CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

mutation of 70nt within segment B containing a possible PRC2-binding motif greatly reduced Ast activity. Mutation of either segment C or D did not have a major effect, whereas concurrent mutation or deletion of both segments did. This suggests that segments C and D cooperatively recruit additional factors. Indeed, MS studies found that Ast interacts with additional transcription and epigenetic repressors such as NuRD, CTCF, YY1, TDP-43. Cell lysate fractionation through a sizing column showed that Ast purifies in high molecular weight fractions of ~2MDa that also contain Ast binding partners. We also found that Ast interacts with members of the C/D box and H/ACA box complexes, which catalyze RNA ribose methylation and pseudouridylation. Indeed, MS analysis of affinity-purified Ast showed that it contains these post-transcriptional modifications in vivo. Conclusion: We identified the PRC2-binding motif of Ast, and showed that Ast binds other transcription repressors. We also found that Ast carries modifications affecting its stability and interaction with protein partners. Our studies suggest that Ast induces HIV-1 latency via multiple pathways. 379 TARGETING SPHINGOSINE-1-PHOSPHATE TO PREVENT LATENT HIV INFECTION Rachel Resop 1 , Rémi Fromentin 2 , Amanda Macedo 1 , Hawley Rigsby 2 , Nicolas Chomont 2 , Alberto Bosque 1 1 George Washington University, Washington, DC, USA, 2 Université de Montréal, Montreal, QC, Canada Background: Sphingosine-1-phosphate (S1P) is an established modulator of cell cycle and chemotaxis and the therapeutic targeting of this pathway has been the subject of investigation for the treatment of multiple sclerosis and other autoimmune diseases. In the context of Human Immunodeficiency Virus 1 (HIV-1) infection, alterations in the expression of S1P receptor 1 in thymocytes as well as impaired S1P signaling in CD4+ T cells have been described. Recently agonists of the S1P receptor 1 have been shown to reverse HIV latency. Here, we sought to determine the role of the S1P in the establishment of latent HIV infection. Methods: We examined whether targeting Sphingosine Kinase (SPK) would alter the establishment of the latent reservoir in memory CD4+ T cells using a primary cell model. Briefly, we isolated naïve human CD4+ T cells from HIV-negative donors, activated and expanded them, and infected themwith NL4-3 virus by spin infection. Three days later, cells were treated with either N,N-dimethyl sphingosine (D.M.S.), a SPK inhibitor; or FTY720, a S1P receptor modulator. We quantified the effects of these inhibitors on latent infection by measuring the frequency of cells harboring total and integrated HIV DNA by qPCR and the ability to reactivate virus from latency following T cell receptor stimulation by intracellular HIV Gag. Results: Treatment with D.M.S. reduced the establishment of latent HIV infection in a dose dependent manner as measured by the frequency of cells producing HIV Gag upon T cell receptor stimulation (6 μM n=9, 95% reduction, p=0.0067; 600 nM n=8, 38% reduction, p=0.0236; 60 nM n=5, 23.3% reduction, p=0.2). Moreover, FTY720, which is used in the clinical setting for the treatment of multiple sclerosis, recapitulated this effect (100nM n=7, 70% reduction, p=0.0425). These inhibitors reduced latent infection during or before reverse transcription since the treatments reduced to a similar extent both total (D.M.S. 600 nM n=4, 34% reduction; FTY720 100nM n=4, 52% reduction) and integrated HIV DNA (D.M.S. 600 nM n=4, 29.5% reduction; FTY720 100nM n=4, 57% reduction). Conclusion: Our results show that targeting S1P has an effect on latent infection. Mechanistically, D.M.S. and FTY720 force CD4+ T cells into a G0 state of the cell cycle as measured by expression of Ki67. Our research suggests that the therapeutic targeting of this pathway early in infection may aid in the development of strategies to promote a functional cure by preventing the establishment of the latent reservoir. 380 RESIDUAL VIREMIA IS DOMINATED BY MONOTYPIC VIRUS RESULTING IN INFECTIOUS PLASMA VIRUS Hadega Aamer 1 , Jan McClure 2 , Sherry McLaughlin 1 , Sheila Styrchak 1 , Daisy Ko 1 , Janine Maenza 2 , Ann Collier 2 , James Mullins 2 , Lisa Frenkel 1 1 Seattle Children’s Research Institute, Seattle, WA, USA, 2 University of Washington, Seattle, WA, USA Background: During suppressive antiretroviral therapy (ART), 1-10 copies of residual viremia (RV) can often be detected per milliliter of plasma in HIV-infected individuals. Several studies have shown monotypic (clonal)

