key: cord-0750726-wqhv8vek
authors: Do, Thuc Nguyen Dan; Chatterjee, Arnab K.; Gallay, Philippe A.; Bobardt, Michael D.; Vangeel, Laura; De Jonghe, Steven; Neyts, Johan; Jochmans, Dirk
title: A robust SARS-CoV-2 replication model in primary human epithelial cells at the air liquid interface to assess antiviral agents
date: 2021-03-26
journal: bioRxiv
DOI: 10.1101/2021.03.25.436907
sha: 48f81f144e4ae64f4cbddb3e1c0499d10b75da8b
doc_id: 750726
cord_uid: wqhv8vek

There are, besides Remdesivir (RDV), no approved antivirals for the treatment and/or prophylaxis of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HAEC) and human small airway epithelial cells (HsAEC) grown at the air/liquid interface (ALI) and infected at the apical side with either one of two different SARS-CoV-2 isolates. The virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35°C than at 37°C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. GS-441524 (the parent nucleoside of Remdesivir), EIDD-1931 (the active metabolite of Molnupiravir) and IFN (β1 and λ1) all resulted in a dose-dependent inhibition of viral RNA and infectious virus titers at the apical side. However, AT-511 (a guanosine nucleotide previously reported to inhibit SARS-CoV-2) failed to inhibit viral replication. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.

Besides Remdesivir (RDV), there are no approved antivirals for the treatment and/or prophylaxis of CoV-2 infections, although the clinical benefit of RDV is still a matter of debate 1 . Major efforts are ongoing 30 to develop novel antiviral drugs. To aid in their development physiological relevant models are needed, in 31 particular because typically immortal cell lines also originating from non-respiratory (and often non-32 human) tissue are being used in early preclinical studies. For example, VeroE6, a widely used cell line in 33 SARS-CoV-2 studies is defective in the expression of main the SARS-CoV-2 receptors (angiotensin-34 converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2)). Hence, screenings 35 campaigns often result in the discovery of antiviral agents that regulate autophagy pathways and 36 endosomal-lysosomal maturation which may not be pertinent or translatable as SARS-CoV-2 therapies 2 . 37

Meanwhile, air-liquid interface of differentiated primary human airway epithelial cells (HAEC) possess the 38 architecture and cellular complexity of human lung tissue and are permissive to variety of respiratory viral 39

infections 3, 4 . Containing all relevant cell types of the lower respiratory tract (ciliated, goblet and basal 40 cells) which includes ACE2 and TMPRSS2 expressing cells, this system allows to dissect the host-pathogen 41

interactions at the molecular and cellular levels and provides a platform for the profiling of antiviral drugs. 42

In this study, we explored the effect of a selected number of reported SARS-CoV-2 inhibitors in HAEC ALI 43 cultures on the replication of different SARS-CoV-2 isolates. Our results provide a reference set of data for 44 the preclinical development of SARS-CoV-2 inhibitors. 45 46 47

The African monkey kidney cell line VeroE6 tagged green fluorescent protein (VeroE6-GFP, kindly provided 50 by M. van in an antiviral assay with hepatitis C (data not shown). IFN λ1 was purchased from R&D Systems and IFN 77 β-1a was a kind gift from the laboratory of Immunobiology (Rega Institute, KU Leuven, Belgium), which 78

were reconstituted in sterile phosphate buffered saline (PBS, Life Technologies) containing at least 0.1% 79 FBS. 80

VeroE6-GFP cells were seeded at a density of 25000 cells/well in 96-well plates (Greiner Bio One, 82 catalogue no. 655090) and pre-treated with three-fold serial dilutions of the compounds overnight. On 83 the next day (day 0), cells were infected with the SARS-CoV-2 inoculum at a multiplicity of infection (MOI) 84 of 0.001 median tissue infectious dose (TCID50) per cell. The number of fluorescent pixels of GFP signal 85 determined by High-Content Imaging (HCI) on day 4 post-infection (p.i.) was used as a read-out. 86

Percentage of inhibition was calculated by subtracting background (number of fluorescent pixels in the 87 untreated-infected control wells) and normalizing to the untreated-uninfected control wells (also 88 background subtracted). The 50% effective concentration (EC50, the concentration of compound required 89

for fifty percent recovery of cell-induced fluorescence) was determined using logarithmic interpolation. 90

Potential toxicity of compounds was assessed in a similar set-up in treated-uninfected cultures where 91 metabolic activity was quantified at day 5 using the MTS assay as described earlier 9 . The 50% cytotoxic 92 concentration (CC50, the concentration at which cell viability reduced to 50%) was calculated by 93 logarithmic interpolation. 94

