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Production and Characterization of Chemically Inactivated Genetically Engineered Clostridium difficile Toxoids

2016; Elsevier BV; Volume: 105; Issue: 7 Linguagem: Inglês

10.1016/j.xphs.2016.04.017

1520-6017

Eugene Vidunas, Antony J. Mathews, Michele Weaver, Ping Cai, Eun Hee Koh, Sujata Patel-Brown, Hailey Yuan, Zi-Rong Zheng, M Carrière, J. Erik Johnson, Jason Lotvin, Justin Moran,

Toxin Mechanisms and Immunotoxins

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.