A variety of techniques, including use of live oral vaccines, have been employed to deliver antigens to the mucosal lymphoid tissues in an attempt to initiate production of specific sIgA. One recent approach has been to employ avirulent mutants of Salmonella as carriers for plasmids which code for virulence determinants of heterologous mucosal pathogens. Antigens expressed by these carrier strains are delivered directly to the antibody- forming cells in the mucosal lymphoid tissues. This has been shown to be an effective means of stimulating significant levels of serum and mucosal antibodies directed against the carrier and against cloned antigens. There are a number of unresolved questions that will influence the utility of this approach to vaccine development. We propose here to address a number of these unresolved questions, using the cholera related enteropathogen, enterotoxigenic E. coli, as a model. We have chosen this system to explore because it is the one with which we are most familiar and within which we have established a large body of preliminary data upon which to build. It is important to note, however, that the questions we propose to address are such that the answers are broadly applicable to those contemplating the use of this system for the development of vaccines against a variety of other human pathogens. Using a murine model, we will examine 1) the effect of multiple use on carrier efficacy, 2) the effect of varying dose and repeated immunization on antibody response, 3) effect of gene location on stability, expression, cellular location, and immunogenicity of cloned antigens, and 4) the immune response to nonimmunogenic peptides when incorporated into genetic fusion polypeptides and delivered as cloned antigens. As a component of this proposal, we will also develop a challenge model system for use in determining the efficacy of this approach to vaccine delivery. This is a technique for vaccine delivery with significant potential to influence the management of infectious diseases on a large scale, not only for vaccines against enteric bacterial pathogens, but also for vaccines against a variety of other bacterial, viral, and parasitic diseases. This proposal examines many of the parameters of the model and will provide valuable, necessary information applicable to the use of these vaccines in humans.
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