title: The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease.
abstract: Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes.
  
  
  
  We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome.
  
  
  
  The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores.
  
  
  
  Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic specific immune responses were elicited by oral administration of B.s-CotC-CsCP spores. The spores effectively promoted intestinal health by inducing secretion of acidic mucins, with no other side effects to the liver or intestine. Oral administration of spores expressing CsCP could provide effective protection against C. sinensis. This study may be a cornerstone for development of antiparasitic agents or vaccines against clonorchiasis based on B. subtilis spore expressing CsCP on the surface.
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Zeli
authors.initials: Z
authors.lastname: Tang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Mei
authors.initials: M
authors.lastname: Shang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Tingjin
authors.initials: T
authors.lastname: Chen
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Pengli
authors.initials: P
authors.lastname: Ren
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Hengchang
authors.initials: H
authors.lastname: Sun
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Hongling
authors.initials: H
authors.lastname: Qu
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Zhipeng
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Lina
authors.initials: L
authors.lastname: Zhou
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Jinyun
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Hongye
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Zhou
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Xuerong
authors.initials: X
authors.lastname: Li
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Xu
authors.affiliation: Department of Parasitology, Zhongshan School of Medicine, Sun Yat-sen University, 74 Zhongshan 2nd Road, Guangzhou, 510080, China. yuhxteam@163.com.|Key Laboratory for Tropical Diseases Control, Sun Yat-sen University, Ministry of Education, Guangzhou, 510080, Guangdong, China. yuhxteam@163.com.
authors.forename: Xinbing
authors.initials: X
authors.lastname: Yu
mesh_terms: D000818:Animals; D000909:Antibodies, Helminth / Q000276:immunology; D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D003003:Clonorchiasis / Q000276:immunology* / Q000469:parasitology / Q000473:pathology / Q000517:prevention & control; D003004:Clonorchis sinensis / Q000201:enzymology* / Q000235:genetics / Q000276:immunology; D057056:Cysteine Proteases / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D005260:Female; D015870:Gene Expression; D015801:Helminth Proteins / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D006801:Humans; D008099:Liver / Q000469:parasitology / Q000473:pathology; D008807:Mice, Inbred BALB C; D019936:Probiotics / Q000008:administration & dosage; D013171:Spores, Bacterial / Q000235:genetics* / Q000378:metabolism; D014612:Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D000909:Antibodies, Helminth; D015801:Helminth Proteins; D014612:Vaccines; D057056:Cysteine Proteases
keywords: Bacillus subtilis spore; Clonorchis sinensis; Cysteine protease; Immunological characteristics; Oral immunization