title: Genetic fusion of human insulin B-chain to the B-subunit of cholera toxin enhances in vitro antigen presentation and induction of bystander suppression in vivo.
abstract: The pentameric B-subunit of cholera toxin (CTB) can be used as an efficient mucosal carrier of either immunogenic or tolerogenic T-cell epitopes. In this study a series of fusions was constructed between the genes encoding CTB and the B-chain of human insulin (InsB). The resulting fusion proteins were expressed in Escherichia coli and isolated as cytoplasmic inclusion bodies that were then dissolved and assembled in vitro. GM1 enzyme-linked immunosorbent assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses showed that the protein construct in which InsB was fused to the C-terminus of a CTB monomer (CI) assembled into structures that both bound to the receptor GM1 ganglioside and reacted with monoclonal antibodies to CTB and insulin. Fusion of InsB to the N-terminus of CTB resulted in protein that could not assemble into pentameric CTB. In vitro assays showed that the CI fusion protein was 300-fold more potent than native insulin at inducing interleukin-2 (IL-2) production by an insulin-specific T-cell hybridoma. When administered orally, the CI fusion protein induced efficient immunological suppression of ovalbumin-specific T-cell responses in mice co-immunized parenterally with insulin and ovalbumin. These results demonstrate the stability, GM1 receptor-binding activity and antigenic authenticity of the CI fusion protein as well as its ability to elicit insulin-specific T-cell responses in vitro. In addition, we demonstrate that the CI fusion protein induces efficient immunosuppression after oral administration, raising the possibility of using such constructs in the treatment of type-1 diabetes.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Göteborg, Göteborg, Sweden.
authors.forename: Hamid
authors.initials: H
authors.lastname: Sadeghi
authors.forename: Søren
authors.initials: S
authors.lastname: Bregenholt
authors.forename: Dale
authors.initials: D
authors.lastname: Wegmann
authors.forename: Jacob S
authors.initials: JS
authors.lastname: Petersen
authors.forename: Jan
authors.initials: J
authors.lastname: Holmgren
authors.forename: Michael
authors.initials: M
authors.lastname: Lebens
mesh_terms: D000818:Animals; D017951:Antigen Presentation / Q000276:immunology*; D024201:Bystander Effect / Q000276:immunology*; D002772:Cholera Toxin / Q000276:immunology*; D005260:Female; D006825:Hybridomas / Q000276:immunology; D007108:Immune Tolerance / Q000276:immunology*; D007328:Insulin / Q000276:immunology*; D007376:Interleukin-2 / Q000096:biosynthesis; D051379:Mice; D008807:Mice, Inbred BALB C; D016688:Mice, Inbred NOD; D010446:Peptide Fragments / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology
chemical_list: D007328:Insulin; D007376:Interleukin-2; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D002772:Cholera Toxin