DNA Prime/Protein Boost Of Japanse Edcephalitis Virus NS1 Protein To Improve Immune Response In Mice

Japanese encephalitis virus (JEV) is a member of the Flavivirus genus of the Flaviviridae family. Japanese encephalitis virus is widely distributed in the south and southeast regions of Asia, including China, India, Japan, et al. It is a mosquito-borne flavivirus responsible for acute encephalitis in humans, with fatality rates ranging from 20% to as high as 50%. Most of the survivors display persistent neurological or psychological sequelae. Therefore, mass vaccination of swine can prevent disease in swine and help to prevent JE epidemics in humans. Thus there is an urgent need to develop more effective JE 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. Vaccination has played an important role in the protection of mammal against virus infections. DNA priming/protein boosting is a vaccine strategy that use plasmid DNA encoding virus protein for the first immunization, then boost with protein-based vaccine as an augmentation. According to strategy of DNA priming/protein boosting, immunogenic efficacy for NS1 protein of Japanese Encephalitis Virus vaccine are tested. The study was unfolded through the following aspects.1. A synthetic NS1 gene of the Japanese encephalitis virus (JEV) was cloned into eukaryotic expression vector pcDNA3.0, named pcDNA-NS1. BHK-21 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 in vitro.2. NS1 gene was amplified and conjuncted to the expression vector pET-28a(+), named as pET-rNS1. Then the plasmid was transformed into host bacterium BL21 (DE3).After IPTG 4h later, cells were suspended in PBS. Cells were treated with ultrasonic, and then centrifuged with 10000rpm for 20min. Both the supernatant and pellet was analyzed by electrophoresis. It showed that the protein was existed in form of inclusion body. The inclusion body was dissolved with 8M urea and purified with His·Bind affinity chromatography according to the instruction manual. We got the 41kD recombinant protein. Western blot showed the specific anti-JEV activity.3. New strategy of immunity was used to investigate the immunogenic efficiency of the pcDNA-NS1 and pET-rNS1, mice (4-week-old, female) were randomly divided into five groups with eight of them each group. Mice were inoculated intramuscularly pcDNA-NS1(Ⅱ) at a dose of 50μg; Mice in group(Ⅰ)were injected with purified rNS1 at a dose of 200 pmol, Mice in group(Ⅲ) were injected with pcDNA-NS1+rNS1; 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-γand IL-4 in splenocytes was detected with commercial ELISA Kit. Data showed that the new vaccine strategy can induce humoral and cellular immune responses. The rank for neutralizing antibodies titers is the attenuated vaccine, pcDNA-NS1+rNS1, rNS1, pcDNA-NS1, and pcDNA3.0 from high to low. The production of gamma interferon (IFN-y) and interleukin-4 (IL-4) was detected from spleen lympholeukocyte of the immunized mice at 45 and 60 days post primary immunization to evaluate the cellular immune response. What is more, mice protection against JEV challenge was test. Results showed that the IgG1 isotype was induced by the rNS1 group alone. In contrast, mice immunized with the attenuated vaccine and pcDNA-NS1 alone elicited predominantly IgG2a.These results suggest that a mixed Th1/Th2 immune response was induced by DNA priming/protein boosting vaccine regimen. The pcDNA-NS1+rNS1 group is sufficient to protect mice against a lethal JEV challenge. Therefore, it can be an attractive candidate vaccine for preventing JEV infection.In summary, the results herein clearly illustrated that DNA/protein prime-boost vaccination strategy can produce high level antibody and trigger significant T cell and B cell responses, highlighting the potential value of such an approach in the prevention of JEV infection.