CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

542

Afucosylation of Broadly Neutralising Antibodies to Eliminate HIV+ CD4+ T Cells and Macrophages Morgane Brunton-O'Sullivan, Olivia Wilhelm, Christine Jordan, Hans Kek, Laura Rikard-Bell, Pradhipa Ramanathan, Amy Chung, Pantelis Poumbourios, Bruce Wines, Anthony Jaworowski, Anna C. Hearps Burnet Institute, Melbourne, Australia Background: The persistence of HIV reservoirs in T cells and myeloid cells precludes HIV cure. Some broadly neutralising anti-HIV envelope (Env) antibodies (bNAbs) can bind HIV-infected cells and elicit natural killer (NK) cell antibody-dependent cellular cytotoxicity (ADCC). Removal of core fucose from the Fc region of antibodies can increase NK cell ADCC, but remains to be fully explored in HIV. The aim of this study was to examine the utility of afucosylated bNAbs to enhance ADCC of multiple HIV-infected cell types by NK cells. Methods: A panel of seven bNAbs were generated in the presence or absence of a fructosyltransferase inhibitor (2F-Peracetyl-Fucose) and purified using protein-G chromatography. Opsonisation of cell-surface Env on primary CD4+ T cells and monocyte-derived macrophages (MDM) infected with HIV BaL in vitro was evaluated by immunolabeling. NK cell ADCC responses to HIV Env expressing targets in the presence of 5µg/mL of bNAbs was assessed via flow cytometry using a CD107a degranulation assay. Elimination of 8E5 cells was assessed as loss of HIV-expressing (p24+) cells after incubation with NK cells +/- unmodified and afucosylated bNAbs. Results: The bNAbs NIH45-46, VRC01, CH103, PGT151 and 35O22 were able to bind Env expressed on the surface of all HIV-expressing cells including macrophages. 8ANC195 had limited ability to opsonise HIV-infected cells, while PGT135 non-specifically opsonised HIV-uninfected MDM. Afucosylated bNAbs demonstrated a significant reduction in core fucosylation compared to unmodified bNAbs (range 31.6 to 38.6% reduction). Afucosylated bNAbs significantly increased ADCC responses from NK cells in response to CD4+ T cells (average 2.1 fold-increase, p<0.05 for all) and MDM (2.6-fold increase, p<0.02 for all) when compared to unmodified bNAbs. A combination of afucosylated NIH45-46, CH103 and 35O22 induced a significant increase in the proportion of non-viable HIV-expressing 8E5 target cells following incubation with either peripheral blood mononuclear cell (2.4-fold increase, p=0.002) or NK cell (1.7 fold increase, p=0.02) effectors. Conclusions: We have identified a panel of anti-Env bNAbs that opsonise HIV-infected CD4+ T cells and macrophages, and show proof of principle that afucosylation modifications of bNAbs can enhance NK cell mediated ADCC in vitro . Together, these findings support the use of anti-Env bNAbs with reduced core fucosylation as a promising immunotherapy tool to achieve ART-free HIV control. Immune Dynamics During Reproductive Aging and Substance Use in Women With HIV Konstantin Leskov 1 , Anna Agaponova 1 , Kathleen M. Weber 2 , Elizabeth Daubert 2 , Antoine Chaillon 3 , Magali Porrachia 3 , Jennifer Kinslow 4 , Eileen Scully 5 , Alan Landay 6 , Saba Valadkhan 1 , Alan Wells 3 , Mary Ann Checkley 1 , Benjamin Luttge 1 , Jonathan Karn 1 1 Case Western Reserve University, Cleveland, OH, USA, 2 Hektoen Institute of Medicine, Chicago, IL, USA, 3 University of California San Diego, La Jolla, CA, USA, 4 Rush University, Chicago, IL, USA, 5 The Johns Hopkins University School of Medicine, Baltimore, MD, USA, 6 University of Texas Medical Branch, Galveston, TX, USA Background: Estrogen depletion during reproductive aging in women with HIV (WWH) affects HIV reservoir burden and activity. Estrogen inhibits HIV transcription and modulates the immune system. Understanding the impact of hormonal changes on the immune system requires single-cell resolution to capture immune heterogeneity. Substance use disorder, prevalent among WWH, can further impact immune function. Methods: Plasma and PBMCs were evaluated across 3 reproductive stages (pre-, peri-, post-menopause) from 12 cisgender WWH in the Chicago Women’s Interagency HIV Study (WIHS), spanning 2009-2019. Participant’s average age at entry was 45 ± 5 years; 67% were Black and 58% reported substance use either close to the time of visit or in the past (e.g., crack/cocaine/ heroin). All participants had sustained viral suppression and CD4>200 cells/ µL. Reproductive stages were determined by sex hormones and self-report. Single-cell RNAseq and ABseq were performed using the BD Rhapsody platform to analyze CD4 and CD8 T-cells, monocytes, NK cells, DCs, B cells, and subpopulations. The inducible HIV RNA reservoir was measured by EDITS;

