ORAL IMMUNIZATION WITH A SALMONELLA-TYPHIMURIUM VACCINE VECTOR EXPRESSING RECOMBINANT ENTEROTOXIGENIC ESCHERICHIA-COLI K99 FIMBRIAE ELICITSELEVATED ANTIBODY-TITERS FOR PROTECTIVE IMMUNITY

Bovine enterotoxigenic Escherichia coli (ETEC) continues to cause mort ality hi piglets and newborn calves. In an effort to develop a safe an d effective vaccine for the prevention of F5(+) ETEC infections, a bal anced lethal asd(+) plasmid carrying the complete K99 operon was const ructed and designated pMAK99-asd(+). Introduction of this plasmid into an attenuated Salmonella typhimurium Delta aro Delta asd strain, H683 , resulted in strain AP112, which stably expresses E. call K99 fimbria e. A single oral immunization of BALB/c and CD-1 mice with strain AP11 2 elicited significant mucosal immunoglobulin A (IgA) titers that rema ined elevated for ttl weeks. IgA and IgG responses in serum specific f or K99 fimbriae were also induced, with a prominent IgG1, as well as I gG2a and IgG2b, titer. To assess the derivation of these antibodies, a K99 isotype-specific B-cell ELISPOT analysis was conducted by using m ononuclear cells from the lamina propria of the small intestines (LP), Peyer's patches (PP), and spleens of vaccinated and control BALB/c mi ce. This analysis revealed elevated numbers of K99 fimbria-specific Ig A-producing cells in the LP, PP, and spleen, whereas elevated K99 fimb ria-specific IgG-producing cells were detected only in the PP and sple en: These antibodies were important for protective immunity. One-day-o ld neonates from dams orally immunized with AP112 were provided passiv e protection against oral challenge with wild-type ETEC, in contrast t o challenged neonates from unvaccinated dams or from dams vaccinated w ith a control Salmonella vector. These results confirm that oral Salmo nella vaccine vectors effectively deliver K99 fimbriae to mucosal indu ctive sites for sustained elevation of IgA and IgG antibodies and for eliciting protective immunity.