Naked DNA encoding vaccine antigens when administered at peripheral sites has proven to be effective to elicit protective immune responses by either T helper Th1- or Th2 cell-dependent responses. These induced immune responses have been limited primarily to peripheral lymphoid tissues, and minimal induction of secretory (S)-IgA responses or immune Th cells from mucosal tissues were evident. To date, limited studies have been performed using DNA to immunize mucosal surfaces. In order to successfully immunize mucosal tissues, efforts must be made to overcome the natural barriers at mucosal surfaces, i.e., mucous, low pH, DNases. In an effort to circumvent these barriers, the applicants have proposed to utilize microencapsulated DNA complexed with an M cell targeting molecule, reovirus sigma 1 (s1) protein. Much like live vector delivery systems which are adept in delivering vaccines to the Peyer's patches (PP), the applicants hypothesize that targeting vaccine antigens to mucosal inductive tissues will stimulate mucosal immune responses. Thus, by encapsulating with the DNA a targeting molecule, namely, reovirus s1 protein, this will direct the DNA to the mucosal inductive sites for the appropriate development of Th cell responses. Subsequent studies will include DNA encoded cytokine genes to facilitate the desired CD4 Th cell response. Microencapsulation will protect the DNA from exposure to mucosal surfaces - such microencapsulation procedures have proven effective in enhancing rotaviral infectivity and antiviral immune responses. One major objective of this application is to render the effectiveness of DNA vaccination of mucosal surfaces to ultimately generate an effective anti-HIV vaccine. In this context, the applicants propose to, first, test the in vitro effectiveness of this delivery system by transfecting mouse L fibroblasts with the microencapsulated DNA-encoded reporter genes complexed with a s1 protein. Once the amount of s1 protein required for optimal transfection is determined, the microencapsulated DNA-s1 protein complexes will be orally administered to follow site and extent of reporter gene expression. Likewise, similar procedure will be applied for delivery of microencapsulated DNA-s1 protein complexes for intranasal administration. Once delivery has been optimized, studies will be performed to determine mucosal immune responses to DNA-encoded gp120 at inductive sites by in vitro antigen-specific restimulation assays, cytokine ELISPOT and ELISA, and by cytokine RT-PCR. Likewise, mucosal immune responses at effector sites will be analyzed for CTL responses. Following these determinations, the applicants propose to test different vaccine DNA formulations to steer host CD4+ and CD8+ T cell responses along a protective immune pathway.
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| Wu, Y; Boysun, M J; Csencsits, K L et al. (2000) Gene transfer facilitated by a cellular targeting molecule, reovirus protein sigma1. Gene Ther 7:61-9 |
| Csencsits, K L; Jutila, M A; Pascual, D W (1999) Nasal-associated lymphoid tissue: phenotypic and functional evidence for the primary role of peripheral node addressin in naive lymphocyte adhesion to high endothelial venules in a mucosal site. J Immunol 163:1382-9 |
