Cannabidiol inhibits SARS-CoV-2 replication through induction of the host ER stress and innate immune responses
The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), a pandemic that continues to cause widespread morbidity and mortality across the globe. SARS-CoV-2 is the seventh species of coronavirus known to infect people. These coronaviruses, which include SARS-CoV, 229E, NL63, OC43, HKU1, and MERS-CoV, cause a range of symptoms from the common cold to more severe pathologies (1). Despite recent vaccine availability, SARS-CoV-2 is still spreading rapidly (2), highlighting the need for alternative treatments, especially for populations with limited inclination or access to vaccines. To date, few therapies have been identified that block SARS-CoV-2 replication and viral production.
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 promote virion budding while also recruiting the nucleocapsid (N) protein and the viral genomic RNA into nascent virions. Like its close relative 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 (3–5), 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. Depending upon the cell type, viral entry can also occur after ACE2 binding, independent of proteolytic cleavage (6–8). Following cell entry, the SARS-CoV-2 genome is translated into two large polypeptides that are cleaved by two viral proteases, Mpro and PLpro (9, 10), 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 (11) 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 (11). Despite numerous studies and many unsubstantiated claims related to CBD-containing products, the biologic actions of CBD itself are unclear and specific targets are mostly unknown (12). However, an oral solution of CBD is an FDA-approved drug, largely for the treatment of epilepsy (13). Thus, CBD has drug status, is viable as a therapeutic, and cannot be marketed as a dietary supplement in the United States (12). Although limited, some studies have reported that certain cannabinoids have antiviral effects against hepatitis C virus (HCV) and other viruses (14).
High purity CBD inhibits SARS-CoV-2 replication in human lung epithelial cells
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) and viral titer. CBD potently inhibited viral replication under non-toxic conditions with an EC50 of ~1 μM (Fig. 1A; fig. S1A). CBD inhibited SARS-CoV-2 replication in human Calu3 lung and Vero E6 monkey kidney epithelial cells as well (fig. S1B), and no toxicity was observed at the effective doses (fig. S1C,D). Finally, we tested three SARS-CoV-2 variants of concern (α, β, and γ) in addition to the original SARS-CoV-2 strain, and their ability to infect cells was comparably inhibited by CBD.
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 (12). To verify that CBD is indeed responsible for the viral inhibition, we analyzed a CBD reference standard as well as CBD from four different sources for purity using 100% quantitative NMR (qNMR). These sources included two chemical vendors (Suppliers A and B) and two commercial vendors (Suppliers C and D). 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% (Fig. 1B) and 2) congeneric cannabinoids were not present at levels above 1.0%. Analysis of these different CBD samples in the viral A549-ACE2 infection assay showed similar EC50s with a range from 0.6–1.8 μM, likely reflecting the intrinsic variability of the biological assay (Fig. 1A). No toxicity was observed for any of the CBD preparations at the doses used to inhibit viral infection (fig. S1 E-G).
The CBD metabolite 7-OH-CBD, but not a panel of closely related CBD congeners, exhibits antiviral activity
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, of this group, only CBD was a potent agent, while no or very limited antiviral activity was exhibited by these 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) (Fig. 2 A,D; see Methods). None of these cannabinoids were toxic to the A549-ACE2 cells in the dose range of interest (fig. S2). Notably, combining CBD with THC (1:1) significantly suppressed CBD efficacy consistent with competitive inhibition by THC.
CBD is rapidly metabolized in the intestine and liver into two main metabolites, 7-carboxy-cannabidiol (7-COOH-CBD) and 7-hydroxy-cannabidiol (7-OH-CBD). The level of 7-COOH-CBD is 40-fold higher, and the level of 7-OH-CBD is 38% of the CBD level in human plasma (15). CBD and its 7-OH-CBD metabolite are the active and equipotent ingredients for the treatment of epilepsy (13). Like CBD, 7-OH-CBD effectively inhibited SARS-CoV-2 replication in A549-ACE2 cells (Fig. 2C) and was non-toxic to cells (fig. S2H,I). Analysis of blood plasma levels in healthy patients taking 1500 mg daily of FDA-approved CBD solution (Epidiolex) showed a maximal concentration (Cmax) at 7 days for CBD and 7-OH-CBD of 1.7 μM and 0.56 μM, respectively; the Cmax can be further increased several-fold by co-administration with a high-fat meal (15). Taken in aggregate, these results suggest the effective plasma concentrations of CBD and its metabolite are within the therapeutic range to inhibit SARS-CoV-2 infection in humans.
