Abha Chopra

Purpose: Despite ART initiation in the acute phase of HIV infection (AHI), the viral reservoir persists. In the NOVA study, the viral reservoir declined between 24 and 156 weeks after ART initiation in individuals treated during AHI and correlated with the presence of HIV-specific CD8+ T-cell responses at 24 weeks. Now we further explore the effect of selective CD8+ T-cell pressure on viral integration sites.

Method: Two individuals participating in the Netherlands Cohort Study on Acute HIV Infection (NOVA study) (P1 and P2) were selected for an in-depth study (clinical characteristics shown in Fig. 1). HIV-specific CD8+ T-cell proliferative responses upon HIV peptide stimulation were determined by flow cytometry. Viral isolates were sequenced and analyzed for the presence of CD8+ T-cell epitopes using a HLA-I class binding prediction tool. HIV proviral structure and integration sites were characterized using a modified Integrated Proviral Sequencing Assay (IPSA).
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Results: In P1, strong, broad HIV-specific proliferative CD8+ T-cell responses were observed at 24 weeks, which corresponded with a substantial number of viral escape mutations in HLA-A*02:01:01, HLA-B*13:02:01, HLA-B*15:01:01 (Gag), HLA-B*13:02:01 and HLA-B*15:01:01 (Nef) restricted epitopes. In P2, low frequencies of proliferating CD8+ T-cells were observed at 24 weeks, which is reflected by a relatively low number of escape mutations at that time point. In P2 viral escape was mostly observed in HLA-B*40:01:02 (Gag) restricted epitopes and in HLA-A*01:01 and HLA-A*02:01:01 (Nef and Pol) restricted epitopes (Fig. 2). IPSA demonstrated a strong decrease in intact HIV DNA between 24 and 156 weeks in both participants. Interestingly, 65-77% of intact proviruses were located in a genic site, of which 33-66% in antisense region implying selection for transcriptionally latent proviruses.
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Conclusions: This study found that broad HIV-specific CD8+ T-cell responses drive viral escape mutations and associates with proviral loss and propensity of antisense (silent) integration.

Background: Co-trimoxazole (Co-T), an antibiotic used globally for bacterial infections and opportunistic infection prophylaxis is one of the most common causes of Stevens-Johnson syndrome/toxic epidermal necrolysis (SJS/TEN); a life-threatening CD8+ T cell-dependent HLA-class I-restricted blistering drug eruption. We aimed to elucidate the differential single-cell immunopathology of Co-T SJS/TEN in persons living with HIV (PLWH) and without HIV (PLWoH).

Method: We obtained blister fluid cells (BC) from three populations: 1) Co-T SJS/TEN PLWH, all of whom were co-infected with tuberculosis (TB) (n = 5); PLWoH (n = 6), or 3) burns patients as control (n = 6). BC were analysed by 5’ scRNA-TCR-CITE-seq with bioinformatic normalization (CellRanger v9.0), transcriptome-based clustering (Seurat v5), and TCRαβ (ClusTCR), differential (VGAS), and proportional analyses (scCODA). Affected skin from two PLWoH with paired BC were analysed by 10x Visium-HD spatial transcriptomics.

Results: Private oligoclonal TCRαβ in blister fluid across Co-T SJS/TEN patients with shared HLA-class I peptide binding specificities were expressed by the same two populations of cytotoxic (GNLY, GZMB, PRF1) and proliferative (STMN1, TUBA1B, H2AFZ) CD8+ T-cells enriched in SJS/TEN and in contact with spatially resolved keratinocytes at the epidermal junction. At late timepoints pathogenic CD8+ T-cells were reduced while M1-like (STAT1, HLA) phagocytes transitioned to M2-like (RNASE1, LIPA) signatures indicative of wound repair. In Co-T SJS/TEN PLWH compared to PLWoH, CD8+ T-cells expressed markers of inactivation (EEF1A1, BTG1, ZFP36L2) and reduced expression of cytotoxic mediators (GNLY, GZMB). PLWH were further discerned by CD1C dendritic cells differentially enriched for markers of activation (CD83, CALR, AIF1), monocytes and macrophages for markers of immune regulation (THBS1, TFGBI, VCAN, STAB1), and CD4+ T-cells for markers of cytotoxicity (GNLY, GZMA), phosphodiesterase-signalling (PDE3B, PDE7B), and immune dysregulation (TFGB1, CRIP1).

Conclusion: We identify putative pathogenic TCR to model shared drug- and HLA-restriction and cell signatures that discern early (cytotoxic) from late (repair) disease processes and a unique cellular sub phenotype of SJS/TEN in PLWH associated with immune dysregulation. These data provide avenues toward personalised treatment approaches for patients with earlier- or later-stage disease and sub phenotypes of SJS/TEN associated with infectious co-morbidities.