Influenza H7N9 Virus Neuraminidase-Specific Human Monoclonal Antibodies Inhibit Viral Egress and Protect from Lethal Influenza Infection in Mice

Abstract

H7N9 avian influenza virus causes severe infections and might have the potential to trigger a major pandemic. Molecular determinants of human humoral immune response to N9 neuraminidase (NA) proteins, which exhibit unusual features compared with seasonal influenza virus NA proteins, are ill-defined. We isolated 35 human monoclonal antibodies (mAbs) from two H7N9 survivors and two vaccinees. These mAbs react to NA in a subtype-specific manner and recognize diverse antigenic sites on the surface of N9 NA, including epitopes overlapping with, or distinct from, the enzyme active site. Despite recognizing multiple antigenic sites, the mAbs use a common mechanism of action by blocking egress of nascent virions from infected cells, thereby providing an antiviral prophylactic and therapeutic protection in vivo in mice. Studies of breadth, potency, and diversity of antigenic recognition from four subjects suggest that vaccination with inactivated adjuvanted vaccine induce NA-reactive responses comparable to that of H7N9 natural infection.

Keywords: B lymphocyte; H7N9; antibodies; epitopes; influenza A virus; monoclonal; neuraminidase; pre-exposure prophylaxis.

Figure 1.. H7N9 Influenza Virus Infection or Vaccination Induces NA- or HA-Reactive B Cells

The frequency of N9-reactive LCLs was compared with the frequency of H7-reactive LCLs. The colors of the bars correspond to LCL specificity: black, N9 NA reactive and white, H7 HA. The gray dotted line indicates the threshold of positive response (0.25%). Arrows indicate donors from which anti-NA mAbs were isolated. Color shapes indicate antigenic exposure history of donors: brown, prime and boost vaccinations with IIV and AS03 adjuvant; red, prime with IIV and AS03 and boost with IIV only; pink, prime with IIV only and boost with IIV and AS03; violet, prime with IIV and AS03 and boost with IIV and MF59 adjuvant; lilac, prime with IIV and ME59 and boost with IIV and AS03; blue, prime and boost with IIV and MF59; yellow, prime and boost with IIV alone; green, H7N9 infection survivors. See also Figure S1 and Table S1.

Figure 2.. Binding of N9-Reactive mAbs Is Subtype-Specific and Is not Affected by Mutations Causing Resistance to Neuraminidase Inhibitors

(A) A panel of 35 human mAbs was isolated on the basis of reactivity to recombinant SH13 N9 NA antigen. Cross-reactivity of N9-reactive mAbs to recombinant NA proteins from group 1 was measured by ELISA. Representative EC₅₀ values (ng/mL) from two independent experiments are plotted as a heatmap. NAs were clustered by amino acid sequence phylogeny. Three mAbs that bind N9 antigen with EC₅₀ value higher than 10 μg/mL (highest tested concentration) excluded from representation. (B) Binding of N9-reactive mAbs to recombinant wild-type N9 NA from A/Shanghai/2/2013 virus or NI-resistant mutants was measured by ELISA. Representative EC₅₀ values (ng/mL) from two independent experiments are plotted as a heatmap. Four mAbs that bind N9 SH13 antigen with EC₅₀ value higher than 10 μg/mL (highest tested concentration) excluded from representation. See also Table S4 and Figure S2.

Figure 3.. Anti-N9 Human mAbs Inhibit Egress of A/Shanghai/2/2013 IDCDC-RG32A H7N9 Virus, Mainly by Blocking the N9 Enzyme Active Site

Individual mAbs were assessed for H7N9 virus neutralization by using hemagglutination inhibition (HAI), egress inhibition, enzyme-linked lectin (ELLA) neuraminidase inhibition (NI) and NA-Fluor NI assays. (A) Egress inhibition of H7N9 virus by mAbs. IC₁₀₀ values (nM) are shown as mean ± SD of three technical replicates. Four mAbs with low expression were excluded from the analysis. (B) Inhibition of N9 enzymatic activity by mAbs measured in ELLA. The assay used fetuin as substrate and H7N9 virus as the enzyme source. Data represent one of two independent experiments, shown as mean ± SD of three technical replicates. (C) Inhibition of N9 enzymatic activity by mAbs measured in NA-Fluor assay. The assay used H7N9 virus as the enzyme source. Data are shown as mean ± SD of three technical replicates. (D) mAbs functional activity comparison. Representative IC₁₀₀ values (nM) from HAI or egress inhibition assays and IC₅₀ values (nM) from ELLA NI or NA-Fluor NI assays are plotted as a heatmap. mAb NA-152 with low expression were excluded from the analysis. See also Table S5.

