High-throughput epitope screening of the Human Cytomegalovirus immediate early protein 2 identifies promising antigenic T cell targets

Human Cytomegalovirus (HCMV) is an omnipresent pathogen that is associated with increased morbidity and mortality of immunocompromised individuals. Studies of T-cell immunity to HCMV primarily reflect anti-CMV pp65 or immediate early antigen 1 (IE-1) activity. Recent evidence highlights the importance of the major immediate-early 2 (IE2) protein, which is expressed early after HCMV infection and reactivation, for regulating the lytic HCMV replication cycle. In this study, we designed a comprehensive screening approach to assess T cell responses against the IE2 HCMV protein in the peripheral blood of 15 HCMV-seropositive and 6 HCMV-seronegative healthy adults using IE2 synthetic long peptide (SLP) pools and cytokine flow cytometry. The T cell response against the IE2 protein was dominated by CD4+ T cells whereas IE2-specific CD8+ T-cell reactivity was measured in only 3 donors. Most of the donors recognized chiefly the IE2351-434 residues, revealing a remarkably immunogenic area of the protein. Numerous novel HLA class I- and II-restricted IE2 T-cell epitopes were identified. Functional characterization of the IE2 CD4+ and CD8+ T cell responses uncovered 5 highly antigenic SLPs, which induced polyfunctional Th1 cytokine (IFN-{gamma}+/ TNF+/ IL-2+) response and could serve as candidate vaccine antigens. Evaluation of these 5 highly antigenic IE2 SLPs in T cell-inducing vaccines aiming to inhibit HCMV infection by targeting the expression of immediate-early genes is warranted.