Research Article

A Highly Conserved Neutralizing Epitope on Group 2 Influenza A Viruses

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Science  12 Aug 2011:
Vol. 333, Issue 6044, pp. 843-850
DOI: 10.1126/science.1204839

Abstract

Current flu vaccines provide only limited coverage against seasonal strains of influenza viruses. The identification of VH1-69 antibodies that broadly neutralize almost all influenza A group 1 viruses constituted a breakthrough in the influenza field. Here, we report the isolation and characterization of a human monoclonal antibody CR8020 with broad neutralizing activity against most group 2 viruses, including H3N2 and H7N7, which cause severe human infection. The crystal structure of Fab CR8020 with the 1968 pandemic H3 hemagglutinin (HA) reveals a highly conserved epitope in the HA stalk distinct from the epitope recognized by the VH1-69 group 1 antibodies. Thus, a cocktail of two antibodies may be sufficient to neutralize most influenza A subtypes and, hence, enable development of a universal flu vaccine and broad-spectrum antibody therapies.

Influenza viruses cause millions of cases of severe illness each year, thousands of deaths, and considerable economic losses. Currently, two main countermeasures are used against flu. First, small-molecule inhibitors of the neuraminidase surface glycoprotein and the viral ion channel M2 have been widely used and proven to be quite effective against susceptible strains (1). However, resistance to these antivirals has reduced their effectiveness, and mutations associated with oseltamivir and amantadine are widespread (24). The second main countermeasure is vaccination. Current vaccines that are based on inactivated viruses elicit a potent immune response against viruses that are closely matched to the vaccine strain (5). However, predicting which strains will dominate annually is difficult, and mismatches between the vaccine and circulating viruses lead to little or no protective effect (6, 7). A vaccine that stimulates production of a robust, broadly neutralizing antibody response would be a considerable advance.

Hemagglutinin (HA) is the major envelope glycoprotein of influenza A viruses and the target of almost all neutralizing antibodies. HA is synthesized as an immature polypeptide chain called HA0, which is activated upon cleavage by host proteases to yield two subunits, HA1 and HA2. The HA1 “head” subunit of HA mediates attachment of the virus to target cells through interactions with sialic acid receptors. After endocytosis of the virus, the low pH triggers conformational changes in HA2, leading to fusion of the viral and endosomal membranes and release of the viral genome into the cytoplasm. Most neutralizing antibodies bind to the exposed loops that surround the receptor binding site and interfere with attachment (812). Because these loops are highly variable, antibodies targeting these regions are strain-specific, explaining why immunity after natural exposure or vaccination is typically restricted to the current circulating strains.

Recently, we described the isolation and characterization of CR6261, a broadly neutralizing antibody with activity against group 1 influenza viruses (1315). Similar antibodies using the same VH1-69 germline heavy chain have also been reported (16, 17). The discovery of such antibodies has raised hopes for the development of monoclonal antibody (mAb)–based immunotherapy and a universal vaccine (1826). Crystal structures of CR6261 in complex with H1 and H5 HAs revealed a highly conserved epitope in the HA stalk (13). CR6261 neutralizes most group 1 HAs, including H1, H5, H9, and some H2s, but has no activity against group 2 viruses (14). Group 2 includes the currently circulating human H3 and avian H7 viruses, which sporadically cross from birds into humans and have the potential to develop into a future pandemic. Consequently, antibodies complementary to CR6261 and related VH1-69 antibodies, but with broad activity against group 2 viruses, are critical for the formulation of antibody-based therapies.

CR8020 activity in vitro. Using a recently described method to generate stable mAb-producing, B cell receptor–positive, human memory B cells, we isolated H3 HA reactive clones from the blood of donors recently vaccinated against influenza (27). Subsequent screening of the resulting immunoglobulins (Igs) for reactivity with other HA subtypes led to identification of mAb CR8020, which recognizes H3 and H7 HAs, as well as representative HAs from other group 2 subtypes, but not group 1 HAs (Fig. 1, A and C). In this sense, CR8020 is complementary to previously described mAbs, such as CR6261, which neutralizes most group 1 HAs (14) but not group 2 HAs (Fig. 1, A and C). CR8020 binds most group 2 HAs with high affinity, including H3 isolates spanning 50 years of virus evolution [dissociation constant (Kd) ~1 to 35 nM for H3, H7, and H10 HAs] (Fig. 1C). Consistent with binding to an apparently highly conserved epitope, CR8020 potently neutralizes a wide spectrum of H3 influenza strains as well as H7 and H10 viruses (Fig. 1B) (28). In contrast, a control mAb against the HA1 head neutralizes only a narrow spectrum of H3 viruses (table S1).

