8 pg/mL, which is similar to that of EHEC-derived Stx2 (2.5 pg/mL), whereas the CD50 of mStx2-His was considerably higher (585 ng/mL). On the other hand, the intraperitoneal MLD of Stx2-His in adult mice (6 weeks of age) was 100 ng, whereas that of mStx2-His was considerably higher (100 μg), indicating that the activities of these mutant toxins are close to non-hazardous when administered

at vaccination dosages. To confirm the effect of mStx2-His as a vaccine antigen, we immunized selleck ICR mice s.c. with 10 μg of mStx2-His containing aluminum hydroxide as a practical adjuvant for vaccine. No mice died of or were weakened by the immunization. As shown in Figure 3a, the IgG antibody titers in mice that were immunized twice with mStx2-His were significantly higher (mean ± SEM 2,206,250 ± 335,643, range 156,250–3,906,250) than those of mice immunized with adjuvant alone (titers of all five were < 10). The neutralizing activities of these antibodies were confirmed by an in vitro neutralization assay using 10 pg/mL of EHEC-derived Stx2 (corresponding to a 20.9% survival concentration in HeLa229 cells). No sera derived from

immunized mice with PBS neutralized the toxicities (mean ± SEM of survival rate 25.7 ± 0.4%), whereas the sera derived from immunized mice with mStx2-His neutralized the toxicities (mean ± SEM survival rate 70.3 ± 7.0%). To investigate the degree of protection HCS assay conferred by antibodies that were induced in mice by immunization with mStx2-His, we divided the mice into three groups and challenged them with different lethal doses of wild-type Stx2. In this

study, we used Stx2-His to challenge mice with high lethal doses of purified toxin on the assumption that a large amount of toxin protein was needed. As shown in Figure 3b, all the mice immunized with mStx2-His survived a challenge of Stx2-His at 10- and 100-fold MLD (1 and Fossariinae 10 μg/mouse, respectively) for at least 1 week with no symptoms, whereas only three of nine mice survived a challenge of 1000-fold MLD (100 μg/mouse). All of the mice immunized with adjuvant alone succumbed to a challenge with 10-fold MLD within 3 days. It is crucial to consider the following three points if toxoids are to have clinical utility. First, the toxoid itself must not be hazardous to humans and animals. Second, the toxoid should induce sufficient antibody production to neutralize an excess amount of wild-type toxin. Third, it should be possible to prepare large amounts of the toxoid antigen easily and cheaply. Taken together, these factors necessitate use of the overexpression method for preparation of antigenic proteins. In the process of constructing the CTB expression plasmid in our previous study [25], we confirmed that the SD sequence derived from LTB worked well for expression of the Vibrio cholerae derived CTB gene in E. coli without any obvious toxicities.