title: Recombinant Bacillus subtilis expressing the Clostridium perfringens alpha toxoid is a candidate orally delivered vaccine against necrotic enteritis.
abstract: Recombinant Bacillus subtilis endospores have been used to vaccinate against tetanus and anthrax. In this work, we have developed spores that could be used to vaccinate against Clostridium perfringens alpha toxin and that could be used to protect against gas gangrene in humans and necrotic enteritis in poultry. The primary active agent in both cases is alpha toxin. A carboxy-terminal segment of the alpha toxin gene (cpa) fused to the glutathione-S-transferase (GST) gene was cloned in B. subtilis such that the encoded GST-Cpa(247-370) polypeptide had been expressed in the following three different ways: expression in the vegetative cell, expression on the surface of the spore coat (fused to the CotB spore coat protein), and a combined approach of spore coat expression coupled with expression in the vegetative cell. Mice immunized orally or nasally with three doses of recombinant spores that carried GST-Cpa(247-370) on the spore surface showed the most striking responses. This included seroconversion with anti-Cpa(247-370)-specific immunoglobulin G (IgG) responses in their sera, a Th2 bias, and secretory IgA responses in saliva, feces, and lung samples. Neutralizing IgG antibodies to alpha toxin were detected using in vitro and in vivo assays, and a toxin challenge established protection. Mice immunized nasally or orally with recombinant spores were protected against a challenge with 12 median lethal doses of alpha toxin. Existing use of spores as competitive exclusion agents in animal feeds supports their use as a potentially economical and heat-stable vaccine for the poultry industry.
authors.affiliation: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom.
authors.forename: Tran H
authors.initials: TH
authors.lastname: Hoang
authors.forename: Huynh A
authors.initials: HA
authors.lastname: Hong
authors.forename: Graeme C
authors.initials: GC
authors.lastname: Clark
authors.forename: Richard W
authors.initials: RW
authors.lastname: Titball
authors.forename: Simon M
authors.initials: SM
authors.lastname: Cutting
mesh_terms: D000284:Administration, Oral; D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood / Q000276:immunology; D000942:Antigens, Bacterial / Q000276:immunology; D001412:Bacillus subtilis / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000276:immunology*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005738:Gas Gangrene / Q000517:prevention & control*; D051379:Mice; D008807:Mice, Inbred BALB C; D011201:Poultry Diseases / Q000517:prevention & control*; D010738:Type C Phospholipases / Q000235:genetics / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D002135:Calcium-Binding Proteins; D010738:Type C Phospholipases; C040649:alpha toxin, Clostridium perfringens
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