SjHSP70, a recombinant Schistosoma japonicum heat shock protein 70, is immunostimulatory and induces protective immunity against cercarial challenge in mice.

High levels of protective immunity can be induced in different animals immunized with radiation-attenuated (RA) Schistosoma cercariae or schistosomula. However, the schistosome-derived molecules responsible for the strong protective effect elicited by RA schistosome larvae have not been identified or characterized. The 70-kDa heat shock proteins of schistosomes are considered major immunogens, and may play an important role in stimulating high levels of innate and adaptive immune responses in an RA schistosome vaccine model. Here, we demonstrate the immunobiological functions of Schistosoma japonicum heat shock protein 70 (SjHSP70) by investigating its expression profile in RA-schistosomula-derived cells, evaluating the protection induced by recombinant SjHSP70 (rSjHSP70) against cercarial challenge, and assaying the humoral and cellular immune responses to rSjHSP70 in BALB/c and C57BL/6 mice. The expression of SjHSP70 on the surfaces of cells from RA or normal schistosomula was determined with flow cytometry. Its expression was significantly higher on early RA schistosomula cells than on the cells from normal parasites. The protection afforded both BALB/c and C57BL/6 mice vaccinated with rSjHSP70 alone, rSj22.6 (a membrane-anchoring protein of S. japonicum) alone, or a combination of rSj22.6 and rSjHSP70 without adjuvant was evaluated. rSjHSP70 alone induced the highest protective effect against S. japonicum cercarial challenge, followed by the rSj22.6 plus rSjHSP70 combination and then rSj22.6 alone, in both mouse strains. Like ISA206 adjuvant, rSjHSP70 enhanced the protective efficacy induced by rSj22.6 in the C57BL/6 mouse strain. Antigen-specific IgG1 and IgG2a responses were detected with enzyme-linked immunosorbent assays in mice immunized with rSjHSP70 alone, rSj22.6 alone, or the rSj22.6 plus rSjHSP70 combination. Immunization with rSjHSP70 or the rSj22.6 plus rSjHSP70 combination induced mixed Th1/Th2-type antibody responses in BALB/c mice and a Th2-type antibody response in C57BL/6 mice. The profiles of cytokine production by splenic lymphocytes in both strains of mice immunized with the antigens described above were detected in vitro using a Cytometric Bead Array. The profiles of the proinflammatory cytokines interferon γ, tumor necrosis factor α, interleukin 6 (IL-6), and IL-17A and the regulatory cytokine IL-10 induced by the rSj22.6 plus rSjHSP70 combination were similar to those induced by rSj22.6 emulsified with the ISA206 adjuvant control. Like the ISA206 adjuvant, rSjHSP70 protein enhanced the proinflammatory and Th2-type or regulatory cytokine production induced by the rSj22.6 antigen. These results indicate that SjHSP70 is exposed on the surfaces of cells from RA schistosomula, and that rSjHSP70 protein is a promising protective antigen with a potential adjuvant function. Thus, SjHSP70 protein might play a key role in the protective immunity elicited by the RA schistosome vaccine.

Molecular characterization and identification of Th1 epitopes of a Schistosoma japonicum protein similar to prosaposin.

The tegument of schistosomula contains T cell antigens that might simulate the protective mechanisms of the radiation-attenuated vaccine in a mouse model of schistosomiasis. Immune mechanisms mediated by the CD4+ Th1 response are important in the RAV model. To rapidly identify Th1 epitopes in molecules from the Schistosoma japonicum schistosomula tegument, this study analyzed S. japonicum proteomics data. Preliminary experiments identified a protein similar to prosaposin (SjPSAP) from the tegument of schistosomula. We confirmed that SjPSAP was present in the tegument of the parasite using an indirect immunofluorescence assay. We then identified Th cell epitopes in SjPSAP using in silico prediction combined with experimental validation. From the SjPSAP sequence, we used several algorithms to predict 11 promiscuous Th cell epitopes that might bind to both murine and human MHC class II molecules. To validate the in silico predictions, proliferation and cytokine production profiles of spleen lymphocytes from BALB/c mice immunized with the 11 predicted peptides were measured in vitro using a modified methyl thiazolyl tetrazolium assay and flow cytometry. The results showed that 4 of the 11 predicted peptides induced a recall CD4+ Th1 response in vitro. We measured direct binding of the four peptides predicted to induce a response to antigen-presenting cells from BALB/c mice using a fluorometric method and found that the peptides bound to both I-Ad and I-Ed mouse molecules. These results demonstrated that potentially protective Th1-type epitopes in SjPSAP molecules could be identified rapidly by combining in silico prediction with experimental validation. This strategy could be a fast method for identifying Th1 epitopes in a schistosoma antigen with features such as large size or poor expression of recombinant antigens.

