A single amino acid substitution determines susceptibility of Clostridioides difficile toxin B variants to monoclonal antibody neutralization.

Monoclonal antibodies have become an integral component of modern therapeutics, however, their efficacy can be diminished by variation in target proteins, emphasizing the need to define these changes to guide the development of effective therapies. In this brief report, we uncover the molecular mechanism underlying a key limitation of bezlotoxumab, a monoclonal antibody targeting Clostridioides difficile toxin B (TcdB), specifically its reduced neutralizing activity against certain TcdB variants. We show that four major toxin B variants, B1-B4 (TcdB1-TcdB4), account for approximately 99.9% of circulating C. difficile strains globally. Bezlotoxumab neutralized TcdB1 and TcdB3 mediated cell death in human colonic epithelial cells, but not TcdB2 or TcdB4. Sequence analysis revealed that TcdB1 and TcdB3 share a conserved residue within a key antibody-neutralizing region, containing glutamic acid at position 2033 (E2033), whereas TcdB2 and TcdB4 contain an alanine at this position (A2033). Substitution of A2033 with E2033 in TcdB2 restored susceptibility to antibody-mediated neutralization. These findings help define the molecular basis of reduced susceptibility among toxin B variants and may guide the design of next-generation vaccines and antibody-based therapeutics against C. difficile.