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. Author manuscript; available in PMC: 2017 Nov 9.
Published in final edited form as: Cell Rep. 2017 Sep 19;20(12):2935–2943. doi: 10.1016/j.celrep.2017.08.084

Antibody 27F3 broadly targets influenza A group 1 and group 2 hemagglutinins through a further variation in VH1-69 antibody orientation on the HA stem

Shanshan Lang 1,2,*, Jia Xie 3,*, Xueyong Zhu 1,*, Nicholas C Wu 1, Richard A Lerner 3, Ian A Wilson 1,2,4
PMCID: PMC5679313  NIHMSID: NIHMS903157  PMID: 28930686

SUMMARY

Antibodies that target both group 1 and group 2 influenza A viruses are valuable for therapeutic and vaccine development, but only a few have been reported to date. Here we describe a new VH1-69 antibody 27F3 that broadly recognizes heterosubtypic HAs from both group 1 and group 2 influenza A viruses. Structural characterization of 27F3 Fab with A/California/04/2009 (H1N1) hemagglutinin illustrates 27F3 shares the key features observed in other VH1-69 antibodies to the HA stem. Compared to other VH1-69 antibodies, the 27F3 VH domain interacts with the HA stem in a distinct orientation, which alters its epitope and may have influenced its breadth. The diverse rotations of VH1-69 antibodies on the HA stem epitope highlight the different ways that this antibody family can evolve to broadly neutralize influenza A viruses. These results have important implications for understanding how to elicit broad antibody responses against influenza virus.

eTOC Blurb

Lang et al. uncover a human VH1-69 antibody that neutralizes group 1 and group 2 influenza A viruses and demonstrate the consensus and differences in the mode of binding of antibodies from this class with the HA stem.

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INTRODUCTION

Influenza virus remains a global health concern due to the ability of seasonal viruses to constantly and rapidly mutate. Furthermore, new pandemic influenza viruses can arise through genetic reassortment with the vast repertoire of avian and other zoonotic influenza viruses. The first influenza pandemic in the 21st century emerged in 2009 from swine-origin H1N1 viruses (Trifonov et al., 2009). These viruses have continued to circulate and have now become seasonal flu strains (Arriola et al., 2014). In 2013, avian H7N9 caused a major outbreak in Eastern Asian, mainly in China with sporadic cases since then (FAO, 2017). To mitigate against infection from new circulating viruses, seasonal influenza vaccines are formulated each year and neuraminidase (NA) inhibitors can be used to ameliorate the symptoms and spread of the virus (Moscona, 2005). However, the seasonal vaccines are not always effective, especially in the elderly, and NA-resistant viruses have begun to emerge over recent years (Chen et al., 2009; Hai et al., 2013; Hayden and de Jong, 2011; Itoh et al., 2015; Moscona, 2005; Ujike et al., 2010; Zhou et al., 2011). Thus, there is constant pressure to discover new therapeutics and to design more universal vaccines for broader and longer-term protection against emerging and seasonal influenza viruses.

