Antibody Recognition of the Pandemic H1N1 Influenza Virus Hemagglutinin Receptor Binding Site

5J8 and CH65 Fab were cloned in a pFastBac dual vector (Invitrogen) with N-terminal gp67 and honeybee melittin secretion signal peptides fused to the heavy and light chains, respectively, and a C-terminal His₆ tag fused to the heavy chain. Recombinant bacmid DNA and baculovirus were generated as previously described (7). The Fabs were purified by Ni-nitrilotriacetic acid (NTA) (Qiagen) and Mono S (GE Healthcare) chromatography. The purified Fabs were then dialyzed into 20 mM HEPES (pH 7.4) and 150 mM NaCl, flash frozen with liquid nitrogen, and stored at −80°C.

The heavy and light chains of the 5J8 and CH65 IgGs were cloned separately in a phCMV3 vector (Genlantis) with an N-terminal Ig kappa secretion signal peptide and a C-terminal His₆ tag fused to the heavy chain. The heavy and light chain plasmids were transiently transfected at a 2:1 ratio into HEK293F suspension cells and incubated for six days. The IgGs were purified by Ni-NTA (Qiagen) and protein A (GE Healthcare) chromatography and then buffer exchanged into 20 mM Tris (pH 8) and 150 mM NaCl.

HA was prepared for binding studies and crystallization as previously described (7, 12). Briefly, each HA was fused with an N-terminal gp67 signal peptide and a C-terminal BirA biotinylation site, a thrombin cleavage site, a trimerization domain, and a His₆ tag. The HAs were expressed as described above for the Fabs and purified by Ni-NTA. The HAs were either matured by trypsin (New England BioLabs) for crystallization or biotinylated with BirA (12) for binding studies.

The HA1 protein was derived from a recombinant fusion protein, STF2.HA1, that contains N-terminal Salmonella enterica serovar Typhimurium flagellin residues 1 to 505 linked to the N terminus of A/California/07/2009 (H1N1) (Cali07/2009-H1) residues 55 to 271 (H3 numbering), which was expressed in batch bioreactor cultures as previously described (13).

Apo 5J8 Fab crystals were grown by sitting drop vapor diffusion at 4°C by mixing 0.5 μl of protein (6.3 mg/ml) with 0.5 μl of reservoir solution (0.2 M calcium acetate, 11% [wt/vol] polyethylene glycol 3350 [PEG 3350]). Crystals were cryoprotected in mother liquor supplement with 20% (vol/vol) ethylene glycol, flash cooled, and stored in liquid nitrogen until data collection.

The 5J8 Fab-STF2.HA1 complex was prepared by mixing individually prepared proteins in a 1.1:1 molar ratio and then purified by gel filtration. Fractions corresponding to the complex were pooled and concentrated to 16 mg/ml for crystallization screening. However, STF2.HA1 degraded over time, likely at or around the linker connecting flagellin and HA1, as only HA1 in complex with 5J8 Fab crystallized. Crystals grew at 23°C by sitting drop vapor diffusion by mixing 0.6 μl of protein solution (16 mg/ml) with 0.5 μl of reservoir solution (0.1 M Tris [pH 8.5], 23% [wt/vol] PEG 8000, 0.2 M magnesium chloride). Crystals were cryoprotected in mother liquor supplemented with 15% (vol/vol) glycerol, flash cooled, and stored in liquid nitrogen until data collection.

The trimer-stabilized A/California/04/2009 (H1N1) (Cali04/2009-H1 HA2 E47G) HA crystals were grown by sitting drop vapor diffusion. The HA was concentrated to 17 mg/ml in 20 mM Tris (pH 8.0), 100 mM NaCl, and 0.02% (vol/vol) NaN₃ and crystallized in 0.1 M Tris (pH 8.8), 25% (wt/vol) methoxypolyethylene glycol 2000 (MPEG 2000) at 23°C. Crystals were flash cooled in mother liquor supplemented with 12% (vol/vol) ethylene glycol and stored in liquid nitrogen until data collection.

X-ray diffraction data for the apo 5J8 Fab were collected to a 1.55-Å resolution at the GM-CA CAT 23ID-D beamline at the Advanced Photon Source (APS). The data were processed in space group P2₁2₁2₁ using XDS (14). The structure was determined by molecular replacement with Phaser (15) by using the variable and constant domains of the anti-HIV-1 V3 Fab 3074 (Protein Data Bank [PDB] accession no. 3MLY, chains H and L) as search models, and two Fab copies were found in the asymmetric unit. The crystal exhibited pseudotranslational symmetry. The model was iteratively built using Coot (16) and refined in Phenix (17). Refinement parameters included rigid-body refinement (set for each Ig domain), simulated annealing, and restrained refinement, including translation/libration/screw (TLS) refinement (for each Ig domain).

