Study On The Multivalent DNA Vaccine Of Schistosoma Japonicum

Up to now, schistosomiasis japonica remains a significant health problem which plague people in endemic regions. Every year the government spends a great deal of material and manpower to prevent and treat the disease. However, the intermediate host (snails), animal reservoir hosts of the disease are still existing in the endemic regions and re-infection is rapid during transmission reason. For these reasons, development of a vaccine for schistosomiasis is considered as a complement measure for chemotherapy on control of the disease. DNA vaccine as a new technique has made more progresses in prevention of schistosome intection. Antigens such as troise-phosphate isomerase (TPI), glutathine-S-transferase (GST), paramyosin, Sm23, fatty acid binding protein and IrV-5 as the candidates of schistosomiasis vaccines have been studied for decades, and their protective effects have been demonstrated by WHO. According to the study of our laboratory, the SjCTPI cDNA fragment was successfully cloned into a eukaryotic expression plasmid pcDNA3.1. The protective experiment of the vaccine in the mouse showed that the worm reduction rate was 27.9%-31.9% in C57BL/6 inbred mouse, and in BALB/c inbred mouse the worm reduction rate and hepatic egg reduction rate were 30.2%-32.7% and 47.0%-52.9%, respectively. It indicated that SjCTPI DNA vaccine was a potential vaccine for schistosomiasis japonica. Guan Xiaohong established a cell line of the monoclonal anti-idiotypic antibody (anti-id) NP30 of Schistosoma japonicum, its isotype was identified to be IgM, and which was testified to be the internal image of GAA. The protective experiment of the NP30 in the mouse showed that the worm reduction rate in C57BL/6 inbred mouse was 42.05%. Through analysis the H chain CDR3 region of NP30 is thought to be an antigenic epitope of monoclonal antibody NP30 and the expression products of H chain 6×CDR3 gene fragment could interact specifically with antibody. So the antigenic epitope vaccine is an important candidate of schistosomiasis japonicum vaccine. In order to enhance the protective effect of DNA vaccine, we constructed multivalent DNA vaccine including TPI gene and (CDR3)6 gene. The immune protective effect was evaluated in BALB/C mice immunized with the multivalent DNA vaccine, and the humoral and cellular responses were tested. Part 1. Construction of multivalent DNA vaccine According to the gene sequence of (Gly4Ser)3 , a pair of single strand of oligodeoxynucleotides was synthesized and annealed to form the double-stranded linker. The linker was cloned into pcDNA3.1(+) to construct the pcDNA3.1-linker, then either the TPI gene or the (CDR3)6 gene of Schistosoma japonicum was inserted before or behind the linker gene to construct the multivalent DNA vaccines: pcDNA3.1-TPI-linker-(CDR3)6 and pcDNA3.1-(CDR3)6-linker-TPI. Part 2. Expression of multivalent DNA vaccine in eukaryotic cells The genes of TPI-linker-(CDR3)6 and (CDR3)6-linker-TPI(stop codon was removed) were cloned into pEGFP-N1 at upstream of the green fluorescent protein (GFP) respectively to construct the eukaryotic expression vector . The recombinants were transfected into COS-7 cells by LipofectamineTM2000. The expression of GFP indicated that the target genes of the multivalent DNA vaccines can be expressed in eukaryotic cells. Part 3. The protective effect of multivalent DNA vaccine in BLAB/c mice Seventy female BLAB/c mice of 5~6 weeks old were randomly divided into 5 groups: pcDNA3.1 group (control group), pcDNA3.1-TPI group (TPI), pcDNA3.1-(CDR3)6 group[(CDR3)6], pcDNA3.1-TPI-linker-(CDR3)6 group (TLC) and pcDNA3.1-(CDR3)6-linker-TPI group (CLT). Each mouse was immunized with 100μg of pcDNA3.1 , pcDNA3.1-TPI , pcDNA3.1-(CDR3)6 , pcDNA3.1-TPI-linker-(CDR3)6 or pcDNA3.1-(CDR3)6-linker-TPI plasmid DNA by intramuscular injection, respectively. All animals were boosted at week 2 and week 4. Four weeks after the 3rd immunization, all mice were challenged with 45±1 cercariae of Schistosomia japonicum by abdominal skin penetration. Forty-five days post-challenge, mice were perfused and the numbers of recovered worms and hepatic eggs were counted. The results