Coden Optimization Of SjCTPI Gene Combined With Electroporation In Vivo Enhancing Immune Protective Effect For Schistosoma Japonicum Challenge In Mice

Schistosomiasis is still a significant public health problem in the world.It is estimated that 779 million people are at risk of infection and 200 million people are infected covered 74 countries.China is one of the most serious countries of schistosomiasis.Although the great effects for control of the disease have been performed in China,the intermediate host(snails),animal reservoir hosts of the disease are still existing in the endemic areas,and re-infection is rapid during transmission season,the endemic situation of schistosomiasis is still severe.Since 1970s the high effective and safe anti-schistosomiasis drug,praziquantel has been used for treatment of the disease leading to dramatic reduction of morbidity in endemic areas.But chemotherapy does not block transmission of the disease and high re-infection rates limit the success by demanding frequently treatments.Since schistosomes do not multiply within the final host,the vaccine could induces reduction,even a partial,in worm burdens could considerably reduce pathology and limit transmission.Thus vaccination is needed to develop to be as a complimentary approach integrated with chemotherapy.The SjCTPI cDNA fragment was successfully cloned into a eukaryotic expression plasmid pcDNA3.1 in our laboratory.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 was 30.2%-32.7%.It indicated that SjCTPI DNA vaccine was a potential vaccine for schistosomiasis japonica.As a candidate of vaccine,antigenic epitopes of SjCTPI is schistosome specific.SjCTPI has no cross-reactive with the mammalian homologues.But the protective effects were not ideal, always lower than 40%.In a wide variety of organisms,synonymous codons are used with different frequencies,as a result,foreign genes can't expressed efficiently in a different host. It has been reported that optimization of G protein-coupled receptor(SmGPCR) gene of S. mansoni can increase its protein expression levels,similar results were found in HIV and malaria.Electroporation in vivo of naked DNA is a novel DNA delivery approach.It has been demonstrated that electroporation of plasmid DNA could increase the DNA uptake leading to enhance expression of protein in the treated cells and result in higher protective efficacy.The technique have been used in influenza,tumor,malaria and hepatitis etc,and the immune protective efficacy of these DNA vaccines could be significantly elevated. Heat-shock proteins(HSPs) are a group of highly conserved proteins.HSP-peptide complexes are taken up by specialized antigen-presenting cells(APCs) and transferred to the MHC molecules inside these APCs for recognition by the T cell,and stimulate T cells. HSP70 molecules have been applied in DNA or protein(peptide)-based vaccines as antigens, chaperones or adjuvants.HSP70-based vaccines have been shown for enhancing the protective effects of vaccines against Schistosoma japonicum infection in water buffalo.In order to enhance the protective effect of DNA vaccine,we constructed DNA vaccines including TPI.opt gene and/or mHSP70 gene.The present study showed that immune protective effect was evaluated in BALB/C mice immunized with the DNA vaccines,and the humoral and cellular responses were tested.The electroporation technology in vivo was applied to further enhance the protective effects.Part 1.Coden optimization of Schistosoma japonicum TPI genesThe gene coding triose phosphate isomerase(TPI) of Schistosoma japonicum was optimized by using the GeneOptimizer software according to the codon usage in mammalian cells.The optimized TP1 gene was synthesised by GENEART and inserted into plasmid pJW4303.Part 2.Construction of coden optimized SjCTPI DNA vaccineThe TPI.opt gene was amplified from pJW4303-TPI.opt and cloned into pcDNA3.1 to construct the pcDNA3.1-TPI.opt.The mHSP70 gene was amplified from pVAX-mHSP70 and cloned into pcDNA3.1 to construct plasmid pcDNA3.1-mHSP70.The TPI.opt gene (removed stop codon) was amplified from pJW4303-TPI.opt and cloned into pcDNA3.1-mHSP70 to construct the plasmid pcDNA3.1-TPI.opt-mHSP70.The TPI gene (removed stop codon) was amplified from pcDNA3.1-TPI and cloned into pcDNA3.1-mHSP70 to construct the plasmid pcDNA3.1-TPI-mHSP70.The recombinant plasmids were identified by sequence analysis. Part 3.Enhancing the protective immunity effect with coden optimized SjCTPI DNA vaccine against Schistosoma japonicum