Background
CD8+ T cell responses to HIV-1 alternate reading frame proteins (ARFP) have been studied, but it is unclear whether CD4+ T cells can recognize these cryptic epitopes. We hypothesized CD4+ T cells in chronic progressors (CPs) would recognize ARFP peptides and that these peptides could be used to reverse latency.
Methods
We performed a 10-day expansion assay using CD8+ T cell-depleted PBMCs from 10 CPs and 6 healthy donors (HDs), stimulating with peptide pools derived from ARFP sequences in Gag (19 peptides), Pol (7 peptides), and Env (3 peptides). Flow cytometry was used to measure ARFP-specific CD4+ T cell responses. A positive response was defined by a stimulation index (SI) greater than 3, calculated by comparing the proportion of CD4+ T cells producing TNF and IFN-gamma in the peptide-stimulated wells to that in the untreated wells. Single peptide stimulations were conducted for responders to identify the most immunogenic peptides and HLA typing was conducted to predict MHCII binding to these peptides. HIV RNA was measured in culture supernatant over a 10-day stimulation period.
Results
Gag ARFP peptide stimulation in CPs resulted in significantly higher SI values compared to the untreated wells (P = 0.002, median SI = 3.9, range 1.0–16.0). In contrast, no significant difference was observed in HDs (P = 0.3125, median SI = 1.1, range 0.1–1.4). Among the 4 CPs who responded to Gag ARFP peptides, single peptide stimulations revealed variable immunogenicity with SIs that ranged from 3.3 to 43.0. One CP had a response to Env ARFP peptides with an SI of 10.3, and single peptide stimulation identified all 3 Env ARFP peptides as potential epitopes. HLA typing predicted strong MHCII binding to these peptides. A marked increase in HIV RNA was observed in the culture supernatant in 2 of the CPs stimulated with Gag ARFP peptides.
Conclusions
Our results provide evidence that ARFP-specific CD4+ T cells can recognize cryptic epitopes in CPs, with Gag and Env ARFP peptides inducing significant responses. The marked increase in HIV RNA during ARFP stimulation suggests that these peptides may contribute to HIV latency reversal. These findings highlight ARFPs as potential targets for future HIV-1 cure strategies.
Table or Figure
