Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA All allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA All-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14-or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA All-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by All-restricted CTL clones at concentrations as low as 5 x 10-14 M. Epstein-Barr virus (EBV) is a widespread lymphotropic herpesvirus which causes infectious mononucleosis and is strongly linked to at least three lymphoid malignancies: endemic Burkitt's lymphoma, immunoblastic B-cell lymphomas of immunosuppressed patients, and a subset of Hodgkin's lymphomas (7-9). Primary EBV infection of immunocompetent hosts is usually asymptomatic and leads to the establishment of a life-long carrier state, whereby the virus persists within the B-cell compartment of healthy carriers (5). These infected B lymphocytes can proliferate in vitro, giving rise to lymphoblastoid cell lines (LCLs) which express at least eight latency-associated viral antigens: the nuclear antigens EBNA1 to -6 and the membrane proteins LMP1 and -2 (reviewed in reference 13). The recent demonstration that the immunoblastic lymphomas occurring in immunosuppressed individuals represent the in vivo outgrowth of EBV-positive LCL-like cells (6) emphasizes the role of immune surveillance in controlling this potentially lymphomagenic virus. EBV induces long-lasting cytotoxic T-lymphocyte (CTL) memory in the infected host. Thus, EBV-specific CTL precursors can be reactivated in relatively large numbers from the T-cell pool of EBV-seropositive donors by challenging in vitro with autologous virus-infected B cell
