Studies On The Recombinant Baculovirus As A Novel Vector For DNA Vaccine Against Schistosoma Japonicum

Currently, DNA vaccine remains the highest priority of vaccine against schistosome. Two general classes of gene-based vaccines are being developed. One is based on the use of plasmid DNA encoding the target antigen. However, the use of plasmid DNA as a gene transfer vehicle for immunization is somewhat limited by the low transduction efficiency that hampers its immunogenic potential. To overcome this problem, numerous strategies are being developed that use electrical stimulation, microparticles, adjuvants, and costimulatory molecules to enhance both the gene transfer capabilities and the intrinsic immunogenicity of plasmid DNA. Mainwhile, the capacity of plasmid vector limits the application of a high molecular weight antigen or multi-antigen DNA vaccine, which is the emphasis to enhance the immune efficacy of anti-schistosome vaccine.The other is based on viral vector. Viruses have highly evolved structures that enable them to bind to cells and deliver genes into the cells that they infect. Thus, it is reasonable to consider using live virus vaccines to induce cytolytic T lymphocytes to treat a disease in which cellular immunity is an absolute requirement. To this end, a variety of viral vectors, primarily based on poxvirus and adenovirus, have been tested, with encouraging results. However, these vectors are themselves antigenic and can cause inflammation, which may constitute a liability for their use as vehiclesfor vaccination. An additional drawback is that existing immunity due to previous infection (as in the case of adenovirus) or to vaccination (smallpox) may limit the potency of the vaccine by clearing the viral vector before it can infect cells and deliver its payload of antigen genes. Thus, there is a need to identify unrelated viral vectors that function efficiently as gene transfer vehicles for immunization whose efficacy is not limited by problems related to toxicity or existing immunity.In recent years, a number of features make baculovirus as attractive potential gene therapy vector. In contrast to other commonly used viral vectors, baculoviruses have the unique property of replicating in insect cells while being incapable of initating a replication cycle and producing infectious virus in mammalian cells, it shows a good biosafety and the baculovirus have absence of pre-existing Abs against of baculovirus in animals. Another this vector can efficiently transduct a variety of cell lines without any apparent cytopathic effect, even at a high multiplicity of infection (MOI). The advantage of baculovirus was also highlighted by the considerably high gene delivery efficiency compared to plasmid tranfection. Mainwhile, manufacture of large quantities of relatively high-titre stock can be easily achieved, and very large insert sizes can be accommodated. The baculovirus as a vector for vaccination was used in pseudorabies virus, hepatitis C virus (HCV) and rodent malaria Plasmodium berghei, which the expression of foreign gene mediated by recombinant baculovirus could elicit antigen-specific immune response in animal experiment. These provide the laboratorial basis that the baculovirus was exploited as vaccine carrier.Furthermore, the co-immunization with DNA vaccine and protein vaccine can enhance protective efficacy. In the present study, we evaluated the feasibility of using baculovirus as a vaccine carrier, the gene encoding 26 kDa glutathione S-transferase of Schistosoma japonicum (Sj26), which is one of promising vaccine candidates selected by WHO, was used as antigen molecular. Whether the synergetic effect of the co-immunization with DNA vaccine and protein vaccine was also observed.The main contents of this thesis include two parts.Part 1: Protective Efficacy of Co-immunization with Sj26 DNA and Recombinant Protein Vaccine against Schistosoma japonicum in MiceObjective: 1) To observe the expression of recombinant plasmid pEGFP-Sj26 with enhanced green fluorescent protein in mammalian cells in vitro and protective immunity of pEGFP-Sj26 DNA vaccine. 2) To study the protective efficacy of co-immunization with pEGFP-Sj2 DNA vaccine and recombinant Sj26 protein (rSj26 GST) vaccine against Schistosoma japonicum in mice.Methods: The Sj26 gene was amplified from the plasmid pGEX-3X by PCR and the product of PCR was ligated with pGEM-T Easy vector. The positive clones were screened and identified by endonuclease digestion and sequencing, the correct recombinant plasmid was named pT1-Sj26. Then the Sj26 gene was cloned into eukaryotic expression vector pEGFP-N3 with enhanced green fluorescence protein (EGFP). The recombinant plasmid pEGFP-Sj26 was transfected into baby hamster kidney (BHK) cells by using cationic lipids (lipofectamine). Both fluorescent microscope and western blot were employed to identify the expressed products. Sixty BALB/c mice were divided randomly into five groups, each consisting of 12 mice, which were immunized three times. The co-immunization group was primed with plasmid pEGFP-Sj26, boosted 2 weeks later and immunized with rSj26 GST 4 weeks later. Two weeks after last immunization, each mouse was challenged with 40±l of cercariae of Schistosoma japonicum Chinese strain. On day 45 post-infection, the mice were sacrificed and perfused from portal vein, then the number of worms and liver eggs were counted. Percent of worm reduction and percent reduction in liver eggs in co-immunization group were compared with that in pEGFP-Sj26 group and rSj26 GST group.Results: Sequence analysis revealed that the full length of Sj26 gene