[Anti-atherogenic effect and its mechanisms of soluble egg antigen of Schistosomia japonicum in ApoE-/- mice].

OBJECTIVE: To study the preventive effects of soluble egg antigen (SEA) of Schistosomia japonicum on atherosclerosis in ApoE-/- mice and its immune modulatory mechanisms. METHODS: ApoE-/- mice were divided into an Experimental Group One and an Experimental Group Two. The mice in the Experimental Group One dividing into a prevention and a control subgroups were fed with high fat diet since the first week, the mice in the former subgroup were injected intraperitoneally with SEA while those in the latter one were injected with phosphate buffered saline (PBS) with 1 week interval for 4 times. The mice in the Experimental Group Two were fed with high fat diet for 14 weeks, and then they were divided into a treatment and a control subgroups, which were injected with SEA and PBS, respectively, since the 14th week with 1 week interval for 4 times. All the mice were killed in the 22nd week, and the atherosclerosis development and the change of levels of cytokines and CD4+CD25+ FoxP3+T cells in mice were observed. RESULTS: Immunization with SEA led to a significant reduction in the levels of cholesterol, TNF-alpha, and IL-10 in all the ApoE-/- mice. The atherosclerosis plaque area of aorta of ApoE-/- mice in the prevention subgroup reduced obviously, while there was no significant change in the treatment subgroup. In the 22nd week, the proportion of CD4+CD25+ FoxP3+T cells population in CD4+ T cells was (4.4 +/- 0.9)% in the prevention subgroup and there was a significant difference compared with that of the control subgroup [(2.6 +/- 0.3)%] (P < 0.05). However, the change of the proportion in the treatment subgroup showed no statistic significance (P > 0.05). CONCLUSION: The preventive injection of SEA in ApoE-/- module mice has the effect of anti-atherosclerosis by increasing the proportion of Treg cells together with the inhibition of inflammatory cytokines at the beginning of disease.

Expression, characterization and therapeutic efficacy of chimeric Fab of anti-idiotypic antibody NP30 against Schistosoma japonicum.

The murine monoclonal anti-idiotypic antibody NP30 is a promising therapeutic antibody against Schistosoma japonicum. However, the immunogenicity of murine NP30 limits its further study and application in humans. Here the chimeric Fab of NP30 (chFab-NP30) comprising the variable regions of murine NP30 and constant regions of human antibody was assembled. chFab-NP30 was expressed and purified as a soluble and functional protein. Administration of chFab-NP30 in vivo increased the survival rate, reduced egg burdens and ameliorated organ pathology of mice with acute schistosomiasis. Our study indicated that chFab-NP30 is a promising candidate to be used as a specific and efficient recombinant antibody against acute schistosomiasis japonica. Further studies on function mechanism of chFab-NP30 needs to be carried out in the future.

[Enhancing protective immunity effects of TPI DNA vaccine against Schistosoma japonicum through codon optimization].

OBJECTIVE: To study the protective effect of codon optimized TPI DNA vaccine against Schistosoma japonicum infection. METHODS: Sixty female BALB/c mice were randomly divided into 5 groups. The mice were injected through musculus quadriceps femoris with 100 microg pcDNA 3.1 control (Group A), pcDNA3.1-TPI (Group B), pcDNA 3.1-TPI-mHSP70 (Group C), pcDNA3.1-TPI.opt (Group D), and pcDNA3.1-TPI.opt-mHSP70 (Group E) respectively. All mice were immunized for three times with an interval of two weeks. The mice were challenged with (40+/-1) cercariae of S. japonicum per mouse by abdominal skin penetration 4 weeks after the last immunization, and sacrificed at 42 days post-challenge, the number of worms or hepatic eggs was counted. Blood was taken for the detection of IgG, IgG1, and IgG2a 2 days before immunization and before challenge, respectively. 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-gamma, and TNF by flow cytometry. RESULTS: ELISA showed that the mice in groups B, C, D, and E produced specific IgG and IgG1, IgG2a antibody isotypes, and the ratio of IgG2a/IgG1 was 1.73, 2.06, 2.44, and 3.09, respectively. The levels of IL-2, IFN-gamma and TNF in groups D and E were higher than that of groups B and C. The worm reduction rate and hepatic egg reduction rate in groups D (36.03%, 41.7%) and E (39.03%, 46.85%) were higher than those of groups B (26.28%, 28.35%) and C (28.38%, 31.39%) (P<0.01) . CONCLUSIONS: The codon optimized TPI DNA vaccine induces higher level of protective effect and Th1-biased cellular immune response than those of non-optimized TPI DNA vaccine.

