Ultra-low level HIV p24 drives immune activation in antiretroviral therapy-treated people living with HIV

BACKGROUND: Despite effective antiretroviral therapy (ART), people living with HIV (PLWH) often exhibit persistent immune activation, the mechanisms of which remain unclear. Increasing evidence suggests that residual low-level viremia and ongoing viral protein expression may persist even under long-term suppressive ART, underscoring the need for a better understanding of residual HIV persistence. METHODS: We therefore optimized a digital single-molecule array (Simoa®) technology to detect ultra-low levels of HIV p24 antigen in plasma, achieving femtogram sensitivity. In addition, we used flow cytometry to analyze HIV-specific T cell responses. RESULTS: Here we show that in a cohort of 108 participants with chronic HIV on long-term ART with HIV-1 RNA < 30 copies/mL for >4 years, p24 is detectable (17 - 370 fg/mL) in 42. Dual protease inhibitor therapy is associated with significantly lower p24 levels (p < 0.05), while age, ART duration or CD4/CD8 ratio show no effect. Monitoring 41 individuals who initiated ART during acute HIV, p24 remains detectable in 20% after two years. Although p24 correlates with viral RNA early in ART (r = 0.83, p < 0.0001), this association is lost after two months (r = 0.20, p = 0.21). Importantly, p24+ individuals show significantly higher frequencies of PD-1 + , CD38 + , and CD38 + HLA-DR + CD8 T cells (p < 0.01; p < 0.05), alongside enhanced TNF-α and CD107a responses to HIV Gag (p < 0.01; p < 0.05). CONCLUSIONS: To best of our knowledge, our findings provide the first large-cohort evidence of low-level p24 persistence during suppressive ART and suggest that ongoing p24 production may contribute to residual immune activation in treated PLWH.