Detoxification of toxin A and toxin B by copper ion-catalyzed oxidation in production of a toxoid-based vaccine against Clostridioides difficile

Clostridioides difficile infections (CDI) has emerged worldwide as a serious antimicrobial-resistant healthcare-associated disease resulting in diarrhea and pseudomembranous colitis. The two cytotoxic proteins, toxin A (TcdA) and toxin B (TcdB) are the major virulence factor responsible for the dise...

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Published in:	Free radical biology & medicine Vol. 160; pp. 433 - 446
Main Authors:	Aminzadeh, Aria, Tiwari, Manish Kumar, Mamah Mustapha, Srwa Satar, Navarrete, Sandra Junquera, Henriksen, Anna Bielecka, Møller, Ian Max, Krogfelt, Karen Angeliki, Bjerrum, Morten Jannik, Jørgensen, René
Format:	Journal Article
Language:	English
Published:	United States Elsevier Inc 20-11-2020
Subjects:	CDI vaccine Clostridioides difficile Metal-catalyzed oxidation Reactive oxygen species Toxoid CDI vaccine Clostridioides difficile Metal-catalyzed oxidation Reactive oxygen species Toxoid
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Summary:	Clostridioides difficile infections (CDI) has emerged worldwide as a serious antimicrobial-resistant healthcare-associated disease resulting in diarrhea and pseudomembranous colitis. The two cytotoxic proteins, toxin A (TcdA) and toxin B (TcdB) are the major virulence factor responsible for the disease symptoms. We examined time-dependent oxidative detoxification of TcdA and TcdB using different molar ratios of protein:Cu2+:H2O2. The metal-catalyzed oxidation (MCO) reaction in molar ratios of 1:60:1000 for protein:Cu2+:H2O2 at pH 4.5 resulted in a significant 6 log10 fold reduction in cytotoxicity after 120-min incubation at 37 °C. Circular dichroism revealed that MCO-detoxified TcdA and TcdB had secondary and tertiary structural folds similar to the native proteins. The conservation of immunogenic epitopes of both proteins was tested using monoclonal antibodies in an ELISA, comparing our MCO-detoxification approach to a conventional formaldehyde-detoxification method. The oxidative detoxification of TcdA and TcdB led to an average 2-fold reduction in antibody binding relative to native proteins, whereas formaldehyde cross-linking resulted in 3-fold and 5-fold reductions, respectively. Finally, we show that mice immunized with a vaccine consisting of MCO-detoxified TcdA and TcdB were fully protected against disease symptoms and death following a C. difficile infection and elicited substantial serum IgG responses against both TcdA and TcdB. The results of this study present copper ion-catalyzed oxidative detoxification of toxic proteins as a method highly suitable for the rapid production of safe, immunogenic and irreversible toxoid antigens for future vaccine development and may have the potential for replacing cross-linking reagents like formaldehyde. [Display omitted] •Clostridioides difficile infection is a serious healthcare-associated disease.•Studies show that a toxoid-based vaccine can protect against C. difficile infection.•Copper ion-catalyzed oxidation as new method for producing immunogenic toxoids.•Detoxification of TcdA and TcdB using copper ion-catalyzed oxidation.•Detoxification by oxidation good alternative to conventional cross-linking reagents.
ISSN:	0891-5849 1873-4596
DOI:	10.1016/j.freeradbiomed.2020.08.021