CROI 2017 Abstract e-Book

Abstract eBook

SEATTLE, WASHINGTON February 13-16, 2017

Abstract eBook

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CONTENTS

ABSTRACT PROCESS

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General Information

ORAL ABSTRACTS

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POSTER AND THEMED DISCUSSION ABSTRACTS

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DISCLOSURE OF FINANCIAL RELATIONSHIPS WITH COMMERCIAL CONCERNS

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AUTHOR INDEX

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KEYWORD INDEX

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©Copyright 2017 CROI Foundation/IAS–USA. All rights reserved. ISBN #978-0-692-84142-6

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ABSTRACT PROCESS

Scientific Categories A.Virology

B.Molecular Epidemiology and HIV/SIV Evolution C. Pathogenesis: Human Studies and Animal Models D.Host Immune Responses to Infection, Vaccines, and Immunotherapy E. HIV Reservoirs, Latency, and All Curative Strategies Including Therapeutic Vaccines and Gene Therapy F. Neuropathogenesis and CNS HIV Complications G.Clinical Pharmacology

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H.Antiretroviral Therapy: Pre-Clinical and Randomized Trials I. Antiretroviral Therapy: Efficacy and Effectiveness Studies J. HIV Drug Resistance K.HIV Diagnostics L. Hepatitis Viruses and Liver Complications M. Malignancies N.Cardiovascular Complications of HIV Infection and Antiretroviral Therapy O.Other Complications of HIV Infection and Antiretroviral Therapy P. Tuberculosis and Other Opportunistic Infections Q.Maternal/Fetal HIV R.Pediatrics and Adolescents S. Epidemiology T. Prevention Interventions U.Prevention Scale-Up

V. Contraceptive and Reproductive Health inWomen W. Implementation and Scale-Up of Treatment and Care

X.Population and Cost Modeling Y. Emerging Viruses: Zika Virus Abstract Content Author names, institutions, abstract titles, and abstracts in the Program and Abstracts eBook are generally presented as submitted by the corresponding author. Abstract Review Process The PC and a panel of volunteer external reviewers reviewed approximately 2000 submitted abstracts. Each abstract was reviewed by 5 to 10 reviewers selected for each abstract category based upon their individual expertise. PC members and external experts in the field reviewed the abstracts for the quality and originality of the work and scored them numerically. All reviewers were instructed to abstain from scoring any abstract on which they are an author or coauthor, have a financial or personal conflict of interest, or do not have the appropriate expertise to evaluate. Scores ranged from 1 (definite oral presentation) to 5 (rejected). Scores for each abstract were averaged and the standard deviation was calculated to assess variability. If variability was high, outlier scores are identified and censored. Abstracts with high variability in scores were discussed individually during a series of conference calls. Abstracts were accepted for oral presentations, for poster presentations, or rejected. Late-breaking abstract reviews included an assessment of the late- breaking nature of the work (versus just being a late submission).

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Common Reasons for Abstract Rejection • Information is not new enough • Methodology is inadequate or insufficient to support conclusions • Background does not summarize the hypothesis • Submission is poorly written • Abstract is duplicative of other submissions

• Abstract is not appropriate for CROI • Controls are absent or inadequate • Statistical evaluation is inadequate or absent

General Information

• Summary of essential results is inadequate or absent • Data are inadequate or insufficient to support conclusions

• Submission reports clinical trial and data from unplanned analysis or incomplete or ongoing studies • Format does not follow guidelines (eg, section[s] missing, more than 1 graphic, table, or figure submitted) Statistics for Abstracts General abstract submitted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1751 General abstracts accepted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 948 General oral abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80 General poster abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .868 Late-breaking abstracts submitted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .159 Late-breaking abstracts accepted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Late-breaking oral abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Late-breaking poster abstracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Total abstracts submitted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1910 Total abstract accepted . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1001 All Authors on Accepted Abstracts Region N Percent Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 . . . . . . . . . . . . . . . . 14 Asia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99 . . . . . . . . . . . . . . . . 5.2 Australia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 . . . . . . . . . . . . . . . . 1.8 Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432 . . . . . . . . . . . . . . . 22.5 Latin and South America . . . . . . . . . . . . . . . . . . . . . . 29 . . . . . . . . . . . . . . . . 1.5 North America . . . . . . . . . . . . . . . . . . . . . . . . . . 1050 . . . . . . . . . . . . . . . . 55

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ORAL ABSTRACTS

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PROGRAM COMMITTEE WORKSHOP FOR NEW INVESTIGATORS AND TRAINEES Conveners: Serena S. Spudich , Yale Univ, New Haven, USA and JohnW. Mellors , Univ of Pittsburgh, Pittsburgh, PA, USA

