A pan-influenza monoclonal antibody neutralizes H5 strains and prophylactically protects through intranasal administration

Abstract

Avian A(H5N1) influenza virus poses an elevated zoonotic threat to humans, and no pharmacological products are currently registered for fast-acting pre-exposure protection in case of spillover leading to a pandemic. Here, we show that an epitope on the stem domain of H5 hemagglutinin is highly conserved and that the human monoclonal antibody CR9114, targeting that epitope, potently neutralizes all pseudotyped H5 viruses tested, even in the rare case of substitutions in its epitope. Further, intranasal administration of CR9114 fully protects mice against A(H5N1) infection at low dosages, irrespective of pre-existing immunity conferred by the quadrivalent seasonal influenza vaccine. These data provide a proof-of-concept for broad, pre-exposure protection against a potential future pandemic using the intranasal administration route. Studies in humans should assess if autonomous administration of a broadly-neutralizing monoclonal antibody is safe and effective and can thus contribute to pandemic preparedness.

Figure 1

CR9114 neutralizes H5 strains belonging to different clades in vitro due to the high level of conservation of its epitope, and even in case of epitope substitutions. (A) CR9114’s epitope on A/Vietnam/1203/2004 HA. The surfaces of HA1 and HA2 subunits are shown in white and grey, respectively. Residues in the epitope are shown in orange, while hotspots, defined as residues forming H-bonds with CR9114, are shown in blue. The numbering of the residues follows the amino acid sequence alignment shown in Supplementary Fig. S1. (B) Phylogenetic tree based on the identity of the hemagglutinin protein sequence, showing the divergence among the strains used in the pseudotype virus neutralization assay (PNA). The selected strains belong to different clades. A/chicken/Scotland/1959 (not shown) was used as a root for building the tree. The full tree is presented in Supplementary Fig. S2. The list of details regarding the H5 strains is reported the Supplementary Table S1. (C) Schematic depiction of the full-length hemagglutinin from A/Vietnam/1203/2004 HA showing the boundaries of the globular head and of the stalk domains. The stalk domain consists of the N- and C-terminal parts of HA1 subunit, and the HA2 ectodomain; the transmembrane domain (TMD), and the cytoplasmic tail (CT) on the HA2 are not shown on the 3D structure. (D) Percent identity of amino acid sequence of the strains tested in the PNA compared to the full-length HA, globular head domain, stalk domain, and CR9114’s epitope of A/Vietnam/1203/2004. (E) Sequence alignment of CR9114’s epitope on H5 HA from A/Vietnam/1203/2004 and the other strains used in the PNA. Hot spots are highlighted in blue. (F) Neutralization values as IC₅₀ values (ng/mL) generated by PNA of CR9114 against ten H5 strains belonging to difference clades. (G) CR9114’s epitope on the HA of the strains with amino acid substitutions compared to A/Vietnam/1203/2004 tested in the PNA. Residues in yellow are fully conserved, whereas residues in red have been substituted in at least one of the tested strains. Figure 1A, C and G have been generated in Mol* Viewer with PDB ID: 4FQI. Single-letter abbreviations for amino acids: A Ala, D Asp, G Gly, H His, I Ile, K Lys, L Leu, M Met, N Asn, P Pro, Q Gln, S Ser, T Thr, V Val, W Trp.

Figure 2

Prophylactic intranasal administration of CR9114 protects mice against A(H5N1), both in the absence and in the presence of pre-existing immunity elicited by the seasonal influenza vaccine. Schematic depiction of the study design testing the prophylactic efficacy of intranasally administered CR9114 versus PBS (-24 h) in mice immunized intramuscularly with Influvac® quadrivalent vaccine (A), or PBS at 28 days before challenge (B). Percentages of survival (top panels) and weight change (bottom panels) of mice challenged with A/HK/156/97 (H5N1) 24 h after intranasal administration with 4 µg CR9114 (0.2 mg/kg assuming each mouse weights 20 g), 100 µg CR9114 (5 mg/kg assuming each mouse weights 20 g) or PBS in the (C) presence of pre-existing immunity from intramuscular Influvac® quadrivalent vaccine, and (D) absence of pre-existing immunity (intramuscular PBS). n = 10 per group, except the vaccinated group receiving 4 µg CR9114, where n = 9. IM intramuscular, IN intranasal.