title: Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies.
abstract: Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA(-) TcdB(+) strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Kasper Krogh
authors.initials: KK
authors.lastname: Andersen
authors.affiliation: Cellular Architecture and Dynamics, Department of Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Nika M
authors.initials: NM
authors.lastname: Strokappe
authors.affiliation: Cellular Architecture and Dynamics, Department of Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Anna
authors.initials: A
authors.lastname: Hultberg
authors.affiliation: Department of Microbiology, University of Tartu, Tartu, Estonia.
authors.forename: Kai
authors.initials: K
authors.lastname: Truusalu
authors.affiliation: Department of Microbiology, University of Tartu, Tartu, Estonia.
authors.forename: Imbi
authors.initials: I
authors.lastname: Smidt
authors.affiliation: Department of Microbiology, University of Tartu, Tartu, Estonia.
authors.forename: Raik-Hiio
authors.initials: RH
authors.lastname: Mikelsaar
authors.affiliation: Department of Microbiology, University of Tartu, Tartu, Estonia.
authors.forename: Marika
authors.initials: M
authors.lastname: Mikelsaar
authors.affiliation: Cellular Architecture and Dynamics, Department of Biology, Utrecht University, Utrecht, The Netherlands.
authors.forename: Theo
authors.initials: T
authors.lastname: Verrips
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Lennart
authors.initials: L
authors.lastname: Hammarström
authors.affiliation: Division of Clinical Immunology and Transfusion Medicine, Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden Harold.Marcotte@ki.se.
authors.forename: Harold
authors.initials: H
authors.lastname: Marcotte
mesh_terms: D000284:Administration, Oral; D000818:Animals; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000276:immunology*; D000992:Antitoxins / Q000008:administration & dosage / Q000276:immunology*; D001426:Bacterial Proteins / Q000276:immunology*; D001427:Bacterial Toxins / Q000276:immunology*; D002161:Camelids, New World; D016360:Clostridioides difficile / Q000276:immunology* / Q000472:pathogenicity; D006224:Cricetinae; D004195:Disease Models, Animal; D004761:Enterocolitis, Pseudomembranous / Q000382:microbiology / Q000517:prevention & control*; D004926:Escherichia coli / Q000235:genetics; D041981:Gastrointestinal Tract / Q000276:immunology / Q000382:microbiology; D007114:Immunization; D007116:Immunization, Passive; D007143:Immunoglobulin Heavy Chains / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D007778:Lactobacillus / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D061905:Single-Domain Antibodies / Q000235:genetics / Q000276:immunology*
chemical_list: D057134:Antibodies, Neutralizing; D000992:Antitoxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D007143:Immunoglobulin Heavy Chains; D011994:Recombinant Proteins; D061905:Single-Domain Antibodies; C031815:toxB protein, Clostridium difficile