CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

light on mechanisms of latency reversal and could guide the selection of new and more effective LRAs. The figure, table, or graphic for this abstract has been removed. Activation of Latent HIV-1 by Retinol Binding Protein 4 Chiara Pastorio 1 , Khumoekae Richard 2 , Luis J. Montaner 2 , Konstantin M. J. Sparrer 1 , Frank Kirchhoff 1 1 Ulm University Medical Center, Ulm, Germany, 2 The Wistar Institute, Philadelphia, PA, USA Background: The persistence of HIV-1 in a transcriptionally silent latent form in long-lasting memory CD4+ T cells remains one of the main barriers to cure AIDS. One strategy to eradicate HIV-1 reservoirs is the so-called “shock and kill” approach. It aims to reactivate latent HIV by administration of latency reserving agents (LRAs) to render infected cells vulnerable to elimination, while preventing de novo infections by cART. Thus far, however, it failed to clearly reduce the majority of latently HIV-infected cells. Thus, novel LRAs are urgently needed. Methods: To discover novel endogenous compounds that reactivate latent HIV-1 proviruses, we screened a human hemofiltrate-derived peptide library containing essentially all the peptides and small proteins circulating in blood. To this purpose, we used J-Lat cells, a very well-established model system for HIV-1 latency involving Jurkat-derived T cell lines containing an integrated HIV-R7/E-/ GFP genome. Viral protein induction in cART-suppressed PBMCs was assessed using HIV-1 Gag-p24 Simoa (PMID: 33796087), following 72 hours treatment with the test agent (RBP4) or anti-CD3/CD28 positive control. Results: Multiple rounds of peptide separation and screening revealed that the human Retinol Binding Protein 4 (RBP4), a small protein that transports vitamin A (retinol) in blood, potently reactivates latent HIV in a variety of Jurkat T cell lines (p<0.001). Carrier-free RBP4 as well as free forms of retinoids (all-trans retinal, retinol and retinoic acid) do not reactivate HIV. Thus, the complex retinol-RBP4 (Holo-RBP4), which is very abundant in the circulation, seems essential for its activity as LRA. Notably, RBP4 significantly reactivates latent HIV-1 in primary T cells isolated from infected individuals under effective cART at physiological concentrations (p<0.05). Finally, JNK signalling pathway was identified to be relevant for RBP4-mediated activation of latent reservoirs. Conclusions: We identified RBP4 as an endogenous and abundant circulating HIV-1 latency reversing agents that may modulate the size of the viral reservoirs in vivo. Zaprinast Reprogrammes Resting CD4+ T-Cell Metabolism and Induces HIV Latency Reversal Ex Vivo Valentin de Masson d'Autume 1 , Valentin LeDouce 1 , Sophie O'Reilly 1 , Nathan François 1 , Matthew Angieladis 1 , Tara McGinty 2 , Jose Carlos Valle Casuso 3 , Alan Macken 2 , Olivier Rohr 4 , Carine Van Lint 5 , Asier Sáez-Cirión 3 , Aoife Cotter 1 , Eoin R. Feeney 1 , Mallon W. G. Paddy 1 , Virginie Gautier 1 1 University College Dublin, Dublin, Ireland, 2 Mater Misericordiae University Hospital, Dublin, Ireland, 3 Institut Pasteur, Paris, France, 4 University Hospital of Strasbourg, Strasbourg, France, 5 Université Libre de Bruxelles, Brussels, Belgium Background: Accumulating evidence supports that the establishment and persistence of HIV latent reservoirs despite suppressive ART are intricately linked to the quiescent metabolism of CD4+ memory T cells. We investigated whether reprogramming T cell metabolism towards aerobic glycolysis could facilitate HIV latency reversal from circulating viral reservoirs. We used Zaprinast, a Mitochondrial Pyruvate Carrier inhibitor, to block pyruvate entry into mitochondria and examined its latency reversal capacity on circulating HIV reservoirs from ART-suppressed individuals and its impact on primary CD4 T cells metabolism. Methods: HIV latency reversal was performed ex vivo on CD4+ T cells or CD8-depleted PBMC from ART-suppressed individuals. HIV gene expression and viral production were measured by RT-qPCR targeting the HIV tat-rev msRNA or HIV gag RNA respectively. Mitochondrial respiration and ATP production were assessed using the Mitostress test and ATP real-time kits (Agilent). Aerobic glycolytic activity was measured using pH Xtra assay (Agilent) and glucose uptake with 2-NBDG. Flow cytometry was used to evaluate mitochondrial mass (MitoView Green, Generon), membrane potential (MitoTracker Red CMXRos, Invitrogen), ROS production (MitoSOX Red, Thermofisher), viability (Viobility 405/452, Miltenyi) and activation (CD69 and CD25 antibodies, Biolegend). Results: Zaprinast induced viral particles release in 65% (11/17) of CD8 deplected PBMCs isolated from ART-suppressed individuals, achieving 69% of

