Monoclonal antibodies specific for discontinuous epitopes direct refolding of influenza A virus hemagglutinin

Abstract

Antibodies (Abs) specific for the globular domain of influenza A virus hemagglutinin (HA) efficiently neutralize viral infectivity and provide the most effective protection against influenza following infection or vaccination. Nearly all neutralizing Abs recognize discontinuous determinants formed by residues present on different stretches of the HA primary structure. Here, I show that approximately 25% of a large panel of neutralizing monoclonal Abs (mAbs), including Abs specific for each of the four major antigenic sites, can bind to completely denatured HA. Binding of these mAbs to denatured HA occurs much more slowly than binding to native HA, but bound mAbs dissociate from denatured and native HA with similar kinetics, and amino acid substitutions that reduce mAb binding to native HA have a similar effect on mAb interaction with denatured HA. HA refolding induced by mAb binding facilitated the binding of mAbs to other antigenic sites, indicating that refolding was not limited to the antibody-interaction domain. These findings validate the localization of antigenic sites by identifying amino acid substitutions selected in mAb escape mutants. Further, they demonstrate that Abs can facilitate the refolding of denatured proteins, which suggests a number of practical applications for optimizing antibody based assays, and also for potentially using Abs as specific chaperones for protein refolding.

Figure 1. Location of mAb Defined Epitopes on PR8 HA

Figure 2. Kinetics of mAb Binding to Native vs. Denatured Immunoadsorbant

The mAbs indicated were incubated for 2, 24, 48 or 72 h with native or denatured PR8 before washing and detection of bound antibody by addition of radioiodinated rat anti-κ chain mAb.

Figure 3. Binding Of Radiolabeled Y8-10C2 to Native or Denatured Virus

Y8-10C2 was radioiodinated and used to measure its affinity for denatured and native immunoadsorbents in direct RIAs. Dotted lines represent 95% confidence limits. Similar findings were made in at least 3 other experiments.