CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

of ten AIDS Clinical Trials Group (ACTG) participants who underwent an ATI and compared their rebounding virus with those from pre-ART plasma, and latent and expressed HIV reservoirs as defined by on-ART PBMC cell-associated DNA and RNA (CA-DNA and CA-RNA). Methods: Ten ACTG participants were identified who enrolled in a first-line ART initiation study and subsequently participated in an ATI study while on suppressive ART. SGS were obtained from pre-ART plasma, PBMCs collected during virologic suppression, and from early post-ATI plasma. The genetics of the HIV populations at each time point and in each compartment were compared phylogenetically and using tests for panmixia. Sequence diversity was calculated by average pairwise distance (APD). Results: PBMCs were collected a median of 4 years after ART initiation and post-ATI plasma were obtained a median of 1 week after initial detectable viremia with a median viral load of 17,286 HIV RNA copies/mL. A median of 25 SGS were obtained from each participant time point. Despite many years of suppressive ART, the viral diversity of on-ART CA-RNA and CA-DNA were similar to that of the pre-ART plasma HIV RNA (median APD of CA-RNA vs. CA-DNA vs. pre-ART plasma RNA: 1.1% vs. 1.2% vs. 1.2%, repeated measures ANOVA P=0.70). Hypermutated HIV sequences were detected in both CA-DNA and CA-RNA. For most participants, the oligoclonal populations of rebounding HIV were found to have a population structure not significantly different than HIV from pre-ART and on-ART samples, although the rebounding virus were most closely related to either CA-RNA or CA-DNA. In one instance, we found CA-RNA that closely matched rebound viremia. The diversity of pre-ART plasma HIV RNA in this participant was low (0.23%), indicating that treatment may have been initiated early after infection. Conclusions: HIV diversity in the latent and expressed PBMC HIV reservoirs were similar to that found in pre-ART plasma RNA despite several years of ART. In some cases, early rebound virus closely matched those found in the expressed PBMC HIV reservoir, but in other cases, rebounding HIV may have originated from sources other than circulating PBMCs. 379 Characterizing the Active HIV Reservoir on ART: Cell-Associated HIV RNA and Viremia Feiyu Hong ; Elizabeth Fyne; Anthony R. Cillo; Margaret A. Bedison; Dianna Koontz; JohnW. Mellors University of Pittsburgh, Pittsburgh, PA, US Background: Despite combination antiretroviral therapy (ART), HIV-1 RNA can be detected in plasma and peripheral blood mononuclear cells (PBMCs), indicating proviral transcription and production of virions, i.e. an active reservoir of HIV. It is not known whether proviral copy number, HIV-1 transcription, and residual plasma viremia on ART are related. Methods: We conducted a cross-sectional study of viremic patients off ART and of virologically suppressed (<50 cps/ml) patients on ART. PBMCs were tested for total cell- associated (CA) HIV-1 DNA and unspliced HIV-1 RNA using sensitive qPCR targeting 3’ pol. Plasma was tested for residual viremia by single copy assay targeting the same pol region. Unpaired t-test was used to compare viremic and patients on ART. Correlations between plasma viremia and cellular nucleic acids were assessed with Pearson’s coefficient. Results: 12 viremic patients and 23 patients on ART were studied. In patients on ART, median CA HIV-1 DNA was 310 copies/10 6 PBMCs (range: 45, 984) and median CA HIV-1 RNA was 59 copies/10 6 PBMCs (range: 1, 454), both were significantly lower than in viremic patients (median 565 [range: 48, 4680], p = 0.033; median 296 [range: 33, 19172], p = 0.030; for CA HIV-1 DNA and RNA, respectively). The 5-fold reduction in CA HIV-1 RNA on ART is small compared with the > 4 log 10 difference in plasma viremia between these two groups (median 0.44 [range: 0.4, 26] vs. 10542 [range: 564, 474211] copies/mL on and off ART, respectively), indicating substantial persistence of HIV-1 transcription despite ART. A strong, positive correlation was detected between cell-associated HIV-1 DNA and unspliced RNA in both viremic (Pearson’s r = 0.974; p < 0.001) and patients on ART (r =0.779; p <0.001). In viremic patients, the levels of plasma HIV-1 RNA also show strong, positive correlations with cell-associated HIV-1 DNA and RNA (Pearson’s r = 0.849 and 0.843, respectively; p < 0.001). By contrast, in patients on ART, residual plasma viremia was not correlated with cell-associated HIV-1 DNA (r = 0.06; p = 0.78) or RNA (r = -0.18; p = 0.39). Conclusions: This is the first study to show i) a strong, positive correlation between the number of HIV-infected cells and the level of cell-associated HIV-1 RNA in patients on ART, and ii) no correlation between cell-associated HIV-1 RNA and the levels of persistent viremia. These findings suggest that most of persistent HIV-1 transcription in patients on ART does not result in viremia. 380 Liver Macrophages and HIV-1 Persistence Abraham J. Kandathil ; Christine M. Durand; Jeffrey Quinn; Andrew Cameron; David L.Thomas; Ashwin Balagopal Johns Hopkins School of Medicine, Baltimore, MD, US Background: Cellular reservoirs of HIV-1 infection that persist despite combination antiretroviral therapy (cART) are impediments to a cure. The extent to which tissue resident macrophages (TRM) sustain infectious HIV-1 in patients on cART is unknown. We hypothesized that liver macrophages (LM),also known as Kupffer cells, comprising 80-90% of all TRM are a reservoir of HIV-1. Methods: To test this hypothesis we used both in vitro and in vivo studies. Purified LM from 3 human donors were infected in vitro with an R5-tropic GFP reporter HIV-1 virus and supernatants were tested periodically for the presence of viral RNA. To assess their role in vivo, LM were purified from liver explants taken from HIV-1 infected persons with uncontrolled (n=1) and cART-suppressed viremia (n=2); viral outgrowth assays were performed on LM isolated from HIV-1 infected persons with cART-suppressed viremia to determine if purified LM contain infectious HIV-1. LM purity was confirmed using sensitive qPCR assays, and when required LM were incubated with a high-affinity T cell immunotoxin. Results: LM were infected in vitro and were found to support infectious virus production for >170 days; LM supernatants were sufficient to propagate HIV-1 in reporter cells. Infectious virus was also recovered during and after in vitro exposure to cART for 24 days. Poly(A) HIV-1 RNA was detectable in LM supernatant from the individual with uncontrolled viremia (N7) 18 days after explantation, indicating virus release. Viral outgrowth assays demonstrated LM supernatants from HIV-1 infected persons (LT01 and LT02) with cART-suppressed viremia could transmit infection to reporter cells: LT01 and LT02 LM were maintained ex vivo for 36 and 95 days, respectively, before stimulation. After stimulation of LM, reporter cells were inoculated with LM supernatants for 36 (LT01) and 11 (LT02) days. Reporter cells were lysed and found to have proviral DNA (LT01: 2.4 cp/10 6 cells; LT02: 4.9 cp/10 6 cells). Reporter cells treated with LM supernatants from LT02 also released HIV-1 RNA at a level of 55 cp/100 μ L on day 11 after inoculation. Notably, LT02 had a pre-cART plasma HIV-1 RNA level of 74 cp/mL (Table).

Poster Abstracts

281

CROI 2015