Fig. 1

In vitro binding and neutralization activity 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, whereas blue indicates positive binding by CR6261. Subtypes that have not been tested are indicated in black. (B) In vitro neutralization of CR8020 against a panel of influenza A viruses as determined by means of microneutralization assay. Concentrations of CR8020 required to reduce virus replication by 50% (IC50) are reported in micrograms per milliliter. (C) Affinity measurements (Kd) 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.

Efficacy of CR8020 in vivo. Prophylaxis using 3 mg/kg CR8020 protected mice against challenge with a high, lethal dose of either mouse-adapted H3N2 or H7N7 virus (Fig. 2, A and B). Consistent with the absence of any signs of respiratory distress, groups of H3N2-challenged mice that received 30, 10, or 3 mg/kg CR8020, and H7N7-challenged mice receiving 10 or 3 mg/kg CR8020 all showed increases in body weight at the study’s end (Fig. 2, A and B) (29). In contrast, animals that received an irrelevant control antibody rapidly lost weight, showed signs of respiratory distress, and succumbed to infection or were euthanized within 2 weeks after challenge. A 1 mg/kg dose was only partially protective against mortality to either virus (Fig. 2, A and B), and the weight loss was not significantly different from the controls (P = 0.666 and P = 0.633 for groups challenged with H3N2 and H7N7 virus, respectively). Therapeutic treatment with 15 mg/kg of CR8020 up to 2 days after infection with H3N2 completely prevented mortality, whereas 50% of the mice treated 3 days after infection were protected (Fig. 2C). Treatment with CR8020 could be started even later after H7N7 challenge because 100% of the mice treated 3 days after infection and 50% treated 4 days after infection survived, although one animal treated 2 days after challenge succumbed to infection (Fig. 2D). Therapeutic treatment did not prevent morbidity as illustrated by initial weight loss, but all surviving animals appeared healthy and were regaining weight at the study’s end.

Fig. 2

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. Shown are 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. Shown are 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-H3 HA crystal structure. To elucidate how mAb CR8020 can neutralize multiple group 2 influenza virus subtypes, we determined the crystal structure of CR8020 Fab in complex with the HA from the 1968 H3N2 pandemic [A/Hong Kong/1/1968 (H3N2), “HK68”] at 2.85 Å resolution (table S2). The overall structure of the HK68 HA in the complex (Fig. 3A) is similar to those of other unliganded H3s (30), although some subtle pH-induced changes are discussed below (31).

Fig. 3

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 purple, 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 β 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. (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). CR8020 binds HA with both chains, using five of the six CDRs (LCDR3 makes no contacts). Key antibody side chains are shown. (D) Footprint of HA on CR8020 combining site, highlighting antibody residues contacting HA and the usage of both the heavy and light chains in recognition (purple and cyan, respectively).

Fab CR8020 binds HK68 HA at the base of the stem in close proximity (~15 to 20 Å) to the viral membrane (Fig. 3, A and B) (32), which is lower down the stalk than any other flu antibody characterized to date (Fig. 4A). Despite its proximity to the viral membrane, this epitope is accessible on virions (fig. S1), which is in accord with the in vitro and in vivo potency of this mAb. The epitope consists of two main components: (i) the outermost strand (HA2 residues 30 to 36) of the 5-stranded β sheet near the base of the stalk and (ii) the C-terminal portion (HA2 residues 15 to 19) of the fusion peptide (Fig. 3B), as well as a few peripheral contacts with other surrounding residues (33). Compared with CR6261, CR8020 recognizes its epitope in a more conventional manner, using both heavy and light chains (Fig. 3, C and D). A total surface area of 1280 Å2 is buried, of which 81% arises from binding of the heavy chain and 19% from the light chain. The fusion peptide component accounts for ~50% of the van der Waals contacts between Fab and HA and is specifically recognized by heavy chain complementarity determining regions (HCDRs) 1 and 3, where Phe32 (HCDR1) and Trp100c (HCDR3) make many of the key hydrophobic interactions (Fig. 3C). HCDR3 also interacts extensively with the outermost strand of the β sheet, in a pseudo β-strand–like interaction with HA2 residues 32 to 36 (fig. S2). Together, the CR8020 interactions with the fusion peptide and edge of the β-sheet account for >80% of the van der Waals contacts and six out of seven hydrogen bonds.

