CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

Results: We detected cytotoxic activity of T and NK cells engineered to express VRC01 and PGT121 CARs against CEM.NKR cells that express Luciferase and HIV-1 AD8ΔCT Envs as initial targets. HIV-1 C-CT of the NL43(AD8) molecular clone, which contains all HIV-1 genes, exhibits significant resistance to bnAbs but could be efficiently blocked by ADCC. Moreover, CAR PBMCs exhibited increased specific killing efficiency across various effector: target cell ratios, particularly after 4-hour incubation. Among the CARs tested, the PGT121 bnAb-based CAR consistently outperformed others in mediating cellular cytotoxicity. Conclusions: These results provide proof of the principle that targeting HIV-1 cell-to-cell transmission through ADCC and CAR NK/T cell therapies can overcome the limitations of bnAbs. This study offers the first evidence to test these approaches to effectively target HIV-1 C-CT, highlighting their potential in studying HIV-1 treatment strategies. Natural Killer Cells Preferentially Kill HIV-Infected CD4 T Cells Using Death Receptors Lesley R. de Armas 1 , Akshay Iyer 1 , Suresh Pallikkuth 1 , Rajendra Pahwa 1 , Paula Vaz 2 , Maria G. Lain 2 , Savita Pahwa 1 1 University of Miami, Miami, FL, USA, 2 Fundação Ariel Glaser Contra o SIDA Pediátrico, Maputo, Mozambique Background: Natural killer (NK) cells provide immune defense against virus infection via surveillance and recognition of virally infected cells. In people living with HIV (PWH) NK cell function has been associated with viral control and is being targeted for immunotherapeutic strategies. Methods: To determine whether NK function is impaired in PWH, in vitro NK functional assays were performed in longitudinal samples from HIV-Exposed Infected (HEI, n=17) and Uninfected (HEU, (n=9) children from Mozambique. NK were enriched from PBMC and co-cultured with CellTrace™ labeled K562, HIV infected HUT78/SF2 (HUT) and NK-resistant CEM.NKR (CEM) cell lines for 6 hours at 4:1 ratio. Death of target cells (viability stain), cytolytic proteins (CD107a, Perforin, GranzymeB, FasL) and cytokine secretion (IFNg, TNF, MIP1b) were assessed at pre-ART (1-2m) and post-ART (12m) by flow cytometry. Expression of activating and inhibitory receptors were evaluated on NK before and after co-culture (NKG2A/C/D, NKp30/46, KLRG1, TIGIT). Adults with and without HIV (n=10 each) at a single timepoint were assessed for comparison. Mixed-effects modelling was used to determine predictors of NK killing. Results: Overall, NK exhibited increased killing of K562 and HUT targets compared to CEM but the magnitudes were not different when comparing HEI and HEU. Paired analysis showed that HEU NK increased killing from 1m to 12m whereas HEI stayed the same or decreased. At 12m only, plasma virus load (VL) had a negative linear relationship with HUT killing (R 2 =0.42, p=0.016) and this was not observed with K562. Expression of the inhibitory receptor, KLRG1 on NK prior to co-culture was associated with reduced killing of K562 and HUT. After co-culture, the expression of CD107a on NK, a marker of degranulation, was highly associated with K562 target cell death but showed no relationship with HUT killing. Meanwhile, expression of the death receptor, FasL, which was present on CD56dimCD16+ NK at 5-30% was positively associated with HUT killing but showed no relationship with K562 killing. Staining of target cells showed that HUT expressed higher levels of surface Fas receptor than K562 or CEM. FasL on NK cells from adults also had a positive association with HUT killing confirming this was not a unique feature of pediatric NK cells. Conclusions: These results suggest that chronic HIV leads to impaired NK cytolytic function against HIV infected CD4 T cells and point to a role for Fas/FasL induced cell death in viral control mediated by NK cells. Tri-Specific Killer Engagers in Macaques Potently Induce NK Cell Responses in Blood and Tissues Julien A. Clain 1 , Ross T. Cromarty 2 , Kevin Nguyen 3 , Hannah Flores 3 , Yvette Soignier 2 , Tumpa Dasgupta 2 , Emma Eungard 2 , Zachary Davis 2 , Timothy Schacker 2 , Justin Harper 3 , Mirko Paiardini 1 , Jeffrey Miller 2 1 Emory University, Atlanta, GA, USA, 2 University of Minnesota, Minneapolis, MN, USA, 3 Emory National Primate Research Center, Atlanta, GA, USA Background: Strategies to enhance immune responses towards HIV-infected cells are needed for eliminating HIV reservoirs. Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) plays a key role in HIV protection. In cancer, Tri-specific Killer Engagers (TriKEs), consisting of a CD16 binding domain, an antigen-targeting domain, and an IL-15 moiety, enhance NK cell-mediated ADCC by forming immune synapses through CD16 and antigen engagement. Building on this strategy, we developed a gp120-targeting TriKE

