CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

365 HIV VIREMIA IS THE PRODUCT OF A SMALL FRACTION OF HIV INFECTED CELLS Elizabeth M. Anderson 1 , Shawn Hill 1 , Jennifer Bell 2 , Catherine A. Rehm 3 , Sara Jones 2 , Rob Gorelick 2 , Mary F. Kearney 1 , John M. Coffin 4 , Frank Maldarelli 1 1 National Cancer Institute, Frederick, MD, USA, 2 Leidos Biomedical Research, Inc, Frederick, MD, USA, 3 NIAID, Bethesda, MD, USA, 4 Tufts University, Boston, MA, USA Background: HIV persistence in replication competent reservoirs during antiretroviral therapy (ART) is a substantial obstacle to HIV cure. Understanding dynamics of HIV viremia and infected T cells is essential to characterizing these reservoirs. Upon initiating ART, plasma HIV RNA undergoes multiphasic decay kinetics reflecting half lives of infected cells. HIV infected cell numbers also decline during ART but kinetics of decay during first weeks of ART have not been well characterized. In particular, it is not known what proportion of infected cells contribute to viremia. To address this issue, we developed sensitive and accurate multiplexed droplet digital approaches (ddPCR) to quantify early HIV-1 decay kinetics. Methods: HIV infected ART-naïve individuals (N=10) enrolled in a clinical trial of 4 drug ART (2 NRTI+NNRTI+PI) at the NIH Clinical Center, were frequently sampled prior to and throughout first and second phase decline of HIV viremia. Cell-associated HIV DNA from PBMCs obtained pre-ART, during first and second phase viral decay, and after viral suppression was quantified using ddPCR assays targeting HIV gag, LTR, and tat/rev ; a host gene ( CCR5 ) was quantified for cell counting. Plasma HIV RNA was measured (bDNA) concurrently. We analyzed the decay kinetics of HIV viremia and of cell-associated HIV DNA during first and second phase decay. Results: All patients had successful suppression of HIV RNA to <50 cps/mL plasma by a median of 139.5 days on ART. Overall HIV DNA/1e6 CD4+ cells decreased for all 3 assays by an average of 4.5 fold from pre-therapy to viral suppression. Strikingly, while HIV RNA in the blood declined on average 96% after 6 days on therapy, HIV DNA declined an average of only 30% (mean 491 cps HIV DNA/ml; range 49-903 cps/ml) indicating that the majority of viremia is produced by a small fraction of HIV infected cells, and that each infected cell is responsible for a median of 104 cps HIV RNA in plasma (range 21.2-8999 cps/ cell). During second phase decline, virus production declined to c. 2.7 cps/cell (range 0.04-13.4 cps/cell). Conclusion: Prior to ART, plasma viremia is the product of only a small fraction of HIV-infected cells. After initiating ART, HIV reservoirs responsible for persistent viremia are relatively limited and exhibit substantial variation in virus production. Analysis of early HIV RNA and DNA decay kinetics will be useful in characterizing patient-specific differences in establishing HIV reservoirs. 366 THE GENETIC TRAITS OF FULL-LENGTH HIV SEQUENCED FROM MEMORY T CELL SUBSETS Bethany A. Horsburgh 1 , Bonnie Hiener 1 , Eunok Lee 1 , John-Sebastian Eden 2 , Timothy E. Schlub 3 , Susanne von Stockenstrom 4 , Jeffery Milush 5 , Teri Liegler 5 , Elizabeth Sinclair 5 , Rebecca Hoh 5 , Rémi Fromentin 6 , Nicolas Chomont 6 , Steven G. Deeks 5 , Frederick M. Hecht 5 , Sarah Palmer 1 1 The Westmead Institute for Medical Research, Westmead, NSW, Australia, 2 The University of Sydney, Sydney, NSW, Australia, 3 University of Sydney, Camperdown, NSW, Australia, 4 Karolinska Institute, Stockholm, Sweden, 5 University of California San Francisco, San Francisco, CA, USA, 6 Centre de Research du Centre Hospitalier de l’Université de Montreal, Montreal, QC, Canada Background: A thorough understanding of the distribution and genetic traits of replication-competent virus will be needed to design future HIV eradication therapies. To address this issue, we used the Full-Length Individual Proviral Sequencing (FLIPS) assay to examine the contribution of genetically identical and intact proviruses within memory CD4+ T cell subsets to the latent reservoir during prolonged ART. Methods: Naïve, central (CM), transitional (TM) and effector (EM) memory CD4+ T cells, as well as CD45RA-HLA-DR+ and CD45RA-HLA-DR- CD4+ T cells were sorted from the peripheral blood of six participants who initiated ART during either acute or chronic infection (n=3 each). Genetic sequences of HIV proviruses from the cell subsets were obtained using the FLIPS assay. FLIPS uses LTR-specific primers to amplify HIV proviruses at limiting dilution followed by next-generation sequencing. Proviruses were characterized as defective (containing INDELs, stop codons or hypermutation) or intact. Expansions of identical sequences (EIS) were determined as ≥2 identical HIV DNA sequences. Results: Of the 728 sequences isolated, only 5%were considered intact. Intact provirus was found in all cell subsets except the CM subset (0/125 isolated

