Human anti-N1 monoclonal antibodies elicited by pandemic H1N1 virus infection broadly inhibit HxN1 viruses in vitro and in vivo

Abstract

Neuraminidase (NA) is one of the two influenza virus surface glycoproteins, and antibodies that target it are an independent correlate of protection. However, our current understanding of NA antigenicity is incomplete. Here, we describe human monoclonal antibodies (mAbs) from a patient with a pandemic H1N1 virus infection in 2009. Two mAbs exhibited broad reactivity and inhibited NA enzyme activity of seasonal H1N1 viruses circulating before and after 2009, as well as viruses with avian or swine N1s. The mAbs provided robust protection from lethal challenge with human H1N1 and avian H5N1 viruses in mice, and both target an epitope on the lateral face of NA. In summary, we identified two broadly protective NA antibodies that share a novel epitope, inhibited NA activity, and provide protection against virus challenge in mice. Our work reaffirms that NA should be included as a target in future broadly protective or universal influenza virus vaccines.

Keywords: antibody; influenza virus; neuraminidase.

Figure 1.. Breadth of binding and neuraminidase inhibition for mAbs 2H08 and 3H03

(A) Heat map of 2H08 and 3H03 mAbs binding to recombinant NA or purified whole virus in an enzyme-linked immunosorbent assay (ELISA). Purified whole virus was used as coating antigen for A/swine/Jiangsu/40/2011 (H1N1), A/rhea/North Carolina/39482/1993 (H7N1), and a cH6/1 virus carrying the N4 NA of A/mallard/Sweden/24/2002 (H8N4) as indicated by the asterisks. (B) Heat map showing 50% inhibitory concentration (IC₅₀) NA inhibition (NAI) for 2H08 and 3H03 mAbs in enzyme-linked lectin assay (ELLA) with fetuin protein. The influenza viruses were clustered by NA gene sequences and the scale bar represents amino acid substitutions per site.

Figure 2.. Functional characterization in vitro for mAbs 2H08 and 3H08.

Neutralization capacity against Singapore/GP1908/2015 (H1N1) was measured by a plaque reduction neutralisation assay (PRNA). PRNA was performed with mAbs present in the overlay to measure inhibition of viral regress (A) and with mAbs both in the inoculum and the overlay to measure inhibition of viral entry and egress (B). Neuraminidase inhibition (NAI) was measured in the NA-Star assay using Singapore/GP1908/2015 (H1N1) virus with mAbs 2H08 and 3H03 (C). 1G01 was used as positive control (pos ctrl) and an irrelevant human IgG mAb as negative control (neg ctrl). Symbols represent mean ± standard deviation. Antibody dependent cellular cytotoxicity (ADCC) activity of mAbs 2H08 and 3H03 was measured in an ADCC reporter assay using Madin Darby canine kidney (MDCK) cells infected with Singapore/GP1908/2015 (H1N1) virus (D). The data are presented as relative luminescent units. For all assays symbols represent mean ± standard deviation. 1G01 was used as positive control and an irrelevant human IgG mAb as negative control in all assays.

Figure 3.. In vivo protection by prophylactic treatment with mAbs 2H08 and 3H03.

Percent of initial body weight (A-D) and survival (E-H) for mice treated prophylactically with mAbs 2H08, 3H03 and an irrelevant human IgG mAb (neg ctrl) before influenza challenge. mAbs were administered intraperitoneally 2 hours before challenge with 5x 50% lethal dose (LD₅₀) H1N1 A/Singapore/GP1908/2015 and H5N1 A/Vietnam/1204/2004 virus. Mice infected with A/Singapore/GP1908/2015 (H1N1) were treated with mAb at 5, 1 and 0.2 mg/kg and mice infected with A/Vietnam/1204/2004 (H5N1, low pathogenic 6:2 reassortant with A/Puerto Rico/8/1934, HA polybasic cleavage site removed) virus were treated with 5 mg/kg. Five mice were used per mAb group. Symbols represent mean ± standard deviation. The dotted lines in (A-D) represent the humane endpoint for euthanasia (75% weight loss). (I) Lung titres of mice treated prophylactically with 5 mg/kg of mAb were measured at 3- and 6-days post infection (dpi) with 0.1 LD₅₀ A/Singapore/GP1908/2015 (H1N1). Three mice were used per group. Symbols represents the mean plaque forming unit per ml (pfu/ml) for each mouse and error bars represent standard deviation. The dotted line in (I) represents the limit of detection.

Figure 4.. In vivo protection by therapeutic treatment with mAbs 2H08 and 3H03.

Percent of initial body weight (A, C) and survival (B, D) for mice treated therapeutically with mAbs 2H08, 3H03 and an irrelevant human IgG mAb (neg ctrl). mAbs were administered intraperitoneally 48 hours post infection with 5x lethal dose 50 A/Singapore/GP1908/2015 (H1N1) and A/Vietnam/1204/2004 (H5N1, low pathogenic 6:2 reassortant with A/Puerto Rico/8/1934, polybasic cleavage site removed) virus. Five mice per mAb group were used. Symbols represent mean ± standard deviation. Arrows indicate time of mAb administration. The dotted line represents the humane endpoint for euthanasia (75% weight loss).

Figure 5.. Binding and NA inhibition against NA with P93L and I117M mutations.

Escape mutant virus with P93L and I117M mutations was generated by passaging A/Singapore/GP1908/2015 (H1N1) virus in MDCK cells under selective pressure of mAb 2H08. NA inhibition (NAI) activity of mAbs 2H08 and 3H03 against unmutated wild-type virus passaged in MDCK cells without mAb (A) and the escape mutant virus (B) was measured by ELLA. Binding against the wild-type NA (C) and recombinant NA proteins with the P93L (D) and I117M (E) mutations individually and in combination (F) was assessed by ELISA. 1G01 was used as positive control (pos ctrl) and a human anti-anthrax IgG mAb was used as negative control (neg ctrl) for the ELLA and ELISA experiments. Symbols represent mean ± standard deviation and the dotted lines in (A, B) represents 50% NAI.

Figure 6.. CryoEM structures of 2H08 and 3H03 Fabs in complex with the N1 NA from A/Brevig Mission/1/1918 (H1N1) (1918 N1).

(A) Structure of 2H08 Fab with 1918 N1 at 3.1 Å resolution. From top to bottom: the NA tetramer in grey with one Fab bound to each NA protomer, the NA protomer bound with one Fab, and the antibody epitope on the NA with the complementarity-determining region (CDR) loops and framework (FR) loops of the heavy and light chains involved in the antibody-antigen interaction superimposed. (B) Structure of 3H03 Fab with 1918 N1 at 2.7 Å resolution. From the top to bottom, as for (A). The 1918 N1 NA in (A) and (B) are shown in the same orientation. One Fab-NA protomer is colored with NA in light grey, Fab light chain (L-chain) in yellow, and Fab heavy chain (H-chain) in orange. The other NA protomers are in dark grey. The molecular surface depicting the epitope is colored in green.

Figure 7.. Epitope comparison of human antibodies 2H08 and 3H03 with 1918 N1 NA versus mouse antibody CD6 with 2009 N1 NA.

(A) The epitope of 2H08 is mapped onto 1981 N1 NA (green). (B) The epitope of 3H03 is mapped onto 1918 N1 NA (green). (C) The epitope of murine mAb CD6 is mapped onto 2009 N1 NA (green, PDB: 4QNP).