaka Ode3, Dr. Yasumasa Iwatani3, Dr. Yasuhiro Yasutomi4, Dr. Tomoyuki Miura5, Dr. Tetsuro Matano1, Dr. Hirofumi Akari2, Dr. Kazuhisa Yoshimura1 1 AIDS Research Center, National Institute of Infectious Diseases, 2Primate Research Institute, Kyoto University, 3National Hospital Organization Nagoya Medical Center, 4Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition, 5Institute for Frontier Life and Medical Sciences, Kyoto University 13 MODELING OF NEUTROPHIL, BASOPHIL, AND CLASSICAL MONOCYTE KINETICS BY BRDU PULSE‐CHASE LABELING IN YOUNG ADULT AND ELDERLY RHESUS MACAQUES Ziyuan He1, Chie Sugimoto1, Carolina Allers1, Hideki Fujioka2, Elizabeth Didier3, Marcelo Kuroda1 1 Division of Immunology, Tulane National Primate Research Center, 2Center for Computational Science, Tulane University, 3Division of Microbiology, Tulane National Primate Research Center 14 PERSISTENT MEMORY RESPONSE ELICITED BY HIV/SIV CONSERVED ELEMENT GAG PDNA PRIMING VACCINE AND RAPID RECALL UPON DNA OR RMVA VECTOR BOOST Xintao Hu1, Antonio Valentin1, Yanhui Cai1, Frances Dayton1, Valerie Ficca1, Margherita Rosati1, Patricia Earl2, Bernhard Moss2, Niranjan Sardesai3, James Mullins4, George Pavlakis1, Barbara Felber1 1 National Cancer Institute At Frederick, 2NIAID, 3Inovio Pharmaceuticals Inc., 4University of Washington 15 IMPROVING CHARACTERIZATION OF THE FULL MHC GENOMIC REGION IN MACAQUES Julie A. Karl1, Hailey E. Bussan1, Trent M. Prall2, Roger W. Wiseman1,2, David H. O'Connor1,2 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, University of Wisconsin‐Madison 16 ISOLATION OF A MONOCLONAL ANTIBODY TO THE RHESUS MACAQUE MHC CLASS I ALLOMORPH MAMU‐A1*002 Laurel Kelnhofer1, Dr. Matthew Reynolds1,2, Jason Weinfurter1 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center 17 A NON‐REDUNDANT, REFERENCE VIRAL DATABASE (RVDB) TO FACILITATE HIGH‐ THROUGHPUT SEQUENCING (HTS) ANALYSIS FOR VIRUS DETECTION Dr. Norman Goodacre1, Ms. Subhiksha Nandakumar1, Ms. Aisha Aljanahi2, Dr. Arifa Khan1 1 CBER/FDA, 2Georgetown University
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18 NEGATIVE ASSOCIATION BETWEEN THE OUTCOME OF VAGINAL SHIV CHALLENGE AND SERUM IGM ANTIBODIES INDUCED BY GP140 VACCINATION IN AGED RHESUS MACAQUES Pam Kozlowski1, Diana Battaglia1, Rafiq Nabi1, Lori Spicer2, Cynthia Derdeyn2, Caroline Petitdemange2, Sudhir Kasturi2, Eric Hunter2, Rama Amara2, David Masopust3, Bali Pulendran2 1 Louisiana State University Health Sciences Center, 2Emory University and Yerkes National Research Primate Center , 3University of Minnesota 19 APPLYING RNA PROFILING AND FUNCTIONAL ANALYSIS APPROACHES TO EVALUATE VACCINE‐INDUCED ADAPTIVE AND INNATE IMMUNE RESPONSES IN NON‐HUMAN PRIMATE SIV CHALLENGE AND PROTECTION STUDIES Dr. Lynn Law1, Richard Green1, Jean Chang1, Elise Smith1, Dr. Connor Driscoll1, Dr. Courtney Wilkins1, Dr. Michael Gale1 1 Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington 20 SECRETED VERSES TRANSMEMBRANE HIV ENV SOSIP DNA VACCINATION INDUCING B CELLS AND PLASMABLASTS IN INDIAN RHESUS MACAQUES Dr. David Leggat1, Dr. Alberto Cagigi1, Dr Luca Schifanella2, Mr. Sandeep Narpala1, Ms. Madhu Prabhakaran1, Ms. Mitzi Donaldson1, Dr. Kathryn Foulds1, Mr. David Ambrozak1, Mr. JP Todd1, Dr. Genoveffa Franchini2, Dr. Mario Roederer1, Dr. Richard Koup1, Dr. Adrian McDermott1 1 Vaccine Research Center, NIH, 2National Cancer Institute, NIH 21 IMMUNE CELLS DISTRIBUTION DURING SIV INFECTION AND TREATMENT: CHARACTERIZATION OF BONE MARROW AND PERIPHERAL BLOOD IN SIV INFECTED CYNOMOLOGUS MACAQUES Dr Julien Lemaitre1 1 CEA ‐ Université Paris Sud 11 ‐ INSERM U1184, Immunology of viral infections and autoimmune diseases 22 DEVELOPMENT AND VALIDATION OF FOUR NOVEL SHIV CHALLENGE STOCKS BEARING TRANSMITTED/FOUNDER TIER 2 HIV‐1 SUBTYPE A, C OR D ENVS Dr. Hui Li1, Ding Wenge1, Dr. Fang‐Hua Lee1, Dr. Beatrice Hahn1, Dr. George Shaw1 1 University Of Pennsylvania 23 ENV375 SHIV DESIGN V2.0 Dr. Hui Li1, Alexander Murphy1, Shuyi Wang1, Wenge Ding1, Dr. Beatrice Hahn1, Dr. George Shaw1 1 University Of Pennsylvania 24 EVOLUTION OF T CELL RESPONSES TO RHCMV‐VECTORED SIV VACCINE IN PREVIOUSLY RHCMV‐NEGATIVE AND ‐POSITIVE YOUNG MACAQUES Kawthar Machmach1,2, Gema Méndez‐Lagares1,2, Amir Ardeshir1,2, Nicole Narayan1,2, William Chang3, Jeff Lifson4, Peter Barry1,3,6, Dennis Hartigan‐O’Connor1,2,5 1 CNPRC, 2Dept of Medical Microbiology & Immunology, UC Davis, 3Center for Comparative Medicine, School of Veterinary Medicine and School of Medicine, UC Davis, 4AIDS and Cancer Viruses Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, , 5Division of Experimental Medicine, Dept of Medicine, UC San Francisco, 6Dept of Pathology and Laboratory Medicine, School of Veterinary Medicine, UC Davis 25 NHP AIDS RESEARCH SERVICES AT THE WISCONSIN NATIONAL PRIMATE RESEARCH CENTER Eileen A. Maher1, Shelby L. O'Connor1,2, Roger W. Wiseman1, Eva G. Rakasz1, Thomas C. Friedrich1,3, David H. O'Connor1,2 1 Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 2Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 3Department of Pathobiological Sciences, University of Wisconsin‐Madison
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26 DELAYED SIV INFECTION IN VACCINATED, RECTALLY‐CHALLENGED MAMU‐B*08+ RHESUS MACAQUES IN THE ABSENCE OF ANTI‐ENV HUMORAL RESPONSES Dr. Mauricio Martins1, Dr. Young Shin1, Mr. Lucas Gonzalez‐Nieto1, Mr. Martin Gutman1, Ms. Aline Domingues1, Ms. Helen Maxwell1, Dr. Diogo Magnani1, Mr. Michael Ricciardi1, Ms. Nuria Pedreño‐ Lopez1, Mr. Varian Bailey1, Ms. Kim Weisgrau2, Dr. John Altman3, Dr. Christopher Parks4, Dr. Keisuke Ejima5, Dr. Brandon George5, Dr. David Allison5, Dr. Eva Rakasz2, Dr. Saverio Capuano III2, Dr. Jeffrey Lifson6, Dr. Ronald Desrosiers1, Dr. David Watkins1 1 University Of Miami, 2University of Wisconsin‐Madison; Wisconsin National Primate Research Center, 3Emory University, 4International AIDS Vaccine Initiative, 5University of Alabama at Birmingham, 6AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research 27 MODELING THE EVOLUTION OF SIV SOOTY MANGABEY PROGENITOR VIRUS TOWARDS HIV‐2 USING HUMANIZED MICE Dr. Kimberly Schmitt1, Dr. Dipu Mohan Kumar1, Mr. James Curlin1, Ms. Stephanie Feely2, Ms. Leila Remling‐Mulder1, Dr. Mark Stenglein1, Dr. Shelby O'Connor3, Dr. Preston Marx2, Dr. Ramesh Akkina1 1 Colorado State University, Dept. of Microbiology, Immunology & Pathology, 2Tulane National Primate Research Center, 3University of Wisconsin School of Medicine and Public Health 28 EFFECTS OF SYSTEMIC AND MUCOSAL IMMUNIZATION OF RHESUS MACAQUES WITH SINGLE‐CYCLE ADENOVIRUS VECTORS AND ENVELOPE PROTEIN VACCINES Mr. William Matchett1, Stephanie S. Anguiano‐Zarate1, Mary E. Barry1, Guojun Yang2, Pramod Nehete2, Siddappa N. Byrareddy3, Delphine C. Malherbe4, Nancy L. Haigwood4, Francois Villinger5, K. Jagannadha Sastry2, Michael A. Barry1 1 Mayo Clinic, 2MD Anderson Cancer Center, 3University of Nebraska Medical Center, 4Oregon National Primate Research Center, 5New Iberia Research Center 29 CHANGES IN THE IMMUNE SYSTEM DRIVEN BY RHCMV AND ANELLOVIRUS INFECTIONS Gema Mendez‐lagares1,2, Nicole Narayan1,2, Amir Ardeshir1, David Merriam1,2, Ding Lu1,2, Eric Delwart3,4, Dennis Hartigan O'Connor1,2,5 1 Uc Davis, 2California National Primate Research Center, 3Blood Systems Research Institute, 4 Department of Laboratory Medicine, 5Division of Experimental Medicine 30 INVESTIGATION OF MACROPHAGES SERVING AS A VIRAL RESERVOIR IN PEDIATRIC SAIDS Dr. Kristen Merino1, Dr. Chie Sugimoto2, Dr. Yanhui Cai3, Dr. Carolina Allers1, Dr. Angela Amedee4, Dr. Christopher Destache5, Dr. Pavan Kumar Prathipati5, Dr. Elizabeth S Didier1, Dr. Marcelo J Kuroda1 1 Tulane National Primate Research Center, 2Laboratory of International Epidemiology, Dokkyo Medical University, 3HIV‐1 Immunopathogenesis Laboratory, Wistar Institute, 4LSU HSC School of Medicine, 5Creighton University, School of Pharmacy and Health Professions 31 RANTES‐DT390 AS A THERAPEUTIC FOR SIV RESERVOIR ELIMINATION Mr. David Merriam1, Ms. Connie Chen1, Dr. Gema Mendez Lagares1, Dr. Francois Villinger2, Dr. Dennis Hartigan‐O'Connor1 1 UC‐Davis, 2University of Louisiana at Lafayette 32 IMMUNOLOGICAL CONTROL OF SIMIAN IMMUNODEFICIENCY VIRUS FOLLOWING AN ADOPTIVE TRANSFER PRIME‐PULL STRATEGY Mariel Mohns1, Dawn Dudley1, Justin Greene2, Eric Peterson3, David O'Connor1,3 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health & Science University, 3Wisconsin National Primate Research Center, University of Wisconsin‐Madison
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33 SCHIV MT145: A NOVEL SHIV BEARING THE ENV OF A PRIMARY CHIMPANZEE SIV STRAIN WITH ANTIGENIC CROSS‐REACTIVITY TO HIV‐1 V1V2 BNAB EPITOPES Alexander Murphy1, Dr. Hui Li1, Wenge Ding1, Jessica Smith1, Dr. Beatrice H. Hahn1, Dr. George M. Shaw1 1 University of Pennsylvania 34 NONINVASIVE MONITORING OF CD4 T CELLS AT MULTIPLE MUCOSAL TISSUES AFTER INTRANASAL VACCINATION IN RHESUS MACAQUES Dr. Pramod Nehete1,3, Dr. Stephanie Dorta‐Estremera2, Dr. Guojun Yang2, Dr. Hong He4, Bharti Nehete1, Dr. Kathryn Shelton1, Dr. Michael Barry5,6,7,8, Dr. Jagannadha Sastry1,2,3 1 The University of Texas MD Anderson Cancer Center, Department of Veterinary Sciences, Bastrop, Texas, TX 78602, 2The University of Texas MD Anderson Cancer Center, Department of Immunology, Houston, Texas, TX 77030, 3The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, 4The University of Texas MD Anderson Cancer Center, Department of Stem Cell Transplantation, Houston, Texas, TX 77030, 5Department of Internal Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN 55902, USA, 6Department of Molecular Medicine,Mayo Clinic, Rochester, MN 55902, USA , 7Department of Immunology,Mayo Clinic, Rochester, MN 55902, USA, 8Translational Immunology virology and Biodefense Program, Mayo Clinic, Rochester, MN 55902, USA 35 INDUCTION OF MUTANT EPITOPE‐SPECIFIC CD8+ T CELLS IS AN INDICATOR OF THE BEGINNING OF VIRAL CONTROL FAILURE IN SIV CONTROLLERS. Dr. Takushi Nomura1,2, Dr. Hiroshi Ishii1, Dr. Sayuri Seki1, Dr. Hiroyuki Yamamoto1, Dr. Kazutaka Terahara3, Dr. Tomoyuki Miura4, Dr. Tetsuro Matano1,5 1 AIDS Research Center, National Institute of Infectious Diseases, 2Center for AIDS Research, Kumamoto University, 3Department of Immunology, National Institute of Infectious Diseases, 4 Institute for Frontier Life and Medical Sciences, Kyoto University, 5Institute of Medical Science, University of Tokyo 36 SIMIAN IMMUNODEFICIENCY VIRUS SIVMAC239 INFECTION AND SIMIAN HUMAN IMMUNODEFICIENCY VIRUS SHIV89.6P INFECTION RESULT IN PROGRESSION TO AIDS IN CYNOMOLGUS MACAQUES FROM ASIAN COUNTRY ORIGIN Dr Tomotaka Okamura1, Dr Yasuhiro Yasutomi1 1 Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition 37 RETINOIC ACID (RA) UPREGULATES Α4Β7 ON CD4+ T CELLS AND ACTIVATES LATENT RESERVOIRS Mr Omalla Olwenyi1,2, Dr Nanda Kishore Routhu1, Dr Neil Sidell2, Dr Aftab A Ansari2, Dr Siddappa N. Byrareddy1 1 University Of Nebraska Medical Centere, 2Emory University School of Medicine 38 EVALUATING VAGINAL FILM FORMULATIONS AS MULTIPURPOSE PREVENTION TECHNOLOGIES (MPT) IN THE MACAQUE MODEL Dr. Dorothy Patton1, Yvonne Cosgrove Sweeney1, Dr Lisa Rohan2 1 University Of Washington, 2Magee Womens Research Institute 39 HIV/SHIV‐SPECIFIC, CCR5‐EDITED CAR T‐CELLS ENGRAFT AND PERSIST IN ACUTELY INFECTED NONHUMAN PRIMATES Christopher Peterson1,2, Courtnee Clough3, Malika Hale3, Taylor Mesojednik3, Bryan Sands3, Hans‐ Peter Kiem1,2, Thor A. Wagner2,3, David J. Rawlings2,3 1 Fred Hutchinson Cancer Research Center, 2University of Washington, 3Seattle Children’s Research Institute
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40 THE OPTIMIZATION OF PEDIATRIC VACCINE REGIMENS TO PREVENT MOTHER‐TO‐CHILD TRANSMISSION OF HIV THROUGH BREAST MILK Dr. Bonnie Phillips1, Genevieve Fouda2, Justin Pollara2, Pamela Kozlowski3, Anthony Moody2, Guido Ferrari2, Sallie Permar2, Kristina De Paris1 1 Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, 2Duke University Medical Center, Duke Human Vaccine Institute, 3Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center 41 FULL‐LENGTH KILLER‐CELL IMMUNOGLOBULIN‐LIKE RECEPTOR TRANSCRIPT DISCOVERY IN INDIAN RHESUS MACAQUES Trent Prall1, Julie Karl2, Michael Graham2, Hailey Bussen2, Cecelia Shortreed2, Roger Wiseman1,2, David O'Connor1,2 1 Wisconsin National Primate Research Center, University Of Wisconsin‐Madison, 2Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison 42 TRACKING NKG2C+ MEMORY AND MEMORY‐LIKE NK CELLS IN SIV AND CMV INFECTIONS OF RHESUS MACAQUES Dr. Daniel Ram1, Dr. R. Keith Reeves1 1 Beth Israel Deaconess Medical Center/Harvard Medical School 43 PRESERVED CYTOKINE AND CYTOTOXIC EFFECTOR FUNCTIONS OF GUT MUCOSAL VΔ1 ΓΔ T CELLS IN SIV‐INFECTED MACAQUES Adrienne S. Woods1, Faith R. Schiro1, Pyone P. Aye1, Stephen E. Braun1, Ronald S. Veazey1, Marcelo J. Kuroda1, Andrew A. Lackner1, Namita Rout1 1 Tulane National Primate Research Center, Tulane University 44 A NOVEL STRATEGY TO ADAPT SHIV‐E1 CARRYING ENV FROM AN RV144 VOLUNTEER TO RHESUS MACAQUES: CORECEPTOR SWITCH AND FINAL RECOVERY OF A PATHOGENIC VIRUS WITH EXCLUSIVE R5 TROPISM Hanna Scinto1,2, Sandeep Gupta1, Swati Thorat3,4, Muhammad Mukhtar1, Anthony Griffiths1, Jennifer Delgado1, Elizabeth Plake1, Hemant Vyas1, Amanda Strickland1, Siddappa Byrareddy3,4, David Montefiori5, Celia LaBranche5, Ranajit Pal6, Jim Treece6, Sharon Orndorff6, Maria Ferrari6, Deborah Weiss6, Agnes‐Laurence Chenine7, Ruth Bonchik7, Robert McLinden7, Nelson Michael7, Jerome Kim7, Merlin Robb7, Supachai Rerks‐Ngarm8, Punnee Pitisuttithum9, Sorachai Nitayaphan10, Ruth Ruprecht1,2,3,4 1 Texas Biomedical Research Institute, 2UT Health San Antonio, 3Dana‐Farber Cancer Institute, 4 Harvard Medical School, 5Duke University Medical Center, 6Advanced Biosciences Laboratories Inc., 7Henry M. Jackson Foundation, 8Department of Disease Control, Ministry of Public Health, 9 Mahidol University, 10Armed Forces Research Institute of Medical Sciences 45 NOVEL FULL‐LENGTH MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I ALLELE DISCOVERY AND HAPLOTYPE DEFINITION IN PIG‐TAILED MACAQUES Matthew Semler1, Roger W. Wiseman1,2, Julie A. Karl1, Michael E. Graham1, Samantha M. Gieger1, David H. O'Connor1,2 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison,, 2Wisconsin National Primate Research Center, University of Wisconsin‐Madison, Madison 46 TARGETING CAR T CELLS TO B CELL FOLLICLES TO CURE HIV INFECTION PJ Skinner1, A Hajduczki2, P Haran1, MS Pampusch1, G Mwakalundwa1, S Bolivar‐Wagers2, DA Vargas‐Inchaustegui2, EG Rakasz3, E Connick4, EA Berger2 1 Department of Veterinary and Biomedical Sciences, University of Minnesota, 2Laboratory of Viral Diseases, National Institutes of Allergy and Infectious Diseases, The National Institutes of Health, 3 Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 4Division of Infectious Diseases, University of Arizona
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47 USE OF QUANTERIX SIMOA ULTRASENSITIVE IMMUNOASSAY FOR ASSESSING RESIDUAL VIRUS IN NON‐HUMAN PRIMATE MODELS OF AIDS Dr. Adrienne E. Swanstrom1, Robert Gorelick1, Guoxin Wu2, Bonnie J. Howell2, Anitha Vijayagopalan1, Gregory Q. Del Prete1, Julian Bess Jr.1, Jeffrey D. Lifson1 1 AIDS And Cancer Virus Program, 2Merck 48 DISTRIBUTION OF LONG‐LIVED AND SHORT‐LIVED MACROPHAGES IN THE INTESTINAL TRACT OF SIV‐INFECTED RHESUS MACAQUES Naofumi Takahashi1, Carolina Allers1, Cecily Midkiff2, Xavier Alvarez2, Elizabeth Didier3, Woong‐Ki Kim4, Marcelo Kuroda1 1 Division of Immunology, Tulane National Primate Research Center, 2Division of Comparative Pathology, Tulane National Primate Research Center, 3Division of Microbiology, Tulane National Primate Research Center, 4Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School 49 DIRECTED HOMING OF ADOPTIVELY TRANSFERRED T‐CELLS THROUGH HOMING MARKER TRANSDUCTION Matthew Trivett1, Daniel Burke1, Lori Coren1, Claire Deleage1, Greg Del Prete1, Jake Estes1, Jeffery Lifson1, David Ott1 1 Leidos Biomedical Research, Inc. 50 FUNCTIONALLY PRESERVED MAIT CELLS ARE ASSOCIATED WITH CONTROLLED SHIV INFECTION Dr Amudhan Murugesan, Dr Chris Ibegbu, Dr Tiffany Styles, Sakeenah Hicks, Mr Micheal Sabula, Dr Pradeep Reddy, Andrew Jones, Dr Rama Amara, Dr. Vijayakumar Velu1 1 Yerkes National Primate Research Center 51 COMPARING THE EFFECTS OF PROGESTIN‐BASED CONTRACEPTION AND LUTEAL PHASE OF THE MENSTRUAL CYCLE ON PUTATIVE HIV SUSCEPTIBILITY GENES IN PIG‐TAILED MACAQUES Dr. Ajay Sundaram Vishwanathan1, Dr. Steven E Bosinger2, Gregory K Tharp2, Nirav B Patel2, Chunxia Zhao1, James Mitchell1, Shanon Ellis1, Ellen N Kersh1, Janet M McNicholl1 1 CDC (Centers For Disease Control & Prevention), 2Yerkes National Primate Research Center, Emory University 52 OPTIMIZED METHOD FOR EXTRACTING HIGH QUALITY RNA FROM FACS‐SORTED, INTRACELLULAR STAINED PRIMARY RHESUS MACAQUE LYMPHOCYTES Dr. Yichuan Wang1, Dr. David Ott1, Matthew Trivett1, Dr. Jeffrey Lifson1 1 Leidos Biomedical Research, Inc. 53 A NOVEL PKC ACTIVATOR, 10‐METHYL‐APLOG‐1 EFFICIENTLY REACTIVATE LATENT HIV‐1 IN COMBINATION WITH A BET INHIBITOR JQ1. Ayaka Washizaki1, Megumi Murata1, Yin Pui Tang2, Yohei Seki1, Kazuhiro Irie3, Hirofumi Akari1 1 Primate Research Institute, Kyoto University, 2Medical School, University of Exeter, 3Graduate School of Agriculture, Kyoto University 54 RESOURCES FOR NON‐HUMAN PRIMATE MODELS OF ZIKA VIRUS Andrea Weiler1, Matthew Aliota2, James Weger‐Lucarelli3, Matthew Semler1,4, Gabrielle Barry1, Dawn Dudley4, Christina Newman4, Shelby O'Connor4, David O'Connor1,4, Gregory Ebel3, Thomas Friedrich1,2 1 WNPRC, 2UW‐Madison Pathobiological Sciences, 3Microbiology, Immunology and Pathology ‐ Colorado State University, 4UW‐Madison Cellular and Molecular Pathology
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55 DOES SIMIAN BETARETROVIRUS (SRV) ANTIBODY IN BABOONS (PAPIO SP.) INDICATE INFECTION? Joann Yee1, Richard Grant2, Koen Van Rompay1, Jeffrey Roberts1, Joe Simmons3, James Papin4 1 California National Primate Research Center, University of California, 2Washington National Primate Research Center, University of Washington, 3Michale E. Keeling Center for Comparative Medicine and Research, University of Texas MD Anderson Cancer Center, 4Department of Pathology, Division of Comparative Medicine, University of Oklahoma Health Science Center 56 A DOSE‐ESCALATION STUDY OF PHARMACOLOGIC INHIBITION OF Β‐CATENIN SIGNALING IN HEALTHY RHESUS MACAQUES PhD Michelle Zanoni1, PhD Maud Mavigner1, Dr. Jakob Habib1, Dr. Cameron Mattingly1, PhD Kirk Easley1, Dr. H Kouji2, Md. PhD. Ann Chahroudi1 1 Emory University, 2PRISM Pharma Co., Ltd. 57 EFFECTS OF RAPAMYCIN ADMINISTRATION ON IMMUNE RESPONSES IN SIV‐INFECTED RHESUS MACAQUES Dr Widade Ziani1, Dr Xiaolei Wang1, Dr Kasi Russell‐Lodrigue1, Dr Ronald S. Veazy1, Dr Huanbin Xu1 1 TNPRC 58 EFFECTS OF RAPAMYCIN ADMINISTRATION ON IMMUNE RESPONSES IN SIV‐INFECTED RHESUS MACAQUES Dr Widade Ziani1, Dr Xiaolei Wang1, Dr Kasi Russell‐Lodrigure1, Dr Ronald S. Veazy1, Dr Huanbin Xu1 1 TNPRC 20:00 – 20:30
Poster removal All posters must be removed at the end of the poster session.
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Thursday, August 24, 2017 7:30 – 8:30
8:30 – 10:15
Scientific Session 3 Virus‐host interactions and immune responses
Chairs: Mario Roederer and Genevieve Fouda 8:30 – 9:00 300 QUANTIFYING LEUKOCYTE TRAFFICKING IN NHP BY SERIAL INTRAVASCULAR STAINING Mario Roederer1, Elizabeth Potter1, Kathy Foulds1, Tricia Darrah1, Robert Seder1, Hannah Gideon2, Joanne Flynn2 1 VRC, NIAID, NIH, 2University of Pittsburgh 9:00 – 9:15 301 FAILURE TO INDUCE CD11C+ B CELLS IS ASSOCIATED WITH A RAPID PROGRESSOR PHENOTYPE IN ORALLY‐INOCULATED SIV‐INFECTED INFANT MACAQUES Matthew Wood1, Megan Templeton1, Adriana Lippy1, Patience Murapa2, Deborah Fuller2, Donald Sodora1,3 1 Center For Infectious Disease Research, 2University of Washington, WaNPRC and Department of Microbiology, 3University of Washington, Department of Global Health 9:15 – 9:30 302 CHARACTERIZING THE CHANGES IN INITIAL HIV/SIV INFECTION BY CELL PHENOTYPING AT ANORECTAL MUCOSA OF RHESUS MACAQUES Dr. Danijela Maric1, Lisette Corbin1, Dr. Ron Veazey2, Dr. Thomas Hope1 1 Northwestern University, 2Tulane University 9:30 – 9:45 303 PARADOXICAL MYELOID‐DERIVED SUPPRESSOR CELL REDUCTION IN THE BONE MARROW OF SIV CHRONICALLY INFECTED MACAQUES Yongjun Sui1, Blake Frey1, Yichuan Wang1, Rolf Billeskov1, Shweta Kulkarni1, Katherine McKinnon1, Tracy Rourke2, Linda Fritts2, Christopher Miller2, Jay Berzofsky1 1 NIH NCI Vaccine Branch, 2Center for Comparative Medicine, University of California Davis 9:45 – 10:00 304 POLYMORPHISMS IN TETHERIN ARE ASSOCIATED WITH DIFFERENCES IN PEAK VIREMIA DURING ACUTE INFECTION OF RHESUS MACAQUES WITH SIV DELTA‐NEF Sanath Kumar Janaka1, William Neidermeyer2, Ruth Serra‐Moreno3, Bin Jia4, James Hoxie5, Ronald Desrosiers6, Paul Johnson7, Jeffrey Lifson8, Steven Wolinsky9, David Evans1 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2 Department of Microbiology and Immunology, Harvard Medical School, 3Department of Biological Sciences, Texas Tech University, 4Pfizer Inc, 5Department of Medicine, University of Pennsylvania, 6 Department of Pathology, Miller School of Medicine, University of Miami, 7Yerkes National Primate Center, 8AIDS and Cancer Virus Program, Leidos Biomedical Research Inc, FNLCR, 9Division of Infectious Diseases, Northwestern University Feinberg School of Medicine 10:00 – 10:15 305 MUTATIONS IN NEF THAT SELECTIVELY DISRUPT TETHERIN ANTAGONISM IMPAIR SIV REPLICATION DURING ACUTE INFECTION OF RHESUS MACAQUES Aidin Tavakoli‐Tameh1, Dr Sanath Kumar Janaka1, Lauren Callahan1, Ksenia Bashkueva1, Katie Zarbock2, Dr Shelby O'Connor1,2, Kristin Crosno2, Saverio Capuano 3rd2, Dr Ruth Serra‐Moreno3, Dr Hajime Uno4, Dr Jeffrey D. Lifson5, Dr David Evans1,2 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, 3Department of Biological Sciences, Texas Tech University, 4 Department of Biostatistics and Computational Biology, Dana‐Farber Cancer Institute, 5AIDS and Cancer Virus Program, Leidos Biomedical Research Inc
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10:15 – 10:45
10:45 – 12:00
10:45 – 11:00
11:00 – 11:15
11:15 – 11:30
11:30 – 11:45
11:45 – 12:00
12:00 – 14:00
Scientific Session 3 Virus‐host interactions and immune responses (continued)
306 NON‐HUMAN PRIMATE MODELS OF HIV MATERNAL AND INFANT IMMUNIZATION Genevieve Fouda1 1 Duke Human Vaccine Institute 307 CD8 T CELLS NOT NECESSARILY REQUIRED FOR CONTROL OF SIV VIREMIA IN MAURITIAN CYNOMOLGUS MACAQUES Matthew Sutton1, Alexis Balgeman1, Amy Ellis1, Gabrielle Barry2, Andrea Weiler2, Benjamin von Bredow1, Dane Gellerup2, David Evans1,2, Thomas Friedrich2,3, Shelby O'Connor1,2 1 Department of Pathology and Laboratory Medicine, UW‐Madison, 2Wisconsin National Primate Research Center, UW‐Madison, 3Department of Pathobiological Sciences, UW‐Madison 308 ASSOCIATION BETWEEN ADIPOSE TISSUE MACROPHAGES AND INFLAMMATION IN SIV‐ INFECTED RHESUS MACAQUES Marissa Fahlberg1, Dr. Elizabeth Didier1, Dr. Marcelo Kuroda1 1 Tulane University 309 SIMIAN IMMUNODEFICIENCY VIRUS INFECTION LEADS TO THE APPEARANCE OF INTERLEUKIN‐18 SECRETING AND CYTOTOXIC NATURAL KILLER‐LIKE B‐CELLS IN THE MUCOSA OF THE COLON Andrew Cogswell1, Moriah Castleman2, Stephanie Dillon2, Cara Wilson2, Ed Barker1 1 Rush University Medical Center, 2University of Colorado 310 INFUSIONS OF IN VITRO EXPANDED AUTOLOGOUS NK CELLS ON VIRAL LOADS IN SIV INFECTED RHESUS MACAQUES Dr. Siddappa Byrareddy1, Robert Russo2, Dr. Dean Lee3, Dr. Aftab Ansari4 1 University of Nebraska Medical Center, 2Emory University School of Medicine, 3Nationwide Children’s Hospital, 4Emory University School of Medicine Lunch (on your own)
14:00 – 15:45pm
Scientific Session 4 Genomics: Host and microbiome
Chairs: Brandon Keele and Jeffrey Rogers 14:00 – 14:30 400 USING MOLECULARLY MODIFIED VIRUSES TO TRACK TRANSMISSION AND LATENCY Brandon Keele1 1 Frederick National Lab 14:30 – 14:45 401 CLONOTYPE LINEAGE TRACING OF VACCINE‐INDUCED B CELLS IN VIVO USING THE BALDR COMPUTATIONAL PIPELINE FOR IMMUNOGLOBULIN RECONSTRUCTION IN SINGLE‐CELL RNA‐SEQ DATA Amit Upadhyay1, Alice Cho2, Amber Wolabaugh1, Robert Kauffman2, Gregory Tharp1, Reem Dawoud1, Nirav Patel1, F. Eun‐Hyung Lee2, Jens Wrammert2, Steven Bosinger1 1 Yerkes Nprc/emory University, 2Emory University
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14:45 – 15:00
15:00 – 15:15
15:15 – 15:30
15:30 – 15:45
15:45 – 16:15
402 DEVELOPMENT OF A RAPID AND SCALABLE METHOD TO BOTH SEQUENCE AND EXOGENOUSLY EXPRESS PAIRED FULL‐LENGTH MACAQUE ANTIGEN‐SPECIFIC T‐CELL RECEPTORS Dr. Shaheed Abdulhaqq1, Dr. Benjamin Bimber1, Dr. Scott Hansen1, Dr. Helen Wu1, Abigail Ventura1, Dr. Karin Wisskirchen2, Dr. Ulrike Protzer2, Alfred Legasse1, Dr. Michael Axthelm1, Dr. Louis Picker1, Dr. Jonah Sacha1 1 Oregon Health & Science University/Vaccine And Gene Therapy Institute, 2Technical University of Munich 403 HIGHLY RESOLVED LONG READ, SINGLE MOLECULE SEQUENCING OF FULL‐LENGTH SIV AND SIV ENV Ms. Alesia Antoine1, Dr. Ismael Ben Farouck Fofana2, Dr. Gintaras Deikus1, Dr. Robert Sebra1, Dr. Welkin Johnson2, Dr. Melissa Laird Smith1 1 Icahn School Of Medicine At Mount Sinai, 2Boston College 404 INFLAMMATORY INSULT PRIOR TO SIMIAN IMMUNODEFICIENCY VIRUS INFECTION INCREASES ACUTE PHASE PATHOGEN BURDEN Dr Adam Ericsen1, Mr Matthew Semler2, Ms Hailey Bussan2, Mr Trent Prall1, Mr Eric Peterson1, Mr Jason Weinfurter2, Dr Roger Wiseman1,2, Prof David O'Connor1,2 1 Wisconsin National Primate Research Center, 2University of Wisconsin 405 IMPACT OF MICROBIOME MANIPULATION ON SHIV ACQUISITION IN RHESUS MACAQUES Dr. Jennifer A. Manuzak1,2, Dr. Tiffany Hensley‐McBain1,2, Charlene Miller1,2, Dr. Alexander S. Zevin1,2, Toni M. Gott1,2, Ernesto Coronado1,2, Ryan Cheu1,2, Andrew Gustin1,2, Dr. Elias K. Haddad3, Dr. Deborah H. Fuller1,2, Dr. Nancy L. Haigwood4,5, Dr. Nichole R. Klatt1,2 1 University of Washington, 2Washington National Primate Research Center, 3Drexel University, 4 Oregon National Primate Research Center, 5Oregon Health and Science University Coffee Break
16:15 – 17:30
16:15 – 16:30
16:30 – 16:45
16:45 – 17:00
17:00 – 17:15
Scientific Session 4 Genomics: Host and microbiome (continued)
406 WHOLE GENOME SEQUENCE DATA FOR MACAQUES: IMPLICATIONS FOR AIDS RESEARCH Jeffrey Rogers1 1 Baylor College of Medicine 407 WHOLE‐TRANSCRIPTOME SEQUENCING TO IDENTIFY IMMUNE GENE VARIANTS Amelia K. Haj1, Julie A. Karl1, Roger W. Wiseman1, David H. O'Connor1 1 UW‐Madison 408 CHARACTERIZATION OF MAJOR HISTOCOMPATIBILITY COMPLEX SEQUENCES IN BABOONS Hailey E. Bussan1, Joe H. Simmons3, Roger W. Wiseman1,2, Julie A. Karl1, Cecilia G. Shortreed1, Michael E. Graham1, David O'Connor1,2 1 Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 3MD Anderson Cancer Center, University of Texas 409 COMPARISON OF THE HOST RESPONSE TO RHCMV/SIV VACCINE VECTORS BETWEEN PROTECTED AND NON‐PROTECTED RHESUS MACAQUES Dr. Courtney Wilkins1, Rich Green1, Dr. Connor Driscoll1, Jean Chang1, Elise Smith1, Dr. Lynn Law1, Dr. Scott Hansen2, Dr. Lewis Picker2, Dr. Michael Gale, Jr.1 1 Department of Immunology, Center for Innate Immunity and Immune Disease, University Of Washington, 2Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health & Science University
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17:15 – 17:30
18:30 – 19:00 19:00 – 19:45 19:45 – 21:00
410 ADAPTATION OF SIV TO BABOON PBMC OR ISOLATED CD4 CELLS: INSIGHTS INTO CELL TYPES REQUIRED FOR BABOON RESISTANCE TO SIV INFECTION Veronica Obregon‐Perko1,2, Laura Parodi2, Vida Hodara2,3, Jason T Ladner4, Michael R Wiley4, Gustavo F Palacios4, Luis D Giavedoni2,3 1 Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Health Science Center, 2Department of Virology and Immunology, Texas Biomedical Research Institute, 3 Southwest National Primate Research Center, Texas Biomedical Research Institute, 4Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases DINNER RECEPTION DINNER DINNER SPEAKER – Dr. Bonnie Mathieson, National Institutes of Health 411 RESEARCH IN NHP ON THE ROAD TO THE END OF AIDS
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Friday, August 25, 2017 7:30 – 8:30
8:30 – 10:15
Scientific Session 5 ‘SIV tools’ for other pathogens
Chairs: Koen Van Rompay and Dawn Dudley 8:30 – 9:00 500 JUMPING FROM HIV INTO ZIKA: RESEARCH INTO HIGH GEAR Dr. Koen Van Rompay1 1 California National Primate Research Center, University of California, Davis 9:00 – 9:15 501 A NEW HIV/HBV CO‐INFECTION MODEL: HEPATOCYTIC EXPRESSION OF HUMAN SODIUM TAUROCHOLATE COTRANSPORTING POLYPEPTIDE (NTCP) ENABLES HEPATITIS B VIRUS INFECTION OF MACAQUES Dr. Benjamin Burwitz1,4, Mr. Jochen Wettengel2, Dr. Martin Muck‐Hausl2, Mrs. Katherine Hammond1, Dr. Marc Ringelhan2,3, Dr. Chunkyu Ko2, Mr. Jason Reed1, Mr. Reed Norris4, Dr. Byung Park5, Dr. Sven Moller‐Tank6, Dr. Knud Esser2, Dr. Justin Greene1, Dr. Helen Wu1, Dr. Shaheed Abdulhaqq1, Dr. Gabriela Webb1, Mr. William Sutton4, Mr. Alex Klug4, Ms. Tonya Swanson4, Mr. Alfred Legasse4, Dr. Aravind Asokan6, Dr. Nancy Haigwood4, Prof. Ulrike Protzer2,7, Dr. Jonah Sacha1,4 1 Vaccine & Gene Therapy Institute, Oregon Health & Science University, 2Institute of Virology, Technical University of Munich, Helmholtz Zentrum München, 3Department of Internal Medicine II, Technical University of Munich, 4Oregon National Primate Research Center, Oregon Health & Science University, 5Public Health & Preventative Medicine, Oregon Health & Science University, 6 Gene Therapy Center, The University of North Carolina at Chapel Hill, 7German Center for Infection Research, Munich partner site 9:15 – 9:30 502 PRE‐EXISTING SIV INFECTION INCREASES SUSCEPTIBILITY OF MAURITIAN CYNOMOLGUS MACAQUES TO M. TUBERCULOSIS Mark Rodgers1, Cassaundra Updike1, Dr. Amy Ellis3, Alexis Balgeman3, Pauline Maiello1, Dr. Tom Friedrich4, Gabrielle Barry4, Dr. Joshua Mattila2, Dr. Shelby O'Connor3, Dr. Charles A. Scanga1 1 University of Pittsburgh School of Medicine, 2University of Pittsburgh Graduate School of Public Health, 3University of Wisconsin‐Madison, 4Wisconsin National Primate Research Center 9:30 – 9:45 503 MTB/SIV CO‐INFECTION INDUCES DIFFERENTIAL T CELL RESPONSES IN RHESUS MACAQUES Miss Allison N. Bucsan1,2, Dr. Taylor W. Foreman1,2, Dr. Shabaana A. Khader3, Dr. Jyothi Rengarajan4, Dr. James A. Hoxie5, Dr. Andrew A. Lackner1, Dr. Deepak Kaushal1,2 1 Tulane National Primate Research Center, 2Tulane University, 3Washington University, 4Emory University School of Medicine, 5UPenn Center for AIDS Research, University of Pennsylvania 9:45 – 10:00 504 DEVELOPMENT OF A NON‐HUMAN PRIMATE MODEL FOR RECTAL SYPHILIS Dr. Ajay Sundaram Vishwanathan1, Dr. Cassandra Tansey1, Ms. Chunxia Zhao1, Mr. Andre Hopkins1, Dr. Yetunde Fakile1, Ms. Tamanna Ahmed1, Dr. Allan Pillay1, Dr. Samantha Katz1, Dr. Ellen Kersh1, Mr. James Mitchell1, Dr. Janet McNicholl1 1 CDC (Centers For Disease Control & Prevention)
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10:00 – 10:15
10:15 – 10:45
505 ALTERNATIVE NK CELL SIGNALING MECHANISMS IN CMV AND SIV INFECTION Spandan Shah1,2, Cordelia Manickam1,2, Daniel Ram1,2, R. Keith Reeves1,2 1 Beth Israel Deaconess Medical Center, 2Harvard Medical School Coffee Break
10:45 – 12:00
10:45 – 11:00
Scientific Session 5B NHP models for other infectious diseases: Using SIV tools to investigate other pathogens
506 NONHUMAN PRIMATE MODELS FOR ZIKA VIRUS INFECTION
Dr. Dawn Dudley1, Christina M. Newman1, Emma L. Mohr2, Matthew T. Aliota3, Sydney M. Nguyen4, Kathleen M. Antony4, Sarah Kohn5, Heather A. Simmons6, Andrea M. Weiler6, Matthew R. Semler1, Mariel S. Mohns1, Meghan E. Breitbach1, Laurel M. Stewart1, Michelle Koenig1, Bryce Wolfe1, M. Shahriar Salamat1, Leandro B. C. Teixeira7, Xiankun Zeng8, Gregory J. Wiepz9, Troy H. Thoong6, Gabrielle L. Barry6, Kim L. Weisgrau6, Logan J. Vosler6, Mustafa N. Rasheed1, Michael E. Graham1, Lindsey Block1, Jennifer Post6, Jennifer M. Hayes6, Nancy Schultz‐Darken6, Michele L. Schotzko6, Josh A. Eudailey10, Jens Kuhn6, Sallie R. Permar10, Eva G. Rakasz6, Saverio Capuano III6, Alice F. Tarantal11, Jorge E. Osorio8, Shelby L. O'Connor1, Thomas C. Friedrich6,3, Thaddeus G. Golos4,6,9, David H. O'Connor1,6 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, Department of Pediatrics, University of Wisconsin‐Madison, 3Department of Pathobiological Sciences, University of Wisconsin‐Madison, 4Department of Obstetrics and Gynecology, University of Wisconsin‐Madison, 5Department of Radiology, University of Wisconsin‐Madison, 6Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 7School of Veterinary Medicine, University of Wisconsin‐Madison, 8Integrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9Department of Comparative Biosciences, University of Wisconsin‐Madison, 10Department of Pediatrics and Human Vaccine Institute, Duke University Medical Center, 11Departments of Pediatrics and Cell Biology and Human Anatomy, University of California‐Davis, California National Primate Research Center 2
11:00 – 11:15
11:15 – 11:30
11:30 – 11:45
507 INFECTION DYNAMICS AND PERSISTENCE OF ZIKA VIRUS IN NEW AND OLD WORLD NON‐ HUMAN PRIMATES Dr Neil Berry1, Dr Deborah Ferguson1, Mrs Claire Ham1, Ms Jo Hall1, Mr Adrian Jenkins1, Mrs Elaine Giles1, Dr Nicola Rose1, Dr Roger Hewson2, Dr Stuart Dowell2, Dr Sarah Kempster1, Dr Neil Almond1 1 NIBSC, 2PHE Porton 508 IMPACT OF RHESUS CYTOMEGALOVIRUS EXPOSURE ON HOST INTESTINAL GENE EXPRESSION Nicole Narayan1,2, Gema Méndez‐Lagares1,2, Kawthar Machmach1,2, David Merriam1,2, Connie Chen1,2, Amir Ardeshir2, W L William Chang3, Peter A Barry3, Dennis J Hartigan‐O’Connor1,2 1 Department of Medical Microbiology & Immunology, University of California, Davis , 2California National Primate Research Center, University of California, Davis, 3Center for Comparative Medicine, University of California, Davis 509 IMPACT OF ANTIRETROVIRAL DRUG THERAPY ON MUCOSAL INFLAMMATORY AND REGULATORY RESPONSES IN SIV INFECTED MACAQUES Megan O'Connor1,2, Paul Munson1,2, Hillary Tunggal1,2, Nika Hajari1,2, Debra Bratt1,2, Drew May2, Solomon Wangari2, Brian Agricola2, Jeremey Smedley2, Deborah Fuller1,2 1 Department of Microbiology, University of Washington, 2Washington National Primate Research Center
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11:45 – 12:00
12:00 – 12:30
510 EVALUATION OF A COMBINATION ANTIRETROVIRAL THERAPY REGIMEN CONTAINING LONG‐ACTING FORMULATIONS OF THE INTEGRASE INHIBITOR CAB‐LA AND THE PROTEASE INHIBITOR GSK385‐MLAP Dr. Gregory Del Prete1, Dr. Adrienne Swanstrom1, Dr. Claire Deleage1, Dr. Jacob Estes1, Dr. Brandon Keele1, Dr. Jerry Jeffrey2, Dr. Jeffrey Lifson1 1 AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., 2GlaxoSmithKline Research & Development, Infectious Diseases Therapy Area Unit Promenade Hall Closing Remarks and Preview of 36th NHP AIDS Symposium SAVE the date of the WaNPRC NHP AIDS 2018 meeting: October 2‐5, 2018
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Oral Abstracts 99 From the “Mississippi Baby” to the Nonhuman Primate Model: Contributions to understanding HIV persistence as a step toward cure Dr. Deborah Persaud1 1
Johns Hopkins University School of Medicine
Nearly 37 million individuals are living with HIV worldwide, including three million children. A cure for HIV would obviate the need for life‐long antiretroviral therapy (ART), which is fraught with drug toxicities, ongoing risk of antiretroviral drug resistance, and stigma. Early speculation on the potential for cure arose following observations of the effectiveness of combination ART to control HIV replication below clinically detectable viral load levels. Optimism for curing HIV quickly dampened, however, with the discovery of the rapid establishment HIV latency in long‐lived, resting memory CD4+ T cells. HIV persistence is enabled by quiescent virus that cannot be targeted by antiretroviral drugs and escape immune‐mediated clearance mechanisms, in part through persistence within immune‐privileged sites within B cell zones of lymphoid follicles. The potential for HIV cure was demonstrated by the “Berlin patient” who remains in remission after receiving treatment for acute myelogenous leukemia that included allogeneic bone marrow transplantation with CCR5‐delta 32 homozygous cells. This single case provided proof‐of‐concept of that HIV could be eliminated from an infected individual and served as the catalyst for a change in the treatment paradigm from life‐long ART to antiretroviral‐free HIV remission and cure. Since then, reports of cases of long‐term HIV remission arising from various treatment approaches, including the case of the “Mississippi baby” who received ART shortly after birth, continue to fuel optimism that early, aggressive treatment will reduce the size of the latent reservoir and permit long‐term remission. The rapidly evolving landscape of the HIV cure clinical trials agenda will be discussed in the context of the seminal studies of HIV/SIV pathogenesis in humans and nonhuman primates.
