STUDIES ON ANTI-FECUNDITY INDUCED BY CATHEPSIN B DNA VACCINE OF SCHISTOSOMA JAPONICUM
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Aim To study the anti-fecundity immunity effect in mice induced by Schistosoma japonicum cathepsin B DNA vaccine(Sj31BIN) Methods The DNA Vaccine of S japonicum was constructed by inserting Sj 319 one into vector VR 1012 The mice were immunized with Sj31BIN and the anti-fecundity immunity was determined by counting worms,and egg burden in liver,intestine and faeces on the 46th day after challenge infection with carcariae of S japonicum Results The immnization with Sj31BIN reduced the worm discovery and egg burden in tissues and faeces The egg reduction rate was 59 3%～65 0% in the liver,57 6%～62 1% in the intestine and 86 5% in the faeces ln addition,the number of eggs in the uteri of female adults was obviously decreased in mice immuicized with Sj31BIN,Conclusion Sj31BIN immunization induces anti-fecundity immunity in miceCite
Schistosoma japonicum glutathione-s-transferase (SjGST), Schistosoma japonicum 32,000 protein (Sj32) and SjGST - Sj32 were expressed and purified to study on protective immunity against Schistosoma japonicum. The result showed that compared with the adjuvant control group, the worm burden was decreased significantly in mice immunized with recombinant SjGST - Sj32, Sj32 and Sj32 + SjGST (P < 0.05). At the same time, the liver egg per gram(EPG) was decreased in mice immunized with SjGST - Sj32, Sj32, Sj32 + SjGST and SjGST. It is concluded that Sj32 significantly induces both protective immunity and anti-fecundity immunity, but SjGST only has an anti-fecundity effect.
Schistosoma
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Objective: To study the effect of recombinant BCG-Sj26GST vaccine of schistosoma japonicum on IL-6 produced by spleen cells of mice. Methods: In the first experiment, mice were immunized subcutaneously by the vaccine, with PBS serving as control, challenged with Sj cercariae after 8w of immunization and killed on 6w of the infection to separate spleens; in the second experiment, after subcutaneous and intravenous immunization by the vaccine respectively, 4 mice were killed to separate spleens on 0,4,8,10,14 and 16w of immunization, spleen cells were stimulated by Sj26 or PHA, and the level of IL-6 in supernatant of spleen cells were detected by ELISA. Results: The level of IL-6 had no obvious change by immunization with the vaccine against challenge with Sj cercariae;dynamic observation showed that IL-6 reached the highest level on 8~10w of immunization. Conclusion: IL-6 may not be related to the protective immunity elicited in mice by vaccination with recombinant BCG-Sj26GST vaccine of schistosoma japonicum.
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To evaluate the potential value of the novel gene of S. japonicum SjCWL01 as a nucleic acid candidate vaccine by the construction of pcDNA3/SjCWL01 followed by observation of its immunological protection effect on mice. A novel gene of S. japonicum SjCWL01 was subcloned into the eukaryotic expression vector pcDNA3. The positive recombinants were identified by PCR and restriction enzyme digestion. Preparation of the DNA vaccine was made by transforming the plasmid pcDNA3/SjCWL01 into E.coli DH5α. The experimental mice were immunized with purified pcDNA3/SjCWL01 DNA vaccine and challenged with 40±1 S. japonicum cercariae percutaneously at the 2nd week after the third immunization. Levels of the specific antibody were detected by ELISA before infection. The reduction rates of the worm burden, liver eggs per gram and fecal eggs per gram were examined 45 days after challenge infection. Compared with the control group, the reduction rates of the worm burden, liver eggs per gram and fecal eggs per gram were 27.6%, 39.5% and 45.9% in the experimental mice, respectively. The pcDNA3/SjCWL01 nucleic acid vaccine can induce partial protective immunity against S. japonicum infection in mice.