CBD acts at an early step after viral entry into cells
CBD could be acting by blocking viral entry to host cells or at later steps following infection. As CBD was reported to decrease ACE2 expression in some epithelial cells, including A549 (16), we first determined whether CBD suppressed the SARS-CoV-2 receptor in the A549-ACE2, Calu-3, and Vero E6 cells. No decrease in ACE2 expression was observed (Fig. 3A; fig. S4A,B). Furthermore, analysis of lentiviruses pseudotyped with either the SARS-CoV-2 spike protein or the vesicular stomatitis virus (VSV) glycoprotein (17) showed that 10 μM CBD only weakly inhibited cell entry by spike-expressing virus, suggesting that other mechanisms are largely responsible for its antiviral effects. The robustness of the assay was confirmed by using anti-spike antibodies that effectively blocked viral infection of lentivirus pseudotyped with spike, but not VSVg (Fig. 3B, and figs. S3 A and B). In contrast to the negligible effect on viral entry, CBD was very effective (~95–99%) at inhibiting SARS-CoV-2 spike protein expression in host cells at 2 and 6 hours after infection post entry (Fig. 3C). This was true even in the presence of antibodies to the spike protein to prevent reinfection (Fig. 3D) suggesting CBD acts early in the infection cycle, in a post entry step. CBD was also partially effective (~60%) at inhibiting SARS-CoV-2 at 15 h after infection (Fig. 3C), suggesting a possible secondary effect on viral assembly and release. To assess whether CBD might be preventing viral protein processing by the viral proteases Mpro or PLpro, we assayed their activity in vitro (fig. S4C,D). CBD did not affect the activity of either protease, raising the possibility that CBD targets host cell processes.
CBD inhibits viral RNA expression and reverses viral-induced changes in host gene expression
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 spike, membrane, envelope and nucleocapsid proteins (Figs. 4 A and B). Both SARS-CoV-2 and CBD each induced significant changes in cellular gene expression (figs. S5 and S6). Principal component analysis (PCA) of host cell RNA shows 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 (Fig. 4C). Clustering analysis using Metascape reveals some interesting patterns and associated themes (Fig. 4D, figs. S7, and S8). 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.
To gain a better understanding of the specific anti-viral action of CBD, we analyzed RNAseq from lysates of uninfected or SARS-CoV-2-infected cells treated for 24 hours with the inactive CBDV homologue. Induction of viral genes for spike, envelope and nucleocapsid proteins is reduced by only 60% with CBDV as opposed to ~99% with CBD (Fig. 5A,B). CBDV treatment causes fewer transcriptomic changes than CBD in A549-ACE2 cells and is largely ineffective at reversing transcriptional changes induced by SARS-CoV-2 (Fig. 5C). Clustering analysis using Metascape reveals only a couple clusters that show CBDV reversal of viral transcriptomic changes (Fig. 5D). These include autophagy and lipid metabolism (Cluster 1) that are induced by CBDV as well as protein translation/cell cycle/DNA replication (Cluster 3) that are suppressed by CBDV.
CBD induces the ER stress response and IRE1α activity as a key mechanism for its anti-viral action
Of particular interest are three sets of genes related to the endoplasmic reticulum (ER) stress response, the unfolded protein response (UPR) and interferon induction that are selectively upregulated by CBD but not CBDV (Fig. 6A). By contrast, genes associated with the oxidative stress response are induced by both cannabinoids. Cells experience ER stress when the workload on the ER protein folding machinery exceeds its capability. Under ER stress, secretory proteins accumulate in unfolded forms within the organelle to trigger a set of intracellular signaling pathways called UPR, which is part of a larger cellular stress response that maintains proteostasis throughout the cell (18). The UPR pathway is controlled by three ER transmembrane proteins – IRE1α, PERK, and ATF6 – that contain an ER luminal domain capable of directly or indirectly sensing misfolded proteins. In response to ER stress, each of these sensors sets in motion transcriptional and translational changes that increase protein folding capacity and attempt to restore homeostasis. However, if the stress on the ER is irremediable, the UPR switches outputs and signals cell death. We validated CBD induction of IRE1α, PERK, and ATF6 gene expression by qRT-PCR (fig. S9A), consistent with previous reports (19). Ingenuity analysis confirmed that CBD induces the UPR significantly more than CBDV (figs. S9B, S10B, S11).
Numerous studies report compelling evidence that the UPR is hyperactivated and required for replication of other closely related coronavirus family members (20, 21). Surprisingly, although GSEA enrichment analysis of the RNA-seq data showed that the IRE1α pathway is strongly activated by CBD in the presence or absence of virus, this pathway was not activated by SARS-CoV-2 alone (Table 1; figs. S12,S13,S14). PERK, by contrast, was functionally activated by both SARS-CoV-2 and CBD. IRE1α is a single pass ER transmembrane protein with bifunctional kinase/endoribonuclease (RNase) activities. In response to ER stress, IRE1α undergoes oligomerization and autophosphorylation, which allosterically activates its RNase to initiate productive splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor that upregulates many host stress responses, including ER chaperone induction and ER-associated degradation (ERAD) components (22) (Fig. 6E).
CBD strongly activates IRE1α RNase activity as shown by analysis of XBP1 splicing using both RNAseq data to quantify spliced XBP1 as well as direct confirmation by qRT-PCR (Fig. 6B; fig. S15). As predicted, CBD induced XBP1 splicing in the presence or absence of virus whereas CBDV had no significant effect and is comparable to virus alone. The time course and dose response curves for CBD induction of XBP1 splicing in the absence of the virus were consistent with the time course and dose responses for CBD inhibition of viral spike protein expression in A549-ACE2 cells (Fig. 6C). Furthermore, while an IRE1α knockout had no significant effect on SARS-CoV-2 infection, it shifted the dose response and significantly reduced the anti-viral effects of CBD, leading to an approximately 2-fold increase in its EC50 against SARS-CoV-2 (Fig. 6D; fig. S16). Together, these results indicate that CBD induction of IRE1α is a critical component of its anti-viral action against SARS-CoV-2.
