The major goal of the proposed work is the development of a safe, effective immunization protocol for long-lasting prevention of enterotoxigenic Escherichia coli (ETEC) diarrhea. ETEC are a common cause of acute diarrhea particularly amongst visitors to, or children resident in, tropical areas. ETEC and rotavirus are considered the principal causes of severe diarrheal disease which results in 5 to 10 million deaths per year of young children in underdeveloped countries. Our rationale is to base the vaccine on the oral administration of purified ETEC proteins responsible for virulence, i.e. CFAs and enterotoxins. The specific objectives of this proposal pertain to the problem of how to produce a protective immunological response using the purified antigens; that is, to produce high levels of antigen-specific secretory IgA. Preliminary work has shown a protective intestinal anti-CFA IgA response in the adult rabbit model. Although immunity against reinfection has been demonstrated with volunteers using the whole bacteria, induction of intestinal IgA in volunteers with purified CFAs has been only partially successful. However, those volunteers that did respond were protected. Future work will concentrate on modifications of the antigen(s); for example, aggregation of CFAs and enterotoxin by heating or using a carrier such as liposomes to overcome the problem of delivering the antigen to the epithelial cell surfaces. Experiments will also be aimed at comparing different forms of the antigen(s) for their ability to stimulate the GALT (gut-associated lymphoid tissue). Experiments will be performed with a mouse model (the Klinman assay) and also by monitoring antigen-primed circulating B cells in the peripheral blood of human subjects. Precursor B cells primed in Peyers' patches circulate while maturing and then """"""""home"""""""" in the lamina propria of the intestine. Pokeweed mitogen will be used to """"""""expand"""""""" the lymphocytes so that these can be assayed for their potential to produce antigen-specific IgA. Antigen preparations shown to consistently produce an IgA response will then be tested in volunteers for protection against ETEC diarrhea in double-blind studies. Another approach will be to genetically engineer E. coli strains which produce CFA(s) but not enterotoxin; such strains will be used to determine if development of anti-CFA immunity alone is sufficiently protective.
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