Synthesis and SAR studies of praziquantel derivatives with activity against Schistosoma japonicum.

The synthesis and structure-activity relationship (SAR) studies of praziquantel derivatives with activity against adult Schistosoma japonicum are described. Several of them showed better worm killing activity than praziquantel and could serve as leads for further optimization.

Characterization of IgG responses of rabbits to Sj14-3-3 protein after experimental infection with Schistosoma japonicum.

An indirect enzyme-linked immunosorbent assay method was developed for detection of IgG against 14-3-3 protein in sera of rabbits. Rabbits infected with 500 cercariae of Schistosoma japonicum were grouped and the characterization of the IgG responses was observed. For the treated group, the IgG could be detected as early as 2-4 weeks post-infection and then their levels rose rapidly and reached a peak at around 6 weeks. After the infected rabbits were treated with praziquantel at 6 weeks post-infection, IgG levels in the sera significantly decreased. While in the untreated group, the IgG levels were constantly very low. For all infected rabbits, 60 % (six of ten) had positive reaction with 14-3-3 protein, and 40 % (four of ten) had high IgG levels. This finding would be more helpful to understand this 14-3-3 protein.

Kinetics of circulating antigen 14-3-3 in sera of rabbits firstly infected with Schistosoma japonicum and treated with/without praziquantel.

A sandwich ELISA was developed for the detection of circulating antigen 14-3-3 in the sera of rabbits. Rabbits that were infected with 500 cercariae of Schistosoma japonicum were grouped and the kinetics of 14-3-3 was observed. For the treated group, the 14-3-3 protein could be detected as early as 2-4 weeks postinfection and then its levels rose rapidly and reached a peak at around 6 weeks. The 14-3-3 levels in the sera significantly decreased after the infected rabbits were treated with praziquantel at 6 weeks postinfection and declined to the initial level about 8 weeks posttreatment. While in the untreated group, 14-3-3 levels reached a peak in 8 weeks postinfection and then remained at plateau level for about 6 weeks. Our findings showed that detection of S. japonicum 14-3-3 has an important value for diagnosis of acute infection of S. japonicum and evaluation of chemotherapy.

Effect of praziquantel prolonged administration on granuloma formation around Schistosoma japonicum eggs in lung of sensitized mice.