Wnt4, the first member of the Wnt family identified in Schistosoma japonicum, regulates worm development by the canonical pathway.

The Wnt signaling pathway is an evolutionarily conserved signal transduction pathway used extensively during animal development. We aim, by increasing our understanding of the Wnt signaling pathway, to find a key gene or protein present in schistosomes that can be developed into vaccine candidate or drug target. We therefore isolated the Wnt4 gene from Schistosoma japonicum. Wnt4 encodes a putative protein of 558 amino acids which contains the conserved functional domain of the Wnt gene family. We suppressed the expression of Wnt4 mRNA in 10-day schistosomulae by RNA interference. Quantitative PCR analysis showed that Wnt4 displayed a 73% reduction in the transcript level. And GSK-3beta and beta-catenin, which are involved in Wnt canonical pathway, showed a 45% and 39% reduction in mRNA levels, respectively. PLC, CaMKII, DVL, and JNK, which are involved in Wnt non-canonical pathway, showed no reduction. These results suggest that the Wnt4 signal protein in S. japonicum regulates downstream genes by a canonical pathway. Wnt4 is the first member of the Wnt family to be identified in S. japonicum. An increased understanding of the Wnt signal transduction pathway will allow us to elucidate further the molecular mechanism of development in schistosomes.

[Immune protection of tegument protein rSj29 against Schistosoma japonicum in mice].

OBJECTIVE: To clone, express and characterize a tegument protein gene of Schistosoma japonicum (Sj29) , and investigate the immune protection of the recombinant protein against S. japonicum in mice. METHODS: The gene coding for Sj29 protein was amplified by PCR, and the sequence was analyzed by bioinformatics tools. Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced the recombinant with IPTG. The recombinant protein (rSj29) was purified by His-binding-resin affinity chromatography and characterized by Western blotting. Three groups each with 10 BALB/c mice were immunized subcutaneously three times (two weeks interval) respectively with 100 microl recombinant rSj29 (0.1 mg/ml) , adjuvant or PBS. At the 15th day after the final inoculation, each mouse was challenged by 40 +/- 2 cercariae of S. japonicum. At the 53rd day after infection, the mice were sacrificed to obtain the number of adult worms, number of eggs in liver and feces. Serum samples were collected at pre-immunization and certain time after immunization, and were analyzed for IgG by ELISA. The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique. mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR. RESULTS: A 576 bp Sj29 gene fragment was obtained. The recombinant protein rSj29 with Mr 22,900 was expressed in the form of inclusion body. The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice. The number of adult worms (15.4 +/- 5.9), number of hepatic eggs (40,143.3 +/- 2,995.9) and number of fecal eggs (3,803.9 +/- 110.9) in recombinant protein group were significantly higher than those of PBS control group (20 +/- 3.4, 49,318.1 +/- 6,648.3, 5,238.1 +/- 303.5, respectively) (P < 0.05) . There was a high level of specific IgG against rSj29 (maximum dilution 1:32000) in recombinant protein group. Immunohistochemical analysis showed the Sj29 protein expressed on the surface of different stages of S. japonicum. mRNA level of Sj29 was the highest at the 32nd day post-infection. CONCLUSION: The recombinant protein rSj29 induces certain degree of protective immunity in mice.

[Prediction for helper T cell epitopes and its application in vaccine development against parasite infection].

