Cannabidiol Inhibits SARS-CoV-2 Replication And Promotes Immune Response
Cannabidiol from the cannabis plant has potential to prevent and inhibit SARS-CoV-2 infection
The rapid spread of COVID-19 underscores the need for new treatments. Here we report that cannabidiol (CBD), a compound produced by the cannabis plant, inhibits SARS-CoV-2 infection. CBD and its metabolite, 7-OH-CBD, but not congeneric cannabinoids, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after cellular infection, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription.
CBD induces interferon expression and up-regulates its antiviral signaling pathway. A cohort of human patients previously taking CBD had significantly lower SARSCoV-2 infection incidence of up to an order of magnitude relative to matched pairs or the general population. This study highlights CBD, and its active metabolite, 7-OH-CBD, as potential preventative agents and therapeutic treatments for SARS-CoV-2 at early stages of infection.
SARS-CoV-2 is a positive-sense single-stranded RNA (+ssRNA) enveloped virus composed of a lipid bilayer and four structural proteins that drive viral particle formation.
The spike (S), membrane (M), and envelope (E) are integral proteins of the virus membrane and serve to drive virion budding, while also recruiting the nucleocapsid (N) protein and the viral genomic RNA into nascent virions. Like SARS-CoV, SARS-CoV-2 primarily enters human cells by the binding of the viral S protein to the angiotensin-converting enzyme 2 (ACE2) receptor, after which the S protein undergoes proteolysis by transmembrane protease, serine 2 (TMPRSS2) or other proteases into two non-covalently bound peptides (S1, S2) that facilitate viral entry into the host cell.
The N-terminal S1 binds the ACE2 receptor, and the C-terminal S2 mediates viral-cell membrane fusion following proteolytic cleavage by TMRSS2 or other proteases. Depending upon the cell type, viral entry can also occur after ACE2 binding, independent of proteolytic cleavage. Following cell entry, the SARS-CoV-2 genome is translated into two large polypeptides that are cleaved by two viral proteases, MPro and PLPro, to produce 15 proteins, in addition to the synthesis of subgenomic RNAs that encode another 10 accessory proteins plus the 4 structural proteins.
These proteins enable viral replication, assembly, and budding. In an effort to suppress infection by the SARS-CoV-2 beta-coronavirus as well as other evolving pathogenic viruses, we tested the antiviral potential of a number of small molecules that target host stress response pathways.
One potential regulator of the host stress and antiviral inflammatory responses is cannabidiol (CBD), a member of the cannabinoid class of natural products. CBD is produced by Cannabis sativa (Cannabaceae; marijuana/hemp). Hemp refers to cannabis plants or materials derived thereof that contain 0.3% or less of the psychotropic tetrahydrocannabinol (THC) and typically have relatively high CBD content.
By contrast, marijuana refers to C. sativa materials with more than 0.3% THC by dry weight. THC acts through binding to the cannabinoid receptor, and CBD potentiates this interaction. Despite numerous studies and many typically unsubstantiated claims related to CBD-containing products, the biology of CBD itself is unclear and specific targets are mostly unknown. However, an oral solution of CBD is an FDA-approved drug, largely for the treatment of epilepsy.
Thus, CBD has drug status, is viable as a therapeutic, and cannot be marketed as a dietary supplement in the United States. Although limited, some studies have reported that certain cannabinoids have antiviral effects against hepatitis C virus (HCV) and other viruses.
To test the effect of CBD on SARS-CoV-2 replication, we pretreated A549 human lung carcinoma cells expressing exogenous human ACE-2 receptor (A549-ACE2) for 2 hours with 0–10 μM CBD prior to infection with SARS-CoV-2. After 48 hours, we monitored cells for expression of the viral spike protein (S).
For comparison, we also treated cells over a similar dose range with an MLK inhibitor (URMC-099) previously implicated as an antiviral for HIV and KPT-9274, a PAK4/NAMPT inhibitor that our analysis suggested might reverse many changes in gene expression caused by SARS-CoV-2. All three inhibitors potently inhibited viral replication under non-toxic conditions with EC50s ranging from 0.2–2.1 μM.