Schistosomiasis remains a major public health problem and it is an immune disease. The schistosome egg is the primary parasite factor responsible for the overt disease. The eggs release the soluble antigen, which induces intensive tissue reaction, a granulomatous reaction to the eggs. If granuloma formation could be suppressed, overt disease might not develop. Praziquantel is an effective antischistosomal drug especially for adult worms. However, whether praziquantel has a suppressing effect on granuloma formation around schistosome eggs directly remains unclear. The purpose of the present study was to investigate the effect of praziquantel, especially administered persistently, on granuloma formation around Schistosoma japonicum eggs in the lung of sensitized mice. Thirty-six mice were divided into three groups averagely. Group A was a control group. First, the mice were injected with schistosomal eggs hypodermically in abdomen, and 10 days later injected with schistosomal eggs intravenously via a tail vein. Group B was a praziquantel short administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 300 mg/kg for 3 days, from 1 day before the intravenous injection of the eggs. Group C was a praziquantel prolonged administration group. In addition to the injections of schistosomal eggs as the same of Group A, the mice were administered with praziquantel in a daily dose of 150 mg/kg for 5 days weekly until the mice were sacrificed. Three mice of each group were sacrificed on days 7, 14, 28, and 56, respectively after the intravenous injection of the eggs, and the lung tissues were fixed with formalin and the slices were HE stained. The granulomas containing eggs in their centers were selected, and 25-30 granulomas from the animals of each group were measured at each time period. The mean areas of egg granulomas of each group were calculated, and the neutrophilic granulocytes, eosinocytes, lymphocytes, fibroblasts, and macrophages within the egg granulomas were counted. The mean numbers of them of each group were calculated. All the data of each group were analyzed and compared statistically. On day 56 after the intravenous injection of the eggs, the mean area of schistosomal egg granulomas in group B was (227.4 ± 728.0) × 10(3) µm(2), less than that of [(297.9 ± 153.3) × 10(3) µm(2)] in group A, and the suppression rate was 23.7% (P < 0.05). On days 7, 14, 28, and 56, the mean areas of schistosomal egg granulomas in group C were (575.8 ± 155.6) × 10(3) µm(2), (310.5 ± 854.0) × 10(3) µm(2), (267.7 ± 513.3) × 10(3) µm(2), and (214.9 ± 446.4) × 10(3) µm(2), respectively, significantly less than those of [(692.7 ± 232.6) × 10(3) µm(2), (439.4 ± 165.0) × 10(3) µm(2), (385.7 ± 129.3) × 10(3) µm(2), and (297.9 ± 153.3) × 10(3) µm(2)] in group A. The suppression rates were 16.9%, 29.3%, 30.6%, and 27.9%, respectively (P values <0.05). On day 56, the mean numbers of neutrophilic granulocytes were 11.4 ± 5.0 in group A and 5.2 ± 3.1 in group C, respectively, with the suppression rate of 54.4% in group C (P < 0.05). On day 56, the mean numbers of eosinocytes within the egg granulomas were 2.3 ± 2.0, 0.1 ± 0.3, and 0.3 ± 0.6 in groups A, B, and C, respectively, with the suppression rate of 95.7% in group B and 87.0% in group C (P values <0.05). On day 56, the mean numbers of macrophages within egg granulomas were 14.3 ± 6.9 in group C, compared with 18.6 ± 8.2 in group A, the suppression rate was 23.1% (P < 0.05). On day 56, the mean numbers of fibroblasts within the egg granulomas were 6.6 ± 4.4 and 5.8 ± 2.6 in groups B and C, respectively, and compared with 14.3 ± 7.8 in group A, the increasing extents decreased by 53.8% and 59.4%, respectively (P values <0.05). Therefore, the administration of praziquantel, especially the prolonged administration, can suppress the formation of schistosomal egg granulomas, including reduction in the areas of granulomas and suppression of the inflammatory cells and the hyperplasia of fibroblasts within granulomas.

[Gene synthesis, expression and characterization of egg protein IPSE of Schistosoma mansoni].

OBJECTIVE: To synthesize and express the gene of egg protein IPSE (IL-4-inducing principle of Schistosoma mansoni eggs) and evaluate its immunologic characteristics. METHODS: The IPSE gene of S. mansoni was synthesized by overlapping PCR, and confirmed by DNA sequencing, The recombinant plasmid IPSE-pET32a(+) was constructed by inserting the gene of IPSE into expression vector pET32a(+) at the downstream of thioredoxin (Trx) coding sequence. The recombinant plasmid IPSE-pET32a(+) was transformed into E. coli BL21(DE3) and followed by expression of the protein induced by IPTG. Large-scale fusion protein was prepared and purified with Ni affinity chromatography gel under denaturing conditions. A small amount of soluble Trx-IPSE was obtained by dialyzing the fusion protein in a large volume of PBS. Western blotting was used to detect if the recombinant IPSE was recognized by the IgG antibody in the pooled patient sera of schistosomiasis japonica and its binding capacity to the non-specific IgE antibody in the sera of healthy persons. RESULTS: DNA sequencing confirmed that the nucleotide sequence of synthesized IPSE gene was completely identical to the native one. SDS-PAGE showed that the recombinant plasmid IPSE/pET32a(+) expressed a fusion protein with an Mr 35700 after being induced by IPTG. The pure fusion protein Trx-IPSE reacted positively with the pooled sera of schistosomiasis patients under either denaturing or renaturing conditions. The protein Trx-IPSE also reacted with the nonspecific IgE in the sera of healthy persons. CONCLUSION: The IPSE gene of Schistosoma mansoni has been synthesized, and the recombinant can react with natural antibody IgG against S. japonicum and non-specifically bind to IgE antibody.

The Differential and Dynamic Progression of Hepatic Inflammation and Immune Responses During Liver Fibrosis Induced by Schistosoma japonicum or Carbon Tetrachloride in Mice.

