CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

393 Kinetics of HIV-1 Gene Expression Following Reactivation in a Primary Cell Model of Latency VictoriaWalker-Sperling ; Joel Blankson Johns Hopkins University School of Medicine, Baltimore, MD, US

Background: CD4+ T cells latently infected with HIV-1 pose a significant barrier to eradication. Proposed “shock and kill” strategies involve using small molecules to reactivate latent HIV-1 without causing the massive toxicity associated with non-specific T cell activation. Previous work suggests that the CD8+ T cell response must be augmented in patients for this strategy to work. While HAART will prevent new rounds of infection when latent virus is reactivated, the immune response must be capable of eliminating the few HIV-infected cells that will inevitably be left when treatment ceases. In order for CD8+ T cells to prevent new rounds of infection following reversal of latency, they must kill infected CD4+ T cells prior to the completion of the viral life cycle. Thus, we examined the kinetics of HIV-1 gene expression in a primary cell model of latency in the context of reactivation to determine the optimal time frame for CD8+ T cell mediated killing. Methods: In order to establish a primary cell model of latency, resting CD4+ T cells were isolated from peripheral blood mononuclear cells (PBMCs) and nucleofected with HIV-1 NL4- 3 reporter-virus DNA that either had GFP replacing env or nef . The cells were then stimulated with anti-CD3/CD28 Dynabeads. Autologous Gag peptide stimulated CD8+ T cells from HAART-suppressed chronic progressors (CP) and elite suppressors (ES) were co-cultured with the resting CD4 T cells to assess susceptibility to killing. FACS analysis was performed at 6, 12, 18, 24, 36, and 42 hours. Results: Env, Gag, and Nef were produced as early as 6 hours post-stimulation. Downregulation of surface MHC-I and CD4 molecules was seen as early as 6 hours post-stimulation. The MFI of these markers on infected cells was 30% less than uninfected cells in the same well, and CD4 was downregulated by 40-50%. In ES and CP, CD8+ T cell-mediated elimination of infected cells was measurable as early as 18 hours post-stimulation.

Downregulation of CD4 and MHC-I in HIV-1 Reactivation . For the virus lacking Nef (∆Nef), HIV-1+ cells display far less downregulation of CD4 and MHC-I than those that retain it (∆Env). GFP indicates HIV+ cells and is expressed instead of Env or Nef. Representative graphs from 12 hours post-stimulation. Conclusions: Both early (Nef) and late (Env and Gag) proteins were produced by six-hours after activation of target CD4+ T cells, and downregulation of CD4 and MHC-I has already begun by that point in time. Despite the downregulation of MHC-I, stimulated CD8+ T cells were capable of modest elimination of CD4+ T cells producing viral proteins beginning as early as 18 hours post-stimulation of the target cells. The results suggest that it may be possible to eliminate recently reactivated, latently-infected cells before new rounds of viral replication occur.

Poster Abstracts

394 Variable HIV Replication Competency Following Latency Disruption in CD4+ T Cells Jason M. Hataye ; Joseph Casazza; David Ambrozak; Eli Boritz;TakuyaYamamoto; Daniel Douek; Richard A. Koup National Institute of Allergy and Infectious Diseases, Bethesda, MD, US

Background: The size of the latent reservoir in a patient with ART induced HIV suppression can be estimated by viral outgrowth in a limiting dilution culture of CD4+ T cells under activating conditions with exogenous cells and IL-2. A culture well containing HIV is typically detected with p24 ELISA, but recently HIV RNA RT-PCR has been shown to be more sensitive. This allowed us to determine the proportion of cells producing viral RNA that resulted in replication competent virus. Methods: Resting memory CD4+ T cells from 9 virally suppressed patients were stimulated with beads coated with antibodies against CD2, CD3, and CD28, and plated in limiting dilution in two conditions: 1) 100,000 MOLT-4/CCR5 cells per well and IL-2 were added on day 1 to facilitate viral outgrowth, or 2) the reverse-transcriptase inhibitor efavirenz (EFV) was present immediately on day 0 to suppress viral replication, with no exogenous cells or IL-2 added. Culture media was collected and replaced every 4 days, and the viral RNA was isolated using a paramagnetic nanoparticle based method. Real time HIV gag RT-PCR was performed and the frequency of HIV RNA producing cells was calculated using the R package for Extreme Limiting Dilution Analysis. Results: The frequency of HIV RNA producing cells following latency disruption was strongly correlated under viral outgrowth vs. viral suppression conditions. In most positive wells under viral suppression, viral RNA was detectable by day 4; some were followed by an increase while others decreased. In some outgrowth wells, the amount of HIV RNA on days 8 and 12 greatly exceeded that in comparable wells in the suppression assay. Culture supernatant from each positive outgrowth well was used to infect new cultures of activated but uninfected allogeneic CD4+ T cells. 33 of the 78 (42%) original positive outgrowth wells contained culture-confirmed replication competent virus, with significant well-to-well and patient-to-patient variability in the amount of virus produced. Conclusions: While HIV gag RNA RT-PCR with a concentrated viral suppression culture was as sensitive for quantifying the frequency of HIV RNA producing cells as a viral outgrowth assay, much HIV RNA recovered in the outgrowth wells, including many wells that had increasing amounts of viral RNA over time, did not represent replication- competent virus.

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CROI 2015