Study On A Multivalent Live Oral Vaccine Against Human Enterotoxigenic Escherichia Coli

Entcroloxigcnic Escherichia coli (ETEC) is a leading cause of dehydrating diarrhea in infant in developing countrics and in travelers to these regions. Two critical steps, fimbria-mediated attachment to enterocytes of the proximal small intestine and enterotoxin production, are involved in the disease and offer targets for immunoprophylaxis. Epidemiological studies have shown that the frequency of CFAs in human ETEC is various in different areas. 20 CFAs have been identified. The most prevalent fimbrial types of them are CFA/I, CS3 and CS6. Two types of enterotoxins have been described. The heat-labile toxin (LT) is a multisubunits protein of form AB5. The A subunit is the active component of the toxin and function to increase the activity of adenylate cyclase. This is delivered into host cells by the B subunits, which bind to gangliosides on the cell surface. The heat-stable toxin (ST) is a small (19-amino-acid) nonimmunogenic polypeptide that has guanylate cyclase-stimulating activity.The determinants of two prevalent CFAs, CFA/I and CS6, were cloned into one plasmid and transformed into an attenuated Shigella flexneri vaccine. The plasmid is steadily maintained by a host-plasmid balanced lethal system based on asd gene. Both CFAs are simultaneously expressed in this vaccine strain and fimbriae assembled on cell surface. The immune responses in serum and local intestine against CFA/I and CS6 were observed following intragastric immunization of Balb/c mice with the recombinant strain. The antibody responses are equivalent as elicited by the strain expressing CFA/I only or CS6 only. Expression of the two antigens does not interfere the growth with each other and the immunogenicity of the host itself.The CSS determinant and a fusion enterotoxin gene, the 5' terminus of the gene encoding mutant attenuated ST and the pro region was genetically fused to the 3' terminus of the gene encoding B unit of LT, were engineered into the attenuated Shigella flexneri vaccine by the same strategy. The results showed that the CS3 is highly expressed by the promoter (re, and expression of the fusion enterotoxin protein bypromoters of ompC and trc is regulated under osmotic pressure. The the vaccine strain grows nearly same as the host strain. Following oral administration of the recombinanl strain to Balb/e mice, serum IgG and mucosal IgA responses were elicited against liTKC antigens and host.A prototype mullivalent live vaccine was constructed by mixing the two recombinant vaccine strains above. The live oral vaccine can elicit Balb/c mice producing mucosal and serum antibody against ETEC antigens of CFA/I, CS6, CS3, LTB and ST, and the O antigen of LPS for S. flexneri carrier. The immunogenicity of the mixed vaccine is indistinctively different from each vaccine strain consisting.