| Up to now, schistosomiasis remains a significant health problem which plague people in endemic regions. Every year the government spends a great deal of material, manpower and money to prevent and treat the disease. Although the great effects have been gained, 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. The endemic situation of schistosomiasis is still severe. After the devolopment of praziquantel since 1970s, chemotherapy becomes the main intervention leading to dramatic reduction of morbidity in endemic areas. However, chemtherapy does not block transmission of the infection and high re-infection rates limit the success by demanding frequently treatments. Besides, the potential risk of development of drug-resistance cannot be roled out. Thus, vaccination is needed as a complimentary approach integrated with chemotherapy. Since schistosomes do not mutiply within the final host, a vaccine that induces reduction, even a partial, in worm burdens could considerably reduce pathology and limit transmission. DNA vaccine, as a new and attractive strategy, has been studied and showed great significance in controlling transmission of schistosomiasis. The DNA vaccines, triose phosphate isomerase and 23 kDa membrane protein of Schistosoma japonicum (chinese strain), have been constructed by our laboratory, also we have constructed bivalent DNA vaccine including TPI gene and (CDR3)6 gene. Through the animal experiments, these DNA vaccines all showed some protective effects, but the effects were not ideal, the worm reduction rates could not stabilize at the level of more than 50%. It is reported that animals immunized with a cocktail vaccine ,which mixed different type of protective antigens, could gain a better protective effects against infection. In the vaccine research of Malaria, Tuberculosis and AIDS, priming with DNA vaccine and boosting with protein vaccine could increase the level of humoral immunity and cellular immunity, also enhance the protective effects. Elecreoporation in vivo, as a new gene delivery system, could increase the efficiency of DNA transfection significantly. In this study, we prepared cocktail DNA vaccines and cocktail protein vaccines, and evaluated the immunol protective effects of vaccines against Schistosoma japonicum infection through prime with cocktail DNA vaccines and boosting with cocktail protein vaccines. In order to further enhance the protective effects, we use the technology of electroporation in vivo to increase the efficiency of gene transfer, and evaluate the value of this technology on enhancing the protective effects. To investigate the mechnism of protection, the humoral and cellular responses were tested.Part 1. Enhancing protective immunity effects of the vaccine against Schistosoma japonicum infection through priming with cocktail DNA vaccines and boosting with cocktail protein vaccinesEach kind of plasmids and proteins for immunization was prepared respectively,and diluted in no bacterial saline solution to final concentration of 1.5mg/ml , mixed with pcDNA3.1-SjC23, pcDNA3.1-SjCTPI, pcDNA3.1-(CDR3)6 plasmid DNAs by equal volume to form the cocktail DNA vaccines, and also mixed with recombinant proteins SjC23-HD, SjCTPI, and NP30 by equal volume to form the cocktail protein vaccines. Seventy female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups (A, B, C, D, E). In Group A (control group) each mouse was immunized with 100μl of saline solution by intramuscular (i.m.); in Group B (pcDNA3.1 control group), each mouse was immunized (i.m.) with 100μl of pcDNA3.1 plasmid DNA for three times at week 0, 3, 6; in Group C (pcDNA3.1 and cocktail protein group) each mouse was immunized (i.m.) with 100μl of pcDNA3.1 plasmid DNA for three times at week 0, 3, 6 and boosted with 100μl of cocktail protein vaccines plus 100μl FCA by subcutaneous at week 9; in Group D (the cocktail DNA vaccines group) each mouse was immunized (i.m.) with 100μl of cocktail DNA vaccines for three times at week 0, 3, 6; in Group E (cocktail DNA vaccines + cocktail proteins) each mouse was immunized (i.m.)with 100μl of cocktail DNA vaccines for three times at week 0, 3, 6 and boosted with 100μl of cocktail protein vaccines plus 100μl FCA by subcutaneous at week 9. Four weeks after the last DNA immunization or two weeks after protein boosting, all the 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 eggs in liver were counted. The blood was collected from the tail veins of all mice two days before the first immunization and challenge, respectively, the serum was prepared for detection of IgG, IgG1 and IgG2a. Two days before the challenge, the spleen cells of two mice from each group were cultured and stimulated with