X-ray diffraction data for the 5J8-Cali07/2009-H1 HA1 complex were collected to 2.25 Å at the Canadian Light Source beamline 08B1-1 (CMGF-BM). The data were processed in space group P3₁21 using XDS (14). The complex was determined by molecular replacement with Phaser (15) by first using one copy of the HA1 from Cali04/2009-H1 (PDB accession no. 3LZG, chain A) residues 55 to 271 (H3 numbering). Next, one copy of the variable and constant domains of the high-resolution 5J8 Fab structure were used as search models after fixing the position and orientation of the HA head. The model was iteratively built using Coot (16) and refined in Phenix (17). Refinement parameters included rigid-body refinement (set for the HA1 and each Ig domain), simulated annealing, and restrained refinement, including TLS refinement (set for the HA1 and each Ig domain).

X-ray diffraction data for the trimer-stabilized HA (Cali04/2009-H1 HA2 E47G) were collected to a 2.20-Å resolution at beamline 12-2 at the Stanford Synchrotron Radiation Lightsource (SSRL). The mutant HA structure was determined by molecular replacement using the program Phaser (15) by using the native Cali04/2009-H1 HA (PDB accession no. 3LZG) as the starting model. The refinement was performed in Refmac5 (18) and Phenix (17), and model building was carried out with Coot (16).

Copper grids (400 mesh) were coated in nitrocellulose and a thin layer of carbon. The grids received samples shortly (<20 min) after glow discharging. Negative staining was performed through application of 4 μl of sample (∼0.02 mg/ml of Fab-HA complex diluted in Tris-buffered saline [TBS]) to the grid, with blotting to remove excess sample, followed by two cycles of staining with 4 μl of “Nano W” stain (2% methylamine tungstate [Nanoprobes, Yaphank, NY]), 20 s of incubation, and blotting to remove excess stain.

Micrographs were acquired on an FEI Tecnai Spirit transmission electron microscope (TEM) operating at an accelerating voltage of 120 kV. A Tietz charge-coupled-device camera was used to record 2,048- by 2,048-pixel images at a magnification of ×52,000 and a defocus range of 900 to 1,300 nm. The stage was tilted in five-degree increments, from 0° to 55°, to increase the number of observed orientations. The pixel size was previously calibrated to be 2.65 Å using a two-dimensional catalase crystal. The Leginon software package (19, 20) was used to automate some steps of data acquisition.

Particles were automatically selected using a difference of Gaussian algorithm (21) provided in the Appion package (22), and most subsequent processing steps were facilitated using Appion. Particle boxing was performed using Eman1.9 (23) and Spider (24). Xmipp (25) was used to normalize the boxed images. There was no correction applied for the contrast transfer function. Initial classification was performed with the CL2D program (26) provided in the Xmipp package (25). Classification steps were followed by manual analysis in which heterogeneities or low-quality picks were excluded.

Images corresponding to homogeneous complexes were inputted into a projection-matching algorithm implemented in Eman 1.9 (23). An unliganded HA (PDB accession no. 4FQV) was low-pass filtered to 30 Å and used as an initial model for the reconstruction by first refining against class averages. The resulting map was then refined against raw particles (128- by 128-pixel box size) for 80 to 90 cycles. Volumes were visualized and interpreted using UCSF Chimera (27). Fourier shell correlation (FSC) curves were calculated using the eotest protocol in Eman 1.9 (23) and were fit to a tanh-based function to estimate the resolutions at an FSC value of 0.5.

Hydrogen bonds and van der Waals contacts were calculated using HBPLUS and CONTACSYM, respectively (28, 29). Surface area upon Fab binding was calculated using MS (30). MacPyMOL (DeLano Scientific) was used to render structure figures. Kabat numbering was applied to the coordinate files using the AbNum server (31). The final coordinates were validated using the JCSG quality control server (version 2.8), which includes MolProbity (32). Structural alignments to calculate root mean square deviation (RMSD) values were performed by iterative fitting on the alpha carbons using the McLachlan algorithm (33) as implemented in the program ProFit (A. C. R. Martin and C. T. Porter, http://www.bioinf.org.uk/software/profit).

The full-length and nonredundant influenza A HA sequences were downloaded from the Influenza Virus Resource at the NCBI database (34). At the time of download (20 March 2013), the data set includes 3,700 human sequences from the H1 subtype. The sequences were aligned using MUSCLE (35) and analyzed using GCG (Accelrys) and custom shell scripts (available from the authors upon request).

