title: Novel modifications to the C-terminus of LTB that facilitate site-directed chemical coupling of antigens and the development of LTB as a carrier for mucosal vaccines.
abstract: To facilitate site-directed chemical coupling of antigens to the heat labile enterotoxin B subunit of Escherichia coli (LTB), a series of genetically modified fusion proteins of LTB were constructed. The LTB fusion proteins had modified versions of a short (14 amino acid) spacer epitope called the Pk tag attached at their C termini. The LTB-Pk.cys mutants differed from each other in the position of a single cysteine residue within the Pk tag. The presence of a cysteine residue at any position within the Pk spacer tag did not prevent the LTB-Pk.cys proteins from forming pentamers or binding to GM1 gangliosides, but the position of the cysteine had variable impact on the yield of the fusion proteins. Following site-directed chemical coupling of antigens to the cysteine residue within the Pk tag, the LTB antigen conjugates retained their ability to bind GM1 on the surface of eukaryotic cells. Intranasal immunisation of mice with an experimental antigen (HRP) chemically linked to LTB-Pk.cys induced high levels of anti-HRP antibodies that could be detected in the serum, saliva and nasal and lung washes. No antibody responses to HRP were detected when HRP was co-administered with, but not linked to, LTB-Pk.cys.
authors: School of Biomedical Sciences, University of St. Andrews, Fife, UK.
authors: A M
authors: AM
authors: O'Dowd
authors: C H
authors: CH
authors: Botting
authors: B
authors: B
authors: Precious
authors: R
authors: R
authors: Shawcross
authors: R E
authors: RE
authors: Randall
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage; D000281:Administration, Intranasal; D000595:Amino Acid Sequence; D000818:Animals; D000917:Antibody Formation; D001427:Bacterial Toxins / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology*; D003545:Cysteine / Q000276:immunology; D004768:Enterotoxins / Q000008:administration & dosage / Q000737:chemistry* / Q000276:immunology*; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005677:G(M1) Ganglioside / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000276:immunology; D018928:Immunity, Mucosal; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013329:Structure-Activity Relationship; D014614:Vaccines, Synthetic
chemical_list: D000276:Adjuvants, Immunologic; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D005677:G(M1) Ganglioside; C027538:heat-labile enterotoxin, E coli; D006735:Horseradish Peroxidase; D003545:Cysteine