The major long-term objective of this project is the development of a live oral vaccine for the prevention of cholera. During the previous period of support, the investigator and his coworkers developed candidate vaccine strains of Vibrio cholerae using recombinant DNA techniques to delete portions of the genes encoding cholera enterotoxin. These strains provide strong protection against challange when tested in volunteers but unexpectedly caused diarrhea in many of them upon initial immunization. In investigating this phenomenon, a new secretogenic toxin in V. cholerae was discovered; it is believed that this toxin is responsible for the observed diarrhea. This toxin affects the tight junctions, or zonula occludens, which are found between epithelial cells. The investigator hypothesizes that this toxin causes diarrhea by the paracellular pathway rather than the transcellular pathway which is affected by cholera enterotoxin. Additional studies have also resulted in several other new findings concerning colonization and immunogenicity which serve as the basis for the propposed future studies. Specifically, Dr. Kaper proposes the following: 1) The new secretogenic toxin (ZOT for zonula occludens toxin), will be characterized by purifying it, raising specific antisera against it, and determining the human immune response to it. Using the zot gene which has already been cloned and sequenced, the distribution and expression of ZOT in other strains will be studied. 2) Attenuated V. cholerae vaccine strains of both classical and El Tor biotypes which are mutated in genes encoding ZOT as well as in genes encoding cholera toxin will be constructed. 3) The genetic defect in the previously constructed vaccine candidate V. cholerae CVD103-HgR which is responsible for decreased intestinal colonization of this strain relative to the parent strain CVD103 will be characterized. 4) The investigator and his coworkers will begin to study the genetic regulation of V. cholerae antigens which are expressed in vivo but not in vitro and are immunogenic in humans. An expression library using a T7 promoter will be constructed and screened with sera and jejunal fluid from convalescent patients to detect genes encoding such antigens. The genetic control of this differential expression will be studied using gene fusions and proteins expressed from cloned genes will be used to study the components of the vibriocidal immune response.
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