Fig. 4

Vandetanib is a potent antiviral agent for SCoV2 propagation. a Apoptosis analysis of SCoV2-infected and uninfected cells treated with vandetanib. HEK293T cells infected with SCoV2 at an MOI of 1 for 4 h were further cultured in the presence of vandetanib (1 or 10 μM) for 44 h. Apoptotic cell death was analysed by flow cytometry as described in Materials and Methods. Staurosporine (200 nM, 44 h) was used as a positive control for inducing apoptotic cell death. Data shown are the representative of two independent experiments. b Dose-dependent antiviral effect of vandetanib against SCoV2 infection. At 2 days post-treatment, whole cell lysates of SCoV2-infected HEK293T cells (MOI of 1) treated with vandetanib at the indicated concentrations were analysed by immunoblotting with SCoV2 N antibody. β-actin, an internal loading control. c Anti-SCoV2 effect by post-treatment of vandetanib. HEK293T cells infected with SCoV2 at an MOI of 1 for 4 h were washed with fresh cell culture media and then treated with vandetanib (0.1, 1, or 10 μM) for 44 h. d Anti-SCoV2 effect by pre-treatment of vandetanib. HEK293T cells were treated with vandetanib (0.1, 1, or 10 μM) for 4 h and then infected with SCoV2 at an MOI of 1 for 44 h. Intracellular SCoV2 RNA level in SCoV2-infected cells was analysed by real-time qRT-PCR using PCR primers set specific to the SCoV2 N gene (c, d). Data shown are the representative of two independent experiments (mean ± SD; n = 2). e–h, In vivo anti-SCoV2 efficacy of vandetanib in hACE2 transgenic mouse model susceptible to SCoV2 infection. e A scheme for analysing clinical disease score and inflammation in SCoV-2-infected mice orally administrated with vandetanib (f, g). Eight-week-old hACE2 transgenic mice (n = 5 per group) were intranasally (IN) inoculated with SCoV2 (2 × 10³ pfu/head, 10 MLD₅₀, clade S). One hour later, they were orally administrated with vandetanib (25 mg/kg) daily. At 6 days post-infection, all mice were terminated for further analyses (f, g). f Clinical scores of SCoV2-uninfected hACE2 transgenic mice (open circle), SCoV2-infected hACE2 transgenic mice administrated with vehicle (black circle) and SCoV2-infected hACE2 transgenic mice administrated with vandetanib (red circle). Vehicle control, PBS with 1% Tween 80 (f–h). Clinical scores for all mice were monitored daily based on ruffled fur (1 point), reduced mobility (1 point), hunched posture (1 point) and death (4 points) as described in Materials and Methods. g Immunohistochemistry analysis showing the effect of vandetanib in SCoV2-induced severe lung injury in hACE2 transgenic mouse. At 6 days post-infection, immunohistochemistry analysis was performed as described in Materials and Methods. Black scale bar, 500 μm (upper), 100 μm (lower). H&E score (right panel, mean ± SD; n = 12; *p < 0.0001). h In vivo anti-SCoV2 efficacy of vandetanib in hACE2 transgenic mice. Eight-week-old hACE2 transgenic mice (n = 3 per group) were intranasally (IN) inoculated with SCoV2 (2 × 10³ pfu/head, clade S). One hour later, they were orally administrated with vandetanib (25 mg/kg) daily. At 3 days post-infection, all mice were terminated for further analyses (upper panel). Intracellular SCoV2 RNA levels of lung tissues isolated from SCoV2-infected hACE2 transgenic mice was analysed by real-time qRT-PCR. Each data point represents the average of two independent experiments (mean ± SD; n = 2, lower panel)