CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

Conclusion: DAG indololactones can stimulate the posttranscriptional expression of P-TEFb in memory T cells at concentrations below the threshold needed to induce T-cell activation through a RasGRP1-mediated ERK1/2 mTORC1-S6K-rpS6 pathway. These agents can synergize with HDAC inhibitors, thereby bolstering the hypothesis that a two-pronged strategy that targets P-TEFb biogenesis and stimulates RNA polymerase II recruitment to the HIV-1 promoter is needed to reverse HIV-1 latency efficiently. Monovalent and Bivalent SMAC Mimetics Reverse HIV Latency and Decreases the HIV Reservoir Youry Kim 1 , Kiho Tanaka 1 , Jesslyn Ong 1 , Carolin Tumpach 1 , James H. McMahon 2 , Ajantha Rhodes 1 , Rebecca Hoh 3 , Steven G. Deeks 3 , Sushama Telwatte 1 , Michael Roche 1 , Sharon R. Lewin 1 1 Peter Doherty Institute, Melbourne, Australia, 2 Alfred Hospital, Melbourne, Australia, 3 University of California San Francisco, San Francisco, CA, USA Background: Latently infected CD4+ T cells persist in people living with HIV (PLWH) despite suppressive antiretroviral therapy (ART). This persistence may be due to the over-expression of pro-survival proteins such as the inhibitors of apoptosis (IAP) proteins. SMAC mimetics (SMACm) are small molecule compounds that inhibit IAPs, leading to their degradation and ultimately the activation of apoptosis. While bivalent SMACm are known to be more potent than monovalent SMACm, they cause significant toxicities, which have not been seen in clinical trials of monovalent SMACm. Here we investigated whether monovalent and bivalent SMACm could reverse latency and/or deplete the reservoir. Methods: Latency reversal by monovalent (GDC0197, GDC0152, LCL161, Xevinapant) and bivalent (AZD5582, BV6) SMACm was assessed in J-Lat 10.6 cells (flow cytometry for GFP expression); the dual-reporter primary CD4+ T cell latency model Morpheus (flow cytometry for productive marker mCherry); and ex vivo CD4+ T cells from PLWH on ART (using HIV transcriptional profiling by digital PCR). Depletion of infected cells in ex vivo cultures was measured by the Intact Proviral DNA assay. Proliferation and function of HIV-specific CD8+ T cells following treatment with SMACm was measured using HIV-specific tetramers and flow cytometry. Results: The bivalent SMACm AZD5582 (100nM) was the most potent latency reversal agent in J-Lat10.6 cells (fold change (FC) reactivation over untreated=22.92, p=0.03) and in the primary cell latency model (FC=1.43, p=0.03). AZD5582 partially overcame all blocks in HIV RNA transcription inducing multiply spliced HIV transcripts (FC over untreated=2.35, p=0.03). AZD5582 treatment also led to a decline in intact HIV provirus (FC=0.51, p=0.02). The monovalent SMACm GDC0197 reversed latency in cell lines (FC=1.82) and primary T cells (FC=1.30, p=0.001); but could only induce HIV RNA transcription initiation (FC=4.26, p=0.03), and not elongation or completion. GDC0197 induced the proliferation of tetramer positive cells (FC=5.63 over untreated, p=0.0001) and enhanced killing of Gag peptide-loaded target cells (FC=1.47 killing over untreated). This was not observed with AZD5582. Conclusion: Bivalent SMACm can reactivate latent HIV and deplete the reservoir. Monovalent SMACm although less potent in latency reversal, may have a novel role in enhancing clearance of the reservoir through altering antigen presentation and inducing greater CD8+ T cell mediated killing. Optimal Administration Timing of Latency Reversal Agents to Reduce Effectively the HIV Reservoir Erick De La Torre Tarazona 1 , Sergio Serrano-Villar 1 , Raúl Vaquer 1 , Marta Rava 2 , Laura Luna Garcia 1 , Sonsoles Sánchez-Palomino 3 , Teresa Aldámiz-Echevarría 4 , Rafael Mican 5 , Adrià Curran 6 , Melchor Riera 7 , José Alcamí 8 , Inma Jarrin 2 , Santiago Moreno 1 1 Hospital Ramón y Cajal, Madrid, Spain, 2 Institute of Health Carlos III, Madrid, Spain, 3 Hospital Clinic of Barcelona, Barcelona, Spain, 4 University Hospital Gregorio Marañon, Madrid, Spain, 5 La Paz University Hospital, Madrid, Spain, 6 Hospital Universitario de la Vall d’Hebron, Barcelona, Spain, 7 Hospital Universitario de Son Espases, Palma de Mallorca, Spain, 8 Institute de Salud Carlos III, Majadahonda, Spain Background: The administration timing of Latency Reversal Agents (LRA) may impact the success of strategies aimed to HIV functional cure. Maraviroc (MVC) is an antiretroviral drug that exhibits HIV latency-reversing properties by activating viral transcription through NF-κB pathway. We aimed to evaluate the efficacy on HIV-DNA reduction following MVC administration at the time of ART initiation (with detectable viral load), or in patients under suppressive ART. Methods: We compared people with HIV initiating ART with a regimen including MVC (Cases, n=12) to two different control groups: Control 1

