Background
HIV-1 persists despite effective ART due to a reservoir of latently infected CD4+ T cells. Previous studies suggest that intact proviruses integrated in heterochromatic regions are progressively enriched during long-term ART. These proviruses may evade immune recognition due to lower viral gene expression, but their clinical significance upon ART interruption is unclear. Given the recent findings that cognate antigen engagement increases HIV-1 RNA expression, we sought to determine if antigen-specific stimulation ex vivo could induce the viral outgrowth of proviruses in regions associated with deeper latency.
Methods
We isolated CD4+ T cells responsive to a protein lysate of the commensal fungus Candida albicans (CA) based on activation-induced markers (CD69, CD154, CD137). HIV-1 populations in CA-reactive cells were analyzed by IPDA, matched integration and proviral sequencing, and integrant-specific digital PCR. We then optimized an antigen-specific quantitative viral outgrowth assay (QVOA) by stimulating CD8-depleted PBMCs with CA lysate for 3 days, followed by co-culture with MOLT-4s for 21 days. To limit non-specific T cell activation, we used a basal culture medium (10% human serum, 10 U/mL IL-2) and omitted irradiated feeders. Phytohemagglutinin (PHA) combined with T-cell activation media was used as a non-specific positive control. Viral outgrowth was monitored via p24 ELISA assay, digital PCR, and full env sequencing.
Results
We identified a study participant on suppressive ART whose CA-reactive cells were highly enriched for intact HIV-1 DNA. A clonally expanded provirus representing 50% of env proviral sequences was confirmed to be genetically intact by full genome sequencing. Integration site analysis showed this provirus was located in a pericentromeric satellite repeat, previously linked to deeper latency. The provirus of interest (PeriC) had a frequency of 930 copies per million CA-responsive cells. In our modified QVOA, we observed exponential outgrowth from multiple wells, with 100% of the outgrowth sequences from the CA lysate condition matching PeriC, compared to 33% in the PHA arm.
Conclusions
Cognate antigen stimulation resulted in the reactivation of a provirus integrated in a pericentromeric region. Although limited to a model culture ex vivo, these findings suggest that CD4+ T cell recognition with ubiquitous antigens can reactivate proviruses in regions previously associated with deeper latency, potentially contributing to viral rebound if ART is discontinued.
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