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Cholera toxin (CT) is the primary virulence factor responsible for severe cholera.  Vibrio cholerae  strains unable to produce CT show severe attenuation of virulence in animals and humans. The pentameric B subunit of CT (CTB) contains the immunodominant epitopes recognized by antibodies that neutralize CT. Although CTB is a potent immunogen and a promising protective vaccine antigen in animal models, immunization of humans with detoxified CT failed to protect against cholera. We recently demonstrated however that pups reared from mice immunized intraperitoneally (IP) with 3 doses of recombinant CTB were well protected against a highly lethal challenge dose of  V. cholerae  N16961. The present study investigated how the route and number of immunizations with CTB could influence protective efficacy in the suckling mouse model of cholera. To this end female mice were immunized with CTB intranasally (IN), IP, and subcutaneously (SC). Serum and fecal extracts were analyzed for anti-CTB antibodies by quantitative ELISA, and pups born to immunized mothers were challenged orogastrically with a lethal dose of  V. cholerae . Pups from all immunized groups were highly protected from death by 48 hours (64–100% survival). Cox regression showed that percent body weight loss at 24 hours predicted death by 48 hours, but we were unable to validate a specific amount of weight loss as a surrogate marker for protection. Although CTB was highly protective in all regimens, three parenteral immunizations showed trends toward higher survival and less weight loss at 24 hours post infection. These results demonstrate that immunization with CTB by any of several routes and dosing regimens can provide protection against live  V. cholerae  challenge in the suckling mouse model of cholera. Our data extend the results of previous studies and provide additional support for the inclusion of CTB in the development of a subunit vaccine against  V. cholerae .

Immunization with Cholera Toxin B Subunit Induces High-Level Protection in the Suckling Mouse Model of Cholera