CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

329 CD127+ LYMPHOID MEMORY CD4+ T CELLS PREFERENTIALLY SUBJECT TO LATENT HIV INFECTION Feng Hsiao 1 , Julie Frouard 1 , Andrea Gramatica 2 , Guorui Xie 1 , Roland Schwarzer 2 , Xiaoyu Luo 2 , Marielle Cavrois 2 , Warner C. Greene 2 , Nadia R. Roan 1 1 University of California San Francisco, San Francisco, CA, USA, 2 Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Background: Lymphoid tissues are a primary site of HIV replication and persistence. We recently demonstrated that tonsillar memory CD4+ T cells expressing CD127, efficiently fused to HIV but did not allow the fused virus to complete a round of productive infection. Mechanisms that prevent transcription off the viral LTR can prevent completion of the viral life cycle, and also promote HIV latency which is one of the main barriers to an HIV cure. In this study, we set out to better characterize the molecular basis for the block in HIV replication in tissue CD127+memory CD4+ T cells by considering two main possibilities: post-entry restriction by SAMHD1, or latent infection of these cells by HIV. Methods: Tonsil cells from uninfected donors were mock-treated or exposed to a CCR5-tropic HIV reporter virus for 3 days. Multiple populations of memory CD4+ T cells were compared for SAMHD1 expression and infection levels by FACS. These subsets were sorted and quantified for levels of integrated HIV-1 provirus using 2-step Alu-gag PCR with ddPCR. Global expression profiling of the sorted subsets was conducted. Results: Lymphoid CD127+mem CD4+ T cells do not exhibit early post-entry restriction by SAMHD1, but rather preferentially undergo latent infection as they harbor high levels of integrated HIV DNA in the absence of reporter gene expression. Relative to other memory CD4+ T cell subsets highly permissive for productive infection, the CD127+ cells preferentially expressed host transcripts associated with cellular quiescence explaining how these cells can preferentially silence the HIV LTR. Latently-infected CD127+memory cells were reactivated by stimulation through the TCR. Conclusion: We identify a population of tissue-specific memory CD4+ T cells expressing CD127 that upon exposure to HIV preferentially supports latent infection. Because these cells can undergo IL7-driven homeostatic proliferation and can be reactivated, they may serve as an important reservoir to target for HIV eradication efforts. They also serve as a useful in vitrotissue model of HIV latency that can be used to investigate multiple aspects of latency establishment and maintenance. Although CD127 has not been found to be preferentially expressed on latent cells in vivo, the data presented herein warrant investigation of this receptor as a potential biomarker of latently infected cells residing in tissues. 330 UNPRIMED CD8+ LYMPHOCYTES PROMOTE THE ESTABLISHMENT OF HIV LATENCY IN CD4+ T CELLS Lavinia Franchitti 1 , Zhan Zhang 1 , Jack Yoon 1 , Mirko Paiardini 1 , Guido Silvestri 1 , Deanna Kulpa 1 1 Emory University, Atlanta, GA, USA Background: The persistence of HIV infection under ART is due to a reservoir of latently infected cells that remain indefinitely despite suppression of virus replication.Defining the mechanisms responsible for the establishment and maintenance of the HIV reservoir under ART has been the focus of efforts aimed at HIV eradication. Several studies have demonstrated that CD8+ T cells inhibit virus replication during untreated HIV/SIV infection; however, the mechanisms responsible for this antiviral effect remain poorly understood. Methods: We used our primary cell based in vitromodel of HIV latency to study the CD8+ T cell mediated suppression of HIV expression. To examine the impact of CD8+ T cells on the establishment of HIV latency, memory CD4+ T cells from HIV naïve donors were infected in vitroand then co-cultured with activated CD8+ T lymphocytes (1:1 or 1:5 target:effector ratios) in the presence of the anti-retroviral compound saquinavir. After three days, we assessed intracellular Gag expression on CD4+ T cells by flow cytometry, and quantified the frequency of integrated HIV DNA by qPCR. To assess the role of CD8+ T cells in latency reversal, latently infected CD4+ T cells generated in our in vitrolatency model were TCR stimulated in the presence or absence of activated CD8+ T lymphocytes (1:1 or 1:5 target:effector ratios). After three days of activation, we again assessed intracellular Gag expression on CD4+ T cells, and quantified the frequency of integrated HIV DNA. Results: In the establishment of HIV latency, we found that HIV expression in CD4+ T cells was reduced when co-cultured with CD8+ T cells an average of 9-fold (p<0.0001) and 18-fold (p<0.0001) at 1:1 or 1:5 ratios respectively,

