Structure and Function Analysis of an Antibody Recognizing All Influenza A Subtypes

Abstract

Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.

Graphical abstract

Figure 1

Developmental Pathway of the MEDI8852 Lineage (A) Alignment of VH and VL amino acid sequences of four mutated antibodies with their UCA and branchpoint (BP) configurations and MEDI8852. Amino acid substitutions are highlighted in red. Residue positions are according to Kabat numbering. Dots indicate identical residues. Boxes indicate CDR borders according to IMGT (solid line) and Kabat (dashed line). (B) Genealogy trees of VH (left) and VL (right) nucleotide sequences generated using dnaml. The number of mutations is indicated on the branches with amino acid substitutions in parentheses. (C) Neutralization of influenza A viruses. IC₅₀ values were determined against a panel of 25 influenza A isolates. Values above 50 μg/ml were scored as negative (dashed line). Average IC₅₀ values were obtained from at least two independent experiments. Full viral strains designations are listed in Table S1. See also Figure S1.

Figure 2

MEDI8852 Binds to All Influenza A HA Subtypes and Exhibits Neutralization of Influenza A Seasonal and Non-seasonal Viral Strains (A) Phylogenetic tree of influenza A HAs. Group 1 and group 2 colored in red and blue are further subdivided into 3 clades (H8, H9, and H12; H1, H2, H5, and H6; H11, H13, and H16) and 2 clades (H3, H4, and H14; H7, H10, and H15), respectively. (B) ELISA binding average EC₅₀ values of FY1 and MEDI8852 to purified recombinant HA proteins. (C) Binding of FY1 and MEDI8852 to surface-expressed HA proteins as determined by flow cytometry. Shown are MFI values. (D and E) FY1 and MEDI8852 neutralization IC₅₀ values were determined against a panel of 36 seasonal influenza A isolates (D) and 13 non-seasonal influenza viruses (E). (F) Neutralization average IC₅₀ values of MEDI8852, 39.29, FI6v3, and CR9114 were determined from at least two independent experiments using a panel of 24 seasonal and non-seasonal influenza viruses and plotted as a single symbol. Full viral strains designations are listed in Tables S1 and S2.

Figure 3

MEDI8852’s Antiviral Mechanisms of Action (A) HA cleavage inhibition assay of uncleaved HA0 recombinant proteins of A/New Caledonia/20/99 (H1N1) or A/Hong Kong/8/68 (H3N2), pre-treated with MEDI8852 or a non-relevant isotype control antibody, MPE8v3, after digestion with TPCK-trypsin for 0, 5, 10, 20, or 40 min. (B) Inhibition of low pH-activated conformational change in HA showing SDS PAGE gels of H5 HA with and without MEDI8852, incubated at decreasing pH values and neutralized after digestion with TPCK- trypsin. (C) Fusion inhibition assay using MEDI8852 (solid) or MPE8v3 (open) incubated with A/Puerto Rico/8/34 (H1N1) virus (red) or A/Aichi/2/68 (H3N2) virus (blue) and human red blood cells and exposed to low pH to induce viral fusion. Percent fusion inhibition was calculated based on the amount of hemoglobin present in the supernatant. (D) ADCC activity on A549 cells infected with A/Puerto Rico/8/34 (H1N1) and incubated with MEDI8852 (red) or MPE8v3 (black) antibody in the presence of human NK cells, antibody-dependent killing was measured in quadruplicate by LDH release. (E) ADCP activity on MDCK cells expressing H1 HA from A/South Dakota/06/2007 that were labeled CFSE and incubated with MEDI8852 (red), or an irrelevant control, R347 (black) antibody in the presence of violet-labeled human macrophages in duplicate. Percent phagocytosis was determined by the amount of total macrophages that were labeled with violet and CFSE. (F) CDC activity on MDCK cells infected with A/Puerto Rico/8/34 (H1N1) and incubated with a serial dilution of MEDI8852 (red) or MPE8v3 (black) antibody in the presence of rabbit complement. Antibody-dependent killing was measured in triplicate by LDH release. Error bars represent two times the SD at each antibody concentration. See also Figure S2.

