(a) The ten antibodies were converted to soluble scFv-Fcs (scFv linked to the hinge, CH2 and CH3 domains of human IgG1) and evaluated for binding to trimeric H5-TH04 or monomeric HA1 of H5-TH04 coated on an ELISA plate. The H5 scFv-Fcs recognize trimeric H5 but not HA1. An antibody raised against HA1 (2A) recognized both. (b) Neutralization of H5-TH04–pseudotyped viruses (virus-like particles with HIV-1 only cores that display H5 on their surface). Percentage of neutralization at two concentrations is shown with s.d. The mAb 80R was used as a negative control (Ctrl.). (c,d) Neutralization of wild-type H5-VN04 and H5-IN05 by the ten scFv-Fcs at three concentrations using a plaque reduction assay. Results are consistent with those obtained from a microneutralization assay (data not shown).

(a,b) Prophylactic efficacy. Percentage of survival of mice treated with anti-H5 nAbs or control mAb 1 h before lethal challenge by intranasal inoculation with H5-VN04 (a) or H5-HK97 (b) viruses. (c–f). Therapeutic efficacy. Mice were inoculated with H5-VN04 and injected with nAbs at 24 h, 48 h of 72 hpi (c,e,f) or with H5-HK97 at 24 hpi (d).

(a) nAbs do not inhibit cell binding of full-length hemagglutinin from H5-TH04–pseudotyped HIV-1 viruses. None of the three nAb-treated viruses inhibited cell binding. Mouse anti-H5 mAb, 17A2.1.2 and ferret anti-H5N1 serum, which inhibit hemagglutination, were used as positive controls. Anti-SARS spike protein (80R) and anti-HA1 (2A) were used as negative controls. Error bars represent s.d. (b) All three nAbs inhibit cell fusion. HeLa cells were transfected with H5-TH04–expressing plasmid and exposed to a pH 5.0 buffer for 4 min in the presence or absence of nAbs. Syncytia formation induced by the brief exposure to pH 5.0 was completely inhibited by D8, F10 and A66, at 20 μg ml⁻¹ (∼0.13 μM), whereas controls (80R and anti-HA1 mAb (2A) at the same concentration had no effect.

(a) Structure of the H5 trimer bound to F10 (scFv). H5 is similar to the uncomplexed structure (pairwise r.m.s. deviation (Cα) = 1.0 and 0.63 Å for two independent trimers). HA1, HA2, the αA helix of HA2, the fusion peptide (FP) and F10 (VH and VL) are color coded. The third F10 molecule is hidden behind the stem. (b) Close-up of the epitope showing H5 as a molecular surface, with selected epitope residues labeled. The fusion peptide is in green. The tip of F10 (red ribbon) and selected CDR side chains are shown. Of 1,500 Ų buried surface at the interface, 43% involves hydrophobic interactions. (c) Surface of the central stem region, showing two H5 monomers. One monomer has HA1 (yellow) and HA2 (blue) colored differently; the path of the FP through the epitope (red) is outlined, and mutations that do not affect binding are colored cyan (Fig. 4d). The fusion peptides (FP and FP′) are labeled in both monomers. Epitope residues are labeled white (HA2) or yellow (HA1), and the position of buried residue H111₂ is shown as a black ball labeled 'H'. (d) Binding of the three nAbs to H5 mutants in the αA helix, transiently transfected into 293T cells. Note the similar response to all mutants tested. Mutations were made either to alanine or to the corresponding H7 residue; 24 h after transfection, nAbs or ferret anti-H5N1 serum was used to stain the transfected cells. Fluorescent intensity was normalized against ferret anti-serum (100%) to account for different expression levels.

Circles below residue numbers indicate estimated contribution to the binding energy at each position: red, strong; yellow, intermediate; blue, neutral. Residues without a circle are not directly involved in the epitope but are discussed in the text. Colored highlighting on the sequences indicates conservation within clusters and groups, with orange indicating high conservation or invariance. Other colors (for example, yellow, cyan and pink) highlight residues that are cluster or subtype specific. The network of interhelical contacts that stabilize the fusogenic structure are indicated below the HA2 sequences. Subtypes that can be recognized/neutralized by F10 are indicated with '+' on the far right. (+) or (−) indicates a predicted positive or negative binding, respectively.

