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Please use this identifier to cite or link to this item: http://repository.li.mahidol.ac.th/dspace/handle/123456789/40662
Title: Structural characterization of humanized nanobodies with neutralizing activity against the bordetella pertussis CyaA-hemolysin: Implications for a potential epitope of toxin-protective antigen
Authors: Aijaz Ahmad Malik
Chompounoot Imtong
Nitat Sookrung
Gerd Katzenmeier
Wanpen Chaicumpa
Chanan Angsuthanasombat
Mahidol University
Biophysics Institute for Research and Development (BIRD)
Keywords: Environmental Science
Issue Date: 1-Apr-2016
Citation: Toxins. Vol.8, No.4 (2016)
Abstract: © 2016 by the authors; licensee MDPI, Basel, Switzerland. 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.
URI: https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84976328152&origin=inward
http://repository.li.mahidol.ac.th/dspace/handle/123456789/40662
ISSN: 20726651
Appears in Collections:Scopus 2016-2017

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