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Reactivation of Human Cytomegalovirus (HCMV) and Human Adenovirus (HAdV) in immunocompromised patients following stem cell transplantation (SCT) or solid organ transplantation (SOT) is associated with high morbidity and mortality. The adoptive transfer of virus-specific CD8 +  and CD4 +  T cells has been shown to re-establish the antiviral T-cell response and improve clinical outcome. The viral load in immunocompromised patients can efficiently be reduced solely by the infusion of virus-specific CD4 +  T cells. The identification of CD4 +  T-cell epitopes has mainly focused on a limited number of viral proteins that were characterized as immunodominant. Here, we used  in silico  prediction to determine promiscuous CD4 +  T-cell epitopes from the entire proteomes of HCMV and HAdV. Immunogenicity testing with enzyme-linked immuno spot (ELISpot) assays and intracellular cytokine staining (ICS) revealed numerous novel CD4 +  T-cell epitopes derived from a broad spectrum of viral antigens. We identified 17 novel HCMV-derived and seven novel HAdV-derived CD4 +  T-cell epitopes that were recognized by > 50% of the assessed peripheral blood mononuclear cell (PBMC) samples. The newly identified epitopes were pooled with previously published, retested epitopes to stimulate virus-specific memory T cells in PBMCs from numerous randomly selected blood donors. Our peptide pools induced strong IFNγ secretion in 46 out of 48 (HCMV) and 31 out of 31 (HAdV) PBMC cultures. In conclusion, we applied an efficient method to screen large viral proteomes for promiscuous CD4 +  T-cell epitopes to improve the detection and isolation of virus-specific T cells in a clinical setting.

Broad and Efficient Activation of Memory CD4+ T Cells by Novel HAdV- and HCMV-Derived Peptide Pools