CHOLERA--PATHOGENESIS AND IMMUNOLOGY

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The goals of this research are to contribute to the understanding of the pathogenesis of cholera, an epidemic diarrheal disease caused by Vibrio cholerae 01, at both the organismal and the molecular levels, with the expectation that this information will lead to the development of rational and effective means of immunoprophylaxis. As cholera is the prototype of an expanding number of diarrheal diseases which are mediated by enterotoxins which are structurally, functionally and immunologically related to the cholera enterotoxin(s), the observations are pertinent to the larger global problem of the billions of cases and millions of deaths annually from diarrheal diseases especially in children in the Third World. Cholera enterotoxin (CT) was discovered in 1959 and it was purified and partially characterized in 1969. In the ensuing period, expectations that, as with diptheria and tetanus, a toxoid vaccine would prevent the disease have not been fulfilled despite early encouraging experimental observations. It is now known that the disease, cholera, is an effective immunizing process in which the human host is presented with a consortium of products elaborated by the cholera vibrios growing in vivo in/on the small bowel. In addition to the enterotoxin, these include colonization factors, such as toxin-coregulated pili (TCP), other surface components including lipopolysaccharide (LPS) and outer membrane proteins, HA/protease and other soluble factors, the cell-associated mannose-sensitive hemagglutinin of the E1 Tor biotype, the flagellum and, possibly, other factors. The solid immunity evoked in people by the disease has not yet been duplicated by non-living preparations, administered parenterally or perorally, or by living attenuated mutants, administered perorally, although some degree of protection has been attained -- even in the absence of toxin antigen. Results of studies with toxin antigen by itself have been even more disappointing. This proposal addresses some of the reasons for previous failures of toxoid vaccines; it addresses the development of a new cholera vaccine to consist of a non-toxic conjugate of de-lipidated LPS and cholera toxin; and, in addition, it will continue to address other factors which may contribute to the virulence and immunogenicity of wild- type cholera vibrios in vivo. The technology includes the synthesis and analysis of sequential continuous overlapping peptides comprising the immunologically dominant B-subunit protein of CT and the use of site- specific mutagenesis to create a V. cholerae strain which produces a CT which is not toxic but expresses the major epitopes of the holotoxin. This CT will also be cross-linked to LPS oligosaccharide to form a conjugate vaccine which will stimulate both antibacterial and antitoxic immunity. We will further examine and define the role of HA/protease and the mannose- sensitive hemagglutinin in attachment and/or detachment of cholera vibrios and examine the significance of phase variation in opacity of V. cholerae colonies.