Live vector delivery systems can effectively target mucosal inductive sites for the efficacious delivery of vaccine antigens to induce both mucosal and systemic immunity. We will employ attenuated Salmonella vectors, which are adept in delivering vaccines to the Peyer's patches (PP) of the small intestine. While these Salmonella vectors typically elicit T helper (Th) 1 cell (IFN-gamma-dependent) immune responses because Salmonella is an intracellular pathogen, our studies have shown that we can obtain Th2 cell (IL-4-dependent) immune responses to colonization factor antigen I (CFA/I) from human enterotoxigenic Escherichia coli (ETEC). Using this expression system, we have devised a novel strategy for vaccine delivery. Following a single, oral delivery of this construct into BALB/c mice, we observed an elevated mucosal and serum IgA, and an elevated serum IgG1 anti-CFA/I fimbriae antibody titers. These responses were supported by the production of Th2 cytokines, IL-4 and IL-5. Thus, in the absence of a Th cell response to Salmonella, an early Th2-type response is elicited to CFA/I fimbriae permitting induction of secretory (S)-IgA memory. With time, the Th2-type response converts to a dominant Th1-type response. We propose that this late induction of Th1-type response regulates the Th2 cell response to minimize toxicities associated with elevated IL-4 production, i.e., elevated IgE production. Consequently, we observed no net increases in IgE generation while sustaining elevated S-IgA antibody levels. The objective of this proposal is to determine how our novel mode of antigen expression will allow future vaccine formulations to elicit the desired Th cell response in a regulated fashion. And this ability to elicit IFN-gamma allows for adequate control of IL-4. We hypothesize that this co-stimulation of IFN-gamma is due to the requisite to eliminate Salmonella vector from the host since in the absence of IFN-gamma, Salmonella vectors are lethal. Thus, we will determine the mechanisms for the preferential induction of PP CD4+ Th2 versus Th 1 cell responses to CFA/I fimbriae. We will determine how the presence of CFA/I fimbriae alters host T and B cell responses to Salmonella antigens. We will evaluate terminal IgA responses in the small intestinal lamina propria and in mucosal secretions for S-IgA anti-CFA/I fimbriae antibodies. We will determine by cytokine ELISPOT and competitive RT-PCR assays the levels of Salmonella-induced cytokine responses. We will assess whether IFN-gamma is required to clear the Salmonella vector in the presence of altered Th cell phenotypes using cytokine-deficient mice. We will determine whether there is preferential support for Th2 cell development by PP antigen presenting cells (APCs). We will examine whether the delayed neutrophil influx supports the development of Th1-type responses to CFA/I fimbriae. Finally, we will determine the efficacy of our vaccine immunization strategy by examining whether passive immunity can prevent colonization of human ETEC in neonatal mice in the presence of elevated immune S-IgA antibodies.
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