Fig. 4

CR8020 binds a unique, highly conserved site in the HA 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 3SDY); purple, CR6261 (PDB codes 3GBM and 3GBN); green, 2D1 (PDB code 3LZF); blue, HC19 (PDB code 2VIR); orange, HC45 (PDB code 1QFU); cyan, HC63 (PDB code 1KEN); pink, BH151 (PDB code 1EO8). mAb F10 (PDB code 3FKU) 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 group 2 and group 1 HAs, respectively. Coloring is similar to that in (A), with CR8020 epitope in red, CR6261 epitope in dark 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. 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 (B), looking from the Fab (not shown) toward the epitope. (D) Location of CR8020 epitope residues in the prefusion and (E) postfusion states, in which they reside on critical regions of the fusion peptide and helix-capping loops 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 postfusion structures). Residue positions were mapped onto the structures from PDB codes 1QU1 and 2KXA.

CR8020 binds an epitope on the HA stem. The crystal structures of CR6261 and F10 revealed a neutralizing epitope on the HA stem that is conserved and accessible in most group 1 influenza viruses (13, 17). However, an N-linked glycosylation at HA1:Asn38 in most group 2 HAs may restrict antibody access to this epitope. Consequently, antibodies with broad neutralizing activity against group 2 viruses would be expected to recognize an epitope that is spatially distinct from that recognized by CR6261. Indeed, of the 15 and 20 residues that constitute the epitopes targeted by CR8020 and CR6261, respectively, only two residues (Asp19 and Leu38) are in common (Fig. 4, A and B). Thus, CR8020 defines a second, neutralizing epitope in the HA stem that is present in all group 2 HAs tested thus far.

To investigate the breadth of CR8020’s cross-neutralizing activity, we examined the epitope conservation across all 16 influenza A virus subtypes by examining all full-length, nonredundant HA sequences in the National Center for Biotechnology Information Flu database (34, 35). Around half of the contact residues are either identical (>95%, HA2 residues 16, 33, 35, and 36) or conserved (>99%, HA2 residues 18, 30, and 146) across all 16 subtypes (Fig. 4C and tables S3 and S4) (36), whereas other contact residues are conserved only across group 2 (>95% identity, HA2 25; >99% conserved, HA1 325 and HA2 15 and 19). Thus, 11 of 15 residues contacting CR8020 are >99% conserved across all group 2 HAs, whereas the remaining residues are more variable (HA2 32, 34, 38, and 150 are conserved in only ~56 to 81% of isolates) (Fig. 4C and table S3). However, virus neutralization and in vitro binding data suggest that most natural variation is well tolerated by CR8020 (Table 1). Essentially, all natural variation in the epitope commonly observed in group 2 viruses is represented in the extensive panel of wild-type H3, H4, H7, H10, H14, and H15 isolates and engineered variants of HK68 that were tested for binding and/or neutralization (Table 1 and Fig. 1, B and C). CR8020 binds nearly all of these naturally occurring HA variants with high affinity (Kd ~1 to 10 nM), similar to the HAs from group 2 viruses that are neutralized by CR8020 (~1 to 35 nM). However, one variant (Asp19Asn) correlates with markedly reduced affinity and loss of neutralizing activity in vitro and is discussed below. Overall, these results show that the core of the CR8020 epitope is highly conserved across all group 2 viruses, whereas natural variation in the surrounding residues is well tolerated and does not adversely affect binding and most likely neutralization.

