Expression And Immune Protective Effecacy Of Immunodominant Epitopes Of Japanese Encephalitis Virus

The Japanese encephalitis virus (JEV), which belongs to the family Flaviviridae,infects the central nervous system of humans and equines, and causes stillbirthsin swine. Japanese encephalitis virus (JEV) is transmitted to humans by persistently-infected mosquitoes and. is maintained in infected vertebrate reservoirs, especially in viremia-amplifying hosts such as swine and avian. In humans, JEV causes acute encephalitis with fatality rates ranging from 20% to as high as 50% and most of the survivors display persistent neurological or psychological sequelae.Swine is an important amplifier of Japanese encephalitis virus (JEV) in the paradomestic environmen.Thus mass vaccination of swine can prevent the disease in swine and help to prevent JE epidemics in humans, In the view of economics, the pig industry suffered serious losses from the viral infection as a result of the reproduce failure in sows and death in piglets. For vaccination, both inactivated and living-attenuated JEVs have been widely used with significant success in providing first-generation vaccines in many Asian countries. However, the former is limited by poor availability, high production cost, lack of long-term immunity, and the possibility of allergic reactions. Living-attenuated vaccine has the possible risk of reverting to virulence. Thus there is an urgent need to develop more effective vaccines and to alter the strategies against this viral disease. In recent years,many studies have demonstrated the efficacy of genetically engineered vaccine for JEV.In this study, we first constructed synthetic multi-epitope by synthesizing E epitopes (six amino acid residues 60-68,327-333,337-345,373-399,397-403 and 436-445 in E,designated TEP).The TEP were expressed in various systems:prokaryoti、eukaryotic and Adenovirus, then evaluated the abilities to induce immune responses in mice and swine.The contents of the paper contain five parts as following:1. Construction and eukaryotic expression for the multiple-epitope gene of Japanese encephalitis virusA synthetic multiple-epitope gene containing the critical epitopes of the Japanese encephalitis virus (JEV) envelope (E) gene was cloned into eukaryotic expression vector pcDNA3.0, named pcDNA-TEP.293 cells were then transfected with this recombinant plasmid. Recombinant protein was showed to transcript and express effectively by indirect immunofluorescence. Collect the cell supernatant after 48h transfection for Western blot, it showed the protein can interact with JEV positive anit-serum, proved its biological activity and antigenicity.2. The prokaryotic expression for the multiple-epitope gene of Japanese encephalitis virusA fragment without TPA signal peptide was amplified by PCR from plasmid pUC-TEP and cloned into pET-32a(+), named pET-rEP. Then the recombinant plasmid was transducted into BL21 Ecoli cells. Recombinant protein was purified as follow:Ecoli cells were induced by IPTG, after 4h, cells were collected by centrifugation,6000rpm 10min, washed twice by PBS, and suspended in PBS. Cells were lysed by ultrasonic, then centrifuged 10000rpm 20min. Both the supernatant and pellet was analyzed by electrophoresis. It showed that the protein was existed in form of inclusion. Dissolved the inclusion with 8M urea and purified by His-Band affinity chromatography, we got the 370Kd recombinant protein. Western blot showed the specific anti-JEV activity.3. The Immunogenic Efficacy for the recombinant DNA vaccine and recombinant protein vaccine on miceIn order to investigate the immunogenic efficiency of the pcDNA-TEP and pET-rEP, forty of JEV negative mice (4-week-old, female) were randomly divided into four groups with eight of them each group. Mice in groupⅠwere inoculated intramuscularly pcDNA-TEP at a dose of 50μg; Mice in groupⅡwere injected with purified pET-rEP at a dose of 200 pmol, Mice in groupⅢwere injected with pcDNA-TEP+pET-rEP; The blank eukaryotic expression plasmid pcDNA3.0 and inactivated vaccine were used as controls. JEV specific neutralizing antibody titer was evaluated with virus neutralization test assay, furthermore, the production of JEV specific IFN-γ, IL-2 and IL-4 in splenocytes was detected with commercial ELISA Kit. Datas showed that both the recombinant protein and the DNA vaccine can induce humoral and cellular immune responses. The rank for neutralizing antibodies titers is inactive vaccine, pcDNA-TEP+pET-rEP, pET-rEP, pcDNA-TEP, and pcDNA3.0 from high to low. No neutralizing antibody was found in the pcDNA3.0 inoculation group throughout the experiment. The production of gamma interferon (IFN-γ), interleukin-2 (IL-2) and interleukin-4 (IL-4) was detected from spleen lympholeukocyte of the immunized mice at 45 and 60days post primary immunization to evaluate the cellular immune response. Results showed that the IgG1 isotype was induced by the inactivated vaccine and rEP alone. In