Despite extraordinary advances in understanding of the pathogenesis, treatment, and prevention of HIV over the past 35 years, research has fallen short of identifying optimal strategies to provide effective antiretroviral treatment to all infected persons, prevent new infections, limit morbidity in individuals on therapy, and achieve a functional ‘cure.’ The annual Program Committee Workshop for New Investigators and Trainees at the Conference for Retroviruses and Opportunistic Infections (CROI) is designed to develop knowledge and discovery across a broad range of basic and clinical HIV investigation. In this half-day session, Program Committee members will succinctly encapsulate current understanding in five distinct topics, specifically identifying areas of controversy or gaps requiring new investigation and ideas. This year, Dr Paul Bieniasz will start the session by reviewing updated knowledge in the field of molecular virology, including advances in understanding of the viral life cycle and host restriction factors. Dr Richard A. Koup will report on advances in antibodies, from knowledge of existing monoclonal antibodies to progress in antibody engineering, antibody functions, and recent trials employing antibodies for HIV protection and treatment. Dr James A. McIntyre will cover recent advances in HIV prevention, reporting on the ongoing challenges of reaching key and vulnerable populations at high risk of infection, and describing evidence of successful implementation of new biomedical prevention strategies. Dr Judith S. Currier will review non-AIDS complications of HIV infection such as cardiovascular, hepatic, malignant and neurologic disease in the setting of aging, highlighting new insights into the epidemiology, pathogenesis, screening, prevention and treatment of these problems. Finally, Dr Nicolas Chomont will summarize state-of-the-art knowledge regarding HIV reservoirs and the potential for HIV cure, explaining how and where HIV persists during antiretroviral therapy, and describing multiple approaches to HIV eradication or remission, including early treatment strategies, latency reversal, immunotherapies, and host genetic modification. Speakers will highlight key work featured at CROI 2017, and participate in moderated question and answer sessions after each talk to stimulate thought and interactions that enhance the attendees’ experience at CROI. 2 MARTIN DELANEY, PRESENTATION FROM LAB TO LICENSURE: THE IMPORTANCE OF GOOD PARTICIPATORY PRACTICE IN RESEARCH Moderator: Mark Hubbard , Tennessee Association of People with AIDS, Nashville, TN, USA Failure to effectively engage stakeholders has historically resulted in the disruption and closure of a number of large HIV prevention trials. There is a growing body of evidence that engaging stakeholders from the earliest stages of research and concept development and beyond trial site communities can strengthen efficient and ethical conduct of research. In 2007, UNAIDS and AVAC developed Good Participatory Practice (GPP) Guidelines to set global standards for stakeholder engagement throughout the research life cycle of biomedical and related HIV prevention trials. GPP establishes guiding principles and promotes the use of a range of stakeholder advisory mechanisms at all levels to help ensure meaningful, collaborative, transparent, and mutually beneficial relationships. GPP provides a flexible and dynamic roadmap for researchers, sponsors, and implementers. Panelists will provide a brief history of events prior to the development of the GPP guidelines, explain GPP and its fundamental principles as they apply to HIV prevention and cure research, and discuss how the guidelines have helped to shape stakeholder engagement in other fields of medical research. Panelists will explore the practical application of GPP to placebo- controlled clinical trials, open-label PrEP demonstration projects, and social research to guide human studies for HIV cure or long-term remission. Insights and concerns expressed by community members and other stakeholders will inform the discussion throughout. This presentation will inform new investigators and community educators about the critical importance of engaging stakeholders and inspire them to consider the use of GPP principles for all stages and types of research. Panelists Stacey Hannah , AVAC: History and Principles of Good Participatory Practice Guidelines Deborah Baron , Wits Reproductive Health and HIV Institute: GPP as a Guide to Biomedical HIV Prevention Trial Design and Implementation David Evans , Project Inform: Applying GPP Principles to Cure Research. 3 PREVENTION TRIAL DESIGN IN THE ERA OF PREP Deborah J. Donnell, Fred Hutchinson Cancer Rsr Center, Seattle, WA, USA We are at a pivotal moment in HIV prevention where daily oral PrEP has been proven effective, yet is not likely to be fully effective for all populations in need. Further advance of HIV prevention requires the development of vaccines, alternative oral regimens, longer acting or coitally dependent prevention strategies. Clinical trials in HIV prevention lack a surrogate and any efficacy study is a resource-intensive undertaking. This session presents the current designs of clinical trials advancing the pipeline of prevention agents, including the statistical issues surrounding the first active-comparator trials in HIV prevention. As we confront a potential future where highly effective prevention reduces the incidence of HIV-endpoints in prevention trials it is important to begin to consider what evidence would be sufficient to definitively establish evidence of prevention efficacy. 4 IMPLEMENTATION SCIENCE TRIALS: DO THE RULES OF RCTs APPLY? James R. Hargreaves, London Sch of Hygiene and Trop Med, London, United Kingdom Implementation science asks questions about the delivery of interventions to those who need them. Technologies such as drugs, vaccines, or surgical procedures may be the “direct mechanism” of the interventions in question. But implementation science trials answer questions about how best to implement these, about the practice of their delivery, or about the systems through which they are delivered. As such, these trials are inevitably more about human behaviour and system function than they are about the biological efficacy of the drug, vaccine or procedure. This presentation will discuss four implications of this perspective for the design of implementation science trials, illustrating key points with examples. First, such trials are usually most appropriately conducted as cluster-level trials since this is the level at which delivery packages and strategies operate. Second they should be “pragmatic”, as defined in the CONSORT guidance for pragmatic trials. One implication of this is that it is important that they enrol study participants who are representative of the intended target population, rather than opportunistically recruiting selected populations as may be appropriate for individual-level efficacy trials. A further implication of “pragmatism” is that, intervention delivery should be monitored and supervised in a realistic way, not closely controlled to ensure ideal implementation as in efficacy trials. Third, integrated process evaluation, ideally guided by the UK MRC Process Evaluation framework for complex interventions, is an essential component of such trials. This framework emphasises the importance of documenting the implementation of interventions, analysing pathways of change and identifying contextual factors relevant to future policy recommendations about scale-up to other settings. Fourth and finally, owing to lack of investigator control, ethical or other reasons, it may not be feasible to undertake randomisation in some implementation science situations. While a full discussion of the range of design alternatives is beyond the scope of this presentation, I will briefly discuss a framework we have developed to map the most rigorous non-randomised impact evaluation designs against constraints to randomisation often described by practitioners in implementation settings. 5 RESPONDENT DRIVEN SAMPLING AND OTHER METHODS FOR RECRUITING HARD TO REACH POPULATIONS Carl A. Latkin, The Johns Hopkins Univ, Baltimore, MD, USA Reaching and enrolling “hidden” populations for infectious disease prevention, testing, and treatment studies and public health programs remains problematic. Adequate sampling strategies are also need to provide infectious disease modelers with accurate data. Two overlapping approaches to sampling hidden populations are respondent