Hemagglutinin (HA) is the most abundant glycoprotein on the influenza virus surface and the primary target of the host humoral immune response. However, the HA is also the most variable influenza protein (Obenauer et al., 2006), and has been classified into eighteen subtypes (H1-H18) that populate the two major phylogenetic groups (Ekiert et al., 2012; Wu et al., 2014). The flu viruses that have caused pandemics or the most lethal sporadic outbreaks in humans come from both group 1 and group 2: e.g. H1, H2, and H5 from group 1 HA, and H3 and H7 from group 2 HA. Most of the antibody responses induced by the seasonal flu vaccine or nature infection target the immunodominant HA head domain (Altman et al., 2015; Angeletti et al., 2017; Caton et al., 1982; Das et al., 2013; Gerhard et al., 1981), which is highly susceptible to antigenic drift. As a result, long-term and more universal protection against influenza A viruses has to date not been achievable. Relatively recent studies have shown that broadly neutralizing human antibodies (bnAbs), against the HA can occasionally be elicted in humans by natural infection or vaccination. These antibodies largely target the two highly conserved functional regions of the HA: the receptor-binding site (Ekiert et al., 2012; Hong et al., 2013; Lee et al., 2014; Schmidt et al., 2015; Xu et al., 2013) and the stem region (Corti et al., 2011; Dreyfus et al., 2013; Dreyfus et al., 2012; Ekiert et al., 2009; Friesen et al., 2014; Kashyap et al., 2008; Sui et al., 2009; Throsby et al., 2008). BnAbs against the HA stem (e.g., A10, CR6261, F10, FI6, CR9114, 3.1 and 39.29) neutralize a broad spectrum of heterosubtypic influenza viruses and, therefore, are exceedingly promising for therapeutic and vaccine development. These antibodies block the viral fusion activity that is triggered in the low pH of endosomes (Corti et al., 2011; Dreyfus et al., 2013; Dreyfus et al., 2012; Ekiert et al., 2009; Nakamura et al., 2013; Sui et al., 2009; Wyrzucki et al., 2014). A better understanding of how these rare potent anti-stem bnAbs are selected and evolve is important for rational design of improved vaccines.

Among the anti-stem HA antibodies discovered so far, the VH1-69 class of antibodies has been the dominant group, except for the heavy chains of bnAbs F16 and MAb 3.1 that are encoded by VH3-30 (Corti et al., 2011; Wyrzucki et al., 2014). More recently, several H5N1 vaccine-induced or genetically optimized broadly neutralizing antibodies against the HA stem were identified that utilize VH1-18 and VH6-1 (Joyce et al., 2016; Kallewaard et al., 2016). These antibodies differ from VH1-69 anti-stem antibodies in that they generally utilize both heavy and light chains for antigen binding as well as a junction-encoded residue in CDR H3 to contact the HA stem and its fusion peptide region (Joyce et al., 2016). Notwithstanding, the VH1-69 class antibodies are still the dominant anti-HA stem lineage with most donors identified from many studies (Avnir et al., 2014; Joyce et al., 2016; Ohshima et al., 2014; Pappas et al., 2014; Throsby et al., 2008; Wrammert et al., 2011), and these antibodies use only their heavy chain to contact the HA stem. Certain germline-encoded residues are also essential for activity and remain selectively conserved during affinity maturation (Lingwood et al., 2012; Pappas et al., 2014). The VH1-69 gene is the only human heavy-chain gene that encodes two hydrophobic residues at the tip of the CDR H2 loop (Huang et al., 2004), which provides the ability to target hydrophobic pockets in the HA stem and the structural basis for their stem recognition. However, the activities of many VH1-69 antibodies, although broad, are generally limited to group 1 influenza A viruses, with the only exception being CR9114, which is able to target both group 1 and group 2 HAs (Corti et al., 2011; Dreyfus et al., 2012; Ekiert et al., 2009).

Here we report on a VH1-69 antibody 27F3 that was discovered from a human combinatorial antibody library by phage display methods (Barbas et al., 1991; Gao et al., 1999; Lerner, 2016). Affinity profiling revealed that antibody 27F3 binds potently to 13 different HAs out of 15 strains tested from both group 1 and group 2 viruses, including pandemic A/California/4/2009 (H1N1), emerging A/Vietnam/04/2004 (H5N1) and A/Shanghai/2/2013 (H7N9), as well as several circulating H3 viruses. Genetic sequence analysis reveals that 27F3 originated from a B cell lineage with a VH1-69 germline gene, likely from the *06 allele (Fig. S1, Table 1). The crystal structure of 27F3 in complex with H1 HA (A/California/4/2009) revealed the typical binding features observed in VH1-69 encoded anti-HA stem bnAbs, but with interesting variation in its angle of approach to the HA surface. We also found that 27F3 and CR9114, the two most broadly neutralizing VH1-69 stem antibodies, share a common mechanism of recognition (particularly for CDR H2), which enables them to expand their reactivity to group 2 viruses. Such structural information is important for design of a more universal flu vaccine and small molecule therapeutics.

