Ma. Gavin et al., ALKALI HYDROLYSIS OF RECOMBINANT PROTEINS ALLOWS FOR THE RAPID IDENTIFICATION OF CLASS-I MHC-RESTRICTED CTL EPITOPES, The Journal of immunology, 151(8), 1993, pp. 3971-3980
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.