Liver fibrosis can result from various causes and could progress to cirrhosis and cancer; however, there are no effective treatments due to that its molecular mechanism is unclear. liver fibrosis model made by Schistosoma japonicum (S. japonicum) infection or Carbon tetrachloride (CCl4) intraperitoneal injection is a conventional model used in liver fibrosis-related studies for mechanism or pharmaceutical research purposes. But the differences in the pathological progression, immune responses and the underlying mechanism between the two liver fibrosis model have not been carefully compared and characterized, which hinders us from correctly understanding and making better use of the two models. In the present study, the pathological changes to the liver, and the cytokines, inflammatory factors, macrophages, and lymphocytes subsets involved were analyzed in the liver fibrosis model of S. japonicum infection or CCl4 intraperitoneal injection. Additionally, the pathological progression, immune responses and the underlying injury mechanism in these two models were compared and characterized. The results showed that the changing trend of interleukin-13 (IL-13), transforming growth factor beta (TGF-ß), inflammatory factors, and M1, M2 macrophages, were consistent with the development trend of fibrosis regardless of whether liver fibrosis was caused by S. japonicum or CCl4. For lymphocyte subsets, the proportions of CD3+ T cells and CD4+ T cells decreased gradually, while proportion of CD8+ T cells peaked at 6 weeks in mice infected with S. japonicum and at 12 weeks in mice injected with CCl4. With prolonged S. japonicum infection time, Th1 (CD4+IFN-γ+) immunity converted to Th2 (CD4+IL-4+)/Th17 (CD4+IL-17+) with weaker regulatory T cell (Treg) (CD4+CD25+FOXP3+) immunity. However, in liver fibrosis caused by CCl4, Th1 cells occupied the dominant position, while proportions of Th2, Th17, and Treg cells decreased gradually. In conclusion, liver fibrosis was a complex pathological process that was regulated by a series of cytokines and immune cells. The pathological progressions and immune responses to S. japonicum or CCl4 induced liver fibrosis were different, possibly because of their different injury mechanisms. The appropriate animal model should be selected according to the needs of different experiments and the pathogenic factors of liver fibrosis in the study.

[Gene synthesis, expression and immunogenicity analysis of TSP2 hydrophilic domain(TSP2HD) of Schistosoma japonicum].

OBJECTIVE: To synthesize and express the gene of TSP2 hydrophilic domain of Schistosoma japonicum, and investigate the immunogenicity of the recombinant TSP2HD protein. METHODS: The whole DNA fragment encoding the TSP2 hydrophilic domain was synthesized by overlapping PCR, and confirmed by DNA sequencing. The recombinant plasmid TSP2HD-PG was constructed by inserting the purified TSP2HD DNA fragment into expression vector pGEX-4T-3 and the GST-TSP2HD fusion protein was expressed by transforming the recombinant plasmid TSP2HD-PG into Escherichia coli BL21 (DE3) and induced the recombinant with isopropyl beta-D-1-thiogalactopyranoside (IPTG). The expressing situation of fusion protein was analyzed by SDS-PAGE. The GST-TSP2HD fusion protein was purified by affinity chromatography with glutathione sepharose 4B gel, and the purified recombinant TSP2HD protein was prepared by digesting the GST-TSP2HD fusion protein with thrombin. The immuno-response of the recombinant TSP2HD recognized by the pool sera of schistosomiasis patients and the pool sera of heavily infected rabbits was explored by Western blotting analysis. The immunogenicity of the recombinant TSP2HD was investigated by comparing the difference of counts per minute (cpm) value of lymphocyte proliferation test between experiment group and control group. RESULTS: A 228 bp of TSP2HD gene fragment was obtained after overlapping PCR of three times and its DNA sequence was confirmed by DNA sequencing, which was same to one of the native TSP2HD. The recombinant containing recombinant plasmid TSP2HD-PG expressed a soluble fusion protein of GST-TSP2HD (Mr approximately 34 000) after being induced with IPTG. The purified recombinant TSP2HD protein was obtained through digesting the GST-TSP2HD fusion protein with thrombin. The recombinant TSP2HD was recognized by pool sera of schistosomiasis patients and pool sera of infected rabbits, indicating that the recombinant TSP2HD has a good response activity. The recombinant TSP2HD also stimulated proliferation of lymphocytes in infected mouse, the cpm value of experiment group was higher than that of the control (P < 0.01). CONCLUSION: The Sj TSP2HD gene has been synthesized and expressed with immunogenicity which is similar to that of the native antigen.