was 654 bp, and showed 100% identical to the sequence reported in literature. The recombinantplasmid pEGFP-Sj26 was successfully constructed, and could be transfected BHK cells in vitro. In BHK cells transfected with the recombinant plasmid pEGFP-Sj26, the bright green fluorescence was observed under fluorescent microcopy, and western blot confirmed that sera from rabbit immunized with rSj26 GST could recognize the band with Mr 53 kDa.Percent of worm reduction in co-immunized group was 50.8%, it was significantly higher than that in pEGFP-Sj26 group (28.0%, P<0.01) and rSj26 GST group (25.5%, P<0.01). Percent of liver egg reduction in co-immunized group, rSj26 GST group and pEGFP-Sj26 group were 32.7%, 33.0% and 20.6% respectively, and percent of liver egg reduction in the former two groups were significantly higher than that in control group (P<0.01), but the difference between pEGFP-Sj26 group and control group was not significant.Conclusion: 1) The recombinant plasmid pEGFP-Sj26 with enhanced green fluorescent protein has been constructed successfully. 2) Eukaryotic expression plasmid pEGFP-Sj26 was expressed successfully in mammalian cells in vitro. 3) pEGFP-Sj26 DNA vaccine could induce partial protective immunity in BALB/c mice. 4) Compared with only Sj26 DNA vaccine or recombinant protein vaccine, the co-immunization with Sj26 DNA vaccine and recombinant protein could enhance protective efficacy in BALB/c mice.Part 2: Protective Efficacy of Co-immunization with Recombinant Baculovirus Carrying Sj 26 DNA and Recombinant Protein Vaccine against Schistosoma japonicum in MiceObjective: 1) To construct the recombinant baculovirus carrying Schistosoma japonicum 26 kDa glutathione S-transferase (Sj26). 2) To explore the feasibility of using baculovirus as DNA vaccine carrier. 3) To study the protective efficacy of recombinant baculovirus carrying Sj26 DNA vaccine. 4) To study the protectiveefficacy of co-immunization with recombinant baculovirus carrying Sj26 DNA vaccine and recombinant protein (rSj26 GST) vaccine against Schistosoma japonicum in mice.Methods: The Sj26 gene was amplified from the plasmid pGEX-3X by PCR and the product of PCR was ligated with pGEM-T Easy vector. The positive clones were screened and identified by endonuclease digestion and sequencing, the correct recombinant plasmid was named pT2-Sj26. Then the Sj26 gene was inserted into the downstream of CMV-IE promoter of donor plasmid pFBDGC, and transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and its transposition was confirmed by PCR. The AcCMVSj26 was transfected into Sf9 cells, green fluorescent was observed under fluorescent microscope. The recombinant baculovirus vAcCMVSj26 was harvested and then identified by PCR, and the titer of vAcCMVSj26 was determined by end-point dilution. After the BHK cells transducted with recombinant baculovirus, the expression of Sj26 GST was identified by western blot.The independent vaccination trials were performed. All animals were inoculated three times with two weeks interval. In trial 1, BALB/c mice were immunized with recombinant baculovirus vAcCMVSj26 by intramuscular injection, baculovirus vAcBW3 and Grace medium as control. In trial 2, the co-immunization group was primed with vAcCMVSj26, boosted 2 weeks later and immunized with rSj26 GST 4 weeks later and only vAcCMVSj26 or rSj26 GST as control. Two weeks after last immunization, each mouse in two trials was challenged with 40±1 of cercariae of Schistosoma japonicum Chinese strain. On day 45 post-infection, the mice were sacrificed and perfused from portal vein, then the number of worms and liver eggs were counted. Percent of worm reduction and percent reduction in liver eggs were calculated.Results: Sequence analysis revealed that the full length of Sj26 gene was 654 bp,and showed 100% identical to the sequence reported in literature. The recombinant donor plasmid pFBDGC-Sj26 was successfully constructed, and PCR confirmed that the gene of interest had transposed to the recombinant bacmid AcCMVSj26. When AcCMVSj26 was transfected into Sf9 cells, green fluorescent was observed under fluorescent microscope during 4-7 days post-transfection, it showed that recombinant baculovirus vAcCMVSj26 had generated. The Sj26 gene in vAcCMVSj26 was confirmed by PCR, and the final titer of vAcCMVSj26 was 1.2×10~8 TCID/ml. The results of western blot showed that the Sj26 GST was expressed in BHK cells transfected with recombinant baculovirus vAcCMVSj26.Animal protection experiment shows in trial 1, vAcCMVSj26 vaccination elicited a 28.3% reduction in worm burdens and 23.6% reduction in liver eggs per female, compared to mice immunized with vAcBW3 baculovirus and Grave medium control. The effect of co-immunization was evaluated in trial 2, in which, percent of worm reduction in co-immunized group was 44.0%, it was significantly higher than that in vAcCMVSj26 group (22.7%, P<0.01) and rSj26 GST group (25.6%, P<0.01). Percent of liver egg reduction in co-immunized group, rSj26 GST group and vAcCMVSj26 group were 25.5%, 33.0% and 23.9% respectively, and it showed the significant difference with PBS control.Conclusion: 1) The recombinant baculovirus carrying Schistosoma japonicum 26 kDa glutathione S-transferase (Sj26) was constructed successfully. 2) The recombinant baculovirus carrying Sj26 were expressed successfully in mammalian cells in vitro. 3) The baculovirus can be used as DNA vaccine carrier. 4) The co-immunization with recombinant baculovirus carrying Sj26 DNA vaccine and recombinant protein vaccine against Schistosoma japonicum in mice could similarly enhance protective efficacy in BALB/c mice.