The Combination of GS-441524 (Remdesivir) and Ribavirin Results in a Potent Antiviral Effect Against Human Parainfluenza Virus 3 Infection in Human Airway Epithelial Cell Cultures and in a Mouse Infection Model

Abstract

Human parainfluenza virus type 3 (HPIV-3) can cause severe respiratory diseases, particularly in young children, the elderly and immunocompromised. There are no approved antiviral drugs against this virus. We report that the combination of ribavirin with either remdesivir or its parent nucleoside GS-441524 results in a pronounced antiviral effect against HPIV-3 in LLC-MK2 cells and in human airway epithelial cells grown at the air-liquid interface. In AG129 mice intranasally inoculated with HPIV-3, the combined treatment with ribavirin and GS-441524 decreased infectious viral lung titers by >2.5 log₁₀ to undetectable levels in 4 out of 11 mice and by 1.6 log₁₀ in the remaining 7 mice as compared with the vehicle. The lungs of all mice that received the combined treatment appeared histologically normal or virtually normal, whereas 8 of 11 vehicle-treated mice presented with bronchopneumonia. By contrast, ribavirin alone did not result in a reduction in infectious viral lung titers; GS-441524 alone reduced infectious viral lung titers by 1.2 log₁₀. Moreover, several mice in the single-treatment groups exhibited severe lung pathology. These findings may warrant exploring this combination in patients with severe HPIV-3 infections and possibly also against infections with other viruses that are susceptible in vitro to these two drugs.

Keywords: AG129 mice; human organoids; human respirovirus 3; obeldesivir; parainfluenza viruses; remdesivir; ribavirin.

Figure 1

The combined antiviral effect of ribavirin with either remdesivir or GS-441524 results in synergistic antiviral efficacy against HPIV-3 replication in LLC-MK2 cells. (A,D) The matrices of inhibition of HPIV-3 replication in the presence of combined treatments. Data are from five independent experiments. (B,E) The interaction between two molecules was determined by using the Bliss independence model. Data are presented as mean values of BLISS scores. Data are analyzed using the one-sample Student’s t-test. p < 0.05, *; p < 0.01, **; p < 0.001, ***. (C,F) 3D synergy maps.

Figure 2

The combination of ribavirin and GS-441524 potently inhibits HPIV-3 replication in nasal human airway epithelial (HnAEC) cultures in a prophylactic setting. (A,B) The antiviral effect of ribavirin and GS-441524 at various concentrations against HPIV-3 replication. Molecules were added to the basal medium of HnAEC cultures 2 h prior to infection, with treatments continuing for 16 consecutive days. Viral RNA levels in the apical washes were quantified by RT-qPCR. Individual data from two inserts of uninfected DMSO control and three inserts of each of the other groups are presented. (C,D) The toxicity of the molecules was determined by LDH assay per day (C) or at the endpoint (D). The maximum LDH activity was determined by lysing two fresh HnAE culture inserts using Triton X-100 for 24 h. Data from four inserts are presented as mean ± SD and analyzed using one-way ANOVA with Dunnett’s multiple comparisons test. ns, nonsignificant. (E) Kinetics of HPIV-3 replication in the presence of single treatments with each of GS-441524, ribavirin or their combination; two independent experiments (Experiment 1 and Experiment 2). Molecules were added for 18 consecutive days, followed by further culturing with basolateral medium that did not contain inhibitors. Levels of viral RNA in apical washes were quantified by RT-qPCR. Data from three inserts of uninfected DMSO control and four inserts from each of the other groups, and data from six inserts of the combination group and four inserts from each of the other groups are presented as mean ± SD in Experiment 1 and Experiment 2, respectively. The grey boxes indicate the treatment window. LLOQ presents the lower limit of quantification. “eq” stands for equivalent, as vRNA was measured but TCID₅₀-equivalent was reported.

Figure 3

The combination of ribavirin and GS-441524 potently inhibits HPIV-3 replication in HnAEC cultures in a therapeutic setting. At day 4 p.i., the HnAEC cultures were treated with GS-441524, ribavirin or their combination. Treatment continued until day 22 p.i., after which the basal medium without molecules was refreshed. (A) HPIV-3 RNA levels in apical washes were quantified by RT-qPCR. LLOQ presents the lower limit of quantification. (B) Infectious virus titers in apical washes were determined by endpoint virus titration assay. The lower limit of detection is −0.02. Experiment 1 and 2 are two independent experiments. Data from 3–4 inserts per group are presented as mean ± SD in Experiment 1 and 2. (C) HPIV-3 RNA levels and infectious virus titers at 14 days post-infection (14 d.p.i.). (D) Infectious virus titers of area under the curve over the treatment period in Experiment 1 and 2. Data from seven or eight inserts per group across two independent experiments are presented as mean ± SD. Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons test. ns, nonsignificant; p < 0.05, *; p < 0.01, **; p < 0.001, ***; p < 0.0001, ****. The grey boxes indicate treatment windows.

Figure 4

The combination of ribavirin and GS-441524 potently reduces viral loads and lung pathology upon intranasal inoculation of HPIV-3 in AG129 mice (A) Setup of the study. AG129 mice were orally dosed with either vehicle, ribavirin (25 mg/kg, per dose), GS-441524 (25 mg/kg, per dose) or their combination twice daily from day 1 p.i. to day 2 p.i. or day 5 p.i. At day 0, the mice were intranasally inoculated with 1.5 × 10⁶ TCID₅₀ of HPIV-3. Lung samples were collected at day 3 p.i. to measure viral loads and at day 6 p.i. to assess lung histopathology. (B) Infectious virus titers are expressed as log₁₀ TCID₅₀ per milligram of lung sample. LLOD presents the lower limit of detection. Data are from two independent experiments with 10 mice in the vehicle-treated group and 11 mice in drug-treated groups. (C) Representative H&E-stained images revealed normal lung parenchyma in uninfected mice, intra-alveolar hemorrhage (left upper corner), significant peri-vascular (red arrows) and peri-bronchial (blue arrows) inflammation, and focus of bronchopneumonia (green arrows) in the vehicle group, focal but significant peri-vascular (red arrows) and peri-bronchial (blue arrows) inflammation in the ribavirin-treated group, very limited peri-vascular (red arrow) and peri-bronchial (blue arrows) inflammation in the GS-441524 group, and very limited and slight peri-vascular inflammation in the combination group. The samples of uninfected mice and infected mice were from the same experiment, but the staining procedure was performed at different moments. The scale bar is 100 µm. (D) Cumulative severity scores of lungs of all infected mice. Data are from two independent experiments with 9 mice in the combination-treated group and 11 mice in all other groups. Individual data and median values (indicated by bars) are presented in all graphs. Data were analyzed with the Kruskal–Wallis test. ns, nonsignificant; p < 0.05, *; p < 0.01, **; p < 0.001, ***. (A) was designed with BioRender.