Identification of Peptide Antagonists to Thioredoxin Glutathione Reductase of Schistosoma japonicum.

Schistosomiasis is one of the world's major public health problems. Praziquantel is currently the only effective drug against schistosomiasis. As resistance of praziquantel has emerged in some endemic areas, development of new antischistosomal agents should be a high priority. In this study, a phage display peptide library was used for screening for peptide antagonists of thioredoxin glutathione reductase of Schistosoma japonicum (SjTGR), which has been identified as an alternative drug target. Three rounds of panning produced four different fusion phages. ELISA proved that all four phages could bind to SjTGR. One peptide, JIPDys1 (aa, WPHNWWPHFKVK), reduced enzyme activity of SjTGR by more than 50%. 2 µM of the synthesized peptide of JIPDys1 inhibited the activity of TrxR, GR, and Grx of SjTGR by 32.5%, 100%, and 100%, respectively. The IC50 values of the synthetic peptide JIPDys1 for TrxR, GR, and Grx were 3.67 µM, 0.11 µM, and 0.97 µM, respectively. Based on computer simulation, it appeared that JIPDys1 binds to the substrate binding sites of glutathione reductase (GR) and glutaredoxin (Grx). Our data show that the peptide, JIPDys1 (aa, WPHNWWPHFKVK), is a promising candidate to develop novel drugs against S. japonicum which acts by binding with SjTGR and reduces enzyme activity of SjTGR.

[Development and identification of the multiple B cell epitope antigens of Schistosoma japonicum].

OBJECTIVE: To develop multiple B cell epitope antigens of Schistosoma japonicum and evaluate their antigenicity. METHODS: Bioinformatics software BioSun was used to predict B cell epitopes from Sj22.6, Sj14-3-3 and Sj26. The predicted epitopes P2, P6 and P7 were ligated to construct P2-P6-P7 and P6-P2-P7 multiepitope in random order, a 6 amino acid linker inserted between epitopes. Recombinant plasmids containing the two multiepitopes identified by enzyme digestion and sequencing were transformed into E. coli BL21. The expressed recombinant fusion proteins of E. coli BL21 induced with IPTG were purified with Ni2+ chelating HiTrap HP column. Their antigenicity was evaluated with Western-blotting. RESULT: The two multiple B cell epitopes P2-P6-P7 and P6-P2-P7 were successfully cloned into pET-32c(+) plasmid and fusion proteins were expressed. SDS-PAGE showed a single band and both of the recombinant fusion proteins were with Mr 20 400. The two proteins reacted with the sera of schistosomiasis patients but not with that of healthy people. CONCLUSION: Two multiple B cell epitope antigens were developed with potential diagnosis value.

Oxadiazole-2-oxides may have other functional targets, in addition to SjTGR, through which they cause mortality in Schistosoma japonicum.

BACKGROUND: Schistosomiasis is one of the world's major public health problems. Besides praziquantel (PZQ), there is currently no other effective treatment against schistosomiasis. The development of new antischistosomal agents to curb the emergence of PZQ resistance should be a high priority. Oxadiazole-2-oxides have been identified as potential antischistosomal reagents, with thioredoxin glutathione reductase (TGR) being one of their molecular targets. METHODS: To develop novel treatment reagents against Schistosoma japonicum, 30 novel oxadiazole-2-oxides were synthesised and their antischistosomal activities on juvenile and adult S. japonicum were evaluated in vitro and in vivo. Their inhibitory activities against S. japonicum thioredoxin glutathione reductase (SjTGR) were also analysed. RESULTS: Most of the oxadiazole-2-oxides showed good juvenile and adult S. japonica killing activities in vitro. However, the antischistosomal effects of these compounds were not positively correlated with either their inhibition of SjTGR, or with nitric oxide (NO) release. Compounds 4a, 4b, 7c, 13, 16 and 20 resulted in 87.7%, 83.1%, 87.1%, 84.6%, 90.8% and 69.5%, respectively, mortality in the adult worms, when used to treat infected mice at schistosomula stage. These mortality rates were similar to or higher than that of artemisinin. Furthermore, compounds 4a and 16 resulted in 66.7% and 69.4% reductions in the worm burdens, respectively, when infected mice were treated at the adult worm stage. These treatment effects were similar to PZQ. No differences in activity of the oxadiazole-2-oxides against female and male adult worms were observed. The toxicity of the oxadiazole-2-oxides on mammalian cells appeared to be similar to, or less than, that of PZQ. CONCLUSIONS: The antischistosomal activity of the oxadiazole-2-oxides does not depend on NO production or the inhibition of SjTGR activity. There may be other functional targets of the oxadiazole-2-oxides in S. japonicum. Several of the novel oxadiazole-2-oxides synthesised in this study could be used to develop novel antischistosomal drugs and explore potential molecular targets.