Eggs per gram
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AIM: To explore the protective immunity against cercariae challenge in mice immunized with Sj32DNA vaccine of Schistosoma japonicum. METHODS: Primers were designed and the full length and part cDNA encoding 32 kDa(Sj32) amplified from pSj32 by PCR were cloned into pBK CMV. Eukaryotic expression vector pBK Sj32 1 containing full length Sj32 cDNA and pBK Sj32 2 containing fragment of Sj32 cDNA were constructed. After selection and identification, two plasmids were transfected into NIH 3T3 cells. The expression of Sj32 in transfected cells was confirmed by IFA. After identification, the two plasmids were prepared and purified on batches. Each BALB/c mouse was vaccinated at weeks 0,3, and 5, by injection with 100 μg of purified Sj32DNA into quadriceps muscle. Mice immunized with constructs and the control mice were challenged four weeks after final DNA injection, and worms and the number of eggs in the liver tissue were counted at 8 weeks after challenge. RESULTS: Perfusion and egg counts showed significant differences in terms of worm reduction rate 38 6%-30 9% and egg reduction rate 55 7%-55 2% between the vaccinated and control mice. CONCLUSION: Sj32 DNA vaccine could result in protection against a subsequent challenge of S.japonicum in mice.
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Abstract The aim of this study was to construct and evaluate the immunity efficacy of the DNA multivalent vaccine pVIVO2SjFABP-23. The vaccine was constructed and produced as follows. Forty BALB/c mice were divided into four groups designated pVIVO2, pVIVO2Sj23, pVIVO2SjFABP and pVIVO2SjFABP-23. Each mouse was immunized with 100 μg of the corresponding plasmid DNA by intramuscular injection. 28 days post-vaccination, the mice were challenged with S. japonicum cercariae, and the worm and egg burdens were determined 42 days post-challenge. Serum samples were collected from all the mice before and after vaccination and at the end of the experiment, and used for antibody detection. The IFN-γ and IL-4 levels were quantified in the supernatants of specifically stimulated spleen cells. The number of worms was reduced by 52%, 40% and 42% in mice respectively immunized with pVIVO2SjFABP-23, pVIVO2Sj23 or pVIVO2SjFABP. A respective 61%, 38% and 39% egg reduction was determined relative to those mice that only received the empty pVIVO2 plasmid. pVIVO2SjFABP-23 immunization increased IgG levels against SWAP and SEA. Increased IFN-γ levels were detected in the supernatant of specific stimulated spleen cells from mice immunized with the 3 different constructs. The multivalent DNA vaccine developed induced higher levels of protection than the two monovalent tested vaccines.
Intramuscular injection
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To observe the effects of anti-fecundity and anti-embryonation immunity of anti-idiotypic monoclonal antibody NP30 of Schistosoma japonicum on female adult worm.The active immunization of C57BL/6 mice was conducted by means of three intraperitoneal injections of NP30. The control group was injected with SP2/0 ascites intraperitoneally.On the twenty-seventh day after challenge infection, the number of eggs in the liver tissue and in uterus of the group immunized with NP30 decreased by 30.91% and by 38.55%, respectively. On the thirty-ninth day after the challenge infection, the number of mature eggs in the liver tissue of the group immunized with NP30 decreased by 66.63% and the number of dead eggs increased by 60.66%.NP30, with which mice were actively immunized, possesses double effects of anti-fecundity and anti-embryonation immunity on female adult worm of Schistosoma japonicum, therefore it can be used as a promising candidate of anti-pathologic vaccine molecule against Schistosomiasis japonica.
Schistosoma
SCHISTOSOMIASIS JAPONICA
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Aim To observe protection effect of BALB/c mouse against Schistosoma japonicum infection through DNA immunization of Schistosoma japonicum novel gene Sj Dad1 Methods First construct eukaryotic expression plasmid pEGFP-N 3-Sj Dad1;Then BALB/c mice were immunized by naked plasmid pEGFP-N 3-Sj Dad1.NS group and plasmid pEGFP-N 3 group were set up as control The mice were charged by Schistosoma japonicum cercaria after twice immunization;finally they were killed and statistical analysis was conducted Results The recombinant plasmid pEGFP-N 3-Sj Dad1 was constructed successfully,It was verified by double restriction,PCR reaction and sequencing Statistical analysis of animal experiment showed:in obtaining adult worms,no significant difference exists between pEGFP-N 3-Sj Dad1 group,pEGFP-N 3 group and NS group(P0 05);In obtaining eggs per gram of liver,significant difference exists between pEGFP-N 3-Sj Dad1 group and NS group(P0 01),egg reduction rate is 65 74 % Conclusions The novel gene Sj Dad1 is anti-reproductive,it is a possible candidate vaccine molecule
Eggs per gram
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Aim To search for the probable mechanism of Sj31B1N anti-fecundity immunity.Methods Mice were immunized with Sj31B1N,and collected blood in tail vein every two weeks.Sera antibody was detected in order to acquire antibody dynamics in mice and anti-fecundity immunity was determined by counting worms,and egg burden in liver and intestine on the 45th day after challenge infection with cercariae of S.japonicum.Results Part of mice immunized with Sj31B1N showed positive antibody on the fourteenth day.The mean OD value and number of positive antibody mice increased gradually.In contrast with negative antibody mice,positive antibody mice's worm reduction rate,egg reduction rate in the liver and intestine was 40.6%,48.0% and 45.4% respectively.Conclusion Sj31B1N could induce mice to produce antibody which had immune protective function.Humoral immunity induced by Sj31B1N.probably was one of mechanism of anti-fecundity immunity.