(a) nAbs D8, F10 and A66 all neutralized indicated pseudotyped viruses (strains described below). Error bars indicate s.d. (b) Microneutralization assay. Neutralization titers (0.1 mg ml⁻¹ antibody stock solution) of nAb F10 against wild-type H5N1, H1N1, H2N2, H6N1, H6N2, H8N4, H9N2 and H3N2 virus strains. 80R is the negative control. Vertical bars and whiskers represent the lowest and the highest neutralization titer (2^χ, values of χ are shown on the y axis), respectively, of two or three independent experiments. (c,d) Prophylactic efficacy against two H1N1 strains in mice. Percentage of survival of mice treated with anti-H5 nAbs or control mAb are shown before lethal challenge by intranasal inoculation with H1-WSN33 (c) or H1-PR34 (d) viruses. Complete viral strain designations are: H1-OH83 (A/Ohio/83 (H1N1)); H1-PR34 (A/Puerto Rico/8/34 (H1N1)); H1-SC1918 (A/South Carolina/1/1918 (H1N1)); H1-WSN33 (A/WSN/1933 (H1N1)); H2-AA60 (A/Ann Arbor/6/60 (H2N2)); H2-JP57 (A/Japan/305/57(H2N2)); H3-SY97 (A/Sydney/5/97(H3N2)); H6-HK99 (A/quail/Hong Kong/1721-30/99(H6N1)); H6-NY98 (A/chicken/New York/14677-13/1998 (H6N2)); H7-FP34 (A/FPV/Rostock/34 (H7N1)); H8-ON68 (A/turkey/Ontario/6118/68); H9-HK(G9)97 (A/chicken/HongKong/G9/97 (H9N2)); H9-HK99 (A/HongKong/1073/99 (H9N2)); H11-MP74 (A/duck/Memphis/546/74 (H11N9)).

Stereo overlay of crystal structures of the five known hemagglutinin subtypes in the region of the F10 epitope, showing conservation and differences between the two phylogenetic groups. H1, H5 and H9 (group 1) are in shades of red and yellow (PDB 1RU7, 2IBX and 1JSD); H3 and H7 (group 2) are in shades of blue (PDB 1MQL and 1TI8). R.m.s. differences for pairwise overlays are 0.56 ± 0.11 Å (observed range, group 1), 0.75 Å (group 2) and 1.21 ± 0.12 Å between groups. Consistent differences between phylogenetic groups include the orientation of Trp21₂ and alternative side chain directions at 18₁ and 38₁, which are linked to the packing of buried His111₂ (the putative pH trigger in group 1; absent in group 2), and the burial of the larger tyrosine (group 1) versus histidine (the putative pH trigger in group 2) at 17₁. Of particular note, Asn38₁ is glycosylated in four members of the group 2 clusters. Other epitope residues are indicated by numbered light blue circles.

(a) The 10 Abs were converted to soluble scFv-Fcs (scFv linked to Hinge, CH2 and CH3 domains of human IgG1) and evaluated for binding to trimeric H5-TH04 or monomeric HA1 of H5-TH04 coated on an ELISA plate. The H5 scFv-Fcs recognize trimeric H5 but not HA1. An antibody raised against HA1 (“2A”) recognized both. (b) Neutralization of H5-TH04-pseudotyped viruses (virus-like particles with HIV-1 only cores that display H5 on their surface). % neutralization at 2 concentrations is shown with standard deviation (s.d.) bars. The mAb 80R was used as a negative control (“Ctrl.”). (c-d) Neutralization of wild type H5-VN04 and H5-IN05 by the 10 scFv-Fcs at three concentrations using a plaque reduction assay. Results are consistent with those obtained from a microneutralization assay (data not shown).

(a and b) Prophylactic efficacy. % survival of mice treated with anti-H5 nAbs or control mAb 1h before lethal challenge by i.n. inoculation with (a) H5-VN04 or (b) H5-HK97 viruses. (c-f). Therapeutic efficacy. Mice were inoculated with H5-VN04 and injected with nAbs at 24, 48, 72h post-inoculation (hpi) (c, e and f) or with H5-HK97 at 24 hpi (d).

(a) nAbs do not inhibit cell-binding of full-length HA from H5-TH04-pseudotyped HIV-1 viruses. None of the 3 nAb-treated viruses inhibited cell binding; mouse anti-H5 mAb, 17A2.1.2, and ferret anti-H5N1 serum, which inhibit haemagglutination, were used as positive controls; anti-SARS spike protein (“80R”) and anti-HA1 (“2A”) were used as negative controls. Vertical bars represent s.d.. (b) All 3 nAbs inhibit cell fusion. HeLa cells were transfected with H5-TH04-expressing plasmid and expose a pH 5.0 buffer for 4 mins in the presence or absence of nAbs. Syncytia formation induced by brief exposure to pH 5.0 was completely inhibited by D8, F10 and A66, at 20 μg ml^-1 (~0.13μM), whereas controls (“80R” and anti-HA1 mAb (“2A”) at the same concentration had no effect.

