title: Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.
abstract: Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity.
authors.affiliation: Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. ajaz_me@hotmail.com.|Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. ajaz_me@hotmail.com.|Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. ajaz_me@hotmail.com.
authors.forename: Aijaz Ahmad
authors.initials: AA
authors.lastname: Malik
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. chompou_ron@hotmail.com.
authors.forename: Chompounoot
authors.initials: C
authors.lastname: Imtong
authors.affiliation: Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. nitat.soo@mahidol.ac.th.
authors.forename: Nitat
authors.initials: N
authors.lastname: Sookrung
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. katzenmeier.ger@mahidol.ac.th.
authors.forename: Gerd
authors.initials: G
authors.lastname: Katzenmeier
authors.affiliation: Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand. wanpen.cha@mahidol.ac.th.
authors.forename: Wanpen
authors.initials: W
authors.lastname: Chaicumpa
authors.affiliation: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand. chanan.ang@mahidol.ac.th.|Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand. chanan.ang@mahidol.ac.th.
authors.forename: Chanan
authors.initials: C
authors.lastname: Angsuthanasombat
mesh_terms: D037361:Adenylate Cyclase Toxin / Q000235:genetics / Q000378:metabolism*; D057134:Antibodies, Neutralizing / Q000235:genetics / Q000378:metabolism*; D000942:Antigens, Bacterial / Q000235:genetics / Q000378:metabolism*; D001886:Bordetella pertussis; D000939:Epitopes / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D061905:Single-Domain Antibodies / Q000235:genetics / Q000378:metabolism*
chemical_list: D037361:Adenylate Cyclase Toxin; D057134:Antibodies, Neutralizing; D000942:Antigens, Bacterial; D000939:Epitopes; D006460:Hemolysin Proteins; D061905:Single-Domain Antibodies
keywords: Bordetella pertussis; CyaA-RTX; CyaA-hemolysin; VH/VHH; intermolecular docking; phage display