Table 1.

Allele variation of known VH1-69 genes

Polymorphism of the VH1-69 gene Derived antibodies against the HA stem
Alleles 33 50 53 54 56 71 73 85
*01 A G I F T A E E CR6261 and others (Avnir et al., 2014; Ekiert et al., 2009; Pappas et al., 2014)
*03 A G I F T A E D Several under study (Pappas et al., 2014)
*05 A G I F T T E E
*07 A R I F T A E E
*12 A G I F T A E E Several under study (Avnir et al., 2014; Pappas et al., 2014)
*13 A G I F T A E E
*06 A G I F T A K E CR9114, 27F3, and others (Avnir et al., 2014; Dreyfus et al., 2012; Pappas et al., 2014)
*14 A G I F T A K E
*02 T R I L I A K E
*04 A R I L I A K E
*08 T R I L T A K E
*09 A R I L I A K E
*10 A G I L I A K E
*11 A R I L T A E E
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RESULTS

Discovery of 27F3, a VH1-69 derived, broadly active anti-HA antibody

The 27F3 antibody was initially selected from a human combinatorial scFv phage display library for its cross-reactivity with both H7 (A/Shanghai/02/2013) and H1 (A/California/04/2009) HAs. To further determine its binding breadth and affinity, 27F3 IgG and Fab were purified and analyzed by bio-layer interferometry (BLI) against a panel of HAs of representative influenza A strains from both group 1 and group 2, as well as flu B viruses. Fab 27F3 shows potent heterotypic activity and binds to nearly all influenza HAs tested (13 of 15), but not flu B HA (Table 2). The bivalency of 27F3 IgG significantly enhances its binding as assessed by BLI against all strains tested (Table 2). Such avidity affects are more commonly observed for antibodies against the HA head (Ekiert et al., 2012; Lee et al., 2014), but not against the stem. For most HAs tested from group 1 viruses (including H1, H5, H6, H9, H11, H12, H13, H16), no dissociation of IgG binding was observed during the assay. Binding of 27F3 Fab to HAs from group 2 viruses is relatively modest with Kd’s around 250 nM, but the 27F3 IgG showed significantly increased avidity to group 2 HAs from various H3 strains with Kd < 10 nM and for H7 from (A/Shanghai/02/2013) with Kd < 1 nM using BLI. Consistent with its binding spectrum, 27F3 neutralized recombinant influenza viruses of H1N1, H5N1, and H6N1 from group 1, and H3N2, H7N9 and H10N8 from group 2 in microneutralization assays (Figs. 1A, S2).

Table 2.

Binding of 27F3 Fab and IgG to various influenza HAs.

Strain 27F3 Fab 27F3 IgG
Influenza A Group 1 A/South Carolina/1/1918/(H1N1) ++++ +++++
A/California/04/2009(H1N1) +++ +++++
A/Japan/305/1957(H2N2) >2μM* +
A/Adachi/2/1957(H2N2) >2μM* +
A/turkey/Massachusetts/3740/1965(H6N2) +++ +++++
A/turkey/Wisconsin/1/1966(H9N2) ++++ +++++
A/duck/England/1/1956(H11N6) ++ +++++
A/duck/Alberta/60/1976(H12N5) + +++++
A/gull/Maryland/704/1977(H13N6) + +++++
A/black-headed gull/Sweden/4/1999(H16N3) + +++++
A/Vietnam/1203/2004(H5N1) ++++ +++++
Influenza A Group2 A/Bangkok/1/1979(H3N2) + +++
A/Hong Kong/1/1968(H3N2) + +++
A/Victoria/361/2011(H3N2) + +++
A/Shanghai/02/2013(H7N9) + ++++
Flu B B/Brisbane/33/2008 >10μM* >10μM*
B/Massachusetts/02/2012 >10μM* ≈ 9 μM
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Kd value < 0.1nM is represented as “+++++”; 0.1-1nM as “++++”; 1nM-10nM as “+++”; 10nM-100nM as “++”; 100nM-500nM as “+”.

