Broadly Reactive Human Monoclonal Antibodies Elicited following Pandemic H1N1 Influenza Virus Exposure Protect Mice against Highly Pathogenic H5N1 Challenge

Abstract

Broadly cross-reactive antibodies (Abs) that recognize conserved epitopes within the influenza virus hemagglutinin (HA) stalk domain are of particular interest for their potential use as therapeutic and prophylactic agents against multiple influenza virus subtypes, including zoonotic virus strains. Here, we characterized four human HA stalk-reactive monoclonal antibodies (MAbs) for their binding breadth and affinity, in vitro neutralization capacity, and in vivo protective potential against an highly pathogenic avian influenza virus. The monoclonal antibodies were isolated from individuals shortly following infection with (70-1F02 and 1009-3B05) or vaccination against (05-2G02 and 09-3A01) A(H1N1)pdm09. Three of the MAbs bound HAs from multiple strains of group 1 viruses, and one MAb, 05-2G02, bound to both group 1 and group 2 influenza A virus HAs. All four antibodies prophylactically protected mice against a lethal challenge with the highly pathogenic A/Vietnam/1203/04 (H5N1) strain. Two MAbs, 70-1F02 and 09-3A01, were further tested for their therapeutic efficacy against the same strain and showed good efficacy in this setting as well. One MAb, 70-1F02, cocrystallized with H5 HA and showed heavy-chain-only interactions similar to those seen with the previously described CR6261 anti-stalk antibody. Finally, we show that antibodies that compete with these MAbs are prevalent in serum from an individual recently infected with the A(H1N1)pdm09 virus. The antibodies described here can be developed into broad-spectrum antiviral therapeutics that could be used to combat infections by zoonotic or emerging pandemic influenza viruses.IMPORTANCE The rise in zoonotic infections of humans by emerging influenza viruses is a worldwide public health concern. The majority of recent zoonotic human influenza cases were caused by H7N9 and H5Nx viruses and were associated with high morbidity and mortality. In addition, seasonal influenza viruses are estimated to cause up to 650,000 deaths annually worldwide. Currently available antiviral treatment options include only neuraminidase inhibitors, but some influenza viruses are naturally resistant to these drugs, and others quickly develop resistance-conferring mutations. Alternative therapeutics are urgently needed. Broadly protective antibodies that target the conserved "stalk" domain of the hemagglutinin represent potential potent antiviral prophylactic and therapeutic agents that can assist pandemic preparedness. Here, we describe four human monoclonal antibodies that target conserved regions of influenza HA and characterize their binding spectrum as well as their protective capacity in prophylactic and therapeutic settings against a lethal challenge with a zoonotic influenza virus.

Keywords: H5N1; HA stalk; hemagglutinin; hemagglutinin stalk; influenza; influenza virus; monoclonal antibody.

FIG 1

Heat map of antibody binding profile by ELISA. Antibodies were tested by ELISA against recombinantly produced HA proteins. A heat map was generated based on minimal binding concentration and plotted next to a phylogenetic tree (based on amino acid difference) to indicate the similarity of HAs that were tested. Each MAb was tested once in duplicate.

FIG 2

H5N1 (A/Vietnam/1203/2004) microneutralization assay on MDCK cells. Antibodies were tested in a neutralization assay against a low-pathogenicity H5 reassortant virus. The x axis shows the amount of monoclonal antibody tested, and the y axis indicates percent inhibition in the assay. Each point shows the mean of data from two replicates, and error bars indicate errors of the means. A nonlinear curve fit was performed for all antibody dilution series (colored lines). Each MAb was tested once in duplicate.

