Epitope analysis of the CS3 fimbrial subunit of human enterotoxigenic Escherichia coli and the construction of novel CS3::ST and CS3::LT-B immunogens

Enterotoxigenic E. coli (ETEC) are the major cause of traveler's diarrhoea and the CS3 fimbriae/fibrillae are expressed by most strains bearing the colonization factor CFA/II. The cstAH gene cluster determining CS3 biosynthesis has been previously cloned and sequenced and it has been shown that cstH encodes the major fimbrial subunit and cstA-G encode an assembly cassette. In the work described here we have sought to define the surface exposed domains on CS3 and to manipulate them so that CS3 can be used as a means of expressing foreign antigenic determinants on the bacterial surface. Using a panel of 21 monoclonal antibodies, which we have used in western blotting, immunofluorescence microscopy and colony blotting, together with computer predictions, we have identified three domains within CstH. Two of these sites were permissive for insertion and we have introduced, in-frame, either an epitope from the B subunit of LT (heat labile toxin) or the entire coding sequence of mature ST (heat stable toxin) to construct hybrid proteins. These proteins could be assembled into hybrid fimbriae which could be recognized by antibodies to both CS3 and the foreign epitope as shown by immunofluorescence microscopy and colony blotting. The immunogenicity of the constructs has been evaluated following both oral and intraperitoneal immunization of mice with the attenuated Salmonella typhimurium strain G30 harbouring the hybrid cst operons. Although plasmid stability is currently a problem, these experiments showed that antibodies to both the carrier and the foreign epitope were generated.