Open AccessNeutralization of Clostridium difficile toxin B with VHH-Fc fusions targeting the delivery and CROPs domains
Author(s) -
Greg Hussack,
Shan Ryan,
Henk van Faassen,
Martín A. Rossotti,
Colin R. MacKenzie,
Jamshid Tanha
Publication year - 2018
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0208978
Subject(s) - toxin , neutralization , single domain antibody , clostridium difficile toxin a , immunogen , clostridium difficile toxin b , epitope , monoclonal antibody , clostridium difficile , antibody , oligopeptide , epitope mapping , microbiology and biotechnology , biology , chemistry , virology , peptide , biochemistry , immunology , antibiotics
An increasing number of antibody-based therapies are being considered for controlling bacterial infections, including Clostridium difficile by targeting toxins A and B. In an effort to develop novel C . difficile immunotherapeutics, we previously isolated several single-domain antibodies (V H Hs) capable of toxin A neutralization through recognition of the extreme C-terminal combined repetitive oligopeptides (CROPs) domain, but failed at identifying neutralizing V H Hs that bound a similar region on toxin B. Here we report the isolation of a panel of 29 V H Hs targeting at least seven unique epitopes on a toxin B immunogen composed of a portion of the central delivery domain and the entire CROPs domain. Despite monovalent affinities as high as K D = 70 pM, none of the V H Hs tested were capable of toxin B neutralization; however, modest toxin B inhibition was observed with V H H-V H H dimers and to a much greater extent with V H H-Fc fusions, reaching the neutralizing potency of the recently approved anti-toxin B monoclonal antibody bezlotoxumab in in vitro assays. Epitope binning revealed that several V H H-Fcs bound toxin B at sites distinct from the region recognized by bezlotoxumab, while other V H H-Fcs partially competed with bezlotoxumab for toxin binding. Therefore, the V H Hs described here are effective at toxin B neutralization when formatted as bivalent V H H-Fc fusions by targeting toxin B at regions both similar and distinct from the bezlotoxumab binding site.