HCMV deletion models enable the identification of physiologically relevant T-cell epitopes

In healthy individuals, immune control of persistent human cytomegalovirus (HCMV) infection is effectively mediated by virus-specific CD4+ and CD8+ T cells. However, identification of the repertoire of T-cell specificities for HCMV is hampered by the immense protein coding capacity of this betaherpesvirus. We applied a novel approach which employs HCMV deletion mutant viruses, lacking HLA class I immunoevasins. Infection of human fibroblast cell lines with those mutant viruses allowed the direct identification of naturally presented HCMV-derived HLA ligands by mass spectrometry. Using this strategy, we identified 368 unique HCMV-derived HLA class I ligands representing an unexpectedly broad panel of 123 HCMV antigens. Functional characterization revealed memory T-cell responses in seropositive individuals for a substantial proportion (28%) of these novel peptides. The unbiased identification of naturally presented viral epitopes enabled a comprehensive and systematic assessment of the physiological repertoire of anti-HCMV T-cell specificities in seropositive individuals. This approach proved to be superior to procedures applying in silico analysis to identify true viral antigens.

健康个体体内，持续性人类巨细胞病毒（human cytomegalovirus, HCMV）感染的免疫控制，可由病毒特异性CD4+及CD8+ T细胞有效介导。然而，该β疱疹病毒（betaherpesvirus）拥有庞大的蛋白编码能力，使得精准鉴定人巨细胞病毒特异性T细胞识别谱的工作始终受阻。本研究采用一种全新策略，使用缺失HLA I类免疫逃逸蛋白（immunoevasins）的人巨细胞病毒缺失突变株。将该突变株感染人成纤维细胞系后，可通过质谱技术直接鉴定出天然呈递的人巨细胞病毒源性HLA配体。借助该策略，本研究共鉴定出368种独特的人巨细胞病毒源性HLA I类配体，对应覆盖123种人巨细胞病毒抗原，其覆盖范围之广超出预期。功能表征实验结果显示，在血清学阳性个体中，有相当比例（28%）的上述新型肽段可触发记忆性T细胞应答。对天然呈递的病毒表位进行无偏倚鉴定，使得我们能够全面、系统地评估血清学阳性个体体内抗人巨细胞病毒T细胞识别特异性的生理谱库。该策略的效果优于通过计算机模拟分析鉴定真实病毒抗原的传统实验流程。