Departmental Papers (Dental)

Document Type

Journal Article

Date of this Version

10-22-2004

Publication Source

Vaccine

Volume

22

Issue

31-32

Start Page

4374

Last Page

4384

DOI

10.1016/j.vaccine.2004.01.069

Abstract

The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene (pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25 μg functional PA/ml. With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field.

Copyright/Permission Statement

© <2004>. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/

Comments

At the time of publication, author Henry Daniell was affiliated with the University of Central Florida. Currently, he is a faculty member at the School of Dental Medicine at the University of Pennsylvania

Keywords

Bioterrorism, GM crops, Anthrax vaccine

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Date Posted: 01 March 2022

This document has been peer reviewed.