CROI 2020 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: Blood samples from 8 individuals on suppressive ART for at least 2 years were collected longitudinally. Clonotype characterization of HIV-infected cells was determined by combining index single-cell sorting of HIV-infected cells by HIV-Flow (which allows recording the memory phenotype of individual p24+ cells, according to their differentiation status: central, transitional, effector memory cells) with multiplex PCR of the V-J junction of the TCRbeta chain (including the CDR3 region) followed by sequencing. A representative subset of p24- cells was analyzed to determine TCR diversity in the CD4+ T-cell compartment. Results: We obtained the TCR sequences from 538 p24+ and 346 p24- single- sorted cells. There was no bias in the selection of V and J segments in p24+ cells when compared to p24- cells. Expanded TCR clonotypes were present in 7/8 individuals and accounted for the majority of reservoir cells (median 89%, range 77-100). These expanded clonotypes were maintained over time on ART in 5 individuals and persisted for up to 6 years. The dynamic of the HIV reservoir on ART greatly varied between individuals, with some participants showing a stable repertoire, whereas others displayed emergence of new clonotypes over time. Expanded infected clones were systematically overrepresented in the most differentiated cells (i.e. transitional and effector memory). Nonetheless, these expanded clones were also identified within the central memory compartment from the majority of the participants, albeit at lower frequencies. Importantly, the memory phenotype of these expanded reservoir cells was maintained over time on ART. Conclusion: Through the repertoire analysis of infected cells, we show that antigen-driven clonal expansion highly contributes to the persistence of the translation-competent HIV reservoir during ART. Our results suggest that infected T cell clonotypes displaying a differentiated phenotype are the progeny of infected central memory cells undergoing clonal expansion during ART 363 ONLY A FEW HIV-1 INTEGRATION SITES CONFER GROWTH ADVANTAGE TO INFECTED CELLS IN VIVO John M. Coffin 1 , Michael J. Bale 2 , Daria W. Wells 3 , Shuang Guo 3 , Brian Luke 3 , Jennifer M. Zerbato 4 , Michele Sobolewski 4 , Twan Sia 2 , Wei Shao 2 , Xiaolin Wu 3 , Frank Maldarelli 2 , Mary F. Kearney 2 , John W. Mellors 4 , Stephen H. Hughes 2 1 Tufts University, Boston, MA, USA, 2 National Cancer Institute, Frederick, MD, USA, 3 Leidos Biomedical Research, Inc, Frederick, MD, USA, 4University of Pittsburgh, Pittsburgh, PA, USA Background: HIV persists during antiretroviral therapy (ART) as proviruses in latently-infected cells that are descendants of a tiny fraction of the CD4+T cells infected prior to ART initiation. We and others previously reported in vivo selection of cell clones with proviruses integrated in several specific genes, based on analyzing small numbers of integration sites. Methods: We compared about 380,000 integration sites in PBMC infected ex vivo to sites combined from 32 individuals on suppressive ART for >1 year. The on-ART dataset comprised about 52,000 sites, of which about 31,000 were unique. The two datasets were compared to look for evidence of selection in vivo, and to infer its mechanism. Results: The overall distribution of unique integration sites was nearly identical between the two datasets. As expected, there was preferential integration in highly-expressed genes (84% of sites) in the ex vivo infected PBMC dataset, and the proviruses were randomly oriented relative to the host gene. By contrast, in the on ART dataset, there was a modest (55%), but significant (P~10-50), bias for integration in the reverse orientation, which was the result of a weak selection acting on a large number of genes, rather than of strong selection acting on a few genes. Proviruses integrated in three genes (MKL2, BACH2, STAT5B), known to be drivers of cell growth or survival, were enriched in vivo (Table 1) and were preferentially integrated in one or two introns in the same orientation as the gene. We detected three more genes (MKL1, IL2RB, MYB) in which the data also suggest proviral effects on cell growth or survival (Table 1). Taken together, the proviruses in the 6 genes comprised only 2.3% of unique integration sites. Outside of these genes there was no evidence of clustering, orientation bias, or local enrichment of clonally amplified proviruses. Conclusion: The primary determinant of the distribution of integration sites in persons on ART is their initial distribution, which is subsequently modified only modestly by selection against proviruses in the sense orientation. Proviruses integrated in the sense integration in any one of 6 genes can enhance cell expansion and/or survival; however, these few selected cells are unlikely to be of major importance to HIV-1 persistence. Other mechanisms driving clonal expansion, for example immune signaling, are more important.