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driven sampling (RDS) and social network sampling. In the field of HIV and STI research, RDS has been successfully utilized to recruit substance users, commercial sex workers, and men who have sex with men. In this workshop, we review of the evidence for the use of RDS as a reliable sampling methodology, assess whether many studies meet the assumptions for RDS in order to claim a representative sample, and identify systematic biases in RDS sampling. In addition, we will review recent studies that have compared RDS to other recruitment approaches or have modified RDS methods. The presentation will also discuss how social network informed sampling may be combined with RDS to improve sampling, what types of studies may be best suited for RDS and social network sampling approaches, and what might be accomplished by an RDS approach versus more conventional sampling strategies. 6 CRISPR/Cas9 gene editing strategies have revolutionized our ability to engineer the human genome for robust functional interrogation of complex biological processes. We have recently adapted this technology to primary human CD4+ T cells to generate a high-throughput platform for analyzing the role of host factors in HIV replication and pathogenesis. Unlike traditional RNA interference or complementation approaches, this technique generates permanent genetic changes to the host genome at high efficiency with only transient expression of the modifying agents. CRISPR/Cas9 ribonucleoproteins (crRNPs) are synthesized in vitro and delivered to activated primary human CD4+ T cells by electroporation. These cells are then expanded and parsed out for validation, cryopreservation, and infection. Our platform supports the arrayed generation of hundreds of specific gene manipulations in only a few hours time and is widely adaptable to an array of culturing conditions, infection protocols, and downstream applications. We first used this platform to perform proof-of-principle experiments targeting host factors with defined roles in HIV replication. As expected, CXCR4 or CCR5 knock-out primary T cells are resistant to HIV infection in a tropism-dependent manner, whereas knock-out of LEDGF or TNPO3 results in a tropism-independent reduction in infection. We next bridged this approach to other proteomic and genomic discovery platforms as a secondary screen for host factor functionality, identifying several candidate dependency and restriction factors for additional mechanistic study. Finally, we found that crRNP multiplexing allows for the editing of multiple genes simultaneously, enabling studies of functional redundancy or epistasis among multiple host and viral factors. This technology should prove useful for not only discovery-based scientific research, but may additionally accelerate target validation for pharmaceutical and cell-based therapies to cure HIV infection. We are currently adapting this technology to achieve efficient genome editing at the level of a single base pair, as well as adapting it to other primary cell types for the study of additional biological processes and disease states. Recent advances with CRISPR interference, CRISPR activation, BaseEditor technology, high-fidelity Cas9 enzymes, and Cas9 inhibitors will also be discussed. 7 IDENTIFYING AND PROFILING VIRUS-SPECIFIC T CELLS USING MASS CYTOMETRY EvanWilliamNewell, Singapore Immunol Network, Singapore Blood and tissue samples taken as part of clinical studies and trials can provide critical information on the roles of the immune response in patient outcome. However, the cellular compositions of these samples are often highly diverse, and important information can be lost if rare cells are overlooked. For instance, antigen specific T cells are critical initiators and orchestrators of the adaptive immune response, but cells specific for any given pathogen or cancer can be exceedingly rare, especially in blood. Here, the utility of high dimensional mass cytometry analysis together with rapidly evolving computational analysis tools will be discussed. A major advantage of this approach is the ability to directly analyze relationships between antigen-specificity and each cell’s phenotypic profile. This is particularly relevant for the study of T cells, whose phenotypic markers can be intuitively segregated into a number of categories such as antigen-specificity, differentiation state, functional capacity, and trafficking receptor profiles. All of these can be measured simultaneously on individual cells through the use of metal-labeled monoclonal antibodies and highly multiplexed combinatorially-coded peptide-MHC tetramers. Our ongoing analysis of T cell responses in chronic hepatitis B viral infection in human patients demonstrate the utility of this approach to identify and profile difficult to study HBV-specific T cells. Our results show that HBV-specific T cell responses are highly diverse in terms of epitopes being targeted and the phenotypes of the corresponding cells. We are investigating the potential utility of T cell phenotypes as biomarkers for patient outcomes. 8 QUANTIFYING HIV-1 mRNA STRUCTURE AND TRANSLATION EFFICIENCY IN CELLS Silvi Rouskin, Whitehead Inst, Cambridge, MA We have recently developed DMS-MaPseq, which allows for targeted RNA structure probing in vivo at single molecule and single nucleotide resolution. First, cells are treated with DMS, which rapidly modifies unpaired adenines and cytosines. Unlike other chemical reagents, DMS is a very small molecule specific to the nitrogen atoms involved in Watson- Crick base-paring, which enables probing of RNA structure in the presence of RNA binding proteins. In the DMS-MaPseq technique, we use DMS concentrations that result in multiple modifications per single molecule. We then use a newly commercialized high fidelity and processive thermostable group II reverse transcriptase (TGIRT) enzyme, which converts DMS modifications into mutations in the cDNA. The cDNA is converted to dsDNA by PCR and then sequenced on an Illumina platformwith long reads. Since the mutation background is negligible, the mutations within a single sequencing read correspond to the open bases within a single RNA molecule. There are two key advantages of using this approach compared to other chemical probing approaches: 1) we can selectively probe the structure of any RNA of interest, even at very low abundance, simply by using a RNA specific RT primer 2) we can analyze multiple DMS modification sites per RNA molecule, which allows us to distinguish heterogeneous RNA structure subpopulations in vivo. We use a combination of DMS-MaPseq and ribosome profiling to quantify the HIV-1 RNA structure and translation efficiency in cells. 9 INTERACTIVE CASE-BASED WORKSHOP ON HEPATITIS C Moderators: Susanna Naggie , Duke Univ, Durham, NC, USA, and DavidWyles , Denver Health, Denver, CO, USA This interactive case-based session is geared toward clinicians who are involved in HCV treatment. Speakers will present cases on: Staging and treatment of early stage HCV ( John D. Scott , Univ of Washington, Seattle, WA, USA), effect of drug resistance on DAA treatment and re-treatment of patients with chronic HCV infection ( Alessandra Mangia , Ospedale Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy), common drug interactions with HIV/HCV coinfection treatments ( Debika Bhattacharya , Univ of California Los Angeles, Los Angeles, CA, USA), and cirrhosis issues ( Sanjay Bhagani , Royal Free Hosp, London, United Kingdom). 10 BERNARD FIELDS LECTURE: INSIGHTS INTO HIV PREVENTION, PATHOGENESIS, AND TREATMENT FROM NONHUMAN PRIMATE MODELS Jeffrey D. Lifson, Frederick National Laboratory for Cancer Research, Frederick, MD, USA Infection of Asian macaques with Simian Immunodeficiency Viruses (SIV) endemic in African nonhuman primates (NHP) can lead to progressive pathogenesis/immunodeficiency that recapitulates key aspects of human HIV infection and AIDS. Different NHP models, employing different naturally occurring or engineered SIVs or related chimeric viruses, used to infect different macaque species, can be used to authentically model relevant features of human HIV infection. Experimental flexibility and control, and opportunities for extensive tissue sampling afforded by NHP models provide advantages for studying virus/host interactions. NHP models have yielded key insights into AIDS virus transmission, allowing characterization of the earliest stages of infection, pathways of early viral spread, and initial host responses, and permit the preclinical safety and activity evaluation of prophylactic vaccines and other prevention approaches. When matched to conditions of clinical vaccine evaluation, results from NHP vaccine studies have been largely congruent. NHP models have also informed our understanding of the pathogenesis of AIDS virus infection, including processes such as early, extensive depletion of intestinal CD4+ T cells, mucosal disruption, microbial translocation and persisting systemic immune activation, along with inflammation related fibrotic disruption of secondary lymphoid tissues. Comparison of SIV infections in Asian macaques and African “natural host” species, where infection can result in extensive viral replication but does not typically lead to progressive disease, highlight the role of host responses in pathogenesis. NHP models have also shed light on mechanisms of viral persistence despite apparently effective viral suppression by antiretroviral drug treatment or potent immune responses, through viral sequestration in relatively immune privileged sanctuary sites such as B cell follicles in HIGH-THROUGHPUT GENOME ENGINEERING IN PRIMARY CD4+ T CELLS Judd F. Hultquist, Univ of California San Francisco, San Francisco, CA, USA

