CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

Methods: CD4+ T-cells were infected with either a WT HIV JRSCF strain (targets) or a variant containing an escape mutation in the Gag-TW10 epitope (bystanders), and co-cultured with TW10-specific CTLs. Infected, HIV-expressing target cells that survived CTL attack (survivors) and bystander infected cells were profiled by ECCITE-seq after sorting based on HIV-Env expression. SCENITH metabolic profiling and DCFH-DA tagging by flow cytometry were used to measure metabolic flux and reactive oxygen species. Killing assays were also performed with pre-treatment of infected cells with the hypoxia-inducing, FDA-approved drug Deferoxamine (DFO) to induce oxidative stress. Memory CD4+ T-cells from PLWH on ART were sorted based on ROS levels and assayed by IPDA. Results: Survivors exhibited reduced expression of aerobic respiration and glycolysis gene-sets among the most differentially expressed pathways (Figure 1A). Markedly reduced bioenergetic capacity and glucose metabolism were confirmed in survivors by SCENITH profiling (Figure 1B). This reduced metabolic flux led to an enrichment of cells expressing lower levels of ROS (P=0.037). Pre treatment of infected cells with DFO enhanced susceptibilities of infected cells to elimination by CTL. IPDA analysis of memory CD4+ T-cells from PLWH sorted based on ROS levels revealed a trend towards increased levels of total and intact HIV DNA in ROS-lo populations (n=4, P = 0.071 and P = 0.078), and this is being extended to additional donors. Conclusions: Oxidative stress contributes to CTL-mediated killing. Our results identify a quiescent metabolic program with low production of ROS as a protective feature of infected, HIV-expressing cells to CTL attack. Treatment with DFO improves CTL-mediated elimination of infected cells. CD4+ T-cells with low oxidative stress are enriched for total and intact proviruses in PLWH on ART and may provide a sanctuary for HIV persistence. Targeting metabolic balance may be a strategy to enhance elimination of persistent HIV-infected cells. The figure, table, or graphic for this abstract has been removed. Engineering Natural Killer Cells With NKp30-Based Chimeric Antigen Receptors (CARs) to Target HIV Nancy Q. Zhao 1 , Ruoxi Pi 1 , Marion N. Santo 1 , David N. Nguyen 2 , Thanmayi Ranganath 1 , Christof Seiler 3 , Susan Holmes 1 , Alexander Marson 2 , Catherine Blish 1 1 Stanford University, Stanford, CA, USA, 2 University of California San Francisco, San Francisco, CA, USA, 3 Maastricht University, Maastricht, Netherlands Background: Natural killer (NK) cells respond rapidly in early HIV-1 infection. HIV-1 suppression and functional cure strategies can be developed by exploiting the intrinsic function of NK cells in recognizing HIV-infected cells. In our previous work, we profiled the phenotype of human primary NK cells responsive to autologous HIV-1-infected CD4 + T cells in vitro . We characterized the patterns of NK cell ligand expression on CD4 + T cells at baseline and after infection with a panel of transmitted/founder HIV-1 strains to identify key receptor-ligand pairings for HIV recognition by NK cells. With CRISPR editing in CD4 + T cells to knockout the NKp30 ligand B7-H6, or the NKG2D ligands MICB or ULBP2, we observed reduction in NK cell responses to HIV-infected cells in some donors. We hypothesized that the function of human primary NK cells of targeting HIV can be enhanced by overexpressing NKp30 and NKG2D and further improved by expressing CARs that are designed based on these two activating NK cell receptors. Methods: We transfected primary NK cells from peripheral blood of health donors with charge-altering releasable transporters (CARTs) to deliver mRNAs that encode unmodified NKp30 or NKG2D and a panel of CARs with the extracellular domain of NKp30. We also co-transfected NK cells with GFP mRNA to distinguish the cells that express the exogenous genes (GFP + ) from the ones that naturally express NKp30 and NKG2D (GFP - ). We evaluated the function of NK cells by co-culturing them with CD4 + T cells and detecting the degranulation marker CD107a and the production of cytokines IFNγ and TNFα in NK cells with flow cytometry and compared the function of GFP + and GFP - NK cells. Results: Overexpression of NKp30 or NKG2D in NK cells enhanced their function of targeting both HIV-infected cells and activated mock-infected autologous CD4 + T cells, but not resting CD4 + T cells, which do not express ligands of NKp30 and NKG2D. Multiple NKp30 CARs outperformed unmodified NKp30. Conclusions: Together with our previous work, the results of our study indicate that receptor-ligand pairs including NKp30:B7-H6 and NKG2D:MICB/ULBP2 facilitate NK cell recognition of HIV-infected cells. Human primary NK cells can be engineered with NKp30-based CARs for improved function of targeting HIV.

