CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

293 INTACT PROVIRUSES ARE UNEQUALLY DISTRIBUTED IN T CELL SUBSETS DURING ART

Bonnie Hiener 1 , Bethany A. Horsburgh 1 , John-Sebastian Eden 2 , Kirston Barton 1 , Eunok Lee 1 , Steven G. Deeks 3 , Jeffery Milush 3 , Nicolas Chomont 4 , Rémi Fromentin 4 , Sarah Palmer 1 1 The Westmead Inst for Med Rsr, Westmead, Australia, 2 The Univ of Sydney, Sydney, Australia, 3 Univ of California San Francisco, San Francisco, CA, USA, 4 Univ de Montréal, Montreal, QC, Canada Background: A thorough understanding of the distribution of replication-competent HIV is needed to design future eradication therapies. We studied the distribution of intact viral genomes in memory T cell subsets in the peripheral blood of individuals on long-term antiretroviral therapy. The distribution of clonal sequences was examined to determine the role of cellular proliferation as a contributor to persistence of intact HIV genomes. Methods: To investigate the proportion of intact proviruses in T cell subsets, we developed an assay utilising Next Generation Sequencing (NGS), to amplify and sequence single near full-length (9kb; 92% of the genome) HIV-1 proviruses within CD4+ T cell subsets (naïve (TNA), central (TCM), transitional (TTM) and effector (TEM) memory) FAC sorted from peripheral blood. Between 30-40 individual proviruses were sequenced per cell subset from 5 participants treated during acute (n=2) and chronic (n=3) infection. NGS was conducted using the Illumina MiSeq platformwith individual proviruses de novo assembled using a specifically designed workflow in CLC Genomics. Proviruses were characterized as defective (containing INDELs, stop codons or APOBEC3G hypermutation) or intact (full-length; lacking defects). Results: The percentage of intact proviruses varied between participants from 6-49%, with no difference in the mean percentage of intact proviruses between participants treated during acute (27%) and chronic (20%) infection (p=0.725). Combining the data for all participants revealed that TEM contained the largest percentage of intact proviruses (8-80%; mean=40%, n=5), compared to TNA (0-22%; mean=3%, n=3), TCM (0%; mean=0%, n=3) and TTM (3-30%; mean=15%, n=4). Clonal expansions of identical proviruses were identified in 2 participants: 1 treated during acute infection, where 66% of TTM proviruses comprised a defective clonal expansion and 1 treated during chronic infection, where 60% of TEM proviruses comprised an intact clonal expansion. Conclusion: Intact proviruses that could potentially contribute to a rebound following a treatment interruption were found unequally distributed across T cell subsets. We identified clonal expansions of intact proviruses indicating that cells that have undergone proliferation contain virus capable of rebound and actively contribute to the latent reservoir. Identification of TEM as the reservoir containing the highest proportion of intact proviruses demonstrates the importance of targeting TEM cells in future eradication strategies. 294 A SMALL FRACTION OF PROVIRUSES IN EXPANDED CLONES EXPRESS UNSPLICED HIV RNA IN VIVO AndrewMusick 1 , Jonathan Spindler 1 , Brandon Keele 2 , Michael J. Bale 1 , Wei Shao 2 , Ann Wiegand 1 , John Mellors 3 , John Coffin 4 , Frank Maldarelli 1 , Mary F. Kearney 1 1 NCI, Frederick, MD, USA, 2 Leidos Biomed, Inc, Frederick, MD, USA, 3 Univ of Pittsburgh, Pittsburgh, PA, USA, 4 Tufts Univ, Boston, MA, USA Background: The vast majority of proviruses that persist on ART are defective. Of the minority that are intact (~2%), the fractions that are latent or transcriptionally active are not known. To address this question, we determined the fraction of proviruses that express HIV RNA in vivo in cell populations carrying either intact or defective proviruses. Methods: PBMC were obtained from Patient #1 in Maldarelli, et al. (Science, 2014). This donor had multiple clones of cells that contain intact or defective proviruses. Proviral expression was determined by single-genome pro-pol sequencing (SGS) of HIV DNA and RNA frommultiple aliquots of PBMC diluted to an endpoint such that each aliquot contained one to a few HIV RNA expressing cells. Intact proviruses were identified using viral outgrowth assays (VOA). The levels and fractions of cells expressing HIV RNA were determined for probable clones (identified by identical sequence matches) carrying intact and defective proviruses. Results: A total of 77 million PBMC were analyzed, of which 10,450 contained HIV pro-pol sequences. Fourteen percent of the infected cells expressed HIV RNA. The median levels of expression in single cells was 1 RNA/cell (ranging from 1-16). We identified 412 different WT or hypermutant RNA species in infected