*

No significant binding was observed at the noted concentration of the Fabs.

Figure 1. Antibody 27F3 neutralizes heterotypic influenza viruses and recognizes the conserved HA stem.

Figure 1

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(A) Neutralization of 27F3 antibodies against representative strains of influenza viruses with the EC50 values. (B) The H1 HA (A/California/04/2009) trimer is shown as solid gray surface with one HA protomer colored with its HA1 in blue and HA2 in green. The 27F3 heavy chain is in pink, and light chain in light grey. Only two Fabs can be seen in this view as the third Fab is hidden behind the trimer at the back. Glycans are not included in the model due to lack of electron density. (C) The 27F3 epitope on H1 HA. The 27F3 heavy chain dominates contacts with the HA stem. The heavy chain CDR loops that contact the HA stem are shown in pink as a backbone trace with side chains of contact residues as sticks. The epitope residues on the HA surface that interact with 27F3 are colored in blue (HA1) and green (HA2) and labeled.

Recognition of pandemic H1 hemagglutinin (2009) by 27F3 Fab

To determine the structural basis of HA stem recognition by 27F3, the crystal structure of Fab 27F3 with H1 (A/California/04/2009) HA was determined to 3.5 Å resolution (Table S1, Fig. 1B, C). Two copies of the H1 HA trimer, each bound by three Fabs, were present in the crystal asymmetric unit. The 27F3 Fab and its interface with the HA stem were well ordered with high quality electron density, while electron density for the HA head domain was not as clear and strong, likely due to the paucity of crystal contacts to the HA head.

Overall, the HA structure targeted by 27F3 shows the critical binding features of other VH1-69 derived, anti-HA stem bnAbs (Dreyfus et al., 2012; Ekiert et al., 2009; Sui et al., 2009). The overwhelming majority of the contacts are mediated by the heavy chain to a hydrophobic groove in the HA stem, which creates an average buried surface of 870Å2 on the HA by the Fab (Fig. 1B, S3E); only ~10 Å2 of that buried surface arises from light chain interactions with the HA stem due to proximity of Tyr33 to HA2 Leu38 (Fig. S5A). Among the heavy-chain contacts, CDRs H2, FR3, and H3 dominate through extensive hydrophobic interactions with the stem groove, while CDR H1 makes two H-bonds with HA2 (Fig. 2A). In CDR H2, Ile53 extends towards Val40 (HA1) and Val52 (HA2), while F54 points in the opposite direction and stacks its aromatic site chain on His18 (HA1) and Trp21 (HA2). In addition, between these hydrophobic contacts, the CDR H2 main chain H-bonds to His38 and Thr318 of HA1 and W21 of HA2 (Fig. 2B). In the FR3 region, the Ile73 somatic mutation contacts a hydrophobic surface between HA1 and HA2. In addition, an H-bond is formed by the germline Asp72 to Ser291 of HA1 (Fig. 2C). CDR H3 contacts to the HA stem are dominated by Tyr100a, which makes aromatic stacking interactions with Trp21 (HA2) and an H-bond from its side-chain hydroxyl to the main-chain carbonyl oxygen of Asp19 (HA2) (Fig. 2D). In addition, Tyr100 H-bonds with Gln42 (HA2) via its main-chain amide, while Phe100b stacks on the aliphatic portion of the Asp19 side chain.

Figure 2. Interaction of the HA stem of A/California/04/2009 (H1N1) by 27F3 heavy-chain CDRs and FR3.

Figure 2

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(A) CDR H1 contacts HA2 mainly though hydrogen bonds. (B) Interactions between CDR H2 and HA are mediated by hydrophobic contacts (side chain) and hydrogen bonding (main chain) of VH1-69 germline (GL) conserved residues Ile53 and Phe54. (C) The somatically mutated Ile73 contacts the hydrophobic HA surface, creating a buried surface area of ~80 Å2. Additionally, the GL conserved Asp72 hydrogen bonds to HA1 Ser291. (D) Tyr100a dominates the CDR H3 contacts with the HA stem via aromatic stacking and hydrogen bonding. Hydrogen bonds are in dotted lines with distances in Å.

