title: Reduction in oral immunogenicity of cholera toxin B subunit by N-terminal peptide addition.
abstract: The mucosal adjuvanticity of cholera toxin and the potential of the B subunit of cholera toxin (CtxB) to serve as an oral vaccine carrier have prompted interest in the coupling of immunogenic peptides to this protein. The purpose of this study was to determine how such fusions affect the function of CtxB. Oligonucleotides were genetically fused to the 5' terminus of the ctxB gene to encode additional amino acids of 8, 12, and 24 residues in length. None of these additions affected the ability of CtxB to oligomerize, as determined by nondenaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Circular dichroism revealed no difference in conformation between the modified B subunits, regardless of the length of the addition. However, when compared with native CtxB, additions to the N terminus induced a consistent change in the net conformation of the protein. By using a competitive enzyme immunoassay, the affinity of the modified B subunits for GM1 ganglioside was shown to gradually decrease with increasing length of the N-terminal addition. A similar pattern was observed for the ability of the chimeras to inhibit proliferation of concanavalin A-stimulated spleen cells in vitro, which is a previously described functional property of CtxB that is dependent on its binding to cells. Lastly, the oral immunogenicity of these chimeras was found to be less than that of native CtxB. These results indicate that large fusions to the N terminus of CtxB can significantly affect its biological properties and could reduce its value as a mechanism for effective mucosal immunization.
authors.affiliation: Division of Gastroenterology, School of Medicine, University of Alabama, Birmingham 35294.
authors.forename: M T
authors.initials: MT
authors.lastname: Dertzbaugh
authors.forename: C O
authors.initials: CO
authors.lastname: Elson
mesh_terms: D000284:Administration, Oral; D000595:Amino Acid Sequence; D000818:Animals; D002772:Cholera Toxin / Q000737:chemistry / Q000276:immunology*; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005677:G(M1) Ganglioside / Q000276:immunology; D007114:Immunization; D008213:Lymphocyte Activation; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013329:Structure-Activity Relationship
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D005677:G(M1) Ganglioside; C050310:cholera toxin, B subunit (50-64); D002772:Cholera Toxin