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Toxins 2016, 8(4), 99; doi:10.3390/toxins8040099

Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen

1
Graduate Program in Immunology, Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
2
Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand
3
Department of Parasitology and Center of Excellence on Therapeutic Proteins and Antibody Engineering, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
4
Department of Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand
5
Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand
*
Authors to whom correspondence should be addressed.
Academic Editor: Shin-ichi Miyoshi
Received: 18 February 2016 / Revised: 17 March 2016 / Accepted: 25 March 2016 / Published: 1 April 2016
(This article belongs to the Section Bacterial Toxins)
View Full-Text   |   Download PDF [2458 KB, uploaded 1 April 2016]   |  

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. View Full-Text
Keywords: Bordetella pertussis; CyaA-hemolysin; CyaA-RTX; intermolecular docking; VH/VHH; phage display Bordetella pertussis; CyaA-hemolysin; CyaA-RTX; intermolecular docking; VH/VHH; phage display
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Malik, A.A.; Imtong, C.; Sookrung, N.; Katzenmeier, G.; Chaicumpa, W.; Angsuthanasombat, C. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen. Toxins 2016, 8, 99.

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