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secondary lymphoid tissues, establishment and expansion of T cell clones bearing clonally integrated proviruses, and other mechanisms, and provide the basis for experiments to evaluate the safety and in vivo activity of strategies to target residual viral reservoirs. Contributions of NHP studies to our understanding in these areas and recent developments will be reviewed, underscoring the key role that NHP models have played and will continue to play in our efforts to develop more definitive approaches for preventing, treating, and attempting to cure HIV infection. 11 N’GALY-MANN LECTURE: HIV/AIDS RESEARCH IN ZIMBABWE: PROVIDING THE EVIDENCE FOR QUALITY CARE James G. Hakim, University of Zimbabwe, Harare, Zimbabwe Following the report of the first case of AIDS in Zimbabwe in 1985 the epidemic escalated rapidly reaching an adult prevalence of 29% in the late 1990s. Given the heterosexual and generalized nature of the epidemic the social, health and economic consequences were devastating. This presentation will describe (a) some historical perspectives of AIDS and the current status of the epidemic in Zimbabwe (b) Zimbabwe’s contribution to cutting edge AIDS research (c) human and research capacity building as an essential aspect of the comprehensive response to the AIDS epidemic. As the health sector and the social fabric of the country reeled under the effects of the epidemic the government took a series of steps which proved visionary given the considerable resource constraints that beset the country. An aggressive behavior change campaign, the introduction of the AIDS levy (domestic financing scheme) and the local manufacture of ARVs defying patent restrictions stand out as significant home-grown solutions complementing the global support the country was receiving. Recent data show an adult prevalence of 14.6%, incidence of 0.48% (a drop of 50% over the past 1½ decades); 900,000 people on ART and a viral suppression rate of 60.4%. Challenges remain and achievement of global targets including ending AIDS are prime aspirations for the country. In the mid-90’s driven by a desire to understand the AIDS epidemic and to contribute to mitigating its effects a small band of researchers with collaborators in the US and UK embarked on high impact research in the face of meager resources and a challenging socioeconomic environment. Data emanating from these efforts have contributed significantly to local and international HIV/AIDS knowledge base and interventions. Personal relations between local and external researchers have been key ingredients in research capacity building and in escalating research in Zimbabwe. HIV prevention and therapeutics research encompassed pMTCT, microbicides, ART strategies and ART in naïve and experienced patients. Other research efforts included tuberculosis, cryptococcal disease, cervical cancer, Kaposi’s sarcoma, mental health and studies among commercial sex workers. In recent years opportunities provided by PEPFAR, NIH, Wellcome Trust and others have enabled the implementation of robust training programs to improve the quality of health delivery and create the next generation of competent researchers and health provider. HIV cure is a desirable goal for children and adults living with HIV who face stigma and life-long antiretroviral therapy (ART) that requires strict adherence. In addition, treatment cost poses a significant burden to national programs and global donors that could be alleviated were a cure available. The ultimate objective is to eliminate all cells capable of producing HIV – a near unattainable goal with current therapies. The field has re-calibrated the HIV cure target to a more achievable one of HIV remission, i.e., the ability to control viral replication after ART interruption to levels below detection. The major obstacle is, however, the seeding of the HIV reservoir that occurs early during acute HIV infection and sets the stage for the establishment of latent reservoirs, particularly in long-lived CD4+ T cells and lymphoid tissues. The unique aspects of the pediatric immune system in the composition of the CD4+ T cell compartment and defense against HIV may influence HIV persistence. Early ART is an important step in the path towards an HIV cure. The pediatric population offers a unique opportunity for immediate treatment because of the known timing of HIV exposure in most newborns. Similarly, acute HIV infection studies have demonstrated the ability to identify and treat very early infection in adults. Both children and adults treated in acute HIV infection achieve significantly smaller HIV reservoirs, preserved immune functions with little viral escape to immune pressure. These qualities could enhance the effects of immune-based