For all infections, SARS-CoV-2 isolate Leiden-0002 (GenBank MT510999) was used (41). Vero E6 cells and Calu-3 2B4 cells (42), were cultured and infected as described previously (41). All experiments with infectious SARS-CoV-2 were performed in the LUMC biosafety level 3 facilities.

Voclosporin (Lupkynis™_, Aurinia Pharmaceuticals Inc.), cyclosporin A (Neoral^®, Novartis), tacrolimus (Prograf^®, Astellas), mycophenolate mofetil (CellCept^®, Roche) or everolimus (Certican^®, Novartis) stock solutions were prepared by dissolving the pharmaceutical formulation of these drugs in dimethyl sulfoxide (DMSO). Placebo capsules and pure VCS powder (Aurinia Pharmaceuticals Inc.), Tacrolimus (PHR1809), cyclosporin A (30024) and mycophenolic acid (M5255) (all from Sigma-Aldrich) and remdesivir (RDV; HY-104077, MedChemExpress) were dissolved in DMSO and stored at −20°C as single-use aliquots. Remdesivir was used as a standard positive control in all experiments.

Before analysis, samples were diluted in methanol and subsequently in blank whole blood to fall within the calibration line of 0-to 600 μg/L of VCS. Human whole blood was added to a final volume of 200 µL and mixed with 200 µL of 0.1 M zinc-sulphate and 500 uL of internal standard solution (32 ug/L of VCS D₄ in acetonitrile). After centrifugation, supernatant was transferred to an autosampler vial after which 20 µL was injected into LC-MS/MS system. Quantification of VCS was performed with LC-MS/MS using a Thermo Quantiva UPLC-MS/MS system (ThermoFisher Scientific) (43), similarly to the validated protocol for measuring CsA and TAC. The equipment consisting of an Ultimate 3000 series UHPLC system, coupled to a TSQ Quantiva triple stage quadrupole mass spectrometer was used. Chromatographic separation was achieved using an Acquity UPLC BEH C18 1.7 µm; 2.1 × 50 mm column coupled to a VanGuard BEH C18 1.7 µm precolumn. Online solid phase extraction was performed using a Xbridge 10 µm 30 × 2.1 mm column. This protocol was validated according to the EMA bioanalytical method validation guideline (44).

CPE reduction assays in Vero E6 cells were performed as previously described (28). Briefly, Vero E6 cells seeded in 96-well cell culture plates were pre-incubated with 2-fold serial dilutions of compounds for 30 min. Subsequently, cells were either mock-infected (to assess cytotoxicity of compounds) or were infected with 300 PFU of SARS-CoV-2 per well. Each well contained a total volume of 150 µL of medium with compound. Plates were incubated for 3 days at 37°C. After, cell viability was determined via a colorimetric method by measuring absorption at 495 nm with an EnVision Multilabel Plate Reader (PerkinElmer). Both EC50 (50% effective concentration, required to inhibit virus-induced cell death by 50%), and CC50 (50% cytotoxic concentration, reduces the viability of uninfected cells to 50% of control) were determined using non-linear regression with GraphPad Prism v8.0. For each compound, at least two independent experiments (each in quadruplicate) were performed.

Compound dilutions were prepared in EMEM-2% FCS to mimic the conditions of cell-based assays. Tween-20 and Tween 40 were diluted in MilliQ water to concentrations lower than 1% as present on the composition of Lupkynis™ capsules (45). Phosphate-buffered saline (PBS) was used as a negative control and 50% ethanol as a positive control. In order to assess its virucidal activity, SARS-CoV-2 (5 × 10⁴ PFU) was incubated with the material for 2 h at 37°C with rocking. Then, serial dilutions (ranging from 10^–1 to 10^–6) of compound mixed with virus were prepared in EMEM-2% FCS and added to Vero E6 monolayers. After 1 h incubation, inoculum was removed and 2 ml/well of overlay containing DMEM, 1.2% Avicel (FMC BioPolymer), 2% FCS, 50 mM HEPES, and antibiotics were added. After a 3-day incubation, monolayers were fixed with 3.7% formaldehyde in PBS, and plaques were visualized using crystal violet staining (41).

Calu-3 cells were seeded in 96-well plates (3 × 10⁴ cells per well). The next day, cells were pre-incubated for 60 min with 2-fold serial dilutions of CsA, TAC or VCS. Subsequently, cells were infected with SARS-CoV-2 (MOI of 1, based on titer determined on Vero E6 cells). After a 1 h incubation at 37°C, cells were washed three times with PBS and medium with compound was added. The medium was harvested at 24-h post-infection (h p.i.) and virus titers were determined by plaque assay on Vero E6 cells as described before (46). In parallel, a cytotoxicity assay with mock-infected cells treated using the same concentration of compounds was performed (Cytopathic Effect Reduction Assay section). VCS concentrations were measured by validated LC-MS/MS.

