CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

564

Ex Vivo Antigen Stimulation Induces HIV-1 Viral Outgrowth From a Pericentromeric Provirus Angelica Camilo-Contreras 1 , Francesco R. Simonetti 1 , Luis J. Montaner 2 , Stuart Ray 1 , Robert F. Siliciano 1 , Janet M. Siliciano 1 , Arturo Casadevall 3 , Daniel Smith 3 , Hao Zhang 3 1 The Johns Hopkins University School of Medicine, Baltimore, MD, USA, 2 The Wistar Institute, Philadelphia, PA, USA, 3 The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA Background: HIV-1 persists despite effective ART due to a reservoir of latently infected CD4+ T cells. Previous studies suggest that intact proviruses integrated in heterochromatic regions are progressively enriched during long-term ART. These proviruses may evade immune recognition due to lower viral gene expression, but their clinical significance upon ART interruption is unclear. Given the recent findings that cognate antigen engagement increases HIV-1 RNA expression, we sought to determine if antigen-specific stimulation ex vivo could induce the viral outgrowth of proviruses in regions associated with deeper latency. Methods: We isolated CD4+ T cells responsive to a protein lysate of the commensal fungus Candida albicans (CA) based on activation-induced markers (CD69, CD154, CD137). HIV-1 populations in CA-reactive cells were analyzed by IPDA, matched integration and proviral sequencing, and integrant-specific digital PCR. We then optimized an antigen-specific quantitative viral outgrowth assay (QVOA) by stimulating CD8-depleted PBMCs with CA lysate for 3 days, followed by co-culture with MOLT-4s for 21 days. To limit non-specific T cell activation, we used a basal culture medium (10% human serum, 10 U/mL IL-2) and omitted irradiated feeders. Phytohemagglutinin (PHA) combined with T-cell activation media was used as a non-specific positive control. Viral outgrowth was monitored via p24 ELISA assay, digital PCR, and full env sequencing. Results: We identified a study participant on suppressive ART whose CA reactive cells were highly enriched for intact HIV-1 DNA. A clonally expanded provirus representing 50% of env proviral sequences was confirmed to be genetically intact by full genome sequencing. Integration site analysis showed this provirus was located in a pericentromeric satellite repeat, previously linked to deeper latency. The provirus of interest (PeriC) had a frequency of 930 copies per million CA-responsive cells. In our modified QVOA, we observed exponential outgrowth from multiple wells, with 100% of the outgrowth sequences from the CA lysate condition matching PeriC, compared to 33% in the PHA arm. Conclusions: Cognate antigen stimulation resulted in the reactivation of a provirus integrated in a pericentromeric region. Although limited to a model culture ex vivo, these findings suggest that CD4+ T cell recognition with ubiquitous antigens can reactivate proviruses in regions previously associated with deeper latency, potentially contributing to viral rebound if ART is discontinued. Multiomics Clustering Reveals Distinct HIV Reservoir Profiles in the 2000HIV Cohort Victoria Rios Vazquez 1 , Mareva Delporte 2 , Vasiliki Matzaraki 1 , Wilhelm A. Vos 3 , Marc J. T. Blaauw 4 , Louise E. van Eekeren 1 , Albert L. Groenendijk 5 , Jéssica dos Santos 1 , Mihai G. Netea 1 , Andre van der Ven 1 , Linos Vandekerckhove 2 , for the 2000HIV Human Functional Genomics Partnership Program 1 Radboud University Medical Center, Nijmegen, Netherlands, 2 HIV Cure Research Center, Ghent University, Ghent, Belgium, 3 OLVG, Amsterdam, Netherlands, 4 Elisabeth-TweeSteden Ziekenhuis, Tilburg, Netherlands, 5 Erasmus University Medical Center, Rotterdam, Netherlands Background: Multi-omics data integration offers potential to uncover hidden patterns among people living with HIV (PLWH). This study aimed to identify clusters of individuals with distinct viral reservoir characteristics and multi omics profiles in the 2000HIV cohort. Methods: Multi-omics data from 1,236 PLWH in the 2000HIV cohort ( NCT03994835 ) were analyzed, including gene expression (58,347 genes, bulk RNA-seq from PBMCs), DNA methylation (793,775 CpG sites, PBMCs), plasma proteins (2,367, Olink Explore), immune cell markers (355, flow cytometry from whole blood), and cytokine production capacity (90 markers, ex vivo stimulation assays from PBMCs). Total viral reservoir (DNA copies/million CD4 T cells) and intactness (intact reservoir DNA copies/million CD4 T cells) were assessed. Data preprocessing included normalization, filtering, and selection of top variable features. Multi-omics clustering was performed using moCluster. Results: Three distinct clusters were identified: High Total Low Intact ( HTLI , n=348), Low Total Low Intact ( LTLI , n=421), and High Total High Intact ( HTHI , n=467), each with distinct viral reservoir profiles and clinical characteristics.

