| The Flavivirus genus consists of more than 70 viruses, many of which are arthropod-borne human pathogens. Japanese encephalitis (JE) is a mosquito-transmitted virus disease that causes 1 case of encephalitis per 10,000 of the world's population and 5~40% deaths , 20~40% sequelae of nerve injury annually. There are several thousand cases annually in China exclude Xinjiang, Tibet, Qinghai which enormously threat people`s health, and JE is defined B infectious diseases by the Ministry of Health.The flavivirus genome has a feature that is the virus RNA is capable of self-replication and capable of infection. When naked RNA is delivered into the cells susceptible for RNA replication, it can self-replicate and translate the corresponding viral proteins, then package into the entire virions in the infectious cells. In the flavivirus replicons, structural protein genes can be partially deleted, and the nonstructural protein genes are remained, thus the subgenome RNAs remain self-replication and are capable of expressing not only the nonstructural but also the remaining structural and/or additional heterologous genes. Theoretically, there are no difference in the replication between the subgenome RNA and the entire genome RNA. The positive-strand RNA can replicate exponentially. When the heterologous genes are inserted into the replicon, amplification of replicon RNA in the cytoplasm of cells makes the heterologous genes express effectively, as a consequence, the replicon RNA can be made the excellent vector for expression of heterologous genes. Nowadays, Replicons of Flavivirus KUN, DEN and YFV, have been described and are being pursued as potentially useful tools for the development of novel vaccines. In this study, we tried to construct the subgenomic replicons of JEV based on the JEV attenuated live vaccine SA14-14-2, and then to construct the JEV replicon system to develop the more new vaccines.We constructed serial of pMW-G2R~ pMW-G4R JEV replicons. The main differences are the types of deletion in the structural protein genes and the adding of MCS, FMDV-2A, et al. among JEV replicons. Stability of the replicons is a major problem in constructing replicons, and we selected several plasmid vectors to make the backbone vectors of the replicons. In order to improve the stability of the replicons, finally we used a very low copy and very stable bacteria plasmid vector pMW-118. In addition, CMV promoter was added the replicon to make our work easy and to improve the expressing, thus we constructed the JEV replicon pCMW-2M. It is confirmed the stability of JEV replicon pCMW-2M during propagation in E. coli strain Top10 at 30℃to have no mutation, insertion nor deletion in the recombinants by sequencing.The expression of virus proteins in transfected BHK-21 cells was analyzed using an indirect immunofluorescent analysis (IFA). The antigens localized mainly in the cytoplasm. These antigen positive-cells showed a similar morphology and viability in comparison with mock cells. Any apparent cytopathic effects (CPE) were not observed in the JEV antigen-positive cells. The difference of approximately 1%~30% of the IFA-positive cells exists among the cells transfected by pMW-G2R~pMW-G4R and pCMW-2M. Thees results show the entire C protein, CMV promoter and the correct expressing of nonstructural genes are the important factors of the expression of replicons.We inserted enhanced green fluorescent protein (EGFP) and luciferase (Luc) reporter gene into JEV replicon pCMW-2M, respectively. Expression of EGFP in BHK-21 cells transfected with p2MEGFP was determined from 24hr post-transfection. EGFP was observed. Morphology and viability of positive cells were not distinguished from mock BHK-21 cells and any CPE was not observed. EGFP positive cells were observed at 72hr post-transfection, whereas the expression was very few at 24hr post-transfection. Just like these, Expression of the luciferase gene could be detected till 7d post-transfection, and the luciferase signals increased exponentially during 24-96hr post-transfection. These results indicate that the JEV replicon inserted foreign gene can effectively express foreign gene.JEV replicon pCMW-2M and pCMW-G2R were evaluated for immunogenicity in mice. Three immunization of 6-week-old female BALB/c mice with JEV replicons pCMW-2M by intramuscular injection at a dose of 50μg per mouse elicited anti-JEV antibody at titers of 1: 1270 and neutralizing (NEUT) antibodies at titers of 1:32(50% plaque reduction) a little higher than with JEV replicon pCMW-G2R. The stimulate index of T lymphocytes (SI) was 2.8 in group pCMW-2M, a little higher than group pCMW-G2R. These rusults show JEV replicons pCMW-2M and pCMW-G2R can induce the antibody and cell immune responses after immunization of mice. In addition, the passive protecton following challenge with JEV demonstrates that the serum of mice immunized by JEV replicons pCMW-2M and pCMW-G2R can effectively protect about 70% mice against challenge of JEV.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. 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 an important area.The fourth domain of PA gene was inserted into JEV replicon pCMW-2M, JEV replicon DNA inserted PA4 gene induced antibody and cell immune responses after immunization of mice.The antibody titers reached to 1: 36200 after the third immunization and the stimulate index of T lymphocytes was 2.6. It is the first research on construction of JEV replicative DNA vector expressing PA from Bacillus anthracis.The domainⅢprotein of Yellow Fever Virus(YFV) was expressed and purified to research on the possibility of developing the DⅢprotein as a subunit vaccine against the YFV and JEV. The recombinant protein was unique in forming a large fraction of the soluble recombinant protein in E. coli. and 50% of total proteins is YFV DⅢ.Rabbits and mice were immunized with the putified DⅢprotein. The antigenicity and immunogenicity of the purified DⅢprotein was tested by Western Blot analysis and ELISA. Rabbits immunized with the purified YFDⅢprotein generated 1:4×105 anti-YFV antibody titers and1:2×104 anti-JEV antibody titers. Mice immunized with the purified YFV E DⅢprotein generated1:7×104 anti-YFV antibody titers and1:2×103 anti-JEV antibody titers . Overall, this study highlighted that recombinant YFV E DⅢprotein delivered in mice and rabbits can generate high antibody titers against YFV, JEV. In addition, the passive protecton following challenge with YFV demonstrates that the serum of mice immunized by YFV E DⅢprotein can effectively protect against challenge of YFV. So it is possible that the recombinant YFDⅢprotein can serve as a potential subunit vaccine, and it is the first E.coli.-synthesized YFDⅢprotein and proved good immunogenicity and protective effect.In conculsion, we successfully constructed new JEV replicon vectors, improved systematic research on the construction, the capacity of expression self and foreign proteins, the immunogenicity of the replicons based on JEV attenuated live vaccine SA14-14-2. These results demonstrate the capacity of delivering the heterologous genes as a good vector, provide the important data for studying the roles of structural proteins and nonstructural proteins in the replication and packaging of virus, provide an assistance for deep research the immunogenicity and protective effect of nonstructural proteins( mainly NS1 protein), establish the foundation of research on the new JEV replicative DNA vector expressing PA from Bacillus Anthracis including divalence or multivalence replicative DNA vectors, as well as establishing a valuable technique platform for further developing the other new DNA vaccines by using the JEV replicon as a vector. |
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