Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2484206 | Journal of Pharmaceutical Sciences | 2016 | 10 Pages |
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.
Keywords
Related Topics
Health Sciences
Pharmacology, Toxicology and Pharmaceutical Science
Drug Discovery
Authors
Eugene Vidunas, Antony Mathews, Michele Weaver, Ping Cai, Eun Hee Koh, Sujata Patel-Brown, Hailey Yuan, Zi-rong Zheng, Marjolaine Carriere, J. Erik Johnson, Jason Lotvin, Justin Moran,