Prophylactic and therapeutic efficacy in mice

We evaluated the protective efficacy of the three IgG1s against H5N1 virus infection in a BALB/c mouse model (Fig. 2). Mice were treated with IgG1s before (prophylactically) or after (therapeutically) lethal viral challenge. Prophylaxis using 10 mg kg⁻¹ of IgG1s effectively protected (80–100%) mice when challenged with a high lethal dose of H5-VN04 (clade 1) or A/HongKong/483/97 ('H5-HK97') (clade 0) (Fig. 2a,b). Therapeutic treatment with 15 mg kg⁻¹ (an achievable dose in humans) of IgG1 24 h post-inoculation (hpi) also protected 80–100% of the mice challenged with either H5-VN04 or H5-HK97 virus (Fig. 2c,d). Mice treated at later times (48 hpi or 72 hpi) with H5-VN04 showed similar or higher levels of protection (Fig. 2e,f). Furthermore, surviving mice remained healthy and showed minimal body weight loss over the 2-week observation period (data not shown).

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Figure 2

Prophylactic and therapeutic efficacy of anti-H5 nAbs in mice.

(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).

Whereas human influenza viruses are typically restricted to the upper respiratory tract, systemic spread is a typical outcome of H5N1 infection in mice, and it has been reported in some humans. We found that the three IgG1s caused potent suppression of viral replication in the lungs (measured 4 d after viral challenge) of mice treated within 48 h of viral challenge; and that two IgG1s, F10 and A66, were effective when given at 72 hpi. The strong impact of antibody therapy on systemic infection was demonstrated by ≥1,000-fold suppression of virus spread to the spleen, even when given 72 hpi (Supplementary Fig. 5 online). We also observed suppression in the brain, but in this case, systemic spread was too low in control animals for accurate quantitation.

nAbs inhibit cell fusion rather than receptor binding

Two ways in which anti-hemagglutinin antibodies can neutralize infection is by blocking the initial binding of hemagglutinin to its cellular receptor (sialic acid) or by interfering with the subsequent step of hemagglutinin-mediated virus-host membrane fusion, which occurs in acidic endosomes^19,20. We found that none of the nAbs inhibited virus binding to cells (Fig. 3a) or hemagglutination of red blood cells (data not shown). However, we were able to show, using a model system of cell fusion, that the nAbs potently inhibited membrane fusion (Fig. 3b).

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Figure 3

Neutralization mechanism.

(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.

Structural characterization of the nAb epitope

To provide a structural basis for neutralization and to explore the prospects for developing even broader-spectrum therapeutics, we determined the crystal structure of F10 (as the scFv fragment) in complex with the H5 (H5-VN04) ectodomain (Fig. 4 and Supplementary Table 2 online). We used H5 activated by cleavage of the single-chain precursor, HA0, into two polypeptides, HA1 and HA2. Cleavage leads to the partial burial of the fusion peptide (the first ~21 residues of each HA2) into the stem^19,21, which also contributes to the formation of each of three hydrophobic 'pockets' located below the large trimeric receptor binding head. In the complex, one F10 nAb binds into each pocket, burying ~1,500 Ų of protein surface. Only the heavy chain (VH) participates directly in binding, using all three of its complementarity-determining regions (CDRs). The light chain (VL) points out into solution and makes only nonspecific contacts with the distal end of the oligosaccharide of glycosylated residue Asn33₁ from a neighboring monomer. The epitope on H5 encompasses the entire pocket, which is formed by the HA2 fusion peptide flanked by elements of HA1 on one side and helix αA of HA2 on the other.

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Figure 4

Structure of the H5–F10 complex.

(a) Structure of the H5 trimer bound to F10 (scFv). H5 is similar to the uncomplexed structure³⁵ (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.