ConA and soluble egg antigen (SEA), and the supernatant was collected for detection of IL-2, IL-4 and IFN-γ. The results showed that the worm reduction rates in Group C, D and E were 17.70%, 32.88% and 45.35%, respectively, compared to the control group. The worm reduction rates in Group D and E were significantly higher than that in Group C (P <0.01), also than in Group E was significantly higher than that in Group D (P <0.01). The egg reduction rates in Group C, D and E were 9.39%, 36.20 and 48.54%, respectively, compared to the control group. The egg reduction rates in Group D and E were significantly higher than that in Group C (P <0.01), also that in Group E was higher than that in Group D (P <0.05). ELISA results showed that the mice in Group C, D and E produced specific IgG, while the mice in the control group(Group A and B) did not. The mice in Group C, D and E also produced IgG1 and IgG2a antibody isotypes, with the ratios of IgG2a/IgG1 0.525, 1.829 and 0.712 respectively. In comparison with the control group (Group A and B), the levels of IL-2 and IFN-γof the mice in Group D and E were obviously augment. The levels of IL-4 of mice in each group were not detected. This study indicated that priming with cocktail DNA vaccines and boosting with cocktail protein vaccines may enhance the protective immunity effects against Schistosoma japonicum infection. It is a new strategy of vaccination for enhancing the protective effect in schistosomiasis.Part 2. Enhancing protective immunity effects of nucleic acid vaccines against Schistosoma japonicum infection through electroporation in vivoOn the fundament of Part 1, in order to improve the protective immunity effects against Schistosoma japonicum infection, we use the technology of electroporation in vivo (E.P.?) for increasing the efficiency of gene transfer, and evaluated the value of the technology on enhancing the protective effects.Plasmids and proteins preparation for immunization were same to Part 1. Seventy female BALB/c mice of 4-5 weeks old were randomly divided into 5 groups (A, B, C, D, E).In Group A (control group), each mouse was immunized with 100μl of saline solution by intramuscular (i.m.); in Group B (pcDNA3.1/E.P. control group), each mouse was immunized (i.m.) with 100μl of pcDNA3.1 plasmid DNA followed by electroporation in vivo (E.P.) for three times at week 0, 3, 6; in Group C (pcDNA3.1/E.P. plus cocktail protein vaccine group), each mouse was immunized (i.m.) with 100μl of pcDNA3.1 plasmid DNA followed by electroporation in vivo for three times at week 0, 3, 6 and boosted with 100μl of cocktail protein vaccines plus 100μl FCA by subcutaneous at week 9; in Group D (cocktail DNA vaccine/E.P. group), each mouse was immunized (i.m.) with 100μl of cocktail DNA vaccines followed by electroporation in vivo for three times at week 0, 3, 6; in Group E (cocktail DNA vaccines/E.P. plus cocktail protein vaccines group), each mouse was immunized (i.m.) with 100μl of cocktail DNA vaccines followed by electroporation in vivo for three times at week 0, 3, 6 and boosted with 100μl of cocktail protein vaccines plus 100μl FCA by subcutaneous at week 9. Four weeks after the last DNA immunization or two weeks after protein boosting, all the 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 eggs in liver were counted. The blood was collected from the tail veins of all the mice two days before the first immunization and challenge, respectively, the serum was prepared for detection of IgG, IgG1 and IgG2a. Two days before the challenge, the spleen cells of two mice from each group were cultured and stimulated with ConA and soluble egg antigen (SEA), and the supernatant was collected for detection of IL-2, IL-4 and IFN-γby flow cytometre. The worm reduction rates in Group C, D and E were 18.09%, 45.00% and 57.09%, respectively, compared to the control group(group A and B). The worm reduction rates in Group D and E were significantly higher than that in Group C (P<0.01), also in Group E was significantly higher than that in Group D (P<0.05). The liver egg reduction rates in Group C, D and E were 12.49%, 50.88% and 59.26%, respectively, compared to the control group. The egg reduction rates in Group D and E were significantly higher than that in Group C (P<0.01), also in Group E was higher than that in Group D (P<0.05). ELISA results showed that the mice in Group C, D and E produced specific IgG, while the mice in the control group(Group A and B) did not. The mice in Group C, D and E also produced IgG1 and IgG2a antibody isotypes, with the ratios of IgG2a/IgG1 0.394, 3.518 and 0.914 respectively. In comparison with the control group (Group A and B), the levels of IL-2 and IFN-γof the mice in