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100 HIV: where did it come from and where is it going? Douglas Nixon1 1
The George Washington University
HIV is a human infectious exogenous retrovirus which is thought to have infected human populations from cross primate species transmissions. Within our genomes are remnants of past infectious retroviruses which have endogenized, called “endogenous retroviruses” (ERVs). We have previously shown that human ERVs (HERVs) can be transcribed in an HIV infected cell, but had not identified which sub‐family or unique HERV was transcribed. Using a novel computational pipeline, Telescope, we have identified which HERVs are expressed in HIV infected and latently infected cells. We have received funding for a Martin Delaney Collaboratory grant called “Believe” in which we research towards HIV eradication based upon enhanced targeted cell therapy. As part of this program, we have investigated whether we can identify markers which uniquely identify the latent reservoir. New data will be presented on two putative markers, CD32a and IFITM1. Funding: The NIH Martin Delaney Collaboratory, “Believe”, NIAID UM1 award AI126617, co‐funded by NIDA/NINDS/NIMH/NIAID; NCI CA 206488; NIAID AI 76059.
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101 Anti‐CD20 antibody mediated B cell depletion enhances viral control in SIV‐infected rhesus macaques Yoshi Fukazawa1,2, Richard Lum1,2, Jin Young Bae1,2, Alden Ho1,2, Scott Hansen1,2, Jeseph Clock1,2, Bryan Randall1,2, Haesun Park1,2, Alfred Legasse1,2, Michael Axthelm1,2, Jeffrey Lifson3, Afam Okoye1,2, Louis Picker1,2 1
Vaccine and Gene Therapy Institute, Oregon Health & Science University, 2Oregon National Primate Research Center, Oregon Health & Science University, 3AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory
We’ve reported that rhesus macaques (RM) with elite control of pathogenic SIV infection show exquisite restriction of replication‐competent virus to CD4+ follicular helper T cells (TFH) resident within B cell follicles of secondary lymphoid tissues, suggesting that the highly effective anti‐viral CD8+ T cell responses in these RM are able to almost completely clear and/or suppress productive SIV infection in extra‐follicular T cell zones but not within B cell follicles. Here we evaluated whether disruption of this B cell follicular sanctuary can facilitate the clearance of persistent virus replication in elite controllers (EC). A total of 7 SIV‐infected EC RM received multiple doses of a B cell depleting anti‐CD20 antibody (Ab), which resulted in a 1‐2 log reduction in the already low plasma viremia found in these RM, indicating that disruption of B follicles by B cell depletion improves overall CD8+ T cell‐mediated viral control. Next we asked whether disruption of B cell follicles in SIV‐infected RM on antiretroviral therapy (ART) would facilitate the clearance of reactivating virus in TFH and enhance virological control after ART cessation. A total of 21 RM selected to be mamu B*08+ (n=9) or B*08‐/B*17‐/A*01‐ (n=11) were intravenously inoculated with SIVmac239 and placed on ART 12 days later. Once all RM achieved stable virus suppression they were randomized into 2 groups and received multiple doses of anti‐CD20 or control Ab starting ~2 weeks before and up to 6 weeks after ART cessation. In comparison with control group, anti‐CD20 treated RM showed enhance control of virus replication immediately after ART cessation and a trend towards lower viral load set points. Overall these data suggests that the B cell follicular sanctuary must be overcome to fully evaluate the ability of virus‐ specific CD8+ T cells to reduce viral reservoirs or control viral rebound to achieve durable viral remission.
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102 Fully MHC‐matched allogeneic hematopoietic stem cell transplantation in SIV‐infected, cART‐suppressed Mauritian cynomolgus macaques Dr. Helen L Wu1, Dr. Benjamin J Burwitz1, Dr. Shaheed A Abdulhaqq1, Christine Shriver‐Munsch2, Tonya Swanson2, Alfred W Legasse2, Katherine B Hammond1, Jason S Reed1, Mina Northrup1, Dr. Stephanie L Junell4, Dr. Gabriela M Webb1, Dr. Justin M Greene1, Dr. Benjamin N Bimber2, Dr. Wolfram Laub4, Dr. Paul Kievit2, Dr. Rhonda MacAllister2, Dr. Michael K Axthelm2, Dr. Rebecca Ducore2, Dr. Anne Lewis2, Dr. Lois Colgin2, Dr. Theodore R Hobbs2, Dr. Lauren D Martin2, Dr. Charles R Thomas4, Dr. Angela Panoskaltsis‐Mortari5, Dr. Gabrielle Meyers3, Dr. Jeffrey J Stanton2, Dr. Richard T Maziarz3, Dr. Jonah B Sacha1,2 1
Vaccine and Gene Therapy Institute, Oregon Health & Science University, 2Oregon National Primate Research Center, Oregon Health & Science University, 3Division of Hematology and Medical Oncology, Oregon Health & Science University, 4 Division of Medical Physics, Oregon Health & Science University, 5Division of Blood and Marrow Transplantation, University of Minnesota
Background: Timothy Brown remains in cART‐free HIV remission following allogeneic hematopoietic stem cell transplant (HSCT), but attempts to recapitulate his cure have been unsuccessful. We recently established a nonhuman primate model of fully MHC‐matched allogeneic HSCT to investigate the mechanism of cure in Timothy Brown. In this model, a reduced intensity conditioning (RIC) regimen consisting of chemotherapy, CD3 depletion, and total body irradiation (TBI) prior to HSCT results in durable, full multi‐lineage donor chimerism in SIV‐naïve HSCT recipients. Here, we investigated (1) if similar results could be achieved without TBI, and (2) the impact of allogeneic HSCT on SIV reservoir size in cART‐ suppressed, SIV‐infected recipients. Methods: Allogeneic HSCT was performed with fully MHC‐matched Mauritian cynomolgus macaque (MCM) donor‐recipient pairs, including two fully cART‐suppressed, SIV‐infected recipients. Mobilized peripheral stem cells collected from donors by leukapheresis were transplanted into recipients following RIC with or without TBI. Donor engraftment was monitored by Illumina sequencing of single nucleotide polymorphisms. Immune subset reconstitution was assessed longitudinally by flow cytometric phenotyping and complete blood counts. Results: We performed two HSCTs without TBI, both resulting in low T cell donor chimerism (<15%). The first, SIV‐naïve recipient experienced incomplete T‐cell rebound resulting in polyoma virus reactivation and euthanasia. The second, cART‐suppressed, SIV‐infected recipient stabilized post‐HSCT, and experienced a reduction in lymph node‐associated SIV DNA despite incomplete T cell donor chimerism. Adding back TBI for HSCT of a second cART‐suppressed, SIV‐infected recipient resulted in stable engraftment with high levels of donor chimerism in whole blood (>95%) and T‐cells (~80%) within 30 days of HSCT. Conclusions: These data demonstrate that TBI is critical to achieving high levels of T cell donor chimerism post‐HSCT. Further, the successful transition of our model of fully MHC‐matched allogeneic HSCT into cART‐ suppressed, SIV‐infected MCM facilitates future studies investigating mechanisms of HSCT‐mediated HIV cure.
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103 The IL‐15 superagonist ALT‐803 decreases plasma viral loads in SIV infected rhesus macaques in the absence of antiretroviral therapy Dr. Amy Ellis1, Alexis Balgeman1, Katie Zarbock2, Gabrielle Barry2, Andrea Weiler2, Thomas Friedrich3, Jeffrey Miller4, Timothy Schacker5, Ashley Haase6, Emily Jeng7, Jack Egan7, Hing Wong7, Eva Rakasz2, Shelby O'Connor1 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, 3Department of Pathobiological Sciences, University of Wisconsin‐Madison, 4Division of Hematology, Oncology, and Medicine, University of Minnesota, 5Department of Medicine, University of Minnesota, 6Department of Microbiology, School of Public Health, University of Minnesota, 7Altor Bioscience Corporation
Developing biological interventions to control HIV replication in the absence of antiretroviral therapy (ART) would contribute to the development of a functional cure. As a potential alternative to ART, the IL‐15 superagonist ALT‐803 has been shown to boost the number and function of HIV‐specific CD8+ T and NK cell populations, in vitro, and can also induce virus replication in HIV‐infected CD4 T cells. In SIV+ rhesus macaques that expressed MHC molecules typically associated with viral control, we tested the hypothesis that ALT‐803 can boost T cell activity and suppress SIV replication. ALT‐803 was administered in three treatment cycles, each consisting of 0.1mg/kg ALT‐803 delivered subcutaneously once a week for four weeks. The first and second cycles of treatment were separated by two weeks, while the third cycle was administered 29 weeks later. ALT‐803 transiently elevated CD8+ effector and central memory T cell and NK cell populations 3‐5 fold, while viral loads decreased by 2‐3 logs in all animals, to undetectable levels. However, viral loads rebounded as T cells became less responsive to ALT‐803 and waned in numbers. No effect on viral loads was observed in the second cycle of ALT‐803, concurrent with downregulation of the IL‐15 common gamma‐C and beta chain receptors on both CD8+ T cells and NK cells. Furthermore, populations of immunosuppressive T cells increased during the second cycle of ALT‐803. A third cycle of ALT‐803 treatment was performed 29 weeks after the first two cycles, and responsiveness to ALT‐803 was restored. CD8+ T cells and NK cells increased again 3‐5 fold, and viral loads transiently decreased again, by 1‐2 logs. Future directions will focus on ALT‐803 dosing to maintain effective control of viral loads. Overall, our data indicates the potential of ALT‐803 to be used in combination with other antiretroviral therapies to combat HIV infection.
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104 The human IL‐15 superagonist complex ALT‐803 activates NK and memory T cells, reactivates latent SIV, and drives SIV‐specific CD8+ T cells into B cell follicles Gabriela Webb1,2, Katherine Hammond1,2, Shengbin Li8, Gwantwa Mwakalundwa8, Justin Greene1,2, Jason Reed1,2, Jeffrey Stanton2, Alfred Legasse1, Byung Park1, Michael Axthelm1, Emily Jeng3, Hing Wong3, James Whitney4,5, Brad Jones6, Douglas Nixon7, Pamela Skinner8, Jonah Sacha1,2 1
Oregon Health & Science University/Vaccine And Gene Therapy Institute, 2Division of Pathobiology and Immunology, Oregon National Primate Research Center, Oregon Health & Science University, 3Altor BioScience Corporation, 4Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, 5Ragon Institute of MGH, MIT, and Harvard, 6Department of Microbiology Immunology and Tropical Medicine, 7School of Medicine & Health Sciences, The George Washington University, 8Department of Veterinary and Biomedical Sciences, University of Minnesota
Background: There is an urgent need for alternate approaches to activate and clear the HIV reservoir that do not negatively impact immune function and are independent of viral sequence. IL‐15 is a key cytokine for homeostatic maintenance, proliferation, and expansion of memory CD4+ T‐cells, the primary HIV cellular reservoir. Here, we explored the human IL‐15 superagonist, ALT‐803, as an immunostimulatory molecule and LRA in cART‐suppressed SIV‐infected macaques. Methods: SIV‐naive and SIV‐infected macaques were administered ALT‐803 and monitored for lymphocyte proliferation. SIV‐infected macaques treated with ALT‐803 were assessed for intrafollicular migration via in situ staining of lymph nodes with MHC‐class‐I tetramers. ALT‐803 was tested as an LRA in vitro with primary CD4+ T‐cells from cART‐suppressed macaques, and in vivo in SIV‐infected, cART‐suppressed macaques. Results: ALT‐803 activated and induced proliferation in NK cells, and T‐cells, both EM and CM. ALT‐803 redirected activated cells to secondary lymphoid tissues, an anatomical location of the viral reservoir, and in situ MHCI tetramer staining showed increased migration into B‐cell follicular sanctuaries. ALT‐803 did not affect viral loads in macaques with uncontrolled SIV infection; instead, ALT‐803 potentiated low‐level viral replication in elite controllers. In experiments using CD4+ T‐cells from cART‐suppressed macaques, ALT‐803 induced viral replication in vitro. In macaques with cART‐suppression of plasma viremia, ALT‐803 treatment resulted in plasma viral “blips” and unlike other common γ‐chain cytokines, ALT‐803 did not cause an increase in the size of the latent viral reservoir. Conclusions: IL‐15 superagonist, ALT‐803, is well tolerated in SIV‐infected, cART‐suppressed macaques and induces virus reactivation in vitro and in vivo. ALT‐803 reactivates quiescent virus, activates NK and CD8+ T‐ cells, which traffic to lymph nodes, entering B‐cell follicles harboring latently‐infected CD4+ TFH cells. The ability of ALT‐803 to potentially mediate the “shock” and “kill” makes it an appealing candidate for studies aimed at durable cART‐free HIV remission.
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105 Next generation gene protection and reservoir targeting approaches for HIV cure Christopher Peterson1,2, Claire Deleage3, Anjie Zhen4, Andreas Reik5, Michael C. Holmes5, Scott Kitchen4, Jacob D. Estes3, Hans‐Peter Kiem1,2 1
Fred Hutchinson Cancer Research Center, 2University of Washington, 3AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., 4UCLA, 5Sangamo Therapeutics
Background: Ten years after the functional cure of the Berlin Patient, a substantial body of evidence suggests that infection‐resistant cells are necessary but insufficient for HIV cure. Active targeting of viral reservoirs at disparate physiological sites will most likely be required; broader cytotoxic approaches such as myeloablative irradiation should be avoided. We have developed active and passive stem cell‐based gene therapies for HIV cure in the nonhuman primate model. In particular, expression of CD4‐based Chimeric Antigen Receptors (CD4CAR) from HSPCs should engender lifelong protection against reactivated reservoir cells. Methods: Pigtail macaques were infected with SHIV‐1157ipd3N4 and suppressed by combination antiretroviral therapy. Following stable suppression, hematopoietic stem and progenitor cells (HSPCs) were either CCR5‐edited with Zinc Finger Nucleases (ZFNs) or transduced with lentiviral vectors expressing an HIV/SHIV‐specific CD4CAR. Peripheral and tissue reservoirs were quantified by cell‐associated SHIV DNA and RNA, RNAscope and DNAscope analyses, and quantitative viral outgrowth assays. Results: Autologous transplantation with CCR5‐edited HSPCs leads to significant decreases in tissue‐ associated SHIV DNA and RNA levels. However, in situ analyses clearly demonstrate that persistent viral reservoirs remain. In animals that receive CD4CAR modified HSPCs, CD4CAR expression persists for years, and is associated with decreased tissue‐associated SHIV RNA levels and higher CD4:CD8 ratios in the gut. The number and function of these cells is proportional to the level of virus antigen. Conclusions: HSPCs can be gene‐modified and rendered HIV/SHIV‐resistant, but gene protection alone may be insufficient for reservoir eradication. Combination approaches such as CD4CAR modification of HSPCs further harness gene‐modified cells to actively target and destroy latently infected cells. Applying these strategies in the setting of reduced intensity conditioning will enable more effective reservoir targeting with substantially less toxicity. Most importantly, HSPC‐based therapies provide a life‐long source of infection resistant, virus‐specific sentinels that will augment the host response to recrudescent viral replication.
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106 Therapeutic vaccines for HIV Dr. Vaiva Vezys1 1
University of Minnesota
A (functional) cure for HIV is thought to be possible, likely through combinatorial interventions, including therapeutic vaccination. The only known person to be cured of HIV infection long‐term is Timothy Brown, who was exposed to aggressive treatment including bone marrow transplantation. I will discuss what features of his treatment may be important for the long‐term eradication or control of HIV and how therapeutic vaccination may or may not be able to recapitulate key aspects of this.
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107 Conserved Elements (CE) DNA vaccination induces CE responses in SIV infected, cART treated Macaques Paul Munson1,2, Hillary Tunggal1,2, Nika Hajari1,2, Megan O'Connor1,2, Debra Bratt2, James T. Fuller1,2, Drew May2, Solomon Wangari2, Brian Agricola2, Jeremy Smedley2, Xintao Hu3, Barbara K. Felber3, George N. Pavlakis4, James I. Mullins1, Deborah Heydenburg Fuller1,2 1
Department of Microbiology, University of Washington , 2University of Washington National Primate Research Center, Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute , 4 Human Retrovirus Section, Vaccine Branch, Center for Cancer Research, National Cancer Institute 3
Background: Therapeutic vaccines could improve HIV specific immunity thus attenuating viral recrudescence after withdrawing combination antiretroviral therapy (cART). However, most vaccines use full‐length (FL) immunogens that bias T‐cell responses to immunodominant epitopes that can incorporate escape mutations without reducing viral fitness. To address these issues, we investigated a gag based conserved elements (CE) DNA vaccine for its ability to re‐direct T‐cell responses to conserved, functionally constrained regions of the viral proteome in the immunodominant setting of viral infection. Materials & Methods: SIV infected macaques initiated cART six weeks post infection then three months post cART initation were vaccinated with Mock (N=8), FL (N=8), or CE (N=6) DNA by gene gun. Vaccines were co‐ formulated with the genetic mucosal adjuvant LT. T‐cell responses were measured by ELISpot and ICS. Viral loads were tested by q‐RT‐PCR. Differences between groups were determined by ANOVA and correlations by a Spearman test. Results: (4/6) CE vaccinated macaques developed significantly broader CE responses with a range of 1‐4 CE targeted, versus none (0/16) in the Mock/FL vaccinated animals. In the CE group, CE breadth was inversely correlated with the frequency of non‐CE Gag T‐cell responses prior to vaccination (P= 0.03, r = ‐0.86), suggesting that pre‐existing responses against non‐CE Gag sequences negatively influence CE DNA vaccine immunogenicity. Viral loads post cART withdrawal were similar between mock and vaccinated animals. Conclusions: Therapeutic vaccination with FL antigens increases virus‐primed T cell responses against variable epitopes whereas a CE DNA vaccine can redirect immune responses toward subdominant sequences. However, as shown here, pre‐existing responses against immunodominant sequences can decrease the potency of a CE vaccine, indicating additional strategies, including longer time on suppressive cART prior to vaccination, may be needed to enhance the effects of CE vaccination.
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108 Comparative assessment of three latent SIV reservoir reactivation strategies in elite controller rhesus macaques Adam Kleinman1, Dr. Ranjit Sivanandham1, Benjamin Policicchio1, Dr. Egidio Brocca‐Cofano1, Kevin Raehtz1, Tianyu He1, Dr. Cui Ling Xu1, Dr. Paola Sette1, Kathryn Martin1, Ellen Penn1, Dr. Ivona Pandrea1, Dr. Cristian Apetrei1 1
University Of Pittsburgh
Background: HIV/SIV persists in latent reservoirs despite effective antiretroviral treatment (ART). Cessation of ART results in virus rebound, calling for an HIV cure. Reservoir elimination/reduction is central to cure approaches, and a strategy to this goal is “shock and kill”: induction of latent provirus, which triggers the elimination of infected cells. HDACis are the main class of latency reversing agents (LRAs). Our group also studies T regulatory cell (Treg) depletion as a strategy to induce both virus reactivation and improve SIV‐ specific mediated viral clearance. We also explore the impact of cytoreductive therapy on the viral reservoir. Here, we compared these three strategies with regard to virus reactivation. Methods: Groups of 3 elite controller SIVagm.sab‐infected RMs were used to assess the efficacy of Romidepsin (RMD, single dose of 7mg/m²), ONTAK (twice, 15 μg/kg/day for 5 days at 21 days interval), and cyclophosphamide (Cy, 50mg/kg/day for 7 days) in inducing viral reactivation. Results: RMD, a potent HDACi, increased histone acetylation for up‐to 5 days post‐treatment, while inducing limited immune activation. ONTAK depleted up‐to 85% of Tregs (CD4+CD25+FoxP3), induced massive activation of CD4+ and CD8+ T cells (as shown by Ki‐67+ increases), and boosted the SIV‐specific immune responses. Cy massively reduced (>99%) total lymphocyte populations, including CD4+ T cells. The most potent virus reactivation occurred with Cy‐mediated cytoreduction; viral loads peaked posttreatment at 10⁷ vRNA copies/mL. After Treg depletion, VLs peaked at 10³ copies/mL Treg while after RMD they rebounded to 10²‐10³ copies/mL. Conclusions: Of the tested strategies, cytoreductive therapy induces the largest viral rebound. In fact, Cy reactivation was 4 orders of magnitude higher than ONTAK and RMD. As such, our results warrant studies aimed at understanding the impact of cytoreductive therapy on viral reservoir and may lead to an effective approach for a functional HIV cure.
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109 SIV‐specific RNA‐guided Cas9 nucleases and paired nickases inhibit SIV replication through proviral genome editing Lisa Smith1, Vida Hodara2, Laura Parodi2, Zhao Lai1, Yi Zou1, Luis Giavedoni2 1
University of Texas Health San Antonio, 2Texas Biomedical Research Institute
As with HIV infections in humans, infection with SIV in Rhesus macaques results in lifelong infection due to the establishment of reservoir cells that harbor the viral genome integrated in the host genome (provirus). CRISPR/Cas9 reagents offer a versatile set of tools for the manipulation of cellular genomes, including the possibility of inactivating integrated proviral DNA. We designed RNA‐guided Cas9 nucleases (RGNu) and nickases (RGNi) targeting highly conserved regions of the SIVmac genome, with the goal of disrupting infectious virus production via insertions and deletions (indels). In vitro assays in HEK293T cells co‐transfected with a plasmid containing the SIVmac239 proviral genome and a plasmid encoding GFP and RGNu were used to select the most effective constructs that inhibited virus replication. Cells from these co‐transfection assays were sorted for GFP expression and their DNA was analyzed with next generation sequencing to characterize indel formation. When RGNu target regions of the long terminal repeat (LTR) or ribosomal slip site (RSS) in our co‐transfection assays, there is a dramatic inhibition of virus production as detected by a reduction of SIV p27 capsid protein assessed by Luminex assays. Paired RGNi targeting the LTR inhibited SIV similarly. RGNu targeting LTRs and RSS showed a remarkable incidence of indels at the Cas9 cleave site with up to a 35% mutation rate. Paired RGNi targeting the LTR had detectable indels that spanned both nickase cleavage sites. Interestingly, single RGNu and RGNi targeting the TAR element inhibited SIV p27 production, but indels at the cleavage site were only detected with RGNu. The mechanism of single TAR‐targeting RGNi inhibition of SIV replication is currently being investigated. In summary, our SIV‐targeting RGNu and RGNi can inhibit viral replication in our co‐ transfection assays which inversely correlates to a high incidence of indels at the Cas9 cleavage sites.
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110 Adoptive T cell immunotherapy using CMV‐specific T cells genetically modified with αHIV‐CAR vectors Chengxiang Wu1, Shan Yu1, Agnus Lo2, Hui Li3, Gautam Sahu4, Preston Marx1, Dorothee von Laer5, Gail Skowron4, George Shaw3, Amitinder Kaur1, Richard Junghans2, Stephen E. Braun1 1
TNPRC, 2Tufts University Medical School, 3University of Pennsylvania, 4Roger Williams Medical Center, 5Medizinische Universität Innsbruck
T cell (Tc) immunotherapy has been successful clinically in advanced ALL using Tc genetically modified to express CD19‐CAR (chimeric antigen receptor). The CD19 extracellular antigen‐binding domain and intracellular Tc‐signaling domain redirect CAR Tc to the tumor antigen. Tc immunotherapy for HIV infection would require homing to residual HIV reservoirs, effective killing of infected cells, and long‐term persistence of CAR transduced Tc in vivo. These studies aim to stimulate CMV‐specific Tc and genetically modify them with αHIV‐CAR vectors, and link HIV killing in vivo to the activation and persistence of CMV antigen presentation. Three rhesus macaque were challenged with the CCR5‐tropic SHIV‐D virus, prior to viral suppression with TFV+FTC+DTG. Autologous rhesus PBMC were stimulated with rhesus CMV‐peptides pools/αCD28 and IL‐2+IL‐15. The expansion of CMV specific Tc was demonstrated by intracellular cytokine staining. CD8+ CMV‐specific Tc were transduced (up to 50%) with MLV‐based αCEA‐CAR control or CD4‐ 28ζ/maC46 vectors. CMV‐specific αHIV‐CAR Tc were expanded up to 120x10⁶ cells and reinfused after structured treatment interruption. After CAR Tc infusion and STI, viral load for the control was sustained at above 1x10⁴ copies/ml 3 weeks post, while viral rebound peaked and lowered to 1.2 x 10³ in one animal and was delayed until week 6 in the other treated animal. CAR Tc peaked at 10% one hour post infusion. The circulating CAR Tc were detectable in the αCEA‐CAR control at 3 weeks, and persisted in the αHIV‐CAR animals at 6 weeks. Thus, rhesus CMV‐specific Tc were expanded and transduced with CAR vectors prior to adoptive Tc immunotherapy in the rhesus SHIV challenge model with cART. Transfer of genetically modified CMV‐specific Tc were detected after 6 weeks and showed evidence of viral control. Long‐term persistence of HIV‐specific Tc in tissues may provide continuous immunosurveillance over HIV replication and residual HIV reservoir.
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200 Overview of vaccine approaches in adult and infant macaques Ann Chahroudi1 1
Nonhuman primates have been used for years as a model to study HIV pathogenesis, prevention, and, more recently, cure approaches. The HIV vaccine research field has benefited from nonhuman primate studies, not only to trial specific immunization strategies and products, but also to better understand the determinants of mucosal transmission and intricacies of immune responses that may or may not be protective. Further, nonhuman primate models are now being utilized to investigate how therapeutic vaccination may contribute to approaches to induce HIV remission. In this talk, the interface between vaccine research, pathogenesis, transmission, and cure will be discussed. Vaccine studies in adult and infant rhesus macaques at the Yerkes National Primate Research Center will be highlighted.
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201 Cross‐species CMV vaccination reveals viral determinants for induction of non‐classical MHC‐E‐restricted T cells Dr. Justin Greene1, Dr. Daniel Malouli1, Dr. Scott Hansen1, Dr. Travis Whitmer1, Abigail Ventura1, Roxanne Gilbride1, Colette Hughes1, Jason Reed1, Dr. Helen Wu1, Luke Uebelhoer2, Jennie Womack1, Matthew McArdle1, Junwei Gao1, Alfred Legasse1, Dr. Michael Axthelm1, Dr. Louis Picker1, Klaus Fruh1, Jonah Sacha1 1
Oregon Health & Science University/Vaccine And Gene Therapy Institute, 2Oregon Health & Science University/ Department of Pediatrics
Rhesus macaques (RM) vaccinated with strain 68‐1 rhesus CMV (RhCMV) vaccine vectors expressing SIV antigens demonstrate unprecedented protection against highly virulent SIVmac239 replication, with protected RM eventually clearing the virus. This protection is associated with unconventional CD8+ T cell responses that are either MHC‐II or MHC‐E restricted. These unconventional CD8+ T cell responses may be the result of the unique MHC complexity present in RM, or the result of conserved immunoregulatory mechanisms utilized by CMV. In order to parse out the importance of host immunogenetics from strain‐ specific CMV mechanisms, additional nonhuman primate models of CMV infection are needed. Mauritian‐ origin cynomolgus macaques (MCM) are a particularly attractive nonhuman primate model due to a significant population bottleneck 400 years ago that resulted in highly limited immunogenetics. We captured MCM CMV (CyCMV) as a BAC and subsequently developed an SIV Gag‐expressing vector with deletions corresponding to those found in RhCMV 68‐1. Vaccination of MCM with “strain 68‐1 like” CyCMV induced unconventional CD8+ T cell responses including MHC‐E and MHC‐II restricted responses, including “supertope” responses that are present in every RhCMV strain 68‐1 vaccinated RM. Interestingly, both RhCMV vaccinated RM and CyCMV vaccinated MCM target identical supertope peptides that are restricted by MHC‐E. As CMV‐vectors advance toward human clinical trials, these results suggest a similarly designed human CMV may induce unconventional CD8+ T cell responses in humans.
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202 A single V2 monoclonal antibody reduces lymphoid tissue viremia and partially protects macaques after repeated SHIV challenges Dr. AJ Hessell1, DC Malherbe1, SM McBurney1, S Pandey1, T Cheever1, P Barnette1, WF Sutton1, S Zolla‐ Pazner2, NL Haigwood1 1
Oregon Health & Science University, Oregon National Primate Research Center, 2Icahn Mt. Sinai School of Medicine
Background. The RV144 human vaccine trial raised interest in discovering specific antibody responses associated with reduced risk of infection that was associated with high levels of V2‐specific antibodies in vaccinee sera. Several V2 specific human mAbs have been isolated and studied. Monoclonal antibody (mAb) 830A binds to a conformational V2 epitope that overlaps the 4 7 integrin binding site located at the Env trimer apex mediates ADCC and phagocytosis. We tested mAb IgG1 and IgG3 830A for protection efficacy in macaques mucosally challenged with SHIV. Methods. Pharmacokinetics studies evaluated the decay rate and achievable plasma concentrations. Thirty male rhesus macaques received 10 mg/kg of mAb 830A or control mAb subcutaneously 48 hours before SHIV exposure that continued weekly for 3 weeks. Macaques received six repeated intrarectal SHIV BaL.P4 exposures twice weekly. Blood draws monitored plasma viremia, transferred antibody kinetics, and neutralization titers at times of challenge. Cell‐associated virus was measured in PBMC, mucosal and lymphoid tissues, and organs at necropsy. Plasma antibodies were measured longitudinally by ELISA to assess the development of de novo antibody responses in infected animals. Results. Plasma concentrations of IgG1 and IgG3 830A averaged 158 g/ml and 118 g/ml, respectively during SHIV challenges. Plasma neutralizing activity at the time of challenges was 100 to 200 (ID50 value). A modest 5 of 18 aminals treated with mAb 830A were protected from infection. However, significant protection efficacy was measured as time to infection comparing IgG1 830A treated macaques vs control animals (P=0.0210) with significantly lower plasma viremia (P=0.0386) and quantified virus in tissues (P=0.0149). Conclusions. V2 mAb IgG1 830A provided a significant effect upon viral acquisition, peak viral loads, and viral integration into lymphoid tissues versus controls. The modest protection from infection in 5/18 macaques seen in these experiments represents the first in vivo experimental evidence that V2 Abs may contribute to a reduced risk of infection. Funding: The International AIDS Vaccine Initiative (IAVI) to N.L.Haigwood.
