CROI 2019 Abstract eBook

Abstract eBook

Poster Abstracts

sorted subset to quantify the copy numbers of intact proviruses. Viral RNA from culture wells was sequenced to assess the clonality of the subset cells. Results: The frequencies of viral outgrowth calculated from p24 ELISAs were compared to the frequencies of intact proviral DNA copies we calculated, allowing us to estimate that on average only 3% of intact proviruses can be induced by multiple rounds of global T-cell activation. Each memory subset contained similar numbers of copies of intact proviruses per million cells. We observed different levels of viral outgrowth from different subsets at different stimulation timepoints, indicating significant patient-to-patient variability and no trend in viral inducibility from each subset. Conclusion: In this study, we investigated different resting CD4+ T cell subsets and their relationships with proviral integration and expression. We observed no enrichment of intact proviruses in any specific subset nor any correlation between the inducibility of intact proviruses and memory subset phenotype. Furthermore, we observed significant plasticity among the canonical defining memory subset surface markers and saw significant patient-to-patient variability that abrogated any potential trend seen between subset and inducibility and complicates the vision for a targeted cure approach based on T-cell subsets. 369 PKC AGONIST EXPOSURE SUFFICIENT TO ACTIVATE T CELLS IN VIVO ALSO CAUSES COAGULOPATHY Chelsea Snyder , Alivelu Irrinki, Ryan McFadden, Jasmine Kaur, Bally Randhawa, Hoa Truong, Daniel Soohoo, Chad Greco, Dymtro Kornyeyev, Eric Hu, Helen Yu, Bernard Murray, Leigh Ann Burns-Naas, Tomas Cihlar, Jeffrey Murry Gilead Sciences, Inc, Foster City, CA, USA Background: Activation of latent HIV reservoir is part of a strategy for HIV cure as it should enable the elimination of infected cells by immune-mediated clearance mechanisms and facilitate long-term remission or cure. Protein kinase C (PKC) agonists are highly effective at activating latent HIV. However, effective use of PKC agonists is limited by their severe toxicity, with a mechanism not clearly elucidated. Methods: A novel small-molecule PKC agonist, C-232A, was identified and characterized in vitro and in vivo. PKC activation was assessed by fluorescent microscopy of GFP-labeled PKC in A549 cells. Resting CD4 T cells from ART- suppressed HIV-infected donors were treated with C-232A and HIV RNA in culture supernatants was assessed by qPCR. Flow cytometry was used to quantify CD62P on platelets and CD69 on T cells in whole blood. Dose escalation studies were performed in both rats and rhesus macaques. Activation markers and cytokines were measured by flow cytometry, quantitative PCR and multiplex immunoassay. Investigational toxicology endpoints were assessed, including hematology, coagulation and anatomic pathology. Results: C-232A induced PKC translocation from the cytoplasm to cellular membranes, consistent with PKC agonist activity. HIV transcription was activated ex vivo to the same magnitude as seen with prostratin, but with 5-fold higher potency. IV infusion of C-232A in rhesus macaques induced dose dependent expression of CD69 on T cells. However, similar to other PKC agonists, dose levels sufficient to activate >50% of T cells in vivo also caused a rapid onset of moribundity in treated animals. Toxicity was mediated by platelet activation and ultimately manifested in disseminated intravascular coagulation, a lethal coagulopathy marked by consumption of clotting factors, thrombus formation and hemorrhage. Using a whole blood in vitro assay, dose-dependent platelet activation has been observed across multiple chemical series of PKC agonists at doses similar to those that activate T cells. Consistent with this data, expression of several PKC isoforms has been confirmed in platelets. Conclusion: Platelet activation is a critical safety liability associated with PKC agonists and should be carefully monitored in any preclinical or clinical studies. In addition, the developed in vitro screening tools should facilitate structure- based design of novel PKC agonists with improved activity in T cells and minimal platelet activation. 370 ACTIVATION OF HIV-SPECIFIC CD8+ T CELLS FROM HIV+ DONORS BY VESATOLIMOD Renee R. Ram , Tiffany Barnes, Paul Duatschek, Michael E. Abram, Romas Geleziunas, Joseph Hesselgesser, Christian Callebaut Gilead Sciences, Inc, Foster City, CA, USA Background: Vesatolimod (GS-9620) is a Toll Like Receptor 7 (TLR7) agonist that directly activates human pDCs, B lymphocytes, and induces the production of cytokines such as type I interferon. GS-9620 is currently being evaluated

