A small fraction of HIV-infected patients known as long-term non-progressors (LTNPs) maintain very low levels of plasma HIV without antiretroviral therapy. The majority of these possess HLA alleles such as HLA-B*57:01 that are thought to offer superior T-cell mediated immunity where intracellular pathogen-derived peptides are presented for scrutiny to CD8+ T cells leading to eradication of infected cells. Here we discuss the identification and detailed characterization of HLA-B*57:01-restricted post-translationally modified (PTM) peptides, specifically focusing on a kynurenine-modified peptide due to its novelty.
Cells expressing HIV envelope (env) were transfected with HLA-B*57:01 followed by immunoaffinity purification of HLA class I molecules. HLA-bound peptides were fractionated and analysed using SCIEX 5600+ TripleTOF. HIV peptides were validated by matching the MSMS and RT to synthetic peptides. HLA-B57 in complex with native and PTM peptides were crystallised to study structural properties followed by biochemical assays to study formation of peptidyl-kynurenine.
Here we report on the identification of over 8700 naturally presented class I HLA-B*57:01-bound peptides including seven derived from HIV-env. Both native and PTM forms of two key peptides were identified. Notably, we identified a kynurenine modified HIV peptide and show using biochemical assays that this can be recapitulated enzymatically from native peptide. We study the binding of such HLA-B*57:01-restricted peptides at a structural level and examine their immunogenicity in preliminary functional studies in HLA-B*57:01+ HIV-infected and HIV-naïve humans.
In summary, we have identified several novel HIV-env-derived peptides including native and PTM forms. Our study demonstrates the potential for kynurenine containing epitopes to be recognized by T-cells, leading the way for other researchers to look for responses against such modified epitopes in their HIV cohorts as well as from other conditions where inflammation may drive the incorporation of kynurenine into other viral, tumour or autoantigenic epitopes.