Cellular immunity plays an important role in defense against diseases, such as pathogenic infection, autoimmunity and tumor. With the progress of molecular immunology, mechanisms of T cellular immunity, and the T cell epitopes and functional genomics, studies on the prediction based on data-derived for T cell epitopes has been highlighted, and could be one of the useful tools for application in vaccine development. This review summarizes theory and methodology of prediction for helper T cell epitopes, and their application in vaccine development against parasites, and new research directions are also discussed.

[Application of reverse vaccinology in Schistosoma vaccine development: advances and prospects].

OBJECTIVE: Current advances in reverse vaccinology based on the principle of "sequence-structure-function" and such integrated platform technologies as immunoinformatics, computer-aid design, and various high-throughput omics (including genomics, transcriptomics and proteomics) may pave a new way for the discovery of candidate vaccine molecules against schistosomiasis. Both theoretical prediction and experimental approaches conventionally used in the field of reverse vaccinology are briefly introduced in this review; and the applications of these approaches to screening and confirming candidate Schistosoma vaccine molecules are also summarized. Furthermore, potential research prospects of the application of reverse vaccinology to Schistosoma vaccine development are discussed by simulating immune effect mechanisms of immunization with radiation-attenuated cercaria vaccine in animal hosts and naturally acquired immunity in human population.

[Cloning, expressing and functional analysis of SjMF4, a novel Schistosoma japonicum gene].

Based on the phenomenon of the natural anti-schistosomiasis in Microtus fortis, the sera from normal Microtus fortis were employed to immunoscreen the cDNA library of adult Schistosoma japonicum (Chinese strain), two positive clones were obtained, RACE technique was further applied to amplify one of the clones, and a cDNA fragment with an ORF was identified. Sequencing revealed that it was a novel gene of Schistosoma japonicum, and it was named SjMF4 (Schistosoma japonicum Microtus fortis 4). Then the structure and functional motifs of SjMF4 were analysed. The gene was subcloned into pET-28a(+) vector; the recombinant protein showed good antigenicity in Western blotting. The gene was further subcloned into eukaryotic expression vector pcDNA3 to construct the DNA vaccine containing SjMF4. Immune experiments in mice showed significant protection that the recombinant plasmid did induce 28.64%+/-3.82% worm reduction and 21.73%+/-3.98% egg reduction than controls against the Schistosoma japonicum cercaria challenge.

Identification of Th1 epitopes within molecules from the lung-stage schistosomulum of Schistosoma japonicum by combining prediction analysis of the transcriptome with experimental validation.

The lung-stage schistosomulum has been regarded as the main target of protective immunity induced by radiation-attenuated vaccines (RAV) in the mouse model of schistosomiasis, and immune mechanisms mediated by the CD4+ Th1 response play a major role in the RAV model. To identify Th1 epitopes rapidly within molecules from the lung schistosomulum of Schistosoma japonicum, in the present study we analyzed transcriptome data from normal and radiation-attenuated lung schistosomula of S. japonicum and Schistosoma mansoni. We selected six genes with high levels of expression of their transcripts as sample sequences from the lung schistosomula. From these six sequences, by using different algorithms, we predicted six promiscuous Th cell epitopes that are capable of binding to both murine and human MHC class II molecules. To validate our in silico prediction experimentally, first, the gene expressions of the six sequences in day 3 lung-stage schistosomula were assessed using reverse-transcription PCR (polymerase chain reaction) analysis. The result showed that all six sequences predicted can be expressed in normal day 3 schistosomula. Second, we measured the direct binding of the four peptides predicted above to APCs (Antigen Presenting Cells) from the BALB/c mouse strain using a fluorometric method, and found that the four peptides could bind to both I-Ad and I-Ed molecules of the mice. Finally, the proliferation and profiles of cytokine production by spleen lymphocytes from the BALB/c mice immunized with the six predicted peptides were detected in vitro using modified MTT (Methyl Thiazolyl Tetrazolium), and flow cytometry methods, respectively. The results showed that three of the six predicted peptides could induce a recall CD4+ Th1 response in vitro. These results demonstrate that potential Th1-type epitopes can be identified rapidly by a combination of in silico analysis of transcriptomes of lung-stage schistosomula with experimental validation.

[Screening and verification on characteristic differentially expressed genes of Schistosoma japonicum from three reservoir hosts].