viral sequences predominate in the RV of suppressed individuals. It is unclear whether specific variants are selectively maintained over time and if they are infectious. We evaluated 3 individuals who were chronically-infected at the time ART was initiated. Banked, longitudinally collected specimens were evaluated from pre-ART, during ART and post-ART-interruption and re-suppression. Quantitative viral outgrowth assay (QVOA) was performed to identify infectious variants with sequences matching the RV. We hypothesized that during long-term ART, prevalent RV will be maintained over time and contribute to infectious viremia, and thus to persistence of the reservoir. Methods: Extracted RNA from at least 4 pre-ART, 4 on-ART (viral RNA <50copies/ml), and 2 post-ART interruption time points was subjected to endpoint-PCR of C2V5env, sequenced, and assembled into a maximum- likelihood tree. QVOA was performed on all 3 subjects from a time point with undetectable viral load (<50copies/ml). Predicted cell tropismwas performed using PSSM. Results: RV variants were often clonal in participants 1 and 2 (n=21/113, n=28/38, respectively), with clonal variants observed for at least 3yrs on ART, but included multiple variants in participant 3. RV across participants 1, 2, and 3 were predominately CCR5(R5)-tropic (57%, 99%, and 84%, respectively), with the remaining being CXCR4-tropic. In participant 1, a RV clone (n=4) had an identical C2V5 to a QVOA variant. This RV clone (R5-tropic) was observed for at least 7yrs sine pre-ART. A match between a QVOA variant and a monotypic plasma pair (n=2) in participant 3 was also observed and maintained for 3yrs on ART. R5-tropic RV monotypic variants detected during ART in participants 1 and 2 were also detected post-ART interruption and re-suppression and these variants were maintained for 7 and 2yrs, respectively. Conclusion: These findings suggest that RV represent a non-latent part of the infectious reservoir that upon ART interruption could fuel new cycles of infection. Furthermore, persistence of certain monotypic clones over time suggests that cells harboring these virions may be resistant to immune clearance or regularly renewed. 381 PD-1/PD-L1 INTERACTION REGULATES HIV TRANSCRIPTION IN LYMPH NODES OF TREATED SUBJECTS Riddhima Banga , Caterina Rebecchini, Francesco Procopio, Alessandra Noto, Olivia M. Monje, Craig Fenwick, Matthias Cavassini, Jean-Marc Corpataux, Laurence de Leval, Giuseppe Pantaleo, Matthieu Perreau Lausanne University Hospital, Lausanne, Switzerland Background: T follicular helper (Tfh) cells expressing high levels of PD-1 were recently shown to serve as a major site of active and persistent HIV transcription despite prolonged ART. The present study aimed to determine the potential role of immune checkpoint (IC)/IC-Ligand (IC-L) interactions on HIV transcription in lymph node (LN) microenvironment. Methods: To address this issue, we assessed the expression of ICs and IC-Ls on LN cell populations and the impact of IC/IC-L interactions on T-cell proliferation, reactivation of HIV production and HIV transcription in LN memory CD4 T-cell populations from viremic and aviremic ART-treated HIV-infected subjects (N=47). Results: We showed that PD-1 and TIGIT are the two major ICs expressed on Tfh cells of healthy, viremic and ART treated HIV-infected subjects ex vivo. We subsequently showed that PD-L1 and to a lower extent CD155 (TIGIT ligand) recombinant proteins significantly reduced TCR-mediated T-cell proliferation and reactivation of HIV production in vitro (P<0.05), demonstrating that PD-1 and TIGIT signaling pathways were 1) functionally active on PD-1+/ Tfh cells and 2) regulate TCR-mediated HIV transcription and production. We therefore explored the phenotype, the frequency and the tissue distribution of IC-L expressing cells and showed that PD-L1 and CD155 were predominantly co-expressed on LN HLA-DRhighCD1chigh dendritic cells (DCs). The frequencies of PD-L1+ DCs in viremic HIV-infected subjects directly correlated with HIV viral load (r=0.93 P=0.0002) and significantly dropped after prolonged ART (P<0.05). Interestingly, PD-L1 expressing cells were detected in both extra- follicular and germinal center (GC) areas of viremic HIV-infected subjects, but were barely detectable in GCs of ART treated subjects, suggesting that ART initiation had a profound impact on IC-L tissue distribution and that PD-1/PD-L1 interactions might be selectively reduced in GCs of ART-treated subjects. Finally, the frequencies of LN PD-L1+ DCs inversely correlated with HIV transcription (r=-0.89; P<0.05) in LN memory CD4 T cells, indicating that PD-L1+ DCs contribute to control HIV transcription in vivo.

Poster Abstracts

CROI 2019 136