Development Of Botulinum Neurotoxin And Anthrax Vaccine Derived From The SFV Replicon Expression Vector System

The clostridial botulinum neurotoxins (BoNT) produced by Clostridium botulinum are the most toxic substances known to science, botulinum toxin has the extremely potential to be used as a terrorist or biological warfare agent. These toxins can be divided into Seven serotypes, designated A through G, which are structurally similar, yet antigenically distinct. A safe and effective vaccine is the only effective measure to prevent BoNT intoxication.The SFV replion DNA vaccine against BoNT serotype A have been developing in our lab,but it's immunogenicity is relatively low for human use ,so several sreateges to optimize this DNA vaccine were accomplished as followed:Three different type CpG sequences were added to the vaccine backbones as bluid-in adjuvants;GM-CSF gene adjuvants were administrated with the DNA vaccine by three different deliver ways;Conventional aluminium adjuvants were applied to enhance the immunogenicity of the DNA vaccine, the antibody level after immunization were detected monthly for one year.The CpG, GM-CSF and aluminium adjuvants were all the first applications to DNA vaccines of BoNT as reports. After immunization of Balb/c mice, the AHc-specific ELISA antibodies and lymphocyte proliferative responses and cytokines induced by DNA vaccine and adjuvants were determined. The antibody and cell immune responses were improved in defrent extend and the antibody level last one year. BoNT/A challenge test confirmed that CpG, GM-CSF and aluminium adjuvants improveded the survival rates after challenge with 104LD50 BoNT/A. So the optimation strateges were effective and could be used to the other vaccine conveniently, the technology platform of SFV replion vaccine reseach were upgrated after optimation.The DNA vaccine based on the SFV system against BoNT serotype B were also constructed, the antibody titers reached to 1:6400 after immunizations, 75% mice survivaled after challenge with 103LD50 BoNT/B.The BoNT/B DNA vaccine based on the SFV replicon were effective and promising candidate vaccine for human use, at the same time establish the foundation for multi-antigen vaccine aganst BoNT and other disease.The fetal disease anthrax is caused by the spore-forming, gram-positive bacterium Bacillus anthracis, and Bacillus anthracis has been postulated to be a potential agent of biowarfare and bioterrorism. The anthrax toxin includes three factors: Protective antigen(PA),Lethal factor(LF) and Edema factor(EF), PA is the main composition of current vaccine and the most effective immunogen to induce immune response. In order to develop safe and effective vaccine againt anthrax, DNA vaccine is a important area.The fourth domain of PA gene was cloned into SFV replicon DNA vector, the PA4 DNA vaccine based on SFV replicon induced antibody and cell immune responses after immunization of mice.The antibody titers reached to 1:25600 after the third immunization. The CpG build-in optimation strateges were applied to the PA4 DNA vaccine based on SFV replicon, the antibody titers were enhanced 2-4 fold after immunization, so the CpG build-in adjuvants were effective in PA4 DNA vaccine based on SFV replicon. Meanwhile the PA4 DNA vaccine can transfect cell with helper system and obtain the RVP with high titers of 107-8U/ml. The RVP vaccine can infect cell in vitro and induce high titer antibody after immunization of mice.The PA4 DNA vaccine and RVP vaccine based on SFV replicon are prominsing candiant vaccines for human use and establish the foundation for deep reseach of multi-antigen vaccine of anthrax.Together, these results showed that our optimation strateges are effective to improve the immunogenicity and survival rates of SFV replicon DNA vaccines.The SFV replicon DNA vaccines and RVP vaccine aganst BoNT/A, B and anthrax could be good candidate vaccines for human use.Our work also provides a valuable technique platform for further developing other multi-antigen vaccines of BoNT and the other bacteria and virus vaccines.