Figure 4.. NI Anti-N9 Human mAbs Targeted Two Main Regions on the N9 Surface

(A) mAbs were assessed for competition binding by surface plasmon resonance by using a Wasatch Microfluidics device. mAbs were judged to compete for the same site if maximum binding of second antibody was reduced to ≤ 49% of its uncompeted binding (shown in black boxes). The mAbs were considered non-competing if maximum binding of second mAb was ≥ 57% of its uncompeted binding (shown in white boxes). Grey boxes indicate an intermediate phenotype (competition between 50% and 56% of uncompeted binding). Blue, green, and orange lines and Roman numerals indicate inferred competition-binding groups. Clones with in vitro inhibitory activity are indicated with bold font. mAb NA-22 did not bind to N9 NA when tested by surface plasmon resonance, so it was excluded from analysis. (B) Binding affinities of two representative mAbs from Group II or Group III for N9 NA from A/Shanghai/2/2013, determined by surface plasmon resonance. See also Figures S3 and S4.

Figure 5.. Epitope Mapping for Anti-N9 mAbs

For a Figure360 author presentation of this figure, see https://doi.org/10.1016/j.chom.2019.10.003. (A) Epitope footprints and HDX-MS profiles of NA-73, NA-45, NA-80, NA-63, or NA-22 mAbs were mapped onto the surface of the N9 NA tetramer (PDB: 4MWL). Lines indicate the epitope footprint of N9-Fab complex structure for mAbs NA-45 (cyan), NA-73 (green), NA-63 (orange), NA-80 (blue), or NA-22 (brown) determined by crystallography or cryo-EM reconstruction. Amino-acid residues within the epitope with decreased deuteration level upon NA-45, NA-73, NA-63, NA-80, or NA-22 mAbs binding is indicated by cyan, green, orange, blue, or brown colors, respectively. Pale cyan color indicates the enzyme active site, and yellow color indicates the hemadsorption site. (B) Two-dimensional class averages of N9 NA alone or in complex with anti-N9 Fabs. Secondary structure features are visible in a variety of different views of the complex. Differences in Fab angles of approach are shown. See also Figures S5 and S6 and Zhu et al., 2019.

Figure 6.. Anti-N9 mAbs Mediate a High Level of Protection In Vivo against Lethal H7N9 Challenge in Mice

Groups of BALB/c mice were inoculated intraperitoneally (i.p.) one day before (A) or one day after (B) virus challenge with 10 mg/kg of anti-N9 mAb or with DENV 2D22 control mAb reactive to an irrelevant antigen (dengue virus) or with 10 mg/kg of a recombinant mAb on the basis of the sequence of the broadly neutralizing influenza stem-targeted mAb rCR9114. On day 0 (d0), mice were challenged intranasally (i.n.) with a lethal dose of influenza A/Shanghai/02/2013 IDCDC-RG32A virus and monitored for protection. The weights and clinical scores are represented as the group mean ± SEM. The lower dotted line indicates the no-recovery threshold (> 30% weight loss) and endpoint for euthanasia. Body weight change curves in (B) were compared by overall test using two-way ANOVA. Survival curves were estimated using the Kaplan-Meier method. Survival of each group that was treated with an anti-NA or anti-HA (rCR9114) mAb was compared with the control group as indicated using log rank (Mantel-Cox) test. A clinical score of 4 corresponds to a moribund state. Data represent one experiment, and “n” symbol in the plots indicates number of mice per group for each treatment condition (5–10 mice per group).

Figure 7.. Contribution of Fc-Region-Mediated Effector Functions to Protection by Strongly or Weakly Neutralizing Anti-N9 mAbs

Groups of BALB/c mice were inoculated by the i.p. route with 10 mg/kg (A and B) or with 2 mg/kg (C and D) of recombinant wild type IgG1 or IgG1 LALA Fc variants of indicated anti-NA mAbs or with DENV 2D22 control mAb or with 10 mg/kg of mAb rCR9114. The next day (d0) mice were challenged by the i.n. route with a lethal dose of influenza A/Shanghai/02/2013 IDCDC-RG32A virus and monitored for protection. The weights (A and C) are represented as the group mean ± SEM. The lower dotted line indicates the no-recovery threshold (> 30% weight loss) and endpoint for euthanasia. Body weight change curves were compared by overall test using two-way ANOVA. Survival curves were estimated using the Kaplan-Meier method, and compared as indicated using log rank (Mantel-Cox) test (B and D). Data represent one experiment, and “n” symbol in the plots indicates number of mice per group for each treatment condition (5–10 mice per group).