{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,31]],"date-time":"2025-12-31T18:10:55Z","timestamp":1767204655422,"version":"build-2238731810"},"reference-count":47,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,4,3]],"date-time":"2025-04-03T00:00:00Z","timestamp":1743638400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100012547","name":"Natural Science Foundation of Guangxi Zhuang Autonomous Region","doi-asserted-by":"publisher","id":[{"id":"10.13039\/100012547","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:sec>\n                    <jats:title>Introduction<\/jats:title>\n                    <jats:p>\n                      Cysteine proteases from\n                      <jats:italic>Clonorchis sinensis<\/jats:italic>\n                      , including various proteins, are essential for its pathogenicity and serve as potential vaccine candidates. This study assesses the protective effects of three\n                      <jats:italic>C. sinensis<\/jats:italic>\n                      cysteine proteases (CsCP1-3).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Mice immunized with recombinant CsCP1-3 and adjuvants were subsequently infected with\n                      <jats:italic>C. sinensis<\/jats:italic>\n                      metacercariae after three immunization rounds. Liver damage was evaluated through hematoxylin and eosin (H&amp;amp;E), Masson\u2019s trichrome, and immunohistochemical analyses. The levels of IgG1, IgG2a antibodies, and cytokines (IFN-g, IL-2, IL-4, and IL-10) were quantified by enzyme-linked immunosorbent assay (ELISA).\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>RT-qPCR revealed that CsCP1-2 exhibited the highest expression in newly encysted larvae (NEL), while CsCP3 was predominantly expressed in adult stages. Immunohistochemical localization confirmed that CsCP1-3 are present in the eggshells, syncytial layers of metacercariae, NEL cuticle, and adult intestines. Histological and immunohistochemical analysis demonstrated that the rCsCP1-3-immunized group displayed reduced liver inflammation and biliary fibrosis compared to the control group. The rCsCP1-3 induced a progressive increase in specific IgG1 and IgG2a antibody titers by the second week post-immunization. In the CsCP1-2 group, cytokines IFN-g, IL-2, IL-4, and IL-10 were elevated relative to the control, with particularly high levels of IFN-g and IL-10 in CsCP1, indicating a strong mixed Th1\/Th2 immune response. In contrast, the CsCP3 immunization group exhibited a transient increase in cytokines (IFN-g, IL-2, IL-4, and IL-10) three days postinfection, which subsided after one to two weeks.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Discussion<\/jats:title>\n                    <jats:p>\n                      These findings suggest that CsCP1-3 elicit robust antibody and cellular immune responses, mitigating liver damage caused by\n                      <jats:italic>C. sinensis<\/jats:italic>\n                      infection. CsCP1, in particular, induces a potent mixed Th1\/Th2 response, positioning it as a promising vaccine candidate.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fimmu.2025.1550775","type":"journal-article","created":{"date-parts":[[2025,4,3]],"date-time":"2025-04-03T03:36:16Z","timestamp":1743651376000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["Characteristics and immunoprotective functions of three cysteine proteases from Clonorchis sinensis"],"prefix":"10.3389","volume":"16","author":[{"given":"Yunliang","family":"Shi","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Xiaoqin","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kai","family":"Liang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ting","family":"Lu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yu","family":"Chen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yashi","family":"Lai","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yaoting","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shuai","family":"Wei","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shanshan","family":"He","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lili","family":"Tang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dengyu","family":"Liu","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yanwen","family":"Li","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2025,4,3]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/j.parint.2011.06.007","article-title":"Clonorchis sinensis and clonorchiasis, an update","volume":"61","author":"Hong","year":"2012","journal-title":"Parasitol