key: cord-0806419-w55gxkck
authors: Volle, Romain; Murer, Luca; Petkidis, Anthony; Andriasyan, Vardan; Savi, Alessandro; Bircher, Cornelia; Meili, Nicole; Fischer, Lucy; Sequeira, Daniela Policarpo; Mark, Daniela Katharina; Gomez-Gonzalez, Alfonso; Greber, Urs F.
title: Methylene blue, Mycophenolic acid, Posaconazole, and Niclosamide inhibit SARS-CoV-2Omicron variant BA.1 infection of human airway epithelial explant cultures
date: 2022-03-31
journal: bioRxiv
DOI: 10.1101/2022.03.30.486461
sha: 1524db4629abd637fcddf57ed8f1f741bb9e4f50
doc_id: 806419
cord_uid: w55gxkck

Sublineages of SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) Omicron variants continue to amass mutations in the spike (S) glycoprotein, which leads to immune evasion and rapid spread of the virus across the human population. Here we demonstrate the susceptibility of the Omicron variant BA.1 (B.1.1.529.1) to four repurposable drugs, Methylene blue (MB), Mycophenolic acid (MPA), Posaconazole (POS), and Niclosamide (Niclo) in post-exposure treatments of primary human airway cell cultures. MB, MPA, POS, and Niclo are known to block infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) with the Wuhan strain, and four variants of concern (VoC), Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28), Delta (B.1.617.2) (1, 2). Our results not only show broad anti-coronavirus effects of MB, MPA, POS and Niclo, but also demonstrate that the Omicron variant BA.1 (B.1.1.529.1) sheds infectious virus from HAEEC over at least 15 days, and maintains both intracellular and extracellular viral genomic RNA without overt toxicity, suggesting viral persistence. The data underscore the broad effects of MB, MPA, POS, and Niclo against SARS-CoV-2 and