Dissociation constant (K_d) values were determined by biolayer interferometry using an Octet RED instrument (ForteBio, Inc.) as previously described (12). Briefly, biotinylated HAs at ∼10 to 50 μg/ml in 1× kinetics buffer (1× phosphate-buffered saline [PBS; pH 7.4], 0.01% bovine serum albumin [BSA], and 0.002% Tween 20) were immobilized onto streptavidin-coated biosensors and incubated with various concentrations of Fab or IgG of 5J8 or CH65. All binding data were collected at 30°C. The k_on and k_off values of each Fab or IgG were measured in real time to determine the K_d values for each HA tested. The sequences of the HA proteins used in the binding studies and the experimental binding curves for each Fab or IgG for fitting k_on and k_off are reported in the supplemental material.

The atomic coordinates and structure factors reported in this paper are deposited in the Protein Data Bank (www.pdb.org; PDB accession no. 4M5Y, 4M5Z, and 4M4Y). The reconstruction data reported in this paper are deposited in the Electron Microscopy Data Bank (www.emdatabank.org; EMDB accession no. EMDB-5731 and EMDB-5733).

To understand the mechanism that antibody 5J8 uses to neutralize a large number of H1 viruses, we determined the crystal structure of its Fab in complex with the globular HA1 head domain of Cali07/2009-H1 (HA1 residues 55 to 271 based on H3 numbering) at a 2.25-Å resolution (Table 1). In addition, the crystal structure of the 5J8 Fab alone was determined at a 1.55-Å resolution and served as a high-resolution starting model for refinement of the complex (Table 1). The asymmetric unit of the complex contains one copy of the 5J8 Fab bound to the monomeric HA1 (Fig. 1). Interestingly, the HA1 was generated from a flagellin-HA1 recombinant fusion protein in Escherichia coli and is a vaccine candidate termed STF2.HA1 (13). However, STF2.HA1 was cleaved over time in our storage buffer and crystallization conditions (Fig. 2), which differ from the vaccine formulation buffer where the vaccine candidates are stable, and only the 5J8-HA1 complex crystallized. In spite of being produced in bacteria and undergoing refolding procedures, the HA1 does indeed properly fold and aligns well with a similar bacterially expressed and refolded HA1 (36) as well as full-length HAs produced from insect cells (4) (Fig. 3A). The lack of carbohydrates on the bacterially expressed HA1 does not affect the folding and antigenic integrity; almost all of the conventional HA antigenic sites, Sa, Sb, Ca1, Ca2, and Cb, which have been classically characterized using polyclonal mouse antisera (37, 38), have good structural integrity and align well with the bacterially expressed and refolded HA1 and full-length HAs from the H1 subtype (4, 36) (Fig. 3A). However, residues in and around the Cb site as well as the eight C-terminal residues, which are located at the opposite end of the RBS, have weak electron density and could not be completely modeled. This conformational heterogeneity is likely attributable to the absence of normal stabilizing secondary structural elements in the minimal construct used. A longer construct may provide additional secondary structural elements that stabilize this region of the structure, as observed in other antibody complexes with HA1 fragments (6, 39) (Fig. 3B).

Consistent with previous epitope mapping and hemagglutination inhibition studies (11), 5J8 recognizes the area in and around the RBS and contacts the Sb and Ca2 antigenic sites, a region of HA that has not previously been observed to be involved in crystal structures of other HA complexes with antibodies (Fig. 4). In particular, 5J8 contacts conserved residues in the structural elements that form the RBS: the 130 loop, 190 helix, and 220 loop. Additional contacts are made outside the RBS at the 140 loop. The antibody recognizes HA using all three complementarity determining region loops (CDRs) of both the heavy and light chains as well as the light chain framework 3 region. A total of 1,298 Ų is buried upon binding (657 Ų on HA and 641 Ų on the Fab). The heavy chain and light chain contribute 56% and 44% of the buried surface area, respectively, and the light chain contributes a larger proportion of the buried surface area than seen for some other Fab-HA complexes that target the RBS (5–7, 9, 10, 39).

The HCDR3 of 5J8 reaches into the HA RBS, makes up 47% of the buried surface area contributed by the antibody, and contacts highly conserved residues that participate in sialic acid receptor binding (Fig. 4). Most notably, the carboxylate moiety of Asp^H100b overlaps closely with the sialic acid carboxylate and utilizes the same network of hydrogen bonding interactions with conserved RBS residues (Fig. 5). In addition, Pro^H100a inserts into a hydrophobic pocket formed by the highly conserved Trp153 and Leu194, which are conserved in 100% and 95.8% of human H1 strains, respectively, in 3,700 sequences from the Influenza Virus Resource at the NCBI database (34). Other RBS-targeted antibodies have also been observed to insert a hydrophobic residue into this RBS pocket, indicating a common recurring recognition motif at this site (5–7, 9, 10).

