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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/V H H 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 V H H-hallmark amino acids as V H H2, VH5, VH18 and V H H37.  In vitro  neutralization assay revealed that all four ~17-kDa His-tagged VH/V H H nanobodies, in particular V H H37, 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 V H H37 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.

toxins

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