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Infection and Immunity, December 2005, p. 8266-8274, Vol. 73, No. 12
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.12.8266-8274.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge

Vijay Koya,1 Mahtab Moayeri,2 Stephen H. Leppla,2 and Henry Daniell1*

Department of Molecular Biology and Microbiology, University of Central Florida, Biomolecular Science Building 20, Room 336, Orlando, Florida 32816-2364,1 Microbial Pathogenesis Section, National Institute of Allergy and Infectious Diseases, NIH, Building 30, Room 303, 30 Convent Dr., Bethesda, Maryland 20892-43492

Received 26 June 2005/ Returned for modification 11 August 2005/ Accepted 4 September 2005

The currently available human vaccine for anthrax, derived from the culture supernatant of Bacillus anthracis, contains the protective antigen (PA) and traces of the lethal and edema factors, which may contribute to adverse side effects associated with this vaccine. Therefore, an effective expression system that can provide a clean, safe, and efficacious vaccine is required. In an effort to produce anthrax vaccine in large quantities and free of extraneous bacterial contaminants, PA was expressed in transgenic tobacco chloroplasts by inserting the pagA gene into the chloroplast genome. Chloroplast integration of the pagA gene was confirmed by PCR and Southern analysis. Mature leaves grown under continuous illumination contained PA as up to 14.2% of the total soluble protein. Cytotoxicity measurements in macrophage lysis assays showed that chloroplast-derived PA was equal in potency to PA produced in B. anthracis. Subcutaneous immunization of mice with partially purified chloroplast-derived or B. anthracis-derived PA with adjuvant yielded immunoglobulin G titers up to 1:320,000, and both groups of mice survived (100%) challenge with lethal doses of toxin. An average yield of about 150 mg of PA per plant should produce 360 million doses of a purified vaccine free of bacterial toxins edema factor and lethal factor from 1 acre of land. Such high expression levels without using fermenters and the immunoprotection offered by the chloroplast-derived PA should facilitate development of a cleaner and safer anthrax vaccine at a lower production cost. These results demonstrate the immunogenic and immunoprotective properties of plant-derived anthrax vaccine antigen.

* Corresponding author. Mailing address: Dept. Molecular Biology & Microbiology, Biomolecular Science Bldg 20, Room 336, University of Central Florida, Central Florida Blvd., Orlando, FL 32816-2364. Phone: (407) 823-0952. Fax: (407) 823-0956. E-mail: daniell{at}mail.ucf.edu.

Editor: J. D. Clements

Infection and Immunity, December 2005, p. 8266-8274, Vol. 73, No. 12
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.12.8266-8274.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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