Identification and profiling of HLA-A*02:01-restricted Toxoplasma gondii peptides through immunopeptidomics in HLA-A2.1 transgenic mice

HLA class I presentation of pathogen-derived peptides is essential for CD8+ T-cell recognition of Toxoplasma gondii. While in vitro MHC ligands have been documented, the in vivo epitope landscape during infection progression remains poorly characterized. Here, we employed an MS-based immunopeptidomics approach to directly profile the T. gondii immunopeptidome presented by HLA-A*02:01 in transgenic mice. Our analysis identified a comprehensive repertoire of 3,744 MHC-I ligands originating from diverse parasite proteins, including a substantial proportion of previously uncharacterized hypothetical proteins. Notably, specific ligands were consistently detected across both acute and chronic stages, suggesting that conserved functional domains may drive MHC-I epitope selection throughout the infection cycle. By integrating in silico predictions with experimental validation, we identified 72 high-affinity candidates, five of which exhibited robust binding capacity in vitro and in vivo. Specifically, we identified glycogen synthase as a novel dominant antigen, yielding a high-affinity ligand (designated PGS) with a favorable HLA-A*02:01 binding architecture. These findings significantly expand the known HLA-A*02:01-restricted antigen landscape of T. gondii and provide critical molecular targets for the rational design of next-generation vaccines against both acute and chronic toxoplasmosis.