caa a22 4500 605499667 CHVBK 20210128100552.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6594-1 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6594-1 Targeting surface-layer proteins with single-domain antibodies: a potential therapeutic approach against Clostridium difficile -associated disease [Elektronische Daten] [Hiba Kandalaft, Greg Hussack, Annie Aubry, Henk van Faassen, Yonghong Guan, Mehdi Arbabi-Ghahroudi, Roger MacKenzie, Susan Logan, Jamshid Tanha] 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 = 3-6nM) 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. The Author(s), 2015 Clostridium difficile nationallicence Surface layer protein nationallicence Single-domain antibody nationallicence VHH nationallicence Nanobody nationallicence Kandalaft Hiba Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut Hussack Greg Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut Aubry Annie Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut van Faassen Henk Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut Guan Yonghong Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut Arbabi-Ghahroudi Mehdi Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut MacKenzie Roger Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut Logan Susan Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut Tanha Jamshid Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8549-8562 0175-7598 99:20<8549 2015 99 253 https://doi.org/10.1007/s00253-015-6594-1 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6594-1 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kandalaft Hiba Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- Hussack Greg Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- Aubry Annie Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- van Faassen Henk Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- Guan Yonghong Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- Arbabi-Ghahroudi Mehdi Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- MacKenzie Roger Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- Logan Susan Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 700 1- Tanha Jamshid Human Health Therapeutics Portfolio, National Research Council Canada, K1A 0R6, Ottawa, ON, Canada aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8549-8562 0175-7598 99:20<8549 2015 99 253