Figure 4

MEDI8852 Provides Dose-Dependent Protection from Lethal Influenza Infection in Mice and Ferrets Even When Treatment Was Delayed (A) Kaplan-Meier survival curves. (B) Lung viral titers on day 5 post infection determined by TCID50 assay after mice were treated with MEDI8852 at 3, 1, 0.3, 0.1, and 0.03 mg/kg (single i.p. dose) and then infected with CA/2009 H1. (C) Kaplan-Meier survival curves. (D) Lung viral titers on day 5 post infection in mice infected with WSN/33 H1 virus, on study day 0, then treated with MEDI8852 or irrelevant control antibody, R347 (single i.p. dose) at 10 mg/kg at various days post infection. (E) Kaplan-Meier survival curves. (F) Lung viral titers on day 5 post infection in mice infected with rHK/68 H3 virus, on study day 0, then treated with MEDI8852 at 10 mg/kg or R347 (single i.p. dose) at various days post infection. (G) Kaplan-Meier survival curves of ferrets infected with 1LD90 of A/Vietnam/1203/2004 H5N1 virus on study day 0. Treatment with MEDI8852 at 25 mg/kg (closed symbols solid line), oseltamivir at 25 mg/kg (open symbol dashed line), or R347 (open symbol solid line) was initiated at the indicated day post infection. (H) Temperature of ferrets treated with MEDI8852, or oseltamivir at various days post infection. Dotted line designates the average normal temperature of a ferret at 38.5°C. Error bars represent the SE of the mean for each determination. ^∗For murine studies, significance was determined compared to control antibody treatment with p < 0.005 for survival (log-rank test) and p < 0.05 for lung viral titers (Student’s t test); for ferret survival studies, significance was determined by comparing to oseltamivir on the indicated initiation day with p < 0.05 for survival (log-rank test). See also Figure S3 and Table S3.

Figure 5

MEDI8852 Binds to a Unique Site within the H5 and H7 HA Proteins (A) Overview of MEDI8852 in complex with H5 hemagglutinin. One HA protomer and the cognate MEDI8852 Fab fragment are highlighted in color, the other two copies in the trimer are colored gray. The HA1 polypeptide is colored blue, the HA2 polypeptide is colored red, with the fusion peptide at the N terminus of HA2 highlighted in yellow. The heavy chain of the MEDI8852 Fab is colored orange, the light chain is colored green. (B) Overlay of MEDI8852 bound to group 1 (H5) and group 2 (H7) HAs. The antibodies are shown in cartoon representation together with Helices A and B of the HA. The components are colored according to (A) and the view orientation is approximately that shown by the black arrow in (A). (C and D) H5 (C) and H7 (D) HAs are shown in surface representation. Only the HA residues in the MEDI8852 binding epitope that differ between H5 and H7 are labeled. The CDR loops of MEDI8852 that are in contact with HA are shown in cartoon and stick representation and colored by element. See also Figures S4, S7, and Table S4.

Figure 6

Binding Epitope of MEDI8852 on H5 HA (A) HA is shown in surface representation and residues that are contacted by MEDI8852 are highlighted in color (blue for HA1, red for HA2 and yellow for fusion peptide residues). Secondary structure elements of HA are shown in cartoon representation. The hydrophobic groove on HA is outlined in gray. The CDR loops of MEDI8852 that are in contact with HA are shown in cartoon representation and colored orange and green for the heavy and light chains, respectively. The colored boxes indicate the three parts of the binding epitope that are shown in more detail in (B), (C), and (D). (B) Interactions of MEDI8852 with the hydrophobic groove of H5 HA. HA is drawn in surface representation, with the main chain shown in cartoon representation and amino acids that are in contact with MEDI8852 shown in stick representation. W21, which adopts different rotamers in group1 and group 2 influenza viruses, is colored magenta. MEDI8852 is also shown in cartoon representation, with contact residues shown in stick representation. Hydrogen bonds and salt bridges are indicated by dashed lines. (C) Interactions of MEDI8852 with the fusion peptide of H5 HA. Shown in the same style as in (B). (D) Interactions of MEDI8852 with the base of helix A of H5 HA. Shown in the same style as in (B). (E) Location of mutations found during affinity maturation of FY1 to MEDI8852. The variable domains of MEDI8852 are shown in cartoon representation, viewed from the direction of HA. Regions of the heavy and light chains in contact with HA are colored orange and green, respectively. Interacting sidechains are shown in stick representation. Residues that differ between the parental and affinity-maturated antibody are shown in sphere representation. (F) Sequences of MEDI8852 variable region framework and CDR residues. CDRs (according to Kabat) are highlighted in orange and green for the heavy and light chains respectively. Residues in contact with HA are colored red and residues changed during affinity maturation from FY1 are colored cyan, with corresponding residues of FY1 indicated. See also Figure S6.