The following coating solutions were prepared fresh before each experiment: BSA, 100 mg/ml bovine serum albumin (Sigma) in PBS; PEG, 1% polyethylene glycol 3350 (Sigma) in MilliQ water; Tween-40, 0.2% polysorbate 40 (Fluka) in MilliQ water; and 500 mM VCS in DMSO (Sigma). All plastic labware, including tubes and tips, was filled with each solution and incubated for 2 h at room temperature with rocking to homogenously coat the surfaces. After rinsing twice with MilliQ water, the items were left to dry at room temperature until further use in experiments.

Borosilicate glass reagent bottles (50-ml) were treated with glacial acetic acid, washed twice with absolute ethanol, dried and UV-sterilized prior to use. Three times concentrated compound solutions were prepared in EMEM-2% FCS using sterile glass culture tubes, a glass syringe (Hamilton) and glass Pasteur pipettes. One ml of each compound dilution was transferred to three different reagent bottles (triplicates). Confluent monolayers of Calu-3 cells grown in culture flasks were infected with SARS-CoV-2/Leiden-002 (MOI of 1). Inoculum was removed after 1 h incubation at 37°C. Cells were washed three times with PBS, trypsinized and resuspended in EMEM-2% FCS. Two ml of this cell suspension (∼10⁶ cells) was transferred to each reagent bottle, containing compound solution. Medium was collected 24 h p.i. and virus titer was determined by plaque assay. VCS concentrations in the medium were determined by LC-MS/MS.

Calu-3 cells were trypsinized and 1.5 × 10⁵ cells in 1 ml of EMEM-2% FCS were divided over glass culture tubes. Two-fold dilutions of VCS, TAC and CsA were prepared in EMEM-2% FCS medium using glass labware, and added to corresponding tubes with cells (three tubes per concentration). After a 24 h incubation, cell viability was determined (see Cytopathic Effect Reduction Assay section).

To compare the antiviral effect of different immunosuppressive drugs commonly used in KTRs, we performed SARS-CoV-2 CPE reduction assays with VCS, cyclosporin, microemulsion (CsA_me), TAC, EVL, and MMF. These experiments were performed using the pharmaceutical formulations of the compounds to ensure optimal solubility and stability. At the start of our study only the pharmaceutical formulation of VCS) was available to us from a previous study. In each experiment, drug cytotoxicity was assessed in parallel, in non-infected cells. RDV was included as a standard positive control for inhibition of viral replication [data not shown (28)].

The EC₅₀ values of VCS, CsA_me and TAC were measured in the low-micromolar range, respectively: 0.22 ± 0.01 µM, 4.3 ± 0.6 µM and 10 ± 1 µM (Figures 1A–C). No inhibitory effect was observed for EVL (Figure 1D). The prodrug MMF (Figure 1E) was included in our comparison, but was not expected to inhibit virus replication, as it is likely not metabolized into its active form MPA (47) in our assay. Thus, we attributed the apparent antiviral effect of MMF to excipients present in the drug formulation (see An Excipient in the Pharmaceutical Formulation of Voclosporin Inhibits SARS-CoV-2 Replication in Cytopathic Effect Reduction Assays section).

Apart from VCS, none of the compounds caused cytotoxicity at tested concentrations (CC₅₀ values >100 µM). Although VCS had a CC₅₀ around 4 µM, its EC₅₀ was also 18–45 times lower than the other compounds tested (Figure 1).

In order to evaluate whether any excipients in the pharmaceutical formulation of VCS contributed to the observed antiviral effect (Figure 1A), VCS capsules and placebo capsules were compared side-by-side using CPE reduction assay. Both capsules provided by Aurinia Pharmaceuticals Inc. had similar composition (45), with exception of the VCS compound. The absence of VCS in placebo capsules was confirmed by LC-MS/MS analysis (not shown). Surprisingly, both the VCS formulation (Figure 2A) and the placebo (Figure 2B) inhibited SARS-CoV-2 replication in a similar dose-dependent manner. This indicated that one or more excipients in the drug formulation might have an antiviral effect in this experimental setup.

Since the pharmaceutical formulation includes surfactants like Tween-20 and Tween-40 that may destroy the viral envelope, the virucidal activity of these reagents, VCS and placebo capsules were analysed. A control treatment with 50% ethanol reduced SARS-CoV-2 titers to below the limit of detection (<100 PFU/ml), while none of the other treatments significantly reduced the number of infectious particles (Figure 3). Therefore, we conclude that excipients in the drug formulation had no virucidal activity or impact on viral infectivity, but that they caused a yet poorly understood antiviral effect in the CPE reduction assays through an unknown mechanism. This invalidated the previously determined EC50 values when calcineurin inhibitors were tested using their pharmaceutical formulations (Figure 1).

