CROI 2017 Abstract e-Book

Abstract eBook

Poster and Themed Discussion Abstracts

314 TLR9 AGONIST TRIGGERS POTENT INTESTINAL ANTIVIRAL RESPONSE IN HIV+ INDIVIDUALS ON ART Astrid R. Krarup 1 , Mariane H. Schleimann 1 , Line K. Vibholm 1 , Mohamed Abdel Mohsen 2 , Anders K. Dige 1 , Lars Østergaard 1 , Thomas Benfield 3 , Satish K. Pillai 2 , Ole S. Søgaard 1 , Paul W. Denton 1 1 Aarhus Univ Hosp, Aarhus, Denmark, 2 Blood Systems Rsr Inst, San Francisco, CA, USA, 3 Copenhagen Univ Hosp, Hvidovre, Denmark Background: HIV persists in infected individuals despite ART. While persistent HIV becomes transcriptionally active in the presence of latency reversing agents, these agents have little effect on the size of the viral reservoir in vivo. Thus, improved killing of infected cells is essential to achieve viral eradication. Towards this goal, we tested whether a TLR9 agonist (MGN1703; lefitolimod) could enhance antiviral immunity in vivo. Methods: Sigmoid biopsies were collected from 11 participants at baseline and 24 hours after the last dose in week 4 of a single-arm phase 1b/2a clinical trial where HIV+ adults received MGN1703 (60 mg s.c.) twice weekly for 4 weeks (NCT02443935). We quantitated IHC signal positive cell profiles (nucleus with associated signal; referred to as cells below) in fixed tissue sections for the antiviral and inflammatory response markers MxA, ISG15, IL-21, CXCL10 or MPO; data were stratified by anatomical location: lamina propria (LP) or epithelium. Remaining biopsy tissue was digested and percoll-enriched intestinal mononuclear cells were obtained for virological and immunological analyses [RNA-seq; HIV DNA/ RNA levels (qPCR); CD4/CD8 T cell frequency and activation (flow)]. Plasma viremia quantitation: Cobas Taqman. Data analyses: Wilcoxon signed rank tests, mean fold change (mfc) and Ingenuity Pathway Analysis right-tailed Fisher Exact Test. Results: RNASeq revealed 248 significantly regulated genes (FDR<0.05) and a potent antiviral response (IPA p=2*10^-14) including many IFN-a stimulated genes. Indeed, MGN1703 dosing increased the number of cells positive for MxA (LP: p=0.001, mfc=8.8; and epithelium: p=0.003, mfc=27.6), ISG15 (LP: p=0.014, mfc=5.6) and IL-21 (LP: p=0.019, mfc=0.5). In contrast, there was no change in the numbers of cells positive for CXCL10 which is IFN-g induced (LP: p=0.97). No cohort-wide changes were observed in: neutrophil infiltration (MPO LP: p=0.97); viral DNA; viral RNA; T cell subset distribution; or T cell activation. However, after stratifying according to MGN1703-induced plasma viremia status, we observed that viremic individuals had a decrease (n=3, mfc=-0.8) in integrated intestinal lymphoid tissue HIV DNA (p=0.042). Conclusion: MGN1703 induced robust antiviral immune responses in the intestines of HIV+ individuals on ART. In addition, the observed lack of neutrophil infiltration suggests that MGN1703 does not cause destructive tissue inflammation. These data support a prominent role for MGN1703 immunotherapy in HIV eradication strategies. 315 SMALL-MOLECULE INHIBITION OF INTRINSIC STRESS PATHWAYS REDUCES HIV TRANSCRIPTION Andrew Timmons , Robert Siliciano, Cynthia K. Bullen The Johns Hopkins Hosp, Baltimore, MD, USA Background: Combination antiretroviral therapy (cART) effectively reduces HIV replication to undetectable levels, but HIV persists as a stable reservoir within resting memory CD4+ T cells. Substantial effort has been applied to identifying small molecules that can perturb the stability of this reservoir. Studies in model systems have identified many latency reversing agents (LRAs), but few are efficacious in cells isolated from HIV-1 infected patients. Dose-response studies in model cells and patient cells exhibit unique patterns across multiple chemical classes of LRAs that suggest that generalized cell stress may play a role in initiating HIV transcription. Methods: Resting T cells were isolated from HIV-1 infected individuals with suppression of viremia on cART. Cells were treated for 24 hours with the protein kinase C (PKC) agonist bryostatin and the synthetic Nrf2 agonist HBB2. Treatment was done the presence or absence of the ROS scavenger N-acetylcysteine (NAc) and the HSF-1 inhibitor KRIB11. Intracellular HIV mRNA was measured by RT-qPCR. HIV-1 RNA levels were compared to levels in T cells activated with phorbol 12-myristate 13-acetate (PMA; a PKC agonist) and ionomycin (a calcium ionophore). Cell surface markers of T cell activation were assessed by flow cytometry. Results: Ex-vivo treatment of resting CD4+ T cells with PKC activators as well as PMA and ionomycin greatly increased levels of HIV-1 mRNAs which were significantly reduced with inclusion of the ROS scavenger NAc. Inhibition of HIV-1 transcription was observed to a much greater extent when the heat shock inhibitor KRIB11 was used instead of N-acetylcysteine with no concomitant change in surface marker expression. Gene expression analysis on cells treated with N-acetylcysteine reveals inhibition of major components of the heat shock response. Conclusion: Inhibitors of the oxidative and heat shock stress responses substantially reduce the amount of HIV expressed in activated T cells by approximately 55 and 80%, respectively. These findings indicate that intrinsic cell stress pathways play a significant role in activating latent HIV-1 transcription in activated T cells. 