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There is still urgent need for a vaccine against schistosomiasis, especially in Schistosoma japonicum endemic areas where even a vaccine that will interrupt zoonotic transmission will be potentially effective as an intervention tool. We had developed a novel nanoparticle gene delivery system, which has proven efficacious in gene transfection to target immune cells with complementary adjuvant effect and high protective efficacy in several diseases. Here, we applied this nanoparticle system in combination with S. japonicum glutathione S-transferase (SjGST) DNA vaccine to show the immunogenicity and anti-fecundity effect of the nanoparticle coated vaccine formulation against murine schistosomiasis. The nanoparticle-coated DNA vaccine formulation induced desired immune responses. In comparison with the nanoparticle coated empty vector, it produced significantly increased antigen-specific humoral response, T-helper 1 polarized cytokine environment, higher proportion of IFN-γ producing CD4(+) T-cells and the concomitant decrease in IL-4 producing CD4(+) T-cells. Although there was no effect on worm burden, we recorded a marked reduction in tissue egg burden. There was up to 71.3% decrease in tissue egg burden and 55% reduction in the fecundity of female adult worms. Our data showed that SjGST DNA vaccine, delivered using the nanoparticle gene delivery system, produced anti-fecundity effect on female adult schistosomes as previously described by using conventional subunit vaccine with adjuvant, proving this DNA vaccine formulation as a promising candidate for anti-pathology and transmission blocking application., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Although schistosomiasis remains a serious health problem worldwide, significant achievements in schistosomiasis control has been made in the People's Republic of China. The disease has been eliminated in five out of 12 endemic provinces, and the prevalence in remaining endemic areas is very low and is heading toward elimination. A rapid and sensitive method for monitoring the distribution of infected Oncomelania hupensis is urgently required. We applied a loop-mediated isothermal amplification (LAMP) assay targeting 28S rDNA for the rapid and effective detection of Schistosoma japonicum DNA in infected and prepatent infected O. hupensis snails. The detection limit of the LAMP method was 100 fg of S. japonicum genomic DNA. To promote the application of the approach in the field, the LAMP assay was used to detect infection in pooled samples of field-collected snails. In the pooled sample detection, snails were collected from 28 endemic areas, and 50 snails from each area were pooled based on the maximum pool size estimation, crushed together and DNA was extracted from each pooled sample as template for the LAMP assay. Based on the formula for detection from pooled samples, the proportion of positive pooled samples and the positive proportion of O. hupensis detected by LAMP of Xima village reached 66.67% and 1.33%, while those of Heini, Hongjia, Yangjiang and Huangshan villages were 33.33% and 0.67%, and those of Tuanzhou and Suliao villages were 16.67% and 0.33%, respectively. The remaining 21 monitoring field sites gave negative results. A risk map for the transmission of schistosomiasis was constructed using ArcMap, based on the positive proportion of O. hupensis infected with S. japonicum, as detected by the LAMP assay, which will form a guide for surveillance and response strategies in high risk areas., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Familial cholesteryl ester transfer protein (CETP) deficiency is more common in some East Asian populations than elsewhere, suggesting the possibility of a selective advantage of this genetic defect against regional infectious diseases. Historically, infection with the Asian blood fluke Schistosoma japonicum has been endemic in these regions, including Japan. We previously reported that eggs of S. japonicum require cholesteryl ester uptake from normal high-density lipoprotein (HDL) but not from CETP-deficient HDL for their maturation to miracidia, a critical step of the hepatic pathogenesis of schistosomiasis. Herein we show that cholesteryl ester uptake is selective from HDL, and identified CD36-related protein (CD36RP) as a candidate to mediate the reaction. CD36RP was cloned from the adult and the egg developmental stages of S. japonicum, with 1880 bp encoding 506 amino acid residues exhibiting the CD36 domains and two transmembrane regions. Using antibodies against recombinant peptides representing the potential extracellular domains of CD36RP, Western blotting detected a protein with a molecular mass of 82 kDa in the particulate fraction of the adult parasite cells, which was reduced to 62 kDa after N-glycanase treatment. The extracellular domain peptide bound human HDL, as established by immunoblots following nondenaturing gel electrophoresis. Antibodies against the extracellular domain suppressed HDL cholesteryl ester uptake and maturation of the eggs in vitro. CD36RP is a candidate receptor on eggs of S. japonicum that facilitates uptake of HDL cholesteryl ester necessary for egg embryonation and maturation.