A novel molluscicidal formulation of niclosamide.

Snail control by molluscicides is an important strategy for schistosomiasis control in China. Currently, only one chemical molluscicide, niclosamide, which is used as 50% wettable powder of niclosamide ethanolamine salt (WPN), is commercially available for field snail control in China. However, WPN is costly, toxic, and has a lower dispersibility and precipitates rapidly. In this paper, we describe the development of a novel formulation of niclosamide, a suspension concentrate of niclosamide (SCN). The efficacy of SCN was evaluated both in the laboratory and field. SCN showed better molluscicidal effects than conventional formulation of WPN, as determined by LC(50) for adult snails, young snails, and snail eggs. The acute toxicity of SCN to Brachdanio rerio hamiton was less than WPN. In conclusion, the novel formulation of SCN suspension is physically more stable, more effective, and less toxic. Therefore, it can be more useful for controlling snails in endemic areas of schistosomiasis in China.

[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.

Epidemiology of schistosomiasis in the People's Republic of China, 2004.

Results from the third nationwide cluster sampling survey on the epidemiology of schistosomiasis in the People's Republic of China, conducted by the Ministry of Health in 2004, are presented. A stratified cluster random sampling technique was used, and 239 villages were selected in 7 provinces where Schistosoma japonicum remains endemic. A total of 250,987 residents 6-65 years of age were included in the survey. Estimated prevalence rates in the provinces of Hunan, Hubei, Jiangxi, Anhui, Yunnan, Sichuan, and Jiangsu were 4.2%, 3.8%, 3.1%, 2.2%, 1.7%, 0.9%, and 0.3%, respectively. The highest prevalence rates were in the lake and marshland region (3.8%) and the lowest rates were in the plain region with waterway networks (0.06%). Extrapolation to all residents in schistosome-endemic areas indicated 726,112 infections. This indicates a reduction of 16.1% compared with a nationwide survey conducted in 1995. However, human infection rates increased by 3.9% in settings where transmission is ongoing.

[Expression and bioactivity of S1 scFv antibody against SAG1 of Toxoplasma gondii fused with green fluorescent protein].

OBJECTIVE: To construct, express and purify human scFv antibody (S1) against the recombinant SAG1 of Toxoplasma gondii and fused to green fluorescent protein (GFP), and observe its binding capacity to tachyzoite of Toxoplasma gondii. METHODS: The GFP gene amplified from vector pEGFP-N1 was subcloned into procaryotic expression vector pET-26b (+), then the S1 scFv antibody gene amplified from phagmid pIT-2-S1 was cloned into downstream of GFP gene. The recombinant plasmid pET-26b-GFPS1 proved by DNA sequencing was transformed into E. coli BL21, and induced for fusion expression of GFPS1 with IPTG, the green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was observed under the fluorescence microscope. The expressed GFPS1 was purified with Ni2+ chelating HiTrap HP column, and detected with SDS-PAGE. Toxoplasma tachyzoites were incubated with the recombinant GFPS1, and the binding bioactivity was observed under the fluorescence microscope. RESULTS: The fused gene of S1 and GFP was successfully cloned into procaryotic expression vector pET-26b proved by DNA sequencing. The green fluorescence of E. coli BL21 harboring plasmid pET-26b-GFPS1 was catched under the fluorescence microscope. The recombinant GFPS1 protein about Mr 53 000 was expressed in E. coli as inclusion body. The immunofluorescence detection verified that anti-rSAG1 scFv antibody S1 could specifically bind outer membrane of Toxoplasma tachyzoite. CONCLUSIONS: The purified rGFPS1 shows a strong binding capacity to outer membrane of Toxoplasma tachyzoite using GFP as a labeling protein, and the constructed pET-26b-GFP can also be used for research on other targeting molecules.

[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.

Immunodiagnosis and its role in schistosomiasis control in China: a review.

