The broad-spectrum antiviral favipiravir protects guinea pigs from lethal Lassa virus infection post-disease onset

Abstract

With up to 500,000 infections annually, Lassa virus (LASV), the cause of Lassa fever, is one of the most prevalent etiological agents of viral hemorrhagic fever (VHF) in humans. LASV is endemic in several West African countries with sporadic cases and prolonged outbreaks observed most commonly in Sierra Leone, Liberia, Guinea and Nigeria. Additionally several cases of Lassa fever have been imported into North America, Europe and Asia making LASV a global threat to public health. Despite this, currently no approved therapeutic or vaccine exists to treat or prevent LASV infections. Here, using a passaged strain of LASV that is uniformly lethal in Hartley guinea pigs, we demonstrate that favipiravir, a broad-spectrum antiviral agent and leading treatment option for influenza, has potent activity against LASV infection. In this model, once daily treatment with favipiravir significantly reduced viral titers in tissue samples and reduced mortality rates when compared with animals receiving vehicle-only or ribavirin, the current standard of care for Lassa fever. Favipiravir remained highly effective against lethal LASV infection when treatments were initiated nine days post-infection, a time when animals were demonstrating advanced signs of disease. These results support the further preclinical evaluation of favipiravir for Lassa fever and other VHFs.

Figure 1. In vitro inhibition of LASV by favipiravir.

Vero cells were infected with wild-type LASV (Josiah, m.o.i. = 0.01) and cultured in the presence of increasing concentrations of favipiravir. Linear regression analysis conducted on infectious LASV titers in supernatants collected at 3 and 5 days post-infection revealed a clear dose response with an EC₉₀ of between 1.7 and 11.1 μg/mL, respectively.

Figure 2. Titration of the GPA-LASV Josiah strain in outbred guinea pigs.

(A) Comparison of survival rates following challenge with wild-type (WT) LASV and GPA-LASV. Groups of eight outbred guinea pigs were infected i.p. with 10,000 TCID₅₀ and monitored for disease progression and survival. (B) In order to determine challenge dose, groups of four outbred guinea pigs were inoculated i.p. with ten-fold serial dilutions of GPA-LASV and monitored for disease progression and survival. The LD₅₀ was estimated at 1,000 TCID₅₀. ***p < 0.001 compared to WT LASV infection.

Figure 3. Treatment of lethal LASV infection in guinea pigs with favipiravir beginning 48 hours after challenge.

Groups of nine guinea pigs were challenged with a lethal dose of GPA-LASV and treated s.c. once daily for two weeks with favipiravir (150 or 300 mg/kg/d), ribavirin (50 mg/kg/d) or vehicle placebo beginning 48 hours after challenge. At a time when control (vehicle treated) animals were demonstrating signs of advanced disease, three animals per group were euthanized for sample collection. The remaining six animals per group were monitored for (A) temperature, (B) body weight, and (C) survival for up to 42 days post-infection (p.i.). *p < 0.05, **p < 0.01, ***p < 0.001 compared to placebo; ^ap < 0.05 and ^cp < 0.001 compared to ribavirin.

Figure 4. Effect of favipiravir treatment on viral loads in guinea pigs.

In the first in vivo efficacy study, three guinea pigs per treatment group (150 or 300 mg favipiravir/kg/d, 50 mg ribavirin/kg/d, or placebo) were euthanized for sample collection on day 12 post-infection, a time when control animals were displaying signs of terminal disease. Infectious titers in (A) Serum, (B) Liver, (C) Spleen and (D) Lung are shown. *p < 0.05, ***p < 0.001.

Figure 5. Immunohistochemical analysis of tissues from animals infected with GPA-LASV and treated daily with favipiravir, ribavirin or placebo.

Tissue samples from infected guinea pigs treated daily with favipiravir (150 or 300 mg/kg/d), ribavirin (50 mg/kg/d) or placebo were collected at 12 days post-infection and examined for the presence of LASV antigen (glycoprotein, GP) using a GP-specific monoclonal antibody according to standard immunohistochemistry techniques.

Figure 6. Favipiravir treatment of advanced LASV infection in guinea pigs.

Groups of six guinea pigs were challenged with a lethal dose of GPA-LASV. Beginning on days 5, 7, and 9 post-challenge, favipiravir treatment (300 mg/kg/d, once daily s.c. for 14 consecutive days) was initiated in the respective group of animals. A control, vehicle placebo treatment group was included with treatments commencing at 5 days post-infection and (A) temperature, (B) body weight, and (C) survival were monitored for up to 42 days post-infection (p.i.). *p < 0.05, ***p < 0.001.