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.

Discrete analysis of camelid variable domains: sequences, structures, and in-silico structure prediction

“…The popular i-TASSER web server was used for V H H designed against PrA (ProteinA) (Fridy et al, 2015) and HCV Non-structural protein NS3/4A (Jittavisutthikul et al, 2015). Raptor-X, another threading-based server, was used to model V H H against adenylate cyclase-hemolysin toxin and the repeats in toxin (CyaA-RTX protein) (Malik et al, 2016) subdomains (see Supplemental Information 1).…”

Immunogenicity and humanization of single‐domain antibodies

“…The degree of FR humanization of library members was variable but was significantly higher for V H s than V H Hs. Surprisingly, despite its unusually small size (1.6×10 5 [145], and inhibited the catalytic activity of the EGFR tyrosine kinase domain [146]. In none of these studies were important properties of the humanized V H /V H Hs including expression, solubility, stability, and affinity assessed.…”

“…The library yielded several humanized V H Hs specific for the light chain of type A botulinum toxin that efficiently neutralized the toxin, one of which had a K D of ~ 12 n m . In subsequent screenings, the library yielded antigen‐specific V H /V H Hs that neutralized monocled cobra venom phospholipase A2 [142], inhibited hepatitis C virus (HCV) RNA‐dependent RNA polymerase [143], inhibited the helicase activity of a C‐terminal NS3 protein [144], inhibited HCV replication by binding to a HCV serine protease [145], neutralized the hemolytic activity of Bordetella pertussis CyaA hemolysin toxin [146], and inhibited the catalytic activity of the EGFR tyrosine kinase domain [147]. In none of these studies were important properties of the humanized V H /V H Hs including expression, solubility, stability, and affinity assessed.…”

VHH Structural Modelling Approaches: A Critical Review

“…Through the screening of a VH/V H H phage display library, two VHs and two V H Hs clones were identified after several optimisation rounds [ 146 , 189 , 190 , 191 ]. Three-dimensional structural models of these proteins were built using RaptorX [ 192 ]. The loop conformations were obtained using the dedicated FALC loop modelling web server [ 193 ].…”