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

Abstract

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.

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression

“…Abbreviations and regulatory elements for plants generated from different plasmids are given in Table II. The plasmids pLD VK1, pLD-Ctv-5CP, and pLSLF CTB-Pins used to generate Nt-Nt-PA, Nt-Nt-CP, and Ls-g10-CP plants have been reported previously (Koya et al, 2005;Ruhlman et al, 2007). In pLD-VK1 (Koya et al, 2005) and pLD-Ctv-5CP (Ruhlman et al, 2007), pagA and CTB-Pins, respectively were regulated by the tobacco psbA promoter and 5# and 3# UTRs.…”

TUBERCULOSIS AND BRUCELLOSIS IN WOOD BISON (BISON BISON ATHABASCAE) IN NORTHERN CANADA: A RENEWED NEED TO DEVELOP OPTIONS FOR FUTURE MANAGEMENT

“…Other countries, realizing the benefits of vaccine development as a viable tool for managing bTB, are investing targeted resources to that end (Gortazar and Boadella 2014;Sheridan 2014) with effective vaccines developed in a 5-10-yr time frame for European badgers and brush-tailed possums. Potential also exists for vaccination against other important bison diseases, such as anthrax (Koya et al 2005;Gorantala et al 2011). …”

Transgene containment by maternal inheritance: Effective or elusive?

“…Therefore, up to 360 million doses of fully functional anthrax protective antigen expressed in tobacco chloroplasts could be harvested from a single acre of this nonfood/nonfeed crop (8). Several vaccine antigens against bacterial (8-10), viral (11, 12), or protozoan (13) pathogens and human therapeutic proteins (14)(15)(16)(17) have already been developed in transgenic tobacco chloroplasts (Table 1).…”