Preconfiguration of the antigen-binding site during affinity maturation of a broadly neutralizing influenza virus antibody

Abstract

Affinity maturation refines a naive B-cell response by selecting mutations in antibody variable domains that enhance antigen binding. We describe a B-cell lineage expressing broadly neutralizing influenza virus antibodies derived from a subject immunized with the 2007 trivalent vaccine. The lineage comprises three mature antibodies, the unmutated common ancestor, and a common intermediate. Their heavy-chain complementarity determining region inserts into the conserved receptor-binding pocket of influenza HA. We show by analysis of structures, binding kinetics and long time-scale molecular dynamics simulations that antibody evolution in this lineage has rigidified the initially flexible heavy-chain complementarity determining region by two nearly independent pathways and that this preconfiguration accounts for most of the affinity gain. The results advance our understanding of strategies for developing more broadly effective influenza vaccines.

Fig. 1.

Inferred CH65–CH67 lineage and sequences. (A) Members of the lineage include the UCA, I-2, and three mature B-cell clones, CH65, CH66, and CH67. (B) Sequence alignment of the heavy- and light-chain variable domains. CDR1, CDR2, and CDR3 loops for each chain are marked. Conservation within the lineage based on the UCA sequence is shown by a period.

Fig. 2.

Crystal structures of free and bound Fabs in the CH65–CH67 lineage. (A) CH65 and CH67 Fabs bound with HA. CH67 Fab (heavy and light chains in green and pale green, respectively), CH65 Fab (heavy and light chains in blue and light blue, respectively, from Protein Data Bank ID code 3SM5), and HA head (silver). The CDR H3 of the Fabs and the 190-helix of HA are marked for reference. (B) Comparison of free and bound Fabs. Alternative conformations of the CDR H3 loop (residues 96–117 are displayed) from the UCA (yellow) and I-2 (silver; constrained by a crystal contact), free CH67 (magenta), bound CH65 (blue), and bound CH67 (green) are displayed. All images created in PyMol.

Fig. 3.

Long time-scale MD simulations. (A) Time required for the CH65 Fab to bind spontaneously to HA starting from various initial separations. Each of these simulations was initiated with the CH65 Fab placed at a different distance, d₀, from its position within the CH65–HA complex. Details of these simulations are described in SI Materials and Methods. Each simulation was 0.5 μs in length, except for those simulations with d₀ > 7.5 Å, which were 5 μs long. To determine whether the Fab had bound to HA, we aligned HA to the CH65–HA crystal structure, computed the Fab backbone rmsd with respect to its position in the complex, and considered the Fab to bind when the computed rmsd first decreased below 1.5 Å. (In all simulations in which the computed rmsd decreased below 1.5 Å, the Fab–HA binding interface remained stable throughout the remainder of the simulation.) Red x indicates a simulation in which the Fab never bound. (B) Fab rmsd from the crystal structure in the simulation of the CH65–HA complex and one simulation of CH65 binding to HA. The blue curve indicates rmsd fluctuations for CH65 in complex with HA. The red curve shows the rmsd for CH65 as it spontaneously binds to HA in the simulation that corresponds to the one indicated by the orange square in A; rmsd is computed for all backbone atoms in the Fab by first aligning HA in the simulation to HA in the crystal structure (Protein Data Bank ID code 3SM5). (C) Superposition onto the CH65–HA crystal structure of the last frame from one of the simulations of UCA binding to HA. CH65 is shown in light blue, UCA is in yellow, and HA is in the surface representation. D107 of CDR H3 forms polar interactions with HA, which is indicated by the cyan patch on the HA surface. V106 and Y109 of CDR H3 make hydrophobic contacts with HA (highlighted green). Images were created in OpenStructure (37). (D) The Fab rmsd from the crystal structure of the CH65–HA complex in the simulation of the UCA–HA complex (blue) and one simulation of the UCA binding to HA (red); rmsd was computed as in B.

Fig. 4.

Conformational dynamics of CDR H3 in the simulations of free antibodies. (A) Time trace of the probability that the conformation is observed in the corresponding simulation of the complex. High values indicate that the CDR H3 loop is conformation ready for binding to HA; low values indicate otherwise. Different colors represent different independent simulations. (B) Probability, in the free Fab simulations, that the CDR H3 loop assumes bound and other conformations.