IEM101, a naturally attenuated Vibrio cholerae strain as carrier for genetically detoxified derivatives of cholera toxin.

Two mutants of cholera toxin (CTS106 containing a Pro106-->Ser substitution and CTK63 containing a Ser63-->Lys substitution) with greatly reduced or no toxicity respectively, were expressed in the naturally attenuated IEM101 Vibrio cholerae strain (El Tor, Ogawa) which does not express cholera toxin (CT). Expression was driven by the natural promoter of CT, or by a promoter known to induce strong in vivo expression such as nirB. In the rabbit ileal loop assay, where 10(4) wild type bacteria were sufficient to induce fluid accumulation, 10(9) IEM101 expressing CTS106 bacteria were needed to induce some fluid accumulation, while IEM101 expressing CTK63 was inactive, even when 10(10) cells were used. When used to immunize mice intranasally, all bacteria induced vibriocidal antibodies; however, anti-CT antibodies were not induced by bacteria expressing low levels of CTK63 under the control of the ct promoter. Anti-CT antibodies were successfully induced by bacteria expressing high levels of CTK63 under the control of the nirB promoter, or by bacteria expressing low levels of CTS106. These data show that antibodies against cholera toxin can be induced in vivo by high level expression of a non toxic mutant, or by using a mutant with residual ADP-ribosyltransferase activity. In conclusion, we have shown that IEM101, a naturally attenuated Vibrio strain known to be safe and immunogenic in humans, can be engineered to express immunogenic levels of CTK63, and may represent a good candidate for vaccination against cholera.