FIG 3

In vivo challenge with highly pathogenic H5N1 after prophylactic treatment. Mice were intraperitoneally injected with antibodies or PBS (no-antibody control) 24 h before infection with highly pathogenic H5N1 virus. Survival was checked daily, and weight was recorded every other day. (A) Weight loss curves after challenge. Each point shows the average percentage of the initial body weight of the group, and error bars indicate the standard errors of the means. (B) Kaplan-Maier curves show the survival rate for each of the groups post-H5N1 challenge. Survival rates of all groups were compared to the rate determined for the group that received the isotype control EM4-C04 in a log rank (Mantel-Cox) test. MAbs 70-1F02 (P = 0.0001), 09-3A01 (P < 0.0001), 1009-3B05 (0.0005), and 05-2G02 (P = 0.0022) all conferred statistically significant protection compared to the isotype control, and no statistically significant difference was observed between mice receiving PBS and those receiving EM4-C04 (Bonferroni-corrected significance threshold = <0.01). Challenge studies were performed once with 8 to 10 mice per group.

FIG 4

Therapeutic treatment postchallenge with highly pathogenic H5N1. Mice were intraperitoneally injected with 70-1F02, 09-3A01, or PBS (negative control) 24 h (A and B) or 72 h (C and D) postinfection with highly pathogenic H5N1 virus. Therapeutic treatment with both antibodies protected mice from weight loss (A and C) and mortality (B and D). As shown in panel B, all groups were compared to the PBS control group in a log rank (Mantel-Cox) test. MAbs 70-1F02 (P < 0.0001) and 09-3A01 (P < 0.0001) both conferred statistically significant protection compared to PBS (Bonferroni-corrected significance threshold = <0.025). In panel D, the same testing showed that MAbs 70-1F02 (P = 0.0002) and 09-3A01 (P = 0.0104) both conferred statistically significant protection compared to PBS (Bonferroni-corrected significance threshold = <0.025). Challenge studies were performed once with 8 to 10 mice per group.

FIG 5

Crystal structure of the 701-F02 Fab/VN/04 HA complex. (A) Overview of the 70-1F02 FAb/H5 complex structure. Ribbons represent a single HA trimer bound to three 70-1F02 FAbs. For clarity, a single HA1 protomer and a single HA2 protomer are colored in magenta and cyan, respectively, with the 70-1F02 FAb heavy and light chains colored in green and blue, respectively. N-carbohydrates are indicated as yellow sticks. (B) The Ab 70-1F02 epitope on H5 HA. Residues on A/Vietnam/1203/2004 (H5N1) HA contacted by 70-1F02 are indicated. The primarily component of the epitope is the helical strand of the HA2 (11 residues). Residues within the 70-1F02 epitope are indicated as sticks on a ribbon representation of the H5 HA. Contact residues in the HA1 and HA2 subdomains are colored in magenta (HA1) and cyan (HA2), respectively. The epitope spans the two HA1 and HA2 subunits in a single HA trimer and does not contact the subunits of adjacent monomers. Residues are numbered consecutively, according to the mature protein.

FIG 6

Overlap of 70-1F02 and CR6261 bound to an H5 monomer. An overlay of the previously crystallized human MAbs CR6261 and 70-1F02 reveals similar binding profiles of these antibodies.

FIG 7

Inhibition of binding to Abs within RDE-treated sera at 1/80 dilution. Recombinant Cal09 HA (A) and VN04 H5 HA (B) were bound, in parallel, to eight Octet Red biosensors (HA binding) and analyzed. Each curve represents a separate biosensor, bound to an equivalent quantity of HA. After washing was performed to remove excess HA, biosensors were incubated with EM4-C04 (see Ab1 [head]; red, blue, and green traces) and then washed with kinetics buffer prior to incubation with Ab2 (stalk), either Ab 70-1F02 (see blue and orange traces) or Ab 05-2G02 (red trace), or 70-1F02 MAb Fab (purple trace). Controls without antibody or serum (black trace) and serum only (yellow trace) were included as well. Biosensors were subsequently washed again in kinetics buffer to remove unbound Ab and then incubated in RDE-treated serum. Following incubation with serum, the biosensors were washed once more to remove unbound IgG prior to data analysis. The instrument detects binding to the biosensor tip, which results in a wavelength shift (measured in nanometers). This experiment was performed once with 5 to 7 different MAb concentrations.