Inhibition of avian-origin influenza A(H7N9) virus by the novel cap-dependent endonuclease inhibitor baloxavir marboxil

Abstract

Human infections with avian-origin influenza A(H7N9) virus represent a serious threat to global health; however, treatment options are limited. Here, we show the inhibitory effects of baloxavir acid (BXA) and its prodrug baloxavir marboxil (BXM), a first-in-class cap-dependent endonuclease inhibitor, against A(H7N9), in vitro and in vivo. In cell culture, BXA at four nanomolar concentration achieved a 1.5-2.8 log reduction in virus titers of A(H7N9), including the NA-R292K mutant virus and highly pathogenic avian influenza viruses, whereas NA inhibitors or favipiravir required approximately 20-fold or higher concentrations to achieve the same levels of reduction. A(H7N9)-specific amino acid polymorphism at position 37, implicated in BXA binding to the PA endonuclease domain, did not impact on BXA susceptibility. In mice, oral administration of BXM at 5 and 50 mg/kg twice a day for 5 days completely protected from a lethal A/Anhui/1/2013 (H7N9) challenge, and reduced virus titers more than 2-3 log in the lungs. Furthermore, the potent therapeutic effects of BXM in mice were still observed when a higher virus dose was administered or treatment was delayed up to 48 hours post infection. These findings support further investigation of BXM for A(H7N9) treatment in humans.

Figure 1

Therapeutic effects of BXM on survival and weight loss in a lethal infection model of mice infected with a low dose of the A(H7N9) virus. Mice were intranasally inoculated with 4.0 × 10⁵ TCID₅₀/mouse (10.4 MLD₅₀) of A/Anhui/1/2013 (H7N9) viruses, and treatment was started immediately after virus inoculation (n = 10/group). Survival time (a) and body weight loss (b) were monitored through a 28-day period after the infection. bid (bis in die): twice a day. The log-rank test was applied for comparison of the survival time between each group (*p < 0.05, **p < 0.001 compared to vehicle, ^†p < 0.01 compared to OSP at 5 mg/kg twice a day, ^§p < 0.05 compared to OSP at 50 mg/kg twice a day). Dunnett’s multiple-comparison method was applied for statistical analysis of body weight changes (*p < 0.01, **p < 0.001 compared to vehicle, ^†p < 0.001 compared to OSP at 5 mg/kg twice a day, ^§p < 0.001 compared to OSP at 50 mg/kg twice a day). The body weights at 5 dpi were calculated from nine mice in vehicle-treated group because one of the ten mice showed more than 30% reduction and was euthanized.

Figure 2

Inhibitory effect of BXM on virus titers in the lungs of mice infected with a low dose of the A(H7N9) virus. Mice were inoculated with 4.0 × 10⁵ TCID₅₀/mouse (10.4 MLD₅₀) of A/Anhui/1/2013 (H7N9) virus and treatment was started immediately after virus inoculation (n = 5/group). The virus titers (TCID₅₀) in lungs of mice at 1, 3 and 5 dpi were measured in MDCK cells. The lower limit of quantification of the virus titer is indicated by a dotted line (1.5 Log₁₀ TCID₅₀/mL). Dunnett’s multiple-comparison method was conducted for statistical comparison (*p < 0.05, **p < 0.001 compared to vehicle, ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 compared to OSP at 5 mg/kg twice a day, ^§p < 0.01, ^§§p < 0.001 compared to OSP at 50 mg/kg twice a day).

Figure 3

Suppressive effects of BXM on proinflammatory cytokine and chemokine production in the lungs of mice infected with a low dose of the A(H7N9) virus. Mice were intranasally infected with 4.0 × 10⁵ TCID₅₀/mouse (10.4 MLD₅₀) of A/Anhui/1/2013 (H7N9) virus, and treatment was started immediately after virus inoculation (n = 5/group). The proinflammatory cytokines and chemokines, IL-6, MCP-1, MIP-1α and IFN-γ, in the lungs at 1, 3 and 5 dpi were quantified. Dunnett’s multiple-comparison method was employed for the comparison (*p < 0.05, **p < 0.01, ***p < 0.001 compared to vehicle).

Figure 4

Effects of BXM on survival, weight loss and virus titers in the lungs in mice infected with a high dose of the A(H7N9) virus. Mice intranasally infected with 1.2 × 10⁶ TCID₅₀/mouse (31.1 MLD₅₀) of A/Anhui/1/2013 (H7N9) virus were administrated with treatment started immediately after virus inoculation, and survival time (a) and body weight loss (b) were analyzed. The log-rank test was applied for survival time (*p < 0.001 compared to vehicle, ^†p < 0.001 compared to OSP at 5 mg/kg twice a day, ^§p < 0.001 compared to OSP at 50 mg/kg twice a day). Dunnett’s multiple-comparison method was employed for body weight changes (*p < 0.001 compared to vehicle, ^†p < 0.001 compared to OSP at 5 mg/kg twice a day, ^§p < 0.001 compared to OSP at 50 mg/kg twice a day). Body weights at 4 and 5 dpi were calculated from nine mice because one of the ten mice in vehicle-treated group showed more than 30% reduction and was euthanized. (c) The virus titers (TCID₅₀) in the lungs of mice at 1 and 5 dpi were measured. The lower limit of quantification of the virus titer is indicated by a dotted line (1.5 Log₁₀ TCID₅₀/mL). Dunnett’s multiple-comparison method was applied for the comparison (*p < 0.05, **p < 0.001 compared to vehicle, ^†p < 0.05, ^††p < 0.01, ^†††p < 0.001 compared to OSP at 5 mg/kg twice a day, ^§p < 0.01, ^§§p < 0.001 compared to OSP at 50 mg/kg twice a day).

Figure 5

Effects of delayed treatment of BXM on a low dose of A(H7N9) infection. Mice were intranasally inoculated with 4.0 × 10⁵ TCID₅₀/mouse (10.4 MLD₅₀) of A/Anhui/1/2013 (H7N9) viruses, and BXM treatment was started at (a) immediately, (b) 24 or (c) 48 hours after virus inoculation (n = 5/group). Vehicle or OSP treatment was started immediately after virus inoculation (n = 5/group). Survival time and body weight loss were monitored through a 21-day period after the infection. The shaded area represents the treatment period. The log-rank test was applied for comparison of the survival time between each group (*p < 0.01 compared to vehicle, ^†p < 0.01 compared to OSP at 5 mg/kg twice a day).