Antiviral Efficacies of FDA-Approved Drugs against SARS-CoV-2 Infection in Ferrets

Abstract

Due to the urgent need of a therapeutic treatment for coronavirus (CoV) disease 2019 (COVID-19) patients, a number of FDA-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. Furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome CoV 2 (SARS-CoV-2), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. To this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for SARS-CoV-2 infection were assessed in the ferret infection model. While the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (PBS)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the PBS-treated control group. Only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at 8 days postinfection (dpi) than the PBS-treated control group. To further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. Compared to the PBS-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (SN) antibody titers. Taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. Despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of COVID-19 patients.IMPORTANCE The SARS-CoV-2 pandemic continues to spread worldwide, with rapidly increasing numbers of mortalities, placing increasing strain on health care systems. Despite serious public health concerns, no effective vaccines or therapeutics have been approved by regulatory agencies. In this study, we tested the FDA-approved drugs lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir against SARS-CoV-2 infection in a highly susceptible ferret infection model. While most of the drug treatments marginally reduced clinical symptoms, they did not reduce virus titers, with the exception of emtricitabine-tenofovir treatment, which led to diminished virus titers in nasal washes at 8 dpi. Further, the azathioprine-treated immunosuppressed ferrets showed delayed virus clearance and low SN titers, resulting in a prolonged infection. As several FDA-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial.

Keywords: COVID-19; antiviral therapeutics; ferrets; immunosuppression; serum neutralization; severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

FIG 1

Schedule of drug treatments and SARS-CoV-2 infection in ferrets. To induce the immunosuppression condition, PBS or azathioprine was orally administered to ferrets for the entire experimental period. All groups of ferrets were administered each drug or PBS via oral gavage once, starting at 1 dpi.

FIG 2

Clinical features of drug-treated ferret groups. Ten ferrets per group were inoculated via the i.n. route with 10^5.8 TCID₅₀ of the NMC-nCoV02 strain. Starting at 1 day of postinfection, lopinavir-ritonavir, hydroxychloroquine sulfate, emtricitabine-tenofovir, or azathioprine was orally administered to each group. Temperature changes (A) and relative weight changes (B) are shown with standard errors of the means. (A) Temperature is represented as relative change (degrees Celsius), and (B) weight change is demonstrated as a percentage of the initial body weight. Nasal wash and fecal samples were collected at 2, 4, 6, 8, 10, 12, and 14 dpi from nasal wash specimens and fecal swabs. (C) Virus titers (TCID₅₀) were measured in nasal wash specimens from each group. (D) The number of viral RNA copies was measured in fecal samples using qRT-PCR. Viral titers and RNA copy numbers are shown as means ± standard errors of the means (SEM) from four animals, and titers below the limit of detection are shown as 1 log₁₀ TCID₅₀/ml or 0.3 log₁₀ viral RNA copy numbers/ml (dashed lines). Asterisks indicate statistical significance between the control and each group as determined by two-way ANOVA and subsequent Dunnett’s test (*, P < 0.05).

FIG 3

Virus titers in respiratory tissues. Groups of ferrets (3/group) were sacrificed at 4 and 8 dpi. Viral titers were measured in turbinate (A) and lung (B) by determining the numbers of TCID₅₀ per gram. The limit of detection is 1 log₁₀ TCID₅₀/g and indicated by the dotted line for each representation.

FIG 4

Comparison of serum neutralization antibody titers of drug-treated ferrets. Blood was collected at 10, 14, and 21 dpi from each group of ferrets (n = 4), and serum neutralization antibody titers were measured in Vero cells. The serum neutralization titer of each ferret is represented by an individual dot in each bar graph. Asterisks indicate statistical significance between the control and each group, as determined by two-way ANOVA and subsequent Dunnett’s test (*, P < 0.05; ***, P < 0.001).