Conflicting reports are found in the literature about the antischistosomal efficacy of Mirazid (MZ), which is a special formulation of myrrh obtained from the stem of the plant Commiphora molmol. This initiated the present study to assess this drug for the first time in experimental schistosomiasis japonicum. Mice were divided into four groups: infected untreated control (I); infected treated with MZ, 500 mg/kg (II); infected treated with MZ, 250 mg/kg (III); and infected treated with praziquantel (PZQ), 200 mg/kg (IV). The drugs were given 7 weeks post-infection for five successive days. All animals were killed 3 weeks posttreatment. Results showed no signs of antibilharzial activity of MZ. Total worms, total tissue egg load, egg developmental stages, and granuloma area were not affected by any of the MZ treatment regimens as compared to the infected untreated group (P > 0.05 for all variables). These results were in contrast to those obtained in PZQ-treated animals in which 82.82 % total worm reduction, 94.62 % egg reduction, and 86.35 % granuloma area reduction were observed. Also, it significantly increased the percentage of dead ova and decreased the percentage of mature ova with complete absence of immature ones in comparison with the control group (P < 0.01 for all variables). In conclusion, the results of the current study raise serious doubts about the antischistosomal activity of MZ.

Polymerase chain reaction (PCR) with the specific primer set amplifying 28S ribosomal DNA (rDNA) of Schistosoma japonicum was able to detect genomic DNA of S. japonicum, but not S. mansoni, at 100 fg. This procedure enabled us to detect the DNA from a single miracidium and a snail infected with one miracidium at just 1 day after infection. We compared these results with those from loop-mediated isothermal amplification (LAMP) targeting 28S rDNA and found similar results. The LAMP could amplify the specific DNA from a group of 100 normal snails mixed with one infected snail A PCR screening of infected snails from endemic regions in Anhui Province revealed schistosomal DNA even in snails found negative by microscopy. PCR and LAMP show promise for monitoring the early infection rate in snails, and they may be useful for predicting the risk of infection in the endemic places.

Three peroxiredoxins (Prxs) are expressed during most of the developmental stages in the schistosome. Prx-1 is localized on the surface of the schistosomula and adults of Schistosoma japonicum, while Prx-2 is localized in the sub-tegumental tissues, parenchyma, vitelline glands, and gut epithelium, but not on the surface of the worms. We applied RNA interference techniques to suppress the specific genes of S. japonicum Prxs. Schistosomula of S. japonicum were cultured together with long-dsRNA encoding Prx-1 and Prx-2 of S. japonicum (the soaking method). The transcription level of each Prx gene was reduced by an RNA interference (RNAi)-mediated effect specifically. Although neither Prx was the essential protein for survival of S. japonicum schistosomula, Prx-1 dsRNA-treated larvae were susceptible to hydrogen peroxide. Moreover, these larvae were also susceptible to t-butyl hydroperoxide and cumene-hydroperoxide. However, the knockdown of neither Prx-1 nor Prx-2 influenced the resistance against nitric oxide generated from DETA/NO. Prx-1 may work as a scavenger against reactive oxygen species (ROS) generated outside of the schistosomes to prevent the oxidation of the bodies and/or the attack by immune cells producing the ROS. These findings suggest that Prx-1 may become a novel target of drugs and vaccines for schistosomiasis.

Calpain is a calcium-dependent cystein protease, and the homologues of schistosome are known as one of vaccine candidate molecules against schistosomiasis. Here, we established two IL-2 producing T cell hybridoma cell lines specific for Schistosoma japonicum calpain, to identify T cell epitope(s) on the molecule. Overlapping 15mer oligopeptides of calpain were synthesized and tested for their stimulatory abilities to the hybridomas. As a result, epitopes recognized by the two hybridoma lines were the same: EQLKIYAQRC. Spleen cells from calpain multiple antigenic peptide (MAP)-immunized BALB/c mice produced IFNgamma upon stimulation with MAP or soluble worm antigen preparation (SWAP). The identification of the T cell epitope to stimulate Th1 response will contribute to the proper design of synthetic vaccines, evaluation of their protective potentials and elucidation of protective mechanisms in murine experimental schistosomiasis.

A monoclonal antibody was generated against the large subunit of Schistosoma japonicum calpain to study the localization and possible function of the molecule in vivo. Mice were immunized with recombinant S. japonicum calpain and polyclonal antisera and a monoclonal antibody specific to schistosome calpain was obtained. In immunohistochemistry, a monoclonal antibody against S. japonicum calpain, KG-2E11, bound weakly to calpain expressed at the surface of adult worm tegument, however, it bound strongly to the cercarial secretions ("footprints") of S. japonicum, emitted from the penetration glands. The present study indicates that calpain is multifunctional as it is expressed at various locations in different developmental stages. Calpain-based vaccines could thus possibly induce protective immunity against cercariae and the following early developing stages.

