key: cord-272626-bw9lbzvt
authors: Pizzorno, Andrés; Padey, Blandine; Julien, Thomas; Trouillet-Assant, Sophie; Traversier, Aurélien; Errazuriz-Cerda, Elisabeth; Fouret, Julien; Dubois, Julia; Gaymard, Alexandre; Lescure, François-Xavier; Dulière, Victoria; Brun, Pauline; Constant, Samuel; Poissy, Julien; Lina, Bruno; Yazdanpanah, Yazdan; Terrier, Olivier; Rosa-Calatrava, Manuel
title: Characterization and treatment of SARS-CoV-2 in nasal and bronchial human airway epithelia
date: 2020-04-02
journal: bioRxiv
DOI: 10.1101/2020.03.31.017889
sha: 
doc_id: 272626
cord_uid: bw9lbzvt

In the current COVID-19 pandemic context, proposing and validating effective treatments represents a major challenge. However, the lack of biologically relevant pre-clinical experimental models of SARS-CoV-2 infection as a complement of classic cell lines represents a major barrier for scientific and medical progress. Here, we advantageously used human reconstituted airway epithelial models of nasal or bronchial origin to characterize viral infection kinetics, tissue-level remodeling of the cellular ultrastructure and transcriptional immune signatures induced by SARS-CoV-2. Our results underline the relevance of this model for the preclinical evaluation of antiviral candidates. Foremost, we provide evidence on the antiviral efficacy of remdesivir and the therapeutic potential of the remdesivir-diltiazem combination as a rapidly available option to respond to the current unmet medical need imposed by COVID-19. One Sentence Summary New insights on SARS-CoV-2 biology and drug combination therapies against COVID-19.

On Dec 31, 2019, a cluster of cases of pneumonia of unknown etiology was reported in Wuhan, China. On Jan 10, 2020, a novel coronavirus, lately named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and classified into the Betacoronavirus genus, was 50 identified as the causative agent (1) . As of Mar 20, 2020, 9 days after the World Health Organization (WHO) declared the COVID-19 a pandemic (2) , the new Coronavirus disease 2019 (COVID-19) had caused approximately 8 800 deaths among more than 210 000 confirmed cases reported mainly in China but also spreading to at least 168 other countries or territories worldwide (3). Compared to the two other coronaviruses responsible for epidemic outbreaks in the past, 55 SARS-CoV and MERS-CoV, the novel SARS-CoV-2 strain shares ⁓79% and ⁓50% genome sequence identity, respectively (4) . Not surprisingly, important differences in terms of the epidemiology and physiopathology between these three viruses have also been observed (5) (6) (7) .

As with most emerging viral diseases, no specific antiviral treatment nor vaccine against any of these three coronaviruses are currently available, with standard patient management relying 60 mainly on symptom treatment and respiratory support when needed. In that regard, and considering that key features of the biology of SARS-CoV-2 and its induced COVID-19 still require further characterization, the scarce readiness of biologically relevant pre-clinical experimental models of SARS-CoV-2 infection as a complement of the African green monkey VeroE6 cell line represents a major barrier for scientific and medical progress in this area. We and others have previously 65 reported the advantage of using more physiological models such as in-house or commercially available reconstituted human airway epithelia (HAE) to isolate, culture and study a wide range of respiratory viruses (8, 9) . Developed from biopsies of nasal or bronchial cells differentiated in the air/liquid interphase, these models reproduce with high fidelity most of the main structural, functional and innate immune features of the human respiratory epithelium that play a central role 70 in the early stages of infection and constitute robust surrogates to study airway disease mechanisms and for drug discovery (10) .

In this study, we initially isolated and amplified in VeroE6 cells a SARS-CoV-2 virus directly form a nasal swab from one of the first hospitalized patients with confirmed COVID-19