The key interactions are as follows (Fig. 4b). (i) CDR-H2 adopts the 'type 2' conformation²². Two hydrophobic residues, Met54 and Phe55, from the tip of H2 insert into the pocket. Phe55 lies across a flat hydrophobic surface formed by the main chain of the fusion peptide, residues 18₂–21₂; it also makes favorable orthogonal aromatic interactions²³ with the side chains of Trp21₂ at the back of the pocket and His18₁ at the front (subscripts 1 or 2 refer to HA1 or HA2, and the numbering scheme follows the structure of H3 (PDB 2HMG)^17,24). The Met54 sulfur makes Π−aromatic interactions²⁵ with the Trp21₂ ring, hydrophobic interactions with Ile45₂ from helix αA and a hydrogen bond between Met54 C=O and the His38₁ side chain. (ii) Tyr102 from CDR-H3 extends from the apex of the H3 loop to a location only ~3 Å from Phe55, and it complements CDR-H2 by cementing together the fusion peptide (via a main chain hydrogen bond to Asp19₂) and the αA helix of HA2 (by intercalating between Thr41₂ and Ile45₂). A large hydrophobic residue at the neighboring position 103 supports the side chain conformation of Tyr102. (iii) The CDR-H1 loop is characterized by small hydrophobic or polar side chains (notably Val27, Thr28 and Ser31) such that CDR-H1 fits snugly beneath the hemagglutinin head while packing against helix αA. A somatic mutation of conserved Gly26, G26E, generates a noncanonical conformation for H1, with Thr27 pointing outward and making contact H5.

An N-terminal hairpin (residues Ile29₂ and Met30₂) from HA2 of the counterclockwise neighbor packs against the other side of helix αA at this point, wrapping around its fusion peptide and further locking it into place (Fig. 4a,c). Thus, the F10 nAb may stabilize the fusion peptide of more than one subunit. One framework (FR3) residue, Gln74, seems to be especially important in stabilizing the CDR-H1 and CDR-H2 loop conformations, by forming hydrogen bonds to the main chain C = O groups of Pro53 and Met54, as well as the side chain of Ser30. The FR3 residue at position 72 is the major determinant of the choice between two distinct conformations of the H2 loop²².

Consistent with the structural data, mutations in three H5 residues on HA2 αA, Val52₂, Asn53₂ and Ile56₂, which make important interactions with F10, greatly reduce or ablate nAb binding, whereas the conservative mutation V52L has no effect (Fig. 4c,d). Mutations to other surfaces of the αA helix either have no effect (typically exposed residues) or lead to increased nAb binding, perhaps by subtly increasing the flexibility of the epitope (Fig. 4d). Notably, the nine other nAbs show similar mutant binding profiles. Together with the cross-competition noted above, this strongly suggests that the epitopes for all ten nAbs overlap very closely indeed, and that the nAbs bind in a similar location and orientation.

Structural basis of H5 neutralization by the nAb panel

The broad neutralizing behavior against H5 may be attributed in part to the exclusive role of VH in antigen binding and the use of a common germline gene, VH1-69, in five out of the six VHs, although their CDR3 loops are variable in sequence and length (13–17 residues) (Supplementary Fig. 6 and Supplementary Table 1 online). In addition, free-energy calculations²⁶ point to dominant binding contributions (~70% of the total favorable free energy) of the three conserved residues in the VH segment (Fig. 4b). In CDR-H2 derived from germline VH1-69, position 55 is always phenylalanine, and position 54 is always hydrophobic (methionine, isoleucine, leucine or valine). In our nAbs, CDR-H3 always has a tyrosine predicted to lie at the tip of the CDR3 loop (conserved at the position 6). The conformation and sequence of the CDR1 loop does not seem to be critical, because the other antibodies we isolated do not contain the somatic mutation (G26E) found in F10 and are predicted to have canonical structures. The sixth VH gene we isolated is derived from the germline gene VH1-2; its H2 loop has the same length as VH1-69, but by virtue of a change from alanine to arginine at position 72 (ref. 22) it is predicted to adopt a distinct conformation ('type 3') that presents loop residues 3 and 4 to the antigen (rather than residues 4 and 5 in type 2 loops). The specific somatic mutation at position 4, from asparagine to methionine, presumably promotes H5 binding. It is not possible to predict the structure of the larger H3 loop, but a tyrosine located at the center of the loop may play an analogous role to that in F10.