CBD inhibited SARS-CoV-2 replication in Vero E6 monkey kidney epithelial cells as well. No toxicity was observed at the effective doses. We also determined that CBD suppressed replication of a related beta-coronavirus, mouse hepatitis virus (MHV), under non-toxic conditions with an EC50 of ~5 μM using A549 cells that express the MHV receptor (A549-MHVR), indicating the potential for more broader viral efficacy.
Cannabidiol (CBD) is a potent inhibitor of SARS-CoV-2 infection in vitro.
(A) A549 cells with ACE2 overexpression (A549-ACE2) were treated with indicated doses of CBD, KPT-9274, or URMC-099 followed by infection with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.5 for 48 hours.
The cells were stained for spike protein and the percentage of cells expressing the spike protein in each condition was plotted. EC50 values are indicated. (B) The 1H qNMR spectra of CBD from a reference material and CBD samples from three different suppliers A, B, and C. (C) A549-ACE2 cells were treated with indicated doses of CBD from three different suppliers followed by infection with SARS-CoV-2 at an MOI of 0.5 for 48 hours.
The cells were stained for spike protein and the percentage of cells expressing the spike protein in each condition was plotted. EC50 values are indicated.
When isolated from its source plant, natural non-synthetic CBD is typically extracted along with other cannabinoids, representing the unavoidable residual complexity of natural products. To verify that CBD is indeed responsible for the viral inhibition, we analyzed a CBD reference standard as well as CBD from three different sources for purity using 100 percent quantitative NMR (qNMR).
These sources included two chemical vendors (Suppliers A and B) and one commercial vendor that used natural materials (Supplier C). The striking congruence between the experimental 1H NMR and the recently established quantum-mechanical HiFSA (1H Iterative Full Spin Analysis) profiles observed for all materials confirmed that 1) the compounds used were indeed CBD with purities of at least 97 percent and 2) congeneric cannabinoids were not present at levels above 1.0 percent.
Analysis of these different CBD preparations in the viral A549-ACE2 infection assay showed similar EC50s with a range from 0.6–1.8 μM likely reflecting the intrinsic biological variability of the assay. No toxicity was observed for any of the CBD preparations at the doses used to inhibit viral infection.
CBD is often consumed as part of a C. sativa extract, particularly in combination with psychoactive THC enriched in marijuana plants. We therefore determined whether congeneric cannabinoids, especially analogues with closely related structures and polarities produced by the hemp plant, are also capable of inhibiting SARS-CoV-2 infection.
Remarkably, only CBD was a potent agent, while limited or no antiviral activity was exhibited by the structurally closely related congeners that share biosynthesis pathways and form the biogenetically determined residual complexity of CBD purified from C. sativa: THC, cannabidiolic acid (CBDA), cannabidivarin (CBDV), cannabichromene (CBC), or cannabigerol (CBG).
None of these compounds were toxic to the A549-ACE2 cells in the dose range of interest. Notably, combining CBD with THC (1:1) significantly suppressed CBD efficacy consistent with competitive inhibition by THC.
Limited or no inhibition of SARS-CoV-2 infection by Cannabinoids other than CBD.
(A) A549-ACE2 cells were treated with indicated doses of various cannabinoids or a CBD/THC 1:1 mixture followed by infection with SARS-CoV-2 at an MOI of 0.5 for 48 hours. The cells were stained for spike protein and the percentage of cells expressing the spike protein in each condition was plotted. All cannabinoids tested were isolated from a hemp extract as described in Methods.
(B) Chemical structures of cannabinoids and 7-OH CBD. (C) A549-ACE2 cells were treated with indicated doses of 7-OH CBD followed by infection with the SARS-CoV-2 at an MOI of 0.5. The cells were stained for spike protein and the percentage of cells expressing the spike protein in each condition was plotted. Representative data of CBD from (Supplier A) is used for comparison. EC50 values are indicated.
CBD is rapidly metabolized in the liver and gut into two main metabolites, 7-carboxy-cannabidiol (7-COOH-CBD) and 7-hydroxy-cannabidiol (7-OH-CBD). Although the levels of 7-COOH-CBD are 40-fold higher than 7-OH-CBD in human plasma, 7-OHCBD is the active ingredient for the treatment of epilepsy. Like CBD but unlike the other cannabinoids, 7-OH-CBD effectively inhibited SARS-CoV-2 replication in A549-ACE2 cells and was non-toxic to cells.
