Combination Therapy with Oseltamivir and Favipiravir Delays Mortality but Does Not Prevent Oseltamivir Resistance in Immunodeficient Mice Infected with Pandemic A(H1N1) Influenza Virus

Abstract

Immunosuppressed individuals can shed influenza virus for prolonged periods of time, leading to the frequent emergence of antiviral resistance. We evaluated the benefits of oseltamivir and favipiravir combination therapy compared to single antiviral agents and monitored the emergence of drug-resistant variants in a pharmacologically immunosuppressed mouse model infected with the A(H1N1) pandemic influenza virus. C57BL/6 mice were immunosuppressed with cyclophosphamide and infected with a lethal dose of pandemic influenza A(H1N1) virus. Forty-eight hours post-infection, mice were treated with oseltamivir (20 mg/kg), favipiravir (20 or 50 mg/kg) or both agents BID for 5 or 10 days. Body weight losses, survival rates, lung viral titers, cytokine levels and emergence of resistant viruses were evaluated. Treatment of immunosuppressed mice with high (50 mg/kg) but not low (20 mg/kg) doses of favipiravir in combination with oseltamivir (20 mg/kg) significantly delayed mortality and reduced lung viral titers compared to treatment with a single drug regimen with oseltamivir but did not prevent the emergence of oseltamivir-resistant H275Y neuraminidase variants. Combination therapy with oseltamivir and favipiravir should be considered for evaluation in clinical trials.

Keywords: combination therapy; favipiravir; immunosuppression; mice; oseltamivir; pandemic influenza virus; resistance.

Figure 1

Weight loss (A) and survival rate (B) of IS C57BL/6 mice intranasally infected with 10² to 10⁶ TCID₅₀ of rec A(H1N1)pdm09 in 30 µL of PBS. Animals were monitored daily for 14 days for clinical signs of illness, including weight loss, ruffled fur, and hunching. Mice were sacrificed when they lost 20% of their original body weight.

Figure 2

Evaluation of five-day single and combined therapy in IS and IC mice. C57BL/6 mice intranasally infected with 10² TCID₅₀ of rec A(H1N1)pdm09 received meglumine (placebo), 20 mg/kg of oseltamivir, favipiravir or the combination by gavage BID for five days, starting at 48 h post-infection (p.i.). An uninfected group (meglumine) was added as control. (A) Animals were monitored daily for 20 days for clinical signs of illness, including weight loss, ruffled fur, and hunching and sacrificed when they lost 20% of their original body weight. (B) Kaplan–Meier survival curves for IS mice were compared using the Log-Rank (Mantel–Cox) test (* p < 0.05, ** p < 0.01 and *** p < 0.001).

Figure 3

Evaluation of ten-day single and combined therapy with low dose of favipiravir in IS mice. C57BL/6 mice intranasally infected with 10² TCID₅₀ of rec A(H1N1)pdm09 received meglumine (placebo), 20 mg/kg of oseltamivir, favipiravir or the combination by gavage BID for ten days, starting at 48 h post-infection (p.i.). An uninfected group (meglumine) was added as control. (A) Animals were monitored daily for 21 days for clinical signs of illness, including weight loss, ruffled fur, and hunching and sacrificed when they lost 20% of their original body weight. (B) Kaplan–Meier survival curves for IS mice were compared using the Log-Rank (Mantel–Cox) test (* p < 0.05, and *** p < 0.001). (C) Lung viral titers ± standard error of mean were determined by TCID₅₀ using ST6-GalI-MDCK cells for groups of four mice euthanized on days 8, 12, and 15 post-infection and compared by one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison post-test (* p < 0.05, ** p < 0.01 and *** p < 0.001). The dotted horizontal line indicates the lower limit of detection; i.e. 10^1,5 TCID₅₀/mL.

Figure 4

Evaluation of ten-day single and combined therapy with high dose of favipiravir in IS mice. C57BL/6 mice intranasally infected with 10² TCID₅₀ of rec A(H1N1)pdm09 received meglumine (placebo), 20 mg/kg of oseltamivir, 50 mg/kg of favipiravir or the combination by gavage BID for ten days, starting at 48 h post-infection (p.i.). An uninfected group (meglumine) was added as a control. (A) Animals were monitored daily for 21 days for clinical signs of illness, including weight loss, ruffled fur, and hunching and sacrificed when they lost 20% of their original body weight. (B) Kaplan–Meier survival curves for IS mice were compared using the Log-Rank (Mantel–Cox) test (** p < 0.01 and *** p < 0.001). (C) Lung viral titers ± standard error of mean were determined by TCID₅₀ using ST6-GalI-MDCK cells for groups of four mice euthanized on days 8, 12 and 15 post-infection and compared by one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison post-test (* p < 0.05, ** p < 0.01 and *** p < 0.001). The dotted horizontal line indicates the lower limit of detection, 10^1,5 TCID₅₀/mL.

Figure 5

Pulmonary cytokine/chemokine levels on day 12 post-infection in IS C57BL/6 mice intranasally infected with 10² TCID₅₀ of rec A(H1N1)pdm09. Mice received meglumine (non-treated), 20 mg/kg of oseltamivir, 50 mg/kg of favipiravir or the combination by gavage BID for ten days, starting at 48 h post-infection (p.i.). Bars represent mean values ± SDs (n = 3/group). One-way analysis of variance (ANOVA) with Tukey’s multiple-comparison post-test (* p < 0.05 and ** p < 0.01) was used for the comparisons.