Huh7 cells were pre-seeded at 6000 cells/well in 96 well-plates (Corning, catalogue no.3300) and 95 incubated overnight at 37•C and 5% CO2. On day 0, cells were firstly treated with the three-fold serial 96 dilution of a potential antiviral, followed by either the inoculation of SARS-CoV-2 at MOI of 0.0037 97 TCID50/cell or addition of fresh medium. After 4 days, differences in cell viability caused by virus-induced 98 cytopathic effect (CPE) or by compound-specific toxicity were evaluated using MTS assays. The EC50 and 99

CC50 were calculated as above-mentioned. 100

Tracheal HAEC (catalogue no. EP01MD) and human small airway epithelium cells (HsAEC) (catalogue no. 102 EP21SA) from healthy donors were obtained from Epithelix (Geneva, Switzerland) in an air-liquid 103 interphase set-up. After arrival, the insert was washed with pre-warmed 1x PBS (Gibco, catalogue no. 104 14190-094) and maintained in corresponding MucilAir medium (Epithelix, catalogue no. EP04MM) or 105

SmallAir medium (Epithelix, catalogue no. EP64SA) at 37•C and 5% CO2 for at least 4 days before use. On 106 the day of the experiment, the H(s)AEC were first pre-treated with basal medium containing compounds 107 at different concentrations for indicated hours, followed by exposing to 100 µL of SARS-CoV-2 inoculum 108 from apical side for 1.5 hours. Then the cultures were incubated at the indicated temperatures. The first 109 apical wash with PBS was collected either right after the removal of viral inoculum (day 0) or 24 hours 110 later (day 1 post-infection (p.i.)). Every other day from day 0, subsequent apical washes were collected 111

whereas compound-containing medium in the basolateral side of the H(s)AEC culture was refreshed. 112

Wash fluid was stored at -80•C for following experiments. 113

Viral RNA in the apical wash was isolated using the Cells-to-cDNA TM II cell lysis buffer kit (Thermo Fisher  115 Scientific, catalogue no. AM8723). Briefly, 5 µL wash fluid was added to 50 µL lysis buffer, incubated at 116 room temperature (RT) for 10 min and then at 75•C for 15 min. 150 µL nuclease-free water was 117 additionally added to the mixture prior to RT-qPCR. In parallel, a ten-fold serial dilution of corresponding 118

virus stock was extracted. The amount of viral RNA expressed as TCID50 equivalent per insert 119 (TCID50e/insert) was quantified by RT-qPCR using iTaq universal probes one-step kit (Bio-Rad, catalogue 120 no. 1725141), and a commercial mix of primers for N gene (forward primer 5'-121

GACCCCAAAATCAGCGAAAT-3', reverse primer 5'-TCTGGTTACTGCCAGTTGAATCTG-3') and probes (5'-122

FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3') manufactured at IDT Technologies (catalogue no. 123 10006606). The reaction (final volume: 20 µL) consisted of 10 µL one-step reaction mix 2X , 0.5 µL reverse 124 transcriptase, 1.5 µL of primers and probes mix, 4 µL nuclease-free water, and 4 µL viral RNA. The RT-qPCR 125 was executed on a Lightcycler 96 thermocycler (Roche), starting at 50•C for 15 min and 95•C for 2 min, 126 followed by 45 cycles of 3 sec at 95•C and 30 sec at 55•C. 127

Titration using a 50% tissue culture infectious dose (TCID50) assay 128

VeroE6 cells were seeded in 96-well tissue culture plates at a density of 1×10 4 cells/180 µL/well. After 24 129 hours, serial 10-fold dilutions of ALI wash fluid were prepared in the plates. Cells were incubated for 3 130 days at 37°C and evaluated microscopically for the absence or presence of virus induced cytopathic effect 131

(CPE). The infectious viral titer was determined by end-point titration, expressed as TCID50/ml. Virus titers 132

were calculated by using the Spearman and Karber method as previously reported 10,11 . 133

All statistical comparisons in the study were performed in GraphPad Prism (GraphPad Software, Inc.). 135

Statistical significance was determined using the ordinary one-way ANOVA with Dunnett's multiple 136 comparison test. P-values of ≤0.05 were considered significant. 137

We first compared the replication kinetics of the Belgian isolate GHB-03021 and the German isolate 141

BavPat1. The main differences in the genomes of these viruses is the D614G amino acid change in the 142 spike-protein of BavPat1 and the deletion of several amino acids near the furin-cleavage site in the GHB-143 03021 isolate (because of extensive passaging in VeroE6 cells). The replication kinetics was investigated 144

at respectively 35 and 37°C in both cultures from tracheal cells (HAEC) or from small airway cells (HsAEC). 145

