Favipiravir antiviral efficacy against SARS-CoV-2 in a hamster model

Abstract

Despite no or limited pre-clinical evidence, repurposed drugs are massively evaluated in clinical trials to palliate the lack of antiviral molecules against SARS-CoV-2. Here we use a Syrian hamster model to assess the antiviral efficacy of favipiravir, understand its mechanism of action and determine its pharmacokinetics. When treatment is initiated before or simultaneously to infection, favipiravir has a strong dose effect, leading to reduction of infectious titers in lungs and clinical alleviation of the disease. Antiviral effect of favipiravir correlates with incorporation of a large number of mutations into viral genomes and decrease of viral infectivity. Antiviral efficacy is achieved with plasma drug exposure comparable with those previously found during human clinical trials. Notably, the highest dose of favipiravir tested is associated with signs of toxicity in animals. Thereby, pharmacokinetic and tolerance studies are required to determine whether similar effects can be safely achieved in humans.

Fig. 1. Virological results with preemptive favipiravir therapy.

a Experimental timeline. Groups of 6 hamsters were intranasally infected with 10⁶, 10⁵ or 10⁴ TCID₅₀ of virus. b Viral replication in lung based on infectious titers (measured using a TCID₅₀ assay) expressed in TCID₅₀/copy of ɣ-actine gene (n = 6 animals/group). c Viral replication in lung based on viral RNA yields (measured using an RT-qPCR assay) expressed in viral genome copies/copy of ɣ-actine gene (n = 6 animals/group). d Relative lung viral particle infectivities were calculated as follows: ratio of lung infectious titer over viral RNA yields (n = 6 animals/group). e Plasma viral loads (measured using an RT-qPCR assay) are expressed in viral genome copies/mL of plasma (the dotted line indicates the detection threshold of the assay) (n = 6 animals/group). f Clinical course of the disease (n = 6 animals/group). Normalized weight at day n was calculated as follows: % of initial weight of the animal at day n. Data represent mean ± SD (details in Supplementary Data 2). Two-sided statistical analysis were performed using Shapiro–Wilk normality test, Student t-test, Mann–Whitney test, Welch’s test, and two-way ANOVA with Post-hoc Dunnett’s multiple comparisons test (details in Supplementary Data 3 and 4). ****, ***, ** and * symbols indicate that the average value for the group is significantly lower than that of the untreated group with a p-value < 0.0001, ranging between 0.0001–0.001, 0.001–0.01, and 0.01–0.05, respectively. Source data are provided as a Source data file.

Fig. 2. Clinical follow-up of animals.

a Experimental timeline. b Evaluation of the toxicity of the three doses of favipiravir (mg/day TID) with groups of four uninfected animals following the experimental timeline described in panel a but without infection. c, d Clinical follow-up with groups of 10 animals infected respectively with 10⁵ and 10⁴ TCID₅₀ of virus and treated with a dose of favipiravir of 37.5 mg/day TID. Normalized weight at day n was calculated as follows: % of initial weight of the animal at day n. Data represent mean ± SD (details in Supplementary Data 2). Two-sided statistical analysis were performed using two-way ANOVA with Post-hoc Dunnett’s multiple comparisons test or Post-hoc Sidak’s multiple comparisons test (details in Supplementary Data 5). ****, ***, ** and * symbols indicate that the average value for the group is significantly lower than that of the untreated group with a p-value < 0.0001, ranging between 0.0001–0.001, 0.001–0.01, and 0.01–0.05, respectively Source data are provided as a Source data file.

Fig. 3. Virological results with preventive favipiravir therapy.

a Experimental timeline. Groups of 6 hamsters were intranasally infected with 10⁴ TCID₅₀ of virus. b Viral replication in lungs based on infectious titers (measured using a TCID₅₀ assay) expressed in TCID₅₀/copy of ɣ-actine gene (n = 6 animals/group). c Viral replication in lungs based on viral RNA yields (measured using an RT-qPCR assay) expressed viral genome copies/copy of ɣ-actine gene (n = 6 animals/group). d Clinical course of the disease (n = 6 animals/group). Normalized weight at day n was calculated as follows: % of initial weight of the animal at day n. e Relative lung virus infectivities were calculated as follows: ratio of lung infectious titer over viral RNA yields (n = 6 animals/group). f Plasma viral loads (measured using an RT-qPCR assay) are expressed in viral genome copies/mL of plasma (the dotted line indicates the detection threshold of the assay) (n = 6 animals/group). Data represent mean ± SD (details in Supplementary Data 2). Statistical analysis were performed using Shapiro–Wilk normality test, Student t-test, Mann–Whitney test, One-sample t-test and two-way ANOVA with Post-hoc Dunnett’s multiple comparisons test (details in Supplementary Data 3 and 4). ****, ** and * symbols indicate that the average value for the group is significantly different from that of the untreated group with a p-value < 0.0001, ranging between 0.001–0.01 and 0.01–0.05, respectively. Source data are provided as a Source data file.