interventions aimed at depleting HIV-infected cells. However, despite these favorable qualities, the majority of early treated individuals do not achieve HIV remission. Therefore, additional therapies will be needed that may include latency reversing agents and passive and active immune therapies. There are exciting new developments of broadly neutralizing antibodies and therapeutic HIV vaccines that could be beneficial to HIV cure. It is highly likely that combination therapies that can generate persistent and effective immune responses to control HIV will be required for a durable HIV remission and cure. In this early discovery phase of HIV cure research that is associated with risks and uncertainties, and further complicated by the difficult concepts of remission and cure, it is also important that ethical, behavioral and social research be conducted in parallel to basic and clinical research. 13 ADVANCES IN CELLULAR THERAPY IN CANCER AND HIV Carl H. June, Univ of Pennsylvania, Philadelphia, PA, USA Chimeric antigen receptor (CAR) T cells have proven that engineered immune cells can serve as a powerful new class of cancer therapeutics. Clinical experience has helped to define the major challenges that must be met to make engineered T cells a reliable, safe, and effective platform that can be deployed against a broad range of tumors. Here I will discuss a road opened CAR T cells for cancer that leads to therapies for HIV. The emergence of synthetic biology approaches for cellular engineering is providing us with a broadly expanded set of tools for programming immune cells that can be used to contain or confer elite controller status on patients with HIV. The convergence of the field of HIV and Cancer is driven in part because both are chronic conditions of antigen overload, where chronic infections and tumors can lead to phenocopies of acquired tolerance and antigen escape. 14LB QUATERNARY CONFIGURATION OF THE FUNCTIONAL CD4-BINDING SITE IN THE HIV-1 ENV TRIMER Qingbo Liu 1 , Priyamvada Acharya 1 , Michael Dolan 1 , Peng Zhang 1 , Aliaksandr Druz 1 , William Rice 2 , Bridget Carragher 2 , Clinton Potter 2 , Peter D. Kwong 1 , Paolo Lusso 3 1 NIH, Bethesda, MD, USA, 2 New York Structural Bio Cntr, New York, NY, USA, 3 NIAID, Bethesda, MD, USA Background: Binding of the gp120 envelope glycoprotein to the CD4 receptor is the first step in the HIV-1 infectious cycle. Although the CD4-binding site has been extensively characterized by mutagenesis and co-crystallization with soluble CD4 (sCD4), most of these studies were performed with monomeric gp120 subunits, thus hindering the evaluation of the role of quaternary elements that may be involved in the initial CD4 interaction. Moreover, the initial receptor interaction has been difficult to study because of major CD4- induced structural rearrangements. Methods: The DS-SOSIP.664 trimer, 4-domain CD4, and PGT145 Fab were expressed in 293FS cells and purified for cryogenic electron microscopy (cryo-EM) analysis; data were acquired using the Leginon system installed on a Krios electron microscope operating at 300kV; for ELISA and SPR, wild-type (WT) and mutated SOSIP trimers and gp120 monomers were expressed in 293FS and extensively purified; WT and mutated full-length gp160 were expressed in 293T cells for flow cytometry and co-transfected with an HIV-1 backbone plasmid to produce infectious pseudoviruses; infectivity assays were performed in TZMbl cells. Results: Cryo-EM analysis of the stabilized DS-SOSIP.664 trimer, which remains in a pre-fusion closed conformation after interaction with CD4, permitted to visualize the initial contact with CD4 at 6.8-Å resolution. We found that the initial CD4-contact site in the HIV-1 Env trimer is constituted by a quaternary surface formed by coalescence of the previously defined CD4-binding region in the outer domain of one gp120 protomer with a second CD4-binding site (CD4-BS2) that encompasses discontinuous elements from the inner domain of a neighboring gp120 protomer. Disruption of CD4-BS2 destabilized CD4-trimer interaction and abrogated HIV-1 infectivity by preventing acquisition of coreceptor- binding competence. A corresponding reduction in HIV-1 infectivity occurred upon mutation of CD4 residues that interact with CD4-BS2. Quaternary interactions were also documented for selected neutralizing antibodies to the CD4 supersite, providing evidence that the CD4-BS2 region is immunogenic in vivo. Conclusion: These results document the critical role of quaternary interactions in the initial HIV-1 envelope-receptor contact, with implications for treatment and vaccine design. 12 THE EMERGING POTENTIAL FOR HIV CURE FOR INFANTS, CHILDREN, AND ADULTS Jintanat Ananworanich, US Military HIV Rsr Prog, USA and The Thai Red Cross AIDS Rsr Cntr, Thailand