Figure 7

MEDI8852 Binds to a Unique Site within the H5 and H7 HA Proteins through CDR-H3 and CDR-L1 Conformational Rearrangements upon Complex Formation (A) Conformational rearrangements in MEDI8852 on complex formation. Conformational change of the CDRH3 and CDR-L1 loops upon HA engagement. The apo structure of MEDI8852 is shown in blue, the bound structure is shown in orange and green for the heavy and light chains, respectively. The beginning and end of the moving regions are indicated with black ovals. HA (H7) is shown as a gray surface. The apo structure does not make interactions with HA and does not fit into its surface features—the conformational change is necessary for productive HA engagement. (B) Epitopes of different broadly neutralizing antibodies on the HA surface. Residues of HA that are in contact with the heavy chain are colored orange, residues that are in contact with the light chain are colored green, and residues that are in contact with both chains are colored yellow. The blue box encases the part of the MEDI8852 epitope (helix A and hydrophobic groove) that can also be found in other broadly neutralizing antibodies as well as group 1 specific ones. The red box encases the part of the MEDI8852 epitope that can also be found in group 2 specific antibodies (middle of fusion peptide). (C) Comparison of the structures of the conserved CDRH3 tetra-peptide in the complexes between MEDI8852 and H7 HA (left panel) and 39.29 and H3 HA (right panel). In both cases the tetra-peptide is shown in stick representation with other loops of the antibody shown as coil, colored as in panel A. The HAs are shown in surface representation. See also Figure S7.

Figure S1

Affinity Maturation Pathway of the FI6 Lineage, Related to Figure 1 (A) Alignment of VH and VL amino-acid sequences of the FI6 lineage mutated antibodies with their UCA and branchpoint (BP) configurations. Residue positions are according to Kabat numbering. Dots indicate identical residues. Boxes indicate CDR borders according to IMGT (solid line) and Kabat (dashed line). (B) Genealogy tree of FI6 lineage VH and VL nucleotide sequences generated using dnaml. The number of mutations is indicated on the branches with amino-acid substitutions in parentheses. Background color and shape identify the origin and the year of isolation. (C) Neutralization of influenza A viruses. Neutralization IC₅₀ values were determined against a panel of 25 seasonal and non-seasonal influenza A isolates. Values above 50 μg/ml were scored as negative (dashed line). Average IC₅₀ values were obtained from at least two independent experiments.

Figure S2

MEDI8852 Fc-Effector Function Activity against H3N3 Viruses, Related to Figure 3 (A) ADCC activity on A549 cells infected with with A/Honk Kong/8/68 (H3N2) and incubated with MEDI8852 (red solid dot) or an irrelevant control, MPE8v3 (black solid dot) antibody in the presence of human NK cells, antibody dependent killing was measured in quadruplicate by LDH release. (B) ADCP activity on MDCK cells expressing H3 from A/Hong Kong/8/68 that were labeled with CSFE and incubated with MEDI8852 (red solid square), or an irrelevant control, R347 (black solid square) antibody in the presence of violet labeled human macrophages in duplicate. Percent phagocytosis was determined by the amount of total macrophages that were labeled with violet and CSFE. Error bars represent two times the standard deviation at each antibody concentration.

Figure S3

MEDI8852 Provides Dose-Dependent Protection from Lethal Influenza Infection in Mice Even When Treatment Was Delayed, Related to Figure 4 (A) Lung function was measured by pulse oximetry on Day 6 post-infection with CA/09 H1 in mice treated with mice were treated with a single IP dose of MEDI8852 at 3, 1, 0.3, 0.1, and 0.03 mg/kg, 3 mg/kg irrelevant control antibody, or non-treated non-infected animals (naive). Error bars represent two times the standard error of the mean at each antibody concentration. ^∗p < 0.05 significance determined by ANOVA with multiple comparison’s test. (B) Kaplan-Meier survival curves. (C) Lung viral titers on Day 5 post infection determined by TCID50 assay after mice were infected with rHK/68 H3 virus on study Day 0, then treatment with MEDI8852 at 10 mg/kg (closed symbols with solid line), Oseltamivir at 25 mg/kg BID for 5 days (open symbol with dashed line, or irrelevant control antibody, R347 (black solid) was initiated at the indicated Day post infection. Error bars represent the standard error of the mean for each determination. ^∗, For survival studies p < 0.005 significance compared to control antibody treatment by Log-Rank test; for lung titers, p < 0.05 significance compared to control antibody treatment by student’s t test.

