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The gram-negative bacterium Vibrio cholerae releases outer membrane vesicles (OMVs) during growth. In this study, we immunized female mice by the intranasal, intragastric, or intraperitoneal route with purified OMVs derived from V. cholerae. Independent of the route of immunization, mice induced specific, high-titer immune responses of similar levels against a variety of antigens present in the OMVs. After the last immunization, the half-maximum total immunoglobulin titer was stable over a 3-month period, indicating that the immune response was long lasting. The induction of specific isotypes, however, was dependent on the immunization route. Immunoglobulin A, for example, was induced to a significant level only by mucosal immunization, with the intranasal route generating the highest titers. We challenged the offspring of immunized female mice with V. cholerae via the oral route in two consecutive periods, approximately 30 and 95 days after the last immunization. Regardless of the route of immunization, the offspring was protected against colonization with V. cholerae in both challenge periods. Our results show that mucosal immunizations via both routes with OMVs derived from V. cholerae induce long-term protective immune responses against this gastrointestinal pathogen. These findings may contribute to the development of "nonliving," OMV-based vaccines against V. cholerae and other enteric pathogens, using the oral or intranasal route of immunization.

Immunization with Vibrio cholerae Outer Membrane Vesicles Induces Protective Immunity in Mice