The applicant proposes to develop the use of Vibrio cholerae, the causative agent of cholera, as a live oral attenuated vaccine vector that can deliver heterologous antigens to stimulate a common mucosal immune response. Bordetella pertussis, the causative agent of whooping cough, is an important pathogen of humans which, like V. cholerae, acts at the mucosal surface. The investigators hypothesize that oral immunization with V. cholerae vector strains expressing protective antigens of B. pertussis may lead to protective immunity against B. pertussis infection in the respiratory tract. Examination of immune responses following infection with the vector strains may aid in elucidating the mechanisms of protective immunity to B. pertussis and may further the understanding of protective immunity at mucosal surfaces. The filamentous hemagglutinin (FHA) of B. pertussis will he used as a model heterologous antigen. FHA is a 220-kDa filamentous protein that is proposed to be one of the major adhesins mediating the interaction of B. pertussis and human cilia. The cloned gene encoding FHA (FhaB) will be placed under the control of an in-vivo expressed V. cholerae promoter. The promoter for the V. cholerae heat-shock gene htpG will be used; this promoter has led to expression of other heterologous antigens from V. cholerae in prior studies. The expression of FHA from htpGp will be compared with that from a constitutively-expressed promoter. The fhaB constructs will be placed onto the chromosome of V. cholerae by in-vivo marker exchange, and the constructs will be tested for expression of the heterologous antigen. A germ-free adult mouse model of colonization by V. cholerae will be developed that can be used to assess the immune response to FHA expressed by the V. cholerae strains, and the induction of protective immunity will be examined using an inhalation challenge model of B. pertussis infection. The Principal Investigator is applying for three additional years of research training in order to further develop the use of V cholerae as a vaccine vector for delivering heterologous antigens. The proposed project will require learning more sophisticated molecular biologic techniques, mastering methods for performing immunologic assays, and working with a variety of new animal models for evaluating colonization, immune response, and protection by the constructed vaccines. The additional research time would allow a smooth transition from post-doctoral training to an independent research career in academic medicine in the specialty of infectious disease. The sponsors are experienced in the necessary techniques and have the resources needed for successful completion of the proposed project.