To avoid interference by excipients, we performed CPE reduction assays with DMSO solutions prepared using high purity powders of the various immunosuppressive drugs. In the case of Neoral (cyclosporin microemulsion), CsA powder was evaluated. VCS solutions prepared from pure powder did not confer the same level of protection to SARS-CoV-2 infected-cells (Figure 4A) as the pharmaceutical formulation (Figure 2A). Additionally, less cytotoxicity was measured/observed CC50 > 50 µM. Similar reductions in antiviral potency were observed for CsA and MPA (Figures 4B,D), suggesting that -also in cell-based assays-these drugs need excipients to ensure solubility/bioavailability or stability for optimal activity. Interestingly, TAC solutions prepared from pure powder inhibited SARS-CoV-2 with similar efficacy as the drug formulations, i.e., with an EC₅₀ of ∼15 µM (compare Figures 1C, 4C), suggesting that the pharmaceutical formulation of TAC does not contain excipients contributing to either antiviral or virucidal effects.

We searched for potential reasons to understand the lower inhibitory effect of VCS active molecule compared to pharmaceutical formulation. Interactions between plastic and lipophilic or hydrophobic compounds, have been described (48–50). Thus, we hypothesized as VCS is highly lipophilic, it may bind to plastic which could compromise its bioavailability in standard cell-based assays using plastic labware. VCS concentrations were measured in medium using LC-MS/MS. Only 27% of the original VCS concentration could be recovered from plates due to loss of compound by binding to pipette tips and tubes during the preparation of dilutions as soon as t = 0 (Table 1).

To test whether we could prevent VCS binding to plastic in our standard antiviral assays, we coated all plastic labware using 3 different agents found in literature: BSA (51), PEG-3350 (52, 53) and Tween 40 (54). Unfortunately, none of the treatments tested was able to reduce binding of VCS to plastic (Table 1), as only 5–7% of its original concentration was recovered in solution. Alternatively, we tested if saturation of binding sites on plastic with a highly concentrated VCS solution (500 mM) prevented loss of compound. Leaching of compound from plastic was observed, resulting in unpredictable concentrations of VCS in solution, e.g., we measured a VCS concentration of >15 µM when a 2 µM stock solution was used (Table 1).

Similarly, TAC and CsA concentrations were measured using the same setup as for VCS. A 76% of the original TAC concentration and 62% of the initial CsA concentration could be recovered in solution (Table 2). This emphasized the need to use different type of materials to perform our experiments to truly evaluate these CNIs antiviral activity.

To evaluate the effect of VCS, CsA and TAC on SARS-CoV-2 replication, viral load reduction assays were performed using human lung epithelial cells (Calu-3). Moreover, we developed custom assays using exclusively glass labware to circumvent the problem of VCS binding to plastic.

Calu-3 cells in glass remained viable and supported SARS-CoV-2 replication as an increase in viral titer was measured at 24 h p.i. (Figure 5A). RDV was included as a positive control for inhibition of SARS-CoV-2 replication and assay validation. Treatment of infected cells with 10 µM RDV inhibited viral replication by > 4log(data not shown), which is in agreement with previously reported data (55). Treatment of cells with 3.2 µM VCS (pure compound) caused a more than 1.5 log reduction in SARS-CoV-2 infectious progeny titers, while an ∼0.5 log reduction was observed when the same concentration of CsA or TAC was used (Figure 5A). However, treatment with 3.2 μM VCS or CsA also caused cytotoxicity, as cell viability dropped to ∼75% (Figure 5C). Therefore, it cannot be excluded that part of the observed antiviral effect is due to pleiotropic effects (toxicity).

In experiments using plastic materials, a dose-dependent reduction in infectious progeny titers was observed when cells were treated with VCS, leading to a more than 1 log reduction at 6.4 µM (Figure 5B). CsA treatment led to a similar reduction at 25 µM. CsA displayed significant cytotoxicity at concentrations of 12.5 µM or above while VCS did not (Figure 5D). In contrast, a higher concentration of TAC (25 µM) was required to reduce the viral titer by more than 1 log. Overall, VCS showed a stronger inhibitory effect in experiments performed with glass instead of plastic labware.

Measurement of the VCS concentration in glass containers without cells demonstrated no significant loss of compound from solution (Table 3). When VCS solutions of 0.2–3.2 µM were used in glass bottles with Calu-3 cells, a ∼75% reduction of the VCS concentration was measured, suggesting the compound was bound or taken up by cells. In contrast, in experiments using standard plastic labware, we measured a 0.68 µM concentration of VCS in medium of cells treated with 25 µM VCS solution. Taking into account a similar reduction in virus titer using 3.2 and 25 µM of VCS in glass and plastic, respectively, this corroborated that when using plastic, the bioavailable amount of VCS is likely only 10% of that in the input solution.