Sulforaphane exhibits antiviral activity against pandemic SARS-CoV-2 and seasonal HCoV-OC43 coronaviruses in vitro and in mice

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease 2019 (COVID-19), has incited a global health crisis. Currently, there are limited therapeutic options for the prevention and treatment of SARS-CoV-2 infections. We evaluated the antiviral activity of sulforaphane (SFN), the principal biologically active phytochemical derived from glucoraphanin, the naturally occurring precursor present in high concentrations in cruciferous vegetables. SFN inhibited in vitro replication of six strains of SARS-CoV-2, including Delta and Omicron, as well as that of the seasonal coronavirus HCoV-OC43. Further, SFN and remdesivir interacted synergistically to inhibit coronavirus infection in vitro. Prophylactic administration of SFN to K18-hACE2 mice prior to intranasal SARS-CoV-2 infection significantly decreased the viral load in the lungs and upper respiratory tract and reduced lung injury and pulmonary pathology compared to untreated infected mice. SFN treatment diminished immune cell activation in the lungs, including significantly lower recruitment of myeloid cells and a reduction in T cell activation and cytokine production. Our results suggest that SFN should be explored as a potential agent for the prevention or treatment of coronavirus infections.

Fig. 1. Antiviral effects of SFN against HCoV-OC43.

Median effect plots and dose-effect curves calculated for a Vero C1008 cells infected with HCoV-OC43 after a 1–2 h incubation with increasing concentrations of SFN; b MRC-5 cells infected with HCoV-OC43 after a 1–2 h incubation with increasing concentrations of SFN; c Vero C1008 cells infected with HCoV-OC43 over 24 h, after which they were incubated with SFN; d Vero C1008 cells incubated with SFN for 24 h, after which the drug was removed, and the cells were infected with HCoV-OC43; e Vero C1008 cells infected with HCoV-OC43 after a 1–2 h incubation with increasing concentrations of remdesivir. f Normalized isobologram showing combination index (CI) for combinations of various doses. Antiviral data is displayed in red; anti-host cell activity (cytotoxicity) is displayed in blue. Synergism (CI < 1); additive effect (CI = 1); antagonism (CI > 1); SFN, Sulforaphane; RDV, remdesivir. Dotted lines represent 95% confidence interval. Experiments were performed a minimum of 2 times (range = 2–7), 3–6 replicates within each experiment, except experiment shown in (d), which was performed once.

Fig. 2. Antiviral effects of SFN against SARS-CoV-2.

Median effect plot and dose-effect curves calculated for a Vero C1008 cells infected with SARS-CoV-2/Wuhan-Hu-1 after 1–2 h incubation with increasing concentrations of SFN; b Vero C1008 cells infected with SARS-CoV-2/Wuhan-Hu-1 for 24 h and then incubated with SFN. Antiviral data is displayed in red; anti-host cell activity (cytotoxicity) is displayed in blue. c The antiviral activity in human Caco-2 cells was determined by measuring viral RNA by qPCR. The cells were incubated with SFN for 1 h before viral inoculation. d Effects of SFN evaluated in Vero C1008 cells exposed to drug for 1 h followed by viral inoculation. A reference strain (USA-WA1/2020) and two 614G+ clinical strains of SARS-CoV-2 were evaluated for CPE using a bioluminescence readout. e Effects of SFN and remdesivir evaluated in Vero C1008 cells exposed to the drug for 1 h followed by viral inoculation. f Normalized isobologram showing combination index (CI) for combinations of various doses of SFN and remdesivir. Synergism (CI < 1); Additive effect (CI = 1); Antagonism (CI > 1); SFN, Sulforaphane; RDV, Remdesivir. Dotted lines represent 95% confidence interval. Experiments were performed a minimum of two times (range = 2–7), 3–6 replicates within each experiment, except the experiment shown in (e), which was performed once.