Thus, the F10-H5 crystal structure suggests a common mechanism of H5 virus neutralization for our nAb panel. They make no contact with the receptor binding sites in the head and so do not inhibit cell attachment. Rather, they lock the fusion peptide and helix αA in place, thereby preventing the large structural reorganizations that are required for membrane fusion^{17,19,27,28,29,30}. Our data point to this event occurring at an early step in infection, although we cannot rule out the possibility that the nAbs act at a later stage, given the close packing of molecules on the surface of the mature virion, which might restrict early access to the epitope. The only previously published crystal structure of a hemagglutinin-nAb complex that inhibits membrane fusion uses a different mechanism: it prevents conformational changes by cross-linking the upper surfaces of adjacent subunits in the head³¹.

NAbs bind and neutralize a broad range of group 1 viruses

Next we examined all of the available hemagglutinin sequences (total 6,360) in the public influenza sequence database (Supplementary Table 3 online). Of note, the sequences of the F10 epitope are nearly always conserved within the H5 subtype. Indeed, many epitope residues, especially in HA2, are highly conserved across all 16 hemagglutinin subtypes (Fig. 5). This high sequence conservation provides a rationale for the cross-neutralization of the H5N1 virus clades described above, and this prompted us to test our antibodies against a broader range of hemagglutinin subtypes.

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Figure 5

Sequence conservation in hemagglutinin groups, clusters and subtypes at the F10 epitope.

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 structure⁶¹ 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.

Group 1 viruses, which contain 10 of the 16 subtypes, are further classified into three 'clusters': H1a, H1b and H9 (refs. 32,33; Fig. 5). We tested nAb binding to eight members of clusters H1a, H1b and H9, which include avian H5 and the most common human influenza subtypes (the major exception is the group 2 subtype, H3). In addition to H5, we found that all three IgG1s bound to cells expressing full-length H1 from three different strains of H1N1, including the 1918 Spanish flu, H2 from H2N2 and H6 from H6N2, the Cluster 1b subtypes H11 from H11N9, H13 from H13N6 and H16 from H16N3, and the Cluster H9 subtypes from three H9N2 strains. However, none of them bound to a group 2 subtype, H7 from H7N1 (Supplementary Fig. 7 online).

The IgG1s also neutralized H5-, H1-, H2-, H6- and H11-pseudotyped virus infections (Fig. 6a). In a microneutralization assay, F10-IgG1 also neutralized H5N1, H1N1, H2N2, H6N1, H6N2, H8N4 and H9N2 influenza viruses (Fig. 6b). However, none of the nAbs neutralized group 2 viruses, for example, H3N2 (Fig. 6b and Supplementary Fig. 8 online). Thus, these nAbs recognize an epitope on hemagglutinin that is conserved among H5 clades and in all members of group 1 viruses. Finally, we demonstrated the in vivo protective efficacy of two of the IgG1s against two lethal H1N1 viral strains in a BALB/c mouse model, using the same protocol as for the H5N1 studies (Fig. 6c,d).

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Figure 6

Cross-subtype neutralization by nAbs.

(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)).

Basis of the group-specific broad-spectrum virus neutralization

The ability of our nAbs to recognize all group 1 (cluster H1a/b and H9) viruses (H12 was not tested) can be attributed to the key conserved features of the nAbs described above in combination with the highly conserved pocket on hemagglutinin (Figs. 4 and ​and5).5). The epitope may be divided into three elements. (i) At its center, the sequence of the N-terminal segment of HA2—fusion peptide residues 18₂–21₂—is conserved across all hemagglutinin subtypes (note that the side chain at position 19₂ does not participate in binding). (ii) A downstream segment of HA2 adopts part of the αA helix (residues 39₂–56₂), which is nearly invariant; the only significant difference is a threonine to glutamine change at position 49₂ in the untested H9 cluster subtype, H12. Thr49₂ lies at the periphery of the epitope and makes one long hydrogen bond (3.5 Å) to Ser31. Simple modeling suggests that there is plenty of space to accommodate the larger glutamine side chain and that it can make comparable hydrogen bonds. (iii) Smaller contributions from segments of the HA1 chain (residues 18₁ and 38₁) and a loop at the base of the head (residues 291₁ and 292₁).

