Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography

By P. Orth, Li Xiao, L. Hernandez, P. Reichert, P. Sheth, M. Beaumont, Xiaoyu Yang, N. Murgolo, G. Ermakov, Edward Dinunzio and 7 more. 2014

124 citations recorded by the source corpus.

Background: Bezlotoxumab is a neutralizing antibody targeting toxin B of Clostridium difficile. Results: The structure of bezlotoxumab bound to a fragment of toxin B reveals its epitopes and mechanism of neutralization. Conclusion: The epitopes overlap with two of the presumed carbohydrate binding pockets, preventing binding of the toxin to target host cells. Significance: The data provide a molecular basis for neutralization by this clinically important antibody. The symptoms of Clostridium difficile infections are caused by two exotoxins, TcdA and TcdB, which target host colonocytes by binding to unknown cell surface receptors, at least in part via their combined repetitive oligopeptide (CROP) domains. A combination of the anti-TcdA antibody actoxumab and the anti-TcdB antibody bezlotoxumab is currently under development for the prevention of recurrent C. difficile infections. We demonstrate here through various biophysical approaches that bezlotoxumab binds to specific regions within the N-terminal half of the TcdB CROP domain. Based on this information, we solved the x-ray structure of the N-terminal half of the TcdB CROP domain bound to Fab fragments of bezlotoxumab. The structure reveals that the TcdB CROP domain adopts a β-solenoid fold consisting of long and short repeats and that bezlotoxumab binds to two homologous sites within the CROP domain, partially occluding two of the four putative carbohydrate binding pockets located in TcdB. We also show that bezlotoxumab neutralizes TcdB by blocking binding of TcdB to mammalian cells. Overall, our data are consistent with a model wherein a single molecule of bezlotoxumab neutralizes TcdB by binding via its two Fab regions to two epitopes within the N-terminal half of the TcdB CROP domain, partially blocking the carbohydrate binding pockets of the toxin and preventing toxin binding to host cells.

Subfields: Immunology, Infectious Diseases

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

1. L. Hernandez
2. F. Racine
3. Edward Dinunzio
4. Li Xiao
5. Nichelle N Hairston
6. P. Sheth
7. N. Murgolo
8. A. Therien

Citations in the corpus

• Broad Coverage of Genetically Diverse Strains of Clostridium difficile by Actoxumab and Bezlotoxumab Predicted by In Vitro Neutralization and Epitope Modeling (2014, methodology): “We showed previously that bezlotoxumab binds to two separate sites in the CROP domain of TcdB, partially overlapping with putative receptor binding pockets and blocking binding of the toxin to host cells (8); actoxumab, which binds to the CROP domain of TcdA (6), is presumed to work in a similar man”