key: cord-0961477-fiuidbfd
authors: Rodriguez, Killian; Saunier, Florian; Josselin, Rigaill; Audoux, Estelle; Botelho-Nevers, Elisabeth; Prier, Amélie; Dickerscheit, Yann; Pillet, Sylvie; Pozzetto, Bruno; Bourlet, Thomas; Verhoeven, Paul O.
title: Evaluation of in vitro activity of copper gluconate against SARS-CoV-2 using confocal microscopy-based high content screening
date: 2021-07-08
journal: J Trace Elem Med Biol
DOI: 10.1016/j.jtemb.2021.126818
sha: b24efd69a659fec743f35c227707861176a3b37f
doc_id: 961477
cord_uid: fiuidbfd

CONTEXT: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that emerged late in 2019 is the etiologic agent of coronavirus disease 2019 (Covid-19). There is an urgent need to develop curative and preventive therapeutics to limit the current pandemic and to prevent the re-emergence of Covid-19. This study aimed to assess the in vitro activity of copper gluconate against SARS-CoV-2. METHODS: Vero E6 cells were cultured with or without copper gluconate 18-24 hours before infection. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Cells were infected with a recombinant GFP expressing SARS-CoV-2. Infected cells were incubated in fresh medium containing varying concentration of copper gluconate (supplemented with bovine serum albumin or not) for an additional 48 -h period. The infection level was measured by the confocal microscopy-based high content screening method. The cell viability in presence of copper gluconate was assessed by XTT and propidium iodide assays. RESULTS: The viability of Vero E6 cells exposed to copper gluconate up to 200 µM was found to be similar to that of unexposed cells, but it dropped below 70% with 400 µM of this agent after 72 hours of continuous exposure. The infection rate was 23.8%, 18.9%, 20.6%, 6.9%, 5.3% and 5.2% in cells treated prior infection with 0, 2, 10, 25, 50 and 100 µM of copper gluconate respectively. As compared to untreated cells, the number of infected cells was reduced by 71%, 77%, and 78% with 25, 50, and 100 µM of copper gluconate respectively (p < 0.05). In cells treated only post-infection, the rate of infection dropped by 73% with 100 µM of copper gluconate (p < 0.05). However, the antiviral activity of copper gluconate was abolished by the addition of bovine serum albumin. CONCLUSION: Copper gluconate was found to mitigate SARS-CoV-2 infection in Vero E6 cells but this effect was abolished by albumin, which suggests that copper will not retain its activity in serum. Furthers studies are needed to investigate whether copper gluconate could be of benefit in mucosal administration such as mouthwash, nasal spray or aerosols.

At the end of 2019, the emergence of a novel coronavirus designated as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a pandemic that threatens human health and public safety [1, 2] . This new virus is highly transmissible and has spread very fast all over J o u r n a l P r e -p r o o f 3/20 the world [1] . Even though the great majority of people (i.e. around 80%) develop mild to moderate coronavirus disease 2019 (Covid- 19) , a significant proportion of cases are severe or critical and can lead to death [1, 3] . So far 3,166,029 people died from Covid-19 worldwide as of April 30th, 2021 [4] .

Thus, there is an urgent need to contain SARS-CoV-2 spread and virulence with effective curative and preventive treatments [2, 3] .

During the early phase of the SARS-CoV-2 outbreak, we have been faced with a strong need for in vitro models able to test the efficacy of compounds against this virus but only a few laboratories were able to do so. In silico studies have identified dozens of drugs potentially active against SARS-CoV-2 [5] [6] [7] [8] [9] [10] . Drug repurposing is one of the most promising strategies for improving the care of Covid-19 patients but published data on the in vitro efficacy of molecules potentially active on SARS-CoV-2 remain very limited to date [11] [12] [13] [14] . Available studies focused mostly on few drugs including hydroxychloroquine, remdesivir, lopinavir, ritonavir, interferon, umifenovir, favipiravir, camostat mesylate and immunomodulatory therapies [10] [11] [12] [13] [14] .

Although some treatments have shown some benefits in patients at later stages of the disease (i.e. dexamethasone, anticoagulation treatments), there are no acknowledged effective antiviral therapies for Covid-19 [1] . Recently, preliminary results of the SOLIDARITY trial showed that hydroxychloroquine, remdesivir, lopinavir/ritonavir, and interferon regimens have no significant effect on the mortality rate nor on the length of hospital stay in COVID-19 patients [15] . Other ongoing clinical trials could provide additional results shortly [16] .

Among the other compounds found to be directly active against SARS-CoV-2, essential minerals may require special attention. Antimicrobial and antiviral activity of copper is well established [17] . Copper ions have been found to elicit a broad action against viruses including coronaviruses [18] [19] [20] [21] . Recently, it has been shown that SARS-CoV-2 can be eradicated from a copper surface within 4 hours while it can survive up to 72 hours on stainless steel and plastic surface [22] .