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203 An oral prime/ boost pediatric vaccine strategy for the prevention of HIV transmission by breast‐feeding Alan Curtis1, Bonnie Phillips1, Neelima Choudhary1, Ryan Tuck1, Koen Van Rompay2, Pamela Kozlowski3, Rama Amara4, Kristina De Paris1 1 4
University Of North Carolina At Chapel Hill, 2California National Primate Research Center, 3Louisiana State University, Emory University
Every day about 400 infants become HIV‐infected, the majority by breast‐feeding. The present study tested the effect of including an oral immunization of SIV DNA together with CD40L+GM‐CSF adjuvants (DNA‐SIV) in a rhesus macaque model of oral SIV infection. The present study t Two groups of 6 neonatal macaques received 2 DNA‐SIV immunizations at weeks 0 and 3, followed by 2 boosts with modified vaccinia Ankara virus (MVA)‐SIVgag/pol/env at weeks 6 and 9. Group A was immunized intramuscularly (IM). Group B received DNA‐SIV both orally (PO) and IM; MVA‐SIV was administered sublingually (SL) and IM. Control animals received PO+IM saline at weeks 0 and 3 and empty MVA vector SL+IM at weeks 6 and 9. Beginning at week 12, all infants were orally challenged weekly with 500 TCID50 of SIVmac251 until infection was achieved. Statistical significance was determined using Mann‐ Whitney or Kruskal‐Wallis tests with p<0.05. SL/IM vaccination resulted in lower per‐exposure risk of oral SIV infection compared to mock‐ and IM/IM‐ vaccinated animals (p<0.0001; log‐ranked Mantel‐Cox test) and lower peak viremia (p=0.03) compared to controls. Control of viremia was associated with higher fecal IgG responses to SIV V1V2 and p55 gag (both epitopes, p=0.0087) at the time of oral challenge initiation. In contrast, plasma SIV‐specific IgG or IgA did not influence challenge outcome. In this study, inclusion of an oral route in a pediatric vaccine to prevent oral SIV infection was superior to an IM only regimen suggesting the importance of enhancing mucosal immune responses via local immunization.
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204 A novel SHIV expressing the B41 HIV‐1 env: Implications for HIV vaccine design and testing Jessica Smith1, Hui Li1, Wenge Ding1, Shuyi Wang, Alexander Murphy, Maho Okumura, Beatrice H. Hahn1, George M. Shaw1 1
Perelman School Of Medicine
Background: The HIV‐1 B41 SOSIP is a prototype soluble stabilized Env trimer that elicits strain‐specific neutralizing antibodies (Nabs) in rhesus macaques (RMs) or in small animals. It is unknown whether this SOSIP has the potential to elicit bNAbs. A limitation of SOSIP immunogens is that, although they resemble native infectious trimers in their structure and antigenicity, they are not identical, and they cannot replicate and coevolve along with NAb responses to stimulate Ig somatic hypermutation. Methods: We used a recently published (PNAS 113:E3413, 2016) Env375 codon substitution strategy to create B41 SHIVs with any of six Env375 residues (S/M/Y/H/W/F). We tested these for sensitivity to a broad panel of neutralizing mAbs,for replication in RM cells in vitro and in vivo, and for the elicitation of NAbs in RMs. Results: SHIV variants Env375 W/H/Y replicated efficiently in primary rhesus T cells in vitro and in vivo while retaining a wild‐type tier 2 neutralization sensitivity and antigenicity to a broad panel of NAbs and bNAbs. Early replication kinetics, peak viremia, and setpoint viremia were indistinguishable from natural human infection by HIV‐1. Longitudinal analysis of autologous and heterologous NAbs, including epitope mapping, is underway. Conclusions: Efficient SHIV B41 replication in RMs extends the paradigm of novel SHIV design based on allelic substitutions at Env codon 375 and demonstrates that HIV‐1 Envs of predetermined specificity can be engineered to replicate as SHIVs. Because of its ability to replicate persistently, SHIV B41 may serve as a “molecular guide” for the development of lineage‐based immunogens designed to engage germline and intermediate ancestor bNAb Igs. In addition, because of its favorable acute and early replication kinetics, SHIV B41 can serve as a valuable subtype B transmitted/founder challenge strain to complement subtype A (BG505), C (CH505 and CH848) and D (D.191859) SHIVs in preclinical protection studies.
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205 Drug distribution at SHIV infection sites in the macaque female reproductive tract Dr. Katarina Halavaty1, Dr. Adina K. Ott1, Dr. Danijela Maric1, Dr. Jonathan T. Su1, Edgar Matias1, Dr. Lara Pereira2, Dr. James M. Smith3, Dr. Patrick F. Kiser1, Dr. Thomas J. Hope1 1
Northwestern University, 2Lifesource Biomedical LLC, 3Centers for Disease Control and Prevention
Worldwide, sexual HIV transmission in young women is double that in young men. Prevention of HIV transmission in the female reproductive tract (FRT) by using a tenofovir disoproxil fumarate (TDF)‐eluting intravaginal ring (IVR) is under investigation. Here we demonstrate the ability to locate the initial sites of infection, and to measure the drug distribution within a TDF‐IVR‐protected macaque FRT. Specifically, we are examining the impact of drug transport on viral spread within the FRT of these animals. Six pigtail macaques were treated with TDF‐IVRs for 28 days, and vaginally challenged with a high dose (~10⁵‐10⁶) of a single round non‐replicative SIV‐based vector expressing HIV envelope and Luciferase and mCherry reporter genes. The FRT was scanned using in vivo imaging (IVIS), fluorescent microscopy and nested PCR (mCherry region) to detect early infection events. TFV tissue concentrations were quantified using LC‐MS/MS, with ¹³C‐labeled TFV used as an internal standard. IVIS revealed infection events in the ovaries of two animals. Fluorescent microscopy revealed transduced cells in ovaries of five of six TDF‐IVR animals. PCR data demonstrated frequent viral infections in the vagina and cervix of four of six animals. However, tenofovir levels were variable throughout the FRT, with the highest concentrations in the upper vaginal/lower cervical area (10⁴‐10⁵ ng/g of tissue), near the site of the ring. Utilizing reporter viruses allows us to detect the initial infection sites within the FRT. We performed complete pharmacokinetic and pharmacodynamic studies at both anatomical and cellular levels. In this ongoing study we are further investigating the IVR drug delivery efficiency and drug transport in tissues compared to the orally administrated TDF in challenged macaques. This novel research is critical in understanding the complex interplay between viral infection events and protective drug levels at the site of initial infection.
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206 Alloimmunization of Mauritian cynomolgus macaques with allogeneic cells fails to protect against repeated, limiting‐dose SIV challenge Dr. Matt Reynolds1 1
The variability of HIV makes it difficult to develop effective vaccines directly targeting viral proteins. An alternative strategy is focusing immune responses against polymorphic host proteins, particularly major histocompatibility complex (MHC) molecules, that are incorporated into virions as they bud from infected cells. However, controlled studies examining the potential of alloreactive immune responses in protecting against immunodeficiency virus infection are difficult. We, therefore, used Mauritian cynomolgus macaques (MCM) that have limited genetic diversity and only express seven major MHC class I haplotypes, designated M1 through M7, to assess the protective capacity of anti‐MHC antibodies against AIDS virus transmission. A cohort of 21 MCM with the M1/M2 MHC class I haplotypes were divided into three groups of 7 animals and received one of the following treatments: (1) immunization with MHC disparate peripheral blood mononuclear cells cells (PBMC) from M4/M5 MCM, (2) immunization with MHC‐identical PBMC from M1/M2 MCM, or (3) no immunization. The alloimmunization regimen induced anti‐MHC binding antibodies in the animals immunized with MHC‐disparate (group 1), but not MHC‐matched (group 2), PBMC. The anti‐ MHC antibodies, however, were unable to neutralize or induce complement‐dependent virolysis of simian immunodeficiency virus (SIV) grown on the PBMC of M4/M5 MCM (SIVmac239‐M4/M5). Approximately three months after the last immunization all of the animals were challenged intrarectally with repeated, limiting doses of SIVmac239‐M4/M5. Vaccination did not significantly affect acquisition or control of infection in the vaccinees in comparison to the controls. These results indicate that alloimmunization alone is not sufficient to protect against immunodeficiency virus infection.
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207 Tracking fluorophore‐conjugated VRC01 following IV injection in the rhesus macaque reveals that tissue distribution is slow and can take approximately 1 week to achieve steady state. Dr. Jeffrey R. Schneider1, Dr. Ann M. Carias1, Dr. Amarendra Pegu2, Dr. Arangassery R. Bastian1, Dr. Gianguido C. Cianci1, Dr. Patrick F. Kiser1, Dr. Ronald S. Veazey3, Dr. John R. Mascola2, Dr. Thomas Hope1 1
Northwestern University, 2Vaccine Research Center, 3Tulane National Primate Research Center
Due to the success of passive infusion of bNAbs in SHIV challenged rhesus macaques to block systemic infection, a new trial was launched this past fall where at risk individuals received an IV infusion of the bNAb, VRC01. However, there are still many questions as to how these specialized antibodies can provide sterilizing immunity, one of which is how long it takes for antibodies to achieve steady state levels in the tissue following IV injection. Current efforts to track antibodies utilize low resolution and sensitivity methods such as ELISA and indirect labeling imaging techniques. Here we demonstrate that it is possible to utilize the fluorophores Cy5 and Cy3 directly conjugated to VRC01‐LS for direct visualization and quantification of passively transferred antibodies in plasma, mucosal secretions, and tissue. VRC01‐LS was formulated with 1‐2 fluorophores per antibody, to minimally influence antibody function, as shown through similar neutralization of HIV‐1 BaL in the TZMB‐l assay and similar binding to SOSIP(gp120) via surface plasmon resonance. In sequential vaginal biopsies acquired from the same animal from 24hr to 8wks post IV‐injection of VRC01‐LS‐Cy5, we found that antibody slowly built up and achieved steady state at 1wk. This was further confirmed by a serial necropsy of VRC01‐LS‐Cy5 IV injected rhesus macaques at 24hrs, 48hrs, 72hrs, 1wk, and 2wks in which biopsies from the FRT and distal sites were harvested and antibody levels were measured. Similar to the findings in a single rhesus macaque, we found that levels of antibody grew steadily in the FRT and at distal sites of these sequentially necropsied animals achieving steady state levels at 1wk and decreasing around week 2. Through tracking antibody in the FRT and at distal sites we will be able to correlate virus replication and antibody levels in these tissues and more importantly identify sites of virus‐antibody interaction.
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208 DNA and protein co‐delivery vaccines using TLR‐4‐based adjuvants induce potent immune responses able to delay heterologous SIV acquisition Barbara Felber1, Shakti Singh1, Antonio Valentin1, Margherita Rosati1, Eric Ramierz1, Rami Doueiri1, Xintao Hu1, Jenifer Bear1, Vanessa Hirsch2, Kate Broderick3, Niranjan Sardesai3, Steven Reed4, Hung Trinh5, Mangala Rao5, David Montefiori6, Guido Ferrari6, Xiaoying Shen6, Georgia Tomaras6, George Pavlakis1 1
National Cancer Institute At Frederick, 2NIAID, 3Inovio Pharmaceuticals Inc., 4IDRI, 5MHRP, 6Duke University Medical Center
Background: HIV/SIV DNA vaccination induces high and durable T cell responses as well as humoral responses, which efficiently disseminate into mucosal sites. We developed a method of simultaneous vaccination with DNA and protein resulting in great increase of humoral responses and improved protection against SIV infection in macaques. Methods: Macaques were divided into 3 TRIM‐genotype balanced experimental groups (N=12/group). Animals were immunized at 0, 2 and 6 months with SIVmac251 gag and env DNA by IM electroporation followed by administration of SIV gp120 Env protein formulated in TLR‐4‐ agonist based liposomes (TLR4+7 or TLR4+QS21) as adjuvants, while the animals in the control group received sham DNA. Vaccine‐induced cellular and humoral immune responses were monitored, and 5 months after vaccination the animals were challenged intrarectally with repeated low dose SIVsmE660. Results: Similar robust levels of cellular and humoral responses, including bAb and NAb to SIVmac251 and SIVsmE660, as well as responses to scaffolded V1V2 and cyclic V2 were found in both vaccine groups. The cellular responses included cytotoxic CD4 and CD8 T cells, and were present at high levels 4 months after the last vaccination, indicating long‐term memory. Animals in the TLR4+7 group showed a trend of increased NAb breath and stronger ADCC activity. Upon heterologous challenge, a trend of delayed viral acquisition was found which was statistically significant in RM with the TRIM5a resistant genotype. Antibody responses to the heterologous SIVsmE660 (NAb, bAb, V1V2 ab) directly correlated with virus acquisition. Upon infection, we observed reduced peak and chronic viremia among animals without the TRIM5a‐ resistant genotype. Reduction of viremia inversely correlated with cellular responses, as well as humoral responses targeting V1 and V2. Conclusions: Combination of DNA and adjuvanted protein using two different formulations induces high, durable and potent cellular and humoral responses. The vaccine induced responses provide protection against heterologous challenge.
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209 Impact of a TLR‐5 Ligand as Aduvant on Immunogenicity and Efficacy of a RhCMV‐SIV Vaccine Dr. Ellen Sparger1, Dr. William Chang1, Dr. Jesse Deere1, Mr. Hung Kieu1, Dr. Diego Castillo1, Dr. Shelley Blozis1, Dr. Jeffrey Lifson2, Dr. Xiaoying Shen3, Dr. Georgia Tomaras3, Dr. Barbara Shacklett1, Dr. Peter Barry1 1
University Of California Davis, 2Frederick National Laboratory, 3Duke Human Vaccine Institute
Previous reports demonstrated that immunization of rhesus macaques with rhesus cytomegalovirus (RhCMV)‐vectored simian immunodeficiency virus (SIV) vaccines was efficacious in 50% of vaccinated animals by demonstration of clearance of SIV infection after mucosal challenge. A RhCMV‐SIV vaccine was modified by incorporation of a SIV Gag‐FliC (flagellin) fusion antigen, validated for replication and TLR‐5 activity in vitro and tested for effects of TLR‐5 activity on immunogenicity and efficacy. Three groups of specific pathogen free (SPF) female rhesus macaques free of RhCMV infection, were immunized with either RhCMV (empty vector), RhCMV vectors expressing Gag, Retanef and Env, or RhCMV vectors expressing Gag/FliC, Retanef and Env by priming and booster immunizations. All animals were examined for systemic and mucosal SIV‐specific immune responses post immunization. All animals subsequently received a multiple low dose SIVmac251 vaginal challenge followed by assessment for plasma virus loads and anti‐viral immune responses. Statistically significant lower virus loads were observed in animals vaccinated with the prototype RhCMV‐SIV vaccine when compared to animals vaccinated with the RhCMV‐SIV vaccine with the TLR‐5 adjuvant or to unvaccinated controls. 50% of animals vaccinated with the prototype RhCMV‐SIV vaccine showed superior control of the SIV challenge infection characterized by declining plasma virus loads to very low or undetectable levels. In contrast, only 17% and 14% of unvaccinated controls and animals vaccinated with adjuvanted vaccine respectively, showed a similar control of virus infection after SIV challenge. Results showing reduced immunogenicity and efficacy for the TLR‐5 adjuvanted RhCMV‐SIV vaccine were unexpected. Results suggested that a robust adjuvant may significantly cripple the RhCMV‐SIV vaccine virus, reduce expression of the vaccine proteins and thereby impair vaccine efficacy. These findings suggest careful consideration of possible impact of the adjuvant activity on host immunity to a CMV‐HIV vaccine virus, which may impair expression of vaccine proteins and therefore vaccine efficacy.
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210 Distinct replication patterns and neutralizing antibody responses in Rhesus Macaques infected by SHIVs bearing 15 different primary or transmitted/founder HIV‐1 envs Dr. Hui Li1, Dr. Fang‐Hua Lee1, Mr. Ryan Roark1, Ms. Jessica Smith1, Ms. Maho Okumura1, Mrs. Shuyi Wang1, Mrs. Wenge Ding1, Dr. Beatrice Hahn1, Dr. George Shaw1 1
University Of Pennsylvania
BACKGROUND: Consistent induction of HIV‐1 tier‐2 neutralizing antibodies (NAbs) in rhesus macaques (RMs) could be key to progress in HIV‐1 vaccine design. We constructed SHIVs containing 15 primary subtype A, B, C, AE or D Envs and tested them for replication and NAb elicitation in 100 RMs. Envs included those that in humans elicited bNAbs against CD4bs, V1/V2 or V3 glycan epitopes. METHODS: HIV‐1 Envs CH505, CH848, BG505, Ce1176, CAP256SU, T250, WITO, ZM233, CH1012, Q23, 1086, 40100, 191859, YU2 and B41 containing substitutions at residue 375 were cloned into SIVmac766 and infections of 100 RMs performed by IR, IVAG or IV routes. Blood was collected weekly or monthly over 6‐24 months. RESULTS: All 15 SHIVs replicated efficiently in human and rhesus CD4 T cells in vitro and in RMs in vivo. Early replication kinetics were indistinguishable from human infection by HIV‐1 with peak viremia generally ranging from 10^6‐10^8 copies/ml ~2 weeks post infection (wpi). Set points of 10^3‐10^5 copies/ml occurred ~12 wpi in most animals. A subset of animals (<15%) controlled viremia to a limit of detection <250 vRNA/ml. Most RMs developed autologous tier 2 neutralizing Ab responses by 12‐24 wpi with reciprocal ID50 titers ranging from 40‐60,000. Remarkably, HIV‐1 Envs that elicited V1/V2, V3 glycan or CD4bs NAbs in humans elicited NAbs of similar specificities in RMs. The CH848 Env that in a naturally infected human elicited bNAbs did so as well in RMs, neutralizing 15 of 19 heterologous tier 2 viruses at titers of 40 ‐ >1000. Neutralization breadth is under study in all animals. CONCLUSIONS: SHIVs bearing primary HIV‐1 envs can be reliably developed by a novel delta Env375 strategy (see Li PNAS 2016 and Li abstract v2.0 this meeting). Env‐Ab coevolution in SHIV infected RMs may serve as a “molecular guide” for HIV‐1 vaccine design.
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300 Quantifying Leukocyte Trafficking in NHP by Serial Intravascular Staining Mario Roederer1, Elizabeth Potter1, Kathy Foulds1, Tricia Darrah1, Robert Seder1, Hannah Gideon2, Joanne Flynn2 1
VRC, NIAID, NIH, 2University of Pittsburgh
The body carefully regulates the number cells in the blood and tissues. For example, in healthy adults, the CD4:CD8 ratio is approximately 2:1; within CD4 or CD8 T cells, the ratio of naïve to memory cells is typically 1:1. Mechanisms that regulate these balances are poorly, if it all, known. Furthermore, disregulation in the homeostasis of these cells often accompanies or leads to disease. We are evaluating the impact of trafficking of leukocytes in the homeostatic maintenance of blood levels. One approach to this end has been to extend the seminal work of Masopust and colleagues, who developed intra‐vital staining of vascular leukocytes in mice to distinguish tissue resident vs. vascular cells in cells take from biopsies or tissues. We adapted and are optimizing this technique to NHP: injecting fluorescent conjugates of CD45 intravenously, a few minutes before necropsy, can distinguish vascular from resident cells as it does in mice. We noted that the amount of mAb injected resulted in uniform staining of only a fraction of surface CD45 molecules on PBMC – thus, the cells could be restained ex vivo using a different color of the same clone. This also means that the intra‐vital staining can be repeated in the same animal at different time points to provide a “pulse‐chase” staining experiment to quantify the entry and exit rates of leukocytes into or out of tissues by marking which cells were in the blood at various times. Combined with 20+ color immunophenotyping, we can quantify the trafficking of virtually any leukocyte population. We are applying this technology to settings of disease and pathogenesis to determine the impact of altered homeostasis on trafficking patterns and vice versa.
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301 Failure to induce CD11c+ B cells is associated with a rapid progressor phenotype in orally‐inoculated SIV‐infected infant macaques Matthew Wood1, Megan Templeton1, Adriana Lippy1, Patience Murapa2, Deborah Fuller2, Donald Sodora1,3 1 3
Center For Infectious Disease Research, 2University of Washington, WaNPRC and Department of Microbiology, University of Washington, Department of Global Health
Background: In the absence of ART HIV infection of infants is associated with bimodal rates of disease progression, with 20% of infants progressing rapidly to AIDS. To discern the immunologic factors that influence infant disease progression rates we utilized SIV‐infected infant Rhesus macaques, half of which exhibited a rapid progressor phenotype. Methods: Macaque infants were orally infected with SIVmac251, and followed longitudinally for 10 to 17 weeks post infection or until euthanasia due to clinical symptoms. Infants were placed into either the High Viremic (HVir) (viral load 7.2×107 – 3.9×108 /mL plasma, n=8) or Low Viremic (LVir) (3.8×105 – 6.2×106 /mL plasma,, n=8) based on a significantly bimodal distribution of chronic‐stage viral set points (after 4 weeks). Circulating B cells, monocytes and T cells were evaluated utilizing flow cytometry. Results: HVir infants exhibited significantly higher levels of IFNa, CD16+ inflammatory monocytes, and were unable to restore CCR5+ and CXCR3+ CD4 T cells following acute infection. Furthermore, HVir infants failed to expand a B cell subset expressing homing markers CD11c and CXCR3+, and activation marker CD80. LVir infants macaques had significantly higher CD11c+ (p<0.0001), CD80+ (p<0.0001), and CXCR3+ (p=0.0003) B cells by 10‐12 weeks post infection compared to HVir infants. This increase in activated B cell levels in LVir infants was associated with higher levels of SIV‐specific antibodies, whereas HVir macaques did not produce detectable levels of SIV‐specific antibodies. In addition, immunofluorescent microscopy provided evidence for increased macrophage infiltration (CD68+) in lymph nodes from HVir infants. Conclusion: Our findings support a model in which the infiltration of inflammatory monocyte/macrophages into lymph nodes inhibits the expansion of activated antiviral B cells resulting in low levels of anti‐SIV antibodies and higher levels of viremia in SIV‐infected infant macaques. These data provide insights into the mechanisms of rapid disease progression in HIV infected infants.
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302 Characterizing the changes in initial HIV/SIV infection by cell phenotyping at anorectal mucosa of Rhesus Macaques Dr. Danijela Maric1, Lisette Corbin1, Dr. Ron Veazey2, Dr. Thomas Hope1 1
Northwestern University, 2Tulane University
We developed methodology to identify transduced cells in anorectal tissues of Rhesus Macaques following rectal challenge with a non‐replicative reporter virus that expresses Luciferase and near‐infrared fluorescent protein 670 (LI670). The phenotypic analysis of the transduced cells revealed that Th17 cells and immature dendritic cells (iDCs) are predominantly targeted. To learn if these cells are targeted simply due to their abundance, or if they are preferentially targeted, we performed a large scale phenotyping analysis of the resident HIV susceptible cells at the anorectal tissue. Interestingly we found that these two cell types are infected at a rate that is five‐fold higher than their relative abundance among target cells in these tissues would suggest. This data indicates that there is a strong preference for infection of Th17 cells and iDCs in the early course of HIV/SIV infection during rectal transmission in our Macaque model. To further advance our understanding of HIV/SIV transmission and the relevant target cells, we next developed methodology to study the infection by the wild type SIVmac239 virus. Macaques were rectally challenged with a mixture of wild‐type SIV and our reporter virus, and necropsied 48, 72, or 96 hours later. Luminescence was used to hone in on small tissue areas which likely contained SIV infected cells. Foci of infected cells are evident as early as 48h post‐challenge and continue to expand by 96h. The spectrum of infected cells changes as infection progresses. Th17 infection rate does not vary much over the first 96h. However, from 48h to 96h, there is a pronounced decrease of iDCs infection rate concurrently with an increase in infection of other T cell types. By studying the changes in infected cell population over time, we will have a better understanding of virus‐host cell interactions which will aid development of more effective HIV prevention strategies.
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303 Paradoxical myeloid‐derived suppressor cell reduction in the bone marrow of SIV chronically infected Macaques Yongjun Sui1, Blake Frey1, Yichuan Wang1, Rolf Billeskov1, Shweta Kulkarni1, Katherine McKinnon1, Tracy Rourke2, Linda Fritts2, Christopher Miller2, Jay Berzofsky1 1
NIH NCI Vaccine Branch, 2Center for Comparative Medicine, University of California Davis
Myeloid derived suppressor cells (MDSCs), which suppress anti‐tumor or anti‐viral immune responses, are expanded in the peripheral blood and tissues of patients/animals with cancer or viral infectious diseases. We here show that in chronic SIV infection of Indian rhesus macaques, the frequency of MDSCs in the bone marrow (BM) was paradoxically and unexpectedly decreased, but increased in peripheral blood. Reduction of BM MDSCs was found in both CD14+MDSC and Lin‐CD15+MDSC subsets. The reduction of MDSCs correlated with high plasma viral loads and low CD4+ T cell counts, suggesting that depletion of BM MDSCs was associated with SIV/AIDS disease progression. Of note, in SHIVSF162P4‐infected macaques, which naturally control viral replication within a few months of infection, the frequency of MDSCs in the bone marrow was unchanged. To investigate the mechanisms by which BM MDSCs were reduced during chronic SIV infection, we tested several hypotheses: depletion due to viral infection, alterations in MDSC trafficking, and/or poor MDSC replenishment. We found that the possible mobilization of MDSCs from BM to peripheral tissues and the slow self‐replenishment of MDSCs in the BM, along with the viral infection‐ induced depletion, all contribute to the observed BM MDSC reduction. Accordingly, we first demonstrate MDSC SIV infection in vivo. Correlation between BM CD14+MDSC reduction and CD8+ T cell activation in tissues is consistent with decreased immune suppression by MDSCs. Thus, depletion of BM MDSCs may contribute to the pathologic immune activation during chronic SIV infection and by extension HIV infection.
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304 Polymorphisms in tetherin are associated with differences in peak viremia during acute infection of Rhesus Macaques with SIV delta‐Nef Sanath Kumar Janaka1, William Neidermeyer2, Ruth Serra‐Moreno3, Bin Jia4, James Hoxie5, Ronald Desrosiers6, Paul Johnson7, Jeffrey Lifson8, Steven Wolinsky9, David Evans1 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Department of Microbiology and Immunology, Harvard Medical School, 3Department of Biological Sciences, Texas Tech University, 4Pfizer Inc, 5Department of Medicine, University of Pennsylvania, 6Department of Pathology, Miller School of Medicine, University of Miami, 7 Yerkes National Primate Center, 8AIDS and Cancer Virus Program, Leidos Biomedical Research Inc, FNLCR, 9Division of Infectious Diseases, Northwestern University Feinberg School of Medicine
Tetherin (BST‐2 or CD317) is an interferon‐inducible protein that interferes with virus release from infected cells. To determine the extent of sequence variation and the impact of polymorphisms in rhesus macaque tetherin on SIV infection, we sequenced full‐length tetherin cDNA clones from 139 rhesus macaques, including 69 animals infected with wild‐type SIVmac239 and 47 animals infected with SIVmac239Δnef. Since Nef is the viral protein of SIV that counteracts rhesus macaque tetherin, these groups afford a comparison of the effects of tetherin polymorphisms on SIV strains that are, and are not, resistant to tetherin. We identified 14 alleles of rhesus macaque tetherin with dimorphic residues at 9 positions throughout the protein. The relationship between these alleles and plasma viral loads was compared during acute infection, prior to the onset of adaptive immunity. Peak viremia did not differ significantly among the wild‐type SIV‐ infected animals; however, differences in peak viremia were associated with polymorphisms in tetherin among the SIVΔnef‐infected animals (p=0.027, Kruskal‐Wallis test). In particular, polymorphisms at positions 14, 43 and 111 (D14, P43 and H111) were associated with significantly lower peak SIVΔnef viral loads (p<0.05, Kruskal‐Wallis test). These observations reveal extensive polymorphism in rhesus macaque tetherin, maintained perhaps as a consequence of variability in the selective pressure of diverse viral pathogens, and identify tetherin alleles that may have an inherently greater capacity to restrict SIV replication in the absence of Nef.
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305 Mutations in Nef that selectively disrupt tetherin antagonism impair SIV replication during acute infection of Rhesus Macaques Aidin Tavakoli‐Tameh1, Dr Sanath Kumar Janaka1, Lauren Callahan1, Ksenia Bashkueva1, Katie Zarbock2, Dr Shelby O'Connor1,2, Kristin Crosno2, Saverio Capuano 3rd2, Dr Ruth Serra‐Moreno3, Dr Hajime Uno4, Dr Jeffrey D. Lifson5, Dr David Evans1,2 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, 3Department of Biological Sciences, Texas Tech University, 4Department of Biostatistics and Computational Biology, Dana‐Farber Cancer Institute, 5AIDS and Cancer Virus Program, Leidos Biomedical Research Inc
Nef promotes the release of SIV from infected cells by counteracting restriction of non‐human primate tetherin (BST‐2 or CD317). Nef also downmodulates cell‐surface CD4 and MHC class I (MHC I) molecules and enhances infectivity by preventing incorporation of SERINC3/5 into virions. We previously demonstrated that tetherin antagonism by Nef is genetically separable from CD4‐ and MHC I‐downmodulation and infectivity enhancement. Here we show that selective disruption of tetherin antagonism impairs virus replication during acute SIV infection of rhesus macaques. Separate groups of four rhesus macaques were infected with either wild‐type SIVmac239 or an SIVmac239 mutant (SIVmac239‐NefAAA) with a combination of three amino acid substitutions in the flexible loop region of Nef that impair tetherin antagonism, but not CD4‐ or MHC I‐downmodulation or infectivity enhancement. Viral RNA loads in plasma were significantly lower during acute infection (weeks 1‐4 post‐infection) for animals infected with SIVmac239‐NefAAA compared to animals infected with wild‐type SIVmac239 (p = 0.0044, mixed‐effects model). Sequence analysis of the virus population in plasma confirmed that the substitutions in Nef were retained during acute infection; however, changes were observed by week 24 post‐infection that either restored the wild‐ type residue or introduced alternative residues at these positions, consistent with selective pressure on Nef to regain anti‐tetherin activity. These observations provide the most direct evidence to date that the ability to counteract restriction by tetherin is important for lentiviral replication in primates.
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306 Non‐human primate models of HIV maternal and infant immunization Genevieve Fouda1 1
Duke Human Vaccine Institute
Despite global scale‐up in antiretroviral‐based prevention of mother‐to‐child transmission services, more than 150,000 infants become infected with HIV‐1 yearly. Immune based interventions including maternal and/or infant immunization will likely be needed to eliminate pediatric HIV. Our group uses non‐human primate models to study passive and active maternal and infant immunization strategies. We have investigated immune responses elicited in breast milk and plasma of hormone‐induced lactating rhesus monkeys after vaccination with a MVA prime protein boost strategy administered either intramuscularly (IM) or intranasally (IN). While the systemic immunization induced functional IgG response in milk, the mucosal immunization induced robust IgA. Interestingly, combining mucosal and systemic immunization led to both functional IgG and IgA responses in breast milk. To determine if maternal immunization can protect infants from virus acquisition, we immunized pregnant rhesus macaques with a MVA prime/ IM+IN protein boost vaccine regimen, then orally challenged their infants starting at 6 weeks of age with a weekly oral low‐dose of the clade C tier 2 SHIV1157ipd3N4. Maternal antibodies were adequately transferred across the placenta, but were not protective as there was no significant difference in the number of infected animals, number of challenges required to achieve viremia or the peak viral load between infants born to HIV vaccinated or placebo mothers. Nevertheless, it is important to note that in general maternal antibody levels were low as only two vaccine doses were administered before delivery and that the challenge and vaccine virus strains were different. As vertical transmission occurs in the presence of maternal antibodies, it is possible that boosting of maternal autologous antibody responses by vaccination could be protective. We have also investigated if passive immunization of infant rhesus macaques with HIV envelope‐specific antibodies isolated from breast milk of HIV‐infected women can protect from a SHIV1157ipd3N4 oral challenge. Interestingly, we observed that administration of an antibody cocktail with neutralizing and ADCC activity led to partial protection with transmission of fewer transmitted/founder (T/F) virus variants compared to control mAb‐treated infants. In future studies, it will be important to determine if boosting of maternal immune responses through optimal immunization with currently available products can impact infant virus acquisition.
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307 CD8 T cells not necessarily required for control of SIV viremia in Mauritian cynomolgus macaques Matthew Sutton1, Alexis Balgeman1, Amy Ellis1, Gabrielle Barry2, Andrea Weiler2, Benjamin von Bredow1, Dane Gellerup2, David Evans1,2, Thomas Friedrich2,3, Shelby O'Connor1,2 1
Department of Pathology and Laboratory Medicine, UW‐Madison, 2Wisconsin National Primate Research Center, UW‐ Madison, 3Department of Pathobiological Sciences, UW‐Madison
There are currently HIV vaccines in development that elicit CD8 T cells targeting conserved regions of HIV, but we don't know whether these CD8 T cells can effectively control virus replication, in vivo. In this study we tested the hypothesis that acute CD8 T cell responses towards conserved regions of SIV could result in virus control. Using SIVmac239Δnef, a virus whose replication is still suppressed in animals without protective MHC alleles, we infected 18 Mauritian cynomolgus macaques (MCMs) with either wild‐type SIVmac239Δnef or a variant (SIVΔnef‐8x) containing mutations in 10 mutable CD8 T cell epitopes restricted by the non‐protective M3 MHC haplotype. We found that virus suppression in M3 homozygous MCMs infected with SIVΔnef‐8x was delayed when compared to M3 homozygous MCMs infected with SIVmac239Δnef, with 5/8 animals able to maintain SIVΔnef‐8x viremia to below 1000 copies/mL. Using IFNγ ELISPOT assays, we defined 8 newly targeted regions, but observed no substantial differences between animals that did or did not control SIVΔnef‐8x replication. We then administered the anti‐CD8β antibody 255R1 during chronic infection to determine if the existing CD8β+ T cells were responsible for virus control. Within one week, we observed a >80% reduction in peripheral CD8β+ T cells that coincided with viral recrudescence. Surprisingly, we found SIV viremia return to pre‐CD8β‐depletion levels in a majority of animals at 3 weeks post‐depletion, despite peripheral CDβ+ T cells remaining >80% depleted. While animals infected with SIVΔnef‐8x lacked broad CD8 T cell responses, this didn't shift the immune response preferentially towards antibody production, as there were no obvious differences in the antibody responses between animals infected with different viruses or that exhibited different outcomes. Together, these data suggest that the existence of a population of CD8 T cells targeting a narrowly defined population of conserved epitopes will be insufficient to control SIV/HIV replication.
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308 Association Between Adipose Tissue Macrophages and Inflammation in SIV‐Infected Rhesus Macaques Marissa Fahlberg1, Dr. Elizabeth Didier1, Dr. Marcelo Kuroda1 1
Background: Persons with HIV infection and treated with ART are developing HIV‐associated non‐AIDS conditions associated with increased circulating inflammatory cytokines such as sCD163, sCD14, and TNFa. Adipose tissue is a driver of low‐grade chronic inflammation during obesity and is comprised of immune cells including macrophages. Adipose also was reported to contribute to the HIV reservoir. We therefore hypothesized that SIV/HIV causes adipose tissue to become pro‐inflammatory, and thereby contributes to systemic inflammation in HIV‐infected persons. Methods: We analyzed historical H&E slides of white adipose tissue from 27 rhesus macaques (11 uninfected, 10 SIV+, and 6 SIV+ART) to examine inflammation. Stromal vascular cells from subcutaneous adipose were harvested from uninfected or infected rhesus macaques (SIVmac 239 or SIVmac251 for one year +/‐ ART) for characterization of cell composition by flow cytometry) and enriched for adherent macrophages to test functional cytokine secretory responses after exposure to IL4 or LPS ex vivo. Results: Among the adipose tissue, 27% of uninfected, 80% with SIV, and 50% with SIV+ART exhibited inflammation. CD163+CD206+ macrophages were the predominant immune cell type in adipose and after SIV infection (+/‐ ART) we observed an increase of CD163+CD206‐ macrophages. Adipose tissue macrophages from uninfected macaques constitutively expressed higher levels of IL‐10, IL‐6, and TNF than SIV+ and SIV+ART macaques (57%, 43%, and 22%, respectively). We also found that after LPS and IL‐4 stimulation, macrophages from SIV+ (+/‐ ART) animals downregulated expression of these cytokines significantly more than from uninfected animals. Conclusions: SIV infection was associated with inflammation in white adipose tissue of adult rhesus macaques and this corresponded with an increase in the CD163+CD206‐ population and decline in the CD163+CD206+ subset of macrophages. We also observed that SIV infection (+/‐ ART) reduced macrophage responsiveness to IL4 or LPS that may contribute to loss of inflammation regulation.
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309 Simian immunodeficiency virus infection leads to the appearance of interleukin‐18 secreting and cytotoxic natural killer‐like B‐cells in the mucosa of the colon Andrew Cogswell1, Moriah Castleman2, Stephanie Dillon2, Cara Wilson2, Ed Barker1 1
Rush University Medical Center, 2University of Colorado
Among lymphocytes that make up the mucosal adaptive immune response there are several that have T‐cell receptors but have innate‐like activity. Here we described the presence of Natural Killer‐like B‐cells (NKB) which co‐express CD20 and CD56 in the lamina propria of the colon from SIV‐infected nonhuman primates (Macaca mulatta). NKBs are absent in uninfected rhesus macaques. However, NKBs are 5‐15% of the CD20+ cells in the colon of infected rhesus macaques. Like B‐cells, NKBs appear to act as antigen‐presenting cells since NKB cells express major histocompatibility complex class II molecules as well as CD40, CD80 and CD86. Moroeover, NKBs differentially express surface Ig including the IgM, IgG, IgA and IgE isotypes. NKBs are CXCR5+, which indicates like B‐cells they are capable of migrating to lymphoid aggregates in the colon. NKBs also express CR2 (CD21), which indicates they may be capable of interacting with C3d bound to antigen. These NKB cells contain NK cell activation receptors, NKG2D, NKp46 and CD16 as well as the inhibitory receptor NKG2A/CD94 and KIR3DL1. One other important feature is NKBs have the potential to be cytotoxic given their expression of perforin and granzyme K, which is present in NK cells but absent in B‐cells. In fact, NKBs, like NK cells, lyse K562 cells while autologous colonic B cells do not. CD21+ NKBs do not possess granzyme K while granzyme K positive cells express NKG2D suggesting distinct subsets within NKBs. Even though NKBs are lytic they are distinct from NK cells and innate lymphoid cells in the colon since they are unable to express interferon‐gamma, interleukin‐22 or interleukin‐26. However, NKB cells did produce IL‐18 but not IL‐1beta. Thus, we have uncovered a unique population of lymphocytes with cytotoxic potential in the colon of SIV‐infected rhesus macaques that are pro‐inflammatory.