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15 EARLY CYTOPLASMIC UNCOATING IS NECESSARY FOR INFECTIVITY OF HIV-1 João I. Mamede , Gianguido C. Cianci, Meegan R. Anderson, Thomas Hope Northwestern Univ, Chicago, IL, USA

Background: After cell fusion, HIV delivers its conical capsid into the cytoplasm. The disassembly of the capsid is termed uncoating and is critical to infection. The understanding of the kinetics, dynamics, and localization of uncoating of infectious particles has been eluded by the unavoidable presence of non-infectious particles. The timing of uncoating remains under discussion with some models proposing that uncoating happens early and other models suggest that the intact capsid docks at the nuclear pore. These different hypotheses formed from diverse assays, lack the information for the kinetics and localization of uncoating of productively infectious viral particles. Methods: We used live-cell fluorescent imaging of intravirion fluid phase markers to determine the integrity of the HIV conical capsid core. To visualize dynamic changes in capsid integrity and composition, we utilized the HIV-iGFP construct. During viral maturation of HIV-iGFP, the GFP is liberated from Gag. A minority population of the free GFP is trapped in the capsid, while the remaining free GFP is located outside of the capsid. With this technique, the loss of the fluid phase GFP occurs in two steps: with fusion and upon the loss of capsid core integrity. Live-cell microscopy of HIV-iGFP virions with a viral complex marker such as Vpr or Integrase allows for the timing of these two steps. Through viral challenge with less than one virion per cell we are able to connect viral particle phenotype to infection. Results: The time between fusion and capsid integrity loss, for both HIV and VSV-G mediated fusion, in tissue culture and primary cells (macrophages and T cells), is approximately 30 minutes. Also, capsid integrity loss occurs entirely in the cytoplasm and co-relates to a big loss of p24CA. With our lowMOI approach, we were able to image individual particle uncoating that produces a viable infection and differentiate between different rates of uncoating. This analysis revealed that all particles associated with cellular infection showed changes in capsid integrity ~29 minutes. We were also able to halt uncoating by blocking specific steps of reverse transcription, linking uncoating to the occurrence of the first-strand transfer step. Conclusion: Together, these observations validate the early cytoplasmic uncoating model. Our live-imaging assay has the ability to follow uncoating at the single infectious particle level providing unprecedented insights into the early steps of HIV infection. 16 ECCENTRIC VIRAL GENOMIC RNA AND INTEGRASE ARE PREMATURELY DEGRADED IN TARGET CELLS Michaela Madison 1 , Dana Q. Lawson 1 , Jennifer Elliott 1 , Ayse N. Ozanturk 2 , Pratibha Chowdary Koneru 3 , James R. Fuchs 3 , Mamuka Kvaratskhelia 3 , Sebla B. Kutluay 1 1 Washington Univ in St. Louis, St. Louis, MO, USA, 2 Bilkent Univ, Ankara, Turkey, 3 Ohio State Univ, Columbus, OH, USA Background: Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome yields aberrant particles, in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the protective capsid core. These particles are non-infectious and blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown, given that eccentric particles appear to retain all components necessary for reverse transcription, i.e. a dimeric viral RNA genome primed with tRNA-Lys, functional RT and normal levels of NC-RNA complexes. In addition, apart from reverse transcription products, the fates of viral core components in cells infected with eccentric particles have not been studied to date. Methods: To determine why the eccentric virus particles, generated with class II IN mutants such as R269A/K273A or ALLINI treatments of the WT virus, fail to support reveres transcription in target cells, we have monitored the fates of eccentric particle components in infected cells. To this end, we took advantage of a previously developed elaborate approach in which we biochemically tracked multiple core components in infected cells. Results: In this study, we show that in target cells eccentrically localized vRNPs and IN are prematurely degraded, whereas reverse transcriptase remains active and stably associated with capsid cores. Importantly, we show that in addition to viral RNAs, IN protein is also mislocalized in particles. Remarkably, the aberrantly shaped capsid cores in the eccentric particles can efficiently saturate and be degraded by a restricting TRIM5 protein, suggesting that TRIM5 recognition does not require the presence of fully formed cores. Conclusion: We propose that IN-RNA interactions allow for packaging of both vRNPs and IN within the protective capsid cores to ensure subsequent reverse transcription and productive infection in target cells. 