Copper has been proposed to prevent transmission of the SARS-CoV-2 in the hospital environment (i.e. to cover door handles) or in application to face masks with the aim of reducing the risk of catching or spreading SARS-CoV-2 [21, 23] .

In eukaryotes, copper acts as an essential cofactor for more than 30 enzymes involved in redox reactions including superoxide dismutase (SOD) and ceruloplasmin. As well as for other trace metal ions (e.g. iron, manganese, zinc, selenium, and cobalt), maintenance of an adequate intracellular concentration of copper is essential to avoid the negative metabolic effects [24, 25] . In humans, the normal cupremia varies from 9.75 to 27.75 µmol/L (650 to 1850 µg/L) in adults [24, [26] [27] [28] and in tissues copper concentration range from 1 to 12 µg/g of tissues [24, 28, 29] . In human cells, copper is internalized by copper transporter 1 and is used for the synthesis of copper-requiring enzymes; it is stored mainly in the mitochondria and secreted by cells in the bloodstream; excess of J o u r n a l P r e -p r o o f 4/20 copper is mostly eliminated by hepatocyte in bile [24] . In physiological conditions, copper is bound to ceruloplasmin (accounting for 40-70% of total plasma copper), albumin, alpha-2 macroglobulin, and other copper-carrying proteins for avoiding uncontrolled redox activity [24, 29] .

To the best of our knowledge, no published study to date have evaluated the effect of copper gluconate using an in vitro cell model of SARS-CoV-2 infection. This study aimed to assess if pre-and post-exposure treatment with copper gluconate could prevent the cells to be infected. For this purpose, we developed an original confocal microscopy-based high content screening (HCS) method using a recombinant GFP expressing SARS-CoV-2. containing viral cDNA of synSARS-CoV-2-GFP clone 6.2 was kindly provide by Volker Thiel [30] . Upon receipt, BAC was stored in Saccharomyces cerevisiae VL6-48N strain [31] , which was grown on YPD Sigma Aldrich) at a density of 20,000 cells per well. Cells were incubated for 18-24h hours at 37°C and in 5% CO2. Then cells were incubated in cell medium with or without copper gluconate for 18-24h hours. Next, the medium was removed before the cell being infected with GFP expressing SARS-CoV-2 at a multiplicity of infection (m.o.i) of 0.005 for 1 hour. After the adsorption step, the medium was removed and cells were incubated at 37°C and in 5% CO2 for another 48 hour-period in fresh medium The improvement of the images for publishing was performed with Fiji software (v1.53c).

Cell toxicity of copper gluconate. Vero E6 cells were treated with gluconate copper concentrations ranging from 0 to 1600 µM for 24, 48 and 72h. Cell viability was determined by measuring the reduction of XTT converted to orange-coloured formazan product using the CyQUANT XTT assay. The viability of Vero E6 treated with copper gluconate up to 200 µM was similar to that of untreated cells. However, the cell viability dropped below 70% at 400 μM after 72h and reached a value close to zero (or zero) for a concentration of 800 μM and 1600 µM after 48h of treatment (Figure 1a, 1b   and 1c) . Similar results were observed by measuring the percentage of dead cells with propidium iodide staining. Copper gluconate toxicity was found to increase over the time (Figure 1d) . At 72 hours, no toxicity was observed up to 100 µM (Figure 1d ). (Figure 2c) . These latter results suggest that copper gluconate might also limit the viral replication inside Vero E6 cells.

To assess the effect of copper gluconate after the cells were infected, the same protocol was used except that Vero E6 cells were maintained in medium without copper gluconate until the infection step was performed. After adsorption, the medium was replaced by a fresh medium supplemented with concentrations of copper gluconate ranging from 0 to 100 µM and cells were incubated for an another 48 hour-period. values were compared by one-way ANOVA with Dunnet correction for multiple comparisons.

For this study, we developed an original confocal microscopy-based HCS using an in vitro model with Vero E6 cells challenged with a recombinant GFP expressing SARS-CoV-2 that was reconstructed using a yeast-based reverse genetics platform [30] . This method has the advantage of being able to analyse each cell individually and to count thousands of cells per well at the same time to increase the reliability of the observations. By using both a motorized stage and a fully automated pipeline for image recording, we virtually eliminated any possible bias that could be linked to the person in charge of image acquisition. The quantitative analysis of images was also fully automated by using an in-house pipeline developed with a plugin of the NIS software to avoid technical bias. A similar experimental setup with Vero E6 cells and the same GFP expressing SARS-CoV-2 clone was found to be suitable for drug screening applications by using the antiviral remdesivir as a reference [30] . Thus, we decided to combine this in vitro model of GFP expressing SARS-CoV-2 infection with confocal microscopy-based HCS to assess the antiviral activity of copper gluconate. Furthers improvements of this technology (e.g. using cell lines with fluorescence reporters) could help to study more easily whether and how drugs or chemical compounds could counteract SARS-CoV-2 infection in mammalian cells.