contrast, mice immunized with pcDNA-TEP alone elicited predominantly IgG2a.These results suggest that a mixed Th1/Th2 immune response was induced by DNA priming/protein boosting vaccine regimen. Therefore, it can be an attractive candidate vaccine for preventing JEV infection.4.Construction of Recombinnat Adenovirus Expression multiple-epitope gene of the Japanese encephalitis virusA synthetic multi-epitope of Japanese encephalitis virus was expressed in adenovirus. The multi-epitope gene was cloned into pAdeno Vator-CMV5-IRES-GFP, named as pA5-TEP. pA5-TEP was Linearized by Pme I, and then Linearized pA5-TEP and pAdeno VatorAEl/E3 co-transformed into E.coli BJ5183 competent cells by electroporation. The homologous recombinant plasmids pA5-TEP was selected and Linearized by Pac I to expose the encapsidation signal. Linearized virus plasmid was transfected into 293A cells by LipofectamineTM-2000, and recombinant virus was selected and named as rAd5-TEP. Infection titer and rate were evaluated through monitoring green fluorescent protein (GFP) expression. Expression of the multi-epitopes of JEV was identified by PCR and western-blot. Results indicated that expression products had specific antigenicity. The titer of rAd5-TEP is 1010..59 TCID50/mL, and the biological characteristics for recombinant virus were stable after passaged for 5 times.5. Recombinant Adenoviruses on Specific Immunization in Mice6 weeks old female BALB/c mice were randomly separated into 4 groups,8 mice per group, mice were celiac immunized with recombinant viruses(rAd5-TEP、inactivated vaccines) three times at 2 weeks intervals, and with PBS as control. Level of IgG1、IgG2a、IL-4、IFN-y and Antibody neutralization test were detected to evaluate humoral immune and cellular immune responses of recombinant viruses. ELISA test of antibody sub-type results indicated that recombinant viruses induced secretion both of IgGl and IgG2a, partial to IgG2a.The seem result elicited from cytokines ELISA test, secretion of both of Thl(y-IFN) and Th2(IL-4) were induced by recombinant viruses, partial to Thl. Furthermore, pcDNA-TEP+rAd5-TEP induces the highest level of the IFN-y and IL-4 and a stronger humoral response against JEV. the highest level of neutralizing antibodies was observed in the group inoculated with the inactive vaccine, which was slightly higher than the pcDNA-TEP+rAd5-TEP group(P>0.05). This promising candidate can be for instance in viral vectors vaccine studies. Thus provide valuable support for further development of JEV genetic engineering vaccines. 6. The Immunogenic Efficacy on Piglets for the Combination application with various vaccines of Japanese encephalitis virusIn order to test the immunogenic efficiency of the pcDNA-TEP+pET-rEP and pcDNA-TEP+rAd5-TEP which was constructed in the fifth chapter and the seventh chapter, twenty of JEV negative piglets (fourth-month-old) were randomly into four groups with ten of them in each group. Piglets in group one were injected (i.m.) with purified recombinant plasmid pcDNA-TEP; piglets in group two were injected with purified recombinant plasmid pcDNA-TEP, piglets in group three were injected with inactivated vaccine; piglets in group four were injected with PBS as control. Injections were done with 200μg purified plasmid via intramuscular (i.m.) route. Four weeks post the primary vaccination, the piglets were boosted with same dose of correspondence pET-rEP, rAd5-TEP, inactivated vaccine and PBS.Neutralizing antibody titers for each group were detected with virus neutralization test assay fortnightly interval after the primary vaccination for six times to evaluate the humoral immune response. The production of gamma interferon (IFN-y), interleukin-2 (IL-2) and interleukin-4 (IL-4) was detected with lympholeukocyte from blood of the immunized piglets at 30,45 and 60 days post immunization to evaluate the cellular immune response. Results showed, both pcDNA-TEP+pET-rEP and pcDNA-TEP+rAd5-TEP can induce both humoral and cellular immune responses, the inactivated vaccine obtained the highest neutralizing antibody titer,however the pcDNA-TEP+rAd5-TEP inoculation group got the earliest immune responses and the most vigorous Thl type response. The strategy of priming with the DNA vaccine and and then boosting the recombinant rAd5-TEP might be a promising JEV vaccine candidate for further.In summary, all the recombinant vaccines can induce humoral and cellular immune responses. The strategy of priming with the DNA vaccine and then boosting the recombinant rAd5-TEP can obtain the higher neutralizing antibody titer and induce the highest level of the IFN-γ、IL-2 and IL-4 production, which suggested that this DNA priming-recombinant rAd5-TEP boosting vaccine can produce high levels of antibody and result in significant T-cell responses both in mices and swine. It might be an attractive candidate vaccine to be tested for preventing JEV infection and the research may lead to a new approach for the generation of JE vaccines.