A common solution to group 2 influenza virus neutralization

Abstract

The discovery and characterization of broadly neutralizing antibodies (bnAbs) against influenza viruses have raised hopes for the development of monoclonal antibody (mAb)-based immunotherapy and the design of universal influenza vaccines. Only one human bnAb (CR8020) specifically recognizing group 2 influenza A viruses has been previously characterized that binds to a highly conserved epitope at the base of the hemagglutinin (HA) stem and has neutralizing activity against H3, H7, and H10 viruses. Here, we report a second group 2 bnAb, CR8043, which was derived from a different germ-line gene encoding a highly divergent amino acid sequence. CR8043 has in vitro neutralizing activity against H3 and H10 viruses and protects mice against challenge with a lethal dose of H3N2 and H7N7 viruses. The crystal structure and EM reconstructions of the CR8043-H3 HA complex revealed that CR8043 binds to a site similar to the CR8020 epitope but uses an alternative angle of approach and a distinct set of interactions. The identification of another antibody against the group 2 stem epitope suggests that this conserved site of vulnerability has great potential for design of therapeutics and vaccines.

Keywords: X-ray crystallography; antibody recognition; electron microscopy.

Fig. 1.

In vitro and in vivo binding and neutralization activity of CR8043. (A) Affinity measurements (K_d) for binding of CR8043 Fab to various H3 HAs and representative members of most of the other group 2 HA subtypes. (B) In vitro neutralization (IC₅₀) of CR8043 IgG against a panel of influenza A viruses as determined by VNA. Prophylactic efficacy of CR8043 IgG against lethal challenge with (C) mouse-adapted H3N2 or (D) A/chicken/Netherlands/621557/2003 (H7N7) viruses. (C and D) Shown are (Left) survival curves and (Right) body weight curves of mice treated with 30, 10, 3, or 1 mg/kg CR8043 or 30 mg/kg unrelated control mAb CR3014 1 d before lethal challenge (at day 0). Body weight curves represent mean ± 95% confidence interval of the mean.

Fig. 2.

Affinity of CR8043 to a panel of HAs of natural group 2 influenza viruses or engineered mutants. The HA residues included compose the CR8043 epitope. K_d values <10 nM, <100 nM or >100 nM are colored in red, yellow or blue boxes, respectively. Residues that differ from the consensus sequence are in gray boxes.

Fig. 3.

CR8043 binds an epitope in the HA stem close to the virus membrane. (A) Crystal structure of CR8043 Fab in complex with HK68/H3 HA. One Fab-HA protomer of the trimeric complex is colored with HA1 in green, HA2 in blue, the Fab heavy chain in red, and the Fab light chain in pink. Glycans are shown as spheres (carbon in orange, oxygen in red, and nitrogen in blue). (B, Left) Side and (B, Right) top views of negative stain EM reconstructions (gray mesh) of CR8043 Fab in complex with HK68/H3 HA with the crystal structure of the complex docked into the EM density. (C) Zoomed-in view of the interaction between CR8043 and HK68/H3 HA. The coloring is similar to A but with the fusion peptide in blue and the three segments of the β-sheet colored purple, orange, and pink (they derive from the C terminus of HA2, the N terminus of HA1, and the N terminus of HA2, respectively). (D) Sequence conservation of the CR8043 epitope across all group 2 HAs. Epitope residues are shown as sticks [carbon in green (HA1) or blue (HA2), oxygen in red, and nitrogen in dark blue], and the weighted percent identity of each residue with the group 2 consensus sequence is indicated, which is not always identical to the residue at that position for HK68/H3.

Fig. 4.

Comparison of CR8043 and CR8020 contacts on HA. (A) CR8043 (red and yellow) and CR8020 (blue and yellow) footprints mapped onto the HA surface, with HA1 colored in light green and HA2 colored in light blue. The yellow areas indicate the overlap in the epitopes. (B) Top view overlay of the crystal structures of the CR8043 (red) and CR8020 (blue; Protein Data Bank ID code 3SDY) Fabs in complex with HK68/H3 showing the different angle of approach to the HA. The interactions of (C) CR8043 or (D) CR8020 CDRs with HA. The antibody footprints are illustrated in dark green (HA1) and dark blue (HA2), and the contacting CDRs are shown as sticks.

Fig. 5.

CR8043 inhibits the fusogenic conformational changes in HA and blocks proteolytic activation of HA0. (A) FACS binding of CR8043 (open bars) and the head binding control mAb CR8057 (solid bars) to various conformations of surface-expressed H3 HAs of A/Hong Kong/1/1968, A/Hong Kong/24/1985, or A/Wisconsin/67/2005. The various conformations are indicated above the corresponding graphs and were as follows: uncleaved precursor (HA0); neutral pH, cleaved (HA); fusion pH, cleaved (pH 4.9); and trimeric HA2 (tHA2). Binding is expressed as the percentage of binding to untreated HA (HA0). Data represent mean + SD of three independent experiments. Ribbon diagrams are modified from ref. . (B) FACS binding of CR8043 (open bars) and CR8057 (solid bars) to surface-expressed H3 HAs as above, except that mAb CR8043 was added before exposure of the cleaved HAs to pH 4.9. (C) Immunoblot of trypsin digestion over time of uncleaved (HA0), soluble H3 HA, which was pretreated without mAb, with CR8043, or with CR8057 and detected using a polyclonal serum against H3 HA.