title: Subcutaneous and intranasal immunization with Stx2B-Tir-Stx1B-Zot reduces colonization and shedding of Escherichia coli O157:H7 in mice.
abstract: The type III secretion system of Escherichia coli O157:H7 is involved in colonization of mammalian hosts by the organism. The translocated intimin receptor (Tir) is inserted into the mammalian host cell plasma membrane in a hairpin loop topology with the central loop of the molecule exposed to the host cell surface and accessible for interaction with an LEE-encoded bacterial outer membrane adhesin called intimin. Shiga toxin type 1 and 2 produced by E. coli O157:H7 are responsible for hemolytic uremic syndrome and able to promote intestinal colonization. Zonula occludens toxin (Zot) is a single polypeptide chain encoded by the filamentous bacteriophage CTXφ of Vibrio cholerae. Zot binds a receptor on intestinal epithelial cells and increases mucosal permeability by affecting the structure of epithelial tight junctions. Because of these properties, Zot is a promising tool for mucosal drug and antigen (Ag) delivery. In the current study, we constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, Tir and Zot, designated Stx2B-Tir-Stx1B-Zot, expressed in the E. coli BL21 and harvested the purified protein by a simple GST·Bind Resin chromatography method. We used a streptomycin-treated mouse model to evaluate the efficacy of subcutaneous vs. intranasal administration of the vaccine. Following immunization, mice were infected with E. coli O157:H7 and feces were monitored for shedding. Immune responses against Stx2B-Tir-Stx1B-Zot, Stx2B-Tir-Stx1B and control agent (GST/PBS) were also monitored. Subcutaneous immunization of mice with Stx2B-Tir-Stx1B-Zot induced significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies but did not significantly induce any antigen-specific IgA in feces, whereas intranasal immunization elicited significant Stx2B-Tir-Stx1B-Zot-specific serum IgG antibodies with some animals developing antigen-specific IgA in feces. Mice that were immunized intranasally with Stx2B-Tir-Stx1B-Zot showed dramatically decreased E. coli O157:H7 shedding compared to those of Stx2B-Tir-Stx1B and control agent following experimental infection. Mice immunized subcutaneously with Stx2B-Tir-Stx1B-Zot or Stx2B-Tir-Stx1B both showed reduced shedding in feces, moreover, Stx2B-Tir-Stx1B-Zot did better. These results demonstrate the perspective for the use of Stx2B-Tir-Stx1B-Zot to prevent colonization and shedding of E. coli O157:H7.
authors.affiliation: College of Veterinary Medicine, Nanjing Agricultural University, Tongwei Road 6#, Nanjing 210095, China.
authors.forename: Xue-Han
authors.initials: XH
authors.lastname: Zhang
authors.forename: Kong-Wang
authors.initials: KW
authors.lastname: He
authors.forename: Shu-Xia
authors.initials: SX
authors.lastname: Zhang
authors.forename: Wei-Cai
authors.initials: WC
authors.lastname: Lu
authors.forename: Pan-Deng
authors.initials: PD
authors.lastname: Zhao
authors.forename: Xiao-Ting
authors.initials: XT
authors.lastname: Luan
authors.forename: Qing
authors.initials: Q
authors.lastname: Ye
authors.forename: Li-Bin
authors.initials: LB
authors.lastname: Wen
authors.forename: Bin
authors.initials: B
authors.lastname: Li
authors.forename: Rong-Li
authors.initials: RL
authors.lastname: Guo
authors.forename: Xiao-Min
authors.initials: XM
authors.lastname: Wang
authors.forename: Li-Xin
authors.initials: LX
authors.lastname: Lv
authors.forename: Jun-Ming
authors.initials: JM
authors.lastname: Zhou
authors.forename: Zheng-Yu
authors.initials: ZY
authors.lastname: Yu
authors.forename: Ai-Hua
authors.initials: AH
authors.lastname: Mao
mesh_terms: D000281:Administration, Intranasal; D000818:Animals; D057138:Bacterial Shedding / Q000276:immunology; D002772:Cholera Toxin / Q000235:genetics / Q000276:immunology*; D004731:Endotoxins; D004927:Escherichia coli Infections / Q000276:immunology / Q000517:prevention & control*; D019453:Escherichia coli O157 / Q000254:growth & development / Q000472:pathogenicity*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000276:immunology*; D022361:Escherichia coli Vaccines / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology*; D007279:Injections, Subcutaneous; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D022622:Shiga Toxin 1 / Q000235:genetics / Q000276:immunology*; D022641:Shiga Toxin 2 / Q000235:genetics / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics / Q000276:immunology
chemical_list: D004731:Endotoxins; D029968:Escherichia coli Proteins; D022361:Escherichia coli Vaccines; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C109287:Tir protein, E coli; D014614:Vaccines, Synthetic; C450841:shiga toxin 2 B subunit; C077728:zonula occludens toxin, Vibrio cholerae; D002772:Cholera Toxin