CROI 2016 Abstract eBook

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Oral Abstracts

82 PD-1+ and Tfh Cells Represent the Major Source of HIV-1 Replication-Competent Virus

Riddhima Banga 1 ; Francesco Procopio 1 ; Matthias Cavassini 2 ; Jean-Marc Corpataux 3 ; Giuseppe Pantaleo 3 ; Matthieu Perreau 1 1 CHUV, Epalinges, Switzerland; 2 Univ Hosp Lausanne, Univ of Lausanne, Lausanne, Switzerland; 3 CHU Vaudois, Lausanne, Switzerland Background: Recent studies have shown that Tfh cells from viremic HIV-1 infected individuals served as the major CD4 T-cell compartment for HIV infection, replication and production . However, their role in long-term treated aviremic HIV-1 infected subjects remains to be established. Methods: In the present study, we have investigated the distribution of HIV-1 infected CD4 T cells containing replication competent HIV within CXCR5 - PD-1 - , CXCR5 + PD-1 - and PD-1 + memory CD4 T-cell populations isolated from blood and lymph nodes (LN) of long-term treated (1.5-14 years) aviremic (<20 HIV RNA copies per ml) subjects (n=11) using virus outgrowth assay (VOA). The LN PD-1 + memory CD4 T-cell population contained about 65% of Tfh cells, i.e . CXCR5 + PD-1 + cells. Results: We demonstrated that the levels of HIV-1 RNA produced in the VOA supernatants were significantly higher in LN PD-1 + CD4 T cells as compared to any other blood or LN CD4 T-cell subset and inversely correlated with the duration of treatment ( P <0.05). In depth analysis showed that the levels of HIV-1 RNA detected in the culture supernatants of LN PD-1 + CD4 T cells were 810-1225 fold higher as compared to the other LN PD-1 - cell populations ( P<0.05 ), and 5755-73123 higher as compared to blood CD4 T-cell populations ( P<0.05 ). In addition, P24 production was clearly detected in culture supernatants of LN memory PD-1 + CD4 T cells in 6 out of the 11 long-term ART treated aviremic HIV-1 infected individuals and border line positive in 1 out of the 11 individuals tested in blood memory PD-1 + CD4 T cells. Interestingly, HIV-1 produced by LN PD-1 + CD4 T cells was also infectious as indicated by the transmission of infection in vitro to CD4 T cells of HIV-negative subjects. Finally, we have evaluated the mean frequencies of inducible replication competent virus from HIV-1 infected cells using both RUPM and IUPM for each sorted blood and LN memory CD4 T-cell populations isolated from 11 aviremic long-term treated patients and showed that LN PD-1 + CD4 T cells were enriched with replication competent and infectious virus and represent the major source of replication competent and infectious virus in long-term treated aviremic HIV-1 infected individuals. Conclusions: Taken together, these results demonstrate that LN PD-1 + CD4 T cells which include Tfh cells serve as the major CD4 T-cell compartment for HIV-1 replication competent and infectious virus in long-term treated aviremic HIV-1 infected subjects. 83 Rapid Accumulation of Defective Proviruses Complicates HIV-1 Reservoir Measurements Katherine M. Bruner 1 ; Ross Pollack 1 ; Alexandra Murray 1 ; Mary Soliman 1 ; Sarah B. Laskey 2 ; Matt F. Strain 3 ; Douglas D. Richman 3 ; Steven G. Deeks 4 ; Janet Siliciano 1 ; Robert Siliciano 5 1 Johns Hopkins Univ Sch of Med, Baltimore, MD, USA; 2 Johns Hopkins Univ, Baltimore, MD, USA; 3 Univ of California San Diego, San Diego, CA, USA; 4 Univ of California San Francisco, San Francisco, CA, USA; 5 Howard Hughes Med Inst, Baltimore, MD, USA Background: HIV-1 establishes latency in resting memory CD4+ T cells, creating a major barrier to eradication. Although defective proviruses predominate in resting CD4+ T cells from patients treated during chronic infection, we hypothesized that the fraction of defective proviruses is not constant and increases over the course of the infection as cells with genetically intact proviruses are eliminated. We sought to determine the time frame of this accumulation as it is relevant to HIV-1 reservoir measurements. We also sought to define the reservoir size in chronic and acutely-treated patients as well as investigate what types of proviruses would be detected by current HIV-1 assays and how a shock and kill strategy would affect those measurements. Methods: We characterized proviruses from patients treated in either acute or chronic infection and untreated viremic patients. We also performed an in vitro infection of CD4+ T cells to determine the fraction of defective proviruses after a single round of infection. Proviruses were analyzed by an unbiased, limiting dilution, full genome PCR and direct sequencing of PCR products. The number of intact proviruses was quantified as a percentage for each patient and compared to total HIV-1 DNA ddPCR values and QVOA IUPMs. Proviruses were also analyzed at the DNA level to predict the likelihood of making HIV-1 RNA or protein, as required for detection by current HIV-1 assays and for