Identification of HLA-A*2402-restricted HCMV immediate early-1 (IE-1) epitopes as targets for CD8+HCMV-specific cytotoxic T lymphocytes

Background: To identify novel HLA-A*2402-restricted human cytomegalovirus ( HCMV) immediate early-1 (IE-1) epitopes for adoptive immunotherapy, we explored 120 overlapping 15-amino acid spanning IE-1. Methods: These peptides were screened by measuring the frequency of polyclonal CD8+ T cells producing intracellular interferon-gamma (IFN-gamma) using flow cytometry and the epitopes were validated with a HCMV-infected target Cr release cytotoxicity assay. Results: Initial screening was performed with 12 mini-pools of 10 consecutive peptides made from 120 overlapping peptides15-amino acids in length that spanned IE-1. When peripheral blood mononuclear cells (PBMCs) from HLA-A*2402 HCMV-seropositive donors were sensitized with each of the 12 mini-pools, mini-pools 1 and 2 induced the highest frequency of CD8+ cytotoxic T lymphocytes (CTLs) producing IFN-gamma. When PBMCs were stimulated with each of the twenty peptides belonging to mini-pools 1 and 2, peptides IE-1(1-15)MESSAKRKMDPDNPD and IE-1(5-19)AKRKMDPDNPDEGPS induced the greatest quantities of IFN-gamma production and cytotoxicity of HLA-matched HCMV-infected fibroblasts. To determine the exact HLA-A*2402- restricted epitopes within the two IE-1 proteins, we synthesized a total of twenty-one overlapping 9- or 10 amino acid peptides spanning IE-1(1-15) and IE-1(5-19). Peptide IE-1(3-12)SSAKRKMDPD induced the greatest quantities of IFN-gamma production and target cell killing by CD8+ CTLs. Conclusion: HCMV IE-1(3-12)SSAKRKMDPD is a HLA-A*2402- restricted HCMV IE-1 epitope that can serve as a common target for CD8+ HCMV-specific CTLs.