CBD induces interferon expression as part of its anti-viral activity
Another mechanism by which CBD could suppress viral infection and promote degradation of viral RNA is through induction of the interferon signaling pathway. Interferons are among the earliest innate immune host responses to pathogen exposure (23). As reported (24), SARS-CoV-2 infection suppresses the interferon signaling pathway (Fig. 7A, and fig. S17). Many genes in the pathway such as ISG15, IFIT1, IFIT3, SOCS1 and OAS1, an interferon-induced gene that leads to activation of RNase L and RNA degradation (25), were moderately up-regulated by CBD alone but highly induced by CBD in the presence of the virus (Fig. 7A and figs. S18,19). These latter results are consistent with the possibility that CBD lowers the effective viral titer sufficiently to enable normal host activation of the interferon pathway. At the same time, CBD effectively reversed viral induction of cytokines that can lead to the deadly cytokine storm at later stages of infection (Fig. 7B). By contrast, the inactive homologue CBDV does not significantly induce genes within the interferon pathway or prevent cytokine induction (Fig. 6A, 7A, 7C, figs S20A,B and S21).
To directly test the possibility that interferons might account in part for the anti-viral activity of CBD, we exposed ACE2-A549 cells to a mixture of antibodies against Type I (α,β,ο) and Type II (γ) interferons prior to 2.5 μM CBD treatment and viral infection. The results show that the anti-interferon antibodies reduce the anti-viral effects of CBD and partially rescue SARS-CoV-2 infection (Fig. 7D). Collectively, these results suggest that CBD inhibits SARS-CoV-2 infection in part by activating IRE1α and the interferon pathways, leading to degradation of viral RNA and subsequent viral-induced changes in host gene expression, including cytokines.
CBD treatment significantly inhibits SARS-CoV-2 replication in mice
As several agents including cationic amphipathic drugs block SARS-CoV-2 replication in cultured cells but not in vivo (26), we determined whether CBD reduces viral titer in female K18-hACE2 mice (27). Mice were injected intraperitoneally twice daily with CBD (20 or 80 mg/kg) for 7 days prior to intranasal challenge with SARS-CoV-2 (2x104 PFU). After the challenge, administration of CBD continued twice daily for an additional 4 days (Fig. 8A). CBD treatment significantly inhibited viral replication in lungs and nasal turbinates at day 5 post-infection in a dose-dependent manner (Figs. 8B-C). The lower dose of CBD reduced viral load by 4.8-fold in lungs and 3.7-fold in nasal turbinates, while the higher dose decreased viral titers by 40- and 4.8-fold in lungs and nasal turbinates, respectively. During this period, the mice showed no signs of clinical disease, and their body weights were not significantly changed (Fig. 8D). These results establish the pre-clinical efficacy of CBD as an anti-viral drug for SARS-CoV-2 during early stages of infection.
CBD usage is negatively associated with indications of SARS-CoV-2 infection in patients
Given that high purity CBD preparations are taken by a large number of individuals, we examined whether medication records of CBD prescriptions or use are associated with indications of SARS-CoV-2 infection (i.e., positive COVID-19 tests and/or COVID-19 diagnoses proximal to COVID-19 tests). An oral solution of CBD 100 mg/mL (CBD100) is often used for the treatment of seizures (see the Patient Analysis Supplement). Analysis of 1,212 patients from the National COVID Cohort Collaborative (N3C) (28) with a history of seizure-related conditions and a medication record of CBD100 revealed 6.2% (75 patients) with an indication of SARS-CoV-2 infection proximal to the dates of their first COVID-19 test in their N3C data. This was a significantly lower rate than the rates of matched control groups of patients that did not have any CBD100 records (e.g., 6.2% for CBD100 patients compared to 8.9% for non-CBD100 patients, p = 0.014; multivariable logit model odds ratio (OR) of 0.65, p = 0.009, 95% C.I. [0.47,0.90]). The demographics and medication history of the CBD100 patients were similar to those of the matched control group. The medical condition history for these patients included seizure-related conditions, the CDC list of at-risk conditions (29) and other potential confounders such as conditions of reduced mobility, chronic pain, or developmental disabilities that can limit public interaction and COVID-19 exposure. The negative association was even more significant in analyses of a subgroup of 531 CBD100 patients who were likely taking CBD100 on the dates of their first COVID-19 tests (e.g., 4.9% among these CBD100 patients compared to 9.0% among 531 matched controls, p = 0.011; OR = 0.48, p = 0.006, 95% C.I. [0.29,0.81])(Fig. 9; Table S4 in Patient Analysis Supplement which describes the patient data analysis methods and findings in detail).