[Fusion expression and antigenicity analysis of miracidial antigen from eggs of Schistosoma japonicum].

OBJECTIVES: To express the miracidial antigen from eggs of Schistosoma japonicum (Chinese mainland strain) (SjMP10), and investigate the role of the miracidial antigen during the hepatic granuloma formation of schistosomiasis. METHODS: A pair of specific primers was designed and synthesized according to the nucleotide sequence of the open reading frame of the miracidial antigen gene. The open reading frame of the miracidial antigen gene was amplified, digested by restrictive enzyme (BamHI, SolI), and cloned directly into the expression plasmid pGEX-4T-3 to construct the recombinant plasmid. The recombinant plasmids were transformed into E. coli BL21(DE3), and induced by IPTG to express the fusion protein of GST-SjMP10. The expressed fusion protein was purified by electric elution method, and its antigenicity was examined by Western blotting and lymphocyte proliferation test. RESULTS: The gene of miracidial antigen was cloned into the expression plasmid pGEX-4T-3. After induced by IPTG, the recombinant expressed a fusion protein of GST-SjMP10, with a molecular weight of 39 000 approximately. The purified fusion protein showed proper antigenicity that could be recognized by the sera of rabbits heavily infected by Schistosoma japonicum and could stimulate the proliferation of splenic lymphocytes of infected BALB/c mice. CONCLUSION: The miracidial antigen from eggs of Schistosoma japonicum was expressed successfully.

[Enhancement of the protective effect of SjC23 DNA vaccine against Schistosoma japonicum infection by immunostimulatory sequence].

OBJECTIVE: To investigate the effect of immunostimulatory sequence on SjC23 DNA vaccine against Schistosoma japonicum infection. METHODS: SjC23 gene fragment was inserted into pcDNA3. 1-CpG to construct pcDNA3.1-SjC23/CpG. BALB/c mice in 4 groups were immunized intramuscularly 3 times at 2 week intervals, with 100 microg plasmid DNA per injection. Four weeks after the 3rd immunization, all mice were challenged with 45 +/- 1 cercariae of S. japonicum by abdominal skin penetration. After 45 days post-challenge, mice were perfused and the number of recovered worms and of eggs in liver was counted. Blood samples were collected from the tail vein of all mice 2 days before the 1st immunization and before challenge respectively. IgG, IgG1 and IgG2a in sera were detected. Three weeks after the 3rd inoculation, the spleen cells of 2 mice from each group were cultured and stimulated with ConA and recombinant peptide. The supernatant was collected to detect IL-2, IL-4 and IFN-gamma. Simultaneously, the cytotoxic activity was detected with 51Cr release assay in vitro. RESULTS: The worm reduction rate in SjC23 group and SjC23/CpG group was 28.1% and 35.1%, the hepatic egg reduction rate was 21.6% and 26.5%, respectively, compared with the control group. The level of protection in SjC23/CpG group was higher than that in SjC23 group (P<0.05). ELISA results indicated that mice immunized with pcDNA3.1-SjC23 and SjC23/CpG produced specific IgG to rSjC23, while mice immunized with pcDNA3.1 and pcDNA3.1-CpG did not. Mice in SjC23 group and SjC23/CpG group also produced IgG1 and IgG2a antibody isotypes, with the ratio of IgG2a/IgG1 10.1 and 12.2, respectively. In comparison with the control, the level of IL-2 and IFN-gamma in mice immunized with pcDNA3.1-SjC23 and pcDNA3.1-SjC23/CpG was augmented. The cytotoxic activity of spleen cells from mice in SjC23/CpG group was augmented from 9.7% to 40.0% compared with that in SjC23 group. CONCLUSION: The study indicates that immunostimulatory sequence appears to increase the level of protection induced by immunization with pcDNA3.1-SjC23 vaccine.