cells. Of these, 3 were from expression of intact proviruses, 81 from obviously defective proviruses, and 328 from proviruses that were likely defective (did not grow out in VOA but did not contain stop codons in the region analyzed). The median fraction of cells in the probable clones (those with matching DNA and RNA) that carried intact proviruses (N=3) and expressed HIV RNA was 2.3% (1.2%- 8.8%). For clones carrying defective proviruses (N=5), the median expressing was 3.5% (0.9%-7.0%), and for presumptive clones carrying likely defective proviruses (N=26), the median was 6.6% (1.3%-66.7%) (p =0.51 for a difference across groups). Conclusion: The large majority (>80%) of infected cells that persist on ART are either latent or incapable of HIV RNA expression. A small fraction of proviruses within infected clones expressed unspliced HIV RNA, but this fraction was not significantly different between clones carrying intact proviruses from clones containing obviously defective proviruses, indicating that HIV RNA expression appears similarly detrimental (or non-detrimental) for infected cells regardless of whether the provirus they carry is intact. 295 PROLIFERATION OF CD4+ T CELLS CONTAINING REPLICATION-COMPETENT HIV-1 Zheng Wang , Ya-Chi Ho, Janet Siliciano, Robert Siliciano, for the Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland Johns Hopkins Univ, Baltimore, MD, USA Background: Persistence of the human immunodeficiency virus type-1 (HIV-1) in a latent form in resting memory CD4+ T cells remains the major barrier to curing HIV-1 infection. The pool of latently infected CD4+ T cells is extremely stable and it has been shown that HIV-infected CD4+ T cells can clonally expand. However, it remains unclear what mechanism drives the expansion of HIV-infected CD4+ T cells and whether HIV-1 proviruses in these clonally expanded cells are replication competent or defective. We hypothesize that clonal expansion of HIV-1-infected CD4+ T cells is caused by physiologic stimulations including T cell activation and homeostatic proliferation and some clonally expanded CD4+ T cells can produce replication-competent virus. Methods: Resting CD4+ T cells from aviremic patients under suppressive antiretroviral therapy were labeled with carboxyfluorescein succinimidyl ester (CFSE) and activated with CD3/CD28 costimulation or stimulated with IL-7/IL15 in the presence of enfuvirtide to prevent new rounds of infection. After 7 days of culture, CD4+ T cells were sorted based on CFSE dilution and plated at 200,000 cells per well in a viral outgrowth assay (VOA) to quantify infectious HIV-1 in both populations. Cells that have proliferated in response to IL-7/IL-15 stimulation were tested in a viral outgrowth assay with or without PHA activation to determine whether cells that proliferate with IL-7/IL-15 stimulation can produce infectious HIV-1 without activation. Results: Our results demonstrate that HIV-infected CD4+ T cells proliferate in response to both CD3/CD28 co-stimulation and cytokine (IL-7/IL-15) stimulations. Some of the cells that have proliferated in response to CD3/CD28 costimulation produce replication-competent virus without additional PHA stimulation. However, additional viral outgrowth is observed with PHA stimulation. Cells that proliferated in response to IL-7/IL-15 treatment are able to produce infectious virus with PHA stimulation. However, no viral outgrowth is observed from this proliferated population in the VOA assay without PHA stimulation. Conclusion: We conclude that T cell activation and homeostatic cytokines were able to induce proliferation of HIV-infected CD4+ T cells containing replication-competent viruses. The results indicate the possibility that both T cell activation and homeostatic proliferation are potential mechanisms that maintain HIV-infected CD4+ T cells containing replication-competent HIV. 296 CYTOTOXIC T LYMPHOCYTES MAY SHAPE THE HIV-1 PROVIRAL LANDSCAPE R. Brad Jones 1 , Mihaela Pertea 2 , Katherine Bruner 2 , Ross Pollack 2 , Adam Capoferri 2 , Robert Siliciano 2 , Ya-Chi Ho 2 1 The George Washington Univ, Washington, DC, USA, 2 The Johns Hopkins Univ, Baltimore, MD, USA Background: The majority of the HIV-1 proviruses are defective, making it irrelevant to use HIV-1 DNA quantitation to measure the size of the latent reservoir. However, a decrease in HIV-1 proviral DNA levels has been reported in vivo using the shock-and-kill strategy. We have previously shown that defective HIV-1 proviruses can be transcribed and translated, making them potential targets for cytotoxic T lymphocyte (CTL) elimination. We hypothesize that HIV-1 proviral landscape is dynamic, and may be shaped by CTL selection pressure.

Poster and Themed Discussion Abstracts

CROI 2017 116