OBJECTIVE: To get the characteristic differentially expressed genes of Schistosoma japonicum from three important reservoir hosts: yellow cattle, water buffalo and goat, so as to find the genetic markers to identify the various sources of the parasite reservoir hosts. METHODS: The 49 d worms were collected from artificially infected animals, and the total RNA(s) of worms were extracted and reverse-transcripted to cDNA, and then hybridized with custom-built microarray to screen characteristic differentially expressed genes of every host, and the microarray results were validated by the real-time PCR method. RESULTS: From results of microarray, we got 3 characteristic differentially expressed genes of S. japonicum from yellow cattle, 4 from water buffalo and 7 from goat. We verified schistosome samples from three reservoir hosts in another experiment, the results showed that 2 in yellow cattle, 3 in water buffalo, and 5 in goat were verified to be consistent with microarray results. CONCLUSIONS: The ten characteristic differentially expressed genes of S. japonicum from three reservoir hosts screened by microarray might be used as genetic markers to identify the various sources of reservoir hosts for S. japonicum.

[Longitudinal observation of epidemic dynamics of schistosomiasis in bovine in two mountainous endemic regions].

OBJECTIVE: To understand the endemic situation dynamics of schistosomiasis in domestic animals (mainly bovine) in mountainous endemic regions, so as to provide the reference for evaluating the control effect and improving control strategy. METHODS: Two representative pilots (Renmei and Dacang) in mountainous schistosomiasis endemic regions were selected for survey. The schistosome infection status of bovine was investigated by the miracidium hatching method, the pasture of bovine were investigated by home visiting, and the distributions of wild feces and Oncomelania snails, and the snail schistosome infection status were also investigated in April and September every year. RESULTS: The schistosome infection rates of bovine reduced by 98.4% and 93.8% in two pilots in 2007 compared with those in 1993, and the infection intensities also showed a decline trend. The infection rate of wild faces was 0 in Renmei pilot since 1995, while in Dacang pilot, the infection rate of wild feces fluctuated in 2007, and the intensities of living snails and infected snails showed a declined trend. CONCLUSIONS: Due to the special natural environment of mountainous endemic regions, there is a dot-like or band-like distribution of endemic areas. The strengthening of schistosomiasis examination and chemotherapy will rapidly reduce endemic situation. However, to completely interrupt the transmission of schistosomiasis, we should emphasize environmental modification and domestic animal management.

In silico prediction of binding of promiscuous peptides to multiple MHC class-II molecules identifies the Th1 cell epitopes from secreted and transmembrane proteins of Schistosoma japonicum in BALB/c mice.

It has not so far been possible to identify rapidly and effectively the anti-schistosomiasis Th cell epitopes that are capable of simulating IFN-γ (Interferon-gamma)-mediated Th1-type protective immunity in response to radiation-attenuated schistosome cercaria. With the advance of the omics studies of schistosomes, an approach that used reverse vaccinology probably resolved the above problems. In this "proof-of-principle" study, first, we selected 31 secreted or transmembrane protein sequences sampled from sequences of the transcriptome of Schistosoma japonicum, and analyzed characteristics of these proteins by using conventional bioinformatics tools. Second, putative promiscuous Th cell epitopes within these proteins were predicted using three to four different immuno-informatics algorithms for the prediction of MHC (Major Histocompatibility Complex) class-II binding peptides. We predicted using these in silico approaches promiscuous Th cell epitopes that are capable of binding to both murine and human MHC class-II molecules. To validate our in silico prediction experimentally, BALB/c mice were immunized with the five predicted peptides, and the proliferative responses and cytokine production of lymphocytes from the immunized BALB/c mice were assessed in vitro by modified MTT (Methyl Thiazolyl Tetrazolium), ELISA (Enzyme-linked Immunosorbent Assay) and flow cytometry methods. The results showed that two of the five predicted peptides could induce a Th1-type response in vitro. These results suggest that promiscuous Th1 cell epitopes from secreted or transmembrane proteins of S. japonicum can be identified using a strategy of reverse vaccinology.

[Cloning, expression and characterization of a gene encoding signal transduction protein Wnt4 of Schistosoma japonicum].