Figure S4

Overview of MEDI8852 in Complex with H7 Haemagglutinin and Unbiased Electron Density Maps, Related to Figure 5 (A) One H7 HA monomer and the cognate MEDI8852 Fab fragment are highlighted in color, the other two copies in the trimer are colored gray. The HA1 polypeptide is colored blue, the HA2 polypeptide is colored red, with the fusion peptide at the N terminus of HA2 highlighted in yellow. The heavy chain of the MEDI8852 Fab is colored orange, the light chain is colored green. (B and C) The MEDI8852 CDR loops H1 and H2 (B) and H3 and L1 (C) are shown in complex with H5 HA. All six instances of the variable domain of the heavy or light chains were omitted from the model after rigid-body refinement and omit maps were calculated. Figures of merit were determined in Sigmaa and six-fold NCS averaging was performed in DM. The maps were sharpened by a negative B-factor of 75 Å². The resulting maps are shown at a σ level of 1.0 within 2.0 Å distance of MEDI8852 atoms. H5 HA is shown in surface representation and colored gray, MEDI8852 is shown in cartoon and stick representation and colored orange for the heavy chain and green for the light chain. MEDI8852 side chains that are not in contact with HA are colored gray.

Figure S5

Sequence Comparison of MEDI8852 Epitope among 18 HA Subtypes and Corresponding Contact Residues in the MEDI8852 Paratope, Related to Figures 5 and 6 The occurrence of amino acid identities was analyzed at different HA residues among each HA subtype, only amino acids with a frequency higher than 1% are shown. In total, 12′472 H1, 404 H2, 12′263 H3, 1’005 H4, 694 H5, 1’152 H6, 119 H7, 119 H8, 1’838 H9, 590 H10, 487 H11, 153 H12, 67 H13, 16 H14, 10 H15, 7 H16, 2 H17 and 1 H18 isolates were analyzed, respectively.

Figure S6

Mutations Associated with Maturation of FY1 to MEDI8852, Related to Figure 6 Heavy chain: Y32 (MEDI8852) instead of N32 (parental): The Y32 sidechain is within hydrogen bonding distance with HA and makes a stabilizing π stacking interaction with R52B, which is in direct contact with V18 in the fusion peptide of HA. G54 (MEDI8852) instead of K54 (parental): This amino acid is located in the middle of the CDRH2 loop in an unusual turn between the two beta strands of CDRH2. The backbone at G54 adopts a conformation that is only possible with a glycine sidechain. The parental K54 would lead to a different backbone conformation. Light chain: T33 (MEDI8852) instead of L33 (parental): This residue is next to CDRL1 and especially Y32, which undergo a conformational change upon binding. The T33 sidechain stacks against the sidechain of F71 in unbound and bound conformations, anchoring the entire CDRL1 region during the conformational change. The leucine in the parental antibody could make a similar stacking interaction, but would anchor CDRL1 in a different conformation due to the additional methyl group of the leucine. R54 and G55 instead of L54 and Q55 (parental): R54 and G55 are in CDRL2 and connected with CDRL1 through backbone hydrogen bonds. The sidechain of Q55 in the parental antibody would sterically clash with L46 and completely alter the backbone and sidechain conformation the LCDR1 and LCDR2 loops. G55 has no sidechain and thus does not clash. Therefore, R54 and G55 enable and stablilize the backbone conformation observed in the complex, which might form a stable platform for the conformational change in CDRL1 necessary for binding.

Figure S7

Comparison between MEDI8852, CR8020, CR9114, and FI6, Related to Figure 7 (A and B) Antibody-HA complex structures were overlayed by aligning their HA2 polypeptides. For clarity, only the HA of the MEDI8852/H5 complex is shown. All proteins are shown in cartoon representation, with HA1 colored blue, HA2 colored red and the fusion peptide colored yellow. The CR8020 (A) and CR9114 (B) Fab fragments are colored light and dark gray, respectively, and the MEDI8852 Fab fragment is colored orange (heavy chain) and green (light chain). (C and D) The antibodies are shown in cartoon representation with group 1 bound antibody colored in magenta and group 2 colored in cyan from the N terminus of the heavy chain to the C terminus of the light chain. Helix A and helix B of HA2 are shown in cartoon representation. FI6v3 is rotated between group 1 and group 2 leading to a 11-13.5 Å distance between the ends of the variable domains. By contrast, for MEDI8852, there is a much smaller rotation of about 1 degree and only 2-1.5 Å displacement between the ends of the variable domains in the complexes with H5 HA (group 1) and H7 HA (group 2).