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310 Infusions of in vitro expanded autologous NK cells on viral loads in SIV infected rhesus macaques Dr. Siddappa Byrareddy1, Robert Russo2, Dr. Dean Lee3, Dr. Aftab Ansari4 1
University of Nebraska Medical Center, 2Emory University School of Medicine, 3Nationwide Children’s Hospital, 4Emory University School of Medicine
While cells of the innate immune system including NK cells play a role during acute SIV infection, their role during chronic SIV infection remains unclear. Our lab has previously shown that the depletion of NK cells using a JAK3 inhibitor during acute infection, led to significant increase of viral loads during chronic infection. In efforts to extend these studies, we successfully employed a protocol of in vitro expansion of CD3‐/CD8a+/NKG2a+ by co‐culture (ratio of 1: 2) with an irradiated (100 cGy) cell surface IL‐21 expressing cell line K562. The expanded NK cells appeared to predominantly consist of the non‐cytolytic cytokine synthesizing α4β7‐expressing subset of NK cells. In vivo intravenous infusion of such autologous NK cells (10‐ 50 X 10^9 cells at weekly intervals for 8 weeks) initiated at day ‐3 prior to infection with 1,000 TCID50 of SIVmac239 led to 1 to 1.5 log reduction (p< 0.01) of plasma viral loads in 6/6 rhesus macaques for up to 20 weeks post infection as compared to 6 controls that received autologous PBMC and infected with same dose of virus. In efforts to better characterize the subset of the infused cells, the expanded and non‐ expanded NK cells were subjected to gene expression analysis using nCounter platform (nanoString Technologies). The differentially expressed genes by in vitro IL‐21‐expanded rhesus macaque NK cells showed a pattern similar to that previously noted for IL‐21‐expanded human NK cells (as compared to IL‐ 15), with the exception of CTLA4 (no change) and SELL (down regulated). These data provide a platform for such innate immune therapies and suggests that the beneficial effects of such NK cell infusions could be due to their selective homing to the gut during acute infection that has the potential to decrease viral load set points.
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400 Using molecularly modified viruses to track transmission and latency Brandon Keele1 1
Frederick National Lab
We developed two distinct virus models for use in nonhuman primates to help clarify the molecular dynamics surrounding transmission and viral rebound following therapeutic release. In transmission study, 15 rhesus macaques were intravaginally challenged with SIVmac239X, a synthetic swarm of sequence tagged but otherwise isogenic SIVmac239 clones that allows for sequence‐based discrimination between ten distinct viral variants. To understand recrudescence following cART release, we generated a genetically tagged virus stock (SIVmac239M) with a 34‐base genetic barcode inserted between the vpx and vpr accessory genes of the infectious molecular clone SIVmac239. Next‐generation sequencing of the virus stock identified at least 9,336 individual barcodes, or clonotypes, with an average genetic distance of 7 bases between any two barcodes. Utilizing these two complementary virus models, we gained a greater understanding of viral transmission and reservoir establishment and maintenance. For the acute serial necropsy study, viremia was first detectable on day 6 with large variation in the time to systemic dissemination between animals infected and necropsied on the same day. The number of viral variants establishing infection was also not consistent between animals, ranging from 2 to 9 variants. Although low levels of vRNA and DNA was detected outside of the female genital tract (FGT) at early time points, accumulated viral RNA and DNA was found within the FGT prior to productive systemic dissemination. More than one viral lineage was found within each focus of infection in the FGT, suggesting independent infection of multiple variants at these sites. Following viral dissemination, next generation deep sequencing and laser capture microdissection (LCM) sequencing demonstrated that viral lineages found in FGT were also detectable in systemic compartments (GI, LN, spleen or plasma) consistent with a lack of restriction by the host to prevent viral spread from the mucosal portal of entry. A significant recruitment of CD4 +T cells and but not myeloid cells was observed at mucosal sites of initial infection as well as a significant upregulation in type 1 IFN responses (e.g. Mx1 and APOBEC), which were only augmented after detection of viral replication and the expression levels were not significantly different within infected and nearby uninfected cells. In this model of vaginal transmission with viral variants with equivalent replication capacity, we find that the mucosal barrier alone constitutes the decisive genetic barrier during transmission. However, variation in viral phenotypes during HIV‐1 transmission might allow for local replication of lineages that fail to disseminate. For the viral reservoir establishment and maintenance study, 6 animals were infected with SIVmac239M and treated with cART beginning on day 4 post‐infection for 305, 374, or 482 days. Upon treatment interruption, between 4 and 8 distinct viral clonotypes were detected in each animal at peak rebound viremia. The relative proportions of the rebounding viral clonotypes, spanning a range of 5 logs, were largely preserved over time for each animal. The viral growth rate during recrudescence and the relative abundance of each rebounding clonotype were used to estimate the average frequency of reactivation per animal. Using these parameters, reactivation frequencies were calculated and ranged from 0.33‐0.70 events per day, likely representing reactivation from long‐lived latently infected cells. The use of SIVmac239M therefore provides a powerful tool to investigate SIV latency and the frequency of viral reactivation after treatment interruption.
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401 Clonotype lineage tracing of vaccine‐induced B Cells in vivo using the BALDR computational pipeline for immunoglobulin reconstruction in single‐cell RNA‐Seq data Amit Upadhyay1, Alice Cho2, Amber Wolabaugh1, Robert Kauffman2, Gregory Tharp1, Reem Dawoud1, Nirav Patel1, F. Eun‐Hyung Lee2, Jens Wrammert2, Steven Bosinger1 1
Yerkes Nprc/emory University, 2Emory University
One of key challenges in designing an effective B cell‐based vaccine in the NHP model is that the tools to study antigenic‐specific or individual clonotypes B cells with “desirable” properties in vivo are limited, thus manipulating the B cell response via vaccination/adjuvantation is a largely an empirical endeavor. Our goal with this study was to develop a tool to dissect the biology of vaccine‐induced B cells at a mechanistic level. To achieve this goal, we developed a protocol to capture single‐cell B cells at various stages of vaccination, and simultaneously determine the Heavy and Light chain sequence and also assay the complete transcriptome using single‐cell RNA‐Seq. Here, we describe the development of a novel bioinformatic pipeline – BALDR(BCR_Assignment_of_Lineage_using_De_novo_Reconstruction) that can reconstruct full‐ length sequences of both immunoglobulin chains in individual cells. We generated Illumina single cell‐RNA‐Seq data for 282 human, plasmablasts induced by seasonal flu vaccination and 88 naïve B cells. Reconstruction was performed using de novo assembly using different filtering strategies. Accuracy of the clonotype‐assignment was validated by comparing reconstructed plasmablast sequences to those by Sanger sequencing of amplicons generated by nested PCR from an aliquot of the single‐cell RNA. In total, we generated matched NGS/PCR data for 220 cells and 396 Ig chains. When compared to PCR/Sanger, clonotype assignment accuracy was 100%. The recovery of unambiguous, IG chain reconstruction was 91%(plasmablasts) and 85%(naive B cells.) We tested BALDR on plasmablasts from macaques induced after vaccination with a Gag/Env protein‐subunit vaccine to test if lacking an annotated rhesus‐specific immunoglobulin genome reference would impair reconstruction. We found a reconstruction efficiency in plasmablasts of 89%, similar to our efficacy in human plasmablasts. In summary, our methodology enables linking of antibodies’ function to the transcriptional signatures of developing B cell lineages which will be important for dissection of vaccine induced humoral immunity.
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402 Development of a rapid and scalable method to both sequence and exogenously express paired full‐length Macaque antigen‐specific T‐cell receptors Dr. Shaheed Abdulhaqq1, Dr. Benjamin Bimber1, Dr. Scott Hansen1, Dr. Helen Wu1, Abigail Ventura1, Dr. Karin Wisskirchen2, Dr. Ulrike Protzer2, Alfred Legasse1, Dr. Michael Axthelm1, Dr. Louis Picker1, Dr. Jonah Sacha1 1
Oregon Health & Science University/Vaccine And Gene Therapy Institute, 2Technical University of Munich
Background: Antigen‐specific CD8+ T‐Cells can mediate powerful anti‐viral and anti‐cancer effects; however, research of CD8+ T‐Cells can be hindered by both the difficulty of sustaining antigen‐specific CD8+ T‐Cells in culture and the often laborious and failure‐prone process of developing peptide‐loaded MHC tetramers. We developed a high through‐put system to single‐cell sort antigen‐specific CD8+ T‐Cells without tetramers, sequence their full length paired TCR chains, and express those TCRs exogenously in naïve T cells. Methods: We stimulated PBMC from SIV Gag vaccinated or SIVmac239 infected animals with peptide minimals for previously identified T‐Cell responses in the presence of TAPI‐0. Cells stained for CD69 and membrane‐bound TNFa were sorted using a FACS aria. Whole transcriptome sequencing was done on either bulk or single cells and used to identify the CDR3 amino acid sequence and V(D)J usage. Paired TCR chains were cloned into a retroviral vector for exogenous expression in allogeneic CD8+ T‐Cells. Results: Using our system we were able to identify, sequence, and subsequently express paired TCRs from both classically and non‐classically MHC‐restricted CD8+ T cells, such as Mamu‐A*01 restricted Gag CM9‐ specific T cells and MR1‐restricted MAITs. We confirmed that allogeneic CD8+ T‐Cells expressing these exogenous TCR secreted effector cytokines upon encountering antigen in the context of their corresponding MHC and were readily stained by tetramers. Discussion: We have developed a system for quickly sequencing and cloning antigen‐specific TCR without the need of peptide‐loaded MHC tetramers. Using this system, we are currently examining the TCRs present in MHC‐E‐restricted T‐Cell responses engendered by RhCMV 68‐1 vaccination. This approach will facilitate the testing of T cell‐based therapies for prophylactic and therapeutic approaches to preventing and clearing the latent viral reservoir.
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403 Highly resolved long read, single molecule sequencing of full‐length SIV and SIV env Ms. Alesia Antoine1, Dr. Ismael Ben Farouck Fofana2, Dr. Gintaras Deikus1, Dr. Robert Sebra1, Dr. Welkin Johnson2, Dr. Melissa Laird Smith1 1
Icahn School Of Medicine At Mount Sinai, 2Boston College
The use of next generation sequencing (NGS) to examine full length SIV and SIV env variants has been limited due to length, compounded by extensive indel polymorphism, GC deficiency, and long homopolymeric regions. We developed and standardized protocols for isolation, amplification and single‐ molecule real‐time (SMRT) sequencing of both near‐full length SIV genomes and SIV env, to facilitate a variety of downstream research applications, including investigating the natural history of non‐human primate lentiviral infections and experimental animal models of SIV vaccination. Near full‐length SIV genomes (~9.6 kb) and SIV env (2.6 kb) were amplified from multiple diverse SIV strains for methods development purposes prior to application using primary samples. SMRTbell libraries were constructed using unsheared amplicons and sequenced on the PacBio RS II using P6/C4 chemistry and 240‐ minute collection. Viral sequences were analyzed using circular consensus sequencing (CCS) reads for the SIV env amplicons and using the cluster consensus algorithm (CluCon) for near‐full length genomes. Viral diversity in mixed samples was determined using a partial order alignment approach, which measures sample diversity as the mean pairwise distance among reads. This study developed standardized molecular input enrichment methods and sequencing protocols to evaluate near‐full length SIV genomes and SIV env molecules using single‐molecule, real time (SMRT) sequencing. The long, accurate reads will greatly simplify downstream bioinformatics analyses for a variety of investigations, as the single molecule nature of these data will support more accurate haplotypic phasing and detection of concurrent or compensatory mutations in the same strand. The sequencing methodology and analysis tools developed here could be successfully applied to any area for which either near full‐length SIV or SIV env analysis would be useful.
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404 Inflammatory insult prior to simian immunodeficiency virus infection increases acute phase pathogen burden Dr Adam Ericsen1, Mr Matthew Semler2, Ms Hailey Bussan2, Mr Trent Prall1, Mr Eric Peterson1, Mr Jason Weinfurter2, Dr Roger Wiseman1,2, Prof David O'Connor1,2 1
Wisconsin National Primate Research Center, 2University of Wisconsin
There is a brief window of opportunity for antiviral immune responses to intercept and contain incipient immunodeficiency virus infection. However, the parameters that influence the outcome of early host‐virus interactions have not been fully elucidated. We recently identified microbial translocation as one of the earliest pathological events to occur during simian immunodeficiency virus (SIV) infection. We also found that treating macaque elite controllers with dextran sulfate sodium (DSS), which increases gastrointestinal permeability and stimulates microbial translocation, provoked a prolonged recrudescent viremia. These data led us to hypothesize that increased inflammation and microbial translocation in early SIV infection compromises host containment of nascent virus replication. To test this hypothesis, we treated a cohort of major histocompatibility complex (MHC)‐identical cynomolgus macaques with DSS approximately 1 month prior to intrarectal inoculation with SIVmac239. Relative to controls, DSS treated animals exhibited significantly higher levels of acute phase viremia. Our data suggest that prior inflammatory insult amplifies acute phase pathogen burden‐ a potentially important consideration in the development of effective HIV prophylactics.
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405 Impact of microbiome manipulation on SHIV acquisition in rhesus macaques Dr. Jennifer A. Manuzak1,2, Dr. Tiffany Hensley‐McBain1,2, Charlene Miller1,2, Dr. Alexander S. Zevin1,2, Toni M. Gott1,2, Ernesto Coronado1,2, Ryan Cheu1,2, Andrew Gustin1,2, Dr. Elias K. Haddad3, Dr. Deborah H. Fuller1,2, Dr. Nancy L. Haigwood4,5, Dr. Nichole R. Klatt1,2 1
University of Washington, 2Washington National Primate Research Center, 3Drexel University, 4Oregon National Primate Research Center, 5Oregon Health and Science University
Background: Given the critical role of mucosal surfaces in susceptibility to HIV infection, it is imperative to induce effective mucosal responses. We have previously shown that microbiome enhancement by probiotic administration in macaques strengthens mucosal and systemic immune function. We hypothesized that probiotic administration would lower frequencies of SHIV target cells, decrease rate of SHIV acquisition and lower viral load (VL) after SHIV infection. Methods: In this ongoing study, male rhesus macaques (n=5) were given continuous oral probiotic therapy (Visbiome) for 31 weeks. Non‐probiotic treated male rhesus macaques were used as controls (n=8). Frequencies of CD4+ T cells and CCR5+ and CCR6+ CD4+ T cells were assessed in colon, rectum and lymph node (LN) throughout probiotic administration. Probiotics were discontinued 40 days prior to challenge, and all animals underwent multiple low‐dose intra‐rectal challenges with SHIV.C.CH505.375H.dCT. Challenge was halted after SHIV RNA was detected in plasma and VLs were monitored for 16 weeks post‐infection. Results: Similar pre‐infection frequencies of CD3+CD4+ T cells, and CCR5+ and CCR6+ CD4+ T‐cells, were found in the colon, rectum and LN of probiotic treated and untreated animals. During SHIV challenge, there was no difference in SHIV acquisition (p=0.6239; Log‐rank test) and no difference in peak VL (p=0.52; Mann‐ Whitney test) or VL kinetics post‐infection between probiotic and untreated animals. Conclusions: Our initial investigations indicate that probiotic administration does not impact mucosal and lymphoid SHIV target cell frequencies and has no impact on SHIV infection, peak VL or viral kinetics post‐ infection. Enrollment of additional animals is underway, which will increase the power of our study. Furthermore, we are assessing the ability of probiotics to enhance SIV/HIV vaccine immunogenicity and efficacy. Our work will provide a comprehensive analysis of probiotic therapy as one method by which to enhance immune defenses and vaccine efficacy at the mucosal portal of entry.
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406 Whole genome sequence data for macaques: Implications for AIDS research Jeffrey Rogers1 1
Baylor College Of Medicine
The response to infection by pathogens can vary significantly among people and among individuals of the nonhuman primate species we use in research. Genetic differences among hosts play an important but poorly understood role in that variability. In my laboratory, we have analyzed whole genome sequences from 530 rhesus macaques (Macaca mulatta), in order to develop a detailed and extensive catalog of genetic variation in this species. We previously analyzed and reported >43 million single nucleotide variants (SNVs) among rhesus macaques, but this is only one component of the functionally significant genetic variation segregating in research colonies. We have now mapped Illumina short read data from 530 rhesus to the current reference genome assembly, and identified insertion/deletion variants using GATK. A subset of 285 animals were also analyzed using SCALPEL. Among the 530 animals, we identified >7 million indel variants, affecting tens of millions of bases. More than 60% of indels are three bases in length or less. Thousands of these variants are predicted by Ensembl VEP to influence gene function. In addition, we used the unmapped reads from these macaques (reads that could not be mapped with high confidence to the reference genome) to identify 364 de novo insertions ‐ sequences found in at least 5 rhesus macaques that are not present in the reference genome. Like the results for SNVs, the average rhesus macaque carries more than twice as many indel variants as the average human. This presentation will describe our findings concerning indels and other complex types of genomic variation among rhesus that may influence gene expression, and hence may eventually help explain individual variation in response to infectious disease. Analyses of genetic variation among research macaques (or any other primate) should not be restricted to SNVs alone.
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407 Whole‐transcriptome sequencing to identify immune gene variants Amelia K. Haj1, Julie A. Karl1, Roger W. Wiseman1, David H. O'Connor1 1
The rhesus MHC class I allele Mamu‐B*08 is associated with control of viral replication, but is likely only one of many genetic modifiers of susceptibility to HIV. Other immune genes, including killer cell immunoglobulin‐like receptor and Fc gamma receptor genes, are also likely to be important in determining the rate of HIV disease progression. Much of the current work in macaque genomics employs whole‐exome or whole‐genome sequencing; however, these approaches rely on flawed reference genomes and annotations for variant calling, and perform especially poorly in complex immune genes. Long‐read RNA sequencing allows for identification of full‐length transcript isoforms and phased SNPs from multiple immune gene families simultaneously, without a reference genome. Here we describe whole‐transcriptome sequencing of peripheral blood mononuclear cells from one rhesus and one cynomolgus macaque using Pacific Bioscience’s long‐read RNA sequencing (Iso‐Seq) technology performed on a PacBio Sequel machine. We obtained over 800,000 consensus reads per animal, with an average read length of approximately 3000 bases. Using data produced from the PacBio Iso‐Seq analysis pipeline (version 4.0.0), we identified reads aligning to expected MHC class I alleles as well as to specific KIR alleles and Fc gamma receptor isoform sequences. We anticipate that this technology will be useful in characterizing cell type‐specific expression of immune gene isoforms in macaques during the course of SIV infection.
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408 Characterization of major histocompatibility complex sequences in baboons Hailey E. Bussan1, Joe H. Simmons3, Roger W. Wiseman1,2, Julie A. Karl1, Cecilia G. Shortreed1, Michael E. Graham1, David O'Connor1,2 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 3MD Anderson Cancer Center, University of Texas
Background: Variation in the major histocompatibility complex (MHC) is known to be responsible for control of infectious agents such as SIV and transplant rejection. When using non‐human primates as models, excluding certain MHC alleles or matching MHC genotypes may be critical for infectious disease challenges, vaccine assessments, and transplantation studies. Although olive baboons (Papio anubis, Paan) are used in various types of biomedical research, their MHC diversity remains poorly characterized. In this pilot study, we used PacBio deep sequencing to define MHC class I alleles in blood samples from twelve baboons at the MD Anderson Keeling Center. Materials and Methods: We amplified full‐length MHC class I transcripts from cDNA using previously validated primers that bind conserved sequences in the 5’ and 3’ UTRs. The resulting 1.1 kb amplicons were pooled and characterized by circular consensus sequencing of a PacBio SMRTbell library. Results: Using an OTU clustering pipeline, we identified 44 novel full‐length class I transcripts. We were able to infer fifteen putative Paan‐A and thirteen putative Paan‐B haplotypes. Of these twelve animals, six expressed Paan‐A*02:02 and five expressed Paan‐B*02:01. Conclusions: Currently, we are extending these analyses to more than 100 additional animals in order to better characterize the MHC allelic diversity within this baboon population and to define MHC‐I haplotypes using parentage analyses. This general allele discovery method can be extended to other non‐human primate species such as African green monkeys and sooty mangabeys, as well as to other complex immune gene families such as KIR and FCGR loci.
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409 Comparison of the host response to RhCMV/SIV vaccine vectors between protected and non‐protected Rhesus Macaques Dr. Courtney Wilkins1, Rich Green1, Dr. Connor Driscoll1, Jean Chang1, Elise Smith1, Dr. Lynn Law1, Dr. Scott Hansen2, Dr. Lewis Picker2, Dr. Michael Gale, Jr.1 1
Department of Immunology, Center for Innate Immunity and Immune Disease, University Of Washington, 2Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health & Science University
Background: The simian immunodeficiency virus (SIV)‐targeted vaccine vectors based on rhesus cytomegalovirus (RhCMV) strain 68‐1 elicit cellular immune responses that are able to stringently control and ultimately clear a highly pathogenic SIV challenge in slightly over half of vaccinated rhesus macaques (RMs). Interrogation of the transcriptomic host response is an important avenue in understanding the immune mechanisms responsible for this protection. Methods: Two groups of RMs were vaccinated with the 68‐1 vaccine vectors, using either subcutaneous or oral delivery. Following vaccination, animals were subjected to repeated limiting dose intrarectal SIVmac239 challenge at week 88 until infected by either detection of plasma virus or de novo development of T cell responses to SIV‐Vif with the following outcome: 53% or 60% of the animals in the subcutaneous and oral groups, respectively, manifested stringent aviremic control of the virus. mRNA‐seq analysis, transcriptional profiling and bioinformatics assessments were performed on blood samples obtained during the vaccination phase to compare the transcriptional profiles between the protected and non‐protected animal groups. These analyses included principal component analysis, differential expression, and both functional and correlation analyses. Results: We have identified distinct gene signatures between the protected and non‐protected animal groups. These included differences in the magnitude and directionality of differentially expressed genes involved in several innate immune networks, including significant differences in genes involved in innate immune activation, inflammation, and immune programming. Conclusions: The knowledge of differences in the transcriptional host response between the protected vs. non‐protected animals will guide efforts to 1) understand the mechanisms responsible for the unique “control and clear” efficacy manifested by RhCMV vectors, 2) define gene correlates and intracellular response pathways of protection, 3) develop a modified vaccine that further enhances these features to achieve efficacy beyond the current ~55%, and 4) translate these vectors from nonhuman primates to people.
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410 Adaptation of SIV to baboon PBMC or isolated CD4 cells: insights into cell types required for baboon resistance to SIV infection Veronica Obregon‐Perko1,2, Laura Parodi2, Vida Hodara2,3, Jason T Ladner4, Michael R Wiley4, Gustavo F Palacios4, Luis D Giavedoni2,3 1
Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Health Science Center, Department of Virology and Immunology, Texas Biomedical Research Institute, 3Southwest National Primate Research Center, Texas Biomedical Research Institute, 4Center for Genome Sciences, United States Army Medical Research Institute of Infectious Diseases
Baboons do not naturally harbor SIVs, despite having overlapping habitats with natural SIV hosts. Previous work has demonstrated baboons are resistant to chronic SIV infection in vivo. The mechanisms underlying resistance remain unknown but their elucidation could provide new targets for antiviral intervention. We sought to identify the major stages of SIV restriction in baboons, using the pathogenic rhesus macaque model for comparison. SIV growth was restricted in baboon PBMC compared to rhesus PBMC; however, SIV replication in isolated CD4 cells was similar between species. Viral loads were reduced in baboon CD4 cells co‐cultured with baboon CD8 T and NK cells. We observed that baboon PBMC produced higher levels of MIP‐1α, MIP‐1β, and RANTES than rhesus PBMC. These chemokines suppress HIV and SIV infection through competitive binding for CCR5. Although these chemokines were produced in baboon CD4 cultures, levels were highest in cultures containing CD8 T and/or NK cells. Inhibition of CCR5‐binding chemokines in baboon PBMC increased viral loads. We generated baboon‐adapted SIVs (SIVbn) by serial passage in baboon PBMC or isolated CD4 cells and analyzed changes to the genome by deep sequencing. In PBMC‐passaged SIVbn, many non‐synonymous substitutions reached 100% frequency by the second passage. By contrast, non‐synonymous substitutions in CD4‐passaged SIVbn occurred gradually and few became fixed by the final passage. Viral titers were also consistent with a more severe population bottleneck during the earliest passages in PBMC compared to CD4 cells. These observations suggest that SIV faces stronger selective pressure in baboon PBMC, a mixed‐cell environment, than in isolated CD4 cells. Indeed, substitutions that occurred during CD4 passage did not increase replication efficiency in baboon PBMC compared to PBMC‐passaged SIVbn or SIVmac. Taken together, we propose that baboon SIV resistance is not mediated by intracellular restriction factors, but by CCR5‐binding chemokines and other mechanisms of cell‐mediated immunity.
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411 Research in NHP on the road to the end of AIDS Dr. Bonnie Mathieson1 1
National Institutes of Health
This talk will cover what I have learned over the years about the general origins of the primate centers in the 1970’s; the discovery of the Simian AIDS models at 3 centers from cross‐species infections and their early applications to AIDS research in the 1980’s; the concerns about retrovirus and herpes B co‐infections that drove the development of the SPF colonies the 1990s; the critical shortage of animals for research that developed as colonies were downsized after the need for live attenuated polio virus vaccine testing decreased and the rapid and emergent need for research animals for AIDS vaccines, microbicides, and other pathogenesis research accelerated in the late 1990s through 2002; the awareness of the need for genetic analyses of the colonies and experimental animals that came with variable results that were seen in vaccine experiments that were paralleling the results in human cohorts; and the complexity that was being observed when investigators improved tools for analyses and sorted out different results. During the presentation, I will attempt to identify some of the unsung heroes that made AIDS research possible at the NPRCs.
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500 Jumping from HIV into Zika: Research into high gear Dr. Koen Van Rompay1 1
California National Primate Research Center, University of California, Davis
Since 2015, Zika virus (ZIKV) has been spreading rapidly over Latin America and other countries. While infection of adults is usually non‐symptomatic, infection during pregnancy can lead to fetal microcephaly and other neurologic defects. Since 2016, a number of research teams have been developing nonhuman primate models of ZIKV that mimic the various routes of transmission, to gain insights in the kinetics of virus replication (including tissue distribution and persistence), immunological responses, and the efficacy of vaccine candidates. Collaborative studies performed at the California National Primate Research Center have focused on developing models that mimic ZIKV infection during pregnancy. A first pilot study, which was initiated when the frequency of natural transplacental transmission was unknown, was geared toward developing a model of fetal neuropathogenesis. To reliably induce fetal infection at defined times, four pregnant rhesus macaques were inoculated intravenously and intraamniotically with a Brazilian ZIKV isolate during the first and second trimester. One animal inoculated at gestational day 41 experienced fetal death 7 days later, with high virus levels in fetal and placental tissues and fluids, implicating ZIKV as cause of death. The other 3 fetuses (inoculated on gestational days 50, 64 or 90) were carried to near term and euthanized. While none exhibited gross microcephaly, all three showed persistent ZIKV RNA in various tissues, especially in the brain, which contrasted with the lymphoid tissue tropism observed in their mothers at time of euthanasia. The fetal and neonatal brains also exhibited calcifications and altered neural precursor cells. As this rhesus macaque model produces neurologic defects similar to human congenital Zika syndrome, it is highly relevant to unravel fetal neuropathogenesis and explore interventions.
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501 A new HIV/HBV co‐infection model: hepatocytic expression of human sodium taurocholate cotransporting polypeptide (NTCP) Enables Hepatitis B Virus Infection of Macaques Dr. Benjamin Burwitz1,4, Mr. Jochen Wettengel2, Dr. Martin Muck‐Hausl2, Mrs. Katherine Hammond1, Dr. Marc Ringelhan2,3, Dr. Chunkyu Ko2, Mr. Jason Reed1, Mr. Reed Norris4, Dr. Byung Park5, Dr. Sven Moller‐Tank6, Dr. Knud Esser2, Dr. Justin Greene1, Dr. Helen Wu1, Dr. Shaheed Abdulhaqq1, Dr. Gabriela Webb1, Mr. William Sutton4, Mr. Alex Klug4, Ms. Tonya Swanson4, Mr. Alfred Legasse4, Dr. Aravind Asokan6, Dr. Nancy Haigwood4, Prof. Ulrike Protzer2,7, Dr. Jonah Sacha1,4 1
Vaccine & Gene Therapy Institute, Oregon Health & Science University, 2Institute of Virology, Technical University of Munich, Helmholtz Zentrum München, 3Department of Internal Medicine II, Technical University of Munich, 4Oregon National Primate Research Center, Oregon Health & Science University, 5Public Health & Preventative Medicine, Oregon Health & Science University, 6Gene Therapy Center, The University of North Carolina at Chapel Hill, 7German Center for Infection Research, Munich partner site
Background: HIV/HBV co‐infected patients exhibit increased liver dysfunction and fibrosis, but the mechanisms contributing to this differential pathology remain poorly understood, and an animal model of HIV/HBV co‐infection is needed. Rhesus macaques are regularly utilized as models for HIV infection, but no endogenous HBV strain has been discovered in this species. We hypothesized that the block to HBV replication in rhesus hepatocytes is due to failure to gain cell entry. To test this hypothesis, we expressed the HBV receptor ‐‐ human NTCP (hNTCP) ‐‐ on rhesus hepatocytes both in vitro and in vivo. We show for the first time that macaques expressing hNTCP can be infected with HBV, paving the way for a physiologically relevant animal model of HIV/HBV co‐infection. Methods: We generated helper‐dependent adenovirus (HDAd serotype 5) and adeno‐associated virus (AAV serotype 8) vectors expressing hNTCP and transduced freshly isolated rhesus primary hepatocytes (PH) in vitro. We then challenged these PH with HBV and monitored in vitro infections by supernatant HBsAg/HBeAg ELISA, HBV DNA qPCR, and electron microscopy. For in vivo studies, macaques were injected with HDAd and AAV vectors expressing hNTCP and challenged with HBV. Results: Rhesus PH expressing hNTCP supported HBV infection, evidenced by increasing supernatant concentrations of both HBsAg and HBeAg. In addition, covalently‐closed circular DNA (cccDNA) qPCR and electron microscopy indicated that all stages of HBV replication are present in rhesus hepatocytes. Expression of hNTCP in vivo facilitated HBV infection, evidenced by detection of serum HBV DNA, HBV DNA/RNA in isolated hepatocytes, anti‐HBV humoral and cellular immunity, and HBcAg expression in the liver. Discussion: We have built a physiologically relevant HBV infection model using rhesus macaques. Given the long history of HIV research in this species, our new data portend the future use of rhesus macaques in HIV/HBV co‐infection research.
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502 Pre‐existing SIV infection increases susceptibility of Mauritian Cynomolgus Macaques to M. tuberculosis Mark Rodgers1, Cassaundra Updike1, Dr. Amy Ellis3, Alexis Balgeman3, Pauline Maiello1, Dr. Tom Friedrich4, Gabrielle Barry4, Dr. Joshua Mattila2, Dr. Shelby O'Connor3, Dr. Charles A. Scanga1 1
University of Pittsburgh School of Medicine, 2University of Pittsburgh Graduate School of Public Health, 3University of Wisconsin‐Madison, 4Wisconsin National Primate Research Center
Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death among HIV positive patients. The precise mechanisms by which HIV impairs host resistance to a subsequent Mtb infection are unknown. We modeled this co‐infection in Mauritian cynomolgus macaques (MCM) using SIV as an HIV surrogate. We infected 7 MCM with SIVmac239 intrarectally and 6 months later co‐infected them via bronchoscope with a low dose (~10 CFU) of Mtb. Another 8 MCM were infected with Mtb alone. TB progression was monitored by clinical parameters, by culturing bacilli in gastric and bronchoalveolar lavages, and by serial 18F‐FDG PET/CT imaging. MCM infected with Mtb alone displayed dichotomous susceptibility to TB, with 4 animals reaching humane endpoint within 13 weeks and 4 animals surviving >19 weeks post Mtb infection. In stark contrast, all 7 SIV‐infected animals exhibited rapidly progressive TB following co‐infection and all reached humane endpoint after just 13 weeks. Serial PET/CT imaging confirmed dichotomous outcomes in MCM infected with Mtb alone and marked susceptibility to TB in all SIV‐infected MCM. Notably, imaging revealed a significant increase in TB granulomas between 4 and 8 weeks post Mtb infection in SIV co‐infected, but not in SIV‐naïve, MCM and implies that SIV impairs the ability of animals to contain Mtb. At necropsy, animals with pre‐existing SIV infection had more extrapulmonary TB disease, more overall pathology, and increased bacterial loads than animals infected with Mtb alone. Immunohistochemistry combined with in situ hybridization identified SIV replication in areas of TB pathology. Immune responses are being compared between the two groups to determine the immunologic mechanism by which pre‐existing SIV impairs control of a subsequent Mtb infection. We thus developed a tractable MCM model in which to study SIV‐Mtb co‐infection and demonstrate that pre‐ existing SIV dramatically diminishes the ability to control Mtb co‐infection.
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503 Mtb/SIV Co‐Infection induces differential T cell responses in Rhesus Macaques Miss Allison N. Bucsan1,2, Dr. Taylor W. Foreman1,2, Dr. Shabaana A. Khader3, Dr. Jyothi Rengarajan4, Dr. James A. Hoxie5, Dr. Andrew A. Lackner1, Dr. Deepak Kaushal1,2 1
Tulane National Primate Research Center, 2Tulane University, 3Washington University, 4Emory University School of Medicine, 5UPenn Center for AIDS Research, University of Pennsylvania
Tuberculosis (TB) is a widespread infectious disease, causing approximately 10.4 million active cases and 1.8 million deaths in 2015. Previously our group has characterized changes to the T cell compartment in latent tuberculosis infection (LTBI) as compared to active TB disease (ATB). We have also described immune responses in Mtb/SIV co‐infected macaques with documented reactivation as well as its absence, likely due to CD4+ T cell independent responses. As HIV significantly increases rates of reactivation of latent TB infection (LTBI) to active disease in humans, we sought to explore the effect of co‐infection in rhesus macaques in greater detail by employing several variations of the LTBI model. One set of animals was co‐ infected with SIVΔGY in a manner comparable to SIVmac239. SIVΔGY results in nonpathogenic infection in macaques, and we are studying if co‐infection with this virus spares lung CD4+ T cell depletion and/or B‐cell follicle disruption. Macaques co‐infected with SIVΔGY did not experience depletion in the CD4+ T cell compartment, contrary to what has been seen with SIVmac239 infection. We have also developed a model of LTBI treatment in NHPs. Rhesus macaques exposed to very low levels of Mtb that develop asymptomatic disease are treated with a weekly oral regimen of LTBI treatment and SIV co‐infection used as a tool to study bacterial killing. We show that in the absence of LTBI antibiotic therapy, Mtb is able to persist in macaque granulomas during long‐term asymptomatic LTBI. We will also investigate the effect of HAART treatment on TB/SIV immune control in Indian rhesus macaques and determine if there is recovery of the CD4+ T cell repertoire and improved immunological function following treatment. By observing changes in CD4+ T cell dependent and independent immune control, it may be possible to develop better treatment strategies for patients suffering from TB/AIDS.
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504 Development of a non‐human primate model for rectal syphilis Dr. Ajay Sundaram Vishwanathan1, Dr. Cassandra Tansey1, Ms. Chunxia Zhao1, Mr. Andre Hopkins1, Dr. Yetunde Fakile1, Ms. Tamanna Ahmed1, Dr. Allan Pillay1, Dr. Samantha Katz1, Dr. Ellen Kersh1, Mr. James Mitchell1, Dr. Janet McNicholl1 1
CDC (Centers For Disease Control & Prevention)
Background: Men who have sex with men are at a higher risk for acquiring HIV if diagnosed with syphilis or other rectal sexual transmitted infections (STIs). This is of concern considering the large increase in reported syphilis cases from 2014 to 2015 in the United States. The effect of rectal STIs like syphilis on biomedical interventions for HIV including pre‐exposure prophylaxis (PrEP) is not clear. A non‐human primate (NHP) model of rectal syphilis would therefore be invaluable to study interventions for syphilis as well as PrEP efficacy against simian/human immunodeficiency virus (SHIV). Methods: We administered Treponema pallidum submucosally (Nichols strain; up to 4 injections sites; 200‐500µL volume; up to 10^8 live organisms/mL) in the rectum of n=2 SHIV‐infected rhesus macaques with a 29‐ gauge needle ~5cm beyond the anorectal opening. Using an image contrast medium, we documented the injection sites and dispersal of administered contents. Rectal lesions were monitored by endoscopy. We assessed T. pallidum presence in the rectum and blood by PCR and/or dark field microscopy. Antibodies in the serum were evaluated by treponemal tests (Treponema pallidum particle agglutination assay, TP‐PA; Syphilis Health Check™; Trep‐Sure™). Results: Rectal lesions were first observed as early as day 3 post‐challenge in 1 animal, and day 16 in another. The serum tested TP‐PA positive between weeks 5 and 6 post‐challenge for both animals with a peak titer of 1:20,480 by week 14 before reaching a plateau. Syphilis Health Check™ and Trep‐Sure™ assays detected antibodies as early as week 5. T. pallidum DNA was detected in blood at week 11 for one animal, indicating active syphilis infection. Conclusions: We have developed the first NHP model for rectal syphilis that reproduces mucosal lesions, systemic dissemination, and seroconversion. The model is being refined for use in the context of other STI co‐ infections and PrEP efficacy studies.
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505 Alternative NK cell signaling mechanisms in CMV and SIV infection Spandan Shah1,2, Cordelia Manickam1,2, Daniel Ram1,2, R. Keith Reeves1,2 1
Beth Israel Deaconess Medical Center, 2Harvard Medical School
Background: Natural killer (NK) cell activation via CD16 initiates a cascade of tyrosine phosphorylation events led by adaptor molecules FceRIγ and Syk and culminating in degranulation and/or cytokine production. However, burgeoning evidence indicates dysregulation or downregulation of these adaptor molecules following CMV or SIV infection. Unfortunately, the full NK signaling mechanisms and the possibility of alternative pathways during infection has not been examined carefully. Methods: A total of sixty rhesus macaques were used in this study: twenty‐one specific pathogen‐free (SPF) rhCMV–; 10 rhCMV+ but otherwise experimentally naïve; and 22 chronically SIVmac‐infected macaques. Viremia and antibody titers were monitored using standard assays. Activated (cross‐linked anti‐CD16) or control cells were analyzed by 18‐color polychromatic and phospho‐ flow cytometry. Results: The γ‐chain adaptor molecule, Syk, was significantly reduced in NK cells during infection or was in an inactive phosphorylated state. Alternatively infection correlated with a significant increase in total levels of CD3ζ‐chain in NK cells, suggesting these cells may exploit the ζ‐chain/Zap70 pathway in the absence of γ‐ chain/Syk to achieve greater functional potency. Phosflow analysis of activated cells showed a significant increase in phosphorylation of CD3ζ in NK cells of CMV infected animals. Surprisingly, SIV NK cells did not seem to utilize the same pathway despite increased total levels of CD3ζ. Conclusion: Collectively, our work presents the first description of alternative signaling cascade to explain the functional potency of NK cells against CMV and SIV in rhesus macaques. Inability of NK cells to activate CD3ζ during SIV infection could be indicative of immune evasion or other pathogenesis induced by SIV. Future studies targeted at harnessing these pathways could open up new modalities for both vaccine and curative therapy approaches.