Fig. 4. Lung histopathological changes with preemptive or preventive favipiravir therapy.

Groups of four animals were intranasally infected with 10⁴ TCID₅₀ of virus and sacrificed at 3 and 5 dpi. Experimental timelines for preemptive (a) and preventive (c) favipiravir therapies. At day of sacrifice, lungs were collected, fixed, and embedded in paraffin. Tissue sections were stained with hematoxylin-eosin (H&E). Based on severity of inflammation, alveolar hemorrhagic necrosis, and vessel lesions, a cumulative score from 0 to 10 was calculated and assigned to a grade of severity (I, II, III, and IV). Scoring of pathological changes for preemptive (b) and preventive (d) favipiravir therapies (n = 4 animals/group) (details in Supplementary Data 7). Two-sided statistical analysis were performed using Shapiro–Wilk normality test, Student t-test, Mann–Whitney test, and two-way ANOVA with Post-hoc Dunnett’s multiple comparisons test (details in Supplementary Data 7 and 8). * Symbol indicates that the average value for the group is significantly different from that of the untreated group with a p-value ranging between 0.01 and 0.05. e Representative images of lung tissue (left lobe) (scale bar: 4 mm): multifocal and extensive areas of inflammation for untreated animal, multifocal but limited areas of inflammation for 37.5 mg/day treated animal and normal lung for 75 mg/day treated animal (n = 4 samples/group). f Representative images of bronchial inflammation (scale bar: 100 µ): severe peribronchiolar inflammation and bronchiole filled with neutrophilic exudates for untreated animal, mild peribronchiolar inflammation for 37.5 mg/day treated animal and normal bronchi for 75 mg/day treated animal (n = 4 samples/group). g Representative images of alveolar inflammation (scale bar: 100 µ): severe infiltration of alveolar walls, alveoli filled with neutrophils/macrophages for untreated animal, moderate infiltration of alveolar walls, some alveoli filled with neutrophils/macrophages for 37.5 mg/day treated animal and normal alveoli for 75 mg/day treated animal. h Representative images of vessel inflammation (scale bar: 50 µ): infiltration of vascular wall with neutrophils/cell debris and endothelial damage (arrow) for untreated animal, moderate endothelial leukocytic accumulation for 37.5 mg/day treated animal and normal vessel for 75 mg/day treated animal (n = 4 samples/group). Clinical courses of the disease are presented in Supplementary Fig. 6. Source data are provided as a Source data file.

Fig. 5. Mutagenic effect of favipiravir.

a Viral genetic diversity in clarified lung homogenates. For each condition, four samples were analyzed. Each triangle represents a mutation (only substitutions with a frequency ≥ 1% were considered). b Patterns of mutation distribution on complete viral genome. Each variable nucleotide position was counted only once when found. The variability was represented using 75 nt sliding windows. For each condition, variable nucleotide positions were determined and represented using a 300 nt sliding window. c Mean number of mutations (n = 4 samples/group). Data represent mean ± SD. d Mutation characteristics (n = 4 samples/group). For each sample, the frequency of a given mutation was calculated as follows: number of this kind of mutation detected in the sample divided by the total number of mutations detected in this sample. Data represent mean ± SD (details in details in Supplementary Data 10 and 13). Two-sided statistical analysis were performed using Shapiro–Wilk normality test, Student t-test, Mann–Whitney test, and Welch’s test (details in Supplementary Data 11 and 12). ***, ** and * symbols indicate that the average value for the group is significantly lower than that of the untreated group with a p-value ranging between 0.0001–0.001, 0.001–0.01, and 0.01–0.05, respectively. e Association between lung infectious titers (measured using a TCID₅₀ assay) and frequency of non synonymous, synonymous and G → A mutations. Each dot represent data from a given animal. Statistical analysis was performed using univariate linear regression. The error band (in gray) represent the 95% confidence interval of the regression line. Source data are provided as a Source data file.