ABSTRACT
Three variants of the cholera toxin B subunit (CTB) were generated by site-specific mutagenesis in which regions of the mature protein were altered to the composition found at the corresponding positions of the closely related B subunit of the heat-labile enterotoxin of enterotoxigenic Escherichia coli (LTB). The mutant proteins were expressed in Vibrio cholerae and purified from the growth medium. In the first of the mutant proteins, the first 25 amino acids corresponded to the sequence found in LTB, and in the second, changes were made at positions 94 and 95 of the mature protein. The third mutant protein combined the changes made in the first two. Analysis of the immunological properties of these novel proteins by using monoclonal antibodies and absorbed polyclonal antiserum demonstrated that they had acquired LTB-specific epitopes. Immunizations with the mutant proteins resulted in antisera containing LTB-specific as well as CTB-specific and cross-reactive antibodies. The sera were also found to be more strongly cross-reactive in the in vitro neutralization of both cholera toxin and heat-labile enterotoxin than were antisera raised against either CTB or LTB. The results suggest that such hybrid CTB-LTB proteins may be useful in a broad-spectrum vaccine against enterotoxin-induced diarrhea.
