key: cord-0254863-oof0nh2u
authors: Fernandes, Maria Fernanda; Chan, John Zewen; Hung, Chia Chun Joey; Tomczewski, Michelle Victoria; Duncan, Robin Elaine
title: Effect of cannabidiol on apoptosis and cellular interferon and interferon-stimulated gene responses to the SARS-CoV-2 genes ORF8, ORF10 and M protein
date: 2022-01-12
journal: bioRxiv
DOI: 10.1101/2022.01.11.475901
sha: 49e53f4240d8b9ea1db32c18ef86c77db23dee50
doc_id: 254863
cord_uid: oof0nh2u

Aims To study effects on cellular innate immune responses to novel genes ORF8 and ORF10, and the more conserved Membrane protein (M protein) from the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes COVID-19, either alone, or in combination with cannabidiol (CBD). Main Methods HEK293 cells were transfected with a control plasmid, or plasmids expressing ORF8, ORF10, or M protein, and assayed for cell number and markers of apoptosis at 24 h, and expression of interferon and interferon-stimulated genes at 14 h. Key findings A significant reduction in cell number, and increase in early and late apoptosis, was found after 24 h in cells where expression of viral genes was combined with 1-2 μM CBD treatment, but not in control-transfected cells treated with CBD, or in cells expressing viral genes but treated only with vehicle. CBD (2 μM) augmented expression of IFNγ, IFNλ1 and IFNλ2/3, as well as the 2’-5’-oligoadenylate synthetase (OAS) family members OAS1, OAS2, OAS3, and OASL, in cells expressing ORF8, ORF10, and M protein. CBD also augmented expression of these genes in control cells not expressing viral genes, without enhancing apoptosis. Significance Our results demonstrate a poor ability of HEK293 cells to respond to SARS-CoV-2 genes alone, but suggest an augmented innate anti-viral response to these genes in the presence of CBD. Furthermore, our results indicate that CBD may prime components of the innate immune system, increasing readiness to respond to viral infection without activating apoptosis, and therefore could be studied for potential in prophylaxis.

transfection in a 96-well plate, 0.1 μg of plasmid DNA and 0.25 μL jetPRIME reagent were 165 mixed with 5 μL buffer and incubated for 10 min at room temperature. For transfection in a 24-166 well plate, 0.5μg of plasmid DNA and 1.25 μL jetPRIME reagent were mixed with 50 μL buffer 167 and incubated for 10 min at room temperature. The incubated solution was diluted in culture 168 medium to a volume of 100 μL (for 96-well plates) or 500 μL (for 24-well plates) and the 169 mixture replaced the culture medium of the cells. experiments, these were averaged to derive single values reported as biological replicates. 230

A concentration response curve was generated by treating cells transfected with the control 233 plasmid (pCMV) or plasmids expressing viral genes with vehicle (0.1% EtOH (i.e. 0 µM CBD)), 234 or with increasing concentrations of CBD (Fig. 1A) . The range of concentrations tested was 235 based on pharmacologically achievable blood concentrations observed in human 236 pharmacokinetic studies [50] . The slopes of lines generated from concentration-responses to 237 CBD in cells expressing viral genes were significantly non-zero, indicating a significant 238 relationship between increasing dose of CBD and relative cell number, while the slope of the line 239 for pCMV was not significantly non-zero. IC50 values for CBD concentrations were 0.89 µM 240 for cells expressing ORF8, 0.91 µM for cells expressing ORF10, 0.99 µM for cells expressing M 241 protein, and 7.24 µM for cells transfected with pCMV. At a treatment level of 2 µM CBD, 242 relative cell numbers in wells transfected with viral genes were reduced by ~55-80% (P<0.0001) 243 relative to cell numbers in wells transfected with viral genes but not treated with CBD or relative 244 to wells transfected with control plasmid and treated with or without 2 µM CBD, among which 245 there were no significant differences. 246 of CBD) did not have higher expression of IFNl1 or IFNl2/3 than controls, although treatment 300 of cells with 2 µM CBD caused a significant induction of IFNl1 and IFNl2/3 by ORF8 (Fig.  301 3D, G). These genes were induced without CBD co-treatment when cells were transfected with 302 ORF10 (by 9.6-fold and 2.4-fold) (Fig. 3E , H) or M protein (by 4.1-fold, for both genes) (Fig.  303 3F, I) and 2 µM CBD strongly augmented the induction of both IFNl1 and IFNl2/3 that 304 occurred when ORF10 or M protein were transfected, by a further 3.8-to 11.2-fold ( Fig. 3 E 

Data are means ± SEM, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. 

Expression of the ISGs IFIT1 and MX1 was not significantly altered by treatment with 2 326 µM CBD, or by expression of the SARS-CoV-2 genes ORF8, ORF10, and M protein, either 327 alone, or in combination (Fig. 4A-F) 

The infectious dose of SARS-CoV-2 required to cause disease in 50% of people exposed 355 has been estimated to be 280 virions apoptosis. This finding suggests that CBD may help limit an initial infection by promoting 394 removal of infected cells, thereby limiting the spread, and therefore also likely raising the 395 necessary infectious titre. This is supported by evidence from users of Epidiolex®, a high-dose pharmaceutical CBD licensed in the United States for use in the treatment of rare types of 397 epilepsy in adults and children [47] . In that study, patients prescribed high-dose CBD had an 398 approximate 10-fold lower risk of testing positive for SARS-CoV-2, even when matched by 399 demographics, recorded diagnoses, and other medications. In those with use of any cannabinoid 400 in their medical record, the positivity rate for SARS-CoV-2 was over 40% lower [47] . Taken 

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