A Novel Multivalent, Single-Domain Antibody Targeting TcdA and TcdB Prevents Fulminant Clostridium difficile Infection in Mice

Abstract

The incidence of Clostridium difficile infection (CDI) and associated mortality have increased rapidly worldwide in recent years. Therefore, it is critical to develop new therapies for CDI. In this study, we generated a novel, potently neutralizing, tetravalent, and bispecific antibody composed of 2 heavy-chain-only VH (VHH) binding domains against both TcdA and TcdB (designated "ABA") that reverses fulminant CDI in mice infected with an epidemic 027 strain after a single injection of the antibody. We demonstrated that ABA bound to both toxins simultaneously and displayed a significantly enhanced neutralizing activity both in vitro and in vivo. Additionally, ABA was able to broadly neutralize toxins from clinical C. difficile isolates that express both TcdA and TcdB but failed to neutralize the toxin from TcdA(-)TcdB(+) C. difficile strains. This study thus provides a rationale for the development of multivalent VHHs that target both toxins and are broadly neutralizing for treating severe CDI.

An Optimized, Synthetic DNA Vaccine Encoding the Toxin A and Toxin B Receptor Binding Domains of Clostridium difficile Induces Protective Antibody ResponsesIn Vivo

“…In both mouse and hamster infection models of CDI, preventing infection-associated mortality is seen as an important metric of vaccine efficacy (12,13,22,23,(58)(59)(60)(61)(62)(63)(64)(65)(66). Upon challenging pRBD-NQ-immunized animals with a lethal dose of spores, we observed 90% and 50% protection against homologous and heterologous strains, respectively.…”

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

“…Heterodimeric VNAs Protect against Anthrax Toxin and Spore Infection in Mice-Linking toxin-neutralizing VHHs into heteromultimeric VNAs has been found to improve toxin affinity and, more importantly, to substantially improve in vivo antitoxin efficacy (11)(12)(13)16). A heterodimeric VNA (VNA2-PA) consisting of the two potent neutralizing VHHs, JIK-B8 and JKH-C7, separated by a short unstructured peptide, was expressed and purified (amino acid sequence shown in Fig.…”

“…32 and 6). We have found that VHH heteromultimer VNAs possess additional potential therapeutic advantages over conventional mAb products such as 1) the option to co-administer an anti-tag effector antibody that binds multiple tags on the VNAs (11,13) to promote antibody Fc effector functions such as serum clearance (33), 2) the ability to target multiple toxins with one biomolecule (12,16), and 3) the use of VNA gene therapy as an effective means to provide prolonged antitoxin protection (34,35). Simple addition of an albumin binding peptide (34,36) can dramatically improve the serum stability of VNAs (34).…”

“…VHHs often target active sites that may be inaccessible to larger conventional antibodies (7,8) and have proven to be effective as toxin neutralizing agents (9 -16). We have found that linking two or more neutralizing VHHs recognizing non-overlapping epitopes into heteromultimers (VHH-based neutralizing agents (VNAs)) often provides major improvements in the in vivo protection from toxin exposure as compared with the unlinked component VHHs (11)(12)(13)16).…”

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

“…V H Hs have been reported in the treatment of toxin-mediated disease (48,49). V H Hs against the two C. difficile toxins have also been generated which possessed toxin-neutralizing activity (42,50) and potent therapeutic efficacy against fulminant C. difficile infection (51).…”