CROI 2018 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: Blood samples from 12 HIV infected individuals on suppressive ART were obtained in three-month intervals for 1 year. Residual plasma viral load (pVL) was assessed with a limit of detection of 1 copy/mL. A fragment of the LTR U3-R region was amplified from total CD4+ T cells in both bisulfite-treated and untreated samples and deep sequenced on a MiSeq instrument. The degree of CpG methylation was assessed as a methylation index defined as a weighed fraction of variants showing methylation in each of the 9 HXB2 canonical CpG sites. Results: Viral LTR contained 4-11 CpG sites. Over time, we observed both loss and gain of LTR CpG sites in predominant variants as well as different numbers of transcription factor binding sites that could potentially be regulated by CpG methylation. In general, LTR methylation was common and was observed in all patients at least at one time point. Nevertheless, LTR methylation showed a dynamic behavior through time. Variation in the degree of methylation between different time points coincided with alternation in predominance of heavily methylated vs. unmethylated variants. In general, variants lacking methylated sites, showed loss of one or more CpG sites. Variants showing 8 methylated CpG sites (CpG2 site was usually mutated) were common (observed in 12/12 patients in at least one time point). We observed a negative correlation between the CpG methylation index and residual pVL in only one patient (r=- 0.9596, p=0.04). Conclusion: We show strong evidence that LTR methylation can vary significantly along time in proviruses from circulating CD4+ T cells, possibly explaining differences between previous cross-sectional studies. We also observed that the association between LTR methylation and residual pVL is weak, but can exist in some virally suppressed patients. Further studies in specific cell subpopulations and in tissues are necessary to assess the role of viral LTR methylation as a latency mechanism.

quantitative PCR following a 4-day treatment with PRRs. Statistical significance was assessed using the Wilcoxon matched pair signed rank test. Results: Agonists of TLR3 (Poly I:C HMW), TLR2/1 (Pam3CSK4), TLR7 (vesatolimod), TLR8 (GSI-288) and NOD2 (romurtide) induced HIV RNA expression of 3.1 (N=15, p < 0.01), 2.4 (N=22, p < 0.001), 2.2 (N=24, p < 0.01), 2.0 (N=26, p ≤ 0.05), and 1.9 (N=15, p < 0.01) fold, respectively, relative to PBMCs treated with the vehicle control. Among the active agonists, only compounds targeting TLR2/1 were directly active in isolated CD4+ T cells (2.2-fold increase, N = 6, p < 0.01). TLR3 and TLR7 agonists had the greatest activity in combination, inducing 5.5-fold activation of HIV relative to vehicle treated control (N = 11, p < 0.001). All agonists tested, regardless of their ability to activate expression induced IL-6 at the concentrations used to assess latency reversal. Conclusion: Agonists of multiple PRRs induced modest, but consistent activation of HIV expression in cells from ART-suppressed HIV infected individuals with TLR3 and TLR2/1 agonists being most active. Notably, these compounds were at least as effective as the TLR7 agonist vesatolimod, which can activate the reservoir in vivo in SIV-infected rhesus macaques. 322 TRANSCRIPTIONAL REPROGRAMMING IN CCR5+ MEMORY CD4+ T CELLS PROMOTES HIV-1 LATENCY Liang Shan 1 , Kai Deng 2 , Adam Capoferri 3 , Robert Siliciano 2 1 Washington University St Louis, St Louis, MO, USA, 2 Johns Hopkins University, Baltimore, MD, USA, 3 Johns Hopkins Hospital, Baltimore, MD, USA Background: Despite extremely effective combination antiretroviral therapy (cART), HIV-1 persists in a small pool of latently infected, resting memory CD4+ T cells. Without elimination of this latent reservoir, patients cannot be cured and must receive lifelong antiretroviral treatment. Current approaches to purging the latent reservoir involve pharmacologic reactivation of HIV-1 transcription by agents that reverse viral latency. To date, no broadly applicable strategy has been developed to effectively clear latent HIV-1 in patients. Although mechanisms for repression of HIV-1 gene expression at the transcriptional and translational levels have been well characterized, it remains unclear how HIV-1 enters a state of latency in vivo. Methods: Peripheral blood for the isolation of primary CD4+ T cells was obtained from HIV-1-infected patients. Genomic DNA was extracted from resting CD4+ T cells from each patient for deep sequencing of HIV-1 env or quantitative measurement of HIV-1 DNA. To measure frequency of latent HIV-1 in various subsets of CD4+ T cells, naive, CCR5+ and CCR5- memory CD4+ T cells (CD4RO+) were purified by FACS and then used for limiting dilution virus outgrowth assay. Results: We performed viral outgrowth assays and deep sequencing to analyze the HIV-1 envelope (env) sequences of the replication-competent viruses from resting CD4+ T cells of cART-treated patients. A dominant presence of replication-competent CCR5-tropic virus was found in most of the patients. We hypothesized that CCR5+ resting memory CD4+ T cells that maintained CCR5 expression throughout the T cell activation and relaxation process should be the most susceptible host cells for HIV-1 latent infection.The frequency of HIV-1 DNA was 10 to 100 fold higher in CCR5+ resting memory CD4+ T cells than other memory or naïve cells. We performed viral outgrowth assay to measure frequency of latent HIV-1. 6 out of 7 patients had a higher frequency of latently infected cells in CCR5+ resting memory CD4+ T cells than in CCR5- resting memory CD4+ T cells (4.8 fold increase, 95% confidence interval 1.752 to 13). Conclusion: In this study, using in vitro cell-based model and patient CD4+ T cells, we demonstrated that the establishment of latent infection y by R5-tropic virus occurs selectively in CCR5-expressing effector CD4+ T cells. 323 LTR METHYLATION AND RESIDUAL VIREMIA IN INDIVIDUALS ON SUPPRESSIVE ART César N. Cortés-Rubio , Santiago Avila-Rios, Gonzalo Salgado-Montes de Oca, Margarita Matías-Florentino, Christopher E. Ormsby, Ana P. Carranco-Arenas, Gustavo Reyes-Terán National Institute of Respiratory Diseases, Mexico City, Mexico Background: There are contrasting results regarding the role of methylation of the HIV LTR promoter region on provirus expression in individuals on suppressive antiretroviral treatment (ART). We longitudinally studied changes in the LTR sequence and methylation status and their possible association with residual viremia in virologically suppressed persons.

Poster Abstracts

324 WITHDRAWN

CROI 2018 113