Humoral immunity
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Objective To observe the protective immunity induced by the DNA vaccines of VR1012 Sj31 and VR1012 SjGST Sj31 of Schistosoma japonicum (Sj)in BALB/c mice.Methods Thirty six BALB/c mice were divided into three groups.Of control group A,each mouse was injected at quadriceps femoris with 100μg VR1012 plasmid DNA;of group B,each mouse was injected intramuscularly with 100μg VR1012 Sj31 plasmid DNA;of group C,each mouse was injected intramuscularly with 100μg VR1012 SjGST Sj31 plasmid DNA.All mice were immunized three times with an interval of 2 weeks and were challenged each with 10 cercariae of Sj at the 21th days after final immunization.At day 42 after challenge,all mice were sacrificed,the numbers of worms and hepatic eggs were counted.Antibody level in the sera of mice before and three weeks after immunization was determined with ELISA.Results There are specific IgG in the sera of mice in the test groups after the 3rd immunization.Compared with the control group,the worm reduction were 25.0% and 30.0%( P 0.05);the egg reduction were 54.90% and 69.74%( P 0.001);the egg reduction rates in the liver per female worm were 47 96% and 54 62%( P 0 001)respectively.Compared with the B group,the egg reduction of C groups were 32 92%( P 0 001),the egg reduction rates in the liver per female worm were 12 79%( P 0 05).Conclusion The DNA vaccines,VR1012 Sj31 and VR1012 SjGST Sj31 could induce partial protective immunity against Sj in mice.The protective immunity induced by bi valent DNA vaccine was better than that induced by monovalent DNA vaccine.
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To study the immune protection of Schistosoma japonicum fatty acid binding protein (Sj14FABP) DNA vaccine enhanced by IL-12.The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed respectively, and were prepared on a large scale after identification. 48 male BALB/c mice were divided into 4 groups randomly. In group A, B, C, and D, each mouse was injected intramuscularly with 100 microl 0.9% NaCl, 100 microg pVIVO2, 100 microg pVIVO2-Sj14FABP and 100 microg pVIVO2-IL12-Sj14FABP respectively. 30 days after immunization each mouse was challenged with 40 +/- 2 cercariae of S. japonicum. On day 45 after challenge, all mice were sacrificed to count the number of recovered adult worms and the hepatic eggs. Sera from mice were used to detect IgG antibody. The production of IL-2, IL-4 and IFN-gamma in the supernatant of spleen cells was observed by means of sandwich ABC-ELISA.The recombinant plasmids pVIVO2-Sj14FABP and pVIVO2-IL12-Sj14FABP were constructed. The worm reduction rate in group C and D was 24.11% and 39.4%, as well as liver egg reduction rate of 27.2% and 32.8% respectively. The level of IL-2 and IFN-gamma in group D increased significantly, while IL-4 secretion decreased (P < 0.01). 30 days after immunization, no higher titer of IgG antibody was shown in all groups. Furthermore, no significant difference on the level of IgG was found among the groups (P > 0.05).Sj14FABP DNA vaccine induces partial protective immunity in BALB/c mice. IL-12 drives the immune response toward a Th1 direction, and enhances the protective immune effect of the vaccine.
BALB/c
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