CROI 2015 Program and Abstracts

Abstract Listing

Oral Abstracts

Conclusions: In this prospective study, presence of genital CMV shedding at baseline was strongly associated with acquisition of syphilis. Lower level of seminal MCP-1 was associated with both presence of genital CMV shedding and syphilis acquisition. Future studies with anti-CMV therapeutics could help determine underlying mechanisms and if causal associations exist. 392 Defective HIV-1 Proviruses Can Be Transcribed Upon Activation

Ya-Chi Ho ; Ross Pollack; PatrickYong; Robert F. Siliciano Johns Hopkins University School of Medicine, Baltimore, MD, US

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. 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. 427 Measurements of Viral Transcription in Elite Suppressor CD4+ T Cells Christopher W. Pohlmeyer ; C. Korin Bullen; Greg Laird; Alyssa R. Martin;VictoriaWalker-Sperling; Stanley U. Chioma; Robert F. Siliciano; Joel Blankson Johns Hopkins University School of Medicine, Baltimore, MD, US Background: Elite suppressors (ES) are patients who control HIV replication without antiretroviral therapy. Prior studies have shown that the frequency of latently infected cells in these patients is much lower than patients on suppressive antiretroviral regimens. However the frequency of CD4+ T cells that express HIV-1 mRNA at baseline and following T cell stimulation is unknown. In this study we compared HIV-1 transcription levels in CD4+ T cells from chronic progressors (CPs) on suppressive antiretroviral regimens and ES. Methods: To measure intracellular HIV-1 mRNA, we isolated CD4 T cells from PBMCs of ES and CPs. Replicates of 5x10 6 cells were stimulated with PMA/ionomycin or DMSO for 24 hours. The cells were collected and lysed in Trizol for RNA extraction and subsequent quantification by qPCR. RNA from supernatants were collected and measured for released HIV-1 mRNA. Results: A comparison of cell associated HIV-1 mRNA in CD4+ T cells of HAART-suppressed CPs and ES shows that ES have significantly less HIV-1 mRNA per 5x10 6 cells before stimulation. HIV1 mRNA was uniformly detected in CPs, but was present at very low levels in just 2 of 8 ES (p>0.05). When 5x10 6 CD4+ T cells were stimulated with PMA/ ionomycin, the levels of cell-associated HIV-1 mRNA increased in 4 of 7 ES. Additionally, when measuring HIV-1 mRNA levels in culture supernatant following stimulation of 5 x 10 6 CD4+ T cells with PMA/ionomycin, we detected release of virus from just 2 of 8 ES compared to 5 of 5 CPs. When more replicates were analyzed, viral release was seen in 4 ES. 2 of these patients showed positivity in 1 of 5 replicates (25x10 6 cells), releasing approximately 3,000 and 5,000 copies HIV-1 RNA each. Poisson statistics suggests an 89% chance that the signal observed reflects a single cell releasing virus, and our HIV-1 mRNA measurements fit with current estimates of the burst size of an infected CD4+ T cell. Conclusions: In the present study, we demonstrate that the baseline levels of cell associated HIV-1 mRNA in ES are significantly lower than those observed in CPs per 5x10 6 cells. However an increase in viral transcription following T cell stimulation was observed. These results further characterize the size of the latent reservoir in ES and confirm earlier studies that suggested that some of these patients are infected with replication-competent virus.

Oral Abstracts

109

CROI 2015