Preparation Of Replicative Alphavirus Particle Vaccine And Dc-targeting Preliminary Research On Alphavirus Particle Vaccine Vector System

In recent years, a kind of new vector system which called DNA replicon vaccine has already been developed. The safety and efficacy of DNA vaccine could be enormously improved by this novel vector system. Meanwhile the DNA replicon vaccine still maintains the stability and the low cost of the tranditional DNA vaccine. So the neotype vector system would be one of the best technology to develop therapeutic vaccine。We have previously developed SFV-based replicon DNA vaccine vector system for the research of new therapeutic DNA vaccines such as tumor vaccine, HBV vaccine and so on. The replicon DNA vaccine was developed based on replicon RNA vaccine. Due to the presence of the replicase genes, the RNA replication will result in superior gene expression compared to conventional DNA vaccine. Moreover, during the RNA self-replication process, double-stranded RNA was produced which proved to be a natural potent immunity adjuvant. So the dosage of replicon DNA vaccine may be reduced below 100 times. Otherwise the self-replication and transcription of the replicon DNA vaccine occurred in the cytoplasm and eventually induced the apoptosis of transfected cells, which could eliminate the risk of integration into the host cell genome and greatly improve the safety of DNA vaccine. Moreover, such vaccines could stimulate the immune system by a pulse form, which can effectively overcome the immune tolerance.In order to improve the efficiency of the vaccine antigen delivery and immune effects, we will prepare Alphavirus-like particle vaccine, while attempts to engineer the surface of Alphavirus-like particle to target DC. The virus-like particle could not only protect replicon DNA from degradation of nuclease but also enhance the efficiency of antigen transfected into cells. And the virus-like particle itself also had immunological characteristics. Meanwhile, after the engineering of DC targeting the recombinant virus particle vaccine can selectively infect DC in vivo with remarkable efficiency and specificity, thus the Alphavirus-like particle can futher raise the DCs'role of uptaking and presenting antigens , accordingly reinforce vaccines'immune effective.Firstly, the aim of the present study was to obtain recombinant virus particles that can express red and green fluorescent protein. Recombinant virus particles are produced by co-transfection of cells with the replicative DNA expression vector pSCK-EGFP-IRES-dsRED, which represents the alphavirus genome in which the region coding for structural proteins has been replaced by the gene of red and green fluorescent protein and the helper vector pSHCAR encoding for structural proteins. At the same time, we compared the expression level of red and green fluorescent protein gene of individual pSCK-EGFP-IRES-dsRED plasmids with the recombinant virus. By adjusting the ratio of expression vector and helper vector during cotransfecting, and calculating the virus titer, we take a preliminary exploration of the optimal experimental conditions of the virus packaging. Preliminary results show that the recombinant virus particles system has a better expression ability through detecting the red and green fluorescent protein expression. The preparation of the recombinant virus titer could reach 6.5×105 IU /mL. Additional, other experimental results obtained are: there will produce a higher titer of virus particles when co-transfected expression vector and helper vector at mass ratio of 1:1 or mole ratio 1:1.Secondly, based on the previously study above, we developed a research on recombinant virus particles package of HBV replicon DNA vaccine pSCK-HBV-Fc-GPI-IRES-GM/B7. HBV replicative virus particles vaccine were produced by co-transfection of BHK21 cells with replicative DNA expression vector pSCK-HBV-Fc-GPI-IRES-GM/B7 and helper vector pSHCAR, then the expression of HBV replicative virus particles was identified by flow cytometry and immunofluorescence. Preliminary results showed that HBV replicative virus particles vaccine could express in cells , and the recombinant virus titer could reach 1.5×105 IU /mL.Finally, we draw on the latest research abroad to attempt to engineer the Alphavirus-like particle vaccine targeting to DC. Here ,we established a new method of DC targeting through the DC-specific surface molecule called DC-SIGN (also known as CD209) by a recombinant SFV helper vector pSHCAR bearing an engineered glycoprotein gene derived from Sindbis virus. By this way, the virus particle vaccine would selectively infect DC, and furtherly improve the efficiency of antigen presentation. Preliminary results showed that the engineered helper vetor pSHCART could express successfully in cells . By RT-PCR detection methods, we found that the engineered helper vetor pSHCART have the ability of packaging virus.To facilitate our study of targeted engeering, we constructed BHK21 cell lines that stably expressed human DC-SIGN (BHK21/DCSIGN) gene. The DC-SIGN gene fragment was then digested with NotI and BamHI, and was cloned into an eukaryotic expression vector pIRES-neo to construct eukaryotic expression plasmid pIRES-neo-DC-SIGN. The recombined plasmid was identified with NotI and BamHI enzyme digestion and sequence test, and then the plasmid was transfected to BHK21 cells by LipofectamineTM2000. After screening by G418, BHK21 cells stably expressing DC-SIGN were established. The detection of flow cytometry showed that the expression ratio of DC-SIGN positive clones was close to 90%. The result of immunofluorescence display that the expression of DC-SIGN was mostly located on the surface of cell membrane. It showed that the BHK21 cells stably expressing DC-SIGN were successfully established. The successful establishment of BHK21 cell lines which stably expressed DC-SIGN provides a solid foundation for further study on the DC targeting vaccines.The final success of replicative Alphavirus particle vaccine targeting of DC may has unmatched technical superiority in the aspects of "granulation,endogenous technology, high efficiency, targeting" of presenting antigens. The application of neotype DC targeted virus like particle need to be researched deeply.