Targeting surface-layer proteins with single-domain antibodies: a potential therapeutic approach against Clostridium difficile-associated disease

• Published in: Applied microbiology and biotechnology 2015-10, Vol.99 (20), p.8549-8562
• Main Authors: Kandalaft, Hiba, Hussack, Greg, Aubry, Annie, van Faassen, Henk, Guan, Yonghong, Arbabi-Ghahroudi, Mehdi, MacKenzie, Roger, Logan, Susan M, Tanha, Jamshid
• Format: Article
• Language: English
• Subjects: Analysis; Anti-Bacterial Agents - isolation & purification; Anti-Bacterial Agents - metabolism; Antibiotics; Antibodies; Antibodies, Bacterial - isolation & purification; Antibodies, Bacterial - metabolism; Bacteria; Bacterial Proteins - antagonists & inhibitors; bacteriophages; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Care and treatment; Clostridium; Clostridium difficile; Clostridium difficile - drug effects; Clostridium difficile - physiology; Clostridium infections; Complications and side effects; death; Disease management; Drug resistance; Enzyme-linked immunosorbent assay; Epidemics; epitopes; Epitopes - metabolism; gastrointestinal system; hamsters; Health aspects; Immunization; Life Sciences; Locomotion - drug effects; melting; Membrane Glycoproteins - antagonists & inhibitors; Microbial Genetics and Genomics; Microbiology; Monoclonal antibodies; Motility; Neutralization; North America; Nosocomial infections; pepsin; Peptide Library; Physiological aspects; Protein Binding; Proteins; relapse; ribotypes; Single-Domain Antibodies - isolation & purification; Single-Domain Antibodies - metabolism; Studies; temperature; Toxins; Virulence
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-015-6594-1

Clostridium difficile is a leading cause of death from gastrointestinal infections in North America. Antibiotic therapy is effective, but the high incidence of relapse and the rise in hypervirulent strains warrant the search for novel treatments. Surface layer proteins (SLPs) cover the entire C. difficile bacterial surface, are composed of high-molecular-weight (HMW) and low-molecular-weight (LMW) subunits, and mediate adherence to host cells. Passive and active immunization against SLPs has enhanced hamster survival, suggesting that antibody-mediated neutralization may be an effective therapeutic strategy. Here, we isolated a panel of SLP-specific single-domain antibodies (VHHs) using an immune llama phage display library and SLPs isolated from C. difficile hypervirulent strain QCD-32g58 (027 ribotype) as a target antigen. Binding studies revealed a number of VHHs that bound QCD-32g58 SLPs with high affinity (K D = 3–6 nM) and targeted epitopes located on the LMW subunit of the SLP. The VHHs demonstrated melting temperatures as high as 75 °C, and a few were resistant to the gastrointestinal protease pepsin at physiologically relevant concentrations. In addition, we demonstrated the binding specificity of the VHHs to the major C. difficile ribotypes by whole cell ELISA, where all VHHs were found to bind 001 and 027 ribotypes, and a subset of antibodies were found to be broadly cross-reactive in binding cells representative of 012, 017, 023, and 078 ribotypes. Finally, we showed that several of the VHHs inhibited C. difficile QCD-32g58 motility in vitro. Targeting SLPs with VHHs may be a viable therapeutic approach against C. difficile-associated disease.