Fig. 3. SFN treatment in SARS-CoV-2 infected K18-hACE2 mice.

a Six- to eight-week-old male K18-hACE-transgenic mice were randomly distributed among treatment groups and inoculated intranasally with SARS-CoV-2/USA/WA1/2020 or vehicle. b Four days post inoculation, there was a marked weight loss in the infected groups, although there was significantly less weight loss in the SFN treated animals. By day 6 post inoculation, the SFN-treated animals had lost 7.5% less bodyweight compared to infected untreated controls (one-way ANOVA, ***P < 0.0001). Data from three independent experiments, uninfected (n = 8), infected untreated (n = 16), infected treated (n = 14). c Bronchoalveolar lavage (BAL) total protein quantification, determined as a surrogate for lung injury, measured 6 days post-infection. Infected untreated animals had significantly higher total protein compared to the infected treated group (one-way ANOVA, ***P < 0.0001). Data from three independent experiments, uninfected (n = 8), infected untreated (n = 16), infected treated (n = 14). d The viral load in the BAL, as determined by qPCR, was significantly higher in infected untreated animals compared to the infected treated group (Mann–Whitney U test, two-tailed, *P = 0.036). Data from two independent experiments, infected untreated (n = 8), infected treated (n = 9). e The viral load in the lungs of infected treated animals, represented as the SARS-CoV-2 N protein copies normalized to Pol2Ra, had a 1.5 log₁₀ reduction compared to infected untreated controls (Mann–Whitney U test, two-tailed, **P = 0.004). Data from two independent experiments, infected untreated (n = 11), infected treated (n = 9). f Hematoxylin and eosin (H&E) staining and immunostaining for SARS-CoV-2 spike protein of histological sections of the lungs of representative uninfected control, infected untreated and infected treated mice. Regions of the lung anatomy where alveolar and peribronchiolar inflammation was assessed are highlighted in boxes. Images show low (left panels; scale bar, 1 mm) and high-power magnification (right panels; scale bar, 50 µm) of the same tissue section. g Histopathological severity scoring was evaluated according to the pathological changes outlined in the methods section. Data from one independent experiment, infected untreated (n = 8), infected treated (n = 5). Mann–Whitney U test, two-tailed, **P = 0.0008. h Quantification of the SARS-CoV-2 spike protein immunostaining showed a 4.41× lower % area in the lungs of SFN-treated mice compared to infected untreated controls (P = 0.01). Data from one independent experiment, uninfected (n = 4), infected untreated (n = 8), infected treated (n = 5). One-way ANOVA, *P < 0.05, **P < 0.005. All the data in this figure are represented as mean ± standard deviation. Each dot represents one animal.

Fig. 4. Effects of SFN treatment in the immune response.

a Uniform Manifold Approximation and Projection (UMAP) was used to visualize all the immune cell populations within the spleen and lung of uninfected (grey), infected untreated (blue), and infected treated (red) mice. The corresponding immune cell populations are presented in multiple colors in the panels on the right (b). Summary of immune cell frequencies out of total CD45⁺ immune cells in spleen and lung of infected treated or untreated mice. c Total cell count of indicated immune cell subset per spleen. Each dot represents one mouse. d Total cell count of indicated immune cell subset per lung. Each dot represents one mouse, data from one independent experiment. Bars represent mean values. DCs, dendritic cells; NK, natural killer, M-MDSC, mononuclear myeloid-derived suppressor cells. Statistical comparisons were made with two-way ANOVA, *P < 0.05, **P < 0.01.

Fig. 5. Functional characterization of the immune response after SFN treatment.

a Myeloid cell subsets shown as percent of total CD45⁺ immune cells within the lung. b Alveolar macrophages (AM) after stimulation with protein transport inhibitors. MFI, mean fluorescent intensity. c Cytokine expression in alveolar macrophages after stimulation with protein transport inhibitors. d T cells were stained ex vivo immediately without further stimulation and evaluated for the expression of Ki67, PD1, and MHC-II. Percent of CD8⁺ or CD4⁺ T cells from spleen or lung expressing indicated marker are shown. e Percent of CD8⁺ or CD4⁺ T cells from spleen or lung expressing indicated marker after stimulation with PMA/ionomycin. Each dot represents one mouse. Data from one independent experiment, uninfected (n = 4), infected untreated (n = 8), infected treated (n = 5). Data represented as mean ± standard error of mean. Statistical comparisons were made with one-way ANOVA, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.