title: Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae.
abstract: There is an urgent need for new adjuvants that are effective with mucosally administered vaccines. Cholera toxin (CT) is the most powerful known mucosal adjuvant but is much too toxic for human use. In an effort to develop a useful mucosal adjuvant we have generated a novel non-toxic mutant CT molecule that retains much of the adjuvant activity of native CT. This was achieved by making the enzymatically active A subunit (CTA) recalcitrant to the site-specific proteolytic cleavage ("nicking") required for toxicity, which was found to require mutations not only in the two residues rendering the molecule resistant to trypsin but also in neighboring sites protecting against cleavage by Vibrio cholerae proteases. This multiple-mutated CT (mmCT) adjuvant protein could be efficiently produced in and purified from the extracellular medium of CT-deleted V. cholerae. The mmCT completely lacked detectable enterotoxicity in an infant mouse model and had >1000-fold reduced cAMP inducing activity compared to native CT in a sensitive mammalian target cell system. It nonetheless proved to have potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to mucosally co-administered antigens including oral cholera and intranasal influenza vaccines. We conclude that mmCT is an attractive novel non-toxic mucosal adjuvant for enhancing immune responses to co-administered mucosal vaccines.
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden. Electronic address: michael.lebens@microbio.gu.se.
authors.forename: Michael
authors.initials: M
authors.lastname: Lebens
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden.
authors.forename: Manuela
authors.initials: M
authors.lastname: Terrinoni
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden.
authors.forename: Stefan L
authors.initials: SL
authors.lastname: Karlsson
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden.
authors.forename: Maximilian
authors.initials: M
authors.lastname: Larena
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden.
authors.forename: Tobias
authors.initials: T
authors.lastname: Gustafsson-Hedberg
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden.
authors.forename: Susanne
authors.initials: S
authors.lastname: Källgård
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden.
authors.forename: Erik
authors.initials: E
authors.lastname: Nygren
authors.affiliation: Department of Microbiology and Immunology and Gothenburg University Vaccine Research Institute (GUVAX), Institute of Biomedicine, Sahlgrenska Academy at University of Gothenburg, Box 435, SE-405 30 Göteborg, Sweden.
authors.forename: Jan
authors.initials: J
authors.lastname: Holmgren
mesh_terms: D000276:Adjuvants, Immunologic / Q000737:chemistry*; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D002772:Cholera Toxin / Q000737:chemistry* / Q000235:genetics; D022121:Cholera Vaccines / Q000276:immunology; D005260:Female; D007111:Immunity, Cellular; D056724:Immunity, Humoral; D018928:Immunity, Mucosal; D007070:Immunoglobulin A / Q000276:immunology; D007074:Immunoglobulin G / Q000097:blood; D007252:Influenza Vaccines / Q000276:immunology; D051379:Mice; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D018675:Toxicity Tests; D014734:Vibrio cholerae / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial; D022121:Cholera Vaccines; D007070:Immunoglobulin A; D007074:Immunoglobulin G; D007252:Influenza Vaccines; D050505:Mutant Proteins; D002772:Cholera Toxin
keywords: Adjuvant; Cholera toxin; Mucosal immunity; Mucosal vaccine