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.

[Toxicity of several drugs against Schistosoma japonicum adult worms in vitro].

OBJECTIVE: To observe the toxicity of auranofin, cisplatin, adriamycin, compounds 4N, H, B, O against Schistosoma japonicum adult worms in vitro and their inhibition on thioredoxin glutathione reductase (TGR). METHODS: The drugs mentioned above with different concentrations were added into RPMI 1640 medium with Schistosoma japonicum adult worms, which had been cultured for 30 - 60 min. The activity, morphological changes and death situation of the worms were observed after 1, 6, 24, 48 h and 72 h, respectively, then the worms were transferred to fresh medium without drugs to observe whether their activity would be recovered, and 50% lethal dose (LD50) of the drugs against adult worms was determined. The TrxR and GR activities of thioredoxin glutathione reductase of Schistosoma japonicum in homogenized supernatant of adult worms processed by drugs were tested following the DTNB reduction and NADPH oxidation methods. RESULTS: The mortality rates of 5 microg/ml of auranofin treating for 24 h, 20 microg/ml of 4N treating for 72 h, 60 microg/ml of H treating for 72 h, and 80 microg/ml of cisplatin treating for 72 h on adult worms were 100%, 60%, 66.7% and 100%, respectively, and there were statistically significant differences compared with the negative control group. LD50(s) of auranofin, 4N, H and cisplatin were 2.56, 17.59, 54.14 microg/ml and 52.87 microg/ml, respectively, but no toxic effects of other drugs on schistosome worms were found. The toxic effects of auranofin, 4N, cisplatin and H on adult worms were irreversible. Auranofin and cisplatin inhibited TGR activity of Schistosoma japonicum, but other drugs had no similar effect. 5 - 30 microg/ml of auranofin, 20 - 30 microg/ml of 4N, 70 - 150 g/ml of cisplatin, and 60 - 220 microg/ml of H caused the morphological changes of the worms after treating for 24 h. CONCLUSIONS: Auranofin, cisplatin and compounds 4N and H have toxicity on Schistosoma japonicum adult worms in vitro, and the schistosomicidal effect of auranofin and cisplatin may be related to the inhibition of TGR activity.

[Application of F-ELISA for immunodiagnosis of schistosomiasis japonica].

OBJECTIVE: To optimize the experimental conditions of fast enzyme-linked immunosorbent assay (F-ELISA) and evaluate its performance for immunodiagnosis of schistosomiasis japonica. METHODS: HRP-SPA was used at different concentrations in the assay to determine the best HRP-SPA concentration by comparing the detection accuracy. The working conditions of F-ELISA were optimized by examining the ratio of absorbance values of positive and negative controls with different incubation time of serum samples and HRP-SPA. To evaluate the performance of F-ELISA in diagnosis of schistosomiasis, the detection accuracy of F-ELISA was compared with routine ELISA and dipstick dye immunoassay (DDIA) by testing serum samples from both healthy subjects and those clinically diagnosed with schistosomiasis and clonorchiasis in parallel. RESULTS: In F-ELISA, the best working concentration of HRP-SPA was 1:2 000 while the optimized incubation condition for serum samples and HRP-SPA was 60 min at 37 degrees C. Using F-ELISA, we identified 93.8% to be schistosomiasis positive among chronic schistosomiasis subjects, 4% within the clonorchiasis group, and a false positive rate of 1.5% in healthy individuals. This diagnostic accuracy for F-ELISA was similar to routine ELISA and DDIA. CONCLUSIONS: F-ELISA combines the 2-steps of routine ELISA into a single step making the assay process faster and simpler without sacrificing the diagnostic accuracy. With further investigations, F-ELISA as an easy and practical method is promising to be applied for schistosomiasis diagnosis in the field.

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.

[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.

[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.

[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.