Group D and E were obviously augment. The levels of IL-4 of mice in each group were not detected. The study showed that the protective immunity effects of nucleic acid vaccines against Schistosoma japinicum infection were greatly improved by electroporation in vivo.Part 3. The further evaluation on application of electroporation in vivo in the DNA vaccines against Schistosoma japonicum infection To further evaluate the stability, reliability and repeatability on application of electroporation in vivo in the DNA vaccines against Schistosoma japonicum infection, we undertook this experiment.The DNA cocktail vaccines and protein cocktail vaccines for immunization were prepared following the above protocol. Ninety-six female BALB/c mice of 5-6 weeks old were randomly divided into 8 groups(A, B, C, D, E, F, G, H), twelve for each group. In group A(pcDNA3.1 control group), each mouse was immunized (i.m.) with 100μl of pcDNA3.1 plasmid DNA for three times at week 0, 3, 6; in group B(pcDNA3.1/E.P. plus cocktail protein vaccine group), each mouse was immunized (i.m.) with 100μl of pcDNA3.1 plasmid DNA followed by electroporation in vivo for three times at week 0, 3, 6 and boosted with 100μl of cocktail protein vaccines plus 100μl FCA by subcutaneous at week 9; in group C(the cocktail DNA vaccines group) each mouse was immunized (i.m.) with 100μl of cocktail DNA vaccines for three times at week 0, 3, 6; in group D(cocktail DNA vaccines + cocktail proteins) each mouse was immunized (i.m.)with 100μl of cocktail DNA vaccines for three times at week 0, 3, 6 and boosted with 100μl of cocktail protein vaccines plus 100μl FCA by subcutaneous at week 9; in group E(cocktail DNA vaccines/E.P. group), each mouse was immunized (i.m.) with 100μl cocktail DNA vaccines followed by electroporation in vivo for three times at week 0, 3, 6; in group F (cocktail DNA vaccines/E.P. plus cocktail protein vaccines group), each mouse was immunized (i.m.) with 100μl of cocktail DNA vaccines followed by electroporation in vivo for three times at week 0, 3, 6 and boosted with 100μl of cocktail protein vaccines plus 100μl FCA by subcutaneous at week 9; in group G(TPI group), each mouse was immunized (i.m.) with 100μl of pcDNA3.1-SjCTPI plasmid DNA for three times at week 0, 3, 6; in group H( TPI/E.P. group) each mouse was immunized (i.m.) with 100μl of pcDNA3.1-SjCTPI plasmid DNA followed by electroporation in vivo for three times at week 0, 3, 6. Four weeks after the last DNA immunization or two weeks after protein boosting, all the 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 eggs in liver were counted. The blood was collected from the tail veins of all the mice two days before the first immunization and challenge, respectively, the serum was prepared for detection of IgG, IgG1 and IgG2a. Two days before the challenge, the spleen cells of two mice from each group were cultured and stimulated with ConA and soluble egg antigen (SEA), and the supernatant was collected for detection of IL-2, IL-4 and IFN-γby flow cytometre. The worm reduction rates of each experimental group(B-H) were 14.77%, 32.54%, 43.95%, 41.47%, 59.38%, 27.23% and 42.37% , respectively, compared to the control group(Group A). The female worm reduction rates of each experimental group(B-H) were 30.69%, 47.19%, 62.60%, 53.80%, 72.28%, 35.64% and 49.60%, respectively, compared to the control group(Group A). The worm reduction rates and the female worm reduction rates of each exprimental group were significantly higher than the control group. The egg reduction rates in liver of each experimental group(B-H) were 20.62%, 44.06%, 57.57%, 52.83%, 67.49%, 27.25% and 42.75%, respectively, compared to the control group, and were higher than that in the control group. ELISA results showed that the mice in each experimental group produced specific IgG, IgG1 and IgG2a, while the mice in control group did not. In camparison with the control group, the IL-2 and IFN-γlevel of the spleen cultured supernatant in each experimental group were obviously augment. The levels of IL-4 of mice in each group were not detected. This study showed that the method of immunization, priming with cocktail DNA vaccines followed by electroporation in vivo and boosting with cocktail protein vaccines, could enhance the protective effects of vaccines against Schistosoma japonicum infection in mice. The worm reduction rates, the female worm reduction rates and the egg reduction rates in liver gained were approximately 60% or more. These results gained were similar to that in Part 2. It is suggested that this method is stable and repeatable as well as feasible for control of schistosomiasis.
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