TY - JOUR
T1 - Detoxification of toxin A and toxin B by copper ion-catalyzed oxidation in production of a toxoid-based vaccine against Clostridioides difficile
AU - Aminzadeh, Aria
AU - Tiwari, Manish Kumar
AU - Mustapha, Srwa Satar Mamah
AU - Navarrete, Sandra Junquera
AU - Henriksen, Anna Bielecka
AU - Møller, Ian Max
AU - Krogfelt, Karen Angeliki
AU - Bjerrum, Morten Jannik
AU - Jørgensen, René
PY - 2020/11/20
Y1 - 2020/11/20
N2 - 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.
AB - 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.
KW - CDI vaccine
KW - Clostridioides difficile
KW - Metal-catalyzed oxidation
KW - Reactive oxygen species
KW - Toxoid
U2 - 10.1016/j.freeradbiomed.2020.08.021
DO - 10.1016/j.freeradbiomed.2020.08.021
M3 - Journal article
SN - 0891-5849
VL - 160
SP - 433
EP - 446
JO - Free Radical Biology & Medicine
JF - Free Radical Biology & Medicine
IS - 160
ER -