316 NNRTIS DECREASE HIV-1 PRODUCTION FROM RCD4+ T CELLS FOLLOWING LATENCY REVERSAL Jennifer M. Zerbato 1 , Gilda Tachedjian 2 , Nicolas Sluis-Cremer 1 1 Univ of Pittsburgh, Pittsburgh, PA, USA, 2 Burnet Inst, Melbourne, Australia Background: Therapeutic strategies which target the latent HIV-1 reservoir in resting CD4+ T cells of infected-individuals are always administered in the presence of combination antiretroviral (ART) therapy. In this study, we evaluated whether the different therapeutic classes of ARV drugs impacted HIV-1 latency reversal. Methods: Methods: Latency reversal was evaluated in a primary cell model of latency that utilizes direct infection of resting CD4+ T cells. Two hours prior to addition of anti-CD3/ CD28 monoclonal antibodies, cells were treated with an entry inhibitor (AMD3100 or maraviroc), a PI (atazanavir, darunavir), NRTI (lamivudine), NNRTI (rilpivirine, efavirenz) or INSTI (raltegravir). Controls included cells that were exposed only to antibody or the ARVs. Seven days post antibody administration, cell-associated HIV-1 DNA and extracellular virion production were quantified. T cell activation and viability were assessed by flow cytometry. Results: Results: NNRTIs were found to decrease both R5- and X4-tropic HIV-1 production (by ≥ 1-log fold-change) in resting CD4+ T cells exposed to anti-CD3/CD28 monoclonal antibodies. This decrease was not due to toxicity, or changes in T cell activation in the presence or absence of NNRTI as assessed by CD25, CD69 or HLA-DR expression. In contrast, none of the other ARVs, including PIs which target the late stages of HIV-1 replication, had a significant impact on virus production. Further analysis in a cell line model of HIV-1 latency (J89GFP cells) revealed that the NNRTIs did not interfere with HIV-1 gene transcription or translation. Instead, consistent with published studies, we propose that the NNRTIs enhance premature activation of HIV-1 PR which results in intracellular processing of Gag and Gag-Pol leading to decreased viral particle production. Conclusion: Conclusions: NNRTIs reduce HIV-1 production from latently infected cells. Ex vivo studies that use NNRTIs to prevent virus spread or cells from donors on NNRTI containing regimens should be cautiously interpreted. 317 INDUCIBLE HIV-1 LATENT RESERVOIR INCREASES AFTER HIV+ OR HIV- SOLID ORGAN TRANSPLANT Alyssa R. Martin 1 , Andrew D. Redd 2 , Gregory Laird 1 , Robert Siliciano 1 , Diane Brown 1 , Darin Ostrander 1 , Thomas Quinn 1 , Dorry Segev 1 , Aaron Tobian 1 , Christine Durand 1 1 The Johns Hopkins Univ, Baltimore, MD, USA, 2 NIAID, Bethesda, MD, USA Background: The latent reservoir (LR) for HIV-1 in long-lived memory CD4+ T cells is a barrier to cure. Measuring the LR is complicated because PCR-based assays for proviral DNA cannot distinguish defective viruses making up the majority of the LR, whereas viral outgrowth assays that measure replication-competent virus have a low dynamic range and are often not feasible due to blood volume requirements. To overcome these limitations, we developed a novel quantitative viral induction assay (QVIA). In an ongoing prospective clinical trial of HIV+ individuals receiving kidney or liver transplant from HIV+ or HIV- donors (NCT02602262), we used QVIA to test the hypothesis that immunosuppressants used after solid organ transplantation may reduce the HIV-1 LR. Methods: Immunosuppression was per transplant center standard of care and for kidney recipients included antithymocyte globulin for induction. The LR was measured before transplant and at week 13 post-transplant using QVIA, in which 4 million resting CD4+ T cells were plated at limiting dilution and treated for 18 hours with PMA and ionomycin or vehicle alone. mRNA isolation, cDNA synthesis, and qPCR specific for HIV-1 mRNA were performed. QVIA uses a cycle threshold cutoff to exclude wells with a low qPCR signal, increasing specificity for intact, induced viral RNA. Number of inducible proviruses per million cells (IPPM) was estimated by qPCR positive wells at each dilution. Pre- and post- transplant IPPMs were compared using paired student’s t-test.

Poster and Themed Discussion Abstracts

CROI 2017 124