Parasitological techniques (stool examination and/or urine filtration) are still the diagnosis of choice in national schistosomiasis control programmes the world over. However, the success of current control efforts, mainly due to the large-scale administration of praziquantel, emphasises the need for a more sensitive approach. In addition, microscopy is labour-intensive and time-consuming, while declining compliance rates after repeated chemotherapy make good coverage for the long-term increasingly problematic. China's success in the control of schistosomiasis is contributing to an enhanced understanding of the need for better and more sensitive screening methods. Immunodiagnostic techniques have a high sensitivity, are easy to perform and are an excellent epidemiological tool for the screening of target populations in schistosome-endemic areas. These assays are also useful for the surveillance of cure after chemotherapy, and for periodic control of transmission of the infection after it has been eliminated in an area. A succinct historical background of using immunodiagnosis for schistosomiasis japonica in China is given, together with a review and evaluation of the relative efficacy of the main techniques applied, i.e. the intradermal test, the circumoval precipitation test, the indirect hemagglutination assay, the dye dipstick immunoassay and different kinds of enzyme-linked immunosorbent assay applications. The important role of immunodiagnosis in the screening for schistosomiasis in China is discussed.

Application of dipstick dye immunoassay (DDIA) kit for the diagnosis of schistosomiasis mekongi.

The dipstick dye immunoassay (DDIA), developed in China for the detection of antibodies against Schistosoma japonicum, relies on soluble egg antigen (SEA) labelled with a colloidal dye. This assay is not only rapid, simple and inexpensive, but also particularly useful for screening in the field. In order to determine whether S. japonicum antigens are sufficiently cross-reactive to make the assay applicable for the diagnosis also of S. mekongi a DDIA approach based on the S. japonicum SEA was tried in cohorts of healthy and infected people living in areas non-endemic and endemic with regard to schistosomiasis mekongi in Cambodia and Laos. A sensitivity of 97.1% was recorded when testing Cambodian subjects, correctly diagnosing 33 out of 34 infected people. When the assay was applied in Laos, a sensitivity of 98.6% (69/70) was found. None of 114 residents living in a non-endemic area in Cambodia tested positive. A cross-reaction of 18.3% was found in patients infected with Opisthorchis viverrini. The results support the notion that the DDIA using S. japonicum SEA antigens can safely be implemented for the diagnosis of schistosomiasis mekongi, but care is needed in the interpretation of results obtained from areas that are co-endemic for O. viverrini.

[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.

Genetic analysis of praziquantel resistance in Schistosoma mansoni.

To determine the genetic basis of the resistance of Schistosoma mansoni to praziquantel (PZQ) and to understand whether the resistance is dominant or recessive trait, a schistosome cross was undertaken between a PZQ-susceptible and a PZQ-resistant isolate using infections of the single-sex cercariae which were identified by a direct W1-specific PCR technique. The resistances of F1 and F2 generation to PZQ were evaluated using in vitro egg, miracidial and cercarial tests. The F1 hybrid progeny from crosses between the susceptible and resistant isolates were resistant to PZQ. The resistant phenotype reappeared in the F2 progeny. It can thus be considered that the PZQ resistance behaves like a dominant trait.

[Sensitivity of Oncomelania snail to niclosamide in China].

OBJECTIVE: To understand the variation in response of Oncomelania hupensis to niclosamide. METHODS: Snails were collected from 37 sampling areas distributed in 10 provinces (municipalities) using random environmental sampling methods in accordance with the different types and categories of snail habitats. In laboratory the snails were immersed in solutions of niclosamide for 24 and 48 hours at 25 degrees C. RESULTS: 1.0 mg/L niclosamide showed 100% killing effect on snails in 24 hours. The LC50 concentrations for snails immersed for 24 hours ranged from 0.0320 to 0.1689 mg/L with a mean value of 0.0920 mg/L. 0.5 mg/L niclosamide showed 100% killing effect on snails in 48 hours. The LC50 values for snails immersed for 48 hours ranged between 0.0299 and 0.1114 mg/L with a mean of 0.0627 mg/L. There is a significant difference in snail sensitivity to niclosamide between sampling areas. CONCLUSION: The sensitivity to niclosamide varied in snails from different sampling fields, but the chemical in a concentration of 1.0 mg/L showed 100% effect of killing snails, which is consistent to the manual of schistosomiasis control.

[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.