Delivery of the cholera toxin B subunit by using a recombinant Yersinia enterocolitica strain as a live oral carrier

The gene ctxB encoding the cholera toxin B subunit was subcloned to design its production by Yersinia enterocolitica. It was joined in two ways to yopH, a gene of the virulence plasmid pYV specific to this genus. This gene encodes one of the major Yop proteins (YopH) secreted by bacteria incubated at 37 degrees C in a Ca(2+)-deprived medium. In a first construction, an operon fusion was obtained between ctxB and yopH so that CT-B and a truncated YopH protein were produced. The recombinant CT-B from Y. enterocolitica was structurally and antigenically similar to CT-B produced by Vibrio cholerae. In another construction, the fusion gene obtained directed the production of YopH'/CT-B hybrid proteins that were secreted by Y. enterocolitica. In both cases, Y. enterocolitica directed the production of the recombinant proteins only when the bacteria were incubated in conditions of Yops production. When bacteria carrying the operon fusion were given orally to mice, a clear serum antibody response against CT-B was detected by ELISA. According to immunoblot analysis, this response was only directed against the polymeric form of the B subunit.