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-γ (IFN-γ) 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-γ. When PBMCs were stimulated with each of the twenty peptides belonging to mini-pools 1 and 2, peptides IE-1_1-15MESSAKRKMDPDNPD and IE-1_{5- 19}AKRKMDPDNPDEGPS induced the greatest quantities of IFN-γ 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_3-12SSAKRKMDPD induced the greatest quantities of IFN-γ production and target cell killing by CD8+ CTLs. Conclusion: HCMV IE-1_3-12SSAKRKMDPD is a HLA-A 2402-restricted HCMV IE-1 epitope that can serve as a common target for CD8+ HCMV-specific CTLs.