Kinetics of HLA antigen presentation in transition from latent to Lytic stage of EBV

Epstein-Barr Virus (EBV) is a gamma herpes virus associated with esophageal cancer, nasopharyngeal cancer and lymphomas. In the latent stage, EBV lies dormant in epithelial and B-cells with eight viral anti-apoptotic and anti-inflammatory proteins lowly expressed . Thus, few latent proteins are presented as peptides by the human leukocyte antigen (HLA). However, in the lytic stage, the cells massively produce virion particles. To our knowledge, there hasn"t been an effort to map out HLA-bound peptides presented in host cells in the various stages of lytic mode. By inducing virus production using 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate (NaBu) in six EBV-positive B-cell lines, we analyzed their antigenic landscape at eight different time points after induction, using sequential HLA-I and HLA-II high-throughput immunopeptidomics aided by the Agilent Assaymap™ automated liquid handling platform. HLA bound peptides were measured on an Eclipse™ mass spectrometer in hybrid DDA/DIA using 1-hour non-linear gradient resulting in more than 30,000 HLA-I and 15,000 HLA-II peptides presented at various stages in virus activation per cell line. We later combined the HLA-I and HLA-II data with transcriptomics and quantitative shotgun proteomics to create the first map of the lytic EBV immunopeptidome from transcript to protein to antigen. Tens of HLA-I peptides derived from key EBV-proteins were detected throughout viral activation. Among others, peptides from lytic proteins such as BCRF1 and HEPA were identified. These assays will shed light on antigen biogenesis in induced cells and unveil novel EBV peptides for development of therapeutic cancer vaccines and T cell-based immunotherapies.