maximal HIV reactivation following TCR activation. Zaprinast also triggered HIV gene reactivation in 50% (4/8) of CD4+ T cell samples isolated from ART-suppressed individuals, confirming that Zaprinast targets CD4+ T cells HIV reservoirs. Metabolic analyses revealed that Zaprinast re-programmed CD4 T cell metabolism in two steps. Initially, Zaprinast transiently inhibited mitochondrial respiration before enhancing both aerobic glycolysis and mitochondrial respiration. This effect was reversible with CD4+ T cells returning to a quiescent metabolism upon removal of the drug. The treatment did not impact cell viability, ROS production, or CD4 T cells activation and proliferation. Conclusions: This study demonstrates for the first time that MPCi represents a potent new class of LRAs capable of inducing robust HIV latency reversal from circulating viral reservoirs ex vivo. This strongly supports the potential of targeting the quiescent metabolism of CD4+ T cells as a promising approach for Shock and Kill strategies. The figure, table, or graphic for this abstract has been removed. Anti-HIV Gene Editing Is Inhibited in Active HIV Provirus Through Long-Terminal Repeat Demethylation Mohamed Bouzidi, Li Du, Prerna Dabral, Manukumar Honnayakanahalli Marichannegowda, Satish Pillai Vitalant Research Institute, San Francisco, CA, USA Background: CRISPR-Cas9 excision of the HIV genome is a promising strategy for an HIV cure. However, epigenetic features, including DNA methylation at the HIV integration site, may impede anti-HIV gene editing. In this study, we investigated the impact of an FDA-approved DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine, on genome demethylation and provirus reactivation in latently infected CD4+ T cells, followed by anti-HIV CRISPR-Cas9 editing. Methods: Primary CD4+ T cells were isolated from three healthy donors and activated using CD3/CD28 activation beads. Cells were then infected with a VSV-G pseudotyped HIV NL4-3 GFP strain, and infected (GFP+) cells were purified using fluorescence-activated cell sorting (FACS). After 7 days of culture, infected CD4+ T cells were sorted again to separate GFP-negative cells (latently infected) from GFP-positive cells (productively infected). Both populations were nucleofected with anti-HIV CRISPR-Cas9 ribonucleoproteins (RNPs) targeting the viral long-terminal repeat (LTR) promoter region. After 48 hours, DNA was extracted, and the LTR region was PCR-amplified, purified, and subject to Sanger sequencing. Sequences were analyzed using Tracking of Indels by DEcomposition (TIDE) to infer editing efficiency. Additionally, HIV-latently infected J-Lat clones 6.3 and 11.1 were treated with DMSO (control) or 5-Aza 2'-deoxycytidine for 72 hours. DNA was then extracted, and the LTR region was PCR-amplified, sequenced, and analyzed using TIDE. Results: Editing efficiency was 1.6-fold higher in latently-infected CD4+ T cells as compared to productively-infected CD4+ T cells (N=3 donors, p<0.05, paired t-test) (Fig.1A). J-Lat 11.1 and 6.3 clones treated with 6.25nM 5-Aza-2'- deoxycytidine showed modest HIV reactivation (~15% GFP-positive). Editing efficiency was 4.8-fold and 3.8-fold lower in 5-Aza-2'-deoxycytidine-treated J-Lat 11.1 and J-Lat 6.3 cells, respectively, compared to their DMSO-treated counterparts (p<0.0001 and p<0.0005, respectively, paired t-test) (Fig.1B). Conclusions: Anti-HIV gene editing efficiency is higher in latently-infected cells as compared to productively-infected cells, and anti-HIV editing is impeded by demethylation of the HIV genome. Our data suggest that the active HIV reservoir may be refractory to excision approaches, warranting investigation into the impact of epigenetic states on anti-HIV gene therapy. Hypermethylating drugs in clinical use, such as budesonide, should be explored in combination with anti-HIV gene editing approaches. Macrophage Training Reactivates Latent HIV-1 From HAART-Suppressed PBMCs of PLWH Sinu P. John, Heera James, Christine Perritano, Iain D. C. Fraser National Institutes of Health, Bethesda, MD, USA Background: HIV virus persists in a latent but activatable chronic state, predominantly through epigenetic modification of resting CD4+ T-cells and other immune cells such as macrophages. This necessitates lifelong combination antiretroviral therapy (cART) for people living with HIV (PLWH). Trained immunity is a myeloid specific phenomenon where chromatin is relaxed to a less condensed state at select immune gene loci, facilitating rapid and robust transcription from these loci upon exposure to an immune stimulus. We hypothesized that trained immunity could be a viable approach to reactivate

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