17 DYNAMICS OF NUCLEAR ENVELOPE ASSOCIATION AND NUCLEAR IMPORT OF HIV-1 COMPLEXES Ryan C. Burdick , Jianbo Chen, Jaya Sastri, Wei-Shau Hu, Vinay K. Pathak NCI, Frederick, MD, USA Background: During productive infection, HIV-1 must enter the nucleus to integrate its DNA into host genome. However, many aspects of nuclear import process are poorly understood because they have been difficult to study using biochemical or imaging assays, and have not been visualized in living cells. Methods: To elucidate critical HIV-1 post-entry events, we analyzed viral complexes labeled with yellow fluorescent protein-tagged APOBEC3F (A3F-YFP), a virion-incorporated host restriction factor, or Vpr-integrase-YFP fusion protein (IN-YFP) using live-cell imaging. Results: We first examined the association between HIV-1 viral complexes and nuclear envelope (NE) by live-cell imaging, and observed that most contacts are transient (<5 sec) and very few are stable (>20 min), suggesting only a subset of viral complexes is competent to stably dock with the NE. Most HIV-1 complexes forming transient interactions with the NE may be encountering regions that did not have any nuclear pore complexes (NPCs). We found that HIV-1-NE stable association is compromised when capsid is mutated to form viral cores that are unstable (K203A) or hyperstable (E128A/R132A), or when host protein Nup358 is knocked down. These findings indicate that HIV-1 capsid and host Nup358 play critical roles in forming the stable associations. To better understand the process of nuclear import, we captured for the first time the translocation of 21 HIV-1 complexes from the cytoplasm to the nucleus and their nuclear movements. Viral complexes labeled with A3F-YFP or IN-YFP behaved similarly, suggesting that the fluorescently tagged proteins did not influence their movements. They exhibited similar long and variable residence times at the NE (1.7 ± 1.7 hours), indicating that the viral core may be undergoing extensive dissociation and/or conformational rearrangements which require an extended period of association with the NPC prior to nuclear import. After import, the viral complexes exhibited a fast phase (<9 min) as they moved away from the point of entry, followed by a slow phase for the rest of the observation time, suggesting that they quickly associate with chromatin and/or other nuclear macromolecules. The viral complexes moved away from the nuclear point of entry, but remained in the nuclear periphery. Conclusion: The tracking of individual HIV-1 complexes provides insights into the dynamics of HIV-1 NE association, nuclear import, and movement inside the nucleus. 18 CYPA REGULATES HIV-1 ACCESS TO AN FG-GUIDED NUCLEAR ENTRY PATHWAY Guangai Xue 1 , Vineet KewalRamani 1 , Shih Lin Goh 2 , Hyun Jae Yu 1 , Anna Gres 3 , KyeongEun Lee 1 , Stefan G. Sarafianos 3 , Jeremy Luban 2 1 NCI, Frederick, MD, USA, 2 Univ of Massachusetts, Worcester, MA, USA, 3 Univ of Missouri, Columbia, MO, USA Background: The choreography of virus infection at the cellular level involves a successive series of host factor interactions to drive the viral replication program. The role of HIV-1 capsid (CA) in the early steps of replication is includes cytoplasmic trafficking, regulation of reverse transcription, interaction with the nuclear pore complex (NPC), and chromatin access during integration. Two host factor interfaces within CA, the CPSF6 and Cyclophilin A (CypA) binding sites, are critical to these steps. The CA binding site for CPSF6 shows selective interaction with protein motifs containing an FG-dipeptide, which is abundant in one-third of nucleoporins. FG-nucleoporins (FG-Nups) maintain the nuclear diffusion barrier and provide docking sites for nuclear transport receptors (NTRs). Notably, Nup153, a makes specific contacts with the same pocket in CA via an FG-containing motif. We sought to understand the respective roles of CPSF6 or CypA binding sites in CA in regulating nuclear entry. Methods: We performed a small interfering RNA (siRNA) screen targeting all known human nucleoporins to assess effects on Wild-type (WT) versus N74D and P90A HIV-1 infection. N74D and P90A are mutations that respectively impair either CPSF6 or CypA binding to CA. We used commercially available pools of siRNAs in the screening. Host factor specificity was confirmed by restoration of expression with siRNA-resistant isoforms of mRNA. Reverse transcription products were measured via qPCR.

