1. Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids
- Author
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Vidunas Eugene Joseph, Ping Cai, Sujata Kaushikbhai Patel-Brown, Marjolaine Carriere, Zi-rong Zheng, Eun Hee Koh, Hailey Yuan, Michele Weaver, Jason Arnold Lotvin, Antony Mathews, Justin Keith Moran, and J. Erik Johnson
- Subjects
0301 basic medicine ,Cell Survival ,medicine.drug_class ,Bacterial Toxins ,030106 microbiology ,Succinimides ,Pharmaceutical Science ,Protein Engineering ,medicine.disease_cause ,Monoclonal antibody ,complex mixtures ,Epitope ,law.invention ,Microbiology ,Enterotoxins ,Epitopes ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Drug Stability ,Ethyldimethylaminopropyl Carbodiimide ,law ,medicine ,Animals ,Cytotoxicity ,Carbodiimide ,chemistry.chemical_classification ,Mesocricetus ,Clostridioides difficile ,Toxin ,Surface Plasmon Resonance ,Toxoids ,Recombinant Proteins ,030104 developmental biology ,Enzyme ,Biochemistry ,chemistry ,Bacterial Vaccines ,Glycine ,Recombinant DNA ,Immunization - 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.
- Published
- 2016
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