Table 1

Most natural variation in CR8020 epitope in group 2 HAs has little effect on binding. Kd, presence of a glycosylation site at HA1 position 21 (predicted to block binding in group 1 HAs), and protein sequence of CR8020 contact residues are shown. Data presented here include targeted mutants of HK68 and wild-type isolates selected to probe the impact of natural variation in epitope on CR8020 activity. Wild-type HK68 is included at the top, as a reference. The bottom row summarizes the most common naturally occurring variants at each position (present in at least >1% of all group 2 sequences), with tested variants in bold and underlined. Residues that differ from the HK68 sequence are in black boxes.

View this table:

The structural constraints imposed by the membrane fusion activity enforce strict conservation of many regions of HA2. To understand why the CR8020 contact residues are so well conserved in group 2 viruses, we examined their potential role in membrane fusion. The fusion peptide (HA2 1 to 25) is critical for membrane fusion and is nearly invariant across all influenza A viruses (fig. S3) (37, 38). The fusion peptide adopts an unusually tight, helical-hairpin conformation (34), which provides an elegant, structural explanation for conservation of most residues, including CR8020 contact residues HA2 16 and 18 (Fig. 4, C to E, and fig. S4A). After exiting the membrane and traversing a short linker region, residues close to HA2:31-38 cap the ends of the long, three-helix bundle of the postfusion structure (Fig. 4, D and E, and fig. S4, A and B), constraining the identity of the amino acids in the cap, which are thought to make a substantial energetic contribution to the membrane fusion process (39). Therefore, elements of the CR8020 epitope probably have critical roles in driving the fusion process.

Group 2 restriction and antibody escape. Antibody escape variants selected by CR8020 in an H3N2 virus exhibited mutations in HA2 Asp19Asn or Gly33Glu close to the CR8020 epitope center. A recombinant H7N7 virus with the Asp19Asn mutation also escaped neutralization by CR8020 (table S5 and S6). This mutation disrupts a possible salt bridge to VL Arg53, presumably leading to destabilization of the antibody-HA interaction (40). The Gly33Glu mutation inserts a large side chain into a highly confined space in the antibody-antigen interface. However, both variants are relatively rare in influenza A viruses (fig. S5), particularly in human isolates. Glycine is strongly preferred at position 33 (8716/8720 sequences, group 1 and 2 HAs), whereas position 19 is an Asp in ~95% of all group 2 viruses and in 1534 of 1537 human H3 viruses (41). Whether these mutations negatively affect viral fitness is not known, but other antibodies may recognize the CR8020 epitope in subtly different ways that would render them less sensitive to these substitutions. Notwithstanding, our results suggest that CR8020 will neutralize most viruses from H3, H7, and H10 subtypes, and possibly H15 (42).

Two avian group 2 subtypes (H4 and H14) are bound by CR8020 with only moderate affinity (Table 1), and neutralization of an H4 virus was undetectable in vitro (43). The H4 and H14 isolates tested differ from HK68 at two contact positions (Glu15Gln and Gln34Thr), and each substitution confers a modest reduction (~10-fold) in CR8020 affinity for HK68 (Table 1) (44). Thus, reduced affinity of CR8020 for H4 and H14 can largely be accounted for by the combined effect of these mutations, but subtle structural differences in noncontact residues surrounding the epitope may also have substantial effects on antibody binding.

Although the HA surface recognized by CR8020 is also relatively well conserved in group 1 viruses, no group 1 viruses tested were bound or neutralized (table S7). Several key differences in group 1 HAs may account for lack of CR8020 reactivity. First, the Gln or Thr that predominates at position 34 in group 2 HAs is substituted by a bulkier Tyr in many group 1 subtypes and would likely clash with HCDR3. CR8020 binding to a nonnatural, Gln34Arg HK68 variant is reduced by more than a factor of 100, further suggesting that larger residues cannot be accommodated. Second, group 1 HAs have a highly conserved N-linked glycosylation site at HA1:Asn21 (5801/5813 group 1 sequences analyzed), which is adjacent to CR8020 HCDR1 (fig. S6). In most configurations, the glycan would conflict with CR8020 VH and probably interfere with antibody binding (45). Lastly, several individual substitutions in CR8020 contact residues that modestly reduce affinity (by a factor of 5–10) are combined in group 1 isolates and may reduce binding below our detection threshold (~10 uM) (Table 1 and table S7).