Three-dimensional comparisons of the epitope in the five known crystal structure subtypes (three from group 1 (H1, H5 and H9) and two from group 2 (H3 and H7)^{21,32,34,35,36}) show that they adopt two distinct structural classes consistent with the phylogenetic groupings^32,33 (Fig. 7). These differences arise from group-specific differences in the location of buried residues, notably histidines (H111₂ is unique to group 1; H17₁ is unique to group 2) that have been proposed to be the 'triggers' for pH-induced conformational changes²⁹. The differences cause the side chain of Trp21₂ to turn through 90° in group 2 subtypes, eliminating favorable binding to Phe55 from our nAb panel. In addition, four out of six group 2 subtypes are glycosylated at position 38₁, at the periphery of the F10 epitope; our modeling studies predict steric clashes with the CDR-H1 loop (data not shown). These structural differences rationalize the observed lack of binding and neutralization of group 2 hemagglutinin subtypes and viruses.

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Figure 7

Three-dimensional comparison of the F10 epitope in group 1 and group 2 hemagglutinins.

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.

Data collection, structure determination and refinement.

We collected diffraction data from crystals that were flash frozen at 100 K in the reservoir buffer at the Stanford Synchrotron Radiation Laboratory beamline 9.2, set at a wavelength of 1.0 Å and processed with XDS⁵² and HKL2000 (ref. 53). The structure was solved at 3.2-Å resolution by molecular replacement with PHASER using the structures of H5 (A/Vietnam/1194/04; PDB 2IBX) and a homology model of F10 based on the structure of SARS nAb 80R (PDB 2GHW)^54,55 as starting models. The asymmetric unit contains two H5 trimers and three F10 molecules per trimer and was refined using REFMAC5 (ref. 56) with simulated annealing in CNS and manual rebuilding with Coot⁵⁷ and Xtalview⁵⁸. The final maps are of high quality, and key features such as the F10 CDR loops and interfacial residues are unambiguous and consistent in the six copies. The final model includes 503, 503, 503, 497, 497 and 497 residues for the six independent copies of H5; 235, 235, 236, 233, 234 and 234 residues for the six F10scFvs; 24 N-acetyl-D-glucosamine and six β-D-mannose units, but no water molecules. The R_free is 0.29 with excellent geometry as assessed with PROCHECK⁵⁹ and Rampage (Table 1): residues in the favored, allowed and outlier regions are 90.0%, 9.5% and 0.5%, respectively.

Phage-display library selection.

We produced recombinant trimeric H5-VN04 ectodomain as for crystallization studies except that we did not use furin co-infection to ensure complete activation. Antibodies were identified by two rounds of selection of a 27 billion–membered human scFv phage-display library against recombinant trimeric H5 immobilized on Immunotube (Nunc), followed by ELISA screening. We identified ten unique anti-H5 antibodies by sequence analysis of 97 H5-positive clones out of 392 clones screened.

Plaque reduction assay.

H5-VN04, H5-IN05 or A/Netherland/219/03 (H7N7) (H7-NL03) viruses (10,000 plaque forming units (p.f.u.)) were incubated with anti-H5 scFv-Fcs at three different concentrations (1 μg ml⁻¹, 10 μg ml⁻¹or 100 μg ml⁻¹) at 37 °C for 30 min. We diluted the virus-antibody mixture logarithmically and transferred the dilutions onto MDCK cell monolayers in 12-well plates, which we then incubated at 37 °C for 1 h. The cells were then washed and overlaid with agar. After 4 d of incubation, we discarded the overlay and the plaques were visualized by crystal violet staining.

Microneutralization assay.

We carried out the assay as described⁶⁰. Briefly, we mixed 100 TCID₅₀ (median tissue culture infectious doses) of virus in equal volume with two-fold serial dilutions of antibody stock solution (0.1 mg ml⁻¹) in 96-well plates and incubated them for 1 h at 37 °C. Indicator MDCK cells (1.5 × 10⁴ cells per well) were added to the plates, followed by incubation at 37 °C for 20 h. To establish the endpoint, we washed the cell monolayers with PBS, fixed the cells in acetone and detected viral antigen by indirect ELISA with a mAb against influenza A NP (A-3, Accurate).

Viral binding inhibition assay.

We incubated 0.5 × 10⁶ 293T cells with H5-TH04–pseudotyped HIV viruses (~500 ng of p24) in the presence of anti-H5 nAbs or control mAbs, or in the absence of antibodies, in PBS buffer containing 0.5% (w/v) BSA and 0.02% (w/v) NaN₃ at 4 °C. After 1 h of incubation, cells were spun down, and the supernatants were tested for p24 levels using an HIV-1 p24^CA capture ELISA kit (National Cancer Institute, US National Institutes of Health) to quantify unbound virus. Cells were then washed once or twice and lysed to quantify the cell-bound virus using the same method.

