ALKALI HYDROLYSIS OF RECOMBINANT PROTEINS ALLOWS FOR THE RAPID IDENTIFICATION OF CLASS-I MHC-RESTRICTED CTL EPITOPES

The characterization of the epitopes recognized by CTL provides insigh ts into the nature of protective immune responses and facilitates the development of methods to enhance immunity to human pathogens. However , no easily applicable approach for CTL epitope identification has bee n developed. We present a rapid and efficient method for locating CTL epitopes within a protein. The gene encoding the protein of interest i s inserted into an inducible prokaryotic expression vector. Random pep tides are then generated by alkali digestion of intact or lysed Escher ichia coli expressing the protein and assayed for the presence of the epitope by coating target cells for a standard CTL targeting assay. A large panel of clones containing serial 3'-deletions of the gene is th en generated by exonuclease III digestion, and the expressed truncated proteins are similarly analyzed for the presence of the antigenic pep tide. The epitope is located by determining the deletion points of clo nes expressing sequential truncations and differing in Ag expression. This technique was used to identify the H-2L(d)-restricted nonamer in E. coli beta-galactosidase, with residues 876-884 representing the nat urally processed epitope. To test the applicability of this method to other proteins, two genes from human CMV, an often fatal pathogen in i mmunocompromised individuals, were screened for HLA class I-restricted epitopes. An HLA-B18-restricted epitope from the CMV major immediate- early protein was found to lie between residues 378 and 389, and an HL A-B35-restricted epitope from the CMV pp65 matrix protein was characte rized as residues 123 to 131. The results demonstrate that this techni que can be used to rapidly identify CTL epitopes within a chosen prote in and should be useful for assaying viral isolates or neoplasms for l oss of epitopes after mutation and selection by host immune responses.