Mechanism of neutralization. Unlike most HA antibodies, which block attachment, CR8020 has no detectable activity in hemagglutination-inhibition (HAI) assays and does not compete with antibodies against the head, which is consistent with CR8020 binding to the stalk region. With the exception of HA1 residue 325, the CR8020 epitope maps entirely to HA2. Upon exposure to low pH, HA2 undergoes extensive conformational rearrangements, bringing the viral and target membranes into close proximity and triggering fusion (Fig. 4, D and E) (39, 46). Consequently, CR8020 is poised to inhibit these conformational changes, blocking membrane fusion and viral entry. Whereas CR8020 binds readily to cell surface–expressed H3 HAs in both its uncleaved (HA0) and cleaved (HA) forms, it did not bind after HA exposure to a pH of 4.9 (Fig. 5A), which is in agreement with previous findings that the epitope structure is not maintained in the fusion-active conformation (46, 47). When CR8020 was added before the pH shift, it remained bound after the pH was lowered (Fig. 5B), indicating that the epitope remains intact at low pH and suggesting that CR8020 inhibits the pH-induced conformational changes in HA. Moreover, CR8020 prevented dithiothreitol (DTT)–mediated dissociation of HA1 and HA2 at low pH (48), which would only be expected if the HA was retained in the prefusion state. Similar results were obtained with H7 and H10 HAs, indicating that the mechanism of inhibition is conserved (fig. S7).

Fig. 5

CR8020 inhibits the fusogenic conformational changes in HA and blocks proteolytic activation. (A) Fluorescence-activated cell sorting (FACS) binding of CR8020 (open bars) and a control mAb against the head (solid 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). 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); and trimeric HA2 (tHA2). Binding is expressed as the percentage of binding to untreated HA (HA0). Error bars represent SD of data obtained in three independent experiments. Ribbon diagrams are adapted from (57). (B) FACS binding of CR8020 (open bars) and a control mAb against the head (solid 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 antihead mAb was added last to detect whether the HA1 heads remained associated with HA2. (C) SDS–polyacrylamide gel electrophoresis results of the protease-susceptibility assay for H3 and H7 HAs. Exposure to low pH converts HAs to the protease-susceptible, postfusion state (lane 3). Pretreatment with CR8020 blocks the pH-induced conformational change, retaining HA in the protease-resistant, prefusion 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% bovine serum albumin. Uncleaved hemagglutinin (HA0) was detected using a polyclonal serum against H3.

Further evidence that this mAb acts by inhibiting conversion from the prefusion to post-fusion conformation is illustrated by lack of protease susceptibility of the HA at low pH in the presence of CR8020. Exposure to low pH followed by trypsin digestion results in complete degradation of H3 and H7 HAs (Fig. 5C, lanes 1 to 4). In contrast, when the HA is pretreated with CR8020, most HA is retained in a protease-resistant, prefusion form (Fig. 5C, lanes 5 to 8). Furthermore, the crystals used for the structural studies were grown below pH 5.4, which is the fusion pH for HK68 HA (fig. S8) (49). Nevertheless, the HA is retained in its prefusion state, even after extended exposure to low pH. However, the HA1 heads have opened up slightly, and the B loop adopts an alternate backbone conformation as compared with neutral pH structures. The head opening is believed to be one of the first steps in the membrane fusion process, and the B loop must refold from an extended random coil to an α helix to deliver the fusion peptide to the target membrane. Thus, our crystal structure appears to have captured an early fusion intermediate (50), trapped by the binding of CR8020. Taken together, our data suggest that CR8020 blocks fusion by sequestering the fusion peptide and preventing its release at low pH.

Furthermore, CR8020 may interfere with fusion by inhibiting activation of HA0 by host proteases. Whereas HA0 was rapidly cleaved by trypsin in vitro into HA1 and HA2, this cleavage was completely inhibited in the presence of CR8020 but not by a control, head-binding mAb (Fig. 5D). Thus, blocking HA0 maturation to HA1/HA2 may represent an additional mechanism by which mAbs can block viral entry.