Our work suggests that NK cell activation through NKp30 and NKG2D can be exploited for designing strategies of cell therapy in conjunction with HIV Env targeting CARs to treat HIV infection. Preclinical Evaluation of Effector Function-Enhanced Variants of N6 bNAb David Wensel 1 , Hossam Omar 2 , Charles Mazzucco 1 , Shreya Pal 1 , Robert Ferris 3 , James Schawalder 3 , Sarah Hindson 2 , Erik Meyer 4 , Lewis Hanham 2 , Hangfei Qi 3 , Allan Tenorio 3 , Umesh Hanumegowda 1 , Richard Dunham 3 , Mark Krystal 1 1 ViiV Healthcare, Branford, CT, USA, 2 GSK, Stevenage, UK, 3 ViiV Healthcare, Durham, NC, USA, 4 GSK, Upper Providence, PA, USA Background: Effector functions of anti-HIV bnAbs may contribute to viral suppression as well as reservoir reduction via recruitment of immune cells and complement fixation to destroy HIV-infected cells. The contribution of native effector functions to the antiviral activity of N6LS has previously been explored (Wang, et al. PNAS. 2020. 117(30):18002-18009) and N6LS is currently in clinical trials (NCT05996471). We wished to determine if the effector functions of N6LS could be further enhanced through modification of the Fc region. Methods: Sixty-one variants of N6 bnAb were expressed and purified. For effector function enhancement, combinations of amino acid substitutions in the Fc region (including S239D, I332E, G236A, A330L in varied combinations; H268F/ S324T; F243L/R292P/Y300L/V305I/P396L) were examined, as were alterations to the N297 glycan. For half-life extension, the M428L/N434S (LS) and L309D/ Q311H/N434S (DHS) substitution sets were tested, as were alterations to the surface charge distribution of the light chain (R18E/R42E/K45E). These bnAbs were assessed for developability properties, antiviral activity, effector functions, immunogenicity risk, and other drug-like characteristics. Results: The R18E/R42E/K45E light chain substitutions generally resulted in poor expression and were not assessed further. The remaining variants all showed similar affinity for gp120 and direct antiviral activity in a pseudovirus assay. No functional difference between the LS and DHS half-life extension substitutions was observed. The S239D/I332E substitutions reduced thermal stability, which could be restored by the addition of T250V/A287F. SPR-based FcGR binding assessment allowed for the binning of variants by FcGR affinity profile; all showed enhanced affinity for the FcGR3a (7- to over 200-fold), which mediates ADCC activity of NK cells, but varied in their affinities for other FcGRs. ADCC assays indicated improved NK-mediated killing of gp160-expressing cells that roughly correlated with FcGR3a affinity improvement. The most potent variants showed both an increase in maximal cell killing capacity as well as ~100-fold greater potency relative to unmodified N6 bnAb. Conclusions: Multiple variants of the N6 bnAb displayed suitable developability properties and varying degrees of effector function enhancement in vitro. Four variants were identified for continued in vitro and in vivo assessment, including initiation of stable cell lines for potential manufacture. Long-Term ART Enhances Cytotoxic Response and Reduces the Reactivation Capacity of HIV-1 Reservoir Alicia Simón Rueda 1 , Guiomar Casado Fernández 1 , Elena Mateos 1 , Mario Manzanares 1 , Virginia Víctor 2 , Noemí Cabello-Clotet 2 , Javier Rodríguez Añover 2 , Reynaldo Homen 2 , Juncal Pérez Somarriba 2 , María José Núñez Orantos 2 , José Sanz 3 , Montserrat Torres 1 , Vicente Estrada 2 , Mayte Coiras 1 , Clara Sánchez Menéndez 1 1 Instituto de Salud Carlos III, Madrid, Spain, 2 Hospital Clinico San Carlos, Madrid, Spain, 3 Hospital Universitario Príncipe de Asturias, Madrid, Spain Background: Long-term antiretroviral therapy (ART) in people with HIV (PWH) has been related to reduced levels of competent proviruses. We aimed to evaluate how time on ART affects reservoir size, reactivation capacity and cytotoxic response. Methods: Cross-sectional study with 64 PWH who were on ART for <1 year (<1y)(n=11), 1 to 10 years (1-10y)(n=20), 10 to 20 years (10-20y)(n=16), and >20 years (>20y)(n=17). HIV-1 reservoir size was quantified by ddPCR in peripheral blood mononuclear cells (PBMCs). Proviral reactivation was analyzed by flow cytometry in purified CD4+ T cells using anti-p24-gag. Immune exhaustion markers, phenotype of cytotoxic cells, and CD4 memory subpopulations were also determined. Direct cell cytotoxicity (DCC) was assessed by measuring caspase-3 levels in TZM-bl cells infected with HIV-1 after co-culturing with participants' PBMCs (1:2). Results: 1) Most participants were male (83%, IQR 77-87). Median age was 30(IQR 24.5-35.5), 42.5(IQR 37.8-49.0), 49.5(IQR 45.8-54.3), and 56(IQR 54-60)

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CROI 2025 143