elimination by a shock and kill strategy. Results: Following one round of in vitro replication, defective proviruses were readily detected and made up 40% of proviruses. Less than 5% of proviruses were intact in both patient groups; the remaining proviruses contained major defects. We also found that the QVOA underestimates the latent reservoir by a median of 64 fold in chronically treated patients and 20 fold in acutely treated patients while total DNA PCR measurements vastly overestimated the reservoir in both patient groups. Importantly, our analysis of the proviral sequences predicts that the majority of proviruses are unable to make protein and are unlikely to be affected by a shock and kill strategy. Conclusions: During acute infection, the fraction of defective proviruses likely increases very rapidly, quickly making up over 95% of proviruses in HIV-1 patients. Our analysis also indicates that most proviruses are unlikely to be affected by a shock and kill strategy. Thus, the high fraction of defective proviruses must be considered when evaluating latency reversing agents. 84 Background: We and others have shown that there is clonal expansion of HIV infected cells in patients, and that, in some cases, the integrated viral DNA can cause the growth and persistence of infected cells. More recently, we showed that a clonally expanded cell carries an intact provirus and produces infectious HIV in a patient. Because there are limits on the samples that can be obtained from patients, we developed a model using SIV-infected rhesus macaques. Methods: The methods of Maldarelli et al (Science 345: 179, 2014) were used to generate an integration site library from rhesus macaque PBMCs infected in culture with SIV. Six additional libraries were generated from two rhesus macaques (two lymph node and one spleen sample from each animal) that were infected with SIV for four weeks and then treated for approximately one year with a cART regiment that fully suppressed the replication of the virus. Samples were taken during necropsy at the end of the treatment period. Results: The distribution of the SIV integration sites in the large integration site library (~50,000 independent sites) prepared from rhesus macaque PBMC infected in vitro was quite similar to the distribution of HIV integration sites in human PBMCs. We obtained approximately 380 independent integration sites from the monkey tissue sample and identified 13 clones of expanded cells. Cells from two of the clones were present in both the spleen and lymph node tissue samples. Conclusions: The presence of expanded clones in two SIV macaques that were treated after 4 weeks of infection shows the cells that are infected early can give rise to expanded clones. Cells from two clones were present in both lymph node and spleen, showing that the distribution of at least some clones was not tissue restricted. Having a large PBMC integration site library makes it possible to look for genes in which the integration of an SIV provirus provides the infected cells with a selective growth advantage in the animal. In this first experiment, there was no evidence for the selection of cells that have integration sites in either BACH2 or MKL2. Our results establish an SIV/macaque model that can be used to study the clonal expansion of infected cells using samples that cannot be obtained from patients. 85 ART Reduces Cellular HIV RNA but Not the Fraction of Proviruses Transcribing RNA Feiyu Hong 1 ; Jonathan Spindler 2 ; Andrew Musick 3 ; Anthony R. Cillo 1 ; Michael Bale 2 ;Wei Shao 4 ; John M. Coffin 5 ; JohnW. Mellors 1 ; Mary F. Kearney 3 1 Univ of Pittsburgh, Pittsburgh, PA, USA; 2 HIV Dynamics and Replication Prog, Natl Cancer Inst, Frederick, MD, USA; 3 NCI, Frederick, MD, USA; 4 Leidos Biomed Rsr, Inc, Frederick, MD, USA; 5 Tufts Univ, Boston, MA, USA Background: Little is known about proviral expression at the single cell level before or during ART. We investigated both the fraction of infected cells that express HIV RNA and the levels of HIV RNA in single cells from untreated, viremic individuals and from those on suppressive ART. Methods: PBMCs from 5 viremic (median VL=5727 c/ml) and 5 ART-suppressed (VL<20 c/ml) individuals were analyzed for expression of unspliced viral RNA in single cells by 2 methods: Cell-Associated RNA (CAR) and DNA single-genome sequencing (CARD-SGS) and fractional proviral expression (fPVE). CARD-SGS was performed by extracting RNA from multiple PBMC aliquots diluted to an endpoint for HIV expressing cells. cDNA was synthesized from each RNA extraction and all cDNA molecules were sequenced by gag-pol SGS. Clones of SIV-Infected Cells Are Present in Spleen and Lymph Nodes in Rhesus Macaques Andrea Ferris 1 ; Gregory Q. Del Prete 2 ; Brandon Keele 2 ; XiaolinWu 2 ; Jeffrey Lifson 2 ; Stephen H. Hughes 1 1 NCI, Frederick, MD, USA; 2 Frederick Natl Lab, Frederick, MD, USA

Oral Abstracts

32

CROI 2016