In preliminary experiments, it was observed that an input of 10 2 , 10 3 or 10 4 TCID50/insert resulted in 146 comparable levels of virus production (data not shown). We therefore selected 2×10 3 TCID50/insert as 147 the viral input for this experiment. Overall, BavPat1 infected the cultures more efficiently than the GHB-148 03021 isolate did (Fig. 1) was surprisingly entirely devoid of antiviral activity (Table 1) . 162

At 10 µM, GS-441524 sterilized the HAEC cultures from the GHB-03021 virus. Indeed, no virus production 163 was detected during the first 9 days of treatment and when treatment was stopped, no rebound was 164 observed over the next 5 days of culturing. When evaluated at a concentration of 1 µM, GS-441524 165 reduced virus yield by ~1 log10 during the time of treatment, but lost activity once the compound was 166 removed from the culture. In a separate experiment, GS-441524 at 3 µM resulted in complete inhibition 167 of virus production upon infection with BavPat1 ( Fig. 2F-H) . Also, 10 µM of EIDD-1931 resulted in a 168 pronounced antiviral effect ( Fig. 3F-J) . AT-511, however, at the various concentrations tested (1 and 10 169 µM) was devoid of an antiviral effect ( Fig. 3A-E) . 170

Human IFN has been used to treat several viral infections 19,20 and recently clinical trialsagainst SARS-CoV-172 2 are ongoing (ClinicalTrials.gov number: NCT04315948, NCT04385095, and NCT04492475). Therefore, 173

we investigated whether IFN β-1a and IFN λ1 exert activity when used as a prophylactic monotherapy. 174

Tracheal cultures were pre-treated with either 5 and 50 ng/mL IFN λ1 (5 ng/mL is the average 175 concentration secreted in the basal medium of infected HAEC cultures 21 ) or 1 and 100 IU/mL IFN β-1a for 176 24 hours, and subsequently infected with BavPat1. Both drugs were able to reduce viral titers in a dose-177 dependent manner (Fig. 4A, 4F ). Viral loads were reduced by 100 IU/mL IFN β-1a (3.3 log10 vRNA 178 reduction, 3.6 log10 titer reduction) and 50 ng/mL IFN λ1 (4.2 logs vRNA reduction, 5.0 log10 titer reduction) 179 on day 4 p.i. (Fig. 4B, 4D nucleoside, has been approved as the first COVID-19 therapy. However, its effectiveness is still a matter 206 of debate 1 . In addition, it has a challenging pharmacological profile allowing intravenous administration 207

only [39] [40] [41] . We demonstrate that the parent nucleoside GS-441524 12-16 can "sterilize" H(s)AEC cultures from 208 SARS-CoV2 as no rebound of the virus was noted several days after removal of the molecule. Differences 209 in antiviral potencies of RDV and GS-441524 have been reported depending on the cell lines used, which 210

correlates with the formation of the biologically active (5'-triphosphate) metabolite 12,13 . Data from a 211 pharmacokinetic study in mice suggests that GS-441524 could possibly be considered as an oral drug 12 . 212 AT-527 is currently being evaluated in phase II clinical trials for . Surprisingly, 213

we did not observe anti-SARS-CoV-2 activity of AT-511, the free base form of AT-527, in VeroE6 and Huh7 214 cells, nor did we observe antiviral activity in the HAEC cultures. This is in contrast with a recent publication 215

where sub-micromolar activities of AT-511 were observed in very similar assay systems 17 . At this moment 216

we have no explanation for this discrepancy. One possibility is that small differences in the assay 217 conditions may influence the metabolization of AT-511 to its active form and thus influence its antiviral 218 activity. As AT-511 is a double pro-drug it may be more susceptible to these nuances. 219

In addition to RDV and AT-511, we also investigated the effect of the nucleoside analogue EIDD-1931 220 which is the active metabolite of the ester prodrug Molnupiravir (EIDD-2801). EIDD-1931 has been 221 reported to exert antiviral activity against various human coronaviruses and Molnupiravir is currently in 222 clinical trials for SARS-CoV-2 17,18 . Initial interim data from a phase II study provides first evidence for 223 antiviral activity in COVID patients (https://www.croiconference.org/abstract/reduction-in-infectious-224 sars-cov-2-in-treatment-study-of-covid-19-with-molnupiravir/). Like GS-441524, EIDD-1931 also results in 225 a pronounced antiviral effect in the human airway epithelium cell cultures, which is consistent with 226 another report 18 . 227 Also, a significant inhibitory effect of IFN β-1a and IFN λ1 was noted, although at high concentrations and 228

in particular during the first days of the treatment. At later time-points, viral replication increased in the 229 treated cultures, suggesting that the virus can escape the effect of IFN. The effective concentration of IFN 230 β-1a used in this study is comparable with the clinically achievable concentration and is in line with other 231

reports 19,20,42 . 232 In conclusion, we assessed ( 

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