title: Vaccine and adjuvant activity of recombinant subunit B of E. coli enterotoxin produced in yeast.
abstract: Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) have been studied intensively as vaccines against diseases caused by those bacteria and as adjuvants for mucosal vaccination. Two major problems interfere with the use of these promising adjuvants: their toxicity and the residual bacterial endotoxins mixed with the desired LT. In this study, subunit B of LT was expressed in Pichia pastoris yeast cells (yrLTB) and the recombinant protein was purified and concentrated by ion-exchange chromatography. The final yield of the recombinant protein was 5-8 mg/l induction medium. The molecule is in pentameric form and binds to GM1 gangliosides. When given orally to chickens, anti-LTB antibodies were produced, exhibiting its ability to cross the digestive system and induce an immune response. The adjuvant activity of yrLTB was proven by fusing it to viral protein 2 (VP2) of infectious bursal disease virus. Birds intramuscularly vaccinated with this molecule exhibit 70-100% protection, in a dose-response-dependent manner. This method eliminated the bacterial endotoxins and enabled the production of large quantities of LTB. Expression in a eukaryotic system allows the production of fusion proteins that require post-translational modifications. This may allow oral vaccination with a protein fused to yrLTB. The approach described in this study will enable the efficient production of a non-toxic, eukaryotically expressed enterotoxin as a vaccine against the toxin itself or as a carrier or adjuvant for foreign vaccine molecules.
authors.affiliation: Migal, Immunology Department, Industrial Zone POB 831, Kiryat Shmona 11016, Israel.
authors.forename: E
authors.initials: E
authors.lastname: Fingerut
authors.forename: B
authors.initials: B
authors.lastname: Gutter
authors.forename: R
authors.initials: R
authors.lastname: Meir
authors.forename: D
authors.initials: D
authors.lastname: Eliahoo
authors.forename: J
authors.initials: J
authors.lastname: Pitcovski
mesh_terms: D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial / Q000096:biosynthesis; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000096:biosynthesis / Q000276:immunology*; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000276:immunology; D014611:Vaccination; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
chemical_list: D000276:Adjuvants, Immunologic; D000907:Antibodies, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C027538:heat-labile enterotoxin, E coli