Comparison of 27F3 with other VH1-69 derived, anti-HA stem antibodies

Fourteen human alleles have been discovered for the VH1-69 gene family so far. The critical difference between these alleles is whether they encode a Phe or Ile at position 54 of CDR H2 (Table 1) (Avnir et al., 2014; Avnir et al., 2016; Pappas et al., 2014). Many antibodies derived from Phe54-encoding alleles of the VH1-69 germline (mainly the *01, *03, *06, *12 alleles) have been found to have heterosubtypic activity against influenza viruses, and likely target the conserved HA stem (Avnir et al., 2014; Pappas et al., 2014). Only three of these antibodies, CR6261, F10, and CR9114, have had their structures published prior to this study. Similar to other VH1-69 derived stem antibodies, 27F3 interacts with the HA stem primarily through its heavy chain. Sequence alignment of the 27F3 VH domain with VH 1-69 germline alleles and other mature VH1-69 antibodies from this HA stem-binding class is shown in Fig. 3A. 27F3 utilizes the VH1-69 (most likely the *06 allele) signature residues I53, F54 in CDR H2, and a Tyr in CDR H3 to contact the hydrophobic groove between HA1 and HA2. Similar contacts have been observed in antibodies CR6261, CR9114 and F10, indicating the essential roles of these binding motifs in the selection and evolution of such stem-binding antibodies (Fig. 3B–E). Furthermore, 27F3 has a distinct three-residue extension in CDR H3 before Tyr100a that directly contacts the HA stem (Fig. 3B, S4). To accommodate the three-residue extension and orient Tyr100a in a similar way to Tyr98 in the other three VH1-69 antibodies, the CDR H3 forms a bulge at Pro98-Pro99 (cis) that bends the main chain by almost 90° after Pro99. This turn is further stabilized by a π-cation interaction between Arg94 and Y100 (Fig. S4B).

Figure 3. Comparison of VH1-69 derived, anti-HA stem antibodies to the HA stem.

Figure 3

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(A) Amino-acid sequence comparison of the 27F3 VH domain with equivalent CR6261, CR9114 and F10 and VH1-69 germline sequences, with residues buried in the antibody-antigen interface marked in red (B–E) The heavy chain CDRs and FR3 of 27F3, CR9114, CR6261 and F10 that interact with the HA stem (HA1 blue, HA2 green) are shown in a backbone trace with side chains in sticks. To calculate the approach angles of different antibodies compared to 27F3, the HAs in the complexes were aligned and the rotation required to superimpose the VH domain of individual antibodies with 27F3 VH were calculated in Coot. A dotted line is drawn from FR3 residue 73 and the structurally conserved Tyr on CDR H3 to more readily visualize the different relative antibody rotations. (C–E) The VH domains of CR9114, CR6261, F10 are rotated by 13°, 29° and 52° away from the HA2 A-helix compared to the 27F3 VH domain. The CR6261, CR9114 and F10 complex coordinates used here are from PDB files 3GBN, 4FQI, and 3FKU, respectively.

