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Clonorchis sinensis secretory protein CsAg17 vaccine induces immune protection.

ABSTRACT: BACKGROUND:Clonorchiasis is endemic in East and Southeast Asian countries. For a preventive strategy against infectious diseases, vaccination is the most effective. Here, we evaluated the molecular characteristics and immune responses of CsAg17 protein from Clonorchis sinensis, and investigated its protective effects against C. sinensis challenge. METHODS:A cDNA clone encoding CsAg17 protein and containing a secretory signal peptide at the N-terminus was retrieved from the C. sinensis transcriptome bank. Recombinant CsAg17 B-cell epitope protein and cDNA vaccines were produced and their immune responses were evaluated in FVB mice. The proportional changes of CD3+/CD4+ and CD3+/CD8+ T cells were detected by flow cytometry, and immune effectors were measured by ELISA. RESULTS:The CsAg17 mRNA was transcribed at a higher level in C. sinensis adults than in metacercariae. The CsAg17 protein was distributed in the sperms, oral and ventral suckers, and mesenchymal tissues of C. sinensis adults. In mice challenged with C. sinensis metacercariae, vaccination with CsAg17 protein and cDNA resulted in a reduction to 64% and 69% in worm burden, respectively. Both CsAg17 protein and cDNA vaccines increased the proportion of CD3+/CD4+ and CD3+/CD8+ T cells and stimulated the production of Th1 type cytokines such as interleukin (IL)-2, IL-12, and interferon-?, while maintaining minimum levels of Th2 cytokines. The levels of IgG specific to CsAg17 protein steeply increased in the two vaccinated groups from 2 weeks after immunization. The liver tissue retained good morphology in the mice vaccinated with CsAg17 protein or cDNA, whereas severe inflammation and large serous cysts were observed in the liver of the unvaccinated mice. CONCLUSIONS:Vaccination with CsAg17 protein and cDNA reduced the pathological changes in the bile duct and liver, and ameliorated the worm burden via cellular and humoral immune responses. Thus, they may serve as good vaccine candidates against C. sinensis infections.

SUBMITTER: Bai X

PROVIDER: S-EPMC7183723 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Clonorchis sinensis secretory protein CsAg17 vaccine induces immune protection.

Bai Xuelian X Song Jin-Ho JH Dai Fuhong F Lee Ji-Yun JY Hong Sung-Jong SJ

Parasites & vectors 20200425 1

<h4>Background</h4>Clonorchiasis is endemic in East and Southeast Asian countries. For a preventive strategy against infectious diseases, vaccination is the most effective. Here, we evaluated the molecular characteristics and immune responses of CsAg17 protein from Clonorchis sinensis, and investigated its protective effects against C. sinensis challenge.<h4>Methods</h4>A cDNA clone encoding CsAg17 protein and containing a secretory signal peptide at the N-terminus was retrieved from the C. sine ...[more]

PMID: 32334611

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Project description:BACKGROUND: Clonorchiasis is a globally important, neglected food-borne disease caused by Clonorchis sinensis (C. sinensis), and it is highly related to cholangiocarcinoma and hepatocellular carcinoma. Increased molecular evidence has strongly suggested that the adult worm of C. sinensis continuously releases excretory-secretory proteins (ESPs), which play important roles in the parasite-host interactions, to establish successful infection and ensure its own survival. Myoglobin, a hemoprotein, is present in high concentrations in trematodes and ESPs. To further understand the biological function of CsMb and its putative roles in the interactions of C. sinensis with its host, we explored the molecular characterization of CsMb in this paper. METHODS: We expressed CsMb and its mutants in E. coli BL21 and identified its molecular characteristics using bioinformatics analysis and experimental approaches. Reverse transcription PCR analysis was used to measure myoglobin transcripts of C. sinensis with different culture conditions. The peroxidase activity of CsMb was confirmed by spectrophotometry. We co-cultured RAW264.7 cells with recombinant CsMb (rCsMb), and we then measured the production of hydrogen peroxide (H2O2) and nitric oxide (NO) in addition to the mRNA levels of inducible nitric oxide synthase (iNOS), Cu-Zn superoxide dismutase (SOD1) and Mn superoxide dismutase (SOD2) in activated RAW264.7 cells. RESULTS: In the in vitro culture of adult worms, the transcripts of CsMb increased with the increase of oxygen content. Oxidative stress conditions induced by H2O2 increased the levels of CsMb transcripts in a dose-dependent manner. Furthermore, CsMb catalyzed oxidation reactions in the presence of H2O2, and amino acid 34 of CsMb played an essential role in its reaction with H2O2. In addition, CsMb significantly reduced H2O2 and NO levels in LPS-activated macrophages, and CsMb downregulated iNOS and SOD expression in activated macrophages. CONCLUSION: The present study is the first to investigate the peroxidase activity of CsMb. This investigation suggested that C. sinensis may decrease the redox activation of macrophages by CsMb expression to evade host immune responses. These studies contribute to a better understanding of the role of CsMb in the molecular mechanisms involved in ROS detoxification by C. sinensis.

Project description:Liver fluke proteins, including excretory-secretory products (ESPs) and tegument proteins, are critical for the pathogenesis, nutrient metabolism, etiology and immune response of liver cancer. To understand the functions of various proteins in Clonorchis sinensis physiology and human clonorchiasis, the ESPs and tegument proteins of C. sinensis were identified. Supernatants containing ESPs from adult C. sinensis after culture for 6 h were harvested and concentrated. The tegument was detached using a freeze/thaw method and successively extracted using various extraction buffers. The outer surface proteins of C. sinensis were labeled with biotin, and the biotinylated proteins were purified. The ESP, tegument and labeled outer surface proteins were identified and analyzed by high-resolution LC-MS/MS. The identified proteins were compared with those of other flukes, and the protein functions associated with pathogenesis, carcinogenesis and potential vaccine antigens and drug targets were predicted and analyzed. A total of 175 proteins were identified after the 6-h culture of C. sinensis ESPs. A total of 352 tegument proteins were identified through sequential solubilization of the isolated teguments, and a subset of these proteins were localized to the surface membrane of the tegument by labeling with biotin. Thirty identified proteins, including annexins, actin and tetraspanins, were identified as potential immunomodulators and promising vaccine antigens. Interestingly, among the 352 tegument proteins, as many as 155 were enzymes, and most were oxidoreductases, hydrolases or transferases. A comparison of the outer surface proteins of C. sinensis with those of other flukes indicated that flukes have some common outer surface proteins, such as actin, tetraspanin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and annexin. Granulin, thioredoxin peroxiredoxin, carbonyl reductase 1 and cystatin were identified in the C. sinensis proteome and predicted to be related to liver disease and cancer. The analysis of the C. sinensis proteome could contribute to a more in-depth understanding of complex parasite-host relationships, improve the diagnosis of clonorchiasis and benefit research on the pathogenesis and development of novel interventions, drugs and vaccines to control C. sinensis infection.

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Clonorchis sinensis secretory protein CsAg17 vaccine induces immune protection.

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Clonorchis sinensis secretory protein CsAg17 vaccine induces immune protection.

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