Fine epitope mapping of monoclonal antibodies against hemagglutinin of a highly pathogenic H5N1 influenza virus using yeast surface display

Biochem Biophys Res Commun. 2011 Jun 3;409(2):253-9. doi: 10.1016/j.bbrc.2011.04.139. Epub 2011 May 5.


Highly pathogenic H5N1 avian influenza viruses pose a debilitating pandemic threat. Thus, understanding mechanisms of antibody-mediated viral inhibition and neutralization escape is critical. Here, a robust yeast display system for fine epitope mapping of viral surface hemagglutinin (HA)-specific antibodies is demonstrated. The full-length H5 subtype HA (HA0) was expressed on the yeast surface in a correctly folded conformation, determined by binding of a panel of extensively characterized neutralizing human monoclonal antibodies (mAbs). These mAbs target conformationally-dependent epitopes of influenza A HA, which are highly conserved across H5 clades and group 1 serotypes. By separately displaying HA1 and HA2 subunits on yeast, domain mapping of two anti-H5 mAbs, NR2728 and H5-2A, localized their epitopes to HA1. These anti-H5 mAb epitopes were further fine mapped by using a library of yeast-displayed HA1 mutants and selecting for loss of binding without prior knowledge of potential contact residues. By overlaying key mutant residues that impacted binding onto a crystal structure of HA, the NR2728 mAb was found to interact with a fully surface-exposed contiguous patch of residues at the receptor binding site (RBS), giving insight into the mechanism underlying its potent inhibition of virus binding. The non-neutralizing H5-2A mAb was similarly mapped to a highly conserved H5 strain-specific but poorly accessible location on a loop at the trimer HA interface. These data further augment our toolchest for studying HA antigenicity, epitope diversity and accessibility in response to natural and experimental influenza infection and vaccines.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies, Monoclonal / immunology*
  • Antibodies, Viral / immunology*
  • Cloning, Molecular
  • Epitope Mapping / methods*
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Hemagglutinin Glycoproteins, Influenza Virus / immunology*
  • Humans
  • Influenza A Virus, H5N1 Subtype / immunology*
  • Mutation
  • Peptide Library
  • Saccharomyces cerevisiae


  • Antibodies, Monoclonal
  • Antibodies, Viral
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Peptide Library