Prophase Research On The Genetic Engineering Combined Vaccine Of Anthrax And Plague

Anthrax and plague are both acute infectious diseases with natural foci. Bacillus anthracis and Yersinia pestis, the etiological agent of anthrax and plague, are the preferred biological terrorism / biological warfare agents. At least three nations (the United States, Russia, and Japan) are known to have developed Bacillus anthracis and Yersinia pestis as biological warfare agents, the mail attacks with B. anthracis spores in the United States on October 2001 underscored the need for better public preparedness for the possible use of bioweapons, there is a wide range natural foci of anthrax and plague in China, we would be stimultaneous up against the threats of anthrax and plague in peacetime and wartime. The abuse or misuse of antibiotics has led to the emergence of multiple antibiotic resistant bacteria. Therefore the importance of infection prevention becomes more and more obvious. A live-attenuated anthrax vaccine, AVA, a killed whole cell plague vaccine and a live-attenuated plague vaccine have been used in humans against the two diseases. However, there have been concerns about the side effects of these vaccines. In the area of new vaccine development, a recombinant PA-based anthrax vaccine and subunit plague vaccines using F1 and V antigens have been approved for use in clinical trials. The protective antigens of Bacillus anthracis and Yersinia pestis are definite, the combined vaccine of anthrax and plague can provide advantages such as simplified the vaccination regimens, increased user compliance, lower production costs, saved human resources for immunization execution, and provide guarantee for the public health emergencies due to the anthrax and plague. The objective of our study is to prepare the protective antigens of plague with genetic engineering technology, evaluate the immune efficacy of the recombinant plague vaccine when co-administered with the sub-unit anthrax vaccine, and lay a foundation for development of the genetic engineering combined vaccine against anthrax and plague.The bacterium Escherichia coli is one of the most popular host for producing recombinant proteins. In addition to its simplicity, safety, and known genetic properties, the major advantage of E.coli is its ability to produce proteins in large quantities and to grow very quickly compared with mammalian cells, which enables excellent space/time yields. Previously, we had prepared recombinant non-tagged PA proteins in Escherichia coli, considering the prokaryotic cell origin and the suitable molecular weight of F1 and V, the thesis selects the E.coli system to high-level express the F1 and V antigen. The hydrophobicity of F1 makes a challenge to the expression and purification. Other study have expressed V antigen as a fusion with glutathione S-transferase (GST) in Escherichia coli and get the protein with affinity purification. However, removal of the GST tag from the target protein is often necessary. In our opinion, the non-tagged recombinant F1 and V were the best choice because of possible advantages of low cost, and ease of quality control. After a number of attempts, we expressed the F1 and V by pET-32a (+) vector in Escherichia coli BL21 (DE3). Results showed that the F1 and V were high level expressed in the E. coli cytoplasm.After scale-up and optimization of both cell culture and fermentation processes in 14L and 42L bioreactor, we get the optimized fermentation process parameters. We perform three check tests on the optimized parameters. The results showed that the fermentation process is stable with a good repeatability. And then we separated proteins according to their different properties. The purification process of V antigen containing connective hydrophobic chromatography, ion exchange chromatography and gel filter chromatography was convenient, economical and robust. The V antigen was captured in the first step and further purified in the second step and finally desalted to suit to the demand. After three steps of purification, rV of up to 95% purity was obtained. The results of western blot showed that V antigen exist as both monomeric and dimeric forms because of a single Cys. Previous research reports that rF1 antigen exists as a multimer of high molecular mass, and mice immunised with monomeric rF1 were significantly less well protected against challenge with Y. pestis than mice immunised with multimeric rF1. In this study, the rF1 was purified by ammonium sulfate fractionation followed by the size exclusion chromatography of HiPrep Superdex 75 column. rF1 of up to 95% purity was obtained and exists as monomer and multimer. The N-terminal sequence analysis of rV and rF1 proved that the N-terminal sequence of the purified F1 and V was identical to the mature F1 and V antigens from Y. pestis.Furthermore, the immunogenicity of the various antigens formulation adsorbed to an aluminum hydroxide adjuvant was evaluated in mice, guinea pigs and rabbits models, humoral immune responses of the combined vaccine were observed and compared with the separate vaccine. Results showed that immunized rF1+ rV and rPA antigen together was as effective as separately for induction of serological antibody response in the three animal models, the antigen-specific IgG antibody can be assayed from 1-2 weeks serum samples post first immunization, which achieved high levels after the booster dose. An isotype analysis of the serum indicates that the co-administration of these antigens did not influence the antigen-specific IgG1/IgG2a ratio which was consistent with a Th2 bias. These IgG antibody titers were maintained for over one year in mice, suggested that the combined vaccine has a good long-term effect.According to the immunogenicity data, we selected mice and guinea pigs which are the sensitive animal model of plague to evaluate the protective efficacy against plague, and rabbits which are the sensitive animal model of anthrax to evaluate the protective efficacy against anthrax. All rabbits immunized the combined vaccine were fully protected at a challenge dose of 80LD50 virulent B. anthracis spores, rabbits of the adjuvant control group all died in 6 days. All mice immunized with the combined vaccine were fully protected 600,000MLD Y.pestis challenge, and mice of the adjuvant control group all died in 4 days. The protective efficacies of the combined vaccine were no less than the separate vaccine in these three animal models.In the present study, we prepared recombinant non-tagged F1 and V protective antigen of plague with genetic engineering technology, evaluated the immunogenicity and protective efficacy of the combined vaccine in mice, guinea pigs and rabbits models. We demonstrated that immunized rF1, rV and rPA antigen together was as effective as separately for induction of serological antibody response. Protection studies suggest that the protective efficacy was not impaired when the antigens were administered in combination. The combined vaccine can protect animals against subcutaneous challenge Bacillus anthracis and Yersinia pesti respectively. These data provide some preliminary evidence for the development of a combinatorial vaccine against anthrax and plague.