CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

ERK1/2 (26.7 fold) and CREB (6.6 fold). ERK and CREB inhibitors reduced Gal-9- mediated viral reactivation (15.8% to 2.9% or 9.2%, respectively; P=0.0001). Given that the immunosuppressive rapamycin uncouples HIV latency reversal from cytokine-associated toxicity [PMID 28094770], we investigated whether rapamycin could uncouple Gal-9-mediated latency reactivation from its concurrent pro-inflammatory cytokine production. Rapamycin reduced Gal-9- mediated secretion of IL-2 (4.4-fold, P=0.001) and TNF (4-fold, P=0.02) without impacting viral reactivation (16.8% compared to 16.4%; P=0.2). Conclusion: Gal-9 modulates HIV transcriptional activity through TCR/Lck- dependent ERK1/2-CREB phosphorylation pathway. Our findings could have implications for understanding the role of endogenous galectin interactions in modulating TCR signaling and maintaining chronic immune activation during HIV infection. In addition, uncoupling Gal-9-mediated viral reactivation from undesirable pro-inflammatory effects, using rapamycin, may increase the potential utility of recombinant Gal-9 within the reversal of HIV latency eradication framework.

survival pathways in GFP+ and mKO2+ cells, which might be driven by the TNFSRs-expressing populations. Conclusion: Multiple TNFSR members are upregulated at the transcriptional and protein level in cells with in vitro-induced productive and latent infection. Increased expression of these markers is associated with transcriptional induction of cell survival signatures. Our data lay the foundation for future investigation on roles of TNFSRs in naturally-infected CD4 T cells to fully appreciate their impact on viral reservoir persistence. 388 HIV PROVIRAL DNA METHYLATION IN SEROCONVERTERS, CONTROLLERS, AND ART-TREATED PATIENTS Sam Kint 1 , Sabine Kinloch-de Loes 2 , Wim Van Criekinge 1 , Linos Vandekerckhove 1 1 Ghent University, Ghent, Belgium, 2 Royal Free Hospital, London, UK Background: DNA methylation is a well-known epigenetic modification that drives gene transcription, but its role in the HIV-1 proviral genome is largely unknown. In latency models, hypermethylation has been linked to silencing, while loss of methylation stimulates reactivation. However, due to low HIV-1 proviral DNA levels and high genomic heterogeneity, obtaining reliable and reproducible patient-derived data has been difficult. This has resulted in the past in conflicting publications. We therefore have performed an in-depth evaluation of the HIV-1 proviral methylation state in a well-characterized HIV-1 patient cohort. Methods: To reliably measure DNA methylation in HIV-1 proviruses from clinical samples, we used a bisulfite-based deep sequencing assay to measure the methylation state of 4/5 CpG Islands (CpGIs) found in the HIV-1 genome (2 in LTR, 2 in env). This assay was used to compare methylation in PBMCs from 72 individuals, divided in four groups: early ART-treated HIV-1 seroconverters (ET, N=15), late ART-treated patients (LT, N=32), ART-naïve seroconverters (SRCV, N=8) and long-term non-progressors (LTNP, N=17). Data was mapped to a reference HIV-1 genome using Bismark (v0.10.1) and analyzed with the methylKit package (version 1.6.2). Results: We show (i) that CpGIs inside the LTR region have low overall methylation level (median <5%) as compared to CpGIs in the env region (median up to 40%), and (ii) that LTR CpGIs are equally methylated in all 4 groups (differential methylation (DM) <5%). (iii) CpGIs in the env region show no DM between patients controlling HIV-1 replication (ET, LT, LTNP), but a decrease of 29.92% in SCRV as compared to these groups. Within the cohorts on long-term ART (median of 10 years), we found no correlation between the time of initiation of therapy and the methylation percentage (Spearman’s rank correlation: p= 0.2131). Conclusion: Our results using this sensitive assay show a paucity of DNA methylation in the HIV-1 promoter region in all patient groups with the absence of ART or different timing of ART initiation suggesting that LTR methylation is not involved in regulating latency, which contradicts the latency model results. Methylation in env on the other hand is higher, and found in all patients who are chronically viral suppressed suggesting that env DNA methylation has latency regulating effect.

Poster Abstracts

387 TNF SURFACE RECEPTORS RELATE TO SURVIVAL PATHWAY ACTIVATION IN HIV RESERVOIR CELLS Hsiao-Hsuan Kuo 1 , Stephane Hua 1 , Ce Gao 1 , Shivaali Maddali 1 , Xu G. Yu 1 , Mathias Lichterfeld 2 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Brigham and Women’s Hospital, Boston, MA, USA Background: HIV reservoir cells possess a remarkable long-term stability and represent the major obstacle to a virological cure. Understanding mechanisms that support reservoir cell survival is of great interest for developing interventions to reduce viral persistence. Previous studies suggest the selective activation of anti-apoptotic host factors in virally infected cells, but the molecular pathways involved remain unclear. Methods: Unstimulated CD4 T cells from HIV-negative donors were infected with a dual-reporter HIV-1, allowing to distinguish productively (GFP) and latently (mKO2) infected cells. Phenotypic properties of infected cells were determined by flow cytometry. Transcriptional programs of cells with latent or productive infection were analyzed by single-cell RNA-Seq, with whole- transcriptome amplification, followed by sequencing on NextSeq 500 system. Results: Compared to uninfected population, both productively- and latently-infected CD4 T cells showed a distinct increase in surface expression of several members of the Tumor Necrosis Factor Surface Receptor (TNFSR) family, including OX40 (p=0.008 both GFP+ and mKO2+ cells), TNFR2 (p=0.003 GFP+ cells; p=0.017 mKO2+ cells) and GITR (p=0.018 GFP+ cells only). Union gating on combinations of the TNFSRs -- OX40, HVEM and TNFR2-- allowed to distinguish mK02+ and GFP+ from uninfected cells at high levels of statistical significance (p=0.003 GFP+ cells; p=0.01 mKO2+ cells). Single-cell RNA-seq data identified several TNFSRs that distinguished transcriptional signatures of GFP+/mKO2+ cells from uninfected cells, and both latently-infected and productively-infected cells contain higher proportions (35-60%) of cells that expressed high-level OX40, TNFR2, or GITR, while frequencies of uninfected cells with high-level expression of these markers were substantially lower (18-24%). Computational analysis of single-cell transcriptomes inferred activation of

389 ANALYTICAL TREATMENT INTERRUPTION (ATI) IN PATIENTS WITH VERY SMALL HIV RESERVOIR Pieter Pannus 1 , Sofie L. Rutsaert 2 , Stephane de Wit 3 , Sabine Allard 4 , Guido Vanham 1 , Coca Necsoi 3 , Joeri Aerts 4 , Ward De Spiegelaere 5 , Achilleas Tsoumanis 1 , Marie Couttenye 6 , Natacha Herssens 1 , Linos Vandekerckhove 2 , Eric Florence 1

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