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506 Nonhuman primate models for Zika virus infection Dr. Dawn Dudley1, Christina M. Newman1, Emma L. Mohr2, Matthew T. Aliota3, Sydney M. Nguyen4, Kathleen M. Antony4, Sarah Kohn5, Heather A. Simmons6, Andrea M. Weiler6, Matthew R. Semler1, Mariel S. Mohns1, Meghan E. Breitbach1, Laurel M. Stewart1, Michelle Koenig1, Bryce Wolfe1, M. Shahriar Salamat1, Leandro B. C. Teixeira7, Xiankun Zeng8, Gregory J. Wiepz9, Troy H. Thoong6, Gabrielle L. Barry6, Kim L. Weisgrau6, Logan J. Vosler6, Mustafa N. Rasheed1, Michael E. Graham1, Lindsey Block1, Jennifer Post6, Jennifer M. Hayes6, Nancy Schultz‐Darken6, Michele L. Schotzko6, Josh A. Eudailey10, Jens Kuhn6, Sallie R. Permar10, Eva G. Rakasz6, Saverio Capuano III6, Alice F. Tarantal11, Jorge E. Osorio8, Shelby L. O'Connor1, Thomas C. Friedrich6,3, Thaddeus G. Golos4,6,9, David H. O'Connor1,6 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Department of Pediatrics, University of Wisconsin‐Madison, 3Department of Pathobiological Sciences, University of Wisconsin‐Madison, 4 Department of Obstetrics and Gynecology, University of Wisconsin‐Madison, 5Department of Radiology, University of Wisconsin‐Madison, 6Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 7School of Veterinary Medicine, University of Wisconsin‐Madison, 8Integrated Research Facility at Fort Detrick, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 9Department of Comparative Biosciences, University of Wisconsin‐Madison, 10Department of Pediatrics and Human Vaccine Institute, Duke University Medical Center, 11Departments of Pediatrics and Cell Biology and Human Anatomy, University of California‐Davis, California National Primate Research Center
The impact of Zika virus infection in utero on a newborn caught the world by surprise. This seemingly harmless and relatively unknown virus suddenly caused severe birth defects and unlike most arboviruses could even be sexually transmitted. We were able to use our extensive knowledge of rhesus macaques as an infectious disease model gained by years of SIV research to quickly set up a robust macaque model to study Zika virus. We quickly put together a team of scientists with expertise in obstetrics and arbovirology to compliment our own expertise in nonhuman primate immunology. We next tested route, dose and strain of virus pertinent to the current epidemics to find an infection model that resulted in similar outcomes in monkeys to what was found in human infections in both non‐pregnant and pregnant rhesus macaques while developing a number of tools required to study this virus. We began sharing our data in real time and informing other scientists setting up similar models about what we learned to perpetuate the collective knowledge about Zika virus and move the field faster in the face of an emerging epidemic. Data will be presented showing what we’ve learned about Zika virus infection in pregnant macaques, alternative routes of Zika virus transmission, and serial infections with dengue virus and Zika virus. The approach we used to develop this model out of the SIV/rhesus macaque model could be easily adapted to study other new and/or emerging infectious diseases.
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507 Infection dynamics and persistence of Zika virus in new and old world non‐human primates Dr Neil Berry1, Dr Deborah Ferguson1, Mrs Claire Ham1, Ms Jo Hall1, Mr Adrian Jenkins1, Mrs Elaine Giles1, Dr Nicola Rose1, Dr Roger Hewson2, Dr Stuart Dowell2, Dr Sarah Kempster1, Dr Neil Almond1 1
NIBSC, 2PHE Porton
Zika virus (ZKV) represents an emergent global pathogen, although the full pathogenesis spectrum in susceptible hosts is not fully understood. To further our understanding of its host range and infection dynamics we undertook a series of studies in different primate species, comparing both New and Old World monkeys. Old World Indian rhesus macaques (Macaca mulatta) and Mauritian cynomolgus macaques (Macaca fascicularis) and New World red‐bellied tamarins (Saquinus labiatus) were inoculated sub‐ cutaneously with the South American/Peurto Rican PRVABC59 strain. Virus kinetics and tissue bio‐ distribution were compared using qRT‐PCR and RNAScope in‐situ techniques in separate time‐course studies with animals euthanased at 3, 42 and 100 days p.i. Productive infection was established in all cases, although notably the highest levels of primary viraemia were in red‐bellied tamarins (~10E6 ZKV RNA copies/ml) typically peaking 2‐4 days p.i. A diverse range of tissue types were recovered post‐mortem, with evidence of widely disseminated ZKV infection in each species determined using complementary qRT‐PCR and RNAScope technologies providing comparative analyses of peripheral and central anatomical sites. Virus was widely disseminated 3 days p.i., present in multiple tissues especially brain, reproductive tissues and lymphoid organs. Our data, extending into two genetically distinct macaque species, furthers comparative understanding of ZKV in cynomolgus macaques as an alternative ZKV model and revealed novel findings of ZKV in New World species. Importantly, we identified widespread persistence of ZKV RNA in distal sites of skin as late as 100 days p.i., particularly around inoculation sites and persisting ZKV RNA in critical anatomical sites of reproductive tissues and brain, in each species. These findings inform the comparative long‐term sequealae of ZKV infection in multiple primate species, demonstrating a more persisting infection than hitherto recognised with implications for New World sylvatic transmission and potential maintenance of reservoirs of ZKV in primate populations in Zika‐endemic regions.
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508 Impact of rhesus cytomegalovirus exposure on host intestinal gene expression Nicole Narayan1,2, Gema Méndez‐Lagares1,2, Kawthar Machmach1,2, David Merriam1,2, Connie Chen1,2, Amir Ardeshir2, W L William Chang3, Peter A Barry3, Dennis J Hartigan‐O’Connor1,2 1
Department of Medical Microbiology & Immunology, University of California, Davis , 2California National Primate Research Center, University of California, Davis, 3Center for Comparative Medicine, University of California, Davis
Environmental factors such as the chronic viral infections have a profound impact on the immune system, especially in infancy. Cytomegalovirus (CMV), for example, is a common usually asymptomatic infection that causes significant changes in the immune system of the immunocompetent host. In addition, rhesus cytomegalovirus (RhCMV) is under consideration as a vaccine vector for immunization against HIV. For this reason, it is important to understand the potential effects of RhCMV exposure on the immune homeostasis of gut tissue, which is one of HIV’s portals of entry. We performed gut biopsies in previously RhCMV‐ negative and RhCMV‐positive infant rhesus macaques before and after vaccination with a RhCMV/SIV recombinant virus. We performed RNA sequencing to assess differential gene expression in gut based on (i) wild‐type RhCMV serostatus or (ii) RhCMV/SIV vaccination. Interestingly, many transcripts were differentially expressed post‐vaccination, including those involved in immune cell pathways, demonstrating a clear change in immune homeostasis. The vaccine responses were remarkably different depending on serostatus of RhCMV. RhCMV‐negative animals manifested greater upregulation in immune response‐ related pathways, including T cell activation and proliferation, while RhCMV‐positive animals had upregulation in gene ontology pathways related to endothelial cell migration and motility. This work adds to the wealth of evidence that environmental factors such as chronic viral infections have a significant impact on the host; in particular, our data suggest an impact of RhCMV‐vectored vaccines on host gut physiology.
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509 Impact of antiretroviral drug therapy on mucosal inflammatory and regulatory responses in SIV infected macaques Megan O'Connor1,2, Paul Munson1,2, Hillary Tunggal1,2, Nika Hajari1,2, Debra Bratt1,2, Drew May2, Solomon Wangari2, Brian Agricola2, Jeremey Smedley2, Deborah Fuller1,2 1
Department of Microbiology, University of Washington, 2Washington National Primate Research Center
Background: The use of a therapeutic HIV/SIV vaccine that improves immune responses in the blood and mucosa has the significant potential to control viral replication in the absence of ART. We previously demonstrated that therapeutic DNA vaccination increased mucosal SIV‐specific T cell responses and potently reduced viral replication in SIV infected macaques following ART withdrawal. The balance between T helper 17 (Th17) and T regulatory (Treg) cells (Th17/Treg ratio) is important in HIV/SIV infection. Higher Th17/Treg ratios in PBMCs are correlated with improved viral‐specific immune responses and better viral control, however the relationship between these two cell types in the mucosa following therapeutic vaccination has not been explored. Increasing mucosal Th17 and decreasing Treg responses (increasing the Th17/Treg ratio), could contribute to improving therapeutic vaccine efficacy by decreasing inflammation and enhancing protective T‐cell responses. Here, we investigated the hypothesis that macaques with higher mucosal Th17/Treg ratios will respond better to ART and therapeutic vaccination. Material & Methods: Rhesus macaques (N=19) were infected intravenously with SIVΔB670 and put on ART 6 weeks post infection (wpi). Mucosal CD4 T‐cell subsets were assessed by intracellular cytokine staining in mesenteric lymph nodes and colonic biopsies Results: Higher acute SIV plasma viral loads positively correlate with higher mucosal Th17/Treg ratios (p=0.0182). At 6wpi infection, Th17 cells are significantly depleted (p=0.0054) and Tregs are significantly increased (p=0.0032) resulting in a significant decrease in the Th17/Treg ratio (p<0.0001) in the colon. Additionally, higher Th17/Treg ratios in the colon at the time of ART initiation associate with increased responsiveness to ART. Conclusions: Mucosal Th17 and Treg cells correlate with peripheral SIV viremia and responsiveness to ART. These results suggest that the Th17/Treg ratio may provide an important indicator of SIV/HIV disease progression, and furthermore may be a factor contributing to the responsiveness to therapeutic interventions.
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510 Evaluation of a combination antiretroviral therapy regimen containing long‐acting formulations of the integrase inhibitor CAB‐LA and the protease inhibitor GSK385‐mLAP Dr. Gregory Del Prete1, Dr. Adrienne Swanstrom1, Dr. Claire Deleage1, Dr. Jacob Estes1, Dr. Brandon Keele1, Dr. Jerry Jeffrey2, Dr. Jeffrey Lifson1 1 2
AIDS and Cancer Virus Program, Frederick National Laboratory for Cancer Research, Leidos Biomedical Research, Inc., GlaxoSmithKline Research & Development, Infectious Diseases Therapy Area Unit
The ability of long‐acting antiretroviral drug formulations to maintain therapeutic drug concentrations with less frequent dosing may be useful for treatment of compliance challenged HIV‐infected populations, for pre‐exposure prophylaxis, and for more feasible drug administration to non‐human primates used for AIDS research. In chronically SIV‐infected rhesus macaques, we evaluated a combination antiretroviral therapy (cART) regimen comprising two long‐acting inhibitors (the integrase inhibitor CAB‐LA [cabotegravir long acting] administered once every three weeks, and the protease inhibitor GSK385‐mLAP [brecanavir] administered once per week, both via intramuscular injection) and two nucleos(t)ide reverse transcriptase inhibitors (NRTIs; tenofovir [TFV]/tenofovir disoproxil fumarate [TDF] and lamivudine [3TC], both administered once daily via subcutaneous injection). Despite suboptimal NRTI plasma concentrations and pretreatment plasma viral loads >10^6 viral RNA (vRNA) copies/ml in several animals, viral loads in six of seven treated animals were suppressed by this cART regimen to <15 vRNA copies/ml after >40 weeks of treatment. One animal was euthanized at 80 weeks of treatment with acute renal failure, which has been previously observed in a subset of rhesus macaques following prolonged TFV administration. After 90 weeks of treatment, cART was withdrawn from the remaining six animals. Although GSK385 plasma concentrations were stably maintained for >14 weeks post‐cART withdrawal, five of six animals rebounded within 10 weeks of cART release with putative protease inhibitor resistance mutations identified in the recrudescent virus of only two animals. In all seven animals, extensive granulomatous myositis was noted at necropsy at long‐ acting drug injection sites, with birefringent drug crystals still present up to 53 weeks after cART discontinuation. The potency and pharmacokinetics of these long‐acting inhibitors are promising for their use in SIV infected rhesus macaques, including as a long‐acting drug‐only maintenance regimen after suppression is achieved, with even more infrequent dosing as may be required to avoid injection site reactions.
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Poster Abstracts 1 The effects of M. tuberculosis on SIV evolution in a Mauritian cynomolgus macaque co‐infection model Alexis Balgeman1, Amy Ellis‐Connell1, Katie Zarbock2, Jaffna Mathiaparanam1, Mark A. Rodgers3, Cassandra Updike3, Tonilynn Baranowski3, Charles A. Scanga3, Shelby O'Connor1 1
University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, 3University of Pittsburgh
For HIV+ individuals the most common cause of morbidity and mortality is co‐infection with Mycobacterium tuberculosis (Mtb). Little is known about the impact of Mtb infection on HIV pathogenesis. This can be modeled in nonhuman primates by co‐infection with SIV and Mtb. We infected seven Mauritian cynomolgus macaques intrarectally with SIVmac239 for six months. The animals then were bronchscopically infected with a low dose (~10 CFU) of Mtb Erdman strain. We found that four animals had an increase of SIV viral loads in plasma by two ‐ three logs after infection with Mtb. Remarkably, SIV viral loads in one MCM spiked three logs after infection with Mtb but the animal then was able to regain viral control back to 10²copies per mL before euthanasia at 11 weeks post Mtb infection. We also isolated viral RNA from lung and lymph nodes of two animals at necropsy, and we found these tissue viral loads largely corresponded to plasma viral loads, although there was no clear association with the number of Mtb bacilli present in the samples. Using these samples from this unique model system, we can now assess whether increases in plasma SIV viremia following Mtb co‐infection corresponds to increased SIV sequence diversity attributed to release of virus from tissue sites.
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2 Novel SHIVs bearing HIV‐1 transmitted/founder envs for cure research: Replication, cART suppression, reservoirs and rebound Dr. Katharine Bar1, Anya Bauer1, Dr. Fang‐Hua Lee1, Dr. Hui Li1, Dr. George Shaw1 1
University Of Pennsylvania
Background: A robust simian‐human immunodeficiency virus (SHIV)‐macaque model of HIV‐1 latency is critical to evaluate eradicative and suppressive strategies that engage Env. We have developed and validated a novel strategy to generate designer SHIVs encoding transmitted/founder (TF) HIV‐1 Envs with tier 2 neutralization that consistently confer productive infection, high peak viremia and desirable early viral kinetics. Methods: We evaluated two promising TF SHIVs, SHIV.D.191859 and SHIV.C.CH848, encoding TF subtype D and C HIV‐1 Envs, respectively, for virus kinetics and persistence on suppressive combination antiretroviral therapy (cART) and following treatment interruption. Results: In 12 non‐CD8 depleted rhesus macaques (RM) inoculated via intrarectal, intravaginal or intravenous routes, SHIV.D.191859 conferred productive infection with peak viremia between 10^5‐ 10^8 copies/ml. In 11 of 12 animals, virus replication was maintained for at least 6 months, with viremia between 10^3‐10^6 copies/ml; one animal spontaneously controlled viremia to at or near undetectable levels by 14 weeks, a second controlled at 48 weeks post‐inoculation. Four viremic animals (viral loads between 10^3‐10^7) were placed cART with daily subcutaneous dolutegravir, emtricitabine and tenofovir and achieved rapid virus suppression. After 24 weeks of virus suppression, cART was interrupted and all four RM experienced viral rebound between day 7 and 17 to levels near pre‐cART viremia. Studies to measure the latent reservoir and kinetics of rebound are ongoing. Eight RM inoculated with SHIV.C.CH848 without CD8 depletion achieved peak viremia between 10^7‐10^8 and setpoint viremia between 10^3‐10^5; one of eight animals spontaneously controlled viremia at 6 months post‐infection. Four viremic RM were placed on cART, rapidly achieved virus suppression and have remained suppressed for three months to date. Conclusions: These studies are the first to show that SHIVs bearing primary, TF HIV‐1 Envs exhibit replication kinetics and persistence typical of HIV‐1 and therefore represent a promising next generation model for latency and cure research.
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3 Simultaneous expression of Interferon‐gamma and interleukin‐22 from innate lymphoid and natural killer cells in the colon of SIV‐infected rhesus macaques Andrew Cogswell1, Moriah Castleman2, Stephanie Dillon2, Cara Wilson2, Dr Ed Barker1 1
Rush University Medical Center, 2University of Colorado
Breakdown of intestinal epithelial integrity occurs during HIV‐infection leading to microbial translocation, which correlates with increased mucosal and systemic immune activation. Maintenance of intestinal barrier integrity relies in part on the presence of interleukin (IL)‐22. Recent findings indicate that innate lymphoid cell type 3 (ILC3) display a degree of functional plasticity and can produce IL‐22 or inflammatory cytokines depending on the local cytokine milieu produced by mucosal myeloid dendritic cells (MDCs). In our studies, colonic MDCs of SIV‐infected rhesus macaques spontaneously produced IL‐ 23, IL‐1β and IL‐12 while the same cells from uninfected animals only produced IL‐23 and IL‐1β. We therefore hypothesized that colonic ILC1s, ILC3s and natural killer cells (NK) cells in context of HIV‐ infection lead to production of IL‐22 and IFN‐γ. Multi‐color flow cytometry was utilized to identify the various ILC and NK cell populations and measure expression of transcription factors (Tbet, RORγt) and frequencies of cytokine‐expressing cells in the absence of stimulation (i. e. , spontaneous production) to best reflect their in vivo cytokine profiles. Regardless if colonic innate lymphocytes were ILC1s, ILC3s or NK cells, the majority co‐expressed both T‐bet and RORγT and co‐produced IFN‐γ and IL‐22 when acquired from colons of SIV‐infected rhesus macaques. These same cells from colons of SIV‐uninfected animals expressed predominately IL‐22 with little to no IFN‐γ. These findings indicate that the presence of inflammatory cytokines not loss of IL‐22 by ILCs and NK cells during SIV infection may increase intestinal epithelial barrier permeability and enhanced microbial product translocation.
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4 Novel features of CRM1‐dependent HIV and SIV RNA nuclear export revealed using live cell imaging Ryan Behrens1, Christina Higgins1, Nathan Sherer1 1
McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin ‐ Madison
Primate lentiviruses including HIV‐1, HIV‐2, and SIV and also deltaretroviruses such as HTLV‐1 have necessarily adapted to overcome profound cell‐ and/or species‐specific barriers to the nuclear export of essential late‐stage, intron‐retaining viral mRNAs. These barriers critically influence virus cell tropism and are key determinants of host immune detection and pathogenic outcomes. Both lentiviruses and deltaretroviruses overcome these barriers using Rev or Rex proteins, respectively, that route unspliced or incompletely spliced viral mRNAs out of the nucleus using the highly specialized cellular CRM1/RanGTP nuclear export machinery. Rev/Rex‐equivalent proteins from diverse retroviral pathogens (e.g., HIV‐1, HIV‐2, SIV, and HTLV) are unified in that they all link viral RNAs to CRM1. However, whether Rev/Rex‐encoded activities have evolved to circumvent identical selective pressures or, instead, govern multiple unique modes of RNA transport and/or other features of regulated gene expression is unknown. Live‐cell imaging represents a powerful new strategy for direct characterization of these pathways. Here, for the first time, we present comparative imaging of Rev/Rex equivalent proteins and RNA trafficking dynamics for HIV‐1, SIV, HIV‐2, HTLV, and 6 additional retroviruses (BIV, EIAV, FIV, Visna, HTLV‐2 and BLV) over >30 hours of continuous imaging in single living cells. We show that the HIV‐1 Rev protein exhibits striking, highly unique subcellular transport dynamics and demonstrate that Rev regulates a novel CRM1‐dependent, “burst‐like” RNA export behavior, activating rapid, late‐stage upregulation of gene expression just prior to the onset of virus particle production. Our comparative experiments reveal both distinct features of Rev/Rex‐regulated export mechanisms and, in some instances, remarkable interoperability of viral elements relevant to natural co‐infections (e.g., for HIV/HTLV or BIV/BLV) and, potentially, cross‐species transmission. Moreover, we discuss the potential utility of next‐generation, low‐toxicity CRM1 inhibitors as broad‐spectrum inhibitors of these viral pathogens, and how we have exploited species‐specific features of CRM1 to expose additional vulnerabilities.
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5 NKTT320‐induced activation of invariant Natural Killer T‐cells (iNKTs) in vivo and implication for its use in modulating AIDS pathogenesis Dr. Nell Bond1, Dr. Shan Yu1, Dr. Namita Rout1, Ms. Dollnovan Tran1, Mrs. Dawn Szeltner1, Dr. Robert Schaub2, Dr. Amitinder Kaur1 1
TNPRC, 2NKT Therapeutics
Background: iNKTs are a unique subset of innate T cells with an invariant TCRα chain that recognize glycolipids presented on the nonpolymorphic MHC class‐I‐like CD1d molecule. Activated iNKTs rapidly secrete a plethora of anti‐inflammatory and pro‐inflammatory cytokines and thus have diverse functions including potentiation of anti‐tumor and pathogen‐specific humoral and cellular immunity and resolution of inflammation. iNKT depletion and dysfunction are common in pathogenic HIV/SIV infection and may hasten AIDS progression. In vivo iNKT activation with glycolipid ligands are being explored as an immunotherapeutic modality in different diseases with varying success. In this study, we tested a novel humanized monoclonal antibody, NKTT320, that specifically binds to the invariant region of the NKT‐TCR as a tool for in vivo iNKT activation in Mauritian‐origin cynomolgus macaques (MCM). Methods: Dose‐escalation NKTT320 pharmacokinetic studies were performed in 11 SIV‐negative MCM. Plasma NKTT320 levels were measured by ELISA, kinetics of iNKT activation and effect on other immune cell subsets were evaluated by flow cytometry. Results: Peak plasma NKTT320 levels ranging between 6.6‐47.2 µg/ml were detected within 24 hours of a single intravenous dose of 100 to 1000 µg/kg and had declined to undetectable levels by 4‐6 weeks. NKTT320 administration resulted in rapid iNKT activation and proliferation of CD8+ iNKTs more than CD4+ iNKTs, without non‐specific T cell activation. Transient elevation of several plasma analytes (IL‐2, IL‐6, IL‐12, MCP‐1, MDC and IP‐10) and increased proliferation of non‐T immune cell subsets, notably B lymphocytes, were observed suggesting downstream potentiation of innate and adaptive immunity. Repeated doses of NKTT320 did not reveal any decrease in iNKT responsiveness or proliferation indicating that NKTT320 treatment did not lead to iNKT anergy. Conclusions: NKTT320‐mediated iNKT activation may be a useful immunotherapeutic tool to preserve and augment iNKT function, and thereby prevent immunodeficiency and aberrant immune activation in pathogenic SIV infection. Grant Support: NIH‐R01‐AI102693(AK), NIH‐P51‐OD011104
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6 Characterization of vaccine‐induced antibody responses Tysheena Charles1, Samantha Burton1, Lori Spicer1, S. Abigail Smith1, Tiffany Styles1, Pradeep Reddy1, Traci Legere1, Vijayakumar Velu1, Dr Rama Amara1, Dr Cynthia Derdeyn1 1
Generating vaccine‐mediated antibody protection against HIV‐1 continues to be a formidable challenge. Our goal is to gain a more complete understanding of how antibody effector functions contribute to protection in a pre‐clinical vaccination setting. Here we evaluated neutralizing antibody (nAb) responses elicited in rhesus macaques (RM) immunized with clade C HIV‐1 envelope (Env) 1086.C K160N‐based DNA/modified vaccinia virus Ankara regimens that also included gp140 protein boosts and CD40‐ligand adjuvant. None of the vaccination regimens provided a significant level of protection against mucosal challenge with heterologous clade C SHIV1157 ipd3N4. To date, serum nAb has been evaluated in the TZM‐bl assay against a heterologous tier 2 CRF01_AE HIV‐1 Env, CNE8, and the autologous Env 1086.C with and without the K160N substitution. Neutralization activity against all of the Envs was modest. Boosting with gp140 protein increased antigen‐specific antibody titers; however, it did not appear to enhance the quality of the nAb response. For a higher resolution analysis, we have characterized antigen‐specific B cells from immunized RM. To date, this analysis has indicated that B cell responses to the 1086.C K160N Env immunogen involved diverse heavy and light chain germline gene usage and pairing. Functional characterization of the monoclonal antibodies produced by these B cells is underway. By characterizing individual vaccine‐induced B cells and antibodies at the monoclonal level, we can gain insight into why these antibodies were not able to provide protection against the SHIV challenge.
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7 Early CNS damage in pediatric SIV infection Heather Carryl1, Bonnie Phillips2, Koen Van Rompay3, Angela Amedee4, Mark Burke1, Kristina DeParis2 1
Howard University, 2UNC Chapel Hill, 3UC Davis‐ CNPRC, 4LSU
Despite the effectiveness of maternal antiretroviral therapy (ART) in reducing mother‐to‐child transmission (MTCT) of HIV, several obstacles limit its efficacy during prolonged breastfeeding, and breast‐milk transmission is now the dominant mode of HIV infection in infants. In the era of ART, these infants can now survive into adolescence and adulthood. However, 70% of HIV‐infected infants experience neurocognitive deficits. To prevent long‐term health sequelae, we need to gain a deeper knowledge of the dynamic interactions between the virus, CNS, and immune development. Utilizing the rhesus macaque model of pediatric oral SIVmac251 infection, we analyzed the impact of SIV infection on CNS development in ART‐naïve animals. Brain SIV RNA and DNA levels were determined by PCR and SIV RNA or SIV p27 positive cells were identified by ViewRNA and immunohistochemistry in the cerebrum and choroid plexus. In addition, we performed IHC for Ki67, GFAP, CD68, and CX3R1 to characterize CNS cell populations. Frequencies of neuronal populations in the hippocampus and a neuroinflammation were also assessed in the hippocampus. Statistical analysis consisted of nonparametric Mann‐Whitney tests between SIV‐infected and uninfected age‐matched controls. Our results demonstrated that SIV entered the brain within 48 hours after oral challenge. SIV infection resulted in reduced stem cell proliferation and differentiation. As a result, we observed decreased pyramidal and immature neuronal populations in the hippocampus of the SIV‐infected compared to uninfected infant macaques. Microglia and astrocytes showed signs of activation. These findings are consistent with neurological features of pediatric HIV‐1 that include dysfunction in the frontal cortex and the hippocampus, global cerebral atrophy, and accelerated neuronal apoptosis in the cortex. Studies in the pediatric model of SIV/ SHIV infection in the presence or absence of ART will provide novel insights into CNS development, identify CNS complications induced by viral infections, and inform novel treatment and cure strategies.
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8 Gut microbiota is associated with the immune response to HIV‐1 envelope vaccination of newborn rhesus macaques Amir Ardeshir1, Holly Heimsath2, Olaf Mueller2, Bonnie Phillips2, Joshua Eudailey2, Erika Kunz2, Genevieve Fouda2, Laura‐Leigh Rowlette2, Holly Dressman2, Kristina De Paris3, Koen Van Rompay1, Sallie Permar2 1
University of California Davis, 2Duke University, 3University of North Carolina
We previously showed that early establishment of the gut microbiota is associated with the immune system development. HIV‐1 envelope epitopes have been reported to mimic microbiota antigens commonly found in the gut. Thus, part of developing a successful vaccine to HIV‐1 may be early immunization of children before the microbiome and microbiome‐specific immunity have fully developed, allowing the immune system to differentiate between helpful commensal bacteria and foreign viral invasion. Our central hypothesis is that the magnitude of immunologic response from distinct vaccination regimens is correlated with the pre‐existing composition of the intestinal microbial communities. Methods: Four vaccine groups (Conventional, Co‐Administration, Protein Only, and Extended Interval), each consisting of 5 infant rhesus macaques were vaccinated in varying HIV Env prime/boost modalities and intervals. Envelope‐specific antibody responses were measured by ELISA, and neutralization assays in TZMbl cells. Phylogenetic profiling of infant microbiomes was conducted by extracting 16S ribosomal RNA from stool samples pre‐ and post‐immunization. 16S rRNA reads were quality filtered, demultiplexed, and clustered into operational taxonomic units (OTUs) using vsearch. Results: The Protein Only regimen yielded the earliest HIV‐1 Env‐specific IgG response to gp120, but the maximal response did not differ between groups. The Protein Only regimen induced the strongest and most persistent Tier 1 neutralizing antibodies. The Extended Interval regimen had the highest mucosal 1086C gp140 IgG and IgA in stool samples. Stool microbiota of the vaccinees demonstrated variable abundance but similar taxonomic profiles by genera. Interestingly, we identified positive correlations between the vaccine‐elicited IgG responses and certain bacterial genera. Conclusions: Abundance of certain bacteria in the infant GI is associated with the magnitude of the IgG response to infant HIV Env vaccination. These bacteria may be used as probiotic to supplement the food in early life to achieve increased IgG response to HIV‐1 Env vaccination.
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9 High‐throughput generation of Simian‐Human Immunodeficiency Virus and simian tropic HIV replication‐competent molecular clones Dr. Debashis Dutta1, Samuel Johnson1, Alisha Dalal1, Martin J. Deymier2, Eric Hunter2, Siddappa N Byrareddy1 1
University of Nebraska Medical Center, 2Emory Vaccine Center, Emory University
Traditional restriction endonuclease‐based cloning has been routinely used to generate replication‐ competent simian‐human immunodeficiency viruses (SHIV) and simian tropic HIV (stHIV). This approach requires the existence of suitable restriction sites, or the introduction of nucleotide changes to create them. Here, using an In‐Fusion cloning technique that involves homologous recombination, we demonstrate the power of this approach to generate SHIVs and stHIVs based on epidemiologically linked transmission‐founder pair HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 generated stHIV. Likewise, exchanging HIV env genes into a SHIVAD8‐EO backbone, coupled with the introduction N375 mutations to each of the Envs to enhance macaque CD4 binding, multiple SHIVs were successfully made. The generated SHIVs and stHIV were infectious in TZM‐ bl cells as well as rhesus and pig‐tailed macaques’ PBMCs. Thus, In‐Fusion based homologous recombination is efficient for generation of SHIV and stHIV including the introduction of select mutations in Env. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV/SHIV based on primary clinical isolates that can serve as novel challenge viruses for HIV vaccine/cure studies.
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10 Impact of maternal IM/IN HIV‐env immunization during pregnancy on postnatal HIV transmission. Maria Dennis1, Josh Eudailey1, Morgan Parker1, Bonnie Philips2, Genevieve Fouda1, Koen Van Rompay3, Kristina DeParis2, Sallie Permar1 1
Duke Human Vaccine Institute, 2UNC, 3UC Davis
We previously reported that immunization of lactating rhesus monkeys with a combined intramuscular (IM)/intranasal (IN) HIV vaccine strategy elicited functional systemic IgG and robust milk IgA responses. In the current study, we tested if vaccine‐elicited maternal HIV‐specific antibodies passively protect infants against postnatal virus challenge. Pregnant rhesus macaques (n=9) were immunized with a HIV transmitted/founder envelope (C.1086C gp120) using a MVA prime/ IM/IN protein boost strategy. The MVA prime was administered at week12 of pregnancy and the protein boosts at week 20 of pregnancy, around delivery and 3 weeks after delivery. A placebo group (n=9) received MVA without the gp120 insert at week 12 of pregnancy and the Respiratory Syncytial Virus protein F at week 12 and 20 of pregnancy. Infants were orally challenged starting at 6 weeks of age with a weekly low‐dose of the clade C tier 2 SHIV1157ipd3N4, which expressed an envelope isolated from an HIV‐infected Zambian infant. Similar to our previous study, this immunization strategy elicited robust cross‐clade specific IgG/IgA Env‐ specific antibodies that mediated tier 1 virus neutralization and ADCC activity against the SHIV challenge virus. Maternal antibodies were adequately transferred across the placenta (transfer efficiency median 131%. Range 17‐384%), but were not associated with protection. There was no significant difference in the number of infected animals, number of challenges required for infection or the peak viral load between infants born to HIV vaccinated or placebo mothers. Interestingly, there was a negative correlation between the percentage of CD69+CD4+T cells at the time point prior to the initial challenge and the number of challenges required for infection (r= ‐0.75, p<0.01). Our results indicate that maternal immunization/breast milk immunity alone did not mediate infant protection, suggesting that active infant HIV vaccination or passive immunization with broadly neutralizing Abs will likely be required to protect infants against HIV‐1 acquisition.
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11 NIAID reagent resource support contract for AIDS vaccine development Dr. Valerie Fremont1, Dr. Rosemarie Mason2, Michael Fisher1, Dr. Mario Roederer2, Dr. Ronald L. Brown1 1
Quality Biological, 2Vaccine Research Center, NIAID, NIH
The NIAID Reagent Resource Support Contract for AIDS Vaccine Development (Contract Number: HHSN272201100023C) was established to facilitate AIDS vaccine development by offering unique reagents and services to investigators. The Program seeks to procure, produce, purify, and test reagents specifically for use in AIDS vaccine development in the preclinical and clinical research arenas. As an example, Quality Biological (QBI) is collaborating with the NIAID Vaccine Research Center (VRC) to produce fully simian antibodies displaying broad and potent neutralization of diverse viral isolates. The program offers NIH‐supported investigators an avenue to advance their research in AIDS vaccine development by suppling large quantities of reagents, viral gene products, peptides, adjuvants, cytokines, virus stocks, expression vectors, monoclonal and polyclonal antibodies, and other novel reagents involved in AIDS vaccine studies and development. The Program supports the use and development of high‐quality, novel, and targeted reagents and assays to better understand the immune system in this endeavor. The QA/QC of these reagents and services assures consistency while affording a level of standardization among laboratories. QBI provides a repository for these reagents with the necessary storage and shipping conditions to maintain their stability and integrity QBI is an ISO 9001 certified, woman‐owned small business manufacturing company that offers a wide range of technical expertise and capabilities required for supporting a program designed for the discovery and development of an AIDS vaccine. Interested investigators please contact Ronald L. Brown, Ph.D., Principal Investigator, Quality Biological, Inc., [email protected], or Jon Warren, Ph.D., Preclinical Research and Development Branch, NIAID, NIH, [email protected]
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12 Bi‐functional entry inhibitors sensitize macaque‐tropic human immunodeficiency virus type 1 (HIV‐1mt) to antibodies generated in HIV‐1mt‐infected macaques Dr. Shigeyoshi Harada1, Yuta Hikichi1, Dr. Yohei Seki2, Dr. Akatsuki Saito2, Dr. Takeshi Yoshida2, Dr. Hirotaka Ode3, Dr. Yasumasa Iwatani3, Dr. Yasuhiro Yasutomi4, Dr. Tomoyuki Miura5, Dr. Tetsuro Matano1, Dr. Hirofumi Akari2, Dr. Kazuhisa Yoshimura1 1
AIDS Research Center, National Institute of Infectious Diseases, 2Primate Research Institute, Kyoto University, 3 National Hospital Organization Nagoya Medical Center, 4Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition, 5Institute for Frontier Life and Medical Sciences, Kyoto University
Background: To date most HIV vaccine candidates have been ineffective at generating broadly neutralizing antibodies (bNAbs). In addition, autologous neutralizing antibodies exert pressure on HIV Env, resulting in neutralization escape. Recently, we have developed bifunctional entry inhibitors (Bf‐ Ent‐Is) with respect to both neutralizing antibodies (NAbs) activation and entry inhibition. We have also generated a series of macaque‐tropic HIV‐1 (HIV‐1mt) to establish non‐human primate models for basic and clinical studies. In the present study, we investigated an approach that increases the sensitivity of the HIV‐1mt to antibodies generated in HIV‐1mt‐infected macaques. Materials & Methods: We passaged HIV‐1mt in cynomolgus macaques for in vivo adaptation. A chimeric clone containing the HIV‐1mt gp160 within a pNL4‐3 backbone was constructed. The neutralization activity in plasma samples collected from different time points was measured using the TZM‐bl assay. Results: We examined the ability of Bf‐Ent‐Is, HTA‐004 and OKS3‐019, to sensitize HIV‐1mt to neutralization by anti‐HIV‐1 monoclonal NAbs, 2F5 (anti‐MPER) and KD‐247 (anti‐V3). The Bf‐Ent‐Is specifically enhanced the neutralizing potency of 2F5 and KD‐247 against HIV‐1mt. We therefore tested the neutralization of HIV‐1mt by the plasma of HIV‐1mt‐infected macaques in the presence of Bf‐Ent‐Is. When HTA‐004 or OKS3‐019 was added at subneutralizing concentrations, HIV‐1mt was efficiently neutralized by the plasma collected 14, 24, 97, 105, or 117 weeks after virus inoculation. Conclusions: In the present study, we found that certain Bf‐Ent‐Is can sensitize HIV‐1mt to antibodies that can be elicited in HIV‐1mt‐infected macaques. Thus, Bf‐Ent‐Is might have therapeutic utility in controlling HIV infection, and might increase the protective efficacy of HIV vaccines.
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13 Modeling of neutrophil, basophil, and classical monocyte kinetics by BrdU pulse‐chase labeling in young adult and elderly rhesus macaques Ziyuan He1, Chie Sugimoto1, Carolina Allers1, Hideki Fujioka2, Elizabeth Didier3, Marcelo Kuroda1 1
Division of Immunology, Tulane National Primate Research Center, 2Center for Computational Science, Tulane University, 3Division of Microbiology, Tulane National Primate Research Center
Myeloid cells are continuously produced via bone marrow myelopoiesis and later are cleared from the circulation to maintain homeostasis. To better understand such cell movement, we applied in vivo 5‐ bromo‐2'‐deoxyuridine (BrdU) pulse‐chase labeling and flow cytometry analysis to follow the cell division kinetics of neutrophils, basophils, and classical monocytes during homeostasis in peripheral blood of rhesus macaques (RMs). BrdU‐labeled neutrophils, basophils, and classical monocytes showed distinct kinetics patterns which indicated different bone marrow transit time. Mathematical modeling was applied to estimate the circulating half‐life and daily production as follows; neutrophils (1.63±0.16 days, 1.42x10⁹ cells/L/day), basophils (1.78±0.30 days, 5.89x10⁶ cells/L/day), and CD14+CD16‐ classical monocytes (1.01±0.15 days, 3.09x10⁸ cells/L/day). We also examined older RMs to examine the effects of natural aging on cell kinetics. Data collected from 32 BrdU‐labeled healthy RMs ranging from 3 to 19 years of age suggested that neutrophils, basophils, and classical monocytes respond differently to aging. Neutrophils had the same circulating half‐lives across all ages, although the older animals produced fewer neutrophils per day. All age groups had similar half‐lives and production of basophils. In the case of classical monocytes, the same numbers of cells were produced but exhibited shorter half‐lives with increasing age. We also examined the alterations in kinetics of neutrophils during SIV and Shigella infection and observed massive turnover during Shigella but not SIV infection. Strikingly, this change could not be observed using the standard quantitation of neutrophils (neutrophilia) in blood. Overall, our results showed the relatively short lifespan and constant replenishment of neutrophils, basophils, and classical monocytes in RMs during homeostasis, suggesting a potential homeostatic role of these cells independent of pathogen clearance. Moreover, our data suggest that dynamic changes in neutrophils during HIV infection could result from bacterial infections due to immunodeficiency rather than directly from HIV infection per se.
