title: Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids.
abstract: A recombinant Clostridium difficile expression system was used to produce genetically engineered toxoids A and B as immunogens for a prophylactic vaccine against C. difficile-associated disease. Although all known enzymatic activities responsible for cytotoxicity were genetically abrogated, the toxoids exhibited residual cytotoxic activity as measured in an in vitro cell-based cytotoxicity assay. The residual cytotoxicity was eliminated by treating the toxoids with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide. Mass spectrometry and amino acid analysis of the EDC-inactivated toxoids identified crosslinks, glycine adducts, and β-alanine adducts. Surface plasmon resonance analysis demonstrated that modifications resulting from the chemical treatment did not appreciably affect recognition of epitopes by both toxin A- and B-specific neutralizing monoclonal antibodies. Compared to formaldehyde-inactivated toxoids, the EDC/N-hydroxysuccinimide-inactivated toxoids exhibited superior stability in solution with respect to reversion of cytotoxic activity.
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Eugene
authors.initials: E
authors.lastname: Vidunas
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Antony
authors.initials: A
authors.lastname: Mathews
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Michele
authors.initials: M
authors.lastname: Weaver
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Ping
authors.initials: P
authors.lastname: Cai
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Eun Hee
authors.initials: EH
authors.lastname: Koh
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Sujata
authors.initials: S
authors.lastname: Patel-Brown
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Hailey
authors.initials: H
authors.lastname: Yuan
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Zi-Rong
authors.initials: ZR
authors.lastname: Zheng
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Marjolaine
authors.initials: M
authors.lastname: Carriere
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: J Erik
authors.initials: JE
authors.lastname: Johnson
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965.
authors.forename: Jason
authors.initials: J
authors.lastname: Lotvin
authors.affiliation: Pfizer Vaccine Research and Development, Pearl River, New York 10965. Electronic address: justin.moran@pfizer.com.
authors.forename: Justin
authors.initials: J
authors.lastname: Moran
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry; D001428:Bacterial Vaccines; D002470:Cell Survival / Q000187:drug effects; D016360:Clostridioides difficile / Q000737:chemistry* / Q000235:genetics*; D004355:Drug Stability; D004768:Enterotoxins / Q000737:chemistry; D000939:Epitopes; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D007114:Immunization; D008647:Mesocricetus; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins; D013388:Succinimides / Q000737:chemistry; D020349:Surface Plasmon Resonance; D014121:Toxoids / Q000737:chemistry* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D004768:Enterotoxins; D000939:Epitopes; D011994:Recombinant Proteins; D013388:Succinimides; D014121:Toxoids; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; C001426:N-hydroxysuccinimide; D005022:Ethyldimethylaminopropyl Carbodiimide
keywords: LC-MS; biopharmaceuticals characterization; chromatography; drug design; high-performance/pressure liquid chromatography; immune response; physical stability; processing; proteins; vaccines