In addition to the RBS contacts, the 140 loop of HA is buried by the antibody and comprises 22% of the buried surface on HA. Although the 140 loop is established as part of the Ca2 antigenic site, no other antibody complex structure has exhibited such extensive contacts with this site. Most contacts between 5J8 and the 140 loop are van der Waals interactions. Additionally, a salt bridge is formed between Lys145 and Asp^L51, as well as a main-chain–main-chain hydrogen bond between Lys145 and Gly^L29 that stabilizes the interaction (Fig. 6A). Three other electrostatic and seven other hydrogen bonding interactions stabilize the interface between 5J8 and Cali07/2009-H1.

Due to the weak association of the HA ectodomain protomers in the wild-type 2009 H1 protein that causes it to have a high propensity to be purified as a monomer (40–42), we investigated mutations that would stabilize the HA stem trimer interface. A mutation from HA2 Glu47 (H3 numbering), which interacts with a γ-turn at HA1 position 30 from a neighboring protomer, to Gly47 was found to stabilize the trimer (Fig. 7). We then determined the stabilized-trimer 2009 H1 crystal structure at a 2.20-Å resolution (Table 1) and observed that the HA2 Glu47Gly mutation appears to relieve steric restraints between HA protomers. We previously reported use of a similar HA2 Ala47Gly mutation to stabilize the bat H17 HA to determine its structure (43). The crystal structures of the native and mutant HA are nearly identical, with Cα root mean square deviation (RMSD) values of 0.5 Å (HA1), 0.4 Å (HA2), and 0.6 Å (HA protomer). We are currently investigating whether this mutation is also beneficial for increased stability of other HA trimers that have weak association between protomers.

Extensive crystallization trials were performed with 5J8 Fab in complex with full-length HAs, and although we were able to grow crystals of the Fab in complex with full-length HAs from 1918, 1986, and 2009 viruses, these crystals diffracted only to ∼8 Å and quickly decayed from radiation damage. Instead, we turned to EM and generated reconstructions of 5J8 Fab in complex with full-length A/Singapore/6/1986 (H1N1) and trimer-stabilized A/California/04/2009 (H1N1) HAs at resolutions of 22 Å and 23 Å, respectively (Fig. 8). These EM reconstructions confirm the binding data and show that 5J8 does indeed target the apex of HA at and around the RBS in the HA trimer, occupying all three potential binding sites.

To further probe the binding specificity and breadth of 5J8, binding studies of Fab and IgG were performed by biolayer interferometry against a panel of 12 H1 strains that spanned from 1918 to 2009. Nine strains were bound by 5J8 ranging from very weak (K_d > 1 μM) to very strong (K_d < 1 nM) affinity (Table 2). Remarkably, 5J8 extends its binding breadth using avidity, as the bivalent binding of IgG has substantially higher apparent affinity to HA than the monovalent Fab. Moreover, the IgG recognizes divergent H1 strains where no detectable binding was observed for the Fab, as for A/AA/Marton/1943, A/Beijing/262/1995, and A/New Caledonia/20/1999. However, these strains are bound by the IgG with a K_d around or greater than 250 nM, which we have previously shown is not sufficient for neutralization, as for antibody S139/1 (7). These binding data are in good agreement with the published neutralization data, which show that 5J8 binds HAs that span decades, including the pandemic strains that circulated in 1918, a sporadic case in 1977, and the most recent 2009 outbreak (11).

Among the pandemic HA strains bound by 5J8, the single-residue insertion at position 133a is in common. This insertion is present in 71.7% of H1 isolates (Fig. 4). The 133a residue is typically lysine (in 97.5% of the human H1 strains that possess the 133a insertion) but can also be a similarly positively charged arginine. The Lys133a amine forms an electrostatic interaction with the Asp^L53 carboxylate and appears to be a common determinant for recognition by 5J8 (Fig. 6B). The importance of this residue has been previously determined from escape mutants and mutagenesis studies, as deletion of the 133a residue, or mutation to Gln or Ile, eliminates binding by 5J8 (11). Another important electrostatic interaction is formed between Lys222 (conserved in 99.7% of human H1 strains) and Asp^L95a, which are both buried in the interface (Fig. 6C). The Lys222Gln escape mutant (11) would disrupt favorable electrostatic interactions and bury a charged residue in the interface and, hence, eliminate recognition by 5J8. In addition, A/duck/Alberta/345/1976 HA possesses Glu222, which would likely form a destabilizing interaction with Asp^L95a. The strains that are not bound by 5J8 contain Glu190 versus Asp190 for the strains that are bound by 5J8. Although Asp190 and Glu190 are both acidic, it is probable that the longer Glu side chain would clash with the antibody (Fig. 6D). Interestingly, Asp190 mediates favorable interactions with α2,6-linked glycans (42) and is highly conserved in 90.7% of human H1 strains.