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14 Persistent memory response elicited by HIV/SIV conserved element gag pDNA priming vaccine and rapid recall upon DNA or rMVA vector boost Xintao Hu1, Antonio Valentin1, Yanhui Cai1, Frances Dayton1, Valerie Ficca1, Margherita Rosati1, Patricia Earl2, Bernhard Moss2, Niranjan Sardesai3, James Mullins4, George Pavlakis1, Barbara Felber1 1
National Cancer Institute At Frederick, 2NIAID, 3Inovio Pharmaceuticals Inc., 4University of Washington
Background: T‐cell based vaccines play a pivotal role in immunotherapeutic approaches against HIV infection. We developed pDNA‐based vaccines to induce robust cytotoxic T cell responses to the conserved elements (CE) of HIV‐1 Gag, selecting regions by stringent conservation, functional importance, independent of ‘protective’ haplotypes associated with virus control, and association with immune control in HIV‐infected LTNP. Based on homology to HIV, an SIV p27gag CE DNA vaccine was also developed. Methods: Macaques were primed with CE DNA followed by boost with full‐length gag DNA via intramuscular injection followed by in vivo electroporation. After a rest period of ~1.5‐2 years, macaques received booster vaccinations with CE DNA, CE+gag DNA or rMVA gag/pol. Cellular immune responses were measured in peptide‐stimulated PBMC by polychromatic flow cytometry. The antiviral activity of the vaccine‐induced SIV‐specific T cells was monitored by a viral inhibition assay (VIA). Results: CE DNA‐immunized macaques showed robust antigen–specific memory responses with a significant fraction of cytotoxic T cells. Full‐length gag or CE+gag DNA vaccine significantly boosted the pre‐existing CE responses, increasing both magnitude, breadth and cytotoxicity. CE responses were found to disseminate to mucosal sites and were able to efficiently inhibit SIV propagation in autologous primary lymphocyte cultures. Persistent CE‐specific T cell responses were observed over ~2‐years of rest following the last vaccination. Subsequent boosting with CE, CE+gag or rMVA significantly increased the CE response magnitude reaching levels higher than those found during the original prime/boost vaccination. Fine mapping showed expanded CE breadth. Conclusions: CE prime‐full‐length DNA vaccine elicited long‐lasting immunity. This vaccine concept is being tested in a clinical trial (HVTN 119). The expanded breadth of the responses targeting invariable regions could provide an advantage in preventing immune escape and suppressing viral propagation. Together, the CE vaccine regimen has promising applications in prevention and therapy.
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15 Improving characterization of the full MHC genomic region in macaques Julie A. Karl1, Hailey E. Bussan1, Trent M. Prall2, Roger W. Wiseman1,2, David H. O'Connor1,2 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center, University of Wisconsin‐Madison
In the last few years, next‐generation sequencing platforms have successfully been used for in‐depth exploration of MHC class I and class II genes. We have used the Illumina MiSeq system to provide highly accurate, ultra‐deep exon 2‐based genotyping results for thousands of macaques annually since 2014. We also developed a robust pipeline for full‐length MHC class I and class II allele discovery using the long‐read PacBio RS II system. However, next‐generation sequencing technologies have not yet been harnessed to explore the full MHC genomic region. Current macaque reference genomes are condensed and inaccurate in this region, making them difficult to use for alignments. The two accurate publically available macaque MHC regions used Sanger sequencing of BAC libraries, which is both labor‐intensive and cost‐prohibitive for multiple animal characterization. We are exploring three third‐generation sequencing technologies for potential usefulness in MHC genomic region characterization – targeted exome sequence capture of the MHC region followed by PacBio Sequel long‐read sequencing, 10X Genomics Chromium system “linked read” technology, and Oxford Nanopore Minion ultra‐long read sequencing. Target capture uses previously designed probes spanning the entire 5 Mb MHC genomic region, then sequences the captured fragments using PacBio single‐molecule real‐time sequencing technology on their new higher‐throughput instrument. The 10X Genomics system essentially barcodes and fragments high molecular weight DNA into pieces that are sequenced on the Illumina platform, then reassembled into chromosome‐phased scaffolds based on those barcodes. The Oxford Nanopore Minion sequences single‐strand high molecular weight DNA to create long scaffolds that can be error‐corrected using higher‐accuracy short reads. We are currently exploring these three technologies using MHC homozygous Mauritian cynomolgus macaques; the most successful techniques will be applied to additional macaques to improve existing reference genomes and expand our knowledge of variation in the MHC region beyond the class I and class II genes. Grant Support NIH NIAID Contract HHSN272201600007C
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16 Isolation of a monoclonal antibody to the rhesus macaque MHC class I allomorph Mamu‐A1*002 Laurel Kelnhofer1, Dr. Matthew Reynolds1,2, Jason Weinfurter1 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Wisconsin National Primate Research Center
Monoclonal antibodies that bind to human leukocyte antigen (HLA) are useful tools for HLA‐typing, tracking donor‐recipient chimerisms after bone marrow transplants, and characterizing specific major histocompatibility complexes (MHC) on cell surfaces. Unfortunately, equivalent reagents are not available for rhesus macaques, which are commonly used animal as models in organ transplant and infectious disease research. To address this deficiency, our lab has successfully isolated an antibody that recognizes the common Indian rhesus macaque MHC class I molecule, Mamu‐A1*001. Using a similar protocol we are currently in the process of isolating a Mamu‐A1*002 antibody. We isolated Mamu‐ A1*002‐binding antibodies from an allosensitized rhesus macaque. A Fab phage display library was constructed with splenocytes and bone marrow from the allosensitized macaque and panned to isolate an antibody that binds to Mamu‐A1*002 but not to other common rhesus macaque MHC class I molecules. Preliminary ELISA screening suggests we have isolated antibody with a high binding affinity to Mamu‐A1*002 and limited binding to the Mamu‐A1*001. Once isolated the Mamu‐A1*002‐specific antibody will be useful for identifying Mamu‐A1*002‐positive rhesus macaques, for detecting Mamu‐ A1*002‐positive cells in populations of Mamu‐A1*002‐negative cells, and for examining disease processes that alter expression of Mamu‐A1*002 on cell surfaces. Moreover, the alloimmunization process will be useful for isolating additional MHC allomorph‐specific monoclonal antibodies or antibodies against other polymorphic host proteins which are difficult to isolate with traditional technologies.
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17 A non‐redundant, reference viral database (RVDB) to facilitate high‐ throughput sequencing (HTS) analysis for virus detection Dr. Norman Goodacre1, Ms. Subhiksha Nandakumar1, Ms. Aisha Aljanahi2, Dr. Arifa Khan1 1
CBER/FDA, 2Georgetown University
BACKGROUND: Genomic and metagenomic analysis using high‐throughput sequencing has resulted in a great expansion of the virosphere. However, there are limitations in the publically available databases for detecting distantly‐related viruses: all viral sequences are not contained in a single database, and virus detection can be obscured by the large amount of cellular sequences in the more diverse NCBI nr/nt collection (Ma et al., J. Virol, 2014). Therefore, we undertook to create a new, non‐redundant, reference viral database (RVDB) that would include all viral, viral‐related, and viral‐like sequences, including retroelements, and have an overall reduced cellular sequence content. MATERIALS and METHODS: The overall strategy included: a) semantic selection of all virus, virus‐related, and virus‐like sequences from GenBank; b) bioinformatics tools (BLAST and HMMER) to confirm viral identity; c) CD‐HIT for clustering at 98% to remove redundancy; and d) testing robustness for HTS analysis using our large in‐house datasets and comparing the results to those obtained using the NCBI databases (RefSeq and nr/nt) based upon detection of a viral hit and length of time for the analysis. RESULTS: The resulting database contains viral genomes and partial viral sequences, as well as endogenous viruses, endogenous retroviruses and retrotransposons. The newly‐created RVDB was more specific and sensitive for virus detection than NCBI Viral, and over 200x faster than nr/nt in run time. A final version has been made publically available to allow scientists to perform more rapid HTS data analysis. The database is meant to be updated on a regular basis to add new viral sequences added to GenBank. CONCLUSION: RVDB is expected to enhance HTS investigations for virus detection with increased efficiency, particularly for novel virus detection. Further efforts toward completing the annotation will result in a high‐quality viral database that will increase confidence in obtaining accurate results from HTS data analysis.
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18 Negative association between the outcome of vaginal SHIV challenge and serum IgM antibodies induced by gp140 vaccination in aged rhesus macaques Pam Kozlowski1, Diana Battaglia1, Rafiq Nabi1, Lori Spicer2, Cynthia Derdeyn2, Caroline Petitdemange2, Sudhir Kasturi2, Eric Hunter2, Rama Amara2, David Masopust3, Bali Pulendran2 1 3
Louisiana State University Health Sciences Center, 2Emory University and Yerkes National Research Primate Center , University of Minnesota
Vaccines that induce gag‐specific T cells in addition to envelope‐specific antibodies may be optimal for protection against HIV. To compare the efficacy of an envelope protein + viral‐vectored gag vaccine approach to protein immunization alone, female rhesus macaques, aged 3‐14 years, were vaccinated 4 times with gp140 C.1086 with or without gag‐expressing viral vectors. Five months after the last gp140 immunization, the animals were vaginally challenged with SHIV‐1157ipd3N4 a total of 12 times or until they became infected. Both vaccines demonstrated similar efficacy, but only in animals less than 9 years of age. To determine why the older animals (9‐14 yrs) were not protected, we compared their antibody responses to those in the younger animals (3‐8 yrs). Vaginal and serum IgA responses to gp140 C.1086 near the time of challenge did not differ between the groups. However, gp140‐specific vaginal and serum IgG responses were significantly lower in the older animals. Serum IgG antibodies to gp41, gp120 (1157ipd3N4), and especially gp70‐V1V2 (1157ipd3N4) were also significantly lower in the older animals. In contrast, serum IgM antibodies to gp140 and gp120 were significantly higher in the older animals. In addition, the concentrations of anti‐gp140 or anti‐gp120 IgM in prechallenge sera were inversely correlated with the number of challenges required for infection. As immunization with C.1086 gp140 did not induce neutralizing antibodies to the tier 2 challenge virus, the IgM antibodies in older animals may have interfered with other beneficial antiviral IgG functions, a possibility under investigation. Overall, these data indicate that age may skew the isotype of vaccine‐induced antibodies toward IgM in female rhesus macaques. Funding: NIH AI096187 and AI24436.
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19 Applying RNA profiling and functional analysis approaches to evaluate vaccine‐induced adaptive and innate immune responses in non‐human primate SIV challenge and protection studies Dr. Lynn Law1, Richard Green1, Jean Chang1, Elise Smith1, Dr. Connor Driscoll1, Dr. Courtney Wilkins1, Dr. Michael Gale1 1
Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington
Background: The NIAID Division of AIDS supports the Nonhuman Primate Functional Genomics Core (NHP‐FGC) at the University of Washington to perform functional genomics studies on samples from nonhuman primate (NHP) trials of HIV‐vaccine candidates. The primary goals of the Core are 1) to ensure standardization and comparability of functional services and assays conducted for the Simian Vaccine Evaluation Unit, 2) to apply and refine high throughput functional genomics approaches to more effectively evaluate vaccine‐induced adaptive and innate immune responses in challenge and protection studies, 3) to perform 16S rRNA sequencing to characterize the changes in microbiome profiles associated with vaccination and challenge and 4) to identify gene signatures that can define correlates of protection and predict vaccine efficacy. Methods: The NHP‐FGC works with collaborating investigators in devising sampling schedules and experimental designs for both host response and microbiome studies. After receiving suitably preserved samples, the Core handles all stages from RNA/DNA isolation, assay measurements, data management, and computational analyses. Depending on the specifics of the individual studies, we utilize microarray analysis, RNA‐sequencing, or 16S rRNA sequencing. We use a variety of computational approaches for data processing and analysis depending on the objectives of the study; these approaches include differential analysis of gene expression, functional annotation, and several types of network analyses. Conclusions: The NHP‐FGC provides a valuable resource to the community of investigators using NHP models to evaluate AIDS vaccine strategies by generating RNA response profiles and performing correlation analysis to determine gene signatures linked to vaccination and infection outcome.
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20 Secreted verses transmembrane HIV ENV SOSIP DNA vaccination inducing B cells and plasmablasts in Indian rhesus macaques Dr. David Leggat1, Dr. Alberto Cagigi1, Dr Luca Schifanella2, Mr. Sandeep Narpala1, Ms. Madhu Prabhakaran1, Ms. Mitzi Donaldson1, Dr. Kathryn Foulds1, Mr. David Ambrozak1, Mr. JP Todd1, Dr. Genoveffa Franchini2, Dr. Mario Roederer1, Dr. Richard Koup1, Dr. Adrian McDermott1 1
Vaccine Research Center, NIH, 2National Cancer Institute, NIH
Understanding B cell immunobiology in Indian rhesus macaques (RM) is fundamental to the development of the RM as a model for vaccines, including HIV. Until recently, specific detection and identification of plasmablasts has been particularly challenging in the RM model. In part due to phenotypic differences compared with similar human cellular populations. However, new clones of antibodies recognizing conserved regions of prototypic B cell, plasmablast, and plasma cell markers between humans and RM, have become available which now allow their characterization in RM. Availability of new clones and the use of the Becton Dickinson Symphony flow cytometer now allow a broader and more comprehensive analysis of these populations for the purpose of evaluating new vaccination strategies. Here we describe a novel study that evaluates transmembrane anchored or secreted versions of HIV Env. We directly compared vaccination using DNA vectors, which encode for either of these forms of HIV‐Env. Evaluation utilized an 18‐parameter flow cytometry panel, which was designed to assess the development of antigen‐specific B cells into long‐lived plasma cells. Analysis of bone marrow resident plasmablasts will be correlated to antigen‐specific B cells and plasmablasts post‐ vaccination to identify characteristics of peripheral blood B cells associated with the generation of plasma cells. The results from this study will be used to evaluate the benefits of using secreted vs. transmembrane forms of HIV‐Env in the context of DNA prime/protein boost and the capacity of HIV immunogens and to elicit long‐lived plasma cell responses in RM and humans.
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21 Immune cells distribution during SIV infection and treatment: Characterization of bone marrow and peripheral blood in SIV infected cynomologus macaques Dr Julien Lemaitre1 1
CEA ‐ Université Paris Sud 11 ‐ INSERM U1184, Immunology of viral infections and autoimmune diseases
Combinational antiretroviral therapy (cART) reduces HIV‐1 associated mortality and morbidity but does not eradicate the virus. As a consequence, lifelong treatment is needed to keep the virus under control. To better understand why the immune system fails in controlling viral replication, we combined the use of a non‐human primate models and high multiparameter single cell analysis of the immune cells by mass cytometry. Indeed, cynomolgus macaque infected by SIVmac251 is the most relevant model to explore the immune system in tissues during both acute and chronic phase, and under cART. Using a mass cytometry we were able to observe global changes induced by SIV infection in T‐cells, B‐cells, NK cells, monocytes and neutrophils. Interestingly, neutrophils distribution in bone marrow and blood was strongly affected by the infection, suggesting a delocalization in the tissues or a massive death. To functionally investigate neutrophils during SIV infection and cART, we used conventional flow cytometry to characterize their maturity and phagocytic activity. SIV infection induce mobilization of immature neutrophils with a specific FC‐receptors profile and phagocytic activity. The present study shows for the first time how SIV infection induces an immune cells redistribution across different tissue and sheds light on the interaction between the virus, the host and the treatment.
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22 Development and validation of four novel SHIV challenge stocks bearing transmitted/founder tier 2 HIV‐1 subtype A, C or D envs Dr. Hui Li1, Ding Wenge1, Dr. Fang‐Hua Lee1, Dr. Beatrice Hahn1, Dr. George Shaw1 1
University Of Pennsylvania
BACKGROUND: High titer SHIV challenge stocks that bear primary or transmitted/founder (T/F) tier 2 Envs of divergent subtypes are needed for preclinical vaccine research. METHODS: SHIVs CH505.375H (subtype C), CH848.375H (subtype C), BG505.332N.375Y (subtype A) and 191859.375M (subtype D) (Li et al., PNAS 2016) were amplified by short‐term (~14d) culture in primary rhesus CD4 T‐cells and frozen in vapor phase N2 in 1000+ vial aliquots. RESULTS: All SHIV stocks had uniformly high infectivity and virion content ranging from 5‐30x106 IU per ml on TZM cells, 80‐200ng p27Ag/ml and 6‐10x108 vRNA/ml. IU:particle ratio was as high as 0.02 on TZM and 0.0005 on primary rhCD4 T‐cells. Single genome sequence analysis of env showed rare random mutations and absence of selection. Neutralization by >20 mAbs targeting bNAb sites and V3 loop and CD4i epitopes was indistinguishable from cloned viruses expressed in 293T cells indicating the retention of native‐like tier 2 NAb sensitivity. IR titration of SHIVs CH505 and BG505 in RMs showed AID50 of 1.0ml challenge stock of ~1:30 and ~1:80, respectively. IVAG infections required ~10‐fold more virus, IV challenges ~1000‐fold less. Early viral replication patterns were indistinguishable following the different infection routes for peak viremia (~10^6‐10^8 vRNA/ml at ~14d post‐inoculation) and setpoint viremia (~10^3‐10^5 vRNA/ml). CONCLUSIONS: Antigenically diverse primary tier 2 SHIV challenge stocks were generated and validated. These have been distributed to five different investigative groups working in different primate facilities and in each instance the infectivity and replication parameters reported here were corroborated. These results indicate the delta Env375 SHIV design strategy yields challenge stocks with consistent infection properties following mucosal and IV routes of administration, making them ideal for preclinical protection studies. Virtually any HIV‐1 primary or T/F Env of predetermined antigenicity can thus be generated as a challenge virus.
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23 Env375 SHIV design v2.0 Dr. Hui Li1, Alexander Murphy1, Shuyi Wang1, Wenge Ding1, Dr. Beatrice Hahn1, Dr. George Shaw1 1
University Of Pennsylvania
Background: Previously, we reported (PNAS 2016) a new strategy for constructing SHIVs that bear primary or transmitted/founder (T/F) HIV‐1 Envs and that replicate consistently and efficiently in rhesus macaques (RMs). We constructed 15 such SHIVs, all with unique primary or T/F Envs, and all 15 replicated efficiently in RMs in vivo (see other abstracts by Li). In longitudinal follow‐up studies, we found that redundant SIV/HIV junctional sequences in tat1 and env (gp41) were spontaneously deleted in vivo, indicating a fitness advantage for the slightly shorter genomes. In addition, we noted that the SHIV construction scheme first reported was cumbersome and fraught with technical challenges since it required tripartite vpu‐env PCR amplifications and ligations. Method: We designed a new, simplified SHIV backbone by removing vpu‐env or env‐only and replacing it with BsmBI restriction enzyme cloning sites. In addition, we deleted redundant 68 nt SIV/HIV tat1 and 21 nt env (gp41) sequences. Results: SHIVs bearing CH505 vpu‐env genes were constructed by either the original v1.0 strategy or the modified v2.0 approach and were compared for virion production in 293T cells, infectivity in TZM cells, and replication in primary RM CD4 T cells in vitro. v2.0 equaled or exceeded v1.0 by all measures. Two RMs were inoculated intravenously with an equal mixture of the two virus stocks (5ng p27Ag each). Analysis is underway to determine which construction scheme yields the most fit virus as determined by plasma viral load kinetics and persistence. Conclusions: We describe here a new design scheme for constructing SHIVs containing primary HIV‐1 delta375 Envs. This new approach reduces the average time for SHIV construction from months to weeks while retaining desirable SHIV properties including wild‐type tier 2 neutralization sensitivity. These improvements in SHIV design can make this important research tool a practicality for many laboratories.
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24 Evolution of T Cell responses to RhCMV‐vectored SIV vaccine in previously RhCMV‐negative and ‐positive young Macaques Kawthar Machmach1,2, Gema Méndez‐Lagares1,2, Amir Ardeshir1,2, Nicole Narayan1,2, William Chang3, Jeff Lifson4, Peter Barry1,3,6, Dennis Hartigan‐O’Connor1,2,5 1
CNPRC, 2Dept of Medical Microbiology & Immunology, UC Davis, 3Center for Comparative Medicine, School of Veterinary Medicine and School of Medicine, UC Davis, 4AIDS and Cancer Viruses Program, Leidos Biomedical Research, Inc., Frederick National Laboratory, , 5Division of Experimental Medicine, Dept of Medicine, UC San Francisco, 6Dept of Pathology and Laboratory Medicine, School of Veterinary Medicine, UC Davis
RhCMV‐vectored SIV vaccines have been shown to provide significant protection against SIVmac239 challenge of juvenile and adult animals. It is unclear if infants will mount similar immune responses to those of adults or if the vaccine will perform similarly for protection against SIV. This question is particularly important because infants are an important target population for vaccination campaigns. In this study, we evaluated (i) T‐cell responses of young (8‐10‐month‐old) animals to RhCMV/SIV Gag vaccine and (ii) whether previous infection with wild‐type RhCMV affects immune responses or protection. We vaccinated 12 naïve and 12 RhCMV‐seropositive rhesus macaques with RhCMV/SIV Gag. The vaccine is based on RhCMV 68‐1 and codes for SIVmac239 Gag p57 protein under control of the EF‐ 1ɑ promoter. Soon after vaccination, naïve animals manifested remarkably higher SIV‐specific T‐cell responses than previously seropositive animals, while at later points this difference was not observed. Seventeen weeks after the first vaccination, 12 animals were challenged orally with low but increasing doses of SIVmac251, of which 11 were eventually infected. Of the 11 infected animals, one maintained low viremia (<700 copies/ml) for several weeks and finally reached undetectable viral load (<15 copies/ml) 22 weeks after first detection of viremia. One animal resisted SIV acquisition for at least seven weeks and remains negative. We also analyzed the class‐Ia‐, class II‐ and Mamu‐E‐restricted responses to SIV Gag‐derived peptides, some of which are known Mamu‐E‐restricted epitopes. The frequency of Mamu‐E‐restricted CD8+ T‐cell responses to SIV‐Gag(69) supertope averaged 0.3% of all CD8+ T cells or 0.4% of CD8+ memory cells just before SIV challenge. Apparent class II‐restricted responses were also detected but not yet confirmed using class II‐expressing cell lines. Our findings show that macaque infants can mount Mamu‐E‐restricted and protective immune responses to RhCMV/SIV vaccines within a short span of time after vaccination.
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25 NHP AIDS research services at the Wisconsin national primate research center Eileen A. Maher1, Shelby L. O'Connor1,2, Roger W. Wiseman1, Eva G. Rakasz1, Thomas C. Friedrich1,3, David H. O'Connor1,2 1
Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 2Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 3Department of Pathobiological Sciences, University of Wisconsin‐Madison
Research Services at the Wisconsin National Primate Center (WNPRC) has developed multiple fee‐for‐ service assays to support investigators studying simian immunodeficiency virus (SIV) in non‐human primates (NHPs). Genetic Services performs SIV deep sequencing and MHC genotyping from cynomolgus, rhesus, and pig‐tailed macaques. SIV stocks that are used to infect animals will be deep sequenced free of charge provided investigators agree to make this important information publicly available. Genetic Services also analyzes additional immune gene loci (killer immunoglobulin‐like receptor [KIR], tripartite motif protein 5α [TRIM5α]) that may affect SIV replication. Immunology Services performs standardized ELISPOT and flow cytometry‐based immune assays (e.g., phenotype and functional capability of memory and naive T cell populations, follicular helper T cells, regulatory T cells), in addition to conducting experiments to evaluate SIV vaccines. Immunology Services develops customized staining panels to characterize the frequency and functional capacity of diverse immune cell populations, such as various NK cell subsets. Virology Services performs quantitative viral load assays and characterized virus stocks for in‐vivo challenge. They also provide a range of assays to measure the SIV latent reservoir. Academic and for‐profit investigators have access to these services by contacting key Research Services staff listed on the WNPRC website (https://www.primate.wisc.edu/?page_id=1461). NHP investigators interested in using WNPRC animal resources are encouraged to contact the Scientific Protocol Implementation (SPI) unit (http://www.primate.wisc.edu/?page_id=1471). SPI staff manage and perform the animal portion of research projects. The overall goal of Research Services and SPI is to advance research through providing high quality state‐of‐the‐science data in a timely way to support investigators and maximize discovery from SIV studies.
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26 Delayed SIV infection in vaccinated, rectally‐challenged Mamu‐B*08+ rhesus macaques in the absence of anti‐Env humoral responses Dr. Mauricio Martins1, Dr. Young Shin1, Mr. Lucas Gonzalez‐Nieto1, Mr. Martin Gutman1, Ms. Aline Domingues1, Ms. Helen Maxwell1, Dr. Diogo Magnani1, Mr. Michael Ricciardi1, Ms. Nuria Pedreño‐Lopez1, Mr. Varian Bailey1, Ms. Kim Weisgrau2, Dr. John Altman3, Dr. Christopher Parks4, Dr. Keisuke Ejima5, Dr. Brandon George5, Dr. David Allison5, Dr. Eva Rakasz2, Dr. Saverio Capuano III2, Dr. Jeffrey Lifson6, Dr. Ronald Desrosiers1, Dr. David Watkins1 1
University Of Miami, 2University of Wisconsin‐Madison; Wisconsin National Primate Research Center, 3Emory University, 4International AIDS Vaccine Initiative, 5University of Alabama at Birmingham, 6AIDS and Cancer Virus Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research
Approximately 50% of rhesus macaques expressing the major histocompatibility complex class I (MHC‐I) allele Mamu‐B*08 control chronic phase simian immunodeficiency virus (SIV) replication. This phenotype is associated with CD8+ T‐cell responses targeting three Mamu‐B*08‐restricted SIV epitopes: Vif RL8, Vif RL9, and Nef RL10. Indeed, we have previously shown that even a poorly immunogenic vaccine encoding these epitopes can increase the incidence of elite control in SIVmac239‐infected Mamu‐B*08+ monkeys. Notably, recent studies suggest that lentiviral infections are vulnerable to early interception by vaccine‐induced T‐cell responses. Given the propensity of Mamu‐B*08+ animals to mount efficacious SIV‐specific CD8+ T‐cells, we hypothesized that vaccine‐elicited CD8+ T‐cell responses targeting the aforementioned epitopes would prevent systemic infection following repeated intrarectal (IR) challenges with SIVmac239. To test this, we delivered genes encoding Vif RL8, Vif RL9, and Nef RL10 to ten Mamu‐B*08+ macaques using a recombinant (r)Ad5 prime followed by boosting with rVSV and rRRV–a persistent herpesviral vector. This new regimen was significantly more immunogenic than our previous vaccine. Six mock‐vaccinated MHC‐I‐matched macaques served as the controls for this experiment. Strikingly, 6/6 control versus 6/10 vaccinated animals became infected after six IR challenges. We kept challenging these uninfected monkeys and, after the 10th exposure, three vaccinees remained aviremic. Though encouraging, these SIV acquisition rates were not significantly different (P = 0.35). Interestingly, the pre‐challenge frequency of vaccine‐induced, SIV‐specific CD8+ T‐ cells in lymph nodes expressing the B‐cell follicle homing marker CXCR5 correlated directly with the number of SIV exposures delivered to each vaccinee (r = 0.7; P = 0.03). Collectively, these data suggest that at least in the context of protective MHC‐I alleles, vaccine‐induced CD8+ T‐cells may be able to prevent systemic lentivirus replication in the absence of Env‐specific antibodies. This potential link between vaccine‐elicited CXCR5+ CD8+ T‐cells and immunodeficiency virus infection requires additional investigation.
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27 Modeling the evolution of SIV sooty mangabey progenitor virus towards HIV‐2 using humanized mice Dr. Kimberly Schmitt1, Dr. Dipu Mohan Kumar1, Mr. James Curlin1, Ms. Stephanie Feely2, Ms. Leila Remling‐Mulder1, Dr. Mark Stenglein1, Dr. Shelby O'Connor3, Dr. Preston Marx2, Dr. Ramesh Akkina1 1
Colorado State University, Dept. of Microbiology, Immunology & Pathology, 2Tulane National Primate Research Center, 3University of Wisconsin School of Medicine and Public Health
Human Immunodeficiency Virus Type 2 (HIV‐2) is thought to have arisen through multiple cross‐species transmission events of Simian Immunodeficiency Virus (SIV) from the sooty mangabey monkey (sm) into humans. Crucial information regarding various factors, such as selective pressure, immune suppression, host restriction factors and viral genomic changes, that led to its emergence in humans is lacking. Furthermore, there has not been an appropriate model to demonstrate SIV viral evolution into HIV‐2. To address this need, we used a humanized mouse (hu‐HSC‐mouse) model to recapitulate the necessary adaptations that SIVsm might have undergone during human transmissions to evolve into HIV‐2. Hu‐HSC mice were infected intraperitoneally with the sooty mangabey primary isolate SIVsmE041 and sequentially passaged for 5 generations in these hu‐mice. Each subsequent generation was monitored for viral load, CD4+ T cell depletion and next generation sequencing to determine nucleotide and amino acid substitutions compared to the stock virus. Plasma viremia was detectable in challenged mice by week 3 and CD4+ T cells gradually declined over each generation, similar to HIV‐2 infection. We also observed several amino acid substitutions that were nonsynonymous and fixed in multiple hu‐HSC mice across each of the 5 generations in the nef, env and vpr regions. The highest rate of substitution occurred in the nef and env regions and most were observed within the first two generations. These data demonstrated the utility of hu‐mice in modeling the SIVsm transmission to the human and evaluate its sequential evolution into a human pathogen like HIV‐2
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28 Effects of systemic and mucosal immunization of Rhesus Macaques with single‐cycle adenovirus vectors and envelope protein vaccines Mr. William Matchett1, Stephanie S. Anguiano‐Zarate1, Mary E. Barry1, Guojun Yang2, Pramod Nehete2, Siddappa N. Byrareddy3, Delphine C. Malherbe4, Nancy L. Haigwood4, Francois Villinger5, K. Jagannadha Sastry2, Michael A. Barry1 1
Mayo Clinic, 2MD Anderson Cancer Center, 3University of Nebraska Medical Center, 4Oregon National Primate Research Center, 5New Iberia Research Center
Background. Most adenovirus vaccines are replication‐defective Ads (RD‐Ads) engineered to prevent adenovirus infections. Unlike RD‐Ads, replication‐competent adenovirus (RC‐Ad) vectors can amplify transgenes up to 10,000‐fold, but they risk causing adenovirus infections. Methods. We engineered “single‐cycle” adenovirus (SC‐Ad) vectors by deleting their capsid protein IIIA. Ads were tested by single immunization in mice and hamsters. Macaques were immunized three times with SC‐Ads by different routes followed by protein boosts. Antibodies were measured by ELISA, ADCC, and neutralization. Cellular immune responses were measured by ELISPOT and flow cytometry. Results. Clade B env sequences G4 and F8 were inserted into SC‐Ad6 and 57. Rhesus macaques were vaccinated by the intramuscular (IM) or intranasal (IN) routes with SC‐Ad6‐G4. 4 weeks after single immunization, binding titers against F8 env were significantly higher in the IM group. Each group was boosted with SC‐Ad6‐F8 by either the IM or the IN route. Binding titers increased in all groups except the IN‐IN group. At week 13, animals were boosted by serotype‐switching with SC‐Ad57‐G4 resulting in binding titer increases for all groups except IN‐IN‐IN. Boosting with gp140 protein increased binding titers by 2 logs in all groups, including the refractory IN‐IN‐IN group. Animals immunized by only the IM route had high Tfh cells in PBMCs and low Tfh in lymph nodes. Conversely, IN‐primed animals had low Tfh in PBMCs and higher Tfh in lymph nodes. Animals immunized by only the IM route had lower neutralizing and ADCC activity than animals immunized by the mucosal IN route. Conclusions. SC‐Ads express more antigen per unit vaccine than RD‐Ads, and do not risk causing adenovirus infections. Mucosal immunization with SC‐Ad induced unique changes in immune correlates when compared to the traditional IM vaccine route. This suggests potential utility in the use of both SC‐ Ads and mucosal immunization routes in HIV vaccine strategies.
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29 Changes in the immune system driven by RhCMV and anellovirus infections Gema Mendez‐lagares1,2, Nicole Narayan1,2, Amir Ardeshir1, David Merriam1,2, Ding Lu1,2, Eric Delwart3,4, Dennis Hartigan O'Connor1,2,5 1
Uc Davis, 2California National Primate Research Center, 3Blood Systems Research Institute, 4Department of Laboratory Medicine, 5Division of Experimental Medicine
The celullar composition and functional capacity of the immune system vary dramatically between inviduals. Recent studies have shown that inter‐individual immunologic variability is driven predominantly by environmental factors including chronic viral infections like human cytomegalovirus (HCMV). HCMV is a common chronic viral infection that is asymptomatic in healthy individuals, yet produces clear and durable immunologic effects. Studies in humans have shown significant changes in adaptive and innate host immunity, such as generation of effector‐memory CTLs and expansion of natural killer (NK) cells, which also adapt their phenotype to the presence of HCMV. However, the mechanisms by which HCMV exerts such a broad influence on immunity are unclear, as are the interactions between effects of HCMV and those of other chronic viral infections. We investigated the effect of rhesus CMV (RhCMV) and other common viral infections on composition of the immune system in rhesus macaques. As in humans, RhCMV infection has massive effects on the frequency and function of both T cells and NK cells. Most impressively, we found that CD4+ T cells with the capacity to synthesize IL‐4 were present in significant numbers only among RhCMV+ animals. The acquisition of this phenotype was associated with and driven in culture by presence of CD83‐deficient antigen‐presenting cells (APCs), especially myeloid dendritic cells (mDCs). Anellovirus infection also impacted the immune system, but to a lesser degree than RhCMV; for example, anellovirus abundance were significantly associated to the frequency of CD8+ memory T cells and the frequency of memory CD8+ T cells producing TNF‐α. Thus, RhCMV and other common viral infections play an important role in modulating T cell homeostasis and have a broader effect on other cell populations of lymphoid and myeloid origin.
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30 Investigation of macrophages serving as a viral reservoir in pediatric SAIDS Dr. Kristen Merino1, Dr. Chie Sugimoto2, Dr. Yanhui Cai3, Dr. Carolina Allers1, Dr. Angela Amedee4, Dr. Christopher Destache5, Dr. Pavan Kumar Prathipati5, Dr. Elizabeth S Didier1, Dr. Marcelo J Kuroda1 1
Tulane National Primate Research Center, 2Laboratory of International Epidemiology, Dokkyo Medical University, HIV‐1 Immunopathogenesis Laboratory, Wistar Institute, 4LSU HSC School of Medicine, 5Creighton University, School of Pharmacy and Health Professions 3
HIV‐infected children have higher viral loads and faster progression to AIDS than adults. Interestingly T‐ cell depletion and degree of immune activation do not consistently correlate with disease progression in children. Our hypothesis is that maturing infant monocytes/macrophages are more susceptible to infection and promote earlier establishment of viral reservoirs. Preliminary data showed increased monocyte turnover with death of short‐lived tissue macrophages predicted SAIDS in SIV‐infected adult macaques. Uninfected newborn macaques exhibited higher monocyte turnover than adults and rates were further increased after SIV infection, remaining high until SAIDS. Infection of 3‐4 month‐old monkeys with declining monocyte turnover, resulted in disease progression that was intermediate to newborns and adults. Our goal is to optimize treatment of pediatric HIV. In rhesus infants infected with SIVmac251, cell turnover was measured using BrdU uptake. Flow cytometry and confocal imaging allowed phenotypic characterization of cells in blood and tissues. PCR and in situ hybridization were used to quantify SIV. Plasma cytokine analysis was performed using Milliplex beads. ART drug concentrations were measured using HPLC. Various inoculation routes and ages showed differences in acute VL and monocyte turnover. None of the i.v.‐infected newborns controlled VL, despite receiving the same ART treatment as adults, and in contrast to orally‐infected newborns, where most controlled viremia but to varying degrees. Additional discrepancies present with age differences in infection and systemic immune responses (plasma cytokines). Infected tissues involved with uncontrolled VL status are macrophage‐rich, with long‐lived macrophage subsets. No clear depletion of CD4+T cells was found for blood or tissues and no tissue pathology was observed. Ongoing works suggest magnitude of long‐lived macrophage infection may dictate effectiveness of ART in pediatric SIV. Infant metabolism and ART pharmacokinetics require further investigation. These results highlight distinctions between pediatric and adult immunology and SIV pathogenesis and stress the value for using age‐matched controls.
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31 RANTES‐DT390 as a therapeutic for SIV Reservoir Elimination Mr. David Merriam1, Ms. Connie Chen1, Dr. Gema Mendez Lagares1, Dr. Francois Villinger2, Dr. Dennis Hartigan‐O'Connor1 1
UC‐Davis, 2University of Louisiana at Lafayette
The HIV reservoir forming at the earliest stages of infection is likely composed of CCR5+ cells, because these cells are the targets of transmissible virus. Restriction of the CCR5+ reservoir, particularly in gut, may be needed for subsequent cure attempts. Strategies for killing or depleting CCR5+ cells have been described, but none have been tested in vivo in non‐human primates and the extent of achievable depletion from tissues is not known. Here we investigate the efficacy of a novel cytotoxic treatment for targeting and eliminating CCR5+ cells in young rhesus macaques, an immunotoxin consisting of the endogenous CCR5 ligand RANTES fused with a truncated Diphtheria exotoxin (RANTES‐DT390). We demonstrate the ability of this exotoxin to deplete CCR5+ cells in Rhesus Lamina Propria Lymphocytes and PBMCs. We compare this treatment’s efficacy with two previous cytotoxic treatments; the first, an immunotoxin using Pseudomonas exotoxin in place of the Diptheria toxin (RANTES‐PE38); the second, a primatized bispecific antibody for CCR5 and CD3. These data open an avenue to investigation of combined early ART treatment and CCR5+ reservoir depletion for cure of HIV‐infected infants.