Despite conservation of these essential HA-targeting motifs in VH1-69-encoded antibodies, only 27F3 and CR9114 bind HAs from both group 1 and group 2 viruses, while CR6261, F10 and others are exclusively against group 1. Moreover, these antibodies all approach the HA stem at different angles, which undoubtedly influence their breadth (see also (Dreyfus et al., 2012)). To estimate the rotational differences, we first superimposed the HA stems bound by these VH1-69 antibodies, and then calculated the rotation required to superimpose the VH domain of 27F3 onto the VH of the other Fabs, or vice-versa. The VH domains of CR9114, CR6261, and F10 are rotated on the HA stem by approximately 13°, 29° and 52° from the HA2 A-helix compared to 27F3 (Fig. 3B–E). Distinct rotations of antibodies with similar genetic origins to an epitope have similarly been observed in our HIV studies (Garces et al., 2015; Garces et al., 2014). However, this phenomenon is relatively underappreciated in other viruses. To more easily visualize these differences, we drew a line between FR3 residue 73 and the conserved Tyr in CDR H3 (Fig. 3B–E). 27F3 has a slightly increased interface with H1 HA2 than CR6261; CR9114 also buries more surface on H5 HA2 than F10 (Fig. S3E). Moreover, F10 has its epitope centered on HA1 His38 (Fig. 3E and Fig. S3D), which might have limited its development against group 2 viruses; in contrast, the VH domains of 27F3 and CR9114 rotate away from His38 and create more space to accommodate the glycosylated Asn38 in group 2 viruses (Fig. 3B, C and Fig. S3A, B).

These different angles of approach correlate well with unique mutations in these different antibodies, and in how they occupy the five distinct hydrophobic subpockets in the HA groove (Kadam, Wilson et al., submitted). For instance, 27F3 and CR9114 evolved an Ile73 near the tip of FR3, which inserts into the second subpocket in the hydrophobic groove between HA1 and HA2, whereas CR6261 evolved Phe29 in CDR H1 to fill this same pocket (Fig. 3B–D). On the other hand, in CR6261 and CR9114, another hydrophobic residue, Phe74, further anchors FR3 in the hydrophobic groove through occupation of the first more distal hydrophobic pocket, while avoiding a clash with the HA2 A-helix (Fig. 3C, D). F10 does not have a hydrophobic residue at FR3 position 73 or 74 and rotates further away from the A-helix so that FR3 does not directly contact the stem groove. Instead, a Val27 somatic mutation in CDR H1 now interacts with more distal fifth pockets in the hydrophobic groove and Thr28 interacts with the second hydrophobic pocket as well as forming hydrogen bonds to the HA2 A-helix (Fig. 3E).

Some differences in interaction of the conserved CDR H2 loop are also observed. Unlike Phe54 of CR6261 and F10 that engage His38 (HA1) and Trp21 (HA2) in the fourth hydrophobic pocket (Fig. 3D, E and Fig. 4C, D), 27F3 F54 primarily targets His18 (HA1) and Trp21 (HA2), while His38 (HA1) hydrogen bonds to the 27F3 P52a carbonyl oxygen (Fig. 2B and Fig. 4A), causing the His38 side chain to flip upwards compared to the apo-HA structure (Fig. 4A). A similar conformational change of His38 was observed when H5 HA was bound by CR9114 (PDB 4N5Y, Fig. 4B). Notably, His38 is largely conserved across group 1 viruses with its side chain pointing downwards, while Asn38 in group 2 HAs is glycosylated in many group 2 strains and points upwards (Fig. S3F, G) (Ekiert et al., 2012). The capability of 27F3 and CR9114 to accommodate an upward conformation of HA1 residue 38 may also allow accommodation of the glycan at Asn38 in group 2 HAs. Moreover 27F3 and CR9114 have similar main-chain conformations for CDR H2, with their carbonyl oxygens pointing directly towards the HA stem so as to form hydrogen bonds (Fig. 5A). In contrast, the main-chain orientations in CDR H2 of CR6261 and F10 make it difficult to make such H-bonds to the HA stem (Fig. 5B). Another significant feature of the HA stem from group 2 viruses is the different orientation of the Trp21 indole (Fig. S3H), which may influence the interaction with the conserved IFY motifs in these different antibodies.

Figure 4. Different targeting mechanisms of CDR H2 to the HA stem.

Figure 4

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(A) In complex with 27F3, His38 (HA1) shifts from the downward apo-conformation (grey) to the upward conformation (cyan) to form a hydrogen bond with the CDR H2 P52a main-chain carbonyl oxygen. Phe54 mainly interacts with His18 (HA1) and Trp21 (HA2). (B) The CR9114-bound HA shows two alternative conformations for His38 (PDB 4FQI). Hydrogen bonds are shown as dotted lines. (C, D) CDR H2 of group 1 specific bnAbs CR6261 and F10 target His38 (HA1) and Trp21 (HA2), and His38 points downwards to contact Phe54 of CDR H2 (PDB 3GBN, 3FKU).