Wnt proteins together with their downstream effectors forms a set of important signal pathways. The Wnt signal pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Wnt4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. With RACE technique based on a EST identified in our lab, a novel gene including a complete open reading frame was cloned and named Sjwnt4 (GenBank accession No. DQ643829). Sequence analyses showed that SjWnt4 had a typical characteristics of Wnt family proteins, sharing 43% similarity to Dugesia japonica and 37% to human Wnt4. The ORF of Sjwnt4 contains 1311 nucleotides, encoding 436 amino acid with 49.6 kD molecular weight. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of Sjwnt4 is highest in the 19 days schistosomula, followed by 44 days female worms, 14 days schistosomula, 31 days adult worms and 44 days male worms, suggesting a stage-and-gender differential express. The Sjwnt4 cDNA fragment was subcloned into a modified expression vector pGEX-4T-2 and transformed into E. coli BL21 (DE3) cells, and the production of recombinant Sjwnt4 protein fused to a GST tag was analysed. In the presence of IPTG, the 76kD fusion protein was expressed in included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the regulation mechanism of the Wnt signaling pathway during the development especially gonadal differentiation processes of Schistosoma japonicum.

Proteomic analysis of differentially expressed proteins between the male and female worm of Schistosoma japonicum after pairing.

Identification of differentially expressed proteins between the male and female worm of Schistosoma japonicum may provide new insights into the development of schistosomes, especially the molecular mechanism of female worm maturation induced by the male worm after pairing. Comparative two-dimensional gel electrophoresis (2-DE) and mass spectrometry were employed to separate and identify differentially expressed proteins between the male and female worm after pairing. Soluble and hydrophobic proteins from egg, schistosomulum (14 days), and female and male worms at adult stage (42 days) were separated by a sequential extraction method followed by 2-DE and 2-DE images were constructed. There were 1016 +/- 67, 1808 +/- 89, 1142 +/- 45 and 1288 +/- 32 spots detected for soluble proteins and 1425 +/- 108, 952 +/- 59, 847 +/- 75 and 965 +/- 69 spots for hydrophobic proteins from egg, schistosomulum, and adult stage female and male worms, respectively. The differentially and uniquely expressed proteins from male and female worms after pairing (42 days) include 41 +/- 4 and 23 +/- 2 unique spots for soluble proteins, and 11 +/- 1 and 26 +/- 3 unique spots for hydrophobic proteins, respectively. Matrix-assisted laser desorption/ionization-time of flight and electrospray ionization-tandem mass spectrometry were employed to analyze 12 unique spots from the female worm and 16 unique spots from the male worm for peptide mass fingerprinting and sequencing. The results showed that the main functions of these differentially expressed proteins were in signal transduction, metabolism and transcriptional regulation etc. Comparison of the schistosomes proteome between male and female worms may permit the identification of protein candidates for the development of vaccines or new targets for drug development against schistosomiasis.

[Protective effect induced by vaccination with a partial cDNA expression library of Schistosoma japonicum in mice].

The cDNA fragments of interest were amplified using Sj lambda ZipLox library as the templates by PCR and then cloned into a eukaryotic expression vector p-CMV-GH; A small number of DNA fragments inserted in the recombinants was identified by restriction cleavage, EST sequencing and bioinformatical analysis; mice were injected intramuscularly with the expression library (L-CMV-SjR) or sublibraries(L-CMV-SjR1, L-CMV-SjR2 and L-CMV-SjR3), immunized mice were challenged with Schistosoma japonicum cercariae on day 35, the levels of IgG antibodies in sera from the immunized mice were detected by ELISA. The results demonstrated that a partial cDNA expression library of S.j, with approximately 10(5) transformants, was constructed, most of the recombinants contained the insert DNA fragments of interest, and these fragments had the features of protein-coding sequences for Schistosome. There were no significant differences for the levels of IgG antibodies in sera from all of the immunized groups. Mice immunized with L-CMV-SjR, L-CMV-SjR1 and L-CMV-SjR2 developed significant protective effect against Sj infection compared to control mice injected with the empty plasmid, the rate of worm reduction was about 30%.