[Establishment and diagnostic performance of biotin-avidin complex enzyme linked immunosorbent assay of detecting specific IgG4 of clonorchiasis].

OBJECTIVE: To explore the performance of the biotin-avidin complex enzyme linked immunosorbent assay of detecting specific IgG4 for the diagnosis of clonorchiasis. METHODS: The avidin-biotin complex enzyme linked immunosorbent assay of detecting specific IgG4 (IgG4-ABC-ELISA)against Clonorchis sinensis was established, and used to detect the serum samples of patients with clonorchiosis sinensis, schistosomiasis japonica, paragonimiasis, toxoplasmosis, echinococcosis, cysticercosis and sparganosis mansoni. At the same time, these sera were analyzed by the ELISA of detecting IgG4 (IgG4-ELISA) and ELISA of detecting the total IgG (IgG-ELISA) as controls. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and the respective diagnostic performance of the three methods were compared. RESULTS: The IgG4-ABC-ELISA for diagnosis of clonorchiasis was established successfully. The sensitivity and specificity of the IgG4-ABC-ELISA for detecting clonorchiasis were 90.0% and 98.2% respectively, and PPV and NPV were 93.8% and 97.0% respectively. Its diagnostic performance was 96.3%. The sensitivity and specificity of the IgG4-ELISA for detecting clonorchiasis were 86.0% and 98.2% respectively, and PPV and NPV were 93.5% and 95.9% respectively. Its diagnostic performance was 95.4%. The sensitivity and specificity of the IgG-ELISA for detecting clonorchiasis were 94.0% and 88.1% respectively, and PPV and NPV were 70.1% and 98.0% respectively. Its diagnostic performance was 89.4%. The sensitivity of IgG4-ABC-ELISA was higher than that of IgG4-ELISA (P < 0.05), and the specificity of IgG4-ABC-ELISA was higher than that of IgG-ELISA (P < 0.05). CONCLUSIONS: IgG4-ABC-ELISA of detecting specific antibody IgG4 against Clonorchis sinensis has high sensitivity and specificity. Therefore, it has a good application value in the diagnosis of clonorchiasis.

[Study on immunologic function of thioredoxin glutathione reductase from Schistosoma japonicum].

OBJECTIVE: To study the immunogenicity and the immuno-protection of thioredoxin glutathione reductase from Schistosomajaponicum (SjTGR) against schistosome infection in mice. METHODS: Seventy-five mice were randomly divided into 5 groups, namely, blank group, PBS group, CpG2 immunized group, TGR immunized group and TGR + CpG2 co-immunized group. Each mouse was immunized for 3 times. The mice were tail bled before the first immunization and 2 weeks after the third immunization. The serum antibody levels of total IgG, IgG1 and IgG2a against SjTGR were assayed by ELISA. Two weeks after the third immunization, each mouse was infected with 40 ± 2 S. japonicum cercariae by abdominal skin penetration. Forty-two days later, all the mice were sacrificed to collect schistosome adult worms and liver eggs. The worm and egg reduction rates were calculated respectively. The single splenocyte of mouse was collected 2 weeks after the third immunization, and the expressions of CD44high, CD4+CD44high or CD8+CD44high on splenocytes of mice were examined by flow cytometry. After 72 h incubation with recombinant SjTGR, the levels of IL-2, IL-4, IL-10, and IFN-γ in the single-cell supernatant were determined by using ELISA kit. RESULTS: Two weeks after the third immunization, the titers of serum IgG against SjTGR in mice immunized with SjTGR and co-immunized with SjTGR and CpG2 were higher than 1:200 000. The IgG2a: IgG1 ratio (IgG2a/IgG1) increased slowly with time in both TGR immunized group and TGR + CpG2 co-immunized group. There were obviously higher levels of IFN-γ and IL-2 in the cell supernatant in the TGR immunized group and TGR + CpG2 co-immunized group compared to the blank, PBS and CpG2 groups (P < 0.05). The increased subpopulations of CD44high, CD8+CD44high and CD4+ CD44high cells in the splenocytes from mice immunized by SjTGR and co-immunized by SjTGR and CpG2 were found comparing to the blank, PBS and CpG2 groups (P < 0.05). The TGR immunization and TGR + CpG2 co- immunization caused 9.4% and 10.5% reductions in the number of adult worms and 9.2% and 32.8% reductions in the number of eggs, respectively. CONCLUSIONS: SjTGR displays strong immunogenicity inducing Th1 type immune response in mice. However, it could not produce protective efficacy against S. japonicum infection. CpG2 ODN may be a broadly effective Th1 adjuvant.