Vaccine development by the use of calpain of Schistosoma japonicum has been tried in our laboratory. We cloned cDNA encoding the heavy chain of S. japonicum calpain, and prepared recombinant molecule of a possible vaccine region of the heavy chain. When BALB/c mice were immunized with our recombinant calpain of S. japonicum with Freund's complete adjuvant, we observed significant reduction in worm burden (41.2% reduction, P<0.05), and also significant anti-fecundity effects. In this sense, calpain of S. japonicum seems to have infection control as well as anti-disease effects. Mechanisms of vaccine effects of calpain remain to be clarified, however, several effector mechanisms are suspected. In immunized mice, raised level of iNos expression was observed, while adhesion of peritoneal exudates cells were also observed in the presence of calpain-immunized sera, suggesting the possibilities of both cellular and humoral protective mechanisms. We examined tissue distribution of calpain in various developmental stages of S. japonicum. Strong signal was observed around excretory grand of cercariae, and they secreted calpain during their migratory movement tested in vitro. Together with the findings, calpain seems to induce larvicidal effects in the immunized mice. We observed time-course kinetics of antibody production against vaccine candidates in experimental S. japonicum infection in pigs. Although significant levels of antibody production were observed for paramyosin and GST, no significant antibody production was observed for calpain. This suggests that calpain is less immunogenic, and route of immunization and/or choice of adjuvant are important in future trials of calpain vaccine.

In our attempt to discover a potential cause for accumulation of cholesteryl ester transfer protein (CETP) deficiency in Eastern Asia, we studied the association of CETP deficiency with pathogenesis of Schistosoma japonicum, a life-threatening parasite peculiar to this region. The eggs of S. japonicum showed slow embryonation when cultured in CETP-deficient human plasma. Restoration of CETP to the deficient plasma rescued it, while inhibition of CETP in normal plasma did not cause slow embryonation of the cultured eggs. The egg embryonation was also retarded in the liver but not in the intestine of wild-type mice in comparison to the CETP-transgenic mice. The granulomatous lesion around the parasite eggs in the liver was less in the wild-type than in the CETP-transgenic mice. Thus, CETP deficiency may act against Schistosomiasis japonica by retarding egg embryonation, a potential cause of liver granulomatosis. It does not seem directly due to the lack of CETP activity in plasma but to abnormal lipoprotein generated by chronic CETP deficiency., (Copyright 2001 Academic Press.)

A large subunit of calpain, a calcium-activated neutral proteinase, from Schistosoma japonicum was cloned and expressed in Escherichia coli. When BALB/c mice were immunized with purified recombinant calpain (r-calpain) emulsified in complete Freund's adjuvant, a significant reduction in the number of recovered worms and also in egg production per female worm was observed (P<0.01). Spleen cells of the immunized mice showed enhanced production of gamma interferon (IFN-gamma) by activated CD4(+) T cells. Considering our observation of elevated expression of inducible nitric oxide synthase mRNA in immunized mice, r-calpain-induced IFN-gamma seemed to upregulate the production of nitric oxide by macrophages and subsequently mediated the killing of schistosomulae in the lung. On the other hand, spleen cells of immunized mice showed only faint interleukin-4 production in response to r-calpain in vitro, suggesting that immunization with r-calpain alters the Th1-Th2 balance in murine hosts even during a Th2-promoting S. japonicum infection. Furthermore, histopathological study of the livers of immunized mice showed that granulomas formed around eggs were diminished in both size and number. Egg production by female worms was clearly decreased in immunized mice, suggesting that r-calpain also has antifecundity effects. Taken together, these results point to S. japonicum calpain as a potential vaccine candidate for both worm killing and disease prevention, possibly through the induction of a strong Th1-dominant environment in immunized mice.

