Allosteric Neutralization by Human H7N9 Antibodies

Abstract

The avian influenza A virus H7N9 causes severe human infections with more than 30% fatality despite the use of neuraminidase inhibitors. Currently there is no H7N9-specific prevention or treatment for humans. From a 2013 H7N9 convalescent case occurred in Hong Kong, we isolated four H7 hemagglutinin (HA)-reactive monoclonal antibodies (mAbs) by single B cell cloning, with three mAbs directed to the HA globular head domain (HA1) and one to the HA stem region (HA2). Two clonally related HA1-directed mAbs, H7.HK1 and H7.HK2, potently neutralized H7N9 and protected mice from a lethal H7N9/AH1 challenge. Cryo-EM structures revealed that H7.HK1 and H7.HK2 bind to a β14-centered surface partially overlapping with the antigenic site D of HA1 and disrupt the 220-loop that makes hydrophobic contacts with sialic acid on the adjacent protomer, thus affectively blocking viral entry. The more potent mAb H7.HK2 retained full HA1 binding and neutralization capacity to later H7N9 isolates from 2016-2017, which is consistent with structural data showing that the antigenic mutations of 2016-2017 from the 2013 H7N9 only occurred at the periphery of the mAb epitope. The HA2-directed mAb H7.HK4 lacked neutralizing activity but protected mice from the lethal H7N9/AH1 challenge when engineered to mouse IgG2a enabling Fc effector function in mice. Used in combination with H7.HK2 at a suboptimal dose, H7.HK4 augmented mouse protection. Our data demonstrated an allosteric mechanism of mAb neutralization and augmented protection against H7N9 when a HA1-directed neutralizing mAb and a HA2-directed non-neutralizing mAb were combined.

Fig. 1. Isolation and characterization of human H7N9 mAbs in vitro.

(A) FACS depicting the staining and selection of H7-specific B cells from donor H7N9.HK2013 PBMCs 1 year post recovery. SSC-A, side scatter area; FSC-A, forward scatter area. (B) ELISA binding curves of the indicated mAbs to soluble recombinant H7N9 HA and H7N7 HA (upper panels), with or without Endo H treatment, to the matching H7N9 HA1 from 2013 or HA1s from 2016 and 2017 (middle panels), and to 6 other non-H7 HA or HA1 proteins (lower panels). (C) Neutralization curves of H7.HK mAbs against H7N9 2013 (left) and 2016 (right) pseudoviruses infecting MDCK cells. Data shown are mean ± SEM.

Fig. 2. Structural analysis of H7.HK1 and H7.HK2 in complex with H7 HA trimer.

(A) Cryo-EM structures of H7.HK1 and H7.HK2 bound to H7 HA in the head region. (B) Top view of alignment of H7.HK1 and H7.HK2 complex structures. (C) Surface presentation of the H7.HK1 epitope (orange) on H7 HA1, with interacting CDRs shown. (D) H7.HK1 heavy chain forms seven hydrogen bonds and one salt bridge with H7 HA1. (E) H7.HK1 light chain forms one additional hydrogen bond with H7 HA1, and the interactions are stabilized by hydrophobic residues on the periphery of the light chain interface. (F) Modeling published structures of H7 HA1-binding antibodies (PDB: 6II4, 6II8, 6II9, 5V2A) onto the H7.HK1 bound structure, with an escape mutation R47K (green) reported for mAb 07–5F01. (G) Modeling the binding site of human receptor analogue LSTc (red) based on a previous crystal structure (PDB: 4BSE) onto H7 from the H7.HK1 complex, showing that H7.HK1 does not compete with sialic acid on the adjacent protomer (black). (H) Alignment of the H7.HK1 complex with a previous crystal structure of H7 (PDB: 4BSE) shows that the 220-loop (pink) required for sialic acid binding (G209-G219) is disorder in the complex structure and would clash with the H7.HK1 light chain if it were present. Green asterisk symbol denotes the <2 Å clash between the CDR L1 N33 and the predicted location of P212 on HA1.

Fig. 3. Prophylactic and therapeutic effects of human H7N9 mAbs in mice i.n. challenged with 10 LD₅₀ of A/Anhui/1/2013 H7N9.

(A) Mice were i.p. injected with 100 μg (equivalent of 5 mg/kg) or 20 μg (equivalent of 1 mg/kg) of the indicated mAbs (as human IgG1 unless otherwise specified) one day before viral challenge; % survival (less than 20% weight loss) and % body weight of survived mice were plotted over time. (B) Mice were i.p. injected with 100 μg of the indicated mAbs one day after viral challenge; % survival and % body weight of survived mice were plotted over time. Arrows indicate the time when mAbs were administered. Control groups of a non-H7 placebo mAb and PBS were included. Data for each group were combined from 1–2 experiments and shown as mean – SEM. Asterisk symbols denote statistical significance with P values < 0.05.