Gene delivery systems represent an effective means to express vaccine antigens in hosts' tissues to induce mucosal and systemic immunity. To this end, the applicant will employ the non-replicating adenovirus 5 (Ade5) vectors to transfect the nasal passages (NP). There has been limited investigation into the immunological consequence of transfecting the NP. Thus, the objectives for this application are to optimize vaccine gene expression in the NP to study T, B, and antigen-presenting cell (APC) function. Further, the applicant will learn whether the NP can effectively stimulate protective immunity at local and distal mucosal immune sites. The Ade5-lacZ construct will be used in preliminary studies to be able to visually follow beta-galactosidase (betagal) expression in the NP. Since it can be demonstrated that gene delivery by Ade5 preferentially induces Th1-dependent responses, the applicant chose a Th-1 dependent antigen, the Brucella abortus L7/L12 ribosomal protein. For subsequent studies, the Ade5-L7/L12 ribosomal protein construct will be used as a candidate vaccine against B. abortus. Mucosal secretions and serum antibody responses to betagal and L7/L12 will be performed to determine how the number of intranasal (IN) administrations affects immunity. From the applicant's studies thus far, a preferential induction of mucosal IgA and serum IgG2a antibody responses following IN delivery of Ade5-lacZ was observed. Together with this evidence, an increase in the number of IFN-gamma producing T cells was obtained upon in vitro stimulation of immune T cells with betagal. Studies proposed here would evaluate whether similar responses are induced in the NALT, lungs, associated respiratory lymph nodes, and at distal mucosal sites, e.g., vagina and small intestine. T cell analysis employing cytokine-specific ELISPOT (single cell analysis) and quantitative RT-PCR will be performed to determine the type of Th cell responses induced at both localized and systemic sites. Cytokine knockout strains (IFN-gamma and IL-4) will also be tested to substantiate the contribution of IFN-gamma, and in the absence of a Th2-type cytokine, the development of immunity to vaccine. It will be ascertained which professional APC or whether transfected nasal epithelia can help support mucosal immunity to L7/L12. Finally, it will be assessed how long term gene expression of L7/L12 may induce tolerance. It will be examined if the nasal epithelia can mimic the effects of oral tolerance by using repeated administration of Ade5-L7/L12 or delivery in combination with anti-B7-2 antibody treatment to block action of B7-CD28 co-stimulation pathway.