Implications for therapy and vaccines. Influenza A viruses responsible for human pandemics have arisen from both group 1 (H1N1 and H2N2) and group 2 (H3N2) viruses (Fig. 1A). In addition, zoonotic viruses from both groups sporadically infect humans and have the potential to trigger future pandemics (including H5N1 and H9N2 from group 1, and H7N2 and H7N7 from group 2). Consequently, an ideal, universal therapy should provide protection against both group 1 and group 2 influenza viruses, as well as influenza B. Attempts to isolate broadly neutralizing antibodies against group 2 viruses from animals have generally yielded antibodies reported as nonneutralizing or of only modest potency or breadth (24, 5155), and broadly neutralizing human antibodies against group 2 viruses have not been previously described. A cocktail of antibodies, such as CR6261 and CR8020, may protect against essentially all influenza A viruses implicated in human disease. Such a therapeutic cocktail would have undisputed benefits for high-risk groups, such as the elderly and immunocompromised, and for severe, life-threatening influenza infections. These antibodies also represent an ideal immunological solution to influenza infection and could serve as a guide for development of vaccines that elicit broader, long-lasting immunity. The identification and characterization of CR6261-like antibodies has already sparked considerable advances, including (i) their detection in some individuals (21) and (ii) design of immunization strategies that efficiently elicit stem antibodies in mice, ferrets, and monkeys (20, 25). Thus, the identification and characterization of CR8020 should facilitate similar advances for group 2 viruses, bringing us one step closer to the ultimate goal of a universal vaccine for influenza.