relative to transcriptionally-active proviruses. Longitudinal analysis in one patient indicated an enrichment of non-genic/pseudogenic integrations after suppressive ART (21%) as compared to pre-ART levels (0%). This trend was paralleled by a 59-fold reduction in the number of transcriptionally-active intact proviruses, and a 7-fold reduction in the number of transcriptionally- silent intact proviruses per million PBMC after suppressive ART. In comparison, transcriptionally-active and -silent defective reservoirs declined 6-fold and 4-fold, respectively. Conclusion: Parallel analysis of proviral sequences, integration sites and viral gene expression from single reservoir cells suggests progressive enrichment of transcriptionally-silent proviruses integrated into non-permissive genomic regions during prolonged ART. Future use of PRIP-Seq will allow profiling of the evolutionary dynamics of viral reservoir cells in great detail. 361 SINGLE-CELL ATLAS AND CLONAL EXPANSION DYNAMICS OF CD4+ T CELLS DURING HIV INFECTION Jack A. Collora 1 , Delia Pinto-Santini 2 , Siavash Pasalar 2 , Ricardo Alfaro 3 , Carmela Ganoza 3 , Jennifer Chiarella 1 , Rachela Calvi 1 , Javier R. Lama 3 , Serena S. Spudich 1 , Ann Duerr 2 , Ya-Chi Ho 1 1 Yale University, New Haven, CT, USA, 2 Fred Hutchinson Cancer Research Center, Seattle, WA, USA, 3 Asociacion Civil Impacta Salud y Educacion, Lima, Peru Background: Despite effective antiretroviral therapy (ART), HIV-1 persists in CD4+ T cells as a major barrier to cure. More than 50% of the HIV-1 latent reservoir is maintained by clonal expansion. HIV-1-infected cells undergo clonal expansion through antigen-driven proliferation, homeostatic proliferation and integration site-driven proliferation. Targeting clonally expanding HIV-1- infected cells without damaging uninfected cells is required to eliminate the latent reservoir. We constructed a single-cell multiomic atlas of CD4+ T cells from HIV-1-infected individuals during acute HIV-1 infection and after viral suppression at its native status without ex vivo stimulation. Methods: We obtained paired CD4+ T cells from three HIV-1-infected individuals from the MERLIN cohort during acute infection (within one month of the estimated day of infection) and after suppressive ART (11 months of ART with viral suppression [plasma viral load <200 copies/ml] within the past 6 months). CD4+ T cells from three uninfected individuals were obtained as negative controls. Using ECCITEseq (Expanded CRISPR-compatible Cellular Indexing of Transcriptomes and Epitopes by sequencing), we captured 1) surface protein expression, including memory phenotypes, activation status and exhaustion markers, 2) transcriptome, 3) HIV-1 RNA and 4) T cell clonality by T cell receptor sequences in the same single cells. We analyzed T cell clonal abundance, repertoire dynamics and clone tracking. Results: We captured an average of 7,950 single cells, 1,504 genes mapped to human genome and 6,110 T cell clones per sample. Among them, we identified a total of 67 HIV-1-infected cells and 25 expanded CD4+ T cell clones harboring HIV-1-infected cells. We mapped the single-cell atlas of CD4+ T cells from HIV-1-infected individuals which is distinct from that of uninfected individuals. We found upregulation of interferon-stimulated genes and T cell activation, reflecting T cell responses to acute HIV-1 infection. We also identified CD4+ T cell clones that persist despite suppressive ART. Even within the same CD4+ T cell clone, CD4+ T cells exhibit heterogeneous transcriptional profiles. Conclusion: We captured the cellular environment of HIV-1-infected cells from HIV-1-infected individuals at the native status without ex vivo stimulation. Transcriptional signatures of HIV-1-infected cells may serve as therapeutic targets for HIV-1 cure strategies. 362 TCR SEQUENCING REVEALS CLONAL EXPANSIONS OF INDUCIBLE RESERVOIRS IN SPECIFIC SUBSETS Pierre Gantner 1 , Amelie Pagliuzza 2 , Marion Pardons 1 , Moti Ramgopal 3 , Jean- Pierre Routy 4 , Rémi Fromentin 2 , Nicolas Chomont 1 1 Université de Montréal, Montreal, QC, Canada, 2 Centre de Recherche du CHUM, Montreal, QC, Canada, 3 Midway Immunology and Research Center, Fort Pierce, FL, USA, 4 McGill University Health Centre Research Institute, Montreal, QC, Canada Background: Clonal expansions occur in the persistent HIV reservoir as demonstrated by the duplication of HIV genes and/or integration sites reported in several studies. However, these approaches do not permit to phenotypically analyze these expanded clones of infected cells nor the inducibility of the proviruses. We took advantage of the uniqueness of the T-cell receptor (TCR) expressed by a given T-cell clone to unravel the phenotype and dynamics of the inducible HIV reservoir.

Poster Abstracts

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