CROI 2016 Abstract eBook
Methods: We analyzed full-length HIV-1 sequences from plasma RNA (6 patients), resting CD4 + T cell proviral DNA (7 patients treated during acute infection, 9 patients treated during chronic infection), and DNA from p24 antigen-negative quantitative viral outgrowth assay culture wells (6 patients). We analyzed between 5 and 121 unique sequences (mean=25, median=16) per patient. We compared the apparent clonality of each patient alignment within previously published PCR amplicons in the gag-pol and envelope V3-V4 regions. We also measured the apparent clonality of each alignment within hypothetical amplicons of various fixed lengths across the genome to identify the optimal genomic region(s) for primer placement and to compare the importance of amplicon location versus amplicon length. Results: Although no short region of the HIV-1 genome is consistently sufficient to distinguish non-clonal full-length viral sequences in all patients, a 1500bp amplicon in the gag-pol region is superior to a 500bp amplicon spanning the envelope V3-V4 region (p<0.001). In sequences from plasma RNA, the sufficiency of a hypothetical amplicon to distinguish non-clonal sequences depends almost entirely on the length of the amplicon. In proviral DNA samples, there is wide variation among patients in terms of which genomic regions work best and in terms of the importance of amplicon length. Conclusions: No short PCR amplicon is sufficient to demonstrate the clonality of HIV-1 genomes within samples from a single patient. Researchers hoping to demonstrate clonality may screen samples with a short PCR but must ultimately sequence full-length viral genomes to demonstrate the clonality of independent samples. 340 Clonal Expansion of Replication-Competent Proviruses is Common in Individuals on ART Michele D. Sobolewski 1 ; John K. Bui 1 ; Jonathan Spindler 2 ; Andrew Musick 3 ; Brandon Keele 4 ;Wei Shao 5 ; AnnWiegand 3 ; John M. Coffin 6 ; Mary F. Kearney 3 ; JohnW. Mellors 1 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 Frederick Natl Lab, Frederick, MD, USA; 5 Leidos Biomed Rsr, Inc, Frederick, MD, USA; 6 Tufts Univ, Boston, MA, USA Background: Clonal expansion of HIV-infected CD4+T-cells in patients on long-term ART was recently described by Maldarelli et. al. and Wagner et. al. (Science 2014), but the replication-competence of such proviruses is controversial. Cohn et. al. (Cell 2015) were unable to identify intact, clonally-expanded proviruses in vivo . Conversely, Simonetti et. al. (CROI 2015) described a case of persistent viremia during ART wherein the primary source was a clonally-expanded, replication-competent provirus. Here, we sought additional evidence of clonally-expanded, replication-competent proviruses in individuals on ART. Methods: Viral outgrowth assays (VOAs) were performed on > 6x10 6 purified CD4 + T-cells from 8 consecutive participants who were on suppressive ART for > 2 years (median 13.5 years) and who had diverse virus populations consistent with long-term chronic infection prior to therapy. Purified CD4+ T-cells were seeded in six replicates of 3-fold serial dilutions beginning with 1x10 6 cells per well. Single-genome sequencing (SGS) targeting p6-PR-RT was performed on supernatants from p24 positive wells and on HIV cell- associated, unspliced HIV RNA (CAR) and HIV DNA (CAD) in PBMCs from the same individuals. Sequences were examined for matches across VOA wells and to PBMC CAR and CAD by neighbor-joining analyses. Results: In 6 of the 8 individuals studied to date, identical sequence matches have been found across multiple VOA positive wells or between VOA positive wells and CAR or CAD SGS. Replication-competent proviruses constituted a median of 1.3% (range 0.7-7.1%) of all unique viral variants and a median of 9.1% (range 7.7-20%) of all clonally-expanded variants detected by SGS. Conclusions: We detected inducible, clonally-expanded, replication-competent proviruses in more than half of individuals studied on long-term suppressive ART. These expanded clones could serve as a persisting source for rebound viremia after interruption of ART and should be targeted by HIV cure strategies. 