CROI 2015 Program and Abstracts

Abstract Listing

Poster Abstracts

to the immune system. Unspliced RNA (usRNA) encoding gag was the predominant HIV RNA form detected in infected resting cells. We designed experiments to ask if nascent transcription occurs in resting CD4+ T cells or if the Gag signal detected is due to an artifact such as read-through transcription or incoming virus. Methods: To address the contribution of incoming virus to Gag signal, we first sorted Gag+ and Gag-negative cells from cultures infected in vitro and measured levels of HIV DNA in both populations, similar to an approach we used in vivo. RT-PCR and FACS analysis were used to determine whether other viral proteins (made from spliced RNA forms) were present in cells infected in vitro and in CD4+ T cells from ART patients. In addition, given that tat/rev is present at very low levels in vivo in patients on ART, we asked if tat/rev is required for LTR driven expression. Results: We found that Gag+ cells were strongly (more than 100-fold) enriched for HIV DNA compared to Gag-negative cells in infected cultures. In addition to spliced HIV RNA forms, further evidence of nascent transcription included direct and indirect evidence of new synthesis of multiple HIV proteins by FACS. Read-through transcripts were detectable but present at low levels compared to gag RNA in both cells infected in vitro and in CD4 cells from ART patients. Stimuli such as IL-7 and Romidepsin preferentially induced gag usRNA over read-through transcripts. In contrast, SAHA induced both read-through and gag usRNA transcription two-fold. Notably, we show that low-level protein expression can occur in the absence of tat/rev using a viral vector with a deletion of tat/rev gene expression. Conclusions: Nascent LTR transcription occurs in HIV-infected resting CD4+ T cells. In vitro and in vivo data suggest that Gag is the predominant transcript (usRNA) and protein expressed in HIV infected individuals on ART. The relative contributions of replication competent and defective proviruses to viral protein expression in vivo remain undefined.

WEDNESDAY, FEBRUARY 25, 2015 Session P-F4 Poster Session

Poster Hall

2:30 pm– 4:00 pm Dynamics of Latency and Reactivation 391 Influenza Vaccination Increases HIV-1 Transcription During Antiretroviral Therapy Christina C. Yek 1 ; Sara Gianella 1 ; Montserrat Plana 2 ; Pedro Castro 2 ; Felipe Garcia 2 ; Marta Massanella 1 ; David M. Smith 1 1 University of California San Diego, San Diego, CA, US; 2 University of Barcelona, Barcelona, Spain

Background: Curative strategies using stimulators such as histone deacetylase inhibitors, disulfiram and IL-7 to reactivate HIV have thus far demonstrated only modest activity. In contrast, transient increases in viremia after administration of standard vaccines have been observed even during antiretroviral therapy (ART). In this study we investigate whether routine influenza vaccination can reactivate HIV. Methods: Eleven HIV-infected individuals on suppressive ART (<50 copies/ml) were selected from the intervention arm of a randomized trial that studied the effects of a vaccination schedule on viral rebound after structured treatment interruption (NCT00329251). Blood samples were obtained at baseline and 1 month after influenza vaccination. DNA and RNA were extracted from cryopreserved peripheral blood mononuclear cells using a Qiagen AllPrep DNA/RNA Mini Kit. Cell-assoiated HIV DNA and RNA transcripts were quantified by droplet digital PCR using primers for gag and 2-LTR (for HIV DNA), unspliced gag RNA (HIV usRNA), multispliced tat-rev RNA (HIV msRNA), polyA and RPP30 (cellular marker for normalization). Values were adjusted for percentage of CD4 T cells as measured by flow cytometry. Results: Nine of 11 subjects showed an increase in HIV usRNA after influenza vaccination despite undetectable viral loads throughout. Median HIV usRNA levels pre- and post- vaccination were 28.7 [4.2-56.4] and 91.0 [43.2-173.1] copies/10 6 CD4 T cells, respectively (p=0.049). Mean increase in HIV usRNA after vaccination ranged from 0 to 49-fold (mean 10.6). No significant changes were observed in HIV msRNA (p=0.25), polyA (p=0.91), total HIV DNA (p=0.15), or 2-LTR circle copies (p=0.74). Conclusions: This study demonstrated a clear increase in cell-associated HIV usRNA 1 month after influenza vaccination, consistent with antigenic stimulation of the HIV reservoir during suppressive ART. The mean 10.6-fold increase in HIV usRNA is comparable to or better than that seen with administration of Vorinostat. Total HIV DNA and 2-LTR circles did not change, suggesting reactivation of replication-incompetent virus and/or ART-mediated suppression of viral propagation. Although we do not propose that standard vaccinations will cure HIV, these findings suggest that a component of immune stimulation could be considered in the development of eradication strategies. 392 Defective HIV-1 Proviruses Can Be Transcribed Upon Activation Background: HIV-1 persists in the latent reservoir, primarily resting memory CD4+ T cells, as integrated proviruses. The majority of these proviruses are defective, containing large internal deletions or APOBEC-mediated G-to-A hypermutations. However, we previously found that even if the HIV-1 genome contains lethal mutations, the LTR promoter may remain intact, indicating that HIV-1 RNA may still be transcribed. The transcription of HIV-1 RNAs from defective proviruses may complicate the measurement of the size of the latent reservoir using latency reversing agents during the shock-and-kill strategy, as measurement of the defective proviral RNA does not indicate the reactivation of the clinically significant replication-competent proviruses. Further, whether the cells harboring defective proviruses would expand upon reactivation, or would be eliminated by cytolytic T cells (CTLs), remains unknown. Methods: Resting CD4+ T cells from aviremic patients under suppressive antiretroviral therapy are activated with CD3/CD28 costimulation under enfurvitide to prevent new rounds of in vitro infection. Autologous CTLs were stimulated with Group M Consensus Gag peptide mixture and IL-2. To examine whether cells containing intact or defective HIV-1 can be eliminated by CTLs, we co-cultured pre-stimulated autologous CTLs with activated CD4+ T cells. Cell-associated RNA and proviral DNA from cells which are resting, activated, and CTL co-cultured was subjected to quantitative PCR and deep-sequencing of the Gag region to examine the start codon of Gag and two tryptophan residues, which are hotspots APOBEC-mediated hypermutations. CTLs were removed by magnetic bead depletion from the CTL-CD4 coculture before qPCR for normalization to CD4 cell count. Results: We found a significant proportion of the HIV-1 RNA in activated patient CD4+ T cells contains lethal mutations. The amount of defective proviruses increased over the course of activation, indicating expansion of cells containing defective proviruses upon stimulation. The percentage of defective proviruses increased, implying the effect of viral cytopathic effects by reactivated intact proviruses. The amount of HIV-1 proviruses, both intact and defective, decreased after addition of CTLs in some patients, indicating possible elimination by CTLs. Conclusions: Defective HIV-1 proviruses may be transcribed during latency reversal. Cells containing defective HIV-1 proviruses may expand under T cell activation. Ya-Chi Ho ; Ross Pollack; PatrickYong; Robert F. Siliciano Johns Hopkins University School of Medicine, Baltimore, MD, US

Poster Abstracts

286

CROI 2015