title: A novel approach to generate a recombinant toxoid vaccine against Clostridium difficile.
abstract: The Clostridium difficile toxins A and B are primarily responsible for symptoms of C. difficile associated disease and are prime targets for vaccine development. We describe a plasmid-based system for the production of genetically modified toxins in a non-sporulating strain of C. difficile that lacks the toxin genes tcdA and tcdB. TcdA and TcdB mutations targeting established glucosyltransferase cytotoxicity determinants were introduced into recombinant plasmids and episomally expressed toxin mutants purified from C. difficile transformants. TcdA and TcdB mutants lacking glucosyltransferase and autoproteolytic processing activities were ~10 000-fold less toxic to cultured human IMR-90 cells than corresponding recombinant or native toxins. However, both mutants retained residual cytotoxicity that could be prevented by preincubating the antigens with specific antibodies or by formalin treatment. Such non-toxic formalin-treated mutant antigens were immunogenic and protective in a hamster model of infection. The remaining toxicity of untreated TcdA and TcdB mutant antigens was associated with cellular swelling, a phenotype consistent with pore-induced membrane leakage. TcdB substitution mutations previously shown to block vesicular pore formation and toxin translocation substantially reduced residual toxicity. We discuss the implications of these results for the development of a C. difficile toxoid vaccine.
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Robert G K
authors: RGK
authors: Donald
authors: Centers for Disease Control, 1600 Clifton Rd, Atlanta, GA 30333, USA.
authors: Mike
authors: M
authors: Flint
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Narender
authors: N
authors: Kalyan
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Erik
authors: E
authors: Johnson
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Susan E
authors: SE
authors: Witko
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Cheryl
authors: C
authors: Kotash
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Ping
authors: P
authors: Zhao
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Shakuntala
authors: S
authors: Megati
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Irina
authors: I
authors: Yurgelonis
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Phillip Kwok
authors: PK
authors: Lee
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Yury V
authors: YV
authors: Matsuka
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Elena
authors: E
authors: Severina
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Anne
authors: A
authors: Deatly
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Mini
authors: M
authors: Sidhu
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Kathrin U
authors: KU
authors: Jansen
authors: Clostridia Research Group, NIHR Biomedical Research Unit in GI Disease, University Park, University of Nottingham, Nottingham NG7 2RD, UK.
authors: Nigel P
authors: NP
authors: Minton
authors: Pfizer Vaccine Research, Pearl River, NY 10654, USA.
authors: Annaliesa S
authors: AS
authors: Anderson
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000276:immunology; D001426:Bacterial Proteins / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics; D001428:Bacterial Vaccines / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology; D002460:Cell Line; D016360:Clostridioides difficile / Q000235:genetics / Q000276:immunology*; D003015:Clostridium Infections / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control*; D006224:Cricetinae; D004761:Enterocolitis, Pseudomembranous / Q000276:immunology / Q000382:microbiology / Q000517:prevention & control; D004768:Enterotoxins / Q000235:genetics; D006801:Humans; D009154:Mutation; D014121:Toxoids / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology; D014614:Vaccines, Synthetic / Q000008:administration & dosage / Q000235:genetics* / Q000276:immunology
chemical_list: D000907:Antibodies, Bacterial; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D014121:Toxoids; D014614:Vaccines, Synthetic; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile
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