showed that the worm reduction rates in TLC group and CLT group were 37.31% and 39.09%, the hepatic egg reduction rates were 41.61% and 49.54% compared with control group, respectively. The level of protection in TLC group and CLT group both were higher than that in TPI group and (CDR3)6 group(P<0.05). However, there was no difference in the worm reduction rate and egg reduction rate between the two multivalent DNA vaccine groups. It indicated that the order of the two antigen molecules has not any influence upon the immune protection of the two multivalent DNA vaccines. Blood was collected from the tail vein of all mice 2 days before the 1st immunization and challenge. Serum was prepared for detection of IgG, IgG1 and IgG2a. ELISA results showed that the mice immunized with pcDNA3.1-TLC and pcDNA3.1-CLT produced specific IgG for rSjCTPI and higher than that of TPI group, while mice immunized with pcDNA3.1 and pcDNA3.1-(CDR3)6 did not. Mice in TPI group, TLC group and CLT group also produced IgG1 and IgG2a antibody isotypes, with the ratios of IgG2a/IgG1 1.71, 2.71 and 4.70 respectively. Three weeks after the 3rd inoculation, the spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI peptide. The supernatant was collected to detect IL-2, IL-4, IFN-γ. In comparison with control group the levels of IL-2 and IFN-γof mice immunized with pcDNA3.1-TPI , pcDNA3.1-TLC and pcDNA3.1-CLT enhanced. It indicated that the multivalent DNA vaccine was induced higher level of protection than that with monovalent vaccine. Part 4. Construction of pcDNA3.1-TLC/CpG and pcDNA3.1-CLT/CpG vaccine and their protective effect in BLAB/c mice pcDNA3.1-TPI-linker-(CDR3)6 and pcDNA3.1-(CDR3)6-linker-TPI constructed in our previous study was digested by restriction enzymes Nhe Ⅰand Xho Ⅰto gain TPI-linker-(CDR3)6 and (CDR3)6-linker-TPI gene fragment. pcDNA3.1-CpG which has been constructed in our lab was digested by the same restriction enzymes as the above. Subsequently, the TPI-linker-(CDR3)6 and (CDR3)6-linker-TPI gene fragments were ligated with the digested pcDNA3.1-CpG to construct pcDNA3.1-TLC/CpG and pcDNA3.1-CLT/CpG vaccine. Eighty female BLAB/c mice of 5~6 weeks old were randomly divided into 6 groups: pcDNA3.1 group (control group), pcDNA3.1-TPI-linker-(CDR3)6 group (TLC), pcDNA3.1-(CDR3)6-linker-TPI group (CLT), pcDNA3.1-TPI-linker-(CDR3)6 /CpG group (TLC/CpG), pcDNA3.1-(CDR3)6-linker-TPI /CpG group (CLT/CpG) and pcDNA3.1-CpG group (CpG). Each mouse was immunized with 100μg of pcDNA3.1 or pcDNA3.1-TLC group, pcDNA3.1-CLT, pcDNA3.1-TLC/CpG, pcDNA3.1-CLT/CpG or pcDNA3.1-CpG plasmid DNA by intramuscular injection, respectively. All animals were boosted at week 2 and week 4. Four weeks after the 3rd immunization, all mice were challenged with 45±1 cercariae of Schistosomia japonicum by abdominal skin penetration. Forty-five days post-challenge, mice were perfused and the numbers of recovered worms and hepatic eggs were counted. The results showed that the worm reduction rates in pcDNA3.1-TLC/CpG group and pcDNA3.1-CLT/CpG group were 40.29% and 40.81%, the hepatic egg reduction rates were 49.29% and 50.41% respectively compared with control group. The worm reduction rates and egg reduction rates of TLC/CpG group and CLT/CpG group seems higher than the rates of TLC group and CLT group, but there was no diffrence by statistical test. Blood was collected from the tail vein of all mice 2 days before the 1st immunization and challenge. Serum was prepared for detection of IgG, IgG1 and IgG2a. Mice in TLC group, CLT group, TLC/CpG group and CLT/CpG group also produced IgG and IgG1, IgG 2a antibody isotypes, with the ratios of IgG2a/IgG1 2.71, 4.70, 3.75 and 3.33 respectively. Three weeks after the 3rd inoculation, the spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI peptide. The supernatant was collected todetect IL-2, IL-4, IFN-γ. In comparison with control, the levels of IL-2 and IFN-γof mice immunized with pcDNA3.1-TLC , pcDNA3.1-CLT, pcDNA3.1-TLC/CpG and pcDNA3.1-CLT/CpG enhanced, but there were no differences between each other. This study indicated that immunostimulatory sequence might not increase the protection level, and the further study need to do in the future.