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32 Immunological control of simian immunodeficiency virus following an adoptive transfer prime‐pull strategy Mariel Mohns1, Dawn Dudley1, Justin Greene2, Eric Peterson3, David O'Connor1,3 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison, 2Vaccine and Gene Therapy Institute and Oregon National Primate Research Center, Oregon Health & Science University, 3Wisconsin National Primate Research Center, University of Wisconsin‐Madison
CD8 T cells are essential for the immunological control of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Adoptive transfers of SIV‐specific lymphocytes in the non‐human primate (NHP) model are a powerful tool for exploring cellular immunity and provide insight into novel strategies for the clinical treatment of HIV. However, CD8 T cells often undergo enlargement with in vitro expansion, which then become entrapped in the lung upon adoptive transfer. Here we demonstrate the utility of a prime‐pull strategy to localize transferred CD8 T cells to mucosal surfaces and reduce frequencies in the lung. Cells were “primed” by expanding SIV Nef₁₀₃‐₁₁₁RM9‐specific lymphocytes from two female Mauritian cynomolgus macaques. Prior to transfer, the chemokines CXCL9 and CXCL10 were administered topically to the vaginal tract to “pull” activated CD8 T cells toward this mucosal surface. Following inoculation with SIVmac239, both animals established control of SIV replication to below 3.5 log₁₀ copies/ml by 7 weeks post‐infection (wpi). We obtained a cervical biopsy at 2wpi, but were unable to isolate lymphocytes from the tissue at that timepoint. Therefore, to assess prime‐pull driven trafficking to mucosal sites of interest, we performed a second prime‐pull transfer in the same animals after 60wpi and mucosal tissues were interrogated at necropsy the following day. While only trace amounts of transferred lymphocytes were detected in the tissues of the reproductive tract, gut mucosa, and lymph nodes, localized populations were well established in the liver and spleen. Large populations were identified in the lung, however, there were 30‐fold fewer cells when compared to our previous adoptive transfer studies without prime‐pull. Given the immunological impact on viral load, the prime‐pull strategy warrants further investigation as an effective method of reducing cellular entrapment in the lung and recruiting cells to mucosal surfaces, as might be desired in cure studies.
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33 SChIV MT145: A novel SHIV bearing the env of a primary chimpanzee SIV strain with antigenic cross‐reactivity to HIV‐1 V1V2 bNAb epitopes Alexander Murphy1, Dr. Hui Li1, Wenge Ding1, Jessica Smith1, Dr. Beatrice H. Hahn1, Dr. George M. Shaw1 1
University Of Pennsylvania
Background: New strategies and new immunogens are needed to elicit broadly neutralizing antibodies (bNAbs) in humans and in animal models. One novel approach is to infect rhesus macaques (RMs) with a persistently replicating viral clone, in this case an infectious SIVmac251‐based chimera that contains the Env of a widely divergent HIV‐1‐related virus SIVcpz MT145 from a wild‐living, naturally‐infected Pan troglodytes troglodytes chimpanzee. Methods: The Env gp 140 of SIVcpz MT145, which shares antigenic cross‐reactivity with HIV‐1 V1V2 bNAb epitopes (mbio 6:e00296,2015), was constructed as a SChIV (simian‐chimpanzee immunodeficiency virus) chimera by methods recently described by our laboratory (PNAS 113:E3413, 2016). SChIV variants were created by substituting residues at Env position 375 (S/M/Y/H/W/F) found naturally across the phylogenetic spectrum of SIVs in infected African primates. Results: SChIV MT145 containing the naturally‐occurring Env375H allele was transfected into 293T cells and found to be efficiently packaged into virions (~1000 ng/ml p27Ag) and infectious on TZM cells (~106 IU/ml), comparable to other SHIVs containing primary HIV‐1 Envs (PNAS ibid). SChIV MT145.375H replicated efficiently in human and rhesus CD4+ T‐cells in vitro. Studies are underway comparing the relative replication rates of the six Env375 variants of SChIV MT145 in rhesus cells in vitro and in RMs in vivo. Conclusion: These findings extend the SHIV Env delta375 design strategy recently described to include novel, antigenically diverse Env immunogens. This will enable immunofocusing strategies to be pursued with the aim of eliciting neutralizing antibodies to predetermined bNAb epitopes.
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34 Noninvasive monitoring of CD4 T cells at multiple mucosal tissues after intranasal vaccination in Rhesus Macaques Dr. Pramod Nehete1,3, Dr. Stephanie Dorta‐Estremera2, Dr. Guojun Yang2, Dr. Hong He4, Bharti Nehete1, Dr. Kathryn Shelton1, Dr. Michael Barry5,6,7,8, Dr. Jagannadha Sastry1,2,3 1
The University of Texas MD Anderson Cancer Center, Department of Veterinary Sciences, Bastrop, Texas, TX 78602, The University of Texas MD Anderson Cancer Center, Department of Immunology, Houston, Texas, TX 77030, 3The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030, 4The University of Texas MD Anderson Cancer Center, Department of Stem Cell Transplantation, Houston, Texas, TX 77030, 5Department of Internal Medicine, Division of Infectious Diseases, Mayo Clinic, Rochester, MN 55902, USA, 6Department of Molecular Medicine,Mayo Clinic, Rochester, MN 55902, USA , 7Department of Immunology,Mayo Clinic, Rochester, MN 55902, USA, 8Translational Immunology virology and Biodefense Program, Mayo Clinic, Rochester, MN 55902, USA 2
Studies in nonhuman primates for prospective immune cell monitoring subsequent to infection and/or vaccination usually rely on periodic sampling of the blood samples with occasional collections of biopsies from mucosal tissues because of safety concerns and practical constraints. Here we present evidence in support of cytobrush sampling of oral, rectal, and genital mucosal tissues as a noninvasive practical approach for the phenotypic analyses of different T cells subsets de novo as well as prospectively after intranasal immunization in rhesus macaques. Significant percentages of viable lymphocytes were obtained consistently from both naive and chronically SIV‐infected rhesus macaques from ongoing studies. The percentages of CD3+ T cells in the blood were significantly higher compared to those in the mucosal tissues analyzed in the naïve animals, while in the SIV+ animals the CD3+ T cells were significantly elevated in the rectal tissues, relative to all other sites analyzed. In the naïve, but not SIV+ macaques, the rectal and vaginal mucosal tissues, compared to oral mucosa and blood, showed higher diversity and percentages of CD4+ T cells expressing the HIV entry co‐receptor CCR5 and mucosal specific adhesion (CD103) as well as activation (HLA‐DR) and proliferation (Ki67) markers. Sequential cytobrush sampling from the oral, rectal, and genital mucosal tissues was performed in SIV+ rhesus macaques from an ongoing study where they were administered intranasal immunization with adenoviral vectored vaccines incorporating the green fluorescent protein (GFP) reporter gene. We detected a transient increase in GFP+ CD4 T cells in only oral mucosa suggesting limited mucosal trafficking. In general, proliferating (Ki67+) CD4+ and CD8+ T cells transiently increased in all mucosal tissues, but those expressing the CCR5, HLA‐DR, and CD103 markers exhibited minor changes. We propose non‐invasive cytobrush sampling as a practical approach for effective and prospective immune monitoring of the oral‐genital mucosal tissues in NHP.
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35 Induction of mutant epitope‐specific CD8+ T cells is an indicator of the beginning of viral control failure in SIV controllers. Dr. Takushi Nomura1,2, Dr. Hiroshi Ishii1, Dr. Sayuri Seki1, Dr. Hiroyuki Yamamoto1, Dr. Kazutaka Terahara3, Dr. Tomoyuki Miura4, Dr. Tetsuro Matano1,5 1
AIDS Research Center, National Institute of Infectious Diseases, 2Center for AIDS Research, Kumamoto University, Department of Immunology, National Institute of Infectious Diseases, 4Institute for Frontier Life and Medical Sciences, Kyoto University, 5Institute of Medical Science, University of Tokyo
[Background] We previously reported CD8+ T cell‐based long‐term aviremic SIV controllers possessing Mamu‐ A1*065:01, indicating that broadening of CD8+ T‐cell targets can be an indicator of residual viral replication (PLoS Pathog 11:e1005247, 2015). In these SIV controllers, Mamu‐A1*065:01‐restricted Gag241‐249 epitope‐specific CD8+ T‐cell responses play an important role in the control. Gag241‐249‐ specific CD8+ T cells frequently select for a viral escape GagD244E mutation in SIV non‐controllers. This mutation does not diminish epitope binding to MHC‐I but results in escape from CD8+ T‐cell recognition. In the present study, we examined changes in cross‐reactivity of Gag241‐249‐specific CD8+ T cells in these SIV controllers. [Materials & Methods] By using the wild‐type Gag241‐249‐Mamu‐A1*065:01 tetramer and the mutant Gag241‐249.244E‐ Mamu‐A1*065:01 tetramer, we examined CD8+ T‐cell responses targeting dominantly wild‐type Gag241‐249 and mutant Gag241‐249.244E, respectively, in long‐term aviremic SIV controllers as well as SIV non‐controllers possessing Mamu‐A1*065:01. [Results] SIV non‐controllers elicited CD8+ T cells dominantly detected by the wild‐type Gag241‐249 tetramer, followed by selection of the GagD244E mutation and induction of CD8+ T cells dominantly detected by the mutant Gag241‐249.244E tetramer in a few months post‐infection. CD8+ T cells dominantly detected by the mutant Gag241‐249.244E tetramer were induced in six months post‐infection in some SIV controllers but not in other controllers. Importantly, the former selected for the GagD244E mutation after one year, but the latter not. [Conclusion] Some SIV controllers elicited CD8+ T cells dominantly targeting the mutant Gag241‐244.244E epitope in six months, which were not induced in other controllers. The GagD244E mutation became dominant and detectable after one year in the former inducing the mutant‐specific CD8+ T cells but not in the latter without detectable mutant‐specific CD8+ T cells. These results suggest that induction of the mutant epitope‐specific CD8+ T cells is an indicator of the beginning of viral control failure in SIV controllers.
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36 Simian immunodeficiency virus SIVmac239 infection and simian human immunodeficiency virus SHIV89.6P infection result in progression to AIDS in cynomolgus macaques from Asian country origin Dr Tomotaka Okamura1, Dr Yasuhiro Yasutomi1 1
Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition
Simian immunodeficiency virus (SIV) infection models in cynomolgus macaques are important for analysis of the pathogenesis of immunodeficiency virus and for studies on the efficacy of new vaccine candidates. Tsukuba Primate Research Center in Japan has a large‐scale breeding colony of experimental cynomolgus macaques that were obtained from Indonesia, Malaysia and Philippines. However, very little is known about the pathogenesis of SIV or simian human immunodeficiency virus (SHIV) in cynomolgus macaques from different Asian country origins. In the present study, we analyzed the infectivity and pathogenicity of CCR5‐tropic SIVmac and those of dual‐tropic SHIV89.6P inoculated in cynomolgus macaques of Indonesia, Malaysia or Philippines origin. The plasma viral loads in macaques infected with either SIVmac239 or SHIV89.6P were maintained at high levels. CD4+ T cells in the macaques infected with SIVmac239 gradually decreased. All of the macaques infected with SHIV89.6P showed precipitously reduced CD4+ T cell numbers within 6 weeks post infection. Eight of the eleven macaques infected with SIVmac239 were euthanized due to AIDS symptoms after 2 to 4.5 years. Four of the five macaques infected with SHIV89.6P were euthanized due to AIDS symptoms after 1 to 3.5 years. We also analyzed cynomolgus macaques infected intrarectally with a repeated low, medium, or high dose of SIVmac239, SIVmac251, or SHIV89.6P. Infection was confirmed by quantitative RT‐PCR at more than 5000, 300 and 500 TCID50 for SIVmac239, SIVmac251 and SHIV89.6P, respectively. The present study indicates that cynomolgus macaques of Asian origin are highly susceptible to SIVmac and SHIV infection by intravenous and mucosal routes. These models will be useful for studies on virus pathogenesis, vaccine, and therapeutics against HIV/AIDS.
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37 Retinoic acid (RA) upregulates α4β7 on CD4+ T cells and activates latent reservoirs Mr Omalla Olwenyi1,2, Dr Nanda Kishore Routhu1, Dr Neil Sidell2, Dr Aftab A Ansari2, Dr Siddappa N. Byrareddy1 1
University Of Nebraska Medical Centere, 2Emory University School of Medicine
Current HIV cure studies are focused on the eradication of the viral reservoir but are limited by low sensitivity and poor reproducibility. This is partly due to the low frequency of cells harboring latent replication competent virus. Furthermore, the process of reactivation of the latent reservoir remains entirely stochastic resulting in an underestimation of the size of the viral reservoir. We have developed an approach that seeks to improve the sensitivity based on upregulating α4β7 on CD4+ T cells. Here we utilized PBMCs from SIV infected rhesus macaques that were a) ART treated, b) those that spontaneously developed low viral loads, and for purposes of control PBMCs from SIV naïve macaques. The PBMCs were activated in vitro using anti‐CD3/CD28 + IL2 in the presence/absence of retinoic acid (RA). Viral loads were measured using RT‐PCR assays, levels of p27 was quantified using ELISA assay and flow cytometry was utilized to measure α4β7 expression levels. The addition of RA led to >2‐fold increase in the density of α4β7 expression by CD4+ T cells in all cultures. Of interest was the finding that the addition of RA to cultures of PBMCs from SIV‐infected/ART‐treated and the spontaneously low viral load macaques led to a 3‐fold and 5‐fold increase, respectively, in p27 levels in RA‐treated activated cells compared to cultures without RA. Finally, the in vivo administration of RA to macaques with low viral loads led to a moderate increase in plasma viral loads. We conclude that targeting the RA pathway can be a useful approach to improve the efficiency of currently used methods to upregulate α4β7 expression and activate latent reservoirs.
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38 Evaluating vaginal film formulations as multipurpose prevention technologies (MPT) in the macaque model Dr. Dorothy Patton1, Yvonne Cosgrove Sweeney1, Dr Lisa Rohan2 1
University Of Washington, 2Magee Womens Research Institute
Background: Various delivery platforms are under investigation as multipurpose prevention technologies (MPT) designed to prevent HIV, sexually transmitted infection (STI) and/or pregnancy. Vaginal films provide a discreet, inexpensive, manipulable base into which multiple drugs can be formulated for quick or extended release. Purpose: We have assessed various film formulations in the macaque model by evaluating film dispersal by MRI imaging (gadolinium contrast), film dissolution by visualization with colposcopy and drug delivery using pharmacokinetic (PK) assays. Methods: Each film formulation was optimized to support its drug (API) component(s). Film platforms were designed to promote quick drug release (within one day), or extended release over several days to weeks. When necessary for visualization, a blue dye was incorporated into the film base to allow for visual determination of film dissolution in the macaque vagina. One film was administered to each macaque, who was monitored by MRI or by colposcopy to document intravaginal film dispersal or dissolution, respectively. Additionally, vaginal swabs were collected for API detection (PK) to establish drug delivery curves for each active drug component. Results: MRI studies determined that vaginal films disperse throughout the vaginal canal, cervix to introitus, as they dissolve. No signal was detected in uterine or intraperitoneal spaces. Sixteen film formulations were monitored for dissolution after vaginal placement. Dissolution characteristics were profoundly affected by components incorporated in film manufacture. These studies revealed dissolution times ranging from 90 minutes to one month, reflective of excipients in the film platforms. Drug release mimicked dissolution rates, as API were detected for similar periods, generally clearing from secretions within days of complete film dissolution. Discussion: Preclinical studies in the pigtailed macaque have established the utility of vaginal films as a customizable delivery platform for products intending to prevent HIV, STI and/or pregnancy.
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39 HIV/SHIV‐specific, CCR5‐edited CAR T‐Cells engraft and persist in acutely infected nonhuman primates Christopher Peterson1,2, Courtnee Clough3, Malika Hale3, Taylor Mesojednik3, Bryan Sands3, Hans‐Peter Kiem1,2, Thor A. Wagner2,3, David J. Rawlings2,3 1
Fred Hutchinson Cancer Research Center, 2University of Washington, 3Seattle Children’s Research Institute
Background: Persistent viral reservoirs are a primary barrier to HIV cure. Although gene and cell therapy approaches protect cells against infection in vivo, recent findings suggest that additional approaches will be required to actively target and destroy latently infected cells in stably suppressed patients. Here, we engineered T‐cells expressing chimeric antigen receptor (CAR) molecules to redirect virus‐specific immunity. We evaluated these products in autologous pigtail macaques infected with simian/human immunodeficiency virus (SHIV) containing an HIV envelope. Methods: T‐cells were isolated from uninfected animals and cryopreserved. Following SHIV infection, cells were thawed and transduced with lentiviral vectors expressing virus‐specific CAR molecules. The CAR extracellular domain consisted of a single chain variable fragment (scFv) derived from the HIV broadly neutralizing antibody VRC07. These cells were also CCR5 gene‐edited to protect against infection. Engineered T‐cells were infused into SHIV+ animals during acute, unsuppressed infection; engraftment and persistence of CAR T‐cells was tracked in peripheral blood and secondary lymphoid tissues longitudinally and at necropsy. Results: Pre‐infusion tests of our T‐cell products showed efficient CAR expression, CCR5 gene disruption, and virus‐specific function. Following infusion into autologous animals, CAR+ cells persisted in peripheral blood and in relevant secondary lymphoid tissues. At necropsy, the percentage of CAR+ T‐cells in various tissues correlated with viral antigen load. Conclusions: We demonstrate that CAR+ T‐cells persist in vivo in nonhuman primates. Our methods, including use of a reduced‐intensity conditioning regimen to increase T‐cell engraftment in these animals, were safe and feasible, even during unsuppressed infection. We are currently evaluating the SHIV‐specific function of CAR+ cells in vivo, optimizing conditioning and dosing parameters to maximize engraftment and persistence of these cells, and initiating studies in SHIV+, combination antiretroviral therapy‐suppressed animals.
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40 The optimization of pediatric vaccine regimens to prevent mother‐to‐ child transmission of HIV through breast milk Dr. Bonnie Phillips1, Genevieve Fouda2, Justin Pollara2, Pamela Kozlowski3, Anthony Moody2, Guido Ferrari2, Sallie Permar2, Kristina De Paris1 1
Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill, 2Duke University Medical Center, Duke Human Vaccine Institute, 3Department of Microbiology, Immunology and Parasitology, Louisiana State University Health Sciences Center
In 2015, half of approximately 150,000 infants infected by mother‐to‐child transmission of HIV acquired the infection through breastfeeding. While ART access significantly reduced the chances of in utero and peripartum HIV transmission, the effect on breastmilk HIV transmission has been less impressive. A pediatric vaccine that rapidly elicits a protective immune response has the potential to prevent breastfeeding infants from orally acquiring HIV. We optimized a pox‐prime/rgp120 pediatric HIV‐1 vaccine in newborn rhesus macaques to induce long‐ lasting, functional HIVenv‐specific antibody responses by testing the following variables: HIVenv immunogen administration, immunization intervals, adjuvant, and the addition of a broadly neutralizing antibody at birth. We compared 4 vaccine regimens: i) Env protein alone, (ii) poxvirus vector expressing HIVenv prime followed by a Env protein boost, or (iii) co‐administration of the poxvirus vector and protein; all groups received three immunizations at three‐week intervals starting at birth. Group 4 received the co‐administration regimen at 6‐week intervals. Env protein immunizations were further tested with and without adjuvants. All vaccine regimens induced Env‐specific plasma binding IgG antibodies and had ADCC activity. The Env protein strategy induced the highest V1V2‐specific IgG and Tier 1 neutralizing antibodies, while the Extended Interval group had higher frequencies of Env‐specific memory B cells and was the only regimen to induce Env‐specific systemic and mucosal antibodies. Currently, we are testing whether the administration of a broadly neutralizing antibody, necessary for protection before the infant has developed vaccine‐induced immunity, interferes with vaccine‐induced induction of Env‐specific antibodies. Few studies have compared pediatric HIV vaccine strategies in parallel to identify a regimen that can induce highly functional and persistent antibody responses in infants to prevent breast milk transmission of HIV and with the potential to boost in adolescence prior to sexual maturity.
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41 Full‐length killer‐cell immunoglobulin‐like receptor transcript discovery in Indian rhesus macaques Trent Prall1, Julie Karl2, Michael Graham2, Hailey Bussen2, Cecelia Shortreed2, Roger Wiseman1,2, David O'Connor1,2 1
Wisconsin National Primate Research Center, University Of Wisconsin‐Madison, 2Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison
Background Killer cell immunoglobulin‐like receptors (KIRs) are a complex family of receptor expressed on the surface of natural killer (NK) cells. Through their interaction with major histocompatibility complex class I molecules, KIRs are able to modulate the immune responses of NK cells in response to viral infection. Because KIR genes are polymorphic and vary in number on different haplotypes within the genome, characterizing KIR genotypes is a challenging task. Understanding KIR variation in Indian rhesus macaques is important because KIR genotypes are associated with protective effects against viral pathogens. Materials and Methods Full‐length KIR1D, KIR2DL, KIR3DS, and KIR3DL transcripts were characterized by cDNA amplicon sequencing using PacBio’s RSII and Sequel platforms. A cohort of 112 Indian rhesus macaques that included 50 parent/offspring pairs was selected for allele discovery and genotyping analyses. Mamu‐KIR haplotypes were defined by identifying shared alleles between pairs of parent and offspring. Results Initial analysis of PacBio data from 44 animals identified 40 novel Mamu‐KIR allelic variants and confirmed/extended 41 Mamu‐KIR alleles to include full open reading frames. In addition, 11 Mamu‐KIR haplotypes were characterized among animals sequenced with sufficient depth of coverage. Conclusions In our initial analysis, we significantly expanded the database of known Mamu‐KIR allele sequences and confirmed the effectiveness of our deep sequencing methodology. As additional animals are sequenced, we expect that the number of characterized novel KIR allele sequences and haplotypes will increase significantly. Funding This research was supported by contract HHSN272201600007C from the National Institute of Allergy and Infectious Diseases, National Institutes of Health.
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42 Tracking NKG2C+ memory and memory‐like NK cells in SIV and CMV infections of rhesus macaques Dr. Daniel Ram1, Dr. R. Keith Reeves1 1
Beth Israel Deaconess Medical Center/Harvard Medical School
Natural killer (NK) cells typify the non‐specific effector arm of the innate immune system. Surprisingly, recent studies report antigen‐specific NK cell memory in mice, humans, and nonhuman primate (NHP) models. Expression of the activating receptor, NKG2C, identifies hCMV‐specific human NK cells, and may also be required for anti‐HIV/SIV memory‐like NK cell responses. NKG2C is highly related to the inhibitory molecule, NKG2A. Despite the critical roles of NHP in modeling HIV (SIV) and hCMV (rhCMV), phenotypic identification of memory counterparts is lacking, primarily due to technical limitations in quantifying NKG2C+ memory cells. We phenotyped peripheral NK cells from rhesus macaques chronically infected with SIV (n=18), rhCMV (n=12) or those negative for both viruses (SPF) (n=24) using flow cytometry (using commercial antibodies against rhesus CD3, NKG2A, CD14 and CD20) and PrimeFlow (Affymetrix), which merges flow cytometry with fluorescence in situ hybridization, in order to identify and phenotype single cells that express NKG2A and NKG2C transcripts (KLRC1 and KLRC2, respectively). Rhesus NK cells were identified using a standard gating strategy: CD3‐CD14‐CD20‐NKG2a+. Frequencies of circulating NK cells were 3‐fold greater in CMV and SIV‐infected compared to SPF animals, suggesting a virus‐specific increase in total NK cells. As expected from previous reports (Labonte et al., Clin Exp Immunol 2004), anti‐NKG2A antibodies could not distinguish between NKG2C and NKG2A. However, via PrimeFlow we found that SPF NK cells had greater frequencies of KLRC1 compared to elevated KLRC2 in NK cells from rhCMV‐/SIV‐infected animals. Our data suggests that KLRC2+ (but not KLRC1+) NK cells expand during CMV and SIV infection – corroborating results from human NK cell studies. Our data confirm for the first time that rhCMV and SIV induce NKG2C+ memory NK cells in NHP models and can be accurately quantified. This technical advance may inform HIV vaccine and cure‐related modalities that rely on macaque models.
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43 Preserved cytokine and cytotoxic effector functions of gut mucosal Vδ1 γδ T Cells in SIV‐infected Macaques Adrienne S. Woods1, Faith R. Schiro1, Pyone P. Aye1, Stephen E. Braun1, Ronald S. Veazey1, Marcelo J. Kuroda1, Andrew A. Lackner1, Namita Rout1 1
Tulane National Primate Research Center, Tulane University
HIV and SIV infections result in perturbations in γδ T cells with a peripheral loss of δ2 subsets and expansion of δ1 subsets. However, the impact of lentiviral infection on the functions of δ1 cells that represent the majority of tissue‐resident γδ T cells remains unclear. In this study, we evaluated δ1 and δ2 γδ T cells in blood, gut mucosa, lung and liver of rhesus macaques with or without SIV infection. Gut mucosal γδ T cells are more skewed towards Th17 function in contrast to the more Th1 and cytotoxic functionality of γδ T cells in the liver of SIV‐naive macaques, suggesting tissue‐specific immune functions. Macaque γδ T cells are predominantly δ1 in the peripheral blood as well as mucosal tissues regardless of SIV infection. Both δ1 and δ2 subsets in the gut mucosa display similar Th17 cytokine responses to mitogen stimulation; however, the δ1 subsets produce significantly higher Th1 cytokines TNFα and IFNγ. In contrast to impaired Th17 cytokine responses by δ2 cells in chronically SIV‐infected macaques, the δ1 subsets maintained similar levels of TNFα, IFNγ, IL‐17, and GranzymeB production to that of uninfected macaques. These results suggest that although SIV infection results in a decline and functional loss of δ2 subsets, the cytokine‐producing and cytotoxic effector functions of δ1 subsets are preserved in early and chronic SIV infection and may be targeted for killing of HIV/SIV‐infected cells, particularly in tissues.
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44 A novel strategy to adapt SHIV‐E1 carrying env from an RV144 volunteer to rhesus macaques: coreceptor switch and final recovery of a pathogenic virus with exclusive R5 tropism Hanna Scinto1,2, Sandeep Gupta1, Swati Thorat3,4, Muhammad Mukhtar1, Anthony Griffiths1, Jennifer Delgado1, Elizabeth Plake1, Hemant Vyas1, Amanda Strickland1, Siddappa Byrareddy3,4, David Montefiori5, Celia LaBranche5, Ranajit Pal6, Jim Treece6, Sharon Orndorff6, Maria Ferrari6, Deborah Weiss6, Agnes‐ Laurence Chenine7, Ruth Bonchik7, Robert McLinden7, Nelson Michael7, Jerome Kim7, Merlin Robb7, Supachai Rerks‐Ngarm8, Punnee Pitisuttithum9, Sorachai Nitayaphan10, Ruth Ruprecht1,2,3,4 1
Texas Biomedical Research Institute, 2UT Health San Antonio, 3Dana‐Farber Cancer Institute, 4Harvard Medical School, 5Duke University Medical Center, 6Advanced Biosciences Laboratories Inc., 7Henry M. Jackson Foundation, 8 Department of Disease Control, Ministry of Public Health, 9Mahidol University, 10Armed Forces Research Institute of Medical Sciences
*This abstract should be considered because of the novel method of SHIV adaptation to rhesus macaques that led to recovery of an R5‐tropic, mucosally transmissible, pathogenic SHIV. In addition, we have new deep sequencing data that identified changes in Env during serial passage and those important for coreceptor switch.* The Phase III RV144 trial conducted in Thailand remains the only one with limited, yet significant efficacy in decreasing the risks of HIV‐1 acquisition. In Thailand, circulating recombinant forms of HIV‐1 clades A/E (CRF01_AE) predominate; in such viruses, env is derived from HIV‐1 clade E (HIV‐E). We constructed a simian‐human immunodeficiency virus (SHIV); env isolated from a RV144 placebo recipient was cloned into SHIV‐1157ipd3N4 that contains long terminal repeats (LTRs) with duplicated NF‐κB sites, thus resembling HIV‐1 LTRs. We devised a novel strategy to adapt the parental infectious molecular clone (IMC), R5 SHIV‐E1, to rhesus macaques: simultaneous depletion of B cells and CD8+ cells followed by intramuscular inoculation of proviral DNA and repeated administrations of cell‐free virus. This led to sustained, high viremia and CD4+ T‐cell depletion. Passage 3 virus unexpectedly caused acute, irreversible loss of CD4+ T cells and turned out to be dualtropic. Virus and IMCs were reisolated from earlier passages, combined, and used to complete the adaptation through two more macaques. The final isolate, SHIV‐E1p5, was exclusively R5‐tropic with a tier 2 neutralization phenotype. When administered intrarectally to a naïve macaque, high viremia and CD4+ T‐cell depletion ensued, indicative of AIDS. Deep sequencing revealed that the Env amino acid sequence was conserved ~99%; X4/dualtropic strains had evolved independently from an early branch of parental SHIV‐E1. In conclusion, R5 SHIV‐E1p5 is mucosally transmissible, replicates robustly, and is pathogenic. Our primate model data reveal that SHIV‐E1 and its progeny recapitulate important aspects of HIV‐1 transmission and pathobiology in humans.
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45 Novel full‐length major histocompatibility complex class I allele discovery and haplotype definition in pig‐tailed macaques Matthew Semler1, Roger W. Wiseman1,2, Julie A. Karl1, Michael E. Graham1, Samantha M. Gieger1, David H. O'Connor1,2 1
Department of Pathology and Laboratory Medicine, University of Wisconsin‐Madison,, 2Wisconsin National Primate Research Center, University of Wisconsin‐Madison, Madison
Background Pig‐tailed macaques (Macaca nemestrina, Mane) are important models for human immunodeficiency virus (HIV) pathogenesis studies. Their ability to be infected with minimally modified forms of HIV makes them a unique animal model to mimic human infection with HIV and progression to acquired immune deficiency syndrome (AIDS). However, compared to the major histocompatibility complex (MHC) of rhesus and cynomolgus macaques, variation in the pig‐tailed MHC and the impact of individual alleles on pathogenesis is understudied. Materials and Methods In this study, we used Illumina MiSeq deep sequencing and circular consensus sequencing with Pacific Biosciences single‐molecule real‐time technology in order to determine full‐length MHC class I transcript sequences for 198 pig‐tailed macaques from three different breeding centers. We then used the newly discovered full‐length alleles to infer Mane‐A and Mane‐B haplotypes containing groups of class I alleles that co‐segregate due to physical linkage. Results In total, we characterized 348 full‐length Mane‐A, Mane‐B, and Mane‐I transcript sequences that were used to define 86 Mane‐A and 106 Mane‐B class I haplotypes. Of the 348 defined transcript sequences, 241 were completely novel sequences and 72 were extensions of previously described alleles to full‐ length. The 35 remaining sequences mapped to previously characterized full‐length transcript sequences. Pacific Biosciences technology allowed us to refine these high‐resolution Mane‐A and Mane‐ B haplotypes to the level of synonymous allelic variants. Conclusions The newly defined Mane‐A and Mane‐B haplotypes and full‐length transcript sequences provide an important resource for infectious disease researchers as certain MHC haplotypes have been shown to provide exceptional control of simian immunodeficiency virus (SIV) replication and prevention of AIDS‐ like disease in nonhuman primates. The increased allelic resolution provided by Pacific Biosciences sequencing also provides a benefit to transplant research since researchers can more specifically match haplotypes between donors and recipients, thus reducing the risk of graft‐versus‐host disease. Funding This research was supported by contract HHSN272201600007C from the National Institute of Allergy and Infectious Diseases and the National Institutes of Health.
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46 Targeting CAR T cells to B cell follicles to cure HIV Infection PJ Skinner1, A Hajduczki2, P Haran1, MS Pampusch1, G Mwakalundwa1, S Bolivar‐Wagers2, DA Vargas‐ Inchaustegui2, EG Rakasz3, E Connick4, EA Berger2 1
Department of Veterinary and Biomedical Sciences, University of Minnesota, 2Laboratory of Viral Diseases, National Institutes of Allergy and Infectious Diseases, The National Institutes of Health, 3Wisconsin National Primate Research Center, University of Wisconsin‐Madison, 4Division of Infectious Diseases, University of Arizona
BACKGROUND There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic disease, HIV replication is concentrated within TFH cells in B cell follicles, where low levels of virus‐ specific CTL permit ongoing viral replication. We have previously shown that elevated levels of SIV‐ specific CTL in B cell follicles is linked to decreased levels of viral replication in follicles, and decreased plasma viral loads. We developed a strategy for targeting follicular viral‐producing TFH cells using anti‐ viral chimeric antigen receptor (CAR) T cells co‐expressing the follicular homing molecule CXCR5. We hypothesize that anti‐viral CAR/CXCR5 expressing T cells when infused into an SIV‐infected animal or an HIV‐infected individual, will home to B cell follicles, suppress viral replication, and lead to long‐term durable remission of SIV and HIV. METHODS We engineered gammaretroviral transduction vectors for co‐expression of an all‐human bispecific anti‐ HIV CAR (designated CD4‐MBL, displaying high potency and breadth) and human CXCR5 on human PMBC‐derived T cells. We also generated vectors for expression of the corresponding rhesus macaque variants of these molecules on rhesus T cells. We measured viral suppression by CAR/CXCR5 T cells in vitro, and the ability of CAR/CXCR5 T cells to migrate to follicles using a novel ex vivo tissue migration assay. RESULTS The CAR/CXCR5 T cells are functional as demonstrated by their potent suppression of SIVmac239 and SIVE660 replication in in vitro. The CAR/CXCR5 T cells also concentrated in B cell follicles ex vivo in tissues. We plan to test this cure strategy in SIV‐infected macaques infused with CAR/CXCR5 T cells. CONCLUSION CAR/CXCR5 T cells exhibit virus suppression and follicular homing suggesting that they could provide long‐term durable remission (functional cure) of HIV and SIV infections.
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47 Use of Quanterix Simoa ultrasensitive immunoassay for assessing residual virus in non‐human primate models of AIDS Dr. Adrienne E. Swanstrom1, Robert Gorelick1, Guoxin Wu2, Bonnie J. Howell2, Anitha Vijayagopalan1, Gregory Q. Del Prete1, Julian Bess Jr.1, Jeffrey D. Lifson1 1
AIDS And Cancer Virus Program, 2Merck
BACKGROUND: Determining the extent of residual virus, including inducible latent virus, during treated HIV/SIV infection is critical for the evaluation of candidate AIDS virus “cure” interventions. Nucleic acid‐ based assays have provided the greatest sensitivity to date, but expression of viral proteins is more immunologically relevant. Thus, we developed assays capable of measuring low levels of viral protein for use in nonhuman primate (NHP) models relevant for evaluating “cure” interventions. MATERIALS and METHODS: Using the Quanterix Simoa platform, we developed an ultrasensitive digital bead‐based immunoassay that detects SIV p27 in culture supernatants expressed from macaque cells infected with SIVs or SHIVs. Additionally, we have used the Quanterix p24 kit to detect viral proteins expressed from macaque cells infected with a minimally chimeric simian tropic HIV‐1 (st‐HIV‐1). RESULTS: In preliminary studies the limit of detection (LOD) for our novel SIV p27 assay is approximately 48 fg/mL, the lower limit of quantification (LLOQ) is approximately 92 fg/mL, with a dynamic range >3 logs. The Quanterix HIV p24 kit has a reported LOD of 2.7 fg/mL and a LLOQ of 10 fg/mL. In conjunction with ultrasensitive qRT‐PCR, we are able to measure levels of viral RNA and Gag protein in supernatants of cultured cells from SIV, SHIV, or st‐HIV‐1 infected rhesus and pigtail macaques, stimulated ex vivo. Enhancement of the SIV p27 assay sensitivity as well as efforts to quantify Gag from additional analytes (e.g. cell lysates, plasma) are underway.
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48 Distribution of long‐lived and short‐lived macrophages in the intestinal tract of SIV‐infected rhesus macaques Naofumi Takahashi1, Carolina Allers1, Cecily Midkiff2, Xavier Alvarez2, Elizabeth Didier3, Woong‐Ki Kim4, Marcelo Kuroda1 1
Division of Immunology, Tulane National Primate Research Center, 2Division of Comparative Pathology, Tulane National Primate Research Center, 3Division of Microbiology, Tulane National Primate Research Center, 4Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School
Background: We previously reported that increased monocyte turnover in blood is a better predictor of AIDS disease progression in SIV‐infected macaques than plasma viral load and CD4+ T cell level. The increased death rate of lung interstitial macrophages caused by SIV infection also directly correlated with the magnitude of lung tissue damage. In contrast to CD163+CD206‐ interstitial macrophages (IM), SIV infection of CD163+CD206+ long‐lived alveolar macrophages (AM) did not induce cell death suggesting these long‐lived AM differ from the shorter‐lived IM and might serve as long‐term viral reservoirs during SIV infection. Since the intestinal tract is also a site of high viral replication, we examined the migration and localization of intestinal macrophages in SIV‐infected vs uninfected macaques. Method: SIV‐infected rhesus macaques were intravenously injected with dextran and BrdU 2‐3 months and 48 hours, respectively, prior to necropsy and collection of the intestinal tissues. We used confocal microscopy to analyze the phenotype and localization of macrophage subpopulations in the colon tissue sections. Results: In lamina propria of SIV‐uninfected macaques, the CD163+CD206+ macrophages subset were dominant, whereas in SIV‐infected animals the majority of the macrophages were CD163+CD206‐. In the submucosa, CD163+CD206+ macrophages were observed equally in uninfected and infected macaques. On the other hand, BrdU‐labeled, newly‐migrated, recently‐dividing, and short‐lived CD163+ macrophages were found in the lamina propria. Conversely, dextran‐labeled long‐lived CD163+ macrophages were observed in submucosa and muscular mucosa. Conclusion: These results suggest that monocytes traffic to the lamina propria of the intestinal tract, differentiate and mature into CD163+CD206+ macrophages, and are then destroyed by SIV infection followed by an accumulation of immature CD163+CD206‐ macrophages. Also, long‐lived CD163+CD206+ macrophages in the submucosa remained for at least two months (based on dextran label retention), suggesting their role as an SIV tissue reservoir.
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49 Directed homing of adoptively transferred T‐cells through homing marker transduction Matthew Trivett1, Daniel Burke1, Lori Coren1, Claire Deleage1, Greg Del Prete1, Jake Estes1, Jeffery Lifson1, David Ott1 1
Leidos Biomedical Research, Inc.
