title: Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain.
abstract: Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (V(H)Hs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four V(H)Hs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when V(H)Hs were administered in paired or triplet combinations at the same overall V(H)H concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of V(H)Hs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific V(H)Hs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these V(H)Hs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract.
authors.affiliation: Institute for Biological Sciences, National Research Council Canada, Ottawa, Ontario K1A 0R6, Canada.
authors.forename: Greg
authors.initials: G
authors.lastname: Hussack
authors.forename: Mehdi
authors.initials: M
authors.lastname: Arbabi-Ghahroudi
authors.forename: Henk
authors.initials: H
authors.lastname: van Faassen
authors.forename: J Glenn
authors.initials: JG
authors.lastname: Songer
authors.forename: Kenneth K-S
authors.initials: KK
authors.lastname: Ng
authors.forename: Roger
authors.initials: R
authors.lastname: MacKenzie
authors.forename: Jamshid
authors.initials: J
authors.lastname: Tanha
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000235:genetics / Q000276:immunology*; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000276:immunology*; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology*; D002161:Camelids, New World; D016360:Clostridioides difficile / Q000235:genetics / Q000276:immunology*; D004761:Enterocolitis, Pseudomembranous / Q000276:immunology / Q000628:therapy; D004768:Enterotoxins / Q000235:genetics / Q000276:immunology*; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology*; D008297:Male; D017434:Protein Structure, Tertiary; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D000907:Antibodies, Bacterial; D057134:Antibodies, Neutralizing; D001427:Bacterial Toxins; D004768:Enterotoxins; D000939:Epitopes; D007143:Immunoglobulin Heavy Chains; D057127:Single-Chain Antibodies; C041348:tcdA protein, Clostridium difficile