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

Abstract

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.

Fig. 1

Display of full-length HA0 and its subunits on yeast cell surface. (A) Schematic of HA proteins displayed on the yeast surface (upper) and the gene construct expressing HA (full-length or each subunit) as a fusion protein with the Aga2 signal peptide (aga2SP), Aga2p and cMyc tag (lower). (B) Display of full-length proteins was confirmed by FACS after indirect immunofluorescent labeling of the C-terminal tag with an anti-cMyc mAb or of HA with a polyclonal anti-H5 antibody. (C) Western blot analysis of total yeast cells showing HA0 or its subunits expressed as a fusion to Aga2p at expected sizes. Lanes 1–3: HA0, HA1 and HA2, respectively.

Fig. 2

FACS analysis of anti-HA mAbs binding to yeast surface-displayed HA0. (A) HA0 is bound by conformationally sensitive mAbs that target the highly conserved stem region of HA, which mediates viral membrane fusion with host cells. The 11A and 80R mAbs were used as negative controls. (B) The F10 mAb was shown to be conformationally sensitive by loss of binding to heat-denatured HA on yeast.

Fig. 3

Binding properties and epitope specificities of H5-2A and NR2728 mAbs. (A) Both H5-2A and NR2728 bound to yeast-displayed HA0 and HA1 but not to HA2. (B) Bindings of H5-2A and NR2728 to heat-denatured HA1 on yeast were both retained, indicating that both mAbs recognize linear HA epitopes.

Fig. 4

Fine epitope mapping of H5-2A and NR2728 mAbs. (A) FACS profiles demonstrating immunoreactivity of HA1 mutant clones displayed on the yeast surface. Clones with single-point mutations that selectively abolished binding of either H5-2A or NR2728 are shown. (B) Crystal structure of H5 HA0 (PDB 3FKU) with indicated epitopes recognized by H5-2A or NR-2728 mAbs to the HA1 monomer on the yeast surface (left) and a close-up view (middle). Top-down view of the HA trimer 3-fold axis shows the surface-exposed NR2728 epitope (yellow) and cryptic H5-2A epitope (gray) at the trimer interface (right). (C) Competition assay between H5-2A and NR-2728 mAbs. FACS histograms show that pre-incubation of HA with unlabeled HA-2A resulted in self-competition with the Alexa Flour 647-labeled H5-2A, while no competition was detected with the labeled NR2728 for the binding of surface-displayed HA1. (D) HI assay showing strong inhibition ability of NR2728, which was demonstrated above to have a epitope adjacent to the RBS, by blocking virus-mediated hemagglutination. Meanwhile, H5-2A showed no HI activity.