Cell fusion inhibition assay.

We transfected HeLa cells, ~90% confluent in six-well plates, with pcDNA3.1-H5-TH04 plasmid (3 μg total DNA per well) using lipofectamine 2000 (Invitrogen). After ~30 h of transfection, the culture medium was supplemented with 1 ml of anti-H5 or control mAbs for 1–2 h, and cells were then washed and incubated with low-pH fusion buffer (150 mM NaCl plus 10 mM HEPES, adjusted to pH 5.0) for 4–5 min. Cells were then returned to the standard culture medium for 2–3 h at 37 °C, and finally fixed with 0.25% (v/v) glutaraldehyde and stained with 0.1% (w/v) crystal violet. Photomicrographs were taken at 10× magnification.

Prophylactic and therapeutic efficacy studies in mice.

We used female 8–10 weeks old BALB/c mice in all experiments. Mice were weighed on the day of virus challenge and then daily for 2 weeks. We used body weight as the clinical endpoint; mice with body weight loss ≥25% of pre-infection values were euthanized. Animal studies were conducted as per the approved Institutional Animal Care and Use Committee protocols.

For the prophylactic efficacy study, three human nAbs (D8-IgG1, F10-IgG1 and A66-IgG1) or control human mAb 80R-IgG1 (ref. 18) at 2.5 mg kg⁻¹ or 10 mg kg⁻¹ were administered into four groups of five mice each by intraperitoneal injection in a 0.5 ml volume. At 1 h after mAb administration, two groups of mice were challenged with H5-VN04 and two groups with H5-HK97 intranasal inoculation with 10 MLD₅₀ in 50 μl volumes per mouse. Mice were observed and weighed daily for 2 weeks after infection. Analogous studies were performed to evaluate the protective efficacy of the nAbs against A/Puerto Rico/8/1934 (H1N1) or A/WSN/1933 (H1N1) viruses.

For the post-exposure therapy efficacy study, the experimental design recapitulates the prophylaxis study, with the following exceptions. We first inoculated 12 groups of ten mice intranasally with 10 MLD₅₀ of VN04. At 24 h, 48 h and 72 h after H5-VN04 infection, the mice received intraperitoneal injections of 15 mg kg⁻¹ body weight of one of the nAbs or control (80R).

We thank J. Appleton (Cornell University) for the gift of mouse mAbs against H5N1, 17A2.1.2 and 22F; A. Klimov (CDC) and A. Balish (CDC) for providing ferret antiserum and virus sequences; R. Webster (St. Jude Children's Research Hospital) for H11N9, H13N6 and H16N3; L. Quynh Mai (National Institute of Hygiene and Epidemiology, Vietnam Ministry of Health) for H5N1; W. Lim (Hong Kong Department of Health) for H5N1 and H9N2, as well as E. Sedyaningsih, T. Soendoro (National Institute of Health Research and Development, Indonesian Ministry of Health) for H5N1 specimens; P. Palese (Mount Sinai School of Medicine) for pCAGGS-H1(SC) plasmid encoding the full-length hemagglutinin protein of H1-SC1918; M. Farzan (New England Primate Research Center, Harvard Medical School) for pCAGGS-H1 (PR) plasmid encoding the hemagglutinin protein of H1-PR34 and X. Yang (Beth Israel Deaconess Medical Center, Harvard Medical School) for pCAGGS-H7 (FPV) encoding H7-FP34 hemagglutinin; and R. Fuller (University of Michigan) for furin cDNA. We thank W. Yuan and W. Li for helpful discussions and Y. Lin for assistance in crystallization and critical discussion. We thank the US National Institutes of Health (NIH) and the Department of Energy (DOE) for access to the Stanford Synchrotron Radiation Facility and the facility staff for assistance in X-ray data collection. Molecular graphics images were produced using the UCSF Chimera package from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIH P41 RR-01081). This work was supported by NIH (U01-AI074518-01) to W.A.M. and in part by NIH (P01-AI055789) to R.C.L.