Mice infected with Schistosoma japonicum were resistant to the intestinal nematode, Strongyloides venezuelensis. The numbers of adult S. venezuelensis recovered from mice were significantly decreased when infections were given from 6 weeks after S. japonicum infection. Larval recovery from the lungs showed that significant numbers of subcutaneously inoculated S. venezuelensis larvae were eliminated by 3 days in S. japonicum-infected mice (P < 0.0001), while histology revealed that this was associated with massive eosinophilic infiltration in the lungs. In addition, adult S. venezuelensis worms implanted in the duodenum of S. japonicum-infected mice could not establish in the intestine. This failure was associated with mucosal mastocytosis. Activation of eosinophils and intestinal mast cells was correlated with elevated expression of mRNA for interleukin (IL)-3, IL-4, and IL-5 in S. japonicum-infected mice. Sera from S. japonicum-infected mice recognized S. venezuelensis larva antigens as strongly as those from S. venezuelensis-infected mice, although transfer of sera from S. japonicum-infected mice to normal recipient mice did not protect them from S. venezuelensis challenge infection. It was concluded that the mechanisms for larval killing and adult worm expulsion of S. venezuelensis in S. japonicum-infected mice were identical to those seen in infections with S. venezuelensis only.

cDNA coding for calpain of Schistosoma japonicum were cloned and sequenced, and serological basis of host responses to calpain were analyzed. cDNA of calpain from S. japonicum of two different isolates, Yamanashi strain (Sj-J) and Hunan strain (Sj-C), were 2, 468 bp and 2, 465 bp in length, including the same number (2, 274) of open reading frame. Nucleotide sequence and amino acid sequence between the two calpains are 99.1% and 98.8% identity, respectively. Sj-J and Sj-C calpains were considered to be translated as a preproenzyme, and a 746-amino acid mature enzyme contains eight motifs without a signal peptide at the N-terminal based on the deduced amino acid sequences. mRNA for calpain were detectable in different developmental stages, however, sera obtained from mice immunized with recombinant calpain showed enhanced binding to cercarial antigen. Human sera from S. japonicum-infected individuals recognized the large subunit of schistosomal calpain, and light-infected sera showed stronger reactivities to the recombinant calpain than moderate/high infection cases. When we tested synthetic peptides, there were four common human B cell epitopes in schistosomal calpain, all of which are shared with S. mansoni. Together with these results, calpain of S. japonicum seems to be not only a vaccine candidate, but also a target antigen for immunodiagnosis of human schistosomiasis.

Serum-mediated regulation of T-cell responses specific for soluble egg antigen (SEA) of Schistosoma japonicum was tested in human hosts. When we added autologous serum to SEA-specific human T-cell lines (CD3+, 4+, 8-), we observed suppression of T-cell proliferation, and this suppressive activity was detected in the immunoglobulin-G2 (IgG2) subclass. Suppression was dose-dependent and antigen-specific. T-cell proliferation induced by only one SEA fraction of greater than 18 kDa was modulated in the presence of 100 micrograms/ml autologous as well as allogeneic infected IgG2. This SEA fraction-driven proliferation was also regulated by suppressor T-cells through distinct suppressive mechanisms. Our results suggest that T-cell responses to a particular component(s) of SEA are strictly regulated through both cellular and humoral mechanisms in human chronic S. japonicum infection.

We analysed regulatory mechanisms of the human T cell response to soluble egg antigen (SEA) of Schistosoma japonicum in vitro. SEA is a crude antigen mixture containing numerous epitopes. We obtained SEA-induced T cell lines from five patients with chronic schistosomiasis japonica, and tested their proliferative response to molecular weight fractions of SEA. Although all T cell lines showed strong responses to crude SEA, there was a heterogeneity in fraction-driven responsiveness. All but one T cell line tested failed to respond to SEA fraction I (mol. wt greater than 18 kD). One patient who was typed as HLA-DQw1/w4, did not show proliferation of CD4+ T cells to fraction I; however, a fraction I-driven helper T cell response was observed when we added HU-11 monoclonal antibody specific for HLA-DQw1/w4. This indicated that the patient had helper T cells to the fraction even though their response was suppressed. Because HLA-DQ had an effect on functional expression of suppressor T cells, it was suggested that there was epitope-specific regulation of the T cell response to SEA, and HLA-DQ-controlled immune suppression might be involved in the regulatory system in human chronic schistosome infection.

T cell lines specific for Schistosoma japonicum egg Ag were established in vitro from patients with chronic schistosomiasis japonica, and investigated their possible immunopathologic roles by testing lymphokines production and in vitro granuloma formation assay. All lines tested had surface phenotypes of CD3+ CD4+ CD8-, and showed S. japonicum soluble egg Ag (SEA)-specific proliferation requiring HLA-DR-restricted Ag presentation. Of these fractions of SEA separated by gel filtration, Fraction II (m.w. 7,000 to 18,000) and III (m.w. 7,000) induced strong proliferation of T cell lines, whereas fraction I (m.w. 18,000+) failed to induce detectable proliferation to any T cell lines tested. One of the T cell lines was cloned by micromanipulation: two of eight clones responded only to fraction II, and six to both fractions II and III. We observed that four of eight clones tested produced IL-2 in response to SEA, and three of them were able to transfer S. japonicum egg-specific granulomatous hypersensitivity in vitro to an HLA haplo-identical individual without previous schistosome infection. These immunopathologic functions of T cell clones seemed to be activated by at least two distinct epitopes of SEA. Our present observations suggest that at least two distinct CD4+ human T cells, both of which recognize epitopes expressed on SEA molecules of less than 18 kDa, might have critical roles in granulomatous hypersensitivity to eggs of S. japonicum in humans.