sequences intact). The proportion of intact provirus was different across the cell subsets (EM>TM>CM and HLA-DR+> HLA-DR-; p=0.001). The frequency of cells infected with intact proviruses was higher in HLA-DR+memory T cells (48 vs <10 infected cells/million cells in HLA-DR+ vs all other subsets). Co-receptor usage was restricted, with 83% of intact proviruses being CCR5 tropic. Overall the percentage of intact and defective sequences contributing to an EIS was 34% (12/35) and 46% (319/693) respectively. In one participant 56 identical sequences contained a deletion in the packaging signal but were intact in the coding region. Despite this defect, the corresponding intracellular RNA sequence was detected. Conclusion: Genetically intact and therefore likely replication-competent CCR5 tropic HIV is enriched in cells expressing HLA-DR and EM cells. This indicates that the latent HIV reservoir is established early and is maintained by T cell proliferation and differentiation. The lack of intact virus in CM cells suggests that the majority of rebound virus will not be derived from these cells. Defective proviruses can produce viral transcripts indicating that RNA quantification will lead to overestimating the genetically intact HIV reservoir. 367 PULMONARY MUCOSAL T CELLS AS POTENTIAL HIV RESERVOIRS DURING LONG-TERM ART Syim Salahuddin 1 , Omar Farnos 2 , Ron Olivenstein 3 , Aurelie Le Page 1 , Amelie Pagliuzza 4 , Christina de Castro 1 , Jean Bourbeau 3 , Petronela Ancuta 4 , Bertrand Lebouché 1 , Jean-Pierre Routy 1 , Nicolas Chomont 4 , Cecilia Costiniuk 1 , Mohammad-Ali Jenabian 2 1 McGill University Health Centre Research Institute, Montreal, QC, Canada, 2 Université du Québec à Montréal, Montreal, QC, Canada, 3 McGill University Health Centre, Glen site, Montreal, QC, Canada, 4 Université de Montréal, Montreal, QC, Canada Background: Cellular and anatomical HIV reservoirs remain the primary challenge towards viral eradication. The lungs are potential but relatively understudied anatomical reservoirs in the ART era. CCR6+ and CD32a+ CD4 T-cells as well as double negative (DN) CD4-CD8- T cells have been described as potential cellular reservoirs for HIV in the blood. Here, we assessed their frequency and distribution in the lungs vs the peripheral blood of HIV-infected adults under long-term suppressive ART. Methods: Cells from bronchoalveolar lavage (BAL), obtained by bronchoscopy, and matched peripheral blood samples were collected from n=16 HIV+ individuals without respiratory symptoms and under long-term suppressive ART (undetectable plasma viral load and CD4 count higher than 350 cells/mm 3 for at least 3 years). T-cell subsets were characterized by flow cytometry, and total and integrated HIV DNA were assessed by ultrasensitive PCR. Paired t-test was used in statistical analyses. Results: A greater frequency of HIV DNA was observed in the BAL cell pellets compared to blood (p=0.006). Higher frequencies of CCR6+memory CD4+ T cells were observed within the lungs vs blood (p=0.003). Importantly, CD4 T-cells expressing the Fc receptor CD32a were highly enriched in the pulmonary cells vs blood (p=0.008). Interestingly, pulmonary CD32a+ CD4 T cells demonstrated higher levels of HLA-DR and CCR5 expression compared to blood. A substantial increase in both CD4-CD8α-CD8β- and CD4-CD8-TCRαβ-TCRγδ- DN T-cells (p<0.0001 and p=0.04) was observed in the lungs vs. blood. Pulmonary DN T-cells were characterized by higher levels of immune activation (HLA-DR+, p=0.02), lower levels of immune senescence (CD28-CD57+, p=0.01) and lower marker of recent thymus emigrants (CD31+, p=0.009). Moreover, memory CCR6+ CD4 T-cells and DN T cells exhibited higher expression of CD32a in the lungs compared to blood (p=0.06 and p=0.04). Conclusion: In virally suppressed HIV+ adults, the lungs contain higher levels of HIV DNA and higher frequencies of various T cell subsets known as preferential HIV reservoirs including CCR6+ and CD32a+ CD4 T cells as well as activated DN T cells when compared to peripheral blood. This particular distribution of mucosal T cells could contribute to the preferential persistence of HIV reservoirs within the lungs. 368 THE BIRC5/OX40 PATHWAY MAINTAINS SURVIVAL OF HIV-1-INFECTED CD4 T CELLS Hsiao-Hsuan Kuo 1 , Rushdy Ahmad 2 , Guinevere Q. Lee 1 , Hsiao-Rong Chen 1 , Matthew J. Szucs 2 , Athe Tsibris 3 , Eric Rosenberg 4 , Steven Carr 2 , Xu G. Yu 1 , Mathias Lichterfeld 3 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Broad Institute of MIT and Harvard, Cambridge, MA, USA, 3 Brigham and Women’s Hospital, Boston, MA, USA, 4 Massachusetts General Hospital, Boston, MA, USA

Poster Abstracts

CROI 2018 128