CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

382 Identical Sequence Expansions Are Predominantly Found in Effector Memory T Cells Susanne von Stockenstrom 1 ; Eunok Lee 2 ; Lina Odevall 1 ; Elizabeth Sinclair 3 ; Hiroyu Hatano 3 ; Peter Bacchetti 4 ; PeterW. Hunt 3 ; Steven G. Deeks 3 ; Frederick M. Hecht 3 ; Sarah E. Palmer 2 1 Karolinska Institutet, Stockholm, Sweden; 2 Westmead Millenium Institute and University of Sydney, Sydney, Australia; 3 Department of Medicine, University of California San Francisco, San Francisco, CA, US; 4 Department of Epidemiology and Biostatistics, University of California San Francisco, San Francisco, CA, US Background: The effects of cellular proliferation, differentiation and expansion on the maintenance and genetic composition of HIV-1 populations within specific T cells is uncertain.To address this issue, we examined the distribution of identical HIV-1 intracellular sequences from peripheral blood and lymph node tissue in order to define the role of cell proliferation as a cause of persistence. Methods: Using single-proviral sequencing, we isolated intracellular HIV-1 genomes derived from defined subsets of CD4+ T cells (central (CM)-, transitional (TM)-, and effector (EM)-memory) from peripheral blood and lymph node tissue. Samples were collected from six subjects on long-term suppressive therapy (6-13 years) treated during chronic infection. Unique clonal populations of sequences were determined (for p6-RT) as > 2 genetically identical sequences among all the viruses analyzed for each subject and we assessed the number of clonal populations found exclusively within a particular cell type. All clonal sequences were compared to pre-therapy plasma-derived single genomes. Results: Phylogenetic analyses revealed that 30-60% of all HIV-1 sequences combined from the cells sorted from blood and lymph node tissue (n=93-243 per person) were clonal in nature, and that the mean number of clonal populations per subject was 14 (range 9 to 26). However, only a few (n=0-4) of these clonal sequences were found in pre-therapy plasma sequences. Clonal populations were more common in EM cells (61%) than other T cell subsets (25-49%). Across all subjects, the percentage of sequences in EM cells that were clonal sequences and found in no other cell types averaged 30%, whereas this average was <7% for CM or TM cells (both p=<0.0001 versus EM cells). The rates of such clonal populations associated with CM versus TM cells were not significantly different (p=0.21). Conclusions: Sequences in EM cells were predominantly clonal. These clones were often not found in less differentiated cells. The source of these cells is not known, but may reflect infection and expansion directly of an infected EM population, or differentiation and expansion of infected progenitor cells in tissue. This suggests that different cellular mechanisms are contributing to the persistence of HIV within different memory T cell subsets and the size and distribution of the latent reservoir during suppressive therapy is likely to be shaped in part by proliferation and expansion of differentiated T cells. 383 Long-Term Effect of Temporary ART During Primary HIV Infection on the Viral Reservoir Alexander Pasternak 1 ; Jan Prins 2 ; Ben Berkhout 1 1 Academic Medical Center University of Amsterdam, Amsterdam, Netherlands; 2 Academic Medical Center University of Amsterdam, Amsterdam, Netherlands Background: Initiation of antiretroviral therapy (ART) during primary HIV-1 infection (PHI) has been proposed to limit the formation of viral reservoirs. However, it remains unknown whether temporary ART during PHI has a long-term effect on the viral reservoir during ART initiated subsequently at chronic HIV infection (CHI). Methods: Levels of cell-associated (CA) HIV-1 unspliced RNA (usRNA) and total CA viral DNA (vDNA) were analyzed in 49 HIV-infected patients who had participated in a randomized controlled trial of 24 or 60 weeks of temporary ART versus no treatment during PHI (Primo-SHM study; PLoS Med 2012;9:e1001196). All 11 patients randomized to the no-treatment arm at PHI, and 19 of 38 patients randomized to receive ART at PHI, subsequently (re)started ART during CHI after a median period of 86 weeks without treatment. HIV nucleic acids were longitudinally quantified in PBMC at ART baseline time points and every 12 weeks thereafter up to week 60 of both PHI and CHI ART by seminested real-time PCR. We used mixed modeling to compare the variables between groups and Spearman tests for correlation analyses. Results: Levels of usRNA and usRNA/vDNA ratios at PHI ART baseline strongly predicted the viral setpoint upon early therapy interruption (p=0.0001 and p=0.00004, respectively), and the slope of the CD4 count decline in the untreated period after interruption of early ART (p=0.009 and p=0.003). The predictive power of CA RNA for both viral setpoint and CD4 count decline was stronger than that of the plasma viremia. Levels of both usRNA (p=0.009) and vDNA (p=0.03) during PHI ART were significantly lower than levels of corresponding markers during CHI ART in patients who were not treated with early ART. However, no significant difference was found in the levels of any marker between the early and chronic therapy periods in the same patients, and strong correlations for all the markers between the two therapy periods were observed (usRNA: p=0.002; vDNA: p=0.0001, usRNA/vDNA: p=0.003). Finally, level of usRNA, measured during CHI ART, was significantly lower in patients who had been pre-treated during PHI than in patients who had not been pre-treated (p=0.018). Conclusions: Level of CA HIV RNA at PHI strongly predicted the CD4 count decline, suggesting that the viral reservoir, established early after infection, influences the disease progression for years afterwards. We observed a long-term suppressive effect of temporary early ART on the viral reservoir during ART initiated subsequently at CHI.

Poster Abstracts

THURSDAY, FEBRUARY 26, 2015 Session P-F3 Poster Session

Poster Hall

2:30 pm– 4:00 pm Cellular Factors of Latency 384 Minor Contribution of Host-HIV Readthrough Transcripts to the Level of HIV-1 gag RNA Alexander Pasternak 1 ; Una O’Doherty 2 ; Ben Berkhout 1 1 Academic Medical Center University of Amsterdam, Amsterdam, Netherlands; 2 University of Pennsylvania, Philadelphia, PA, US

Background: Cell-associated (CA) HIV-1 unspliced RNA is an important marker of the viral reservoir and the response to antiretroviral therapy (ART). Recently it has been used in clinical trials as a measure of virus activation by latency-reversing agents. Primers specific for the HIV gag regions are frequently used in PCR-based assays that quantify unspliced RNA. However, because HIV-1 integrates within actively transcribed host genes, it has been suggested that some of the transcripts detected by the gag -specific assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. To properly interpret the results of the gag assays, it is necessary to determine the relative contribution of such readthrough transcripts to the HIV gag RNA in ART-treated patients. Methods: We developed a sensitive nested real-time PCR assay that amplifies the 5’ LTR-encoded U3 – packaging signal region (U3-Psi) of HIV-1. This assay specifically measures host-HIV readthrough transcripts but does not detect genuine HIV-1 unspliced RNA (Fig. 1). Total DNA and total RNA were isolated from PBMC samples of 48 ART-treated patients whose plasma viremia had been undetectable (<40 copies/ml) for ≥ 1 year prior to the study. CA HIV-1 DNA and RNA were separately quantified in these samples using both the U3-Psi assay and the seminested real-time PCR assay specific for the HIV-1 gag region. The sensitivity of both assays is 4 copies/reaction. The same inputs of DNA or RNA were used for both assays.

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CROI 2015