To characterize the mechanisms of induction and regulation of the cell population involved in granuloma formation around eggs of Schistosoma japonicum, we utilized a simple method of in vitro experiments. Lyt1+2-T cells were essential for in vitro responses to the intact S. japonicum eggs, which were assumed to be comparable to in vivo granulomatous responses. T-cell responses seemed to be macrophage-dependent, and responding T cells produced IL-2-like activities in the culture supernatant. Sera taken from chronically infected mice acted as regulatory factors to these T-cell responses as was the case in in vivo granuloma formation. The method used here was simple and highly informative for the studies on pathogenesis of schistosomiasis japonica.

We tested effects of praziquantel, an antischistosomal compound, on clinical and immunological parameters of chronic schistosomiasis japonica. Two Japanese patients, who had high antibody titers to Schistosoma japonicum antigens but no fecal schistosome eggs or no or mild symptoms complained, were treated with praziquantel. Within two years after treatment, anti-schistosome antibodies in sera from the patients became negative in enzyme-linked immunosorbent assay. There was no significant alteration in cellular immunity to the parasite. Although S. japonicum infection is believed to have been eradicated in Japan, our present results seem to suggest the possibility that a few Japanese individuals, who have high anti-schistosome antibody, still harbor live parasites.

Immune responsiveness to schistosomal worm antigen was investigated in the individuals infected with Schistosoma japonicum by measuring the antigen-specific proliferative response of the peripheral T lymphocytes in vitro. Out of 57 infected individuals, 10 (17.5%) were low responders, whereas 47 (82.5%) were high responders to this antigen. The strong association between the HLA-Aw24-Bw52-Dw12 haplotype and the low responder group was demonstrated. Because the association of the low responder group was strongest with the HLA-D specificity, Dw12, and because the HLA-D region was assumed to be comparable to the I region of the murine H-2 complex, it was suggested that the low responsiveness to schistosomal worm antigen was controlled by a single dominant immune suppression gene that was in strong linkage disequilibrium with HLA-Dw12.

T cell lines (TCLs) specific for Schistosoma japonicum egg antigen were established from a patient with chronic schistosomiasis japonica to investigate the regulatory mechanism of S.japonicum egg antigen-driven T cell responses in man. All five TCLs tested were CD2+, CD4+, CD8-, and were strongly proliferative only to S. japonicum egg antigen in the absence of exogenous IL-2. All but one TCL produced IL-2-like lymphokines in vitro, indicating their helper T cell functions. One TCL, SjE-3, failed to produce IL-2-like lymphokines. Moreover, this TCL suppressed the specific proliferation of autologous peripheral blood lymphocytes to S. japonicum egg antigen. This TCL produced a soluble suppressor factor(s). These functional diversities among established TCLs were also confirmed by cloned T cells. Our observations might suggest that the regulatory system through helper and suppressor T-T interactions somehow involved in T cell responses to the egg antigen in human chronic schistosomiasis japonica.

We analysed regulatory mechanisms of the human T cell response to soluble egg antigen (SEA) of Schistosoma japonicum in vitro. SEA is a crude antigen mixture containing numerous epitopes. We obtained SEA-induced T cell lines from five patients with chronic schistosomiasis japonica, and tested their proliferative response to molecular weight fractions of SEA. Although all T cell lines showed strong responses to crude SEA, there was a heterogeneity in fraction-driven responsiveness. All but one T cell line tested failed to respond to SEA fraction I (mol. wt> 18 kD). One patient who was typed as HLA-DQwl/w4, did not show proliferation of CD4+ T cells to fraction I: however, a fraction I-driven helper T cell response was observed when we added HU-11 monoclonal antibody specific for HLA-DQwl/w4. This indicated that the Patient had helper T cells to the fraction even though their response was suppressed. Because HLA-DQ had an effect on Functional expression of suppressor T cells, ii was suggested that there was epitope-specific regulation of the T cell response to SEA, and HLA-DQ-controlled immune suppression might be involved in the regulatory system in human chronic schistosome infection. [ABSTRACT FROM AUTHOR]