CROI 2016 Abstract eBook

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Poster Abstracts

NKT and CD8 T cells, there were no differences found between the autogeneic and allogeneic groups. However, CD8 T cell activation was strongly correlated with HIV production (R 2 = 0.975). Conclusions: Our findings suggest that lymphocytes, including NK and NKT cells, may play an important role in surveillance and clearance of residual HIV-infected cells following HSCT. 368 WITHDRAWN

369 Differential Effect of Selenium on HIV Replication and Reactivation in Primary Cells Christina Gavegnano 1 ; Raymond F. Schinazi 1 ; SallyYuan 2 ; Deanna Kulpa 3 ; Nicolas Durier 4 ; Ariana Santos 1 1 Emory Univ Sch of Med, Atlanta, GA, USA; 2 Janssen Pharmaceuticals, Springhouse, PA, USA; 3 Southern Rsr Inst, Frederick, MD, USA; 4 TREAT Asia/amfAR, Bangkok, Thailand Background: Sodium Selenite (Na 2 SeO 3 ) can modulate cellular dynamics that may alter viral replication, and has been correlated with CD4 + T cell recovery in HIV-infected persons in vivo , however cellular mechanisms responsible for these findings are poorly understood. Understanding these factors could elucidate key viral/host cell events controlling viral replication, persistence/reactivation that can be explored as a therapeutic intervention. Methods: Human macrophages (MΦ) and peripheral blood mononuclear (PBM) cells were treated with various concentrations of Na 2 SeO 3 for 2hr prior to infection (HIV-1 baL ) . Cells were maintained for 6 days before viral quantification (p24-ELISA and FACS). MΦ or PBM cells were treated with various concentrations of drug for 6 days and stained with Zombie- Violet live/dead dye and quantified by FACS (MΦ) or MTT (PBM cells). Resting memory CD4-T cells were infected with 89.6 prior to IL-7/TGF-b-induced latency and reversal was quantified (p24 + cells, FACS). Latent MΦ were reactivated with various concentrations of drug with or without PMA+TNF-alpha and extracellular virus was quantified (p24-ELISA). Results: Na 2 SeO 3 selectively inhibited HIV-1 replication in MΦ (EC 50/90 3.9±1.7 /19.2± 4.1µM), without apparent cytotoxicity (IC 50 > 100µM), but did not inhibit HIV in lymphocytes (EC 50 >40 µM). Na 2 SeO 3 significantly reduced (p<0.01) levels of reactivation from in vitro latently infected CD4-T cells versus untreated controls at 5µM, resulting in reactivation levels similar to non-reactivated controls, without apparent toxicity (IC 50 > 100µM in uninfected lymphocytes). Na 2 SeO 3 induced reactivation of latent HIV-1 in MΦ (EC 50 3.9µM) without apparent toxicity (IC 50 >100 µM) and did not inhibit PMA+TNF-alpha induced reactivation. Conclusions: Na 2 SeO 3 inhibited HIV-1 replication in MΦ, but not lymphocytes, and inhibited reactivation of latent HIV-1 from resting CD4-T cells, but surprisingly induced reactivation from latent MΦ. These findings suggest the effects of Na 2 SeO 3 on viral replication and reactivation are distinct across CD4-T cells versus MΦ, and that its antiviral potency/reactivation profile could be determined by cell-type specific milieus. These data suggest that Na 2 SeO 3 could represent an add-on therapy to existing ARV to 1)reactivate latent HIV-1 in MΦ, 2)specifically inhibit viral replication in MΦ, and 3)inhibit reactivation in CD4 T cells, which could also prevent viral reseeding and reduce the viral reservoir. 370 Kinetics of HIV-1 Latency Reversal Measured by a New Flow-Based Technique Gloria Martrus ; Annika Niehrs; Anne Rechtien; Marcus Altfeld Heinrich Pette Inst, Hamburg, Germany Background: During HIV-1 infection, the virus early establishes a pool of latently infected cells. While current antiretroviral treatment can suppress viral replication, HIV-1 persists in cellular reservoirs. New therapeutic approaches aim to diminish the reservoir by reactivation of latently infected cells. However, the reactivation kinetics of viral mRNA and viral proteins production remain largely unknown. We designed a methodology to assess the dynamics of HIV-1 latency reactivation on the single cell level by flow cytometry to differentiate cells in which only viral mRNA is being produced from cells in which viral proteins or novel HIV-1 viruses are being produced. Methods: J89 cells, a Jurkat cell line containing a single full copy of HIV-1, were used as an HIV-1 latency model. J89 cells were stimulated at several time points (ranging from 0 hr to 48 hrs) with different concentrations of hTNFα cytokine and Romidepsin. Using a new flow cytometry technique that allows for synchronized detection of mRNA targets and intracellular proteins, as well as cell surface markers, combined intracellular staining for p24 Gag protein and gag mRNA was performed. Results: After stimulation of J89 cells with 10 ng/mL of hTNFα for 3h, moderate HIV-1 gag mRNA expression with no intracellular Gag protein was detected. Longer incubation times with hTNFα lead to an increased expression of HIV-1 gag mRNA as well as intracellular Gag protein synthesis. Three distinct populations were identifiable by flow cytometry: only HIV-1 gag mRNA positive cells, HIV-1 gag mRNA and Gag protein double-positive cells, and double negative cells. Romidepsin stimulation (5nM) induced gag mRNA production only after 12h. However, the overall stimulation of J89 with RMD induced a faster gag mRNA and Gag protein production at 18h and 24h compared to hTNFa. Using both treatments CD4 receptor was downregulated on the cell surface following HIV-1 reactivation. Conclusions: We here describe a novel method allowing for the first time to quantify the kinetics of HIV-1 mRNA and protein synthesis after latency reactivation on the single cells level using flow cytometry. This new technique will enable the phenotypic characterization of latently infected cells at different stages of latency reversal, and the identification of specific surface markers from these cells as targets for innate and adaptive immune responses. 371 Reactivation of Latent HIV-1 in J-Lat T Cells by Type I Interferon Agonists Melanie Alvarado 1 ; Mark A. Alday 1 ; Priscilla Natcher 1 ;Vanessa Muhlenbruch 1 ; Audrey M. Rutz 1 ; Jonathan C. Rupp 1 ; Benjamin R. Harrison 1 ; Faye D. Schilkey 2 ;Viviana A. Simon 3 ; Eric M. Bortz 1 1 Univ of Alaska Anchorage, Anchorage, AK, USA; 2 Natl Cntr for Genome Resources, Santa Fe, NM, USA; 3 Icahn Sch of Med at Mount Sinai, New York, NY, USA Background: We are investigating innate antiviral immune responses to target and eradicate latent HIV reservoirs. Type I interferon pathways are triggered when cellular receptors bind and recognize pathogen-associated molecular patterns (PAMP) in viral RNA, activating transcription factors NF-kB and IRF3/7 to induce antiviral interferon-

Poster Abstracts

140

CROI 2016