Salmonella typhimurium G30 was used as a vector to express the ETEC (enterotoxigenic Escherichia coli) fimbrial antigens K88 and K99. Two plasmids encoding K88 or K99 production and having a non-antibiotic selection marker (thyA+) were constructed. These were introduced into a thyA G30 derivative to give the vaccine strains EX841 and EX603, which were shown to express surface K88 or K99, respectively. When administered orally to adult pigs, a dose of 1011 vaccine organisms elicited significant serum antibody responses to the respective fimbrial antigens. Two such immunizations with EX841 generated serum antibody levels comparable to those obtained with intramuscular injection of killed organisms. Attenuated salmonellae can thus be used to deliver ETEC fimbrial antigens to the porcine intestinal immune system.

The construction of live oral carriers based on attenuated Salmonella strains as vectors offers a new approach to vaccine development. We have constructed a set of plasmid vectors which have the thyA gene of Escherichia coli (encoding thymidylate synthetase) as the marker for selection and maintenance of plasmid clones. The thyA system offers an alternative to antibiotic-resistance selection markers. It can be easily adapted to a particular host-vector combination since thyA chromosomal mutations can be readily introduced by trimethoprim selection. We also describe the construction of thyA-based plasmids with the Vibrio cholerae rfb genes (encoding O-antigen biosynthesis of the Inaba serotype). These have been found to be useful in the construction of candidate bivalent cholera-typhoid vaccines.