to activate NK cells toward targeting HIV-infected cells. A version specific for rhesus macaques (RMs) has been generated to conduct an in vivo dose-response study to evaluate TriKE safety and impact on NK cell responses. Methods: TriKE was administered subcutaneously 5 times per week for 3 weeks in 3 uninfected RMs, with doses from 100 to 300 µg/kg (Figure 1). Complete blood counts and blood chemistry tests were performed; whole blood and lymph nodes (LNs) were collected longitudinally for immunophenotyping. Results: TriKE treatment, even at the higher dose, was well-tolerated; blood biochemistry parameters remained normal throughout the study, with slight and transient increases in transaminases (AST/ALT) and creatine kinase levels following the first administration of each cycle. TriKe treatment significantly increases NK cell frequencies in blood and LNs. Specifically, CD16+CD56- and CD16+CD56+ NK cell subsets peaked in LNs on day 11, coinciding with the final administration in week 2. NK cell expansion was accompanied by increased NK cell activation (HLA-DR+CD69+) and proliferation (Ki-67+). Following the third week’s dosing (300 µg/kg, day 14), the frequency of NK cells and their activation and proliferation levels decreased. Interestingly, PD-1 expression significantly increased on day 4 (end of the first week’s dosing) in both blood and LNs, normalizing by day 11; this may reflect an immunological response to prevent excessive NK cell activation. Conclusions: Our study demonstrates, for the first time, that the gp120 targeting TriKE safely and potently engages NK cell responses in RMs, with their substantial activation, proliferation, and expansion in blood and lymphoid tissues. Furthermore, blocking PD-1 upregulation could synergize with TriKE treatment to enhance TriKE-mediated NK cell function. These findings provide strong rationale to further characterize TriKE-based therapies as a strategy for targeting HIV/SIV reservoirs, including in lymphoid tissues. Extracellular Acyl-CoA-Binding Protein Prevents Autophagy and T-Cell Function in People With HIV Stephane Isnard 1 , Nazanin Gharari 2 , Léna Royston 1 , Tsoarello Mabanga 1 , Carolina A. Berini 1 , Julien van Grevenynghe 2 , Guido Kroemer 3 , Jean-Pierre Routy 1 1 McGill University Health Centre Research Institute, Montreal, Canada, 2 Centre Armand-Frappier Santé-Biotechnologie, Laval, QC, Canada, 3 University of Paris, Paris, France Background: Through the degradation of cytosolic structures and proteins, autophagy generates macromolecules for energy production which allows efficient anti-HIV T-cell responses. Extracellular Acyl-CoA-Binding Protein (ACBP) inhibits autophagy, tricarboxyclic acid (TCA) cycle and oxidative phosphorylation in mouse models, mainly through binding to the GABAA receptor. Herein, we assessed the levels of circulating ACBP and its influence on T-cell function in people living with HIV (PLWH) on antiretroviral therapy (ART) and tested whether inhibition of ACBP-receptor binding could be used in preclinical models to improve the immune function in PLWH on ART. Methods: Plasma ACBP and cytokines were quantified by ELISA in 50 PLWH on effective ART (mean duration of 14.7 years) and 30 controls with similar age. In vitro , recombinant ACBP (recACBP) was added at increasing concentrations up to 10 µ g/mL on PBMCs from PLWH on ART and controls, T-cell responses and autophagy levels were assessed by flow cytometry. A synthetic GABAAreceptorg2 chain peptide was used to prevent binding of ACBP to this receptor, as described by Dr Kroemer’s group. Results: ACBP levels were higher in PLWH on ART compared to controls (median 127.5 vs 78.1 ng/mL, p=0.03), independently of age and sex. In ART-treated PLWH, plasma ACBP levels correlated with levels of pro inflammatory Th1 and Th2 cytokines, angiogenesis and innate inflammation. Conversely, ACBP levels were inversely associated with plasma IL21 levels (r=-0.54, p<0.01). ACBP levels were associated with secretion of energy production intermediary macromolecules glutamate and α-ketoglutarate.

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