Adoptive cellular immunotherapy holds great promise, but optimizing persistence and trafficking of infused cells remain challenging. Directed localization of T‐cells to immunologically relevant sites would increase the utility of autologous T‐cell transfer for investigation of in vivo antiviral activity or other therapeutic applications. Past nonhuman primate (NHP) studies with SIV‐specific, but otherwise unmodified cells, showed no significant homing to or retention in lymphoid tissue sites of viral replication and no effect on host virus burden. We therefore evaluated the ability of transduction with retroviral vectors to achieve constitutive expression of different homing markers to enhance preferential trafficking to specific tissues in vivo after adoptive transfer. We showed recently that engineering cells for CXCR5 expression facilitates localization of infused CD8+ effector T‐cells into the B‐ cell follicles of lymphoid tissues. Building on this work we now demonstrate that T cells engineered to express CCR9 localize to and are retained in GALT tissue after infusion at a much higher rate than co‐ infused non‐CCR9 transduced cells and can be detected for at least 4 months after transfer. Preliminary results with cells engineered to express CD62L and CCR7 show homing to and retention of infused T‐ cells in lymph nodes. Together, these results suggest it should be possible to direct infused T‐cells engineered for co‐expression of SIV‐specific T‐cell receptors and various homing receptors to different tissues. Facilitated entry into B‐cell follicles will allow us to ascertain the ability of such infused T‐cells to reduce or eliminate virus reservoirs in this immunological sanctuary in chronically infected macaques. Further, establishment of adoptively transferred SIV‐specific T‐cells in LN and GALT tissues prior to SIV challenge will enable us to examine the quantitative or qualitative aspects of SIV specific T‐cell immunity necessary to blunt primary viremia or potentially prevent infection. Contract HHSN261200800001E ‐ Funded by the National Cancer Institute
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50 Functionally preserved MAIT cells are associated with controlled SHIV infection Dr Amudhan Murugesan, Dr Chris Ibegbu, Dr Tiffany Styles, Sakeenah Hicks, Mr Micheal Sabula, Dr Pradeep Reddy, Andrew Jones, Dr Rama Amara, Dr. Vijayakumar Velu1 1
Yerkes National Primate Research Center
Mucosa‐associated invariant T‐cells (MAIT) home to mucosal sites, exert antimicrobial activity against microorganisms and maintains gut homeostasis. Here, we studied the distribution, phenotype, and function of MAIT cells during chronic SHIV infection using rhesus macaques (RM). Two groups of RM, healthy and SHIV infected were studied. Lymphocytes from various tissues were stained for MAIT cell markers (CD161++,TCR7.2+ and MR1 tetramer) and analyzed using flow cytometry for their association with SHIV infection. Similar to humans, we found significant fraction of (~2%) naive RM CD8 T cells co‐express MAIT cell markers TCR7.2, CD161 including MR1. Phenotypically these cells express high levels of IL‐18R, CCR6 and display central memory phenotype (CD28+CD95+) and produce higher levels of IL‐17, TNF‐α, IFN‐γ compared to non‐MAIT cells. During chronic SHIV infection, the frequency of MAIT cells are unchanged in the blood but are decreased in the rectum. Tissue analysis showed a significant enrichment of MAIT cells in liver (~8%), BAL (3%) than in blood (1%),spleen(0.5%), lymph node(0.13%) and gut (0.2%). Importantly, tissue resident CD69+MAIT cells expressed higher levels of T cell exhaustion marker PD‐1 compared to blood. These PD‐1+ MAIT cells lack proliferating capacity and cytokine production during progressive infection. Interestingly, during controlled SHIV infection (<1000 copies), MAIT cells were functionally preserved in blood, which correlated inversely with viral load and associated directly with CD4 T cells. In addition, these preserved MAIT cells express higher levels of memory differentiation marker IL‐7Rα and treatment with IL‐7 in vitro enhance the proliferation of MAIT cells during chronic infection. These data demonstrate that MAIT cells are decreased during chronic SHIV infection and express PD‐1 but preserved cytokine+ MAIT cells correlated inversely with viral levels. Future studies focused on blocking the PD‐1 pathway to retrieve MAIT cell function during chronic SHIV infection may yield therapeutic benefit to HIV and other coinfection.
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51 Comparing the effects of progestin‐based contraception and luteal phase of the menstrual cycle on putative HIV susceptibility genes in pig‐ tailed Macaques Dr. Ajay Sundaram Vishwanathan1, Dr. Steven E Bosinger2, Gregory K Tharp2, Nirav B Patel2, Chunxia Zhao1, James Mitchell1, Shanon Ellis1, Ellen N Kersh1, Janet M McNicholl1 1
CDC (Centers For Disease Control & Prevention), 2Yerkes National Primate Research Center, Emory University
Background: In women and macaques, the progesterone‐rich luteal phase of the menstrual cycle is associated with vaginal changes that may increase HIV risk compared to the follicular phase. We hypothesized that progestin‐based contraception including depot medroxyprogesterone acetate (DMPA) and levonorgestrel (LNG)‐containing combined oral contraceptives (COC) might exacerbate expression of genes affecting HIV susceptibility more than the luteal phase. Methods: Archived vaginal pinch biopsies were obtained from two separate studies in pig‐tailed macaques. In one study, animals were given two doses of DMPA (0.5, 1.5, or 2.5 mg/kg; n=3 per group) 4 weeks apart. In another, they were given daily high (n=4) or low (n=3) dose COC for at least 60 days. The high dose COC was 0.066mg LNG + 0.0132mg ethinyl estradiol (EE); low dose: 0.033mg LNG + 0.0066mg EE. The menstrual cycle was divided into early (days 1‐8) and late (days 9‐16) follicular, early (days 17‐24), and late (days 25‐32) luteal phases based on progesterone levels. Pre‐ and post‐treatment time‐points were analyzed via Affymetrix Genome Arrays. Downstream analyses were performed using Partek Genomics Suite software version 6.5. To determine differentially expressed genes (DEGs), we combined data from all DMPA or COC doses and used a paired t‐test, requiring a ≥2.0‐fold difference, followed by gene set enrichment and principal component analyses. Results: There was no significant dose effect on gene expression. Compared to the late luteal phase, we observed 570 DEGs post‐DMPA and 571 post‐COC; of these 240 DEGs were common. There was reduction in antiviral gene expression by DMPA (serpin B1, IL‐12) and COC (SLPI, cystatins, β‐defensin); also, there was enhanced expression of HIV‐promoting genes including integrin ITGB7 (post‐COC) and tetraspanins (post‐DMPA). Conclusions: When compared to gene expression in the luteal phase, the use of progestin‐based contraception triggers greater reduction of antiviral genes and enhancement of HIV‐promoting genes.
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52 Optimized method for extracting high quality RNA from FACS-sorted, intracellular stained primary rhesus macaque lymphocytes Dr. Yichuan Wang1, Dr. David Ott1, Matthew Trivett1, Dr. Jeffrey Lifson1 1
Leidos Biomedical Research, Inc.
High throughput gene expression analysis has become a key technique to characterize transcription profiles of different cell types that are usually isolated by Fluorescence‐Activated Cell Sorting (FACS). FACS allows identification of distinct lineages, subtypes, or functional states of cells based on the expression of surface and intracellular molecules stained with various fluorescence‐labeled antibodies. Analysis of intracellular markers by FACS typically requires fixation and permeabilization of cells, procedures which can compromise RNA integrity, which is critical for successful downstream gene expression assays, using techniques such as RNA‐seq, Microarray, Nanostring and RT‐qPCR. We developed a method, based on optimized working conditions for cell thawing, surface staining, fixation, permeablization, RNA stabilization and enzyme digestion, enabling consistent extraction of high quality RNA (RIN≥7.5) from frozen/thawed, fixed/permeablized and FACS sorted primary rhesus macaque lymphocytes. RT‐qPCR showed comparable gene expression levels in FACS sorted, fixed/permeabilized cells and fresh cells. This approach for extracting intact high quality RNA from FACS‐sorted, intracellular stained cells could serve as a basic technique used across multiple immunobiological disciplines.
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53 A novel PKC activator, 10‐methyl‐aplog‐1 efficiently reactivate latent HIV‐1 in combination with a BET inhibitor JQ1. Ayaka Washizaki1, Megumi Murata1, Yin Pui Tang2, Yohei Seki1, Kazuhiro Irie3, Hirofumi Akari1 1
Primate Research Institute, Kyoto University, 2Medical School, University of Exeter, 3Graduate School of Agriculture, Kyoto University
Shock and kill therapy attracts attention as a promising strategy leading to sterilizing cure. Here we show a novel protein kinase C (PKC) activator, 10‐methyl‐aplog‐1 (10MA‐1) as a promising latency‐ reversing agent (LRA). 10MA‐1 is a simplified analog of aplysiatoxin, of which tumor promoting and proinflammatory activities have successfully been removed. The activity of 10MA‐1 per se to reactivate latently infected HIV‐1 is modest, but is greatly augmented (up to 100‐fold) as a synergetic effect in combination with a BET inhibitor, JQ1. It is notable that 10MA‐1 exhibits no significant cytotoxic activity even when 10MA‐1 is used with JQ‐1. Together with the notion that 10MA‐1 can be synthesized at reasonable cost, 10MA‐1 appears to be advantageous as compared with other PKC activators. We previously reported a novel nonhuman primate model of HIV latency (Seki et al., 34th NHP symposium for AIDS, 2016). In this model, cynomolgus macaques experimentally infected with macaque‐tropic HIV‐1 carrying the R5‐tropic Env developed acute viremia, followed by long‐term latent infection. However, depletion of CD8+ T cells induces detectable viremia, indicating the presence of reservoir cells. Importantly, follicular helper T lymphocyte in germinal center of lymph nodes appears to be the major reservoir as well as the site of HIV‐1 replication. Thus, this model shares characteristics of HIV‐1 latency in humans and it is therefore suitable for the study of shock and kill therapy. Presently, we are further seeking to examine if the combination of 10MA‐1 and JQ‐1 efficiently reactivate the latently infected HIV‐1 in the macaque’s lymph nodes ex vivo. Based on these data, we hope to proceed to proof‐of‐concept study of the therapy in our macaque model.
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54 Resources for non‐human primate models of Zika virus Andrea Weiler1, Matthew Aliota2, James Weger‐Lucarelli3, Matthew Semler1,4, Gabrielle Barry1, Dawn Dudley4, Christina Newman4, Shelby O'Connor4, David O'Connor1,4, Gregory Ebel3, Thomas Friedrich1,2 1
WNPRC, 2UW‐Madison Pathobiological Sciences, 3Microbiology, Immunology and Pathology ‐Colorado State University, 4UW‐Madison Cellular and Molecular Pathology
Zika virus (ZIKV), the causative agent of Zika fever, is causing an ongoing outbreak in the Americas. Congenital ZIKV infection is associated with a range of birth defects collectively termed congenital Zika syndrome (CZS), for which there is no treatment or cure. Because ZIKV was understudied prior to the American outbreak, there is a great need to better understand ZIKV infection and how it affects pregnancy outcomes. Non‐human primates are susceptible to ZIKV infection, and their reproductive physiology closely resembles that of humans. We have developed a suite of resources to facilitate studies of ZIKV pathogenesis and preclinical testing of vaccines and therapeutics in non‐human primates, particularly in pregnancy. For example, we have developed a “universal” QRT‐PCR assay that can be used to quantify vRNA from both African‐ and Asian‐lineage ZIKV in plasma, other body fluids, and tissues. This assay established that pregnant dams infected with Asian‐lineage ZIKV at the Wisconsin National Primate Research Center exhibit prolonged viremia as compared with non‐pregnant animals. We are also producing ZIKV stocks for NHP studies. An extensively characterized large‐scale stock of Puerto Rican ZIKV (ZIKV‐PRVABC59, 2015) is available for in‐vivo studies. We are also developing and characterizing a “barcoded” ZIKV‐PRVABC59 derivative, a molecularly cloned virus bearing a set of synonymous mutations that can be used to track viral genetic diversity in vivo, e.g., for studies of tissue compartmentalization. Preliminary experiments suggest that the “barcoded” virus can replicate in NHP with kinetics similar to those of wild type ZIKV.
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55 Does Simian Betaretrovirus (SRV) Antibody in Baboons (Papio sp.) Indicate Infection? Joann Yee1, Richard Grant2, Koen Van Rompay1, Jeffrey Roberts1, Joe Simmons3, James Papin4 1
California National Primate Research Center, University of California, 2Washington National Primate Research Center, University of Washington, 3Michale E. Keeling Center for Comparative Medicine and Research, University of Texas MD Anderson Cancer Center, 4Department of Pathology, Division of Comparative Medicine, University of Oklahoma Health Science Center
The accurate serological detection of simian betaretrovirus (SRV) infection is an ongoing diagnostic challenge. The existence of antibody positive / virus negative time points over the course of infection have been documented in some macaque monkeys; however, reproducible antibody positivity with no virus detection at multiple time points have been observed in others. Similar SRV antibody reactivity has been observed in various species of baboons (Papio sp.); for which the scientific literature includes reports of endogenous but not exogenous betaretrovirus. These studies aim to either isolate an exogenous SRV from seropositive baboons or demonstrate that this antibody reactivity may not indicate SRV infection. In our initial experiments, peripheral blood mononuclear cells (PBMC) from 6 baboons demonstrating indeterminate or positive SRV antibody reactivity were isolated, stimulated and co‐cultured with known susceptible, uninfected macaque PBMCs, Raji, or SupT cells. Although some mild cytopathologic effects were noted in a few cultures, all were negative for SRV1‐5 DNA when tested by PCR for 8 weeks. In a second set of experiments PBMCs from 2 SRV seronegative baboon donors were isolated, stimulated, and inoculated with SRV1 or SRV 2 tissue culture virus. The cultures were observed and tested positive by SRV1‐5 PCR for 2 weeks. To ensure that the virus detected was not residual input, the cells were washed and sub‐cultured with fresh, uninfected PBMCs for 2 additional weeks. SRV1 PCR remained positive for both donors and SRV2 for one. Further analysis of the cells and supernatants is in progress. Results thus far indicate that although baboon PBMCs can be infected in vitro with SRV, virus has not been cultured from PBMCs from SRV antibody positive baboons. Future plans include transfusion studies to determine whether or not SRV virus stocks or SRV infected macaque blood can infect baboons in vivo.
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56 A dose-escalation study of pharmacologic inhibition of β-catenin signaling in healthy rhesus macaques PhD Michelle Zanoni1, PhD Maud Mavigner1, Dr. Jakob Habib1, Dr. Cameron Mattingly1, PhD Kirk Easley1, Dr. H Kouji2, Md. PhD. Ann Chahroudi1 1
Emory University, 2PRISM Pharma Co., Ltd.
Signaling pathways required for maintenance of long‐term CD4+ T‐cell memory are potential targets for an HIV cure. Besides its role in homeostasis of hematopoietic and cancer stem cells, the Wnt/β‐catenin signaling pathway regulates the balance between self‐renewal and differentiation of T memory stem cells (TSCM) and central memory T cells (TCM). Pharmacological manipulation of this pathway may offer an opportunity to reduce persistence of latently‐infected memory CD4+ T‐cells. To evaluate the safety profile of pharmacologic inhibition of β‐catenin signaling, we treated 11 healthy rhesus macaques with PRI‐724, an inhibitor of the β‐catenin co‐activator CBP, in a dose‐escalation study. Three dosing groups were treated with 20, 40, and 80 mg/kg s.c. of PRI‐724 daily for 12 weeks. Safety and toxicity monitoring was assessed using complete blood cell counts, serum chemistries and bone marrow reviews. T‐cell phenotyping was evaluated by flow cytometry in blood, lymph nodes, bone marrow and rectal biopsies at baseline, 4 week intervals during PRI‐724, and 4 weeks post‐PRI‐724 interruption. No clinical adverse events occured in the low dose group, with one injection site abscess observed in the intermediate dose group. Significant adverse effects were observed in the high dose group: severe weight loss (2/4 animals), elevated liver transaminases (3/4 animals) and injection site abscess (2/4 animals). Complete blood counts were not affected during PRI‐724, with the exception of high WBC count in one animal with an abscess. No interruption in trilineage hematopoiesis was observed in bone marrow. Flow cytometric analysis of CD4+ memory T‐cells subsets did not reveal a consistent reduction in the frequency of either TSCM or TCM, however. This work supports the safety of PRI‐724 at doses of 20 and 40 mg/kg and suggests that a combination of approaches may be more effective in promoting long‐lived memory CD4+ T‐cell differentiation as an HIV/SIV reservoir reduction strategy.
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57 Effects of rapamycin administration on immune responses in SIV‐ infected rhesus macaques Dr Widade Ziani1, Dr Xiaolei Wang1, Dr Kasi Russell‐Lodrigue1, Dr Ronald S. Veazy1, Dr Huanbin Xu1 1
The elimination of HIV latency and induction of neutralizing antibodies (nAb) represent the major obstacles in a cure for HIV infection. The immunosuppressive rapamycin inhibits PI3K‐AKT‐mammalian target of rapamycin (mTOR) signaling, which is reported to regulate follicular T helper cell accumulation and germinal center formation and improve immune protection against influenza in mice through dominant IgM responses, suggesting its potential therapeutic application in HIV infection. In this study, rapamycin was orally administrated for 14 days to evaluate the side effects and effects of rapamycin treatment in rhesus macaques with or without SIV infection. Our results indicated that rapamycin treatment had limited impact in the levels of alanine aminotransferase (ALT) and CD8+ T cells in peripheral blood, yet the percentage of B and CD4+ T cells is significantly reduced during rapamycin treatment. Notably, rapamycin did not increase levels of total or SIV‐specific IgM responses, but suppressed SIV‐specific IgG production in plasma. Rapamycin administration, albeit low to no cytotoxicity, resulted in rapid disease progression in SIV‐infected macaques, as indicated by all of the SIV‐infected macaques on rapamycin treatment whom maintained high viremia throughout viral infection and developed to final stage of AIDS within one year. Rapamycin, as a critical immunomodulatory compound, will need further investigation to define its optimal use either alone or in combination with antiretroviral therapy (ART).
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58 Effects of rapamycin administration on immune responses in SIV‐ infected rhesus macaques Dr Widade Ziani1, Dr Xiaolei Wang1, Dr Kasi Russell‐Lodrigure1, Dr Ronald S. Veazy1, Dr Huanbin Xu1 1
The elimination of HIV latency and induction of neutralizing antibodies (nAb) represent the major obstacles in a cure for HIV infection. The immunosuppressive rapamycin inhibits PI3K‐AKT‐mammalian target of rapamycin (mTOR) signaling, which is reported to regulate follicular T helper cell accumulation and germinal center formation and improve immune protection against influenza in mice through dominant IgM responses, suggesting its potential therapeutic application in HIV infection. In this study, rapamycin was orally administrated for 14 days to evaluate the side effects and effects of rapamycin treatment in rhesus macaques with or without SIV infection. Our results indicated that rapamycin treatment had limited impact in the levels of alanine aminotransferase (ALT) and CD8+ T cells in peripheral blood, yet the percentage of B and CD4+ T cells is significantly reduced during rapamycin treatment. Notably, rapamycin did not increase levels of total or SIV‐specific IgM responses, but suppressed SIV‐specific IgG production in plasma. Rapamycin administration, albeit low to no cytotoxicity, resulted in rapid disease progression in SIV‐infected macaques, as indicated by all of the SIV‐infected macaques on rapamycin treatment whom maintained high viremia throughout viral infection and developed to final stage of AIDS within one year. Rapamycin, as a critical immunomodulatory compound, will need further investigation to define its optimal use either alone or in combination with antiretroviral therapy (ART).
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Author Index Poster abstract authors are numbered 1‐58. Oral abstract authors are numbered 99‐510.
102, 402, 501
13, 30, 48
Almond , N
6, 18, 50, 203
8, 24, 29, 508
101, 102, 104, 201, 402
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1, 103, 307, 502
54, 103, 307, 502, 506
24, 209, 508
Bess Jr., J
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15, 41, 404, 408
9, 28, 37, 44, 310
Capuano III, S
26, 305, 506
Castleman , M
24, 209, 508
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Cosgrove Sweeney, Y
De Paris, K
8, 40, 203
Del Prete, G
47, 49, 510
49, 105, 510
13, 30, 48, 308
23, 33, 204, 210
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Dowell , S
32, 54, 506
103, 307, 502
49, 105, 510
8, 10, 506
304, 305, 307
14, 107, 208
Ferguson , D
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8, 10, 40, 306
25, 54, 103, 307, 502, 506
107, 301, 405, 509
Gale, Jr., M
Giles , E
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41, 45, 408, 506
32, 102, 104, 201, 501
22, 23, 33, 204, 210
28, 202, 405, 501
Hall , J
Ham , C
102, 104, 501
101, 201, 402, 409
Hartigan O'Connor, D 24, 29, 31, 508 Hayes, J
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Hewson , R
205, 207, 302
14, 107, 208
Jenkins , A
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15, 41, 45, 407, 408
Kempster , S
Kishore Routhu, N
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18, 40, 203
Kumar Janaka, S
13, 30, 43, 48, 308
2, 22, 210, 401
101, 102, 104, 201, 402, 501
2, 22, 23, 33, 110, 204, 210
24, 26, 47, 49, 52, 101, 209, 304, 305, 510
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Mendez Lagares, G
24, 29, 31, 508
29, 31, 508
Mohan Kumar, D
23, 33, 204
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24, 29, 508
15, 25, 32, 41, 45, 54, 404, 407, 408, 506
1, 25, 27, 54, 103, 305, 307, 502, 506
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Panoskaltsis‐Mortari, 102 A Papin, J
14, 107, 208
8, 10, 40, 506
7, 8, 10, 40, 203
101, 201, 402, 409
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51, 41, 404
25, 26, 46, 103, 506
102, 104, 201, 501
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11, 20, 300
Rose , N
Russo , R
102, 104, 201, 402, 501
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45, 54, 404, 506
2, 22, 23, 33, 110, 204, 210
33, 204, 205, 210
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Van Rompay, K
7, 8, 10, 55, 203, 500
43, 207, 302
von Bredow, B
von Laer, D
23, 204, 210
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102, 104, 501
54, 103, 307, 506
15, 25, 41, 45, 404, 407, 408
102, 201, 402, 501
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1, 103, 305
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Registrant List First Name Shaheed Debra Matthew Ali Hanne Alesia Cristian Amir Michael Pyone Pyone Alexis Priyankana Katharine Ed Michael Ryan Elena Neil Benjamin Nell Steven Stephen Ronald Allison Benjamin Hailey Siddappa Jessica Ann Tysheena Elizabeth Andrew Yvonne Kristen Anne Alan Kristina Gregory Cynthia Connor Saritha
Last Name Abdulhaqq Adams Aliota Andalibi Andersen Elyard Antoine Apetrei Ardeshir Axthelm Aye Balgeman Banerjee Bar Barker Barry Behrens Bekerman Berry Bimber Bond Bosinger Braun Brown Bucsan Burwitz Bussan Byrareddy Callery Chahroudi Charles Church Cogswell Cosgrove Crosno Cunanan Curtis De Paris Del Prete Derdeyn Driscoll D'Souza
Organization Oregon Health & Science University CDC UW‐Madison George Mason University
BIOQUAL Inc. Icahn School of Medicine‐Mount Sinai University of Pittsburgh University of California Davis Oregon Health & Science University Tulane National Primate Research Center UW‐Madison UW‐Madison AIDS Vaccine Research Lab University of Pennsylvania Rush University Medical Center Mayo Clinic UW‐Madison Gilead Sciences NIBSC Oregon Health & Science University TNPRC Yerkes NPRC/Emory University TNPRC Quality Biological, Inc Tulane National Primate Research Center Oregon Health & Science University UW‐Madison University of Nebraska Medical Center Texas Biomedical Research Institute Emory University Emory University NIH/Office of AIDS Research Rush University Medical Center University of Washington UW‐Madison Roche Sequencing Solutions, Inc. University of North Carolina at Chapel Hill University of North Carolina‐Chapel Hill Leidos Biomedical Research, Inc. Emory University University of Washington WNPRC
Dawn Debashis Amy Adam David Marissa Barbara Courtney Joanne Genevieve Valerie Thomas Sandra Yoshi Deborah Michael Gallya Luis Theresa Jonathan Glen Richard Justin Michael Shoko Nancy Amelia Katherine Shigeyoshi Anna Ann Sheri Raphaël Xintao Sanath Kumar Paul Samuel Julie Sudhir Pai Amitinder Brandon Laurel Arifa Adam Katarina Pam
Dudley Dutta Ellis Ericsen Evans Fahlberg Felber Ferrebee Flynn Fouda Fremont Friedrich Fuentes Fukazawa Fuller Gale Gannot Giavedoni Glissman Glock Grandea Green Greene Grunst Hagen Haigwood Haj Hammond Harada Heffron Hessell Hild Ho Tsong Fang Hu Janaka Johnson Johnson Karl Kasturi Kaur Keele Kelnhofer Khan Kleinman Kotnik Halavaty Kozlowski
UW‐Madison University of Nebraska Medical Center UW‐Madison WI National Primate Research Center WI National Primate Research Center Tulane University National Cancer Institute at Frederick Emory Vaccine Center University of Pittsburgh Duke Human Vaccine Institute Quality Biological UW‐Madison FDA Oregon Health & Science University University of Washington, WA NPRC University of Washington NIH/NIDCR Texas Biomedical Research Institute Tulane National Primate Research Center NIAID/DMID UW‐Madison Department of Pathology University of Washington Oregon Health & Science University UW‐Madison Oregon Health & Science University Oregon National Primate Research Center UW‐Madison Oregon Health & Science University National Institute of Infectious Diseases UW‐Madison Oregon Health & Science University DCM/ORIP/NIH CEA/IDMIT National Cancer Institute at Frederick UW‐Madison Yerkes National Primate Research Center University of Nebraska Medical Center UW‐Madison Yerkes NPRC/Emory University Tulane National Primate Research Center Frederick National Lab UW‐Madison CBER/FDA University of Pittsburgh Northwestern University LSU Health Sciences Center
Stephanie Lillian Celia Lynn David Julien Hui Jeffrey Ma Hailun Kawthar Eileen Natalia Matthew Jennifer Danijela Mauricio Rosemarie Tetsuro William Bonnie Bonnie George Margaret Arthur Janet Gema Kristen David Mariel Emma Paul Alexander Chitra Nicole Pramod Christina Douglas Takushi Veronica David Shelby Megan Tomotaka Afam Christian
Krislov Kuo Labranche Law Leggat Lemaitre Li Lifson Luo Ma Machmach Maher Makarova Mannino Manuzak Maric Martins Mason Matano Matchett Mathieson Mathieson Mayhew McCluskey Mcmillan McNicholl Mendez‐lagares Merino Merriam Mohns Mohr Munson Murphy Narayan Narayan Nehete Newman Nixon Nomura Obregon‐Perko O'Connor O'Connor O'Connor Okamura Okoye Olsen
UW‐Madison NIAID Duke University University of Washington National Institutes of Health IMVA, UMR1184, CEA‐INSERM‐UPSUD University of Pennsylvania Leidos Biomedical Research, Inc. University of Manitoba FDA CNPRC WI National Primate Research Center CDC WNPRC University of Washington Northwestern University University of Miami VRC ARC, National Institute of Infectious Diseases Mayo Clinic National Institutes of Health National Institutes of Health Roche Sequencing Solutions, Inc. US Agency for International Development DHVI CDC UC Davis Tulane National Primate Research Center UC‐Davis UW‐Madison UW‐Madison University of Washington University of Pennsylvania Roche Sequencing Solutions, Inc. University of California‐Davis MD Anderson Cancer Center UW‐Madison The George Washington University National Institute of Infectious Diseases UT Health Science Center‐San Antonio UW‐Madison UW‐Madison WA National Primate Research Center Tsukuba Primate Research Center Oregon Health & Science University Pactific Biosciences
Omalla Antonito Matthew Jean Dorothy Deborah Chris Trent Eva Daniel Jason Matt Mark Jeffrey Namita Nanda Kishore Jonah Jagan Charles Kimberly Jeffrey Nancy Hanna Yohei Matthew Spandan George Shaunna Nathan Joe Anjali Pam Jessica Lisa Scott Don Ellen Priya Yongjun Matthew Adrienne Andrew Naofumi David Aidin Belete
Olwenyi Panganiban Parsons Patterson Patton Persaud Peterson Prall Rakasz Ram Reed Reynolds Rodgers Rogers Rout Routhu Sacha Sastry Scanga Schmitt Schneider Schultz‐Darken Scinto Seki Semler Shah Shaw Shen Sherer Simmons Singh Skinner Smith Smith Smith Sodora Sparger Srinivasan Sui Sutton Swanstrom Sylwester Takahashi Tampa Tavakoli‐Tameh Teferedegne
University of Nebraska Medical Center Tulane National Primate Research Center University of Melbourne NIH/Office of AIDS Research University of Washington Johns Hopkins University School of Medicine Fred Hutchinson Cancer Research Center UW‐Madison UW‐Madison Beth Israel Deaconess Medical Center Oregon Health & Science University UW‐Madison University of Pittsburgh Baylor College of Medicine TNPRC‐Tulane University University of Nebraska Medical Center Oregon Health & Science University MD Anderson Cancer Center University of Pittsburgh Colorado State University Northwestern University WNPRC UT Health San Antonio Kyoto University UW‐Madison Beth Israel Deaconess Medical Center University of Pennsylvania Duke University UW‐Madison UT MD Anderson Cancer Research Center NIAID/NIH University of Minnesota Perelman School of Medicine Texas Biomedical Research Institute Beth Israel Deaconess Medical Center Center For Infectious Disease Research University of California‐Davis CDC NIH NCI Vaccine Branch UW‐Madison AIDS and Cancer Virus Program Oregon Health & Science University Tulane National Primate Research Center Promega UW‐Madison FDA
Bargavi Gregory Matthew Koen Ronald Vijayakumar Vaiva Ajay Sundaram Agneta Yichuan Jonathan Ayaka Andrea Carol Ryan Roger Matthew Helen Huanbin Yasuhiro Joann Michelle Widade
Thyagarajan Timmel Trivett Van Rompay Veazey Velu Vezys Vishwanathan von Gegerfelt Wang Warren Washizaki Weiler Weiss Westergaard Wiseman Wood Wu Xu Yasutomi Yee Zanoni Ziani
Global HIV Vaccine Enterprise Oregon National Primate Research Center Leidos Biomedical Research, Inc. California National Primate Research Center Tulane National Primate Research Center Yerkes National Primate Research Center University of Minnesota CDC Bioqual Inc. Leidos Biomedical Research, Inc. National Institute for Health Kyoto University WNPRC FDA/CBER UW‐Madison UW‐Madison Center for Infectious Disease Research Oregon Health & Science University Tulane National Primate Research Center Tsukuba Primate Research Center California National Primate Research Center Emory University TNPRC
Things to do, places to see… Downtown Dining Opportunities The unique food scene in Madison has endless options that are all very close. We would like to provide you some of the local favorites all within walking distance. Old Fashioned ‐ Wisconsin‐themed, retro‐style tavern offering beers, brats & cheese curds (all sourced in‐ state). o http://www.theoldfashioned.com/lunch‐dinner‐menus/ Great Dane ‐ A changing roster of craft brews & pub eats served in a lively venue with beer garden & pool tables o http://www.greatdanepub.com/menus Sardine ‐ Hip bistro with creative French fare & a lively bar in a former warehouse with Lake Monona views o http://sardinemadison.com/dinner Tornado Steakhouse ‐ Old‐school steakhouse with a rustic, supper‐club vibe serving classic chops, seafood & cocktails o http://www.tornadosteakhouse.com/menu/ Graze ‐ Lively gastropub plating farm‐to‐table comfort fare in a modern glass venue with Capitol views o http://www.grazemadison.com/#!menu/c5a2 Harvest ‐ Upscale spot overlooking Capitol Square offering farm‐to‐table New American fare. o http://www.harvest‐restaurant.com/menu.php DLUX ‐ Upscale haunt serving gourmet burgers & artisanal cocktails in a modern, trendy setting o http://dluxmadison.com/menu/?menu=Main Pig in a Fur Coat ‐ Hip, cozy bistro serving small & large Mediterranean plates, beer & wine at butcher‐ block tables o http://apiginafurcoat.com/menu/ Tipsy Cow ‐ Comfortable brewpub with outdoor seating, a big beer selection & artful menu of burgers & bar bites o http://www.tipsycowmadison.com/menus/tipsymenuf11.pdf Downtown Madison Nightlife Not only is the downtown scene a place to experience during the day, but the nightlife is sure to not to disappoint. The possibilities are just a few minutes’ walk away. Blue Velvet Lounge ‐ Plush cocktail lounge in dimly lit, bi‐level digs with a big martini menu & happy‐hour deals. o www.thebluevelvetlounge.com/ Madison’s ‐ Casual, 2‐level bar with artisanal cocktails & pub grub plus TV sports & a cozy downstairs lounge o www.madisonsdowntown.com Vintage Spirits & Grill ‐ Bar & grill with a retro vibe with a big craft beer list including house brews & an outdoor patio. o http://www.vintagemadison.com/ Nitty Gritty ‐ Longtime bar & grill featuring free birthday beer, cocktails or soda plus pub grub & TVs. o www.thegritty.com/ The Brink Lounge ‐ Nightlife & event venue featuring live jazz, blues & other music, plus bar bites, wine & martinis. o https://www.thebrinklounge.com/
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Downtown Madison Nightlife (continued) Comedy Club on State Street ‐ Comedy club with a lounge vibe, a roster of nationally known comics & open‐mike nights. o http://www.madisoncomedy.com/ State Street Brats ‐ Wisconsin Badger sports bar featuring red bratwurst, burgers & beer plus a front patio. o www.statestreetbrats.com/ High Noon Saloon ‐ Large rustic venue offering nightly live music in a variety of genres plus beer & pizza slices o www.high‐noon.com/ The Frequency ‐ Small, funky venue with local, national & global acts performing nightly plus drinks & pizza. o www.madisonfrequency.com/ Liquid ‐ International DJs spin at this spacious nightclub with an elevated dance floor, 4 bars & a VIP area. o www.liquidmadison.com/
Brewery Tours/Tastings http://hopheadtours.com/ (this is a bus tour that will hit three breweries) http://www.aleasylum.com/ http://www.newglarusbrewing.com/ https://www.karben4.com/ http://capitalbrewery.com/ Around Madison Set on an isthmus between two scenic lakes, Madison offers a never‐ending variety of opportunities for everyone to enjoy should time permit. * Denotes a 10 or less minute walk. ** Denotes a 10 minute or less drive. Wingra Boats and Brittingham Boats **‐ Stand Up Paddle Boards (SUP), SUP Yoga, Lakeside Yoga, Canoe/Kayak rental o http://www.brittinghamboats.com/Brittinghamrentals.html o http://www.wingraboats.com/Ice‐Cream‐Boat‐Float.html Biking and Hiking * Bikes can be rented from a Madison B Cycle station 1 block from the hotel o https://madison.bcycle.com/ Henry Vilas Zoo ** One of the nation's few admission‐free zoos, Vilas Park Zoo is open year‐round and is a great spot for kids and adults of all ages. http://www.vilaszoo.org/ Madison Children’s Museum‐* Play, learn, imagine and create at the award‐winning Madison Children's Museum. o http://www.madisonchildrensmuseum.org/ Madison Museum of Contemporary Art (MMoCA), 227 State Street, 608‐257‐0158 o http://www.mmoca.org/ Wisconsin Historical Museum, 30 North Carroll Street, 608‐264‐6555 o http://www.historicalmuseum.wisconsinhistory.org/ Wisconsin Veterans Museum, 30 West Mifflin Street, 608‐267‐1799 o http://www.wivetmuseum.com/
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Around Madison (continued) State Street * This seven‐block bustling pedestrian only street runs between the Capitol Square and the University of Wisconsin always buzzes with activity. o http://www.visitdowntownmadison.com/shop/search.php Capitol Square * Amazing views and accessibility of Wisconsin’s State Capitol are just a few steps away. In addition to some top notch restaurants, there are plenty of special events held on the Square. o Dane County Farmer’s Market‐The largest producer‐only Farmers' Market in the country! Wisconsin agricultural products direct from producers featuring an outstanding market of over 300 vendors is held on Wednesday/Saturday http://dcfm.org/ o State Capitol Tours‐ Free tours are held year round. http://tours.wisconsin.gov/ Olbrich Botanical Gardens ** Stroll 16 acres of outdoor gardens, from a Midwest‐hardy Rose Garden to the only Thai Pavilion in the continental U.S. The Bolz Conservatory, an indoor tropical paradise, features exotic plants, flowers, birds and fish. o http://www.olbrich.org/ Bicycle Rental right on bike trail along Lake Monona o http://machineryrowbicycles.com/ Paddling Rentals on Lake Mendota ‐ Madison’s favorite landmark, offers food, live music, film, art galleries, open arts studios and courses, and outdoor recreation. o https://union.wisc.edu/events‐and‐activities/outdoor‐uw/outdoor‐rentals/paddling‐ rentals/ UW‐Madison Campus Campus Walking Tours https://info.wisc.edu/campus‐tours/daily‐walking‐tour/ **Reservations are recommended** Tours leave from Union South, 1308 West Dayton Street Explore the University of Wisconsin‐Madison through a guided walking tour. These 100‐minute tours of campus are led by experienced tour guides and highlight campus life, academics and the history of the university. They are offered various weekdays and on the weekends. Tours leave from Union South, 1308 West Dayton Street. See website for more details and to make reservations. Bascom Hill Historic Architecture Walking Tour June 16 – 6PM – 7:30PM http://www.madisonpreservation.org/#!2016‐tour‐info/ppop1 **Reservations are NOT necessary** Starting Location: Library Mall entrance to the Wisconsin Historical Society headquarters on the campus end of State Street Chazen Museum of Art www.chazen.wisc.edu 800 University Avenue Madison, WI 53706 608‐263‐2246 UW Geology Museum www.geoscience.wisc.edu/museum_wp 1215 West Dayton Street Madison, WI 53706 608‐262‐1412
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UW‐Madison Campus (continued) Babcock Hall Dairy Store http://babcockhalldairystore.wisc.edu/ 1605 Linden Drive Madison, WI 53706 608‐262‐3045 Allen Centennial Garden http://allencentennialgarden.org/ 620 Babcock Drive Madison, WI 53706 Memorial Union Terrace ‐ Madison’s favorite landmark, offers food, live music, film, art galleries, open arts studios and courses, and outdoor recreation. https://union.wisc.edu/ 800 Langdon Street Madison, WI 53715 608‐890‐3000 Lakeshore Path and Picnic Point http://lakeshorepreserve.wisc.edu/ UW Arboretum ‐ The birthplace of ecological restoration, the Arboretum is an outdoor laboratory and popular destination. Explore prairies, woodlands, wetlands and gardens while taking tours, classes, and more. https://arboretum.wisc.edu/
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Where are we? Map of downtown/capitol area.
Overture Center for the Arts, 201 State Street (registration, breakfasts and all sessions)
Concourse Hotel, 1 West Dayton Street (primary conference site hotel)
Monona Terrace Community and Convention Center, 1 John Nolen Drive (dinner banquet)
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Schedule at a Glance Tuesday, August 22, 2017 17:00 – 22:00