Phosphoproteomic landscaping of HIV-1 <i>in vitro</i> identified phosphorylated epitopes capable of eliciting CD8<SUP>+</SUP> cytotoxic T cell (CTL) responses in patient samples <i>ex vivo</i>

Abstract Significant progress has been made in recent years in our abilities to simultaneously detect proteome and posttranslational modifications (PTMs), including phosphorylation responsible for regulating protein functions. We have an incomplete understanding of the role of phosphorylation in host-pathogen interaction. The objective of this study was to fine-map phosphorylation of HIV-1 proteins and subsequently study the consequences of these PTMs. Using mass-spectrometry, we identified over 30 unique phosphorylated sites on HIV-1 proteins detected in infected primary CD4+ T cells and in cell-free virus, with the majority being present on the viral proteins Gag, Pol, and Rev proteins. Interestingly, many of these phosphorylated sites were within previously identified optimal CTL epitopes. While CTL responses to phospho-neoepitopes is subject to recent research in cancer immunology, we lack any understanding of how phosphorylation of virus affects antiviral immune responses. We stimulated PBMCs from HIV-1 infected patients ex vivo in the presence of HIV-1 epitopes with or without phosphorylation, followed by assessment of IFN-γ, TNF-α, MIP1β, and CD107a expressions by flowcytometry. Two Gag-derived phospho-epitopes were able to elicit immune responses in CTLs, potentially indicating the genesis and functional responses to such modified epitopes in vivo in HIV-1 infected patients. Moreover, CTL clones of HLA B*5701 background responded with significantly more cytotoxic marker without any effector cytokine expression, suggesting that phospho-epitopes can modulate the quality of CTL responses. Taken together, our studies shed light on a new way of evaluating host-pathogen interaction with potential for vaccine research.