Development of a Delta glnA balanced lethal plasmid system for expression of heterologous antigens by attenuated vaccine vector strains of Vibrio cholerae

We have previously shown that more prominent immune responses are induced t o antigens expressed from multicopy plasmids in live attenuated vaccine vec tor strains of Vibrio cholerae than to antigens expressed from single-copy genes on the V. cholerae chromosome. Here, we report the construction of a Delta glnA derivative of V. cholerae vaccine strain Peru2, This mutant stra in, Peru2 Delta glnA, is unable to grow on medium that does not contain glu tamine; this growth deficiency is complemented by pKEK71-NotI, a plasmid co ntaining a complete copy of the Salmonella typhimurium glnA gene, or by pTI C5, a derivative of pKEK71-NotI containing a 1.8-kbp fragment that directs expression of CtxB with a 12-amino-acid epitope of the serine-rich Entamoeb a histolytica protein fused to the amino terminus. Strain Peru2 Delta glnA( pTIC5) produced 10-fold more SREHP-12-CtxB in supernatants than did ETR3, a Peru2-derivative strain containing the same fragment inserted on the chrom osome. To assess immune responses to antigens expressed by this balanced le thal system in vivo, we inoculated germfree mice on days 0, 14, 28, and 42 with Peru2 Delta glnA, Peru2 Delta glnA(pKEK71-NotI), Peru2(pTIC5), Peru2 D elta glnA (pTIC5), or ETR3, All V. cholerae strains were recoverable from s tool for 8 to 12 days after primary inoculation, including Peru2 Delta glnA ; strains containing plasmids continued to harbor pKEK71-NotI or pTIC5 for 8 to 10 days after primary inoculation. Animals were sacrificed on day 56, and serum, stool and biliary samples were analyzed for immune responses. Vi briocidal antibody responses, reflective of in vivo colonization, were equi valent in all groups of animals. However, specific anti-CtxB immune respons es in serum (P less than or equal to 0.05) and bile (P less than or equal t o 0.001) were significantly higher in animals that received Peru2 Delta gln A(pTIC5) than in those that received ETR3, confirming the advantage of high er-level antigen expression in vivo. The development of this balanced letha l system thus permits construction and maintenance of vaccine and vector st rains of V. cholerae that express high levels of immunogenic antigens from plasmid vectors without the need for antibiotic selection pressure.