infection.Sixty female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups:group A (pcDNA 3.1,control group),group B(pcDNA3.1-TPI),group C(pcDNA 3.1-TPI-mHSP70),group D(pcDNA3.1-TPI.opt) and group E (pcDNA3.1-TPI.opt-mHSP70).Each mouse was immunized with 100μg of plasmid DNA by intramuscular injection,respectively.All mice were boosted at week 3 and week 6 with the same dosage and same method.Four weeks after the 3rd immunization,all mice were challenged with(40±1) cercariae of Schistosoma japonicum by abdominal skin penetration. Forty-two days post-challenge,the mice were sacrificed and perfused,and the numbers of recovered worms and hepatic eggs were counted.The blood was collected from the tail veins of all mice 2 days before immunization and challenge,respectively.Serum was prepared for detection of IgG,IgG1 and IgG2a.Two weeks after the 3rd immunization,the spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI peptide,and the supernatant was collected for detection of IL-2,IL-4,IL-5,IFN-γand TNF by flow cytometre.The worm reduction rates in B,C,D,E group were 26.28%,28.38%, 36.03%and 39.03%,respectively,the hepatic egg reduction rates were 28.35%,31.39%, 41.71%and 46.85%respectively compared with the control group.The levels of protection in group D and E both were significantly higher than those in group B and C(P＜0.01). ELISA results showed that the mice in group B,C,D,E produced specific IgG and IgG1, IgG2a antibody isotypes,with the ratios of IgG2a/IgG1 1.73,2.06,2.44 and 3.09, respectively.The levels of IL-2,IFN-γand TNF in growp B,C,D,E were augmented all,but IL-4,IL-5 were not detectable.In comparison with the mice in group B,C,the levels of IL-2,IFN-γand TNF of mice in group D,E were augmented.The experiment results showed that the conden optimized TPI DNA vaccine could induce higher level of protective effect than that with non-optimized TPI DNA vaccine,and induce high level of cellular immunity which is Th1-biased responses.It indicates that the conden optimized DNA vaccines is a good,novel potential vaccine for schistosomiasis. Part 4.Enhancing protective immunity effects against Schistosoma japonicum infection with coden optimized SjCTPI DNA vaccine combined with electroporation in vivoIn order to further enhance the protective immunity effects,the coden optimized SjCTPI DNA vaccines combined with the electroporation in vivo(E.P.~*) was performed.Sixty female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups: group A(pcDNA3.1,control group),group B(pcDNA3.1-TPI.opt),group C(pcDNA 3.1-TPI.opt-mHSP70),group D(pcDNA3.1-TPI.opt/E.P) and group E (pcDNA3.1-TPI.opt-mHSP70/E.P).Each mouse was immunized with 100μg of plasmid DNA by intramuscular injection,at week 0,3 and 6 with the same dosage and same method. In group D and E each mouse was followed by electroporation in vivo(E.P.) while vaccinate with plasmid DNA.Four weeks after the 3rd immunization,all mice were challenged with(40±1) cercariae of Schistosoma japonicum by abdominal skin penetration. Forty-two days post-challenge,the mice were sacrificed and perfused,and the numbers of recovered worms and hepatic eggs were counted.The blood was collected from the tail veins of all mice 2 days before immunization and challenge,respectively.Serum was prepared for detection of IgG,IgG1 and IgG2a.Two weeks after the 3rd immunization, the spleen cells of 2 mice from each group were cultured and stimulated with ConA and rSjCTPI peptide,and the supernatant was collected for detection of IL-2,IL-4,IL-5,IFN-γand TNF by flow cytometre.The worm reduction rates in B,C,D,E group were 36.03%, 39.03%,49.74%and 51.64%,respectively,the hepatic egg reduction rates were 41.71%, 46.85%,57.04%and 61.42%respectively compared with the control group.The levels of protection in group D and E both significantly were higher than those in group B and C(P< 0.01).ELISA results showed that the mice in group B,C,D,E produced specific IgG and IgG1,IgG2a antibody isotypes,with the ratios of IgG2a/IgG1 2.44,3.09,3.82 and 4.06,respectively.The levels of IL-2,IFN-γand TNF in gropus B,C,D,E were augmented all,but IL-4,IL-5 were not detectable.In comparison with the level of mice in group B,C,the levels of IL-2,IFN-γand TNF of mice in group D,E were augmented.The study showed that the protective immunity effects of conden optimized TPI DNA vaccines combined with electroporation in vivo against Schistosoma japinicum infection were greatly improved.Worm reduction and hepatic egg reduction rates were all more then 50%.