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Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-labile enterotoxin (LT) ofEscherichia coli , the latter when expressed inV. cholerae . Both toxins are also potent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed byE. coli have been developed. One such mutant LT molecule has the substitution of a glycine residue for arginine-192 [LT(R192G) ]. Live attenuated strains ofV. cholerae that have been used both asV. cholerae vaccines and as vectors for inducing mucosal and systemic immune responses directed against expressed heterologous antigens have been developed. In order to ascertain whether LT(R192G) can act as an immunoadjuvant when expressed in vivo byV. cholerae , we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains ofV. cholerae , Peru2, ETR3, and JRB14; the latter two strains contain genes encoding different heterologous antigens in the chromosome of the vaccine vectors. We found that LT(R192G) was expressed from pCS95 in vitro by bothE. coli andV. cholerae strains but that LT(R192G) was detectable in the supernatant fraction ofV. cholerae cultures only. In order to assess potential immunoadjuvanticity, groups of germfree mice were inoculated with the threeV. cholerae vaccine strains alone and compared to groups inoculated with theV. cholerae vaccine strains supplemented with purified CT as an oral immunoadjuvant orV. cholerae vaccine strains expressing LT(R192G) from pCS95. We found that mice continued to pass stool containingV. cholerae strains with pCS95 for at least 4 days after oral inoculation, the last day evaluated. We found that inoculation withV. cholerae vaccine strains containing pCS95 resulted in anti-LT(R192G) immune responses, confirming in vivo expression. We were unable to detect immune responses directed against the heterologous antigens expressed at low levels in any group of animals, including animals that received purified CT as an immunoadjuvant. We were, however, able to measure increased vibriocidal immune responses against vaccine strains in animals that receivedV. cholerae vaccine strains expressing LT(R192G) from pCS95 compared to the responses in animals that receivedV. cholerae vaccine strains alone. These results demonstrate that mutant LT molecules can be expressed in vivo by attenuated vaccine strains ofV. cholerae and that such expression can result in an immunoadjuvant effect.

infection and immunity

In Vivo Expression and Immunoadjuvancy of a Mutant of Heat-Labile Enterotoxin of <i>Escherichia coli</i> in Vaccine and Vector Strains of <i>Vibrio cholerae</i>

In Vivo Expression and Immunoadjuvancy of a Mutant of Heat-Labile Enterotoxin of Escherichia coli in Vaccine and Vector Strains of Vibrio cholerae