A pan-H1 anti-hemagglutinin monoclonal antibody with potent broad-spectrum efficacy in vivo

Abstract

Seasonal epidemics caused by antigenic variations in influenza A virus remain a public health concern and an economic burden. The isolation and characterization of broadly neutralizing anti-hemagglutinin monoclonal antibodies (MAb) have highlighted the presence of highly conserved epitopes in divergent influenza A viruses. Here, we describe the generation and characterization of a mouse monoclonal antibody designed to target the conserved regions of the hemagglutinin of influenza A H1 viruses, a subtype that has caused pandemics in the human population in both the 20th and 21st centuries. By sequentially immunizing mice with plasmid DNA encoding the hemagglutinin of antigenically different H1 influenza A viruses (A/South Carolina/1/1918, A/USSR/92/1977, and A/California/4/2009), we isolated and identified MAb 6F12. Similar to other broadly neutralizing MAb previously described, MAb 6F12 has no hemagglutination inhibition activity against influenza A viruses and targets the stalk region of hemagglutinins. As designed, it has neutralizing activity against a divergent panel of H1 viruses in vitro, representing 79 years of antigenic drift. Most notably, MAb 6F12 prevented gross weight loss against divergent H1 viruses in passive transfer experiments in mice, both in pre- and postexposure prophylaxis regimens. The broad but specific activity of MAb 6F12 highlights the potent efficacy of monoclonal antibodies directed against a single subtype of influenza A virus.

Fig 1

MAb 6F12 recognizes a panel of H1 influenza A viruses. (A) MDCK cells were infected at an MOI of 5 with USSR77 (H1), TX91 (H1), NC99 (H1), Bris07 (H1), rCal09 (H1), HK68 (H3), or rVN04 (H5) viruses and at 12 hpi fixed with 0.5% paraformaldehyde. Reactivity was detected using immunofluorescence with MAb 6F12, 7B2 (Cal09 specific), or E10 at 5 μg/ml. MAb E10 is an M2-specific MAb that is used as an infection control. EIA/RIA plates were coated with baculovirus-expressed HA proteins of PR8 (B), Cal09 (C), JP57 (D), VN04 (E), HK99 (F), HK68 (G), or Yam88 (H) or purified preparation of whole virus of rCal09, Bris07, and USSR77 (I) in duplicate at a starting concentration of 100 or 30 μg/ml for MAb 6F12. Sera from infected mice or MAb PY102, 8, G1-26, or XY102 were used as positive controls.

Fig 2

MAb 6F12 binds to the stalk domain of HA. (A) EIA/RIA plates were coated with baculovirus-expressed Cal09 HA and then incubated with MAb CR6261(human) or C179 (mouse) at a concentration of 100 μg/ml. An ELISA using biotinylated MAb 6F12 at a starting concentration of 100 μg/ml was performed with a streptavidin antibody conjugated to HRP, used as a secondary antibody. Positive competition was detected by an increase in the EC₅₀ of samples preincubated with MAb CR6261 or C179 over samples that did not show increases. (B) Wild-type HA (PR8, HK68, and HK99) or chimeric HA (cH9/1) was expressed in High Five insect cells using a recombinant baculovirus vector and fixed with 0.5% PFA at 48 hpi. Reactivity was detected by immunofluorescence with MAb 6F12 or pan-H3 12D1 at 1 μg/ml. (C) Eight chicken hemagglutination units (4 wells) of rCal09 virus was preincubated with MAb 6F12 or 7B2 with a starting concentration of 50 μg/ml before addition of 50 μl of 0.5% chicken red blood cells. PBS with virus and no MAb was used as a negative control, while PBS with no virus and MAb served as a background control.

