Study On Immunological Characterization And Function Of Excretory-secretory Antigen Of Echinococcus Granulosus

Cystic echinococcosis (CE) is caused by infection with the Echinococcus granulosus and severely impairs both human and livestock health, especially in western China. The E. granulosus have a complex life cycle involving two hosts. The definitive hosts are primarily dogs, which harbor adult worms in their small intestines. Human and herbivores, particularly sheep and cattle, are intermediate hosts of this parasite. Intermediate hosts become infected by ingesting the eggs released in the feces of definitive hosts or comsuming food contaminated with the eggs. Dogs, as definitive hosts, are pivotal in the transmission of CE., and dog vaccination provides a very practical and cost-effective prevention strategy, because there are far fewer dogs than sheep or cattle in endemic areas. However, lack of understanding about host immune response to infection and immunomodulatory mechanism restrict the development of vaccine against CE. Excretory secretory products (ES) are known to modulate both the innate and adaptive host immune responses and appear to target both cellular and humoral responses. Additionally, ES may interfere with other processes of parasite-host interaction. Therefore, investigate the mechanism of ES effect on host immune response and identify protein content of ES could provide valuable insights into host-parasite interaction and contribute to development of vaccine against E. granulosus immunized in dogs in the endemic areas. PartⅠStudy on immunological function of excretory-secretory antigens from Echinococcus granulosusES are exposed to host immune system directly and play a key role in modulate immune response of host. Dendritic cells (DCs) are currently known to be the most potent professional antigen-presenting cells (APCs), which have the unique function of stimulating naive T cells and control the development of immunity. Consequently DC is one of targets that parasite used to modulate host immune response. In this study, we assessed the effects of the adult E. granulosus ES on the functions of DC from BALB/c mice, with the aim of investigating parasite-host interactions as well as parasite survival mechanismsThe murine bone marrow cells of BALB/c mice were collected and cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) to harvest murine bone marrow-derived dendritic cells (BMDC). The DC were treated with ES or adult worm antigen (AWA) of E. granulosus in vitro, phenotype and cytokine secretion were analyzed to investigate the ability to induce DC maturation by the ES or AWA antigens. The DC were pre-treated with ES or AWA, and then be co-cultured with T cell, the phenotype and cytokine secretion of T cells were analyzed to investigate the effect of ES on the ability of DC to active T cells. And the effect of ovalbumin (OVA)-pulsed BMDC pre-treated with ES on the potent Thl promoting effect of CpG was investigated.The results showed that the DC stimulated with AWA presented higher levels of expression of co-stimulatory molecules (CD40, CD80, CD86and MHC class II) and cytokine secretion (IL-6, IL-10, IL-12p40, IL-12p70and TNF-a). However, expression of co-stimulatory molecules and cytokine secretion of DC stimulated with ES was similar to that observed for the PBS control. Data demonstrated that AWA induced maturation of DC, while ES failed. T cell co-cultured with DC pre-treated with AWA, the production of IFN-y was increased whereas IL-4was reduced. However, ES pre-treated BMDC showed impaired capacity to prime CD4+T cells to secrete these cytokines, and increased percentage of CD4+T cells expressing CD25and Foxp3. CpG, which is a potent Thl adjuvant, induced higher levels of CD40, CD80, CD86and MHC class II expression. But ES treatment could reduced all of these co-stimulatory molecule expression induced by CpG, showed strong suppressive effect.Part IIProteomic analysis of excretory-secretory antigen of adult Echinococcus granulosusProteomic analysis was performed to obtain protein profiles and antigenic protein profile of ES and worms from adult stage E. granulosus, the results of analysis will provide valuable information for screenning candidate protein molecules for vaccination, immunodiagnosis and drug development.Samples of worms and ES from adult E. granulosus were prepared and subjected to IEF and SDS-PAGE. Gels were stained with Coomassie blue and scanned with ImageScanner. For immunoblot analysis, proteins were electrotransferred from2-DE gel to PVDF membranes. Membranes were blocked and incubated with serum of E. granulosus infected dog. The2-DE blots were revealed with DAB reagent. Protein spots were manually excised and digested for MALDI-TOF MS. All data were searched against database to identify protein.More than200protein spots were presented on the gels of worm total protein sample, most of these spots distributed in pI4.5-9,192spots were excised for mass spectrometry. Approximately50protein spots were were presented on the gels of ES sample, most of these spots distributed in molecular weight20-100kDa, pI4.5-9,48spots were excised for mass spectrometry.36antigenic protein spots were revealed by immunoblotting. A series of protein associated to survival, development, movement and modulation of parasite were identified by MALDI-TOF MS.61spots of worm total protein were identified, corresponding to32different proteins.34spots of ES were identified, corresponding to9different proteins.21spots of antigenic protein were identified, corresponding to13different proteins,7of these were described here for the first time. According KOG functional classification, most of the identified proteins are related to Z (cytoskeleton), O (post-translational modification, protein turnover, and chaperones) and G (Carbohydrate transport and metabolism).Part IIICloning, expression and analysis of proteins from excretory-secretory antigen of adult Echinococcus granulosusBase on the results of part I and part II, EgEno and EgCyp were chosed from antigenic proteins indwell in both worm and ES of E. granulosus to clone, expression and bioinformatic analysis, the result will provide basis for further research on mechanisms and potential as vaccine candidates of these proteins.Total RNA of adult E. granulosus was extracted and reversedly transcripted to cDNA. EgEno and EgCyp gene were amplified from cDNA and inserted into vector pET28a. Recombinant plasmids pET28a-EgEno and pET28a-EgCyp was transformed into E. coli BL21(DE3) for expression under the induction of IPTG The expressed products waere identified by SDS-PAGE and Western blotting. EgEno and EgCyp were analyzed by the bioinformatics software.The EgEno gene, which has1302bp ORF encoding433amino acids, and EgCyp gene, which has489bp ORF encoding162amino acids, were successfully amplified from cDNA of adult E. granulosus. The recombinant expression system was constructed and fusion proteins were expressed in E.coli BL21(DE3). SDS-PAGE showed that the molecular weights of expressed proteins of EgEno and EgCyp were50k Da and22kDa, respectively. The Western blotting by serum of E. granulosus infected dog indicated that the antigenicity of the proteins was specific. The bioinformatics analysis revealed that there were16antigen epitopes in EgEno and7antigen epitopes in EgCyp, suggested the potential of these proteins as vaccine candidats and diagnosis markers.Conclusions1. ES of adult E. granulosus downregulated host immune responses by inability to induce maturation of DC, impairment of DC function and induction of CD4+CD25+Foxp3+T cells.2. Presented protein profiles of ES and worms from adult stage of E. granulusus and antigenic protein profile of E. granulosus adult worms. A series of protein associated to survival, development, movement and modulation of parasite were identified.3. EgEno and EgCyp of adult E. granulosus, which were antigenic protein in both worms and ES, were cloned and expressed in E. coli BL21(DE3) successfully and showed the potential for immunodiagnosis and vaccine development.