Figure 5. Different conformations of CDR H2 in VH1-69 anti HA-stem antibodies.

Figure 5

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Pro52a, Ile53 and Phe54 of CDR H2 in various VH1-69 antibodies to the HA stem are shown in sticks with main-chain carbonyl oxygens labeled. (A) The group 1 and 2 binding antibodies 27F3 and CR9114 share similar main-chain conformations that enable CDR H2 carbonyl oxygens to H-bond with the HA stem. In addition, Phe54 stacks on Trp21 in a relatively planar orientation. (B) The group 1 antibodies CR6261 and F10 have their CDR H2 main chains oriented differently, making it less favorable to form H-bonds with the HA stem.

The disparate orientations of the VH domain relative to the HA stem also affect the relative disposition of the light chain with respect to the stem surface. As VH rotates from its orientation in F10 to that in 27F3, the closest main-chain distance between the stem surface and CDR L3 decreases from 15.5 Å to 8.9 Å (Fig. S5). Furthermore, it appears that the different orientations of these antibodies allow enough distance and space to accommodate the conserved N154 glycan without steric interference. As for side-chain contacts, the closest distances between the HA stem and 27F3 or CR9114 (5.0 Å and 4.9 Å, respectively) are shorter than for CR6261 and F10 (8.6 Å and 6.6 Å), which makes it possible for the light chain of 27F3 and CR9114 to contribute to a small extent (~10 Å2) to the HA buried surface (Fig. S5). Although the light-chain interaction is minor, and likely not essential for binding, the light-chain interface could potentially be evolved further to improve antibody binding.

DISCUSSION

The discovery, isolation and characterization of broadly neutralizing antibodies against influenza virus has provided a great stimulus for the design of novel vaccine and therapeutics against these highly variable pathogens (Impagliazzo et al., 2015; Laursen and Wilson, 2013; Yassine et al., 2015). Prior to this study, CR9114 was the only known anti-HA stem, VH1-69 antibody that is effective against both group 1 and group 2 influenza A viruses. Here, we report the structure and properties of another VH1-69 derived anti-stem antibody 27F3 that neutralizes both group 1 and group 2 influenza A viruses. Overall, 27F3 shows the now classic signature features of VH1-69 anti-HA stem antibodies. Namely, 27F3 binds to the HA stem exclusively through heavy-chain contacts, similar to other VH1-69 derived antibodies against the HA stem (although a very small buried surface between the light chain and HA stem is observed with 27F3 and CR9114, as well as a possible long-range electrostatic interaction in CR9114). 27F3 harnesses highly conserved hydrophobic CDR H2 and H3 residues, especially Ile53, Phe54 and Tyr100a (Tyr98 in the other VH1-69 antibodies) (the IFY motif) to engage a hydrophobic groove in the HA stem.

Despite these conserved contacts among VH1-69-encoded stem antibodies, their variable domains show significantly different rotations with respect to the HA stem (Fig. 3B–E). Moreover, 27F3 utilizes F54 to primarily engage His18 (HA1) and Trp21 (HA2), while inducing a rotamer change of the His38 imidazole (HA1). Similar conformational changes of His38 are observed in H5 HA upon binding of CR9114 (Dreyfus et al., 2012). In addition, 27F3 and CR9114 show similar conformations of the CDR H2 main chain that expose the carbonyl oxygens for H-bond interactions with the HA stem. Such overall similarities appear to allow these two antibodies to accommodate the glycosylated Asn38 in group 2 HAs and attain greater breadth. In summary, the previous finding that the IFY motif of VH1-69 antibodies is essential for their development into broadly neutralizing anti HA-stem antibodies is strongly reinforced here in this study ((Pappas et al., 2014; Tung et al., 2015). Furthermore, the similarities and differences observed in the evolution of these antibodies broaden our understanding of how the VH1-69 class of anti-stem human antibodies are selected and evolve that should accelerate development of immunogens to elicit immune responses against the HA stem.