Fig 3

MAb 6F12 neutralizes H1 viruses, but not an H5 virus, in a plaque reduction neutralization assay. Sixty to 80 PFU of PR8 (H1), sw30 (H1), USSR77 (H1), TX91 (H1), NC99 (H1), rCal09 (H1), or rVN04 (H5) virus was preincubated with dilutions of MAb 6F12 at a starting concentration of 100 μg/ml at room temperature prior to infection of a monolayer of MDCK cells. The agar overlay was also supplemented with the proper dilutions of MAb 6F12. At 48 hpi, the monolayers were fixed with 4% PFA and permeabilized with 0.5% Triton X-100. Plaques were visualized either through immunostaining using MAb HT103 (anti-PR8 nucleoprotein), sera (anti-USSR77), or crystal violet staining. The IC₅₀ was calculated by fitting data with a nonlinear regression curve using GraphPad Prism. An isotype IgG2b (22A6) control was also tested in parallel, with no dose-dependent inhibition observed (data not shown).

Fig 4

MAb 6F12 binds to the prefusion conformation of rCal09 HA. EIA/RIA plates were coated with purified preparations of whole rCal09 virus at 5 μg/ml and exposed to buffered solutions of pH 7.0, 5.6, 5.0, and 4.4 for 30 min before performing an ELISA with MAb 7B2 (A), 6F12 (B), or C179 (C) at a starting concentration of 100 μg/ml. To remove the globular head (HA1), the whole-virus preparation was exposed to 0.1 M DTT after exposure to pH-buffered solutions prior to the ELISA. Purified preparations of whole rCal09 virus were preincubated with MAb 6F12 (IgG2b) at a concentration of 100 μg/ml before exposure to acidic buffer (pH 4.4) and then reduced with 0.1 M DTT. (D) An ELISA with MAb 7B2 (IgG2a) was then performed using an isotype-specific secondary antibody.

Fig 5

An alanine-to-valine mutation at position 44 in the HA2 subunit of Cal09 HA abrogates MAb 6F12 binding. (A) 293T cells were transfected with wild-type Cal09 (H1), Cal09 A44₂V (H1), or wild-type HK68 (H3) HA, and at 24 h posttransfection cells were fixed with 0.5% paraformaldehyde. Reactivity was detected by immunofluorescence using Cal09-infected sera or MAb 6F12 or 7B2 at 5 μg/ml. (B and C) PyMOL was used to model the location of residue 44 on the HA2 subunit of A/South Carolina/1/1918 (H1) HA (1RD8). Residue 44 of HA2 is indicated in red in the model of the homotrimeric molecule of HA (B) or a monomer of HA2 (C). The HA1 is shown in light gray, while the HA2 is shown in light blue. HA molecules are not drawn to scale.

Fig 6

MAb 6F12 provides preexposure protection against several H1 virus strains. Six- to 8-week-old BALB/c or DBA.2 mice were intraperitoneally administered 30, 15, 7.5 3.0, 1.0., or 0.5 mg/kg of MAb 6F12 2 h prior to challenge with 5 mLD₅₀ of PR8 (A), sw30 (B), SI06 (C), or NL09 (D) virus. PBS and an isotype IgG2b (22A6) at a concentration of 30 mg/kg served as negative controls. Mice were monitored daily for signs of illness and weight change. The ratios beside the legends indicate the number of survivors over the total number of animals in each group. Mice were administered 15 mg/kg of MAb 6F12 or 22A6 2 h before infection with 5 mLD₅₀ of PR8 or NL09 virus. At 3 and 6 days postinfection, three mice from each group were randomly sacrificed and lungs were harvested. Lungs were then homogenized, and viral lung titers were determined by plaque assay (E). Undetectable viral lung titers are indicated by an asterisk.

Fig 7

MAb 6F12 provides postexposure protection against NL09 virus. Six- to 8-week-old BALB/c mice were infected with 5 mLD₅₀ of NL09 virus and treated intraperitoneally with 30 mg/kg of MAb 6F12 at 24, 48, 72, 96, 120, or 144 hpi. Mice were monitored daily for clinical signs of illness and weight change. The ratios beside the legend indicate the number of survivors over the total number of animals in each group.