title: Intranasal immunization using the B subunit of the Escherichia coli heat-labile toxin fused to an epitope of the Bordetella pertussis P.69 antigen.
abstract: The plasmid pBRD026, which directs expression of the B subunit of the Escherichia coli heat-labile toxin (LTB), was modified so that DNA encoding epitopes could be inserted at the 3' end of the gene. An oligonucleotide linker containing restriction sites for BglII and SpeI was inserted at the SpeI site at the 3' end of the LTB gene to form plasmid pFV1. This linker also encodes the amino acid sequence Gly-Pro-Gly-Pro which we propose acts as a 'hinge' between the LTB and the foreign epitope. Oligonucleotides specifying an epitope from the Bordetella pertussis P.69 outer membrane protein were cloned into pFV1 to form pFV169. The resultant fusion protein (LTB69) was partially purified from the periplasm of E. coli strains in a soluble pentameric form which could bind GM1 gangliosides. Mice immunized intranasally with purified LTB69 produced antibodies against both LTB and the P.69 protein. In addition, ELISPOT assays demonstrated the presence of LTB-specific and P.69-specific antibody-secreting cells in the lungs of immunized mice.
authors.affiliation: Department of Molecular Biology, Wellcome Research Laboratories, Beckenham, Kent, UK.
authors.forename: M
authors.initials: M
authors.lastname: Lipscombe
authors.forename: I G
authors.initials: IG
authors.lastname: Charles
authors.forename: M
authors.initials: M
authors.lastname: Roberts
authors.forename: G
authors.initials: G
authors.lastname: Dougan
authors.forename: J
authors.initials: J
authors.lastname: Tite
authors.forename: N F
authors.initials: NF
authors.lastname: Fairweather
mesh_terms: D000281:Administration, Intranasal; D000595:Amino Acid Sequence; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D001886:Bordetella pertussis / Q000235:genetics / Q000276:immunology*; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D029968:Escherichia coli Proteins; D005260:Female; D005677:G(M1) Ganglioside / Q000378:metabolism; D007114:Immunization; D007136:Immunoglobulins / Q000096:biosynthesis; D008168:Lung / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D010567:Pertussis Vaccine / Q000235:genetics / Q000276:immunology*; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014614:Vaccines, Synthetic / Q000235:genetics / Q000276:immunology; D010566:Virulence Factors, Bordetella
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; D029968:Escherichia coli Proteins; D007136:Immunoglobulins; D010567:Pertussis Vaccine; D011993:Recombinant Fusion Proteins; D014614:Vaccines, Synthetic; D010566:Virulence Factors, Bordetella; D005677:G(M1) Ganglioside; C068929:pertactin; C027538:heat-labile enterotoxin, E coli