CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

as the primary source of IFNγ. Finally, IFNγ was found to trigger rapid HSPC proliferation, leading to the exhaustion of the HSPC pool over time. Conclusions: Using this humanized model, we identified IFNγ signaling as a key factor in driving bone marrow failure post-acute HIV infection. Importantly, blocking IFNγ signaling rescued HSPC defects, thereby protecting the hematopoietic system during the early stages of infection. These findings offer valuable insights into the clinical management and treatment of PLWH. HIV Drives CARD8 Inflammasome Activation and Proinflammatory Cytokine Release by Myeloid Cells Marilia R. Pinzone 1 , Francesco R. Simonetti 2 , Liang Shan 1 1 Washington University in St Louis, St Louis, MO, USA, 2 The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Inflammasome activation in response to “danger-associated stimuli” including HIV can lead to the release of inflammatory cytokines such as IL-1ß and IL-18 as well as pyroptotic cell death. The HIV-1 protease can activate the CARD8 inflammasome in CD4 T cells, resulting in T-cell loss during untreated HIV infection. Since myeloid cells are equipped to mount an inflammatory response upon inflammasome activation, we hypothesize that CARD8 activation in these cells might be driving IL-18 production upon HIV infection. Methods: We tested our hypothesis using an in vitro , ex vivo , and in vivo model. In vitro , we cocultured monocyte-derived macrophages (MDMs) with HIV-infected autologous CD4 T cells (CD4s) or HIV viral particles. We used either CARD8 knockout (KO) or Cas9 control cells. Ex vivo , we co-cultured MDMs with autologous CD4s from people living with HIV (PLWH) in which latent HIV-1 was reactivated by CD3/CD28 antibodies. For in vivo experiments, humanized mice were engrafted with either Cas9 control or CARD8 KO CD34+ cells, and then infected with HIV at week 9. Plasma or supernatant levels of IL-18 were measured by ELISA. P values were calculated using t test or ANOVA test. Results: Coculture of MDMs with autologous CD4s infected with CCR5-tropic HIV or from PLWH resulted in higher levels of IL-18 compared to controls. Cytokine release was not triggered by free viral particles or X4-infected CD4s. Coculture with CARD8 KO CD4s resulted in significantly lower levels of IL-18 compared to Cas9 only controls. In humanized mice, CARD8 KO did not affect overall human CD45 engraftment, CD4 development or HIV viral loads. CARD8 KO and Cas9 control mice had similar IL-18 at baseline. However, CARD8 KO mice had a blunted inflammatory response to HIV compared to controls. While IL-18 levels trended up after infection in both groups, this increase was significant only in the Cas9 control group. Conclusions: CARD8 inflammasome activation drives the release of inflammatory cytokines from autologous myeloid cells in vitro and ex vivo . Moreover, CARD8 KO decreases IL-18 surge during untreated HIV infection in humanized mice. These findings offer novel insights into HIV pathogenesis since they provide a mechanism for myeloid cell activation during acute infection. Since inflammation appears to persist even during effective antiretroviral therapy, understanding the contribution of CARD8 activation in PLWH could offer new strategies to decrease chronic inflammation. Genome-Wide Association of NLRP3 Inflammasome Plasma Cytokines in Virally Suppressed People With HIV Ryan Chung 1 , Sonia Savur 2 , Sannidhi Sarvadhavabhatla 2 , Alton Barbehenn 2 , Caroline H. Sheikhzadeh 2 , Maria Sophia Donaire 2 , Vivian Pae 2 , Xiuping Chu 3 , Daniel Winder 4 , Colin T. Maguire 4 , Rafick P. Sekaly 5 , Brian Agan 6 , Jeffrey A. Tomalka 5 , Nilah Ioannidis 1 , Sulggi Lee 2 1 University of California Berkeley, Berkeley, CA, USA, 2 University of California San Francisco, San Francisco, CA, USA, 3 Infectious Disease Clinical Research Program, Bethesda, MD, USA, 4 University of Utah, Salt Lake City, UT, USA, 5 Emory University, Atlanta, GA, USA, 6 Uniformed Services University of the Health Sciences, Bethesda, MD, USA Background: Despite antiretroviral therapy (ART), people with HIV (PWH) experience higher rates of morbidity and mortality than people without HIV (PWoH), which may be driven by chronic inflammation due to persistent virus. Inflammasome activation leading to proinflammatory cytokine production may be a major driver of this risk. Identification of genes associated with signaling cytokines could advance our understanding of the role genetic variation plays in inflammatory activation. Methods: We performed whole genome sequencing of 1000 PWH from the U.S. Military HIV Natural History cohort in relation to circulating plasma levels of NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome cytokines (IL-1β, IL-6, IL-18) during ART suppression. We performed