EXPERIMENTAL PROCEDURES

Screening of broad anti-HA antibodies

A human antibody scFv library with an estimated diversity of 109 was screening with biotinylated H7 HA (A/Shanghai/2/2013 (H7N9) (PMID 24311689). 27F3 was selected for its cross-binding activities to both H7 (A/Shanghai/2/2013) and H1 (A/California/04/2009).

Fab and IgG cloning, expression and purification

27F3 Fab and IgG were expressed in 293F mammalian cells as previously described (Garces et al., 2015; Irimia et al., 2016). The Fabs or IgG were purified from the supernatant by Ni-NTA Superflow (Qiagen), monoS chromatography (GE Healthcare).

Kd determination

Bio-layer interferometry (BLI) was used to evaluate the binding breadth and Kd values of 27F3 Fab/IgG against a panel of HAs (Figs. S6, S7), which were purified and biotinylated as previously described (Lee et al., 2014).

Crystallization and structure determination of the complex of 27F3 Fab with HA (A/California/04/2009(H1N1))

The H1 HA (A/California/04/2009(H1N1)) with a stabilization mutation HA2 E47G was expressed and purified as in previous protocols (Hong et al., 2013; Xu et al., 2012). HA was partially digested with trypsin (molar ratio of protein to enzyme was about 1000:1, at 4°C overnight) to cleave off the tags and convert HA0 into HA1 and HA2. The Fab-antigen complex was crystallized with 1.6M ammonium sulfate, 0.1 M citric acid pH 4.0 as mother liquor. The complex structure was determined by molecular replacement in Phaser (McCoy et al., 2007) and further refined in Refmac (Skubak et al., 2004) and Phenix (Adams et al., 2010).

Structural analysis

Hydrogen bond and buried molecular surface analyses were calculated using the PDBePISA server of EMBL-EBL. Structure figures were generated by MacPyMol (DeLano Scientific LLC).

Supplementary Material

supplement

Highlights.

  • Antibody 27F3 was isolated from a human combinatorial scFv phage display library

  • 27F3 targets most group 1 and group 2 influenza A viruses

  • 27F3 possesses the IFY motif, the signature of VH1-69 Abs that bind the HA stem

  • Variable VH1-69 VH orientations on HA stem evolve via distinct somatic mutations

Acknowledgments

We thank Henry Tien from the Wilson lab and robotics core at the Joint Center for Structural Genomics (JCSG) for automated crystal screening, Wenli Yu and Yuanzi Huang (Wilson lab, TSRI) for excellent technical support, and Dr. Jesse Bloom (Fred Hutchinson Cancer Research Center) for the pHH21-PB1flank-eGFP plasmid, 293T-CMV-PB1 cells, and MDCK-SIAT1-CMV-PB1 cells. X-ray diffraction data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL). Use of SSRL SLAC National Accelerator Laboratory is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The research was supported by R56 AI117675 (to IAW) and the Skaggs Institute for Chemical Biology. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS, NIAID or NIH. This is manuscript 29507 from The Scripps Research Institute.

Footnotes

ACCESSION NUMBERS

The atomic coordinates and structure factors will be available immediately upon publication in the Protein Data Bank (PDB) under accession code 5WKO for Fab 27F3 in complex with H1 HA (A/California/4/2009).

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures and one table and can be found within this article online.

AUTHOR CONTRIBUTIONS

J.X and R.A.L. isolated and sequenced the 27F3 antibody. S.L. performed protein purification and biological assays. S.L and X.Z. collected X-ray diffraction data, and determined the structure. N.C.W. performed the microneutralization assay. S.L., X.Z., and I.A.W. analyzed the structural data. S.L., J.X, and I.A.W wrote the paper. All authors edited the manuscript.

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