Clostridium difficile infections (CDIs) are the leading cause of 
hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA 
and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that 
neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a 
phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate 
of CDI recurrence in patients who were also treated with standard-of-care 
antibiotics. However, it is not known whether the antibody combination will be 
effective against a broad range of C. difficile strains. As a first step toward 
addressing this, we tested the ability of actoxumab and bezlotoxumab to 
neutralize the activities of toxins from a number of clinically relevant and 
geographically diverse strains of C. difficile. Neutralization potencies, as 
measured in a cell growth/survival assay with purified toxins from various C. 
difficile strains, correlated well with antibody/toxin binding affinities. 
Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all 
clinical isolates tested, including multiple isolates of the BI/NAP1/027 and 
BK/NAP7/078 strains, at antibody concentrations well below plasma levels 
observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor 
binding domain across known TcdB sequences and found that key substitutions 
within the bezlotoxumab epitopes correlated with the relative differences in 
potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 
and 078. Combined with in vitro neutralization data, epitope modeling will 
enhance our ability to predict the coverage of new and emerging strains by 
actoxumab-bezlotoxumab in the clinic.