Study On The Recombinant Baculovirus Vaccine Of Japanese Encephalitis

Japanese encephalitis is a serious enzootic disease, caused by infection with Japanese encephalitis virus (JEV), a member of the mosquito-borne flavivirus. The pig industry suffered significant losses because of infectious reproductive failure in swine caused by JEV. Also, JEV is transmitted to humans and causes an acute infection of the central nervous system and encephalitis. A high proportion of the survivors exhibit neurogical and psychiatric sequelae. With no specific effect on preventing Japanese encephalitis by mosquito control, vaccination is the most effective prophylactic measure to prevent and control these diseases. However, there are problems in both inactivated and attenuated JEV vaccines that are used currently. Therefore, to explore the new vaccine design against JEV, recombinant baculovirus expressing E protein of Japanese encephalitis virus was constructed, and the immune efficacy of the recombinant baculovirus was investigated. The main projects are:1. Construction of recombinant baculovirus expressing E protein of Japanese encephalitis virusTo construct the transfer plasmid pFast-G-E, the DNA fragment containing the E expression cassette (CMV-E-BGH poly (A)) was released from pc-E and inserted into pFastBac-VSV-G, in which the VSV-G gene is under the control of the polyhedron promoter of pFastbac^TM (Invitrogen). Transfer vector was transformed into E. coli DH10Bac containing baculovirus shuttle vector (bacmid) and helper vector. Within E. coli DH10Bac, the E gene was transposed into the bacmid. The colonies of E. coli containing recombinant bacmid (Bacmid-G-E) were collected three times by blue/white selection. After isolation and purification, Bacmid-G-E DNA was transfected into sf9 cells to yield recombinant baculovirus BV-G-E. To investigate the obtained baculovirus BV-G-E's transduction efficiency and level of E protein expression with in mammalian cells, PK-15 cells were transduced with BV-G-E. Expressed E protein was confirmed with indirect immunofluorescent assay and western blotting.2. Immune effect of recombinant baculovirus BV-G-ETo characterize the induction of antigen-specific immune response mediated by baculovirus, mice were subjected to intramuscular with different doses of baculovirus vectors ranging from 10⁸ to 10¹⁰ pfu/mL in a 100μL volume. At 6 weeks after primary immunization, all mice immunized with BV-G-E, even at the low dose used in this experiment (10⁸ pfu/mL), developed higher JEV-specific ELISA and neutralizing antibodies when compared with mice vaccinated with pc-E, and mice immunized with 10¹⁰ pfu/mL of BV-G-E produced significantly higher antibody titers than mice received10⁹ or 10⁸ pfu/mL of BV-G-E, exhibiting evident dose-dependent pattern. In agreementwith the humoral immune response, at 6 weeks after primary immunization, the highestIFN-γlevel was found in restimulated splenocytes from mice immunized with BV-G-E10¹⁰ pfu/mL.3. Protective efficacy of BV-G-E against wild-type JEV challengeTo evaluate the efficacy of BV-G-E immunization against JEV infection, mice were challenged (i.p.) with JEV strain P3 at 6 weeks after the primary immunization and checked daily for survival. 3 weeks after challenge, the highest suvival rate (80%) was observed in the group of mice immunized with BV-G-E 10¹⁰ pfu/mL. However, significantly lower survival rate of 50% was observed in the group of mice immunized with pc-E. To evaluate the efficacy of BV-G-E immunization against JEV entry into the mouse brain, the virus contents in the brain tissues of surviving mice were analyzed by real-time RT-PCR. The lowest viral content was detected in surviving mice receiving 10¹⁰ pfu/mL BV-G-E. The above results revealed that, BV-G-E with the dose of 10¹⁰ pfu/mL shows the best immunogenicity and protective efficacy against lethal JEV challenge, and this vaccine may be used as a strategy to develop a new genetation of vaccine against JEV in the future.