[Protective immunity induced by the nucleic acid vaccine of SjC 21.7 in mice].

OBJECTIVE: To observe the protective immunity induced by the nucleic acid vaccine of 21.7 kDa membrane protein molecule of Schistosoma japonicum Chinese mainland strain (SjC 21.7) in BALB/c mice. METHODS: A pair of primers (P1 and P2) was synthesized according to the DNA sequence of the SjC21.7. The ORF sequence of SjC21.7 was amplified by PCR, and the Kozark sequence was added to the position of initiator. The gene fragment was inserted into the eukaryotic expression plasmid pcDNA3.1 to form the recombinant plasmid SjC21.7-pcDNA3.1. Forty-eight BALB/c mice were divided into three groups: control, test and boost. Each mouse was injected in quadriceps femoris with plasmid pcDNA3.1 (control) or recombinant plasmid SjC21.7-pcDNA3.1 (test, boost); for the boost group, with additional P35-pcDNA3.1 and P40-pcDNA3.1. All mice were immunized three times with an interval of 2 weeks, challenged each with 45 cercariae of S. japonicum at the 30th day after final immunization. At day 45 after challenge, all mice were sacrificed, the numbers of worms and hepatic eggs were counted. Antibody level in the sera of mice before and two weeks after immunization was determined with ELISA. The expression of the target gene in quadriceps femoris was observed with immunohistochemistry. RESULTS: The immunohistochemistry analysis showed that there were specific antigens expressed in the local tissue of the test group mice. There was specific IgG in the serum of partial mice in test and boost groups. Compared with the control group, the worm reduction rate was 29.9% and its egg reduction rate 13.8% in the test group; 31.9% and 28.0% respectively in the boost group. The egg reduction rate in the boost group was higher than that of the test group (P < 0.05). CONCLUSION: The SjC21.7 nucleic acid vaccine could induce partial protective immunity against Schistosoma japonicum in BALB/c mice.

[Expression of nitric oxide synthase (NOS) mRNA in the gonad of Oncomelania hupensis cultured in different temperature].

OBJECTIVE: To study the expression of nitric oxide synthase (NOS) mRNA in the gonad of Oncomelania hupensis in different temperature. METHODS: The snails were cultured at temperature of 0 degree C, 15 degrees C and 25 degrees C for 1 month. The total RNA of each group was extracted with the RNA extraction kit. A pair of degenerate primers was designed from conserved regions of mammalian NOSs, and the expression of NOS mRNA in the gonad was measured by means of reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The target genes of NOS were detected in the gonad of snails. The level of expression of snail NOS mRNA in 25 degrees C group and 0 degree C group was significantly higher than that in the control group(P < 0.01), there was no significant difference for the expression products between 15 degrees C group and control group (P > 0.05). CONCLUSION: The designation of primers of the snails was validated. The impact of temperature on the expression of snail NOS mRNA was determined, which suggested that NO plays an important role in the physiological and pathological modulation.

[Cloning and function analysis of high mobility group box 1 (HMGB1) protein of Schistosoma japonicum (Mainland strain)].