In the present study, we observed that the combination of pre-and post-exposure treatment of Vero E6 cells with copper gluconate at a concentration as low as 25 µM led to a 71% reduction of J o u r n a l P r e -p r o o f 12/20 the cell infection rate. When the cells were treated after the infection step, the concentration of copper gluconate needed for reducing by 70% the infection rate was 100 µM. In any case, we could not observe a complete inhibition of the viral infection in this in vitro model. It must be acknowledged that the effect we observed with 25 µM of copper gluconate is not as powerful as that described with antiviral drugs [32] . In humans, the concentration of copper in whole blood is approximately 15 µM (1000 µg/L) [24, [26] [27] [28] [29] . Depending on the tissue, the copper concentration range from 1 to 12 µg/g, which is 1000-fold lower than the serum concentration [28, 29] . It is important to note that almost all of the copper in the blood is tightly bound to components and not free to interact with cells, except to enter through specific transporters. In our in vitro model, the addition of albumin at physiological concentration (40 mg/L) and lower (20 mg/L and 10 mg/L) abolished the antiviral effect of copper gluconate suggesting that complexed copper no longer had an effect on the virus. Because of its high affinity for copper, albumin is known to be one of the main copper-binding components in mammalian blood plasma [29] . We observed that the addition of BSA inhibits antiviral activity of copper gluconate probably because free copper ions are bound by albumin and cannot be effective against SARS-CoV-2 viruses. In vivo, copper can also encounter various other proteins that can bind copper, and making it unavailable as an antimicrobial [29, 45, 46] .

Even if copper seems to act against the virus by decreasing the number of infected cells, the mechanism of action is still far from being understood. The antiviral effect observed in Vero E6 cells with copper gluconate is probably multifactorial and certainly much more complex in vivo. Warnes et al. showed that copper ions can damage virus membranes and destroy the viral genome of human coronavirus 229E [33] . In our study, a direct effect of copper cannot be excluded because copper gluconate was maintained in the culture medium as long as the infection lasted. We also observed that the increase of copper gluconate concentration up to 100 µM is associated with a statistically significant decrease of MFI. Because GFP expressed by the recombinant SARS-CoV-2 is fused to the non-structural protein 7, we can speculate that copper might limit the synthesis of viral proteins. This hypothesis is supported by in silico studies predicting that metal ions such as cobalt(III) or copper(II) could inhibit the SARS-CoV-2 main protease [34, 35] . However, we found only two in vitro studies corroborating that copper ions could inhibit the synthesis of viral proteins or the replication cycle [36, 37] . Thus, whether copper ions may limit the synthesis of viral proteins in mammalian cells is still far from being understood. In vitro studies showed that an increase of SOD1 expression is associated with a decrease in viral replication [38, 39] . Further studies are needed to investigate whether the copper gluconate supplementation in culture media increases the internalization of copper and if intracellular copper is required to struggle against viral infection. Last, it was well established that the coronavirus replication complex requires autophagy-associated cellular components [40] . Because copper is known to be able to modulate autophagy, copper induced-autophagy could limit the J o u r n a l P r e -p r o o f 13/20 availability of autophagy associated cellular components that are required for viral replication [19] .

Whether one of these mechanisms more than another could be involved in the antiviral effect observed in our study remains unclear.

In vitro, we found that copper gluconate is well tolerate by Vero E6 cells even at strong concentration. It could be interesting to confirm these results with others cell lines but also with primary cells and human reconstructed epithelium. In our hand, we observed that very high concentrations over a limited period of 2 hours could be used with no evidence of cytotoxicity after 24h (Rigaill J, personal data). If the safety of local administration could be confirmed using in vivo models, it could be considered that copper gluconate delivered locally by mouthwashes or sprays could help to tackle the viruses produced by infected cells. By contrast, it is unlikely that copper keep an antiviral activity if administered by intravenous injection because copper is bound by albumin, ceruloplasmin and many other proteins [29, 45, 46] . The animal models of SARS-CoV-2 infection that have been developed worldwide [44] seems to be the most appropriate approach to assess the effect of copper administration during SARS-CoV-2 infection in vivo.

In humans, a retrospective observational study showed that zinc and selenium transporter selenoprotein P and zinc deficiency was associated with the worst outcomes in elderly Covid-19 patients [42] . A meta-analysis in Chinese children reported that copper deficiency is associated with recurrent respiratory tract infection [43] . Thus, it could be interesting to study copper concentrations in serum but also tissues such as nails and hairs in asymptomatic, mild, and severe Covid-19 patients.

In conclusion, our findings showed that copper gluconate is able to mitigate SARS-CoV-2 infection in Vero E6 cells but this effect was abolished by albumin, which suggests that copper will not retain its activity in serum. In the current context of active virus transmission, it is undoubtedly interesting to pursue the development of new therapeutic strategies in addition to vaccination efforts Furthers studies are still needed to investigate whether copper gluconate could be of benefit in mucosal administration such as mouthwash, nasal spray or aerosols 

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