The Hemagglutinin Stem-Binding Monoclonal Antibody VIS410 Controls Influenza Virus-Induced Acute Respiratory Distress Syndrome

Abstract

Most cases of severe influenza are associated with pulmonary complications, such as acute respiratory distress syndrome (ARDS), and no antiviral drugs of proven value for treating such complications are currently available. The use of monoclonal antibodies targeting the stem of the influenza virus surface hemagglutinin (HA) is a rapidly developing strategy for the control of viruses of multiple HA subtypes. However, the mechanisms of action of these antibodies are not fully understood, and their ability to mitigate severe complications of influenza has been poorly studied. We evaluated the effect of treatment with VIS410, a human monoclonal antibody targeting the HA stem region, on the development of ARDS in BALB/c mice after infection with influenza A(H7N9) viruses. Prophylactic administration of VIS410 resulted in the complete protection of mice against lethal A(H7N9) virus challenge. A single therapeutic dose of VIS410 given 24 h after virus inoculation resulted in dose-dependent protection of up to 100% of mice inoculated with neuraminidase inhibitor-susceptible or -resistant A(H7N9) viruses. Compared to the outcomes in mock-treated controls, a single administration of VIS410 improved viral clearance from the lungs, reduced virus spread in lungs in a dose-dependent manner, resulting in a lower lung injury score, reduced the extent of the alteration in lung vascular permeability and protein accumulation in bronchoalveolar lavage fluid, and improved lung physiologic function. Thus, antibodies targeting the HA stem can reduce the severity of ARDS and show promise as agents for controlling pulmonary complications in influenza.

FIG 1

Efficacy of VIS410 prophylaxis on survival of mice inoculated with A(H7N9) influenza virus. BALB/c mice (n = 12/group) were treated with a single i.p. dose of VIS410 (50 mg/kg) or PBS (control) 12 h before intranasal inoculation with 10 MLD₅₀ of the NAI-susceptible A/Anhui/1/2013(H7N9) influenza virus. Mock-inoculated mice received a single i.p. administration of PBS (mock). Body weights were monitored daily (A); animals that lost ≥25% of their initial body weight (those above the dotted line) were euthanized, and the survival rates were determined (B). The error bars represent the standard deviations.

FIG 2

Efficacy of VIS410 treatment on survival of mice inoculated with A(H7N9) influenza viruses. BALB/c mice (n = 10/group) were inoculated intranasally with 10 MLD₅₀ of either NAI-susceptible A/Anhui/1/2013 (H7N9) (A and B) or NAI-resistant A/Shanghai/1/2013 (H7N9) (C and D) influenza virus. Mice were treated i.p. with a single dose of VIS410 (2, 10, or 50 mg/kg), PBS (control), or oseltamivir (100 mg/kg/day, twice daily for 5 days by oral gavage), with all treatments being initiated 24 h after virus inoculation. Mock-inoculated mice received a single i.p. administration of PBS (mock). The animals' body weights were monitored daily (A and C); animals that lost ≥25% of their initial body weight (those above the dotted line) were euthanized, and the survival rates were determined (B and D). The error bars indicate the standard deviations (SDs).

FIG 3

Effect of VIS410 treatment on lung damage of mice infected with A(H7N9) influenza viruses. BALB/c mice were inoculated intranasally with 10 MLD₅₀ of either NAI-susceptible A/Anhui/1/2013 (H7N9) or NAI-resistant A/Shanghai/1/2013 (H7N9) influenza virus and treated i.p. with a single dose of VIS410 (2, 10, and 50 mg/kg), PBS (control), or oseltamivir (100 mg/kg/day, twice daily for 5 days by oral gavage), with all treatments being initiated 24 h after virus inoculation. Mock-inoculated mice received a single i.p. administration of PBS (mock). The lungs were harvested from mice (n = 3/group) at 7 dpi, weighed, and then dried and weighed again. Wet-to-dry lung weight ratios are shown (A and B). BALF was harvested from the lungs of mice (n = 3/group) at 7 dpi, and the total protein (μg/ml) (C and D) and LDH levels (E and F) were determined. The oxygenation level was determined in mice inoculated with A/Anhui/1/2013 (G) or A/Shanghai/1/2013 (H7N9) (H) at 6 dpi as a ratio of the arterial partial pressure of oxygen (PO₂) to the fraction of inspired oxygen (FIO₂) using the i-STAT CG8+ system. Values between 200 and 300 mm Hg indicate mild ARDS, as defined by the ARDS Working Group (18). The mean values for each group are indicated by solid lines. The dotted line indicates the normal oxygenation levels in healthy uninfected mice. The statistical significance of the differences between the treatment and control groups was determined using one-way ANOVA, followed by Dunnett's post hoc test (a, P < 0.001; b, P < 0.05). OSE, oseltamivir phosphate.