341 A Novel Assay to Quantify Replication-Competent Latent HIV-1 From rCD4+T Cells Phalguni Gupta 1 ; Anwesha Sanyal 1 ; Nicolas Sluis-Cremer 2 ; Deena Ratner 1 ; Ming Ding 1 ; Robbie B. Mailliard 2 ; Charles Rinaldo 2 ; Jennifer M. Zerbato 2 ; Amanda Chargin 3 ; Bruce K. Patterson 3 1 Univ of Pittsburgh, Grad Sch of PH, Pittsburgh, PA, USA; 2 Univ of Pittsburgh, Pittsburgh, PA, USA; 3 IncellDx, Inc, Menlo Park, CA, USA Background: The latent HIV-1 reservoir that resides in resting CD4+ (rCD4+) T cells of infected individuals on combination antiretroviral therapy (cART) is a major obstacle to a cure. A primary challenge in defining this reservoir is the lack of a high-throughput, sensitive and robust assay that can quantify the size of the inducible replication-competent pool of latent HIV-1. Currently, the quantitative viral outgrowth assay (Q-VOA) that is widely used is labor intensive, time consuming and expensive, and is not amenable to a high- throughput format. Here we report on the development of a TZM-bl cell based assay (TZA) for the quantification of replication-competent latent HIV-1, which is fast, sensitive, robust and amenable to a high-throughput format. Methods: The strategy for quantifying inducible replication competent HIV-1 in rCD4+T cells by TZA involves two steps: (i) induction of latent HIV-1 from rCD4+T cells by anti-CD3/CD28 antibodies; and (ii) quantification of induced replication competent HIV-1 in the TZM-bl cell based assay . Using TZA, Q-VOA and a simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI) assay we quantified the size of the inducible replication-competent pool of latent HIV-1 in rCD4+ T cells isolated from 13 aviremic HIV-1 infected individuals on suppressive cART from the Pittsburgh Multicenter AIDS Cohort Study. Results: For the TZA, the amount of replication competent HIV-1 was found to range 1.2-141 (mean 46.4) infectious units per million (IUPM). By contrast, the IUPM values for the Q-VOA ranged 0.28-2.37 (mean 0.70), which is almost 70-fold lower (p=0.0029). To confirm the high levels of IUPM obtained from the TZA assay we applied the SUSHI assay in parallel to anti-CD3/CD28 activated rCD4+ T cells from 10 out of 13 donors. The number of HIV-1 RNA positive cells per million rCD4+T cells in the SUSHI assay was similar in magnitude to the TZA assay ranging from 10 to 240 (mean 75). In subsets of HIV-infected subjects, we also evaluated fractional HIV-1 provirus expression (fPVE). For 9 subjects the fPVE as measured by the TZA ranged from 0.12-13.93% (mean 4.03%), while fPVE as measured by the Q-VOA ranged from 0.02- 0.9% (mean 0.14%). Conclusions: Using the novel TZA assay, we show that the size of the inducible latent HIV-1 reservoir in virally suppressed individuals on cART is significantly larger than previous estimates using the Q-VOA, which could pose a challenge for the irradication of latent virus in rCD4+ T cells in these patients. 342 Cellular HIV-1 RNA/DNA As Biomarkers of Inducible Virion Production Anthony R. Cillo 1 ; Feiyu Hong 1 ; AngelaTsai 2 ; Alivelu Irrinki 2 ; Jasmine Kaur 2 ; Jacob Lalezari 3 ; Derek Sloan 2 ; Jeffrey P. Murry 2 ; JohnW. Mellors 1 1 Univ of Pittsburgh, Pittsburgh, PA, USA; 2 Gilead Scis, Inc, Foster City, CA, USA; 3 Quest Clinical Rsr, San Francisco, CA, USA Background: Simple biomarkers of the inducible HIV-1 reservoir have not been identified. We investigated whether the frequency of infected cells in blood and their transcriptional activity is related to the inducible HIV-1 reservoir from resting CD4+ T cells in persons on virologically-suppressive antiretroviral therapy (ART). Methods: Total blood mononuclear cells (PBMC) were isolated from leukapheresis product obtained from volunteers on suppressive ART for ≥1 year. Cellular unspliced HIV-1 RNA (CA-RNA) and proviral HIV-1 DNA (CA-DNA) in uncultured PBMC were quantified by qPCR assays targeting 3’ integrase. Resting CD4+ T (rCD4) cells were isolated from PBMC by negative selection, and were then activated with PMA/ionomycin in the presence of 300 nM efavirenz. On day 6 of culture, supernatants were collected from replicate wells, centrifuged (500 x g for 5 min), and stored at -80°C. HIV-1 RNA in supernatants (surrogate for virion production) was quantified by COBAS Roche TaqMan v2.0. Correlations between virion production and cellular HIV-1 RNA/DNA were assessed using Spearman’s correlation coefficient. Results: A total of 22 donors were evaluated. Levels of HIV-1 RNA produced after 6 days of treatment with PMA/ionomycin varied >1000 fold among the donors; the median was 4406 copies/mL of culture supernatant, ranging from of 38 to 42756 copies/mL. The median level of CA-RNA was 38 (range: <1 to 355) copies per 10 6 PBMC, and the median level of CA-DNA was 286 (range: 7 to 2972) copies per 10 6 PBMC. CA HIV-1 RNA and DNA levels were strongly correlated with each other (rho=0.80, p<0.001), and both were strongly correlated (see figure) with inducible virion production (rho=0.76, p<0.001 for CA-RNA; rho=0.75, p<0.001 for CA-DNA).
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