The high-resolution cryo-EM of small antigen–antibody complexes remains a major challenge due to low molecular weight, conformational flexibility, and particle heterogeneity. To address this, we engineered a bivalent Fab from the broadly neutralizing antibody HC84.26.5D, which targets the hepatitis C virus (HCV) E2 glycoprotein. By deleting a single residue (VHSer113) in the Fab elbow region, we induced stable dimerization (~80% in solution) and confirmed dimer formation by solving its crystal structure at 1.8 Å resolution. The resulting Fab dimer adopts a domain-swapped conformation with ~25 Å separation between the VL/VH domains, forming a doughnut-shaped molecule with a rectangular central hole. This architecture enables the simultaneous binding of two E2 molecules and improves symmetry, effective mass, and particle alignment—key parameters for high-resolution cryo-EM reconstruction. Using this fiducial marker approach, we solved cryo-EM structures of E2 bound to three monoclonal antibodies with distinct neutralization profiles, a broadly neutralizing, moderately neutralizing, and non-neutralizing antibody, at resolutions of 3.8 Å, 3.3 Å, and 3.7 Å, respectively. Epitope mapping revealed that the broadly neutralizing antibody targets the conserved front layer of E2, while the non-neutralizing antibody binds to the back layer. Notably, the moderately neutralizing antibody spans both regions, defining a previously unrecognized cross-layer epitope associated with partial neutralization. This work establishes a generalizable strategy for domain-swapped Fab design that preserves antigen-binding integrity while enabling the structural analysis of small or flexible antigen–antibody assemblies by cryo-EM. In addition, it provides mechanistic insights into how epitope location influences neutralization potency, offering a molecular blueprint for rational HCV vaccine design focused on conserved, functionally critical epitopes of E2.
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Cryo-EM Structures of HCV E2 Bound to Neutralizing and Non-Neutralizing Antibodies Using Engineered Domain-Swapped Bivalent Fabs as Fiducial Markers
Published:
07 October 2025
by MDPI
in The 1st International Online Conference by Antibodies
session Antibody-based Therapeutics
Abstract:
Keywords: Antibody engineering, Crystallography, CryoEM, HCV, epitope mapping, Vaccine