OBJECTIVE: To clone and express a high mobility group box 1(HMGB1) protein of Schistosomajaponicum (Mainland strain) and analyze its function. METHODS: The DNA fragment of open reading frame encoding Sj HMGB 1 protein was amplified by RT-PCR from the mRNA of S. japonicum worms, then it was subcloned into the expression vector pET28a(+) to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3), and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombinant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding capacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 protein and infected with 45 +/- 2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection, the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue, respectively. The worm and egg reduction rates were calculated respectively. RESULTS: A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR, which was the open reading frame (ORF) encoding SjHMGBlprotein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA, and the recombinant protein immunized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abundantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effective immune protection against S. japonicum. CONCLUSION: The gene encoding HMGB1 from S. japonicum and the soluble recombinant SjHMGB1 protein with natural functional activity are obtained, and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

Identification of proteins inducing short-lived antibody responses from excreted/secretory products of Schistosoma japonicum adult worms by immunoproteomic analysis.

The excretory/secretory antigens of Schistosoma japonicum (Sj ESAgs) play important roles in host-parasite immune interactions. In this study, the antibody response patterns to Sj ESAgs in sera of individual rabbits at the healthy stage, 2-6 weeks post-infection and 4-16 weeks after treatment were examined. Antigens inducing short-lived antibody responses were selected by comparing differences in immune recognition of proteins in sera across the different stages by Western blotting and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS). The diagnostic value of these short-lived antibody responses for schistosomiasis was investigated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as a major antigen inducing a short-lived antibody response in Sj ESAgs. The antibody response against Sj GAPDH decreased at week 4 and disappeared between weeks 8-12 after effective chemical treatment of rabbits, and this response declined to negative levels in schistosomiasis patients one year after treatment. The intensity of the antibody response against Sj GAPDH was dependent on parasite load in mice. The sensitivity and specificity of IgG antibodies against recombinant Sj GAPDH for schistosomiasis diagnosis were 82.5% and 91.3%. Our findings suggest that Sj GAPDH induces short-lived antibody responses in the host, and detecting IgG against this antigen provides the basis for developing a potential method for diagnosis and evaluating treatment effects for schistosomiasis japonicum. BIOLOGICAL SIGNIFICANCE: Schistosomiasis is one of the world's major public health problems. Developing effective diagnostic methods for detecting schistosome-specific antibodies to effectively identify active infections is part of a critical strategy for blocking transmission of the parasite and eradicating schistosomiasis. The excretory/secretory antigens of S. japonicum (Sj ESAgs) play important roles in host-parasite immune interactions. In our study, we examine the antibody response patterns to Sj ESAgs within individual rabbits at the healthy, schistosome infection and post-treatment stages by Western blotting. Proteins among the Sj ESAgs inducing short-lived antibody responses were identified by Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS), and their potential as immune markers for diagnosis and evaluating therapeutic effects in schistosomiasis was evaluated. Our findings suggest that S. japonicum glyceraldehyde-3-phosphate dehydrogenase (GAPDH) induces short-lived antibody responses in the host, and detecting IgG against this antigen provides the basis for developing a potential method for diagnosis and evaluating treatment effects for schistosomiasis japonicum.

[Epitope identification of monoclonal antibody 5C6 against 14-3-3 protein of Schistosoma japonicum].

OBJECTIVE: To identify the epitope of monoclonal antibody (McAb) 5C6 against 14-3-3 protein of Schistosoma japonicum by phage display peptide library. METHODS: The phage display 12-peptide library was screened with purified McAb 5C6 against 14-3-3 protein of S. japonicum three rounds of bio-panning "adsorption-elution-amplification" to enrich the specific binding phages. The single phage clones selected randomly were amplified, their genomic DNA were extracted and sequenced. The immune response characterization of phages with the same or high homologous foreign inserted DNA sequence was identified by ELISA and Western blotting for further defining the epitope recognized by McAb 5C6. RESULTS: A total of 33 single phage clones were selected and sequenced. Among them, 25 shared the same foreign inserted DNA sequence of 5'-CCACCTAGTAGCAGACCGATTCTCAGTCGAAGGAAA-3', encoding a deduced peptide PPSSRPILSRRK. This peptide was not homologue to Sj14-3-3 protein or any other known native protein in the world. The results of Western blotting showed that this peptide could be recognized by the sera of patients with schistosomiasis, but not by those of healthy persons. CONCLUSION: The mimic antigen epitope of McAb 5C6 against 14-3-3 protein of S. japonicum, which is a conformational epitope, has been defined successfully in this study.