A Potent Germline-like Human Monoclonal Antibody Targets a pH-Sensitive Epitope on H7N9 Influenza Hemagglutinin

Abstract

The H7N9 influenza virus causes high-mortality disease in humans but no effective therapeutics are available. Here we report a human monoclonal antibody, m826, that binds to H7 hemagglutinin (HA) and protects against H7N9 infection. m826 binds to H7N9 HA with subnanomolar affinity at acidic pH and 10-fold lower affinity at neutral pH. The high-resolution (1.9 Å) crystal structure of m826 complexed with H7N9 HA indicates that m826 binds an epitope that may be fully exposed upon pH-induced conformational changes in HA. m826 fully protects mice against lethal challenge with H7N9 virus through mechanisms likely involving antibody-dependent cell-mediated cytotoxicity. Interestingly, immunogenetic analysis indicates that m826 is a germline antibody, and m826-like sequences can be identified in H7N9-infected patients, healthy adults, and newborn babies. These m826 properties offer a template for H7N9 vaccine immunogens, a promising candidate therapeutic, and a tool for exploring mechanisms of virus infection inhibition by antibodies.

Keywords: ADCC; H7N9 influenza; germline; hemagglutinin; monoclonal antibody.

Graphical abstract

Figure 1

The Schematic Biopanning Process and the Characterization of H7 HA-Specific mAb from the Antibody Library (A) The biopanning strategy of H7 HA1-specific mAbs from the naive antibody phage display library. (B) Polyclonal phage ELISA showing the binding of the first to fifth rounds (Rd 1 to Rd 5) of phages to HA and HA1. Bound phages were detected with anti-M13-HRP conjugate. (C) Binding of Fab m826 to four different subtypes of influenza virus HA. The error bars reflect the SD. (D) Immunogenetic analysis of the heavy- and light-chain variable regions of m826 using the IMGT tool. (E) Germline-rooted circular phylogenetic tree of m826-like antibody sequences found in IgM libraries derived from healthy human adults, neonates, and H7N9-infected patients. The sequences of m826 and the clone identical to m826 are shown in red. Sequence ID started with CB represents sequences derived from the neonates, started with HH represents sequences derived from the healthy adults, and started with H7N9 represents that from the H7N9-infected patients. The phylogenetic tree was constructed by the Neighbor-Joining method.

Figure 2

Binding Profiles of m826 mAb to HA and HA1 Measured by BLI in OctetRED96 The m826 mAb was immobilized on activated AR2G biosensors. The analytes consisted of serial dilution (between 100 and 1.2 nM) of trimeric H7N9 HA at pH 5.0 (A) or pH 7.4 (B), H7N9 HA1 at pH 7.4 (C), or 100 nM H1N1 and H3N2 HA at pH 7.4 (D). Binding kinetics was evaluated using a 1:1 Langmuir binding model by ForteBio Data Analysis 7.0 software.

Figure 3

Analysis of Detailed Interactions between the m826 Fab and H7N9 HA (A and B) The crystal structures of Fab m826 in complex with H7N9 HA1; view from the side (A) and the top (B). (C) Close-up of the three CDRs of heavy chain from the m826 bound to HA. Side and main chains involved in hydrogen bonding are shown as sticks, with hydrogen bonds depicted as black dotted lines. Residues of the HA protein are labeled. CDR regions are shown as tubes. (D) The three CDRs of light chain also make direct contacts with HA to form several hydrogen bonds (black dashed lines) with the HA residues. (E) Amino acid sequences of m826 are shown with Kabat numbering. The CDRs are colored and amino acids involved in HA binding are labeled with ^∗.

Figure 4

m826 mAb Recognizes a Unique Epitope on H7 HA (A) Docking of the m826-HA1 complex onto the crystal structure of monomeric H7 HA. The epitope of m826 is distinct from the RBS. (B) Superposition of the antibody epitope which was partially buried in the trimeric HA structure. (C) Sequence alignment of HA1 from the H7N9, H7N7, and H7N4, and highlighting of structurally defined influenza antibody epitopes. The conventional antigenic sites (Sa, Sb, Ca1, Ca2, and Cb) are shaded in blue, the epitope of m826 is shaded in red, and RBS is shown in the black box.

Figure 5

m826 mAb Does Not Neutralize H7 Virus but Mediates Potent ADCC (A) The immunostaining-based neutralization assay for m826 mAb against H7N4 virus. Immunostaining was conducted using influenza NP-specific mAbs to detect virus-infected cells. H7N4-specfic Ab was used as positive control. (B) Confocal images of pCMV3-H7N9-HA-GFP transfected 293T cells stained with 100 nM m826 IgG or an unrelated anti-hepatitis B virus human IgG G12 as the control. Green signal, GFP; red signal, detecting with Alexa Fluor 647-conjugated goat anti-human IgG. Scale bar, 25 μm. (C) ADCC-related signaling in Jurkat T cells engineered to express human FcγRIIIa triggered by m826 and CR9114 IgG in the presence of H7N9 HA-expressing 293T cells. An unrelated anti-MERS-CoV IgG m336 was used as negative control. (D) ADCC activity of m826 and CR9114 IgG against H7N9 HA-expressing 293T cells mediated by freshly prepared human PBMCs. The percentage (%) of specific lysis was calculated as described in the STAR Methods. All error bars reflect SD.

Figure 6

Prophylactic and Therapeutic Efficacy of mAb m826 in Protecting Mice against Lethal Dose H7N9 Challenge (A–D) For prophylactic efficacy study, mice were treated (intraperitoneally [i.p.]) with m826 antibody 12 hr before viral challenge (intranasally [i.n.]) with 10 × LD₅₀ of H7N9 virus and were monitored daily for 14 days for the accumulated mortality (A) and weight loss (B), expressed as percent (%) weight loss and survival, respectively (n = 5 per group). PBS was used for the control group. For therapeutic efficacy study, mice were treated (i.p.) with m826 antibody 12 hr after viral challenge (i.n.) with 10 × LD₅₀ of H7N9 virus and were monitored daily for 14 days for the accumulated mortality (C) and weight loss (D). The weight loss of (B and D) represents mean change in body weight per group and SD was also shown. (E) Evaluation of histopathological changes in the pulmonary tissues of uninfected mice and mice from therapeutic, prophylactic, and control group. The black, yellow, and blue arrows represented the H7N9 infection-induced inflammation of blood vessel, alveoli, or capillary, respectively. (F) Evaluation of histopathological changes in the bronchial tissues. The black, yellow, blue, and green arrows represented bronchial epithelial cell, bronchial alveoli, capillary, and interstitial tissue, respectively.