Development Of Vaccine Research Derived From JEV Replicon Expression Vector System

Japanese encephalitis virus (JEV) is an emerging threat throughout Southern and Southeast Asia and Australia. Although most JEV infections are asymptomatic, the estimated 0.3% that lead to disease result in over 35,000 cases including 10,000 deaths annually worldwide, and many of the remaining cases produce permanent sequelae. Japanese encephalitis virus is one of globally important human pathogens. Despite an emergence and resurgence of JEV-mediated disease,specific therapies are not yet available.Subgenomic replicons of positive-stranded RNA viruses , which contain all the non-structural proteins required for amplifying themselves but lack some or all of the structural proteins,have been shown to be useful tools to study viral replication in the absence of virion assembly and maturation。Several of the flaviviruses, including JEV, display a number of characteristics that make them useful for the development of delivery vectors to express foreign genes。In the past few years, heterologous genes have been expressed in the context of (i) an infectious virus genome, in the case of WNV and TBEV; or (ii) a replicon lacking viral structural proteins (C, prM, and E), in the case of several flaviviruses, including DENV,YFV, WNV,KUNV and TBEV. JEV has not been used for this purpose to date. Here we report three strategies for utilizing JEV replicon vectors to express foreign genes in a variety of cell types, taking advantage of a full-length infectious JEV cDNA that we have previously constructed. These JEV replicon expression systems can potentially serve as powerful tools in both in vitro and in vivo applications. The large packaging capacity of JEV offers a distinct advantage with regard to expressing larger genes and the treatment of associated diseases.We report the construction of subgenomic replicons derived from a full-length cDNA clone of the SA14-14-2 strain of JEV. Using multiple strategies, various reporter genes including green fluorescence protein (GFP) and Lac Z were expressed from these replicons in a DNA replication-dependent manner. That expressed the JEV structural proteins were also constructed and could be used to package JEV replicons into pseudo-infectious virus-like particles (PIPs). We inserted enhanced green fluorescent protein (EGFP) and Lac Z reporter gene into JEV replicon pCMW-2M, pCMW-G2R respectively,expressions of EGFP in BHK -21 cells transfected with pCMW-2MEG were determined from 24h post-transfection. EGFP was observed in cytoplasm. EGFP positive cells were seen at 72h post-transfection, whereas the expression was very few at 24h post-transfection. These results indicate that pCMW-2MEG can express foreign gene of EGFP. Just like these, Expression of the Lac Z gene could be detected at 4 days after transfection. The 53-kDa envelope (E) glycoprotein of JEV has an important role in virus adhesion and entry into target cells through receptor binding and, therefore, in inducing neutralizing antibodies that protect hosts against JEV infection. The higher expression of the E-VLP antigen should be more toxic to the expressing cells, resulting in cell fusion, apoptosis, and death.The presence of the capsid protein was found to be absolutely required for packaging. Moreover, the ability to efficiently package JEV replicons indicates that any potential RNA sequences within JEV genome, which might function as packaging signals, are not contained within the JEV structural protein coding sequence.Based on the infectious clone of JEV vaccine strain SA14-14-2, prM-E genes and C-prM-E genes were cloned into pCDNA3.1 vectors. The recombinant plasmid pCJEME was transfected into BHK-21 cells, the expressed proteins were toxic to the cell growth and accelerated the cell death. But when transfected with the plasmid pCJECME, the cell lines continuously expressing structural proteins can be selected with G418. And the expression products of pCJECME vector could be detected by ELISA,Western Blot and IFA assay. It shows that the JEV capsid protein can enhance the stability of the cell lines expressing the structural proteins. The established cell lines can make the acquirement of the virus-like particles much easier.To facilitate our future investigations of virus assembly, expressing the JEV structural proteins were constructed and used to package JEV replicons into PIPs. With the application of recombinant DNA technology and reverse genetics, PIPs of several flaviviruses thus established have been shown to be valuable for studying viral replication and inhibitors and promising for developing vectors of gene therapy and vaccines。In this study, we have established a cell line stably producing structural proteins of JEV, which can successfully incorporate JEV replicon and produce single-round infectious PIPs. A closer examination of the expression of replicon and the infectivity of PIPs containing replicon revealed that the infectivity of PIPs correlated with the expression of the replicon, The highest titer of JE PIPs in this study, was 1.6×105 U/ml.After immune with JE PIPs induced specific antibody after immunization of mice.The antibody titers reached to 1:2560 after the twice immunization,Further improvement in the efficiency of RNA transfection is needed to increase the titers of our PIPs.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. The fourth domain of PA gene was cloned into JEV replicon DNA vector, then transfect packaging cell lines with and obtain the PIPs with high titers of 5×105U/ml. The PIPs can infect cell in vitro and induced 1:1280 titer antibody after immunization of mice.The PA4 PIPs based on JEV replicon are prominsing candiant vaccines for human use and establish the foundation for deep reseach of multi-antigen vaccine of anthrax.Here we report the development of a JEV-based vector system for foreign gene expression that utilizes a full-length infectious JEV cDNA. involved a variety of replication-competent, propagation-deficient viral replicon vector DNAs showing a spectrum of DNA replication efficiencies. The other procedure made use of stable packaging cell lines for the production of single-round infectious, propagation-deficient JEV PIPs. These data strongly suggest that our JEV replicon vector systems represent attractive potential candidates for foreign gene expression and antiviral compound screening in a wide variety of cells in vitro,and possibly in vivo for immunization applications.The E protein of Japanese encephalitis virus (JEV) is the major antigen used to elicit neutralizing antibody response and protective immunity in hosts. In this study, the domain III protein of the attenuated strain SA14-14-2 was cloned to the pET32a expression vector and expressed as a thioredoxin (Trx) fusion protein in Escherichia coli. The recombinant protein was unique in forming a large fraction of the soluble recombinant protein in E. coli. The antigenicity and immunogenicity of the purified DⅢprotein was tested by Western Blot analysis .The purified domain III protein was emulsified in Freund's adjuvant (FA) for immunization in mice and rabbits. After 3 times immunized,Rabbits'serum generated 1:4×105 anti-JEV antibody titers .Mice'serum generated 1:7×104 anti-JEV antibody titers and1:2×103 anti-YFV antibody titers .Then resulted in eliciting neutralizing antibodies and protective immunity in ICR mice. These studies can provide useful information for further developing the domain III recombinant protein vaccine against JEV.