In vitro binding and neutralization of CR8020 and complementarity with CR6261. (A) Phylogenetic tree showing the relationships between the 16 HA subtypes and a summary of CR8020 and CR6261 activity. Red indicates positive binding by CR8020 while blue indicates positive binding by CR6261. Subtypes that have not been tested are indicated in black. (B) In vitro neutralization (IC₅₀ in μg/ml) of CR8020 against a panel of influenza A viruses as determined by microneutralization assay. (C) Affinity measurements (K_D) for binding of CR8020 and CR6261 to various H3 HAs and representative members of most of the other HA subtypes. nb indicates no detectable binding. Lowest affinity detectable under the experimental conditions was ~10⁻⁵ M.

CR8020 binds an epitope in the HA stem close to the virus membrane. (A) Crystal structure of HK68/H3 HA in complex with broadly neutralizing antibody CR8020. One HA/Fab protomer of the trimeric complex is colored with HA1 in yellow, HA2 in green, the Fab heavy chain in blue, and the Fab light chain in cyan. The other two protomers are colored gray. Glycans are shown as spheres (carbon in light pink, oxygen in red and nitrogen in blue). (B) Closer view of the interaction between HK68 HA and CR8020. The coloring is essentially as in (A,) but with the fusion peptide, which forms part of the epitope, in magenta, and the three segments of the small -sheet in purple, orange, and light blue (derived from the C-terminus of HA2, the N-terminus of HA1, and the N-terminus of HA2, respectively). On the Fab, HCDR1 is blue, HCDR2 is green and HCDR3 is red. For clarity, light chain CDRs are not highlighted here (see part C). (C) Interaction of CR8020 CDRs with the membrane proximal region of HA. CDRs are shown as ribbons and sticks (H1 in purple, H2 in green, H3 in red, L1 in orange, and L2 in blue. L3 makes no contacts). CR8020 binds HA with both chains, using 5 of the 6 CDRs (LCDR3 makes no contacts). Key antibody side chains are shown. (D) Footprint of HA on CR8020 combining site, highlighting antibody residues contacting HA. Note the usage of both the heavy and light chains in recognition (blue and cyan, respectively).

CR8020 inhibits the fusogenic conformational changes in HA and blocks proteolytic activation. (A) FACS binding of CR8020 (open bars) and a control mAb against the head (closed bars) to various conformations of surface-expressed H3 HAs from A/Hong Kong/1/1968 (1968), A/Hong Kong/24/1985 (1985) or A/Wisconsin/67/2005 (2005). Note that the control mAb has a narrow spectrum of neutralization and only binds A/Wisconsin/67/2005. The various conformations are depicted above the binding data and are as follows: uncleaved precursor (HA0); neutral pH, cleaved (HA); fusion pH, cleaved (fusion pH); trimeric HA2 (tHA2). Binding is expressed as the percentage of binding to untreated HA (HA0). Error bars represent SD of data obtained in 3 independent experiments. Ribbon diagrams are adapted from (57). (B) FACS binding of CR8020 (open bars) and a control mAb against the head (closed bars) to surface-expressed HA of A/Hong Kong/1/1968 (1968), A/Hong Kong/24/1985 (1985) or A/Wisconsin/67/2005 (2005) as above, except that mAb CR8020 was added before exposure of the cleaved HAs to a pH of 4.9. The anti-head mAb was added last to detect whether the HA1 heads remained associated with HA2. (C) SDS-PAGE results of the protease-susceptibility assay for H3 and H7 HAs. Exposure to low pH converts HAs to the protease-susceptible, post-fusion state (lane 3). Pre-treatment with CR8020 blocks the pH-induced conformational change, retaining HA in the protease-resistant, pre-fusion state (lane 7). (D) Immunoblot of uncleaved (HA0), recombinant soluble H3 HA after digestion with trypsin at pH 8.0. Digest reactions contained either HA alone, or HA pretreated with CR8020 or a control mAb against the head. Digestion was stopped at several time-points by adding 1% BSA. Uncleaved hemagglutinin (HA0) was detected using a polyclonal serum against H3.

Prophylactic and therapeutic efficacy of CR8020 in mice. Prophylactic efficacy of CR8020 against lethal challenge with (A) mouse-adapted A/Hong Kong/1/1968 (H3N2) or (B) A/Ck/Netherlands/621557/2003 (H7N7) viruses. Survival curves (left) and weight loss (right) of mice treated with 30, 10, 3, or 1 mg/kg of CR8020 or 30 mg/kg control mAb 24 hours before lethal challenge by intranasal inoculation (at day 0). Therapeutic efficacy of CR8020 against lethal challenge with (C) mouse-adapted A/Hong Kong/1/1968 (H3N2) or (D) A/Ck/Netherlands/621557/2003 (H7N7) viruses. Survival curves (left) and weight loss (right) of mice treated with 15 mg/kg CR8020 at various time-points after inoculation (at day 0).

CR8020 binds a unique, highly conserved site in the stem. (A) Comparison of CR8020 binding site to the epitopes of all other structurally characterized antibodies. All antibody footprints are mapped onto the surface of HK68 HA, although they are derived from different structures (and subtypes): Red, CR8020 (PDB code: XXXX); Purple, CR6261 (PDB codes 3GBM and 3GBN); green, 2D1 (PDB code: 1LFZ); blue, HC19 (PDB code: 2VIR); orange, HC45 (PDB code: 1QFU); cyan, HC63 (PDB code: 1KEN); pink, BH151 (PDB code: 1EO8). Note that mAb F10 and CR6261 bind the same epitope in the stem, but only CR6261 is illustrated here for clarity. (B) Comparison of the broadly neutralizing CR8020 and CR6261 epitopes, which constitute discrete surfaces on on group 2 and group1 HAs, respectively. Coloring is similar to (A), with CR8020 epitope in red, CR6261 epitope in blue, and shared epitope residues in yellow. (C) Conservation of CR8020 epitope across group 2 HAs. Residues comprising the epitope are shown as sticks (carbon in light pink, oxygen in red and nitrogen in blue). Percent identity with the group 2 consensus sequence is indicated alongside each residue. Note that the residue label reflects the most common residue across group 2 HAs, which is not always identical to the residue at that position in the HK68 crystal structure. View is similar to that of Fig. 4B, looking from the Fab (not shown) towards the epitope. (D) Location of CR8020 epitope residues in the pre-fusion and (E) post-fusion state, where they reside on critical regions of the fusion peptide and helix-capping region of HA2. For D and E, CR8020 epitope residues are indicated as spheres, with green mapping to the fusion peptide and N-cap region. Additional contact residues are purple (HA2) and gray (HA1, not present in available post-fusion structures). Residue positions were mapped onto the structures from PDB codes 1QU1 and 2KXA.