modulation of innate immunity could impact viral latency and contribute to the clearing of HIV reservoir. Our previous data indicated that IRF7 expression correlates with HIV latency reversal. Here, we demonstrate the key role of IRF7 in HIV-1 transcription and latency, identifying also novel therapeutic agents useful in HIV eradication. Methods: Latency was evaluated by non-clonal models drug-treated, alone or in combination with known LRAs. qPCR and WB were used to assess gene and protein expression. IRF7 loss and gain-of-function models were developed by siRNA or plasmid overexpression. IRF7 role in HIV-1 transcription was evaluated by measuring LTR-driven transactivation in TZM-bl cells and co-immunoprecipitation. Immunophenotyping of ex vivo treated CD4+ T-cells from ART-suppressed HIV+ subjects was performed by flow cytometry and latency reactivation/promoting activity was measured by qPCR in cell supernatant. Results: IRF7 expression correlates with latency reversal/promoting capacity of LRAs/LPAs, including PMA, PNB, JQ1 and the JAK2inhibitors fedratinib and pacritinib among others (r2= 0.8; p-value=0.0012). Downregulation of IRF7 impaired the latency reversal capacity of LRAs (p=0.005). On the contrary, overexpression of IRF7 enhances Tat-mediated transactivation of integrated HIV in the presence of LRAs (at least 50% increase, p=0.05). Coimmunoprecipitation studies showed physiological interaction between Tat protein and IRF7, relating IRF7 to HIV-1 transcription control. The JAK2 inhibitor pacritinib dowregulated IRF7 expression and thus, was used to further explore IRF7 role in HIV latency. Pacritinib significantly blocked HIV-1 reactivation both alone or in combination with PMA or VOR in non-clonal models of HIV-1 latency (50% reduction, p=0.05) and ex vivo in CD4+ T cells from ART-suppressed HIV+ subjects (1log reduction). Immunophenotypic characterization of pacritinib-treated primary CD4+ T cells from PLWH showed no major changes on CD4+ T cell subsets nor activation markers. Moreover, pacritinib significantly reduced the presence of multispliced HIV transcripts in primary CD4 T cells. Conclusion: IRF7 controls latent HIV-1 transcription and plays a role in both HIV-1 reactivation or blockade. Moreover, modulation of IRF7 expression through pharmacological agents might represent an asset to current HIV-1 cure strategies. Reversing HIV-1 Latency by Targeting RasGRP1-Dependent Biogenesis of P-TEFb Uri Mbonye 1 , Ana Bellomo 2 , Eleonora Elhalem 2 , Lucia G. Donadio 2 , Maria J. Comin 2 , Jonathan Karn 1 1 Case Western Reserve University, Cleveland, OH, USA, 2 National Institute of Industrial Technology, Buenos Aires, Argentina Background: The deliberate reactivation of latent HIV-1 to enable clearance of persisting latently infected memory T cells – the Shock and Kill strategy – has had limited success because efficient, non-toxic latency-reversing agents (LRAs) remain to be discovered. We are taking a direct approach to LRA development based on the principle that proviral reactivation is tightly coupled to the posttranscriptional biogenesis of P-TEFb, a cellular transcription elongation factor whose expression is highly restricted in resting memory T cells. Recently, we reported that naturally occurring diacylglycerol (DAG)-mimicking protein kinase C (PKC) agonists stimulate the posttranscriptional expression of the cyclin T1 (CycT1) subunit of P-TEFb to reactivate latent HIV-1 primarily via the RasGRP1-Ras-Raf-MEK-MAPK ERK1/2 signaling pathway rather than through PKC enzymes. Here we describe synthetic agonists that preferentially bind RasGRP1 over PKC and can safely activate P-TEFb to reverse HIV-1 latency in primary CD4+ T cells. Methods: Synthetic DAG indololactones, with known differential affinities for PKC and RasGRP, were tested for their ability to stimulate P-TEFb expression in healthy donor-derived memory CD4+ T cells. Combinatorial LRA studies were performed in a primary CD4+ T-cell latency model to examine the effectiveness of DAG indololactones at synergizing with LRAs that target proviral transcription initiation. Results: Three of the 4 DAG indololactones we tested were more effective at inducing CycT1 and active P-TEFb than the T-cell activation markers CD69 and CD25. The DAG indololactone 2A-127, which preferentially binds RasGRP1 over PKC more than 60-fold, synergized with the HDACi SAHA in reactivating latent HIV-1 in primary T cells. DAG indololactones and other DAG-mimicking agonists stimulate CycT1 protein synthesis by activating mTORC1 kinase through ERK1/2 MAPK signaling.

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