Supporting Online Material

www.sciencemag.org/cgi/content/full/science.1204839/DC1

Materials and Methods

Figs. S1 to S11

Tables S1 to S7

References

References and Notes

  1. The most common vaccine formulations include influenza A H1N1 and H3N2 and influenza B components.
  2. HAs cluster into two distinct groups on the basis of their primary sequence. Group 1 HAs include 10 of the 16 subtypes: H1, H2, H5, H6, H8, H9, H11, H12, H13, and H16. Group 2 HAs account for the remaining 6 subtypes: H3, H4, H7, H10, H14, and H15.
  3. Materials and methods are available as supporting material on Science Online.
  4. Although CR8020 binds to H4 and H14, the affinity is probably too low to result in in vitro neutralization, at least at the concentration tested here. In vitro neutralization of H14 and H15 viruses was not tested.
  5. All increases in body weight were statistically significant (P ≤ 0.018), except for the H7N7 challenge group treated with 3mg/kg CR8020 (P = 0.168).
  6. The final model includes HA1 residues 11 to 328, HA2 residues 1 to 175, CR8020 heavy chain residues 2 to 222, and light chain residues 1 to 212. The asymmetric unit contains one HA protomer bound to one CR8020 Fab, with the additional protomers in the trimer generated by crystallographic symmetry operations.
  7. In this regard, CR8020 can be thought of as being analogous to the envelope antibodies to HIV 2F5, 4E10, and Z13. These antibodies recognize the membrane-proximal external region (MPER), a short helical peptide from the gp41 subunit that is closely associated with the viral envelope.
  8. CR8020 also contacts (i) HA1 residue 325, near the C terminus of the chain; (ii) HA2 residue 25, in the second strand away from CR8020 in the small sheet; (iii) HA2 residue 38, at the bottom of helix A; and (iv) HA2 residues 146 and 150, in a short helix adjacent to the sheet. In addition to protein-protein interactions, the Fab also makes contacts with the core fucose of a universally conserved glycan linked to Asn154 in HA2, although the extent of the contribution of this interaction to the overall binding energy is unclear.
  9. Details of the data set and analysis can be found in the supporting online material on Science Online.
  10. In this analysis, residues are considered conserved if they fall within one of the following groups: Asp/Asn/Glu/Gln, Phe/Tyr, Ile/Leu/Val/Met, Lys/Arg, or Ser/Thr.
  11. Twenty of the first 23 positions in HA2 are well conserved across all subtypes (fig. S3). Two of the remaining positions (HA2 positions 12 and 15) have differing, group-specific residues. The final position, HA2 residue 19, is also conserved as Asp or Asn across most subtypes from both groups. However, we regard this substitution as nonconservative in the context of CR8020 because Asp19Asn mutations escape virus neutralization.
  12. Although there is clear density for the VL:Arg53 side chain placing the guanidinium moiety in close proximity to HA2:Asp19, the preferred rotamer for the Arg side chain cannot be assigned unambiguously. Various rotamers consistent with the observed electron density result in charged atom contact distances of ~4 to 4.5 Å. Although somewhat longer than expected for a salt bridge that would make a major contribution to antibody binding, this discrepancy may be in part due to shielding by a nearby sulfate from the crystallization solution, which is sandwiched between the VL:Arg53-Arg54 side chains.
  13. Most of the group 2 HAs with Asp19Asn are restricted to a single, geographically restricted lineage of the H7 subtype, and disproportionate sampling in birds in this region may exaggerate the prevalence of Asp19Asn substitutions in the H7 population (35).
  14. Neutralization of H15 has not been tested, but the Kd for CR8020 binding is comparable with that of H7, which is neutralized.
  15. Neutralization of H14 viruses has not been tested, but comparable binding of CR8020 to H4 and H14 suggests that H14 will not be neutralized.
  16. H14 also has a Glu325Gly mutation in HA1, which has a negligible effect on CR8020 binding in a HK68 background.
  17. This scenario is reminiscent of the group 1 restriction of CR6261, which cannot interact with group 2 viruses, such as H3 and H7, at least in part due to a conserved glycan at HA1:Asn38 and the more general and well-documented use of glycans to mask and unmask surfaces to evade immune recognition, such as vividly illustrated in the evolution of human H1N1 viruses (12, 56).
  18. In contrast, a control mAb against the HA1 head bound to A/Wisconsin/67/2005 HA in all three conformations, and binding was only lost after DTT treatment, which dissociates HA1 from HA2 in the postfusion state.
  19. This is evidenced by the fact that, in this case, treatment with DTT did not diminish CR8057 binding to A/Wisconsin/67/2005 HA. In line with its narrow spectrum of neutralization (table S1), CR8057 did not bind to any conformation of the HAs of A/Hong Kong/1/1968 or A/Hong Kong/24/1985.
  20. Because the initial crystals only appeared between 3 and 7 days after the start of the experiment, CR8020 must be capable of retaining HA in the prefusion state for several days at low pH.
  21. Acknowledgments: We thank H. Tien and D. Marciano of the Robotics Core at the Joint Center for Structural Genomics for automated crystal screening; T. Doukov and the staff of the SSRL BL9-2 for beamline support; X. Dai and R. Stanfield for excellent assistance with data collection, processing, and analyses; R. Lerner, J. Paulson, and D. Burton for valuable comments and insightful discussion; E. Geelen, D. Spek, and V. Klaren for excellent assistance and advice; K. Hegmans, A. Lourbakos, J. Meijer, and A. Apetri and their teams for producing the mAbs and Fabs; C. Y. H. Leung for providing the A/WF/Hong Kong/MPA892/06 virus; E. de Boer-Luijtze and technicians in the groups of P. van Rossum and S. Riemersma for assistance with the animal experiments; E. Brown from Ottawa University, Canada for the mouse-adapted A/Hong Kong/1/68 strain; and A. Dingemans for critical review of the manuscript. This project has been funded in part by the National Institute of Allergy and Infectious Diseases, NIH, Department of Health and Human Services, USA, under contract HHSN272200900060C; the Area of Excellence Scheme of the University Grants Committee, Hong Kong (grant AoE/M-12/06); a predoctoral fellowship from the Achievement Rewards for College Scientists Foundation (D.C.E.); grant GM080209 from the NIH Molecular Evolution Training Program (D.C.E.); and the Skaggs Institute (I.A.W.). Portions of this research were carried out at the Stanford Synchrotron Radiation Lightsource, a national user facility operated by Stanford University on behalf of the U.S. Department of Energy (DOE), Office of Basic Energy Sciences. The Stanford Synchrotron Radiation Lightsource (SSRL) Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research and by NIH, National Center for Research Resources, Biomedical Technology Program, and the National Institute of General Medical Sciences. This is publication 20951 from the Scripps Research Institute. Coordinates and structure factors have been deposited in the Protein Data Bank (PDB code 3SDY). Nucleotide sequences for the CR8020 variable regions have been deposited in GenBank (accession nos. JN093122, JN093123). A patent application relating to antibody CR8020 has been filed (International Publication Number WO2010/130636). Sharing of materials will be subject to standard material transfer agreements.
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