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Infection and Immunity, November 2008, p. 5257-5265, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00686-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Recombinant Bacillus subtilis Expressing the Clostridium perfringens Alpha Toxoid Is a Candidate Orally Delivered Vaccine against Necrotic Enteritis{triangledown}

Tran H. Hoang,1,{dagger} Huynh A. Hong,1,{dagger} Graeme C. Clark,2 Richard W. Titball,3 and Simon M. Cutting1*

School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom,1 Defence Science and Technology Laboratory, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom,2 School of Biosciences, University of Exeter, Exeter, Devon EX4 4QD, United Kingdom3

Received 31 May 2008/ Returned for modification 1 August 2008/ Accepted 27 August 2008

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-Cpa247-370 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-Cpa247-370 on the spore surface showed the most striking responses. This included seroconversion with anti-Cpa247-370-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.

* Corresponding author. Mailing address: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom. Phone: 44-(0)1784-443760. Fax: 44-(0)1784-414224. E-mail: s.cutting{at}rhul.ac.uk

{triangledown} Published ahead of print on 8 September 2008.

Editor: S. R. Blanke

{dagger} These authors contributed equally to this work.

Infection and Immunity, November 2008, p. 5257-5265, Vol. 76, No. 11
0019-9567/08/$08.00+0     doi:10.1128/IAI.00686-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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