in HIV-1 infected participants as part of an HIV remission strategy. Here we investigated the potential of GS-9620 to trigger indirect activation of HIV- specific CD8 T cells, using immune cell cultures derived from HIV+ donors. Methods: Peripheral blood mononuclear cell (PBMC) cultures derived from HIV+ donors virologically suppressed on stable antiretroviral therapy (n=39) were profiled. PBMCs were collected by leukapheresis, separated by Ficoll centrifugation, and treated with GS-9620 (20 and 1000 nM) or vehicle alone for 24 hours. HIV pentamers (Proimmune) composed of five MHC Class I peptide-complexes were used to detect CD8 T cell HIV specificity. Pentamers were selected according to donors’ HLA type(s). Cells were incubated with HIV specific pentamers, surface stained with anti-CD3, CD4, CD8, CD69 fluorescent conjugated antibodies, stained intracellularly with anti-CD107a, TNF-α, and IFN-γ fluorescent conjugated antibodies, and analyzed by flow cytometry (FACS). Donors with GS-9620 activated HIV-specific CD8 T cells were scored as positive using a cut-off of 0.5% Pentamer binding. Results: In vitro treatment of PBMCs with GS-9620 resulted in all 39 donor cultures demonstrating an increase in CD8+ T cell activation of up to 80% as measured by CD69 expression compared to no treatment. Of these, 17/39 donors showed HIV-specific CD8+ T cell activation with 5/17 donors positive at 20 nM, and 17/17 donors positive at 1000 nM GS-9620. Intracellular staining was done in a subset of donors (n=13), resulting in 4 donors showing HIV-specificity, 2 of which were positive for degranulation (CD107a), 3 positive for TNF-α, and none positive for IFN-γ. Conclusion: Vesatolimod treatment of HIV+ donor derived PBMCs resulted in robust activation of CD8+ T cells as demonstrated by expression of the activation marker CD69. Furthermore, HIV-specific CD8+ T cell activation was observed in approximately half of the donors tested, with 3 of the donors’ CD8 T cells also up-regulating expression of CD107a and/or TNF- α. These data support the in vivo potential of Vesatolimod to induce HIV-specific CD8 mediated killing of latently infected cells as part of an HIV remission strategy. 371 DEBIO 1143 IS AN ATTRACTIVE HIV-1 LATENCY REVERSAL CANDIDATE Michael Bobardt 1 , Udayan Chatterji 1 , Sumit Chanda 2 , Susan J. Little 3 , Norbert Wiedemann 4 , Gregoire Vuagniaux 4 , Philippe Gallay 1 1 The Scripps Research Institute, La Jolla, CA, USA, 2 Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA, 3 University of California San Diego, La Jolla, CA, USA, 4 Debiopharm, Lausanne, Switzerland Background: Antiretroviral therapy (ART) suppresses HIV replication, but does not cure the infection because replication-competent virus persists within latently infected CD4+ T cells throughout years of therapy. These reservoirs contain integrated HIV-1 genomes and can replenish active virus. Thus, the development of strategies to eliminate the reservoir of latently infected cells is a research priority of global significance. Results: D1143 activates HIV transcription via NF-kB signaling by degrading the ubiquitin ligase baculoviral IAP repeat-containing 2 (BIRC2), a repressor of the non-canonical NF-kB pathway. D1143-induced BIRC2 degradation results in the accumulation of NF-κB-inducing kinase (NIK) and proteolytic cleavage of p100 into p52, leading to nuclear translocation of p52 and RELB. D1143 greatly enhances the binding of RELB to the HIV-1 LTR. These data indicate that D1143 activates the noncanonical NF-kB signaling pathway by promoting the binding of RELB:p52 complexes to the HIV-1 LTR, resulting in the activation of the LTR-dependent HIV-1 transcription. Importantly, D1143 reverses viral latency in HIV-1 latent T cell lines. Using knockdown (siRNA BIRC2), knockout (CRIPSR NIK) and proteasome machinery neutralization (MG132) approaches, we found that D1143-mediated HIV latency reversal is BIRC2 degradation- and NIK stabilization-dependent. D1143 also reverses HIV-1 latency in resting CD4+ T cells derived from ART-treated patients or HIV-1-infected humanized mice under ART. D1143 has been tested as cancer therapy in various human clinical trials. Interestingly, daily oral administration of D1143 in cancer patients at well-tolerated doses elicited pharmacodynamic effects on BIRC2 in PBMCs and induced a moderate increase in cytokine and chemokine that are mechanistically related to NF-kB signaling modulation. Conclusion: We provide strong evidence that the IAPa D1143, by initially activating the noncanonical NF-kB signaling and subsequently reactivating HIV-1 transcription, represents a new and attractive viral latency reversal agent. Methods: We tested the ability of a new inhibitor of apoptosis protein antagonist (IAPa) called Debio 1143 (D1143) at reversing HIV latency and investigated its mechanisms of action.

Poster Abstracts

CROI 2019 133