Parenteral immunization with either formalin-fixed whole cells of the fimbriate Bgdl7 strain or purified fimbriae protected against Vibrio cholerae 01 infection in rabbits, independent of biotype and serotype. Parenteral immunization of adult rabbits with purified fimbriae prior to V. cholerae 01 challenge resulted in a reduction of 2 to 3 orders of magnitude in the number of bacteria recovered from the small intestines of immunized rabbits in comparison to non-immunized controls. IgG and IgA antibodies against fimbrillin of V. cholerae 01 were detected in the convalescent sera of patients with cholera; however, little fimbrial antigen was detected in the commercially available cholera vaccines when examined by polyclonal and monoclonal antibodies against fimbriae. These data suggest that fimbrial hemagglutinin is a major adhesin of V. cholerae 01 and that parenteral immunization with fimbriae generates a specific immune response in the gut that may serve as one means of mitigating subsequent V. cholerae 01 gut infection.Key words: Cholera, Fimbriae, Vaccine Cholera is an acute diarrheal disease that is caused by the Gram-negative bacillus Vibrio cholerae 01. V. cholerae 01 infects via uptake of contaminated water and foods and preferentially colonizes the upper small intestine.Fimbriae of V. cholerae 01 consist of cell-associated hemagglutinin that is proposed to be one of the major adhesins mediating the interaction of V. cholerae 01 and human epithelial cells in the small intestine (1). Passive protection tests with anti-mannose-binding hemagglutinin pili antibodies (polyclonal and monoclonal) have been proven effective in protection against experimental cholera caused by El Tor vibrios in infant mouse and in rabbit intestinal loop models (7): but to date, there has been no correlation between serum antibody titers to known antigens purified from V. cholerae 01 and clinical protection. Naturally occurring V. cholerae 01 infection confers longlasting protection against reinfection, but such effective V. cholerae 01 antigens remain to be elucidated.Experimentally infecting rabbits with V. cholerae 01 using the ligated ileal loop test has shown that the bacteria are associated with small intestinal epithelial cells (4,5,8). Therefore, small intestinal infection of rabbits may be used to analyze parameters of immunity that interfere with the persistence of V. cholerae 01 at the intestinal epithelium. As previously reported (2, 3), fimbriate V. cholerae 01 strain of Bgd17 (classical biotype, Inaba serotype) was selectively induced and hydrophobic fimbriae were purified from the fimbriate strain.
The genome of the filamentous phage of Vibrio cholerae fs2 was found to contain rstC and rstB1 (truncated) genes downstream of ORF500. att-fs2-dir and att-fs2-rev sequences homologous to that of att-CTXphi were found between orf500 and rstC of the fs2 genome. This prompted us to search for the integration site of fs2 in the genomes of V. cholerae O1 and O139. The genome of fs2 was found to integrate downstream of attRS of the CTXphi phage, which integrated into chromosome I of V. cholerae O1 and O139. When infected with fs2, a fimbriate strain of V. cholerae O1 appeared to reduce fimbrial production in an adult rabbit ileal loop assay.
The Vibrio cholerae O1 and O139 fimbrillin genes (fimA or mshA) were amplified by polymerase chain reaction and cloned into an Escherichia coli pCRTm vector. These clones were sequenced. The fimA sequences were found to be identical between V cholerae O1 and O139. One of the plasmids was digested with EcoR I and inserted into the EcoR I site of pGEX-3X. The plasmid pVPP thus obtained was transferred into strains of wild-type V cholerae O1 Bgd17 (classical in biotype) and its fimbriated strain by electroporation. The recombinant plasmid pVPP overexpressed mature fimbriae following induction of the tac promoter with isopropyl-beta-D-thiogalactopyranoside. The cloned gene product was purified to homogeneity by sucrose-linear gradient centrifugation (7.8 mg of fimbriae/L-culture). All the properties of the recombinant fimbriae (e.g., subunit structure, hydrophobicity, hemagglutinating activity sensitive to D-mannose and D-glucose and immunogenicity) were identical to those of the wild-type fimbriae.This overexpression system will be extremely useful for rapid, inexpensive preparation of large amounts of fimbriae for vaccine design and development.
Several fimbriated phases of Vibrio cholerae O139 strains were selectively induced and compared immunologically and biochemically with those of V. cholerae O1. Fimbrial antigens were detected on the surfaces of vibrio cells colonizing the epithelial cells of a rabbit small intestine. Convalescent-phase sera from six individuals infected with V. cholerae O139 revealed the development of antibody against the fimbrillin. These findings suggest that the fimbriae of V. cholerae O1 and O139 are expressed in vivo during infection and that consideration must be given to the use of fimbrial antigens as components of vaccines against cholera.
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