In vivo expression and immunoadjuvancy of a mutant of heat-labile enterotoxin of Escherichia coli in vaccine and vector strains of Vibrio cholerae

Vibrio cholerae secretes cholera toxin (CT) and the closely related heat-la bile enterotoxin (LT) of Escherichia coil, the latter when expressed in V. cholerae. Both toxins are also patent immunoadjuvants. Mutant LT molecules that retain immunoadjuvant properties while possessing markedly diminished enterotoxic activities when expressed by E. coli have been developed. One s uch mutant LT molecule has the substitution of a glycine residue for argini ne-192 [LT(R192G)]. Live attenuated strains of V. cholerae that have been u sed both as V. cholerae vaccines and as vectors for inducing mucosal and sy stemic immune responses directed against expressed heterologous antigens ha ve been developed. In order to ascertain whether LT(R192G) can act as an im munoadjuvant when expressed in vivo by V. cholerae, we introduced a plasmid (pCS95) expressing this molecule into three vaccine strains of V. cholerae , Peru2, ETR3, and JRB14; the latter two strains contain genes encoding dif ferent heterologous antigens in the chromosome of the vaccine vectors. We f ound that LT(R192G) was expressed from pCS95 in vitro by both E. coli and V . cholerae strains but that LT(R192G) was detectable in the supernatant fra ction of V. cholerae cultures only In order to assess potential immunoadjuv anticity, groups of germfree mice were inoculated with the three V. cholera e vaccine strains alone and compared to groups inoculated with the V. chole rae vaccine strains supplemented with purified CT as an oral immunoadjuvant or V. cholerae vaccine strains expressing LT(R192G) from pCS95. We found t hat mice continued to pass stool containing V. cholerae strains with pCS95 for at least 4 days after oral inoculation, the last day evaluated. We foun d that inoculation with V. cholerae vaccine strains containing pCS95 result ed in anti-LT(R192G) immune responses, confirming in vivo expression, me we re unable to detect immune responses directed against the heterologous anti gens expressed at low levels in any group of animals, including animals tha t received purified CT as an immunoadjuvant. We were, however, able to meas ure increased vibriocidal immune responses against vaccine strains in anima ls that received V. cholerae vaccine strains expressing LT(R192G) from pCS9 5 compared to the responses in animals that received V. cholerae vaccine st rains alone. These results demonstrate that mutant LT molecules can be expr essed in vivo by attenuated vaccine strains of V. cholerae and that such ex pression can result in an immunoadjuvant effect.