Cross-neutralization of influenza A viruses mediated by a single antibody loop

Abstract

Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens.

Figure 1. C05 neutralizes multiple influenza virus subtypes from groups 1 and 2

Phylogenetic tree with the two main viral lineages indicated with group 1 in the upper blue circle and group 2 in the lower green circle. Strains from subtypes circled in red are bound or neutralized by C05.

Figure 2. C05 protects mice from lethal virus challenge

Survival and weight loss were monitored in response to varying amounts of C05 IgG administered prophylactically to mice 24 hours before challenge with (a) 25x the 50% mouse lethal dose (MLD₅₀) of A/Memphis/3/2008 (H1N1) or (b) 33 MLD₅₀ of A/Aichi/2/X-31/1968 (H3N2) viruses. A single therapeutic dose of 15 mg/kg C05 IgG was delivered 1, 2, 3, 4, or 5 days post-challenge with (c) 25 MLD₅₀ of A/Memphis/3/2008 (H1N1) or (d) 33 MLD₅₀ of A/Aichi/2/X-31/1968 (H3N2) viruses.

Figure 3. C05 binds the receptor binding site on the HA1 head

a, Crystal structures of C05 Fab in complex with trimeric HA and an HA1 fragment reveal the location of the C05 epitope in the HA1 “head” region. The C05 Fab heavy (orange) and light (yellow) chains and the HA1 (blue) and HA2 (red) subunits are depicted as ribbons. b, C05 inserts its long HCDR3 (red) into the receptor binding site. c, C05 binds HA using only its heavy chain. d, Interaction of the HA receptor binding pocket (in a solid electrostatic surface representation) with HCDR3 from C05 (in yellow backbone with side chains labeled by residue type and number) and e, with an α2,6 sialoglycan receptor (derived from PDB entry 1MQN), with an additional carbohydrate residue modeled in gray. The binding site for the C05 H3 loop and the glycan receptor overlap extensively. f, 3D EM reconstructions of C05 Fab (red) bound to H1 (blue), H2 (light blue), and H3 (cyan) HAs. Fitting of H1, H2, and H3 crystal structures into the reconstructions support a common binding mode of C05 to each of the HAs studied. g, In addition to its long HCDR3 (red), C05 has a 5-residue insertion in HCDR1 (yellow), extending the tip of this loop relative to the germline-encoded canonical conformation. For comparison, a sequence-related Fab structure (PDB code 2VXS) without an insertion in HCDR1 (blue loop) is superimposed on C05.

Figure 4. C05 epitope conservation across influenza A viruses

a, Due to the functional constraints imposed on the receptor binding site by interaction with sialyoglycans, many of residues that make up the C05 epitope are conserved among human influenza viruses. C05 contact residues are depicted as sticks and the percent conservation of each position across human H1, H2, and H3 viruses, the subtypes that have caused pandemics and epidemics in humans, is shown. b, Similar to the view in (a), but includes the position of C05’s HCDR3 (yellow ribbon and sticks) relative to the epitope variation that is color coded on the HA backbone (with warmer colors indicating higher conservation). Contact residues on HA are depicted with a thick tube for their backbone, while non-contacting regions of HA are shown as a thin white ribbon.