Identification of pp65-derived CTL epitopes for which a computer algorithm was not applicable. To narrow down the region containing the epitope, the linear expression fragments encoding further truncated forms of the deletion mutant, Δpp65(1-310) for HLA-Cw*1202 or Δpp65(1-210) for HLA-Cw*0801 and -Cw*1502, were generated and transfected into 293T cells together with restricting HLA cDNA. (A) Recognition by the pp65-specific CTL lines was evaluated 48 hours later by ELISPOT assay. (B) Amino acid sequence of pp65 around the defined region. (C) Based on the results shown in the upper panels, various linear expression fragments within the region were generated, transfected into 293T cells together with restricting HLA cDNA, and then tested for the recognition by corresponding pp65-specific CTL line using ELISPOT assay. Each bar represents the number of spots per 10⁴ cells.