Identification of novel canonical and cryptic HCMV-specific T-cell epitopes for HLA-A*03 and HLA-B*15 via peptide-PRISM.

Human cytomegalovirus (HCMV) reactivation poses a substantial risk to transplant patients. Effective risk stratification and vaccine development is hampered by a lack of HCMV-derived immunogenic peptides in patients with common HLA-A*03:01 and HLA-B*15:01 haplotypes. This study aimed to discover novel HCMV immunogenic peptides for these haplotypes by combining Ribo-seq and mass spectrometry with state-of-the-art computational tools, Peptide-PRISM and PRICE. Furthermore, employing machine learning, an algorithm was developed to predict immunogenicity based on translational activity, binding affinity and peptide localization within small open reading frames to identify the most promising peptides for in vitro validation. Immunogenicity of these peptides was subsequently tested by analyzing peptide-specific T-cell responses of HCMV-seropositive and -seronegative healthy donors as well as transplant patients. This resulted in the direct identification of three canonical and one cryptic HLA-A*03-restricted immunogenic peptides as well as five canonical and one cryptic HLA-B*15-restricted immunogenic peptide, with a specific IFNγ+/CD8+ T-cell response of ≥ 0.02%. High T-cell responses were detected against two HLA A*03-restricted and three HLA-B*15-restricted canonical peptides with frequencies of up to 8.77% IFNγ+/CD8+ T cells in patients post alloSCT. Therefore, our comprehensive strategy establishes a framework for efficient identification of novel immunogenic peptides from both existing and novel Ribo-seq datasets.