in France (11) . The complete genome sequence of the isolated SARS-CoV-2 virus was deposited 75 in the GISAID EpiCoVTM database under the reference BetaCoV/France/IDF0571/2020 (accession ID EPI_ISL_411218). Phylogenetic analysis confirmed that the isolated virus is representative of currently circulating strains (12) . We first characterized the replicative capacities of this viral strain in VeroE6 cells at different multiplicities of infection (MOIs) (Fig. 1A) , using both classic infectious titer determination in cell culture (TCID50) and molecular semi-quantitative 80 methods, the latter based on ORF1b-nsp14-specific primers and probes designed by the School of Public Health/University of Hong Kong (details in Supplementary Materials). This double approach was facilitated by the appearance of clearly observable characteristic cytopathic effect from 48 hpi (Fig. 1B) , and enabled the validation of a large interval (range 1-8 log10(TCID50)) with high correlation (R-squared 0.94) between molecular and infectious viral titers (Fig. 1C) . 85 In parallel, we successfully inoculated nasal MucilAir™ HAE on the apical surface directly with nasal swab samples, as confirmed by transmission electron microscopy observations (Fig.   S1 ). Characteristic features of coronavirus-induced cell ultrastructure remodeling were easily distinguishable in both the apical and basal sides of the HAE at 48 hpi, notably the high accumulation of progeny virions in mucus-producer goblet cells. Then, we advantageously 90 exploited the MucilAir™ HAE model and in-house adapted protocols previously optimized for different respiratory viruses (13) to perform experimental infections with SARS-CoV-2. Viral replication was monitored through repeated sampling and TCID50 titration at the apical surface of HAE (Fig. 1D) . Trans-epithelial electrical resistance (TEER), considered as a surrogate of epithelium integrity, was also measured during the time-course of infection (Fig. 1E) . In parallel, 95 comparative molecular viral genome quantification was performed at the three levels of the air/liquid HAE interphase: in apical washes (Fig. 1F, Apical) , total cellular RNA (Fig. 1G , Intracellular) and basal medium (Fig. 1H, Basal) . SARS-CoV-2 viral production at the epithelial apical surface increased sharply at 48 hpi, reaching 5.8 and 6.3 log10 TCID50/mL in nasal and bronchial HAE, respectively. The peak of viral replication was reached earlier in bronchial (48-72 100 hpi) than in nasal HAE, in which a progressive increase in infectious viral titers was observed until at least 96 hpi (Fig. 1D) . This replication kinetics was validated by molecular viral genome quantification at the apical pole ( Fig. 1F) . High viral replication correlated with a reduction in epithelium integrity at 48 hpi, reflected by more than 2.8-and 4-fold decreases in bronchial and nasal HAE TEER values, respectively, followed by a partial recovery in the case of bronchial HAE 105 (Fig. 1E) . Moreover, viral production at the apical pole was well correlated with intracellular viral genome detection during infection, except for the nasal HAE at 48 hpi, in which a strong relative increase of nsp14 RNA was observed (Fig. 1F) . Interestingly, viral genome was detected in the basal medium from 48 hpi, with the peak observed at 72 hpi ( Fig. 1H ) coinciding with the highest impact of SARS-CoV-2 infection on epithelium integrity. 110 To further characterize the biology of the SARS-CoV-2, we inoculated both nasal ( Fig.   2A , B) and bronchial (Fig. 2C, D) HAE and analyzed the infection-induced remodeling of the cellular ultrastructure using transmission electron microscopy. At 48 hpi, both HAE exhibited a well-established infection, with ciliated, goblet and to a lesser extent basal cells showing active production of viral progeny. This observation is accordance with viral replication results described in Fig. 1 and with a recent study reporting high expression levels of the SARS-CoV-2 cell receptor angiotensin-converting enzyme-2 (ACE2) in both ciliated and goblet respiratory cells (14) . As previously observed in structural studies of other coronaviruses, notably SARS-CoV and MERS-CoV (15-18), we distinguished characteristic clusters in the perinuclear region of infected HAE cells. These clusters are mainly composed of numerous viral single-and double-membrane 120 vesicles (DMV) and mitochondria ( Fig. 2A, A1 We therefore evaluated in both VeroE6 and HAE model the antiviral potential against SARS-CoV-2 of remdesivir monotherapy but also in combination with diltiazem. Diltiazem is a voltage gated 170 Ca2+ channel antagonist currently used as anti-hypertensive for the control of angina pectoris and cardiac arrhythmia (26) , which we have recently repurposed as an effective host-directed influenza inhibitor due to its so far undescribed capacity of inducing the interferon (IFN) antiviral response, particularly type III IFNs (Fig. S2) (8) . Additionally, the rationale of testing such virus-directed plus host-directed drug combination is consistent with a novel study describing hypertension as a 175 potential risk factor observed among a cohort of inpatients with COVID-19 (19), and two reports not anticipating potential adverse effects of diltiazem (27) or negative pharmacological interactions of between remdesivir and diltiazem for the treatment of COVID-19 (28) . cells has been proved functional, this cell line cannot produce type I IFNs (29, 30) . This incomplete IFN response most likely accounts for the lack of significant antiviral effect observed with diltiazem monotherapy in our experimental conditions. Nonetheless, addition of 11.5 µM diltiazem significantly potentiated the antiviral effect of remdesivir ( Fig. 4A-C) , inducing 68% and 50% 185 reductions in remdesivir IC50 values at 48 and 72 hpi, respectively. Comparably, daily treatment with 20 µM remdesivir resulted in 7.3 log10 and 7.9 log10 reductions of intracellular SARS-CoV-2 viral titers at 48 hpi in nasal and bronchial HAE, respectively (Fig. 4D, upper panel) . Not surprisingly for a model with a completely functional IFN response, daily treatment with 90 µM diltiazem resulted in moderate yet substantial (0.4 log10 and 0.8 log10, respectively) reductions of 190 intracellular viral titers in nasal and bronchial HAE at the same time-point (Fig. 4D, upper panel) .

On top of that, we observed an additional 1.3 log10 reduction in nasal HAE viral titers for the remdesivir-diltiazem combination when compared with remdesivir monotherapy. In all cases, the antiviral effects induced by remdesivir, diltiazem or the remdesivir-diltiazem combination translated into a protection of the nasal but not the bronchial HAE barrier integrity, preventing the 195 drop on TEER values induced by the infection (Fig. 4D, lower panel) . Importantly, remdesivir also showed a strong antiviral effect at 72 hpi (Fig. 4E, upper panel) . This time, the ⁓2 log10 reductions in nasal and bronchial HAE viral titers observed for the remdesivir and remdesivirdiltiazem treatments correlated higher TEER values in both HAE compartments (Fig. 4E 

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Scale bar: 0.1 µm. (D2) Enlargement of a double-membraned spherule containing virions (V), double-membrane vesicles and electron-dense viral materials