without significantly reducing the frequency of HIV-infected cells (n=21). We also observed a significant suppression of HIV latency reversal, a 6-fold decrease at 1:1 target: effector ratio (p= 0.0156) and 14-fold decrease at 1:5 ratio (p= 0.0156). Conclusion: Our studies demonstrated a CD8+ lymphocyte mediated suppression of HIV expression in CD4+ T cells that functions to induce the establishment as well as maintain latency in the presence of activation signaling. Understanding the mechanisms by which CD8+ lymphocytes suppress virus transcription and ultimately promote HIV latency in ART-treated HIV-infected individuals may provide critical insight to support the design HIV eradication approaches. 331 THE HIV ANTISENSE TRANSCRIPT AST INDUCES VIRAL LATENCY VIA SEVERAL SILENCING PATHWAYS Rui Li 1 , Zahra Gholizadeh 1 , Kaveh Daneshvar 2 , Michelle Pleet 3 , Fatah Kashanchi 3 , Luigi Marchionni 4 , Alan Mullen 2 1 University of Maryland, Baltimore, MD, USA, 2 Massachusetts General Hospital, Boston, MA, USA, 3 George Mason University, Fairfax, VA, USA, 4 Johns Hopkins University, Baltimore, MD, USA Background: The HIV-1 antisense transcript (Ast) induces the establishment and maintenance of HIV-1 latency via recruitment of the Polycomb Repressor Complex 2 (PRC2) to the HIV-1 5’LTR, leading to trimethylation of lysine 27 on histone H3 (H3K27me3), nucleosome assembly and transcriptional silencing. Methods: Ast mutants were tested after stable transduction in Jurkat E4 cells. To identify new binding partners, Ast was fused to a streptavidin-binding RNA aptamer, expressed in 293 cells, affinity-purified by streptavidin, and binding proteins identified by mass spectrometry (MS). For RNAseq, differential analysis was performed with edgeR with negative binomial distribution using FANTOM- CAT permissive set as reference transcriptome. Results: We produced a panel of substitution and deletion mutants. A 376-nt segment at the 5’ end of Ast (5AST, from the 3’LTR) mediates binding of Ast to the proviral 5’LTR via sequence homology. We divided the Ast sequence downstream of 5AST into four segments (A through D). Substitution of segment A or B reduces Ast function. Substitution of 70nt in segment B containing a putative PRC2-binding motif also reduces Ast activity decreasing H3K27me3 levels at Nuc-1. Concurrent substitution or deletion of segments C and D also impacted Ast activity, suggesting the recruitment of additional factors. We found that Ast interacts with several repressors such as NuRD, CTCF, YY1, TDP-43, forming a complex of ~2MDa. To assess off-target effects of Ast, we performed RNAseq in cells stably transduced with Ast compared to cells stably transduced with empty lentivirus and parental cells, and using three different cellular backgrounds. Only 7 and 16 host genes differentially expressed in Ast-expressing cells compared to parental cells and empty lentivirus cells, respectively. To gain insight into Ast transcriptional regulation, we measured Ast expression in response to a panel of LRAs and found that all agents induced antisense transcription to similar or greater extent than sense HIV-1 transcription. Conclusion: We identified the LTR- and PRC2-binding regions of Ast, and new Ast-binding partners. We found that Ast does not affect host gene expression and is highly specific for HIV-1. These results identify Ast an ideal tool for the development of a functional cure. Induction of Ast to greater extent than sense HIV transcripts in response to LRA may explain their limited efficacy in HIV reactivation. 332 LUNG DOUBLE NEGATIVE T CELLS HARBOR HIV IN ACUTE INFECTION AND DURING LONG-TERM ART Oussama Meziane 1 , Syim Salahuddin 1 , Tram Pham 2 , Omar Farnos 3 , Amelie Pagliuzza 4 , Nicolas Chomont 4 , Elaine Thomson 5 , Ron Olivenstein 6 , Marianna Orlova 5 , Erwin Schurr 5 , Eric A. Cohen 2 , Cecilia Costiniuk 5 , Mohammad-Ali Jenabian 3 1 Research Institute of McGill University Health Centre, Montreal, QC, Canada, 2 Université de Montréal, Montreal, QC, Canada, 3 Université du Québec à Montréal, Montreal, QC, Canada, 4 Centre de Recherche du CHUM, Montreal, QC, Canada, 5 McGill University Health Centre Research Institute, Montreal, QC, Canada, 6 McGill University Health Centre, Glen site, Montreal, QC, Canada Background: The lungs are relatively unexplored reservoirs in the ART era. Double negative (DN) T-cells originate either from the thymus by escaping negative selection, or in the periphery following CD4 downregulation by HIV Nef/Vpu. As circulating DN T-cells have been described as cellular HIV reservoirs,

Poster Abstracts

CROI 2020 114