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Refractory HIV-Expressing CD4+ T Cells Retain Infectivity After NNRTI-Induced CARD8 Activation
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CROI 2026 Feb 22-25 Denver
A. Ochoa-Ortiz1, Q. Sun1, J. Wickramasinghe1, J. Ye1, C. Tomescu1, U. Zankharia1, C.J. Balibar2, B. Howell2, L.J. Montaner1
1. HIV Immunopathogenesis Laboratory, BEAT-HIV Delaney Collaboratory, Wistar Institute, Philadelphia, PA. 2. Merck & Co, Inc., Rahway, NJ.
Program Abstract
Background: NNRTI-based Targeted Activators of Cell Kill (TACK) selectively induces pyroptosis in HIV-expressing cells via premature viral protease activation and CARD8 activation raising its potential use in cure-directed strategies. TACK molecules such as PYR01 rapidly eliminate >95% of HIV-infected CD4+ T cells. However, whether refractory populations persist and retain infectivity remains unclear, and whether potentiation with the DPP9 inhibitor Val-boroPro (VbP) can further reduce these cells is unknown.
Methods: Primary CD4+ T cells were infected and treated after p24 frequency exceeded >20%. Cultures received 1 mM PYR01 or 2 uM Raltegravir (RAL) or 3 uM efavirenz (EFV) À} indinavir (IND) and À} Val-boroPro (VbP; DPP9 inhibitor). p24, viability, and subsets were quantified by flow cytometry; cell-to-cell infectivity of viable p24+ survivors after drug washout was tested. Bulk RNA-seq on PYR01 with/without RAL was compared against controls (RAL, untreated DMSO). Group comparisons for flow-cytometry endpoints were analyzed in GraphPad Prism using one-way ANOVA with appropriate multiplecomparison corrections across conditions. Differential expression was computed with DESeq2 pathway analysis, which used GSEA (MSigDB Hallmark/Reactome; FDR ≤ 0.05) and IPA for upstream regulators and canonical pathways.
Results: PYR01 reduced the p24+ fraction >90%; abrogated by the protease inhibitor IND, confirming the role for HIV protease as expected. EFV also reduced p24+ levels but to a lesser extent than PYR01. DPP9 inhibitor VbP increased clearance by left-shifting dose responses (greater potency) for both PYR01 and EFV, with a greater impact on EFV; however, the refractory p24+ plateau at saturating drug remained unchanged. Caspase 1 (CASP1) activation was linked to TACK ART but absent in residual p24+ cells. Co-culture of persistent p24+
cells with new target cells after TACK removal maintained cell-to-cell infectivity. Transcriptional analysis of persistent cells indicated retention of an survival and effector cytotoxic phenotype with lower CASP1 and CARD8 expression in the presence of TACK ART (Figure).
Conclusions: NNRTI TACK or EFV ART, alone or with DPP9 inhibition, markedly reduces HIV-infected CD4+ T cells expressing viral proteins, but residual populations persist and remain infectious. Cure strategies including NNRTI-induction of pyroptosis will require combination approaches that extend beyond CARD8-mediated activation to eliminate refractory reservoirs.
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