Int"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1016\/j.actatropica.2019.105320","article-title":"Surveillance of clonorchiasis in China in 2016","volume":"203","author":"Zhu","year":"2020","journal-title":"Acta Trop"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.1016\/s1470-2045(09)70096-8","article-title":"A review of human carcinogens\u2013Part B: biological agents","volume":"10","author":"Bouvard","year":"2009","journal-title":"Lancet Oncol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.5483\/bmbrep.2016.49.11.109","article-title":"Clonorchis sinensis, an oriental liver fluke, as a human biological agent of cholangiocarcinoma: a brief review","volume":"49","author":"Kim","year":"2016","journal-title":"BMB Rep"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/j.actatropica.2019.105309","article-title":"Clonorchis sinensis and clonorchiasis","volume":"203","author":"Na","year":"2020","journal-title":"Acta Trop"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1016\/s0140-6736(15)60313-0","article-title":"Clonorchiasis","volume":"387","author":"Qian","year":"2016","journal-title":"Lancet"},{"key":"B7","doi-asserted-by":"publisher","first-page":"71","DOI":"10.1186\/s40249-016-0166-1","article-title":"Current status and perspectives of Clonorchis sinensis and clonorchiasis: epidemiology, pathogenesis, omics, prevention and control","volume":"5","author":"Tang","year":"2016","journal-title":"Infect Dis Poverty"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1128\/cmr.00009-23","article-title":"Clonorchiasis and opisthorchiasis: epidemiology, transmission, clinical features, morbidity, diagnosis, treatment, and control","volume":"37","author":"Qian","year":"2024","journal-title":"Clin Microbiol Rev"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.13350\/j.cjpb.200326","article-title":"The epidemiology of clonorchiasis and the status of its treatment","volume":"15","author":"Yao","year":"2020","journal-title":"J Pathogen Biol"},{"key":"B10","doi-asserted-by":"publisher","first-page":"1","DOI":"10.18388\/abp.2001_5108","article-title":"Structural studies of cysteine proteases and their inhibitors","volume":"48","author":"Grzonka","year":"2001","journal-title":"Acta Biochim Pol"},{"key":"B11","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0006512","article-title":"Cysteine proteases in protozoan parasites","volume":"12","author":"Siqueira-neto","year":"2018","journal-title":"PloS Negl Trop Dis"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0005919","article-title":"Cysteine proteases during larval migration and development of helminths in their final host","volume":"12","author":"Grote","year":"2018","journal-title":"PloS Negl Trop Dis"},{"key":"B13","doi-asserted-by":"publisher","first-page":"1937","DOI":"10.3390\/microorganisms11061397","article-title":"Roles of cysteine proteases in biology and pathogenesis of parasites","volume":"11","author":"Yang","year":"2023","journal-title":"Microorganisms"},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1016\/j.micpath.2021.104859","article-title":"Vaccination with cathepsin L mimotopes of Fasciola hepatica in goats reduces worm burden, morphometric measurements, and reproductive structures","volume":"155","author":"Villa-mancera","year":"2021","journal-title":"Microb Pathog"},{"key":"B15","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1016\/j.jare.2018.12.008","article-title":"Role of T lymphocytes and papain enzymatic activity in the protection induced by the cysteine protease against Schistosoma mansoni in mice","volume":"17","author":"Tallima","year":"2019","journal-title":"J Adv Res"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0005443","article-title":"Protective immune responses against Schistosoma mansoni infection by immunization with functionally active gut-derived cysteine peptidases alone and in combination with glyceraldehyde 3-phosphate dehydrogenase","volume":"11","author":"Tallima","year":"2017","journal-title":"PloS Negl Trop Dis"},{"key":"B17","doi-asserted-by":"publisher","first-page":"580","DOI":"10.1186\/s13071-021-05010-y","article-title":"Immune reactivity and host modulatory roles of two novel Haemonchus contortus cathepsin B-like proteases","volume":"14","author":"Bakshi","year":"2021","journal-title":"Parasit Vectors"},{"key":"B18","doi-asserted-by":"publisher","first-page":"433","DOI":"10.1186\/s13071-023-06051-1","article-title":"Trichinella spiralis cathepsin L induces macrophage M1 polarization via the NF-\u03baB pathway and enhances the ADCC killing of newborn larvae","volume":"16","author":"Liu","year":"2023","journal-title":"Parasit