Analysis of blood plasma levels in healthy patients taking FDA-approved CBD (Epidiolex®) shows a maximal concentration (Cmax) for CBD in the nM range whereas 7-OH-CBD had a Cmax in the μM range, similar to that observed in cultured cells.
These results suggest that CBD itself is not present at the levels needed to effectively inhibit SARS-CoV-2 in people. By contrast, the plasma concentrations of its metabolite 7-OH-CBD, whose Cmax can be increased several-fold by co-administration of CBD with a high-fat meal, are sufficient to potentially inhibit SARS-CoV-2 infection in humans.
CBD could be acting to block viral entry to host cells or at later steps following infection. As CBD was shown to decrease ACE2 expression in some epithelial cells including A549, we first determined whether CBD suppressed the SARS-CoV-2 receptor in our A549-ACE2 overexpressing cells.
No decrease in ACE2 expression was observed. Furthermore, analysis of lentiviruses pseudotyped with either the SARS-CoV-2 spike protein or the VSV glycoprotein showed that antibody to the spike protein effectively blocked viral infection of the SARS-CoV-2, but not VSV-G expressing viruses. However, 10 μM CBD only partially inhibited cell entry by spike-expressing virus, suggesting that other mechanisms were largely responsible for its antiviral effects.
By contrast, antibodies to the spike protein effectively blocked viral infection of the SARS-CoV-2 but not VSV-G expressing viruses. Consistent with this, CBD was also effective at inhibiting SARS-CoV-2 spike protein expression in host cells even 2 hours after infection in the presence of antibodies to the spike protein to prevent reinfection during this time period.
To assess whether CBD might be preventing viral protein processing by the viral proteases Mpro or PLpro, we assayed their activity in vitro. CBD did not affect the activity of either protease, raising the possibility that CBD targets host cell processes.
CBD inhibits viral replication after SARS-CoV-2 entry into the host cell.
(A) Immunoblots of ACE2 protein expression from A549-ACE2 cell lysates either untreated or treated with vehicle or CBD at indicated doses (n=3). Blots were probed with antibodies against ACE2 and tubulin. ACE2 protein expression levels were normalized to the tubulin signal within each sample. ACE2 expression levels were plotted relative to untreated samples. Expression levels were compared to vehicle by one-way ANOVA.
(B) 293T-ACE2 cells were infected by spike or VSV-G pseudovirus for 72 hours with the indicated doses of CBD treatment, and the percentage of infected cells plotted. (C) A549-ACE2 cells were either pre-treated or not with 10 μM CBD for 2 hours, then infected with SARS-CoV-2 at an MOI of 0.5 for 2 hours. Cells were then treated with 10 μM CBD or DMSO for 16 hours with the spike neutralizing antibody to prevent reinfection.
Spike positive cells were quantified and normalized to the virus-infected only sample. (D) Validation of neutralizing antibody efficacy. 400 pfu of SARS-CoV-2 virus was incubated with or without 100 μM of neutralizing antibody for 1 hour. A549-ACE2 cells were treated with the mixture for 16 hours and Spike positive cells were quantified.
Consistent with this interpretation, RNA-seq analysis of infected A549-ACE2 cells treated with CBD for 24 hours shows a striking suppression of SARS-CoV-2-induced changes in gene expression. CBD effectively eradicated viral RNA expression in the host cells, including RNA coding for a spike, membrane, envelope and nucleocapsid proteins.
Both SARS-CoV-2 and CBD each induced significant changes in cellular gene expression, including a number of transcription factors. Principal component analysis of host cell RNA shows an almost complete reversal of viral changes but, rather than returning to a normal cell state, the CBD+virus infected cells resemble those treated with CBD alone. Clustering analysis using Metascape reveals some interesting patterns and associated themes.
For example, viral induction of genes associated with chromatin modification and transcription (Cluster 1) is reversed by CBD although CBD alone has no effect. Similarly, viral inhibition of genes associated with ribosomes and neutrophils (Cluster 3) is largely reversed by CBD, but the drug alone has no effect. This contrasts with Clusters 5 and 6 where CBD alone induces strong activation of genes associated with the host stress response.
Together these results suggest that CBD acts to prevent viral protein translation and associated cellular changes.