Study Of A Novel Anthrax Vaccine

Anthrax is an ancient disease, for which the causative bacterium, Bacillus anthracis, was the firstly and positively identified in bacteriology. Bacillus anthracis is a Gram-positive spore-forming, rod-shaped bacterium. The major virulence factor of Bacillus anthrax is a tripartite anthrax toxin, consisting of three proteins:protective antigen (PA), lethal factor (LF), and edema factor (EF). The three proteins are encoded by the virulence plasmid pXO1 and nontoxic individually. However, they act in binary combinations to form two active toxins:lethal toxin (PA plus LF) and edema toxin (PA plus EF). Once inside the body, PA binds to its cell surface receptor which is found on most cell types and is then cleaved at the consensus 164RKKR167 sequence by furin-like proteases. This cleavage generates a 20KDa amino-terminal fragment, PA20, which dissociates into the medium, and a 63KDa carboxy-terminal fragment, PA63, which self-assembles into a ring-shaped heptamer that inserts into the membrane to form a pore. Subsequently EF, LF or both binds to the PA63 heptamer and is translocated into the cytosol through the pore, where they exert a cytotoxic effect on the cell. Of the three proteins, EF, LF and PA, only PA elicits antibodies that are protective against the disease.Therefore, protective antigen-is a major immunogen of the vaccine against anthrax.In the past, PA was prepared from Bacillus anthracis, requiring stringent safety measures for culturing, and the removal of EF and LF residual impurities was difficult. Now E. coli provides an attractive alternative to produce rPA, and many researchers have expressed and purified PA from E. coli. In our study, a new expression system E. coli strain Rosetta 2(DE3) was used, which supported a high expression of PA without the need to resynthesize the PA gene with altered codon bias, whose gene contains 66 rare E. coli codons (9.0%of 733 total PA gene codons). The rPA-formed inclusion bodies in Rosetta 2(DE3) were washed by Triton X-100 and 2 M urea and solubilized in 5 M urea, followed by a 60% ammonium sulfate precipitation. Finally, the untagged rPA was efficiently and rapidly purified by single-step hydrophobic interaction chromatography using Phenyl Sepharose High Performance on an AKTA Purifier 10 system. The purity of purified rPA was determined by SDS-PAGE and reached 99%while the yield was about 13mg from an initial 1 liter of culture. The biological activity of rPA was determined by a toxin neutralization assay. rPA along with rLF in vitro was able to lyse the macrophage cells and therefore was biologically active.The rPA protein was combined with different treated spores or thallus to construct rPA-based vaccines and administered to Balb/c mice by intramuscular injection to assess their immunogenicity. The main conclusion includes:(1) After vaccinating with Formaldehyde-inactivated spores (FIS), the antibody titer, stimulating index of proliferation of splenic lymphocyte and ELISPOT positive conversion rate are superior to immunization with autoclaving or Cobalt 60-irradiated spores. (2) Immunization with rPA plus AI adjuvant can induce B cells to elicits antibodies but cannot promote the splenic lymphocyte to proliferate and elicit cytokine IFN-y. Therefore, rPA alone mainly induces the humoral immunity not the cellular immunity and resulting higher PA-neutralizing antibody titers can play an important role in the protection from anthrax. (3) Immunization with rPA plus different treated spores or thallus,both humoral immunity and cellular immunity of spores or thallus are strong, and spores or thallus can promote the cellular immunity of rPA, leading to rPA antigen-specific T lymphocyte that elicits cytokine IFN-γ. However, the rPA antibody titer was not stable.(4) Among the challenge of Balb/c mice with low virulent strain A16R, protective efficacy of rPA-based vaccines was almost 100% and after immunization with FIS alone protection was also 100%. However, protection was 0% after immunization with thallus alone in mice intraabdominal infected with twice the minimum lethal dose of strain A16R. Although the thallus had cross antigen of the spores, the cross and thallus-specific antigen did not protect mice against challenge.That is to say, the immune response against FIS is sufficient to protect against infection with A16R and the inclusion of killed spores can enhance the protective efficacy of a rPA-based vaccine. In summary, rPA plus FIS could be an appropriate vaccine component and the results provied a feasible paradigm for the study of a novel anthrax vaccine.