Vectors"},{"key":"B19","doi-asserted-by":"publisher","first-page":"15","DOI":"10.1081\/imm-120027681","article-title":"Immunization with Trichinella spiralis Korean isolate larval excretory-secretory antigen induces protection and lymphocyte subset changes in rats","volume":"33","author":"Quan","year":"2004","journal-title":"Immunol Invest"},{"key":"B20","doi-asserted-by":"publisher","first-page":"383","DOI":"10.1186\/s13071-019-3639-9","article-title":"Molecular characterization and functional analysis of the Schistosoma mekongi Ca(2+)-dependent cysteine protease (calpain)","volume":"12","author":"Chaimon","year":"2019","journal-title":"Parasit Vectors"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.1007\/s00253-021-11126-z","article-title":"The storage stability of Bacillus subtilis spore displaying cysteine protease of Clonorchis sinensis and its effect on improving the gut microbiota of mice","volume":"105","author":"Tang","year":"2021","journal-title":"Appl Microbiol Biotechnol"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.1002\/pmic.200700613","article-title":"Identification of a serodiagnostic antigen, legumain, by immunoproteomic analysis of excretory-secretory products of Clonorchis sinensis adult worms","volume":"9","author":"Ju","year":"2009","journal-title":"Proteomics"},{"key":"B23","doi-asserted-by":"publisher","first-page":"7","DOI":"10.1016\/j.molbiopara.2009.11.006","article-title":"A family of cathepsin F cysteine proteases of Clonorchis sinensis is the major secreted proteins that are expressed in the intestine of the parasite","volume":"170","author":"Kang","year":"2010","journal-title":"Mol Biochem Parasitol"},{"key":"B24","doi-asserted-by":"publisher","first-page":"647","DOI":"10.1186\/s13071-015-1248-9","article-title":"Identification and characterization of Clonorchis sinensis cathepsin B proteases in the pathogenesis of clonorchiasis","volume":"8","author":"Chen","year":"2015","journal-title":"Parasit Vectors"},{"key":"B25","doi-asserted-by":"publisher","DOI":"10.1007\/s00436-019-06223-y","article-title":"Identification and characteristics of a cathepsin L-like cysteine protease from","volume":"118","author":"Ma","year":"2019","journal-title":"Clonorchis sinensis Parasitol Res"},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1016\/j.vaccine.2005.11.062","article-title":"Vaccination with DNA encoding cysteine proteinase confers protective immune response to rats infected with Clonorchis sinensis","volume":"24","author":"Lee","year":"2006","journal-title":"Vaccine"},{"key":"B27","doi-asserted-by":"publisher","first-page":"648","DOI":"10.1186\/s13071-016-1928-0","article-title":"The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease","volume":"9","author":"Tang","year":"2016","journal-title":"Parasit Vectors"},{"key":"B28","doi-asserted-by":"publisher","DOI":"10.1016\/j.fsi.2017.03.030l","article-title":"Oral delivery of Bacillus subtilis spores expressing cysteine protease of Clonorchis sinensis to grass carp (Ctenopharyngodon idellus): Induces immune responses and has no damage on liver and intestine function","volume":"64","author":"Tang","year":"2017","journal-title":"Fish Shellfish Immunol"},{"key":"B29","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1186\/s13071-023-05891-1","article-title":"Multilayer omics reveals the molecular mechanism of early infection of Clonorchis sinensis juvenile","volume":"8","author":"Wu","year":"2023","journal-title":"Parasit Vectors"},{"key":"B30","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2023.1280358","article-title":"Clonorchis sinensis calcium-binding protein Cs16 causes acute hepatic injury possibly by reprogramming the metabolic pathway of bone marrow-derived monocytes","volume":"10","author":"Li","year":"2023","journal-title":"Front Cell Infect Microbiol"},{"key":"B31","doi-asserted-by":"publisher","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"12","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.1002\/(ISSN)1527-3350","article-title":"A histological semiquantitative scoring system for evaluation of hepatic fibrosis in needle liver biopsy specimens: comparison with morphometric studies","volume":"8","author":"Chevallier","year":"1994","journal-title":"Hepatology"},{"key":"B33","doi-asserted-by":"publisher","DOI":"10.1016\/0168-8278(95)80226-6","article-title":"Histological grading and staging of chronic hepatitis","volume":"6","author":"Ishak","year":"1995","journal-title":"J Hepatol"},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1007\/s00436-016-5012-7","article-title":"Interleukin-13 is involved in the formation of liver fibrosis in Clonorchis