LTLI exhibited the lowest total reservoir DNA and intactness. HTHI revealed high total reservoir DNA and highest intactness, while HTLI showed high total reservoir DNA but low intactness. These differences were statistically significant (P<0.05). Top 10 driver markers were identified in each omics layer, with bulk transcriptomics showing significant contributions to intercluster differences. Key driver genes included ACVR1C , AK5 , LRP6 , MMP28 , SLC22A17 , SLC16A10 , OBSCN , MAN1C1 , EDAR , and LEF1 , which were highly expressed (P<0.05) in LTLI compared to HTLI and HTHI . These genes are involved in processes relevant to control HIV infection, including cell signaling, T cell development, immune response and apoptosis regulation, macrophage function, iron homeostasis, mitochondrial biogenesis, and HIV-1 transcriptional regulation. Conclusions: Multi-omics clustering revealed distinct molecular signatures associated with viral reservoir size and intactness in PLWH, identifying three clusters with different characteristics, particularly distinct in the LTLI group. These findings suggest potential for personalized therapeutic strategies informed by biomarkers from distinct multi-omics profiles. Further investigation of these and other omics markers could provide insights into HIV reservoir dynamics and tailored interventions for HIV cure research. The figure, table, or graphic for this abstract has been removed. Sex-Specific Immune Responses Shape Proviral Landscapes in Individuals on Long-Term Suppressive ART Toong Seng Tan 1 , Ce Gao 1 , Aischa Niesar 1 , Chloe M. Naasz 1 , Samantha K. Marzi 1 , Alex Hochroth 1 , Sruthi Kalavacherla 1 , Mathias Viard 2 , Steven G. Deeks 3 , Michael Peluso 3 , Jeffrey M. Jacobson 4 , Mary Carrington 2 , Mathias Lichterfeld 1 , Xu G. Yu 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 3 University of California San Francisco, San Francisco, CA, USA, 4 Case Western Reserve University, Cleveland, OH, USA Background: Our previous work revealed sex-specific selection and evolution of HIV-1 reservoir cells in individuals on long-term suppressive ART (LT-ART), and demonstrated more efficient selection of intact proviruses in heterochromatin locations in females. However, immune mechanisms responsible for viral reservoir cell selection remain elusive. Methods: We selected a total of 20 participants on LT-ART and categorized them into 4 study groups based on their HIV-1 integration site profiles: females and males with viral reservoirs dominated by genome-intact proviruses integrated in transcriptionally-repressed heterochromatin (HTC) sites (HTC high ; n=5 females, n=5 males) and those without evidence of selection for intact proviruses at HTC (HTC low/neg ; n=4 females, n=6 males). We conducted global immunophenotyping studies using multidimensional spectral flow cytometry with 27 colors for innate immunity and 18 colors for T cell adaptive immune responses. Results: A global linear discriminant analysis demonstrated distinct phenotypic signatures between males and females in innate immune cells, but not in adaptive HIV-specific T cell responses. In particular, we observed that proportions of CD64+PD-L1+ myeloid dendritic cells were higher in females (p < 0.05), specifically in the HTC high group. In addition, NK cells in the HTC high group were characterized by a more limited ensemble expression of inhibitory KIR receptors and TIGIT compared to other groups, particularly relative to the group of males from the HTC low/neg group (p < 0.01). Statistical correlations supported a negative association between expression levels of these NK cell markers and the frequencies of clonal intact proviruses (r = -0.46, p < 0.05) and the proportions of intact proviruses in HTC locations (r = -0.71, p < 0.001). Cytokine-producing HIV-1 specific T cells were detectable in most LT-ART individuals albeit at a relatively low level (< 1% of both total CD4 and CD8); however, T cell responses did not significantly differ between males and females, and statistical analyses failed to demonstrate any associations between HIV-specific T cell responses and the clonality or HTC locations of intact proviruses. Conclusions: These data suggest that innate immune responses may drive efficient immune selection of intact proviruses in heterochromatin locations and suggest that females may be better candidates to explore innate immunity-dependent strategies for targeting HIV-1 reservoir cells.

566

Poster Abstracts

565

CROI 2025 149