FIG 4

Histopathologic changes in the lungs of infected mice treated with VIS410 or that were mock treated. Representative features in lungs of BALB/c mice (n = 3/group) infected with 10 MLD₅₀ of the NAI-resistant A/Shanghai/1/2013 (R292K NA) influenza virus and treated i.p. with 50 mg/kg of VIS410 (A, C, and E) or mock treated (B, D, and F) on day 6 postinoculation are shown. In contrast to the clear alveoli in the lungs of antibody-treated mice (A), there was an increase in intra-alveolar protein/fibrin (black arrowheads) and thickened septa (red arrowheads) in the lungs of mock-treated animals (B). Influenza antigen-positive cells (black arrows) were rare and weakly stained in lungs of mice treated with 50 mg/kg of antibody (C and E), in marked contrast to the large numbers of intensely stained antigen-positive cells in pulmonary lesions of mock-treated animals (D and F). Magnification: 20×, A to D; 40×, E and F.

FIG 5

Quantitative comparisons of the extent of pulmonary lesions in inoculated mice treated with VIS410. BALB/c mice were inoculated with 10 MLD₅₀ of either NAI-susceptible A/Anhui/1/2013 (A, C, D, G, H, K, and L) or NAI-resistant A/Shanghai/1/2013 (R292K NA) (B, E, F, I, J, M, and N) influenza virus. The areas of lung with both active (defined as lesions containing cells that are strongly positive for viral antigen) and inactive (containing only a few positive cells with only weak staining) were outlined and quantified using image analysis software (Aperio ScanScope). The ratios shown are relative to those from lung tissues of PBS-treated (control) animals (dotted lines) (A and B). Mock-inoculated mice received a single i.p. administration of PBS (mock). The values represent the means ± SDs from 3 animals. Statistically significant differences between VIS410-treated mice inoculated with A/Anhui/1/2013 (H7N9) or A/Shanghai/1/2013 (H7N9) virus and control mice were determined by one-way ANOVA, followed by Dunnett's post hoc test (a, P < 0.001). Representative images of lung tissues from control mice (C and E), animals treated with 50 mg/kg of VIS410 (D and F), 10 mg/kg of VIS410 (G and I), 2 mg/kg of VIS410 (H and J), 100 mg/kg/day (d) oseltamivir (K and M), or mock (L and N) are shown.

FIG 6

Effect of VIS410 prophylaxis and treatment on development of anti-HA antibody response in mice. BALB/c mice were inoculated intranasally with 10 MLD₅₀ of either NAI-susceptible A/Anhui/1/2013 (H7N9) or NAI-resistant A/Shanghai/1/2013 (H7N9) influenza virus and treated i.p. with a single dose of VIS410 (2, 10, and 50 mg/kg), PBS (control), or oseltamivir (100 mg/kg/day, twice daily for 5 days by oral gavage) at the indicated times pre- or postinoculation. At 21 days after the initial virus inoculation, the HI titers were determined from the surviving mice (A to C). None of the animals inoculated with A/Shanghai/1/2013 (H7N9) influenza virus and treated with oseltamivir survived the infection (Fig. 2C and D). All surviving mice were then reinoculated with 25 MLD₅₀ of the same virus used for the initial inoculation. The weight loss of naive and VIS410-treated mice after reinfection with A(H7N9) influenza viruses is shown (D and E); animals that lost ≥25% of their initial body weight (above the dotted line) were euthanized. The error bars indicate SDs. Oseltamivir-treated mice that survived the initial A/Anhui/1/2013 (H7N9) influenza virus challenge were not included in the analysis. In panels A to C, the horizontal solid lines visually divide the data obtained for animals at day 21 postinoculation and day 40 postinoculation.