(a) Structure of the H5 trimer bound to F10 (scFv). H5 is very similar to the uncomplexed structure (pairwise RMSD (Cα) = 1.0 and 0.63 Å for 2 independent trimers). HA1, HA2, the αA-helix of HA2, the “fusion peptide” (FP), and F10 (VH and VL) are color-coded. The third F10 molecule is hidden behind the stem. (b) Close-up of the epitope showing H5 as a molecular surface, with selected epitope residues labeled. The fusion peptide is in green. The tip of F10 (red ribbon) and selected CDR side-chains are shown. Of 1500 Å buried surface at the interface, 43% involves hydrophobic interactions. (c) Surface of the central stem region, showing two H5 monomers. One monomer has HA1 (yellow) and HA2 (blue) colored differently; the path of FP through the epitope (red) is outlined, while mutations not affecting binding are in cyan (see Fig. 4d). The fusion peptides (FP and FP') are labeled in both monomers. Epitope residues are labeled white (HA2) or yellow (HA1), the position of buried residue H111₂ is shown as a black ball labeled “H”. (d) Binding of the 3 nAbs to H5 mutants in the αA helix. Note the very similar response to all mutants tested. Mutations were made either to alanine or to the corresponding H7 residue. 293T cells were transiently transfected with mutants; 24 hours after transfection, nAbs or ferret anti-H5N1 serum were used to stain the transfected cells. Mean fluorescent Intensity (MFI) was normalized against ferret anti-serum (100%) to account for different expression levels.

Circles below residue numbers indicate estimated contribution to the binding energy at each position: strong=red, intermediate=yellow; neutral=blue. Residues without a circle are not directly involved in the epitope but are discussed in the text. Colored highlighting on the sequences indicates conservation within clusters and groups, with orange indicating high conservation/invariance. Other colors (eg. yellow, cyan, pink) highlight residues that are cluster/subtype specific. The network of inter-helical contacts that stabilize the fusogenic structure are indicated below the HA2 sequences. Subtypes that can be recognized/neutralized by F10 are indicated with “+” on the far right. “(+) or (-)” indicates a predicted positive/negative binding.

(a) nAbs D8, F10 and A66 all neutralized H5-TH04, H1-SC1918, H1-PR34, H1-WSN33, H2-JP57, H6-NY98 and H11-MP74 (strains described below) pseudotyped viruses. (b) Microneutralization assay. Neutralization titers (0.1 mg ml^-1 Ab stock solution) of nAb F10 against two wild-type H5N1, three H1N1, one H2N2, one H6N1, one H6N2, one H8N4, two H9N2 and one H3N2 virus. 80R is the negative control. Vertical bars and whiskers represent the lowest and highest neutralization titer (2^χ, values of χ are shown on the y-axis) of 2-3 independent experiments. (c-d) Prophylactic efficacy against two H1N1 strains in mice. % survival of mice treated with anti-H5 nAbs or control mAb are shown before lethal challenge by i.n. inoculation with (c) H1-WSN33 or (d) H1-PR34 viruses. Complete viral strain designations are: H1-OH83 (A/Ohio/83 (H1N1)), H1-PR34 (A/Puerto Rico/8/34 (H1N1)), H1-SC1918 ((A/South Carolina/1/1918 (H1N1)), H1-WSN33 (A/WSN/1933 (H1N1)), H2-AA60 (A/Ann Arbor/6/60 (H2N2)), H2-JP57 (A/Japan/305/57(H2N2)), H3-SY97 (A/Sydney/5/97(H3N2)), H6-HK99 (A/quail/Hong Kong/1721-30/99(H6N1)), H6-NY98 (A/chicken/New York/14677-13/1998 (H6N2)), H7-FP34 (A/FPV/Rostock/34 (H 7 N 1)), H 8-ON68 (A/turkey/Ontario/6118/68), H9-HK(G9)97 (A/chicken/HongKong/G9/97 (H9N2)), H9-HK99 (A/HongKong/1073/99 (H9N2)), H11-MP74 (A/duck/memphis/546/74 (H11N9)).

Stereo overlay of crystal structures of the 5 known HA subtypes in the region of the F10 epitope, showing conservation and differences between the 2 phylogenetic groups. H1, H5 and H9 (Group 1) are in shades of red/yellow (PDB codes 1RU7, 2IBX and 1JSD); H3 and H7 (Group 2) are in shades of blue (PDB codes 1MQL and 1TI8). RMS differences for pair-wise overlays are 0.56±0.11 Å (observed range, Group 1); 0.75 Å (Group 2); and 1.21±0.12 Å between groups. Consistent differences between phylogenetic groups include the orientation of W21₂ and alternative side-chain directions at 18₁ and 38₁, which are linked to the packing of buried His111₂ (the putative pH trigger in Group 1; absent in Group 2); and the burial of the larger tyrosine (Group 1) versus histidine (the putative pH trigger in Group 2) at 17₁. Of particular note, N38₁ is glycosylated in 4 members of the Group 2 clusters. Other epitope residues are indicated by numbered light blue circles.