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Abstract eBook

Oral Abstracts

Results: In addition to previously known NPC co-factors, we identify Nup35 and POM121 to aid HIV-1 infection. HIV-1 reliance on Nup35 or POM121 is linked to CA. Nup35 and POM121 are FG-Nups. Preliminary examination indicates that a C-terminal FG-motif of Nup35 is required for HIV-1 infection. Notably, HIV-1 interaction with CypA regulated dependence on the FG-Nups. Disruption of the interaction between CA and CypA either by cyclosporine A (CsA) treatment or CypA knockdown restored WT HIV-1 infectivity in Nup35 knockdown cells. Conclusion: We hypothesize that CypA use by HIV-1 prevents access to the N74 pocket in CA until the virus docks at the NPC. We propose that the HIV-1 core, comprised of hundreds of CA molecules, directly functions as a NTR and exploits successive FG interactions to achieve nuclear entry. Thus, the development of pharmacological compounds targeting the CPSF6-binding site in CA have the potential to disrupt interactions with multiple co-factors and significantly impair HIV-1 infection. 19 COMPREHENSIVE CRISPR SCREEN IDENTIFIES NOVEL HIV RESTRICTION FACTORS Molly Ohainle , Louisa Pendergast, Jolien Vermeire, Michael Emerman Fred Hutchinson Cancer Rsr Cntr, Seattle, WA, USA Background: Interferon-Stimulated Genes (ISGs) inhibit HIV replication by inducing an array of antiviral effectors known as HIV restriction factors. Restriction factors, such as TRIM5, APOBEC3s, Tetherin and MxB, have previously been discovered one-by-one through classic techniques such as cDNA library screening and comparison of RNAseq data in permissive versus non-permissive cells. Here we describe a comprehensive novel CRISPR gene knockout screen to identify HIV restriction factors. Methods: A CRISPR knockout screen was performed for wild type HIV-1 in THP-1 cells, a human monocytic cell line with strong Interferon-induced inhibition of HIV-1 replication. An available whole-genome CRISPR library was modified to allow for packaging of lentiCRISPR genomes in trans into budding HIV-1 particles after infection. Known and novel, candidate restriction factors are identified by measuring the relative enrichment of gRNAs in HIV-1 viruses released from cells (MAGeCK gRNA and gene analyses). Further, we have designed an ISG-specific gRNA CRISPR library targeting ~2000 human ISGs allowing for validation of hits from the whole-genome screen as well as identification of additional restriction factor candidate genes. Results: Our screen identified known restriction factors, such as MxB, ZAP and ISG15. Potential previously-uncharacterized HIV restriction factors were also identified such as Nedd4-binding protein 1 (N4BP1), a nucleolar protein previously identified as an ISG that participates in PML bodies and is an inhibitor of the ubiquitin ligase ITCH. Conclusion: We have developed a novel method to uncover HIV restriction factors. Both known and novel HIV restriction factors were identified. Our studies show that a subset of these genes can explain most of the inhibitory effects of interferon on HIV replication in THP-1 cells. Background: Most enveloped viruses, including HIV-1, exploit the endosomal sorting complexes required for transport (ESCRT) pathway to bud from cells. Owing to its essential functions in membrane fission events such as cytokinetic abscission and closure of the post-mitotic nuclear envelope, the ESCRT machinery is conserved and evolutionarily constrained. These properties pose a challenge for cells in trying to adapt to pathogens that exploit the ESCRT pathway. Methods: Using phylogenetic analyses, we identified retrotransposed copies of numerous ESCRT factors in primate genomes including a retrogene encoding a truncated version of the ESCRT-III protein CHMP3 (retroCHMP3) in squirrel monkeys. Truncated CHMP3 proteins had previously been shown to block HIV-1 budding, apparently by dominantly inhibiting the ESCRT pathway (Zamborlini et al., 2006). We therefore hypothesized that retroCHMP3 might also inhibit the budding of HIV-1 and other enveloped viruses, and tested this hypothesis by expressing retroCHMP3 in human cells and measuring inhibition of retrovirus budding and infectivity. Evolutionary reconstruction and functional analysis of chimeric proteins showed crucial differences between retroCHMP3 and the parental CHMP3 protein. Results: We found that both squirrel monkey retroCHMP3 and the analogously truncated endogenous CHMP3 protein potently inhibit budding of HIV-1 and other retroviruses. Importantly, however, retroCHMP3 exhibited much less cellular toxicity than the truncated parental protein. Characterization of chimeric constructs revealed that just seven amino acid changes were largely responsible for retroCHMP3 detoxification. Cytotoxicity correlated with the formation of cytoplasmic CHMP3 punctae, suggesting that the truncated parental CHMP3 protein may block both viral and cellular ESCRT pathways by sequestering essential ESCRT factors into insoluble aggregates, whereas retroCHMP3 has evolved to maintain viral inhibition without sequestering essential ESCRT factors. Conclusion: Our studies identify retroCHMP3 proteins as broad-spectrum inhibitors of enveloped virus budding in NewWorld monkeys. Moreover, retroCHMP3 has retained the ability to inhibit viral budding while apparently evolving to lose cellular cytotoxicity, revealing unexpected separation of cellular and viral ESCRT functions. More generally, our work illustrates how retrotransposition can create opportunities for cells to evolve new antiviral activities and counteract pathogen exploitation of essential host pathways. Background: The HIV-1 Gag polyprotein contains membrane binding, assembly and budding activities that are required to create extracellular virions that can transfer the viral genome from infected producer cells to new, uninfected target cells. These activities also underlie the efficacy of retroviral vectors, which have been explored extensively as potential therapeutic delivery vehicles. However, safety concerns, immunogenicity and inefficient packaging of non-nucleic acid cargoes limit their potential use. To overcome some of these limitations and to test our understanding of the fundamental principles of virion assembly and release, we have undertaken the design of new proteins that can self-assemble into enveloped protein nanocages (EPNs) and induce their own release from human cells. Methods: We characterized: 1) cellular release of EPNs using western blot assays of cell culture supernatants, 2) membrane integrity of released EPNs using antibody and protease susceptibility, 3) EPN vesicle architectures using cryo-EM tomography, 4) nanocage structures using high resolution single particle cryo-EM reconstructions, and 5) target cell fusion and enzymatic cargo delivery by VSV-G pseudotyped EPNs using colorimetric assays of packaged β-lactamase-Vpr fusion proteins. Results: We observed robust EPN assembly and release, which required all three design elements: membrane binding, self-assembly, and ESCRT factor recruitment. The overall strategy was very general and we have identified 16 different combinations of membrane binding, assembly and ESCRT recruiting elements that can produce EPNs. Detailed analyses of one EPN design (termed EPN-01) revealed that the protein nanocages assembled precisely as designed, and were released within membrane vesicles (110 nm average diameter), each of which contained multiple nanocages (14 nanocages/vesicle average). Pseudotyping with VSV-G allowed the EPNs to fuse with new target cells and deliver β-lactamase-Vpr cargoes. Conclusion: We have used enveloped virus assembly principles to design new proteins that can induce the formation extracellular vesicles and transfer their contents between cells. 20 A RETROTRANSPOSED ESCRT-III FACTOR BLOCKS HIV-1 BUDDING WITHOUT INDUCING CYTOTOXICITY Lara Rheinemann , Diane Downhour, Gaelle Mercenne, Alesia McKeown, John McCullough, Wes Sundquist, Nels Elde Univ of Utah, Salt Lake City, UT, USA 21 DESIGNED PROTEINS INDUCE THE FORMATION OF NANOCAGE-CONTAINING EXTRACELLULAR VESICLES Joerg Votteler 1 , Cassie Ogohara 2 , Sue Yi 2 , Yang Hsia 2 , Una Nattermann 2 , David M Belnap 1 , Neil P King 2 , Wes Sundquist 1 1 Univ of Utah, Salt Lake City, UT, USA, 2 Univ of Washington, Seattle, WA, USA

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