integration site sequencing (Illumina) assessed HIV DNA clonal expansion in six women across reproductive stages. Results: EDITS showed an 18% increase in the HIV RNA reservoir after menopause with evidence of increased clonally expanded HIV+ cells during perimenopause; these expanded clones persisted during postmenopause. Analysis of >900,000 single-cell transcriptome profiles identified a statistically significant ~5-fold enrichment in TNFα signaling via NFκB in premenopausal vs postmenopausal CD4 T-cells. In perimenopausal stages, IFNA and IFNG pathways were similarly enriched, indicating inflammatory responses. Ligand receptor analysis predicted increased IFNA/IFNAR activity in CD4 T-cells as well as HLA-D/CD4 interactions between DCs and CD4 T-cells during the menopausal transition. Reported substance use was associated with unique transcriptomic changes, including the overexpression of MTRNR2L1 and other humanin paralogs in myeloid and T-cells. These markers decreased after reported substance cessation. Conclusions: Reproductive aging in WWH shifts immune transcriptomes toward innate antiviral pathways, likely due to the rise in the inducible HIV reservoir and proviral clonal expansion. Substance use exacerbates immune dysregulation. The role of humanin paralogs in opioid response warrants further investigation regarding HIV persistence. Latency Reversal Induced by CD4+ T-Cell Recognition of HIV-1 Alternate Reading Frame Proteins Joel Sop, Tyler Beckey, Joel Blankson The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: CD8+ T cell responses to HIV-1 alternate reading frame proteins (ARFP) have been studied, but it is unclear whether CD4+ T cells can recognize these cryptic epitopes. We hypothesized CD4+ T cells in chronic progressors (CPs) would recognize ARFP peptides and that these peptides could be used to reverse latency. Methods: We performed a 10-day expansion assay using CD8+ T cell-depleted PBMCs from 10 CPs and 6 healthy donors (HDs), stimulating with peptide pools derived from ARFP sequences in Gag (19 peptides), Pol (7 peptides), and Env (3 peptides). Flow cytometry was used to measure ARFP-specific CD4+ T cell responses. A positive response was defined by a stimulation index (SI) greater than 3, calculated by comparing the proportion of CD4+ T cells producing TNF and IFN-gamma in the peptide-stimulated wells to that in the untreated wells. Single peptide stimulations were conducted for responders to identify the most immunogenic peptides and HLA typing was conducted to predict MHCII binding to these peptides. HIV RNA was measured in culture supernatant over a 10-day stimulation period. Results: Gag ARFP peptide stimulation in CPs resulted in significantly higher SI values compared to the untreated wells (P = 0.002, median SI = 3.9, range 1.0–16.0). In contrast, no significant difference was observed in HDs (P = 0.3125, median SI = 1.1, range 0.1–1.4). Among the 4 CPs who responded to Gag ARFP peptides, single peptide stimulations revealed variable immunogenicity with SIs that ranged from 3.3 to 43.0. One CP had a response to Env ARFP peptides with an SI of 10.3, and single peptide stimulation identified all 3 Env ARFP peptides as potential epitopes. HLA typing predicted strong MHCII binding to these peptides. A marked increase in HIV RNA was observed in the culture supernatant in 2 of the CPs stimulated with Gag ARFP peptides. Conclusions: Our results provide evidence that ARFP-specific CD4+ T cells can recognize cryptic epitopes in CPs, with Gag and Env ARFP peptides inducing significant responses. The marked increase in HIV RNA during ARFP stimulation suggests that these peptides may contribute to HIV latency reversal. These findings highlight ARFPs as potential targets for future HIV-1 cure strategies. Reduced Metabolic Flux Protects HIV-Expressing CD4+ T-Cells From CTL-Mediated Elimination Alberto Herrera, Louise Leyre, Tan Thinh Huynh, Feng Wang, Paul Zumbo, Maider Astorkia Amiama, Doron Betel, R. Brad Jones Weill Cornell Medicine, New York, NY, USA Background: HIV latency is a driver of persistence; however, cells expressing HIV RNA and proteins can still be detected after years of antiretroviral therapy (ART) and appear to drive ongoing stimulation of HIV-specific T-cells – indicating that some infected cells may be recognized by cytotoxic T lymphocytes (CTL) but are resistant to elimination. In this study, we use a primary in vitro infection model and cells from people living with HIV (PLWH) to evaluate mechanisms of resistance.

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CROI 2025 142