Construction And Immunogenicity Evaluation Of Janpanese Encephalitis Virus RNA Vaccine

Japanese encephalitis virus(JEV)is a zoonosis pathogen,which can cause Japanese encephalitis(JE)after infection,with a fatality rate of 30%.In addition to causing serious harm to human life and health,JEV can also infect pigs and cause reproductive disorders,resulting in significant economic losses to the pig breeding industry.Currently,the vaccines against JEV in the market include inactivated and live attenuated vaccines,in which inactivated vaccines have disadvantages such as poor immunogenicity and high side effects due to multiple doses of vaccination,while live attenuated vaccines have the potential threat of virulence enhancement.Therefore,there is an urgent need to develop a new type JEV vaccine with safety and good immunogenicity.E protein is the envelope glycoprotein of JEV and the main antigenic protein for the induction of neutralizing antibodies.The third domain(E domain,EDⅢ)of this protein contains the main neutralizing epitope.pr M protein is essential for proper folding,membrane binding,and assembly of the E protein and is an important cooperative component of virusinduced protective immunity,so the pr M-E fusion protein may be more immunologically advantageous.RNA vaccines such as mRNA and CircRNA vaccines have significant advantages over other new vaccines,such as unintegrated into the genome,carrier-free immunogenicity and easier to produce with high purity.Therefore,in this study,pr M-E and EDⅢ of JEV were used as antigen proteins to study the preparation and immunogenicity of mRNA and CircRNA vaccines.The details of the study are as follows:1.Construction and expression verification of mRNA vaccine encoding JEV pr M-E gene.The sequences of UTR,poly A,Kozak and signal sequence were introduced into p GEM-3zf(+)vector by molecular cloning technique,and the pr M and E genes of JEV were selected as antigen genes.The plasmid p GEM-IVT-pr M-E encoding pr M-E gene was constructed and the corresponding mRNA(pr M-E-mRNA)was obtained by in vitro transcription.Western blot assay showed that HEK-293 T cells transfected with pr M-E-mRNA could effectively express pr M and E proteins,and could secrete to the supernatant.At the same time,it was found that modified nucleotides could reduce the activation of pr M-E-mRNA to cellular inflammation.Finally,lipid nanoparticles(LNP)and pr M-E-mRNA were mixed with microfluidic platform to prepare LNP-encapsulated mRNA vaccine(LNP-pr M-E-mRNA).2.Immunogenicity of JEV LNP-pr M-E-mRNA vaccine and evaluation of its protective effect in mice.LNP-pr M-E-mRNA vaccine was administered intramuscularly to 6-week-old C57BL/6female mice,and the control groups were respectively injected with LNP,attenuated live vaccine SA14-14-2 and DMEM.After a booster immunization,blood was collected from the mice and neutralizing antibody levels were measured by plaque reduction neutralization assay.The result showed that the level of neutralizing antibody in serum of mice immunized with mRNA and attenuated live vaccine was significantly improved.Subsequently,the results of ELISA and flow cytometry experiments showed that the level of IFN-γ in serum and the number of CD8+T cells in spleen of mice immunized with mRNA vaccine increased significantly,which proved that mRNA vaccine could also activate cellular immune response.Then the mice were challenged with lethal dose of JEV virulent strain.The results showed that mice injected with LNP and DMEM showed typical clinical symptoms on day 5 after challenge,and all of them died,while mice immunized with mRNA and attenuated live vaccine only showed mild clinical symptoms and finally 100% survived.Moreover,compared with mice injected with LNP and DMEM,the viral load and inflammatory factors in the brain of mice immunized with mRNA and attenuated live vaccine were significantly reduced.This indicates that the mRNA vaccine with pr M-E as the antigenic protein has good immunogenicity and virus protection effect.3.Construction and expression verification of CircRNA vaccine encoding JEV EDⅢ gene.In this study,the modified class I intron of Anabaena was used as a cyclization strategy,and IRES,spacer sequence and Kozak sequence were introduced into the vector pcircRNA to construct an efficient CircRNA vector.Considering the efficiency of cyclization,we chose JEV EDⅢ as the antigen,which has a short sequence but contains more neutralizing epitopes.The methods of formaldehyde denaturing gel,reverse transcription,and sequencing proved that the construction of CircRNA encoding JEV EDⅢ(EDⅢ-CircRNA)was successful.Subsequently,Western blot and indirect immunofluorescence experiments proved that EDⅢ-CircRNA could effectively express EDⅢ protein in HEK-293 T cells and the CircRNA had good stability.Finally,LNP was mixed with EDⅢ-CircRNA to prepare LNP-encapsulated CircRNA vaccine(LNP-EDⅢ-CircRNA).4.Immunogenicity study of JEV LNP-CircRNA vaccine and protective effect in mice.LNP-EDⅢ-CircRNA vaccine was administered intramuscularly to 6-week-old C57BL/6female mice,and the control groups were injected with LNP,and attenuated live vaccine SA14-14-2.After a booster immunization,blood was collected from the mice and the neutralizing antibody level was measured by plaque reduction neutralization assay.The results showed that the neutralizing antibody level in serum of mice immunized with attenuated live vaccine was significantly improved,while the neutralizing antibody level stimulated by CircRNA vaccine was relatively low.In the challenge experiment,LNP-injected mice showed typical clinical symptoms and eventually all died.The overall symptoms of mice immunized with CircRNA vaccine were milder than those of LNP-injected mice and the final survival rate was 40%.Moreover,compared with LNP injected mice,the viral load in the brain of mice immunized with CircRNA vaccine decreased to some extent.The above results showed that the CircRNA vaccine encoding EDⅢ antigen still needs further improvement and optimization to improve its immunogenicity and protective effect.In summary,the recombinant plasmid containing complete elements was constructed to prepare mRNA and CircRNA in this study.The RNA vaccine was prepared and its protective effect on mice was evaluated.The study lays a foundation for the subsequent development of safer and more effective Japanese encephalitis virus RNA vaccine.