The exotoxins toxin A (TcdA) and toxin B (TcdB) are produced by the bacterial 
pathogen Clostridium difficile and are responsible for the pathology associated 
with C. difficile infection (CDI). The antitoxin antibodies actoxumab and 
bezlotoxumab bind to and neutralize TcdA and TcdB, respectively. Bezlotoxumab 
was recently approved by the FDA for reducing the recurrence of CDI. We have 
previously shown that a single molecule of bezlotoxumab binds to two distinct 
epitopes within the TcdB combined repetitive oligopeptide (CROP) domain, 
preventing toxin binding to host cells. In this study, we characterize the 
binding of actoxumab to TcdA and examine its mechanism of toxin neutralization. 
Using a combination of approaches including a number of biophysical techniques, 
we show that there are two distinct actoxumab binding sites within the CROP 
domain of TcdA centered on identical amino acid sequences at residues 2162-2189 
and 2410-2437. Actoxumab binding caused the aggregation of TcdA especially at 
higher antibody:toxin concentration ratios. Actoxumab prevented the association 
of TcdA with target cells demonstrating that actoxumab neutralizes toxin 
activity by inhibiting the first step of the intoxication cascade. This 
mechanism of neutralization is similar to that observed with bezlotoxumab and 
TcdB. Comparisons of the putative TcdA epitope sequences across several C. 
difficile ribotypes and homologous repeat sequences within TcdA suggest a 
structural basis for observed differences in actoxumab binding and/or 
neutralization potency. These data provide a mechanistic basis for the 
protective effects of the antibody in vitro and in vivo, including in various 
preclinical models of CDI.