genome-wide association (GWAS), rare variant association, and gene-based transcriptome-wide analyses (TWAS). Results: After filtering for sequencing read depth and quality, a total of 993 participants and a total of 66,084,459 variants passed quality control. Participants were mostly of European (n=417) and African (n=387) ancestry. We identified 14 significant GWAS signals (p<5x10 -8 ). Our GWAS and rare variant association signals including host genes involved in myeloid immune responses ( ETV1 ), HIV replication ( DDX17 , DDX41 , BCL11a ), HIV cell-cell transmission ( EEA1 ), HIV protease inhibitor transport ( ABCC1 ), cardiovascular disease risk ( KLHL29 ), angiogenesis ( RNF213 ), and cholesterol metabolism (e.g., ABCA12 , ABCA1 , AGMO ). Three GWAS loci were identified as expression quantitative trait loci (eQTLs) in coronary artery tissue (GTEx). Our TWAS identified several innate and adaptive immune related genes known to regulate the expression of NLRP3 cytokines. Conclusions: In the first genome-wide and transcriptome-wide analyses of circulating NLRP3 inflammasome cytokines, we found several host genes involved in immune responses to HIV and cardiovascular disease pathogenesis associated with plasma IL-1β, IL-6, and IL-18 in PWH on ART. These data suggest that host genetics may contribute to the elevated risk of vascular events and inflammation in this population, even during suppressive ART; additional analyses are ongoing. Further studies are needed to validate these findings both within populations of people with and without HIV, and in relation to risk of incident vascular events. Extracellular Vesicles From the Cells Harboring HIV Defective Provirus Initiate Inflammatory Cascade Hongyan Sui 1 , John G. Bernbaum 2 , Hiromi Imamichi 3 , H. Clifford Lane 3 , Tomozumi Imamichi 1 1 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 2 National Institutes of Health, Bethesda, MD, USA, 3 National Institute of Allergy and Infectious Diseases, Baltimore, MD, USA Background: It has been demonstrated that over 95% of provirus present in the peripheral blood of people living with HIV on combination antiretroviral therapy are defective. Defective viruses have long been considered biologically irrelevant, as they cannot encode infectious viruses. However, recent studies have shown that these defective viruses are translationally active, raising potential concerns for current HIV therapies.Therefore, there is a need for a better understanding of the biological function of defective HIV proviruses. Methods: Previously subcloned cell lines derived from H9MN were used in this study. The H9-FI harbored an intact full-length HIV-1 provirus, the H9-FD contained a full-length defective provirus with a 1-bp inserted frameshift, and the H9-SD possessed a 2.4-kb internal deletion. Cytopathic effect was determined using H9-FD or H9-SD cells co-culturing with MT-2 cells. Extracellular vehicles (EVs) were isolated from cell culture supernatants of H9-FD or H9-SD using ultracentrifugation. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analysis were used to characterize the EVs. A CodePlex secretome proteomics workflow was used to measure cytokine inductions. Results: SEM analysis confirmed 25% of viral particles budding from H9-FD cells, with 75% of these particles being fully released, a level similar to that observed in H9-FI. Additionly, TEM images confirmed that more than 90% of EVs from H9-FD were virus-like particles (VLPs), with the remainder being typical EVs. Of the VLPs, approximately 5 to 10% were mature, while 90 to 95% were immature particles. In contrast, no VLPs were identified in the EVs from H9-SD. Of interest, cell killing of MT-2 cells was observed only when co-cultured with H9-FD, not with H9-SD cells, indicating that VLPs are critical in causing the cytopathic effect. Stimulating human primary T cells with a mixture of EVs, but not with individual EVs, was found to induce the production of multiple cytokines, including IFN-γ, IL-10, IL-15, IL-17A, IL-2, IL-4, IL-5, IP-10, sCD137, and TNF-α. Conclusions: The present study demonstrated that a mixture of EVs produced by cells harboring defective HIV provirus is a potent stimulator of human primary T cells and that even EVs without VLPs can still contribute to initiating inflammatory cascades in the human body. This finding provides new insight: HIV-1 defective proviruses are not silent and may play a critical role in stimulating innate immune responses.

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CROI 2025