sinensis-infected mice","volume":"115","author":"Xu","year":"2016","journal-title":"Parasitol Res"},{"key":"B35","doi-asserted-by":"publisher","DOI":"10.1002\/iid3.235","article-title":"A 21.6\u2009kDa tegumental protein of Clonorchis sinensis induces a Th1\/Th2 mixed immune response in mice","volume":"6","author":"Chung","year":"2018","journal-title":"Immun Inflammation Dis"},{"key":"B36","doi-asserted-by":"publisher","first-page":"626","DOI":"10.1186\/s13071-017-2526-5","article-title":"Virus-like particles vaccine containing Clonorchis sinensis tegumental protein induces partial protection against Clonorchis sinensis infection","volume":"10","author":"Lee","year":"2017","journal-title":"Parasit Vectors"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pntd.0006251","article-title":"Clonorchis sinensis adult-derived proteins elicit Th2 immune responses by regulating dendritic cells via mannose receptor","volume":"12","author":"Zhao","year":"2018","journal-title":"PloS Negl Trop Dis"},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0033703","article-title":"Identification and characterization of paramyosin from cyst wall of metacercariae implicated protective efficacy against Clonorchis sinensis infection","volume":"7","author":"Wang","year":"2012","journal-title":"PloS One"},{"key":"B39","doi-asserted-by":"publisher","DOI":"10.1016\/j.biopha.2019.109110","article-title":"Resveratrol improves CCL4-induced liver fibrosis in mouse by upregulating endogenous IL-10 to reprogramme macrophages phenotype from M(LPS) to M(IL-4)","volume":"117","author":"Yu","year":"2019","journal-title":"BioMed Pharmacother"},{"key":"B40","doi-asserted-by":"publisher","DOI":"10.1155\/2019\/4652596","article-title":"Interleukin 10 gene-modified bone marrow-derived dendritic cells attenuate liver fibrosis in mice by inducing regulatory T cells and inhibiting the TGF-\u03b2\/smad signaling pathway","volume":"2019","author":"Xu","year":"2019","journal-title":"Mediators Inflamm"},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1038\/emm.2013.144","article-title":"Regulation of anti-inflammatory cytokines IL-10 and TGF-\u03b2 in mouse dendritic cells through treatment with Clonorchis sinensis crude antigen","volume":"46","author":"Jin","year":"2014","journal-title":"Exp Mol Med"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1016\/j.intimp.2023.111045","article-title":"MMP10 alleviates non-alcoholic steatohepatitis by regulating macrophage M2 polarization","volume":"124","author":"Chang","year":"2023","journal-title":"Int Immunopharmacol"},{"key":"B43","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbrc.2005.08.085","article-title":"Interleukin-10 gene therapy reverses thioacetamide-induced liver fibrosis in mice","volume":"336","author":"Hung","year":"2005","journal-title":"Biochem Biophys Res Commun"},{"key":"B44","doi-asserted-by":"publisher","first-page":"215","DOI":"10.1186\/s13071-020-04083-5","article-title":"Clonorchis sinensis secretory protein CsAg17 vaccine induces immune protection","volume":"13","author":"Bai","year":"2020","journal-title":"Parasit Vectors"},{"key":"B45","doi-asserted-by":"publisher","first-page":"156","DOI":"10.1186\/s13071-018-2757-0","article-title":"Bacillus subtilis spore with surface display of paramyosin from Clonorchis sinensis potentializes a promising oral vaccine candidate","volume":"11","author":"Sun","year":"2018","journal-title":"Parasit Vectors"},{"key":"B46","doi-asserted-by":"publisher","first-page":"88","DOI":"10.1038\/nrgastro.2015.200","article-title":"Immunology in the liver\u2013from homeostasis to disease","volume":"13","author":"Heymann","year":"2016","journal-title":"Nat Rev Gastroenterol Hepatol"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-immunol-051116-052415","article-title":"Immune responses in the liver","volume":"36","author":"Kubes","year":"2018","journal-title":"Annu Rev Immunol"}],"updated-by":[{"DOI":"10.3389\/fimmu.2025.1663826","type":"correction","label":"Correction","source":"publisher","updated":{"date-parts":[[2025,9,15]],"date-time":"2025-09-15T00:00:00Z","timestamp":1757894400000}}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1550775\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,9,15]],"date-time":"2025-09-15T10:30:49Z","timestamp":1757932249000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1550775\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,4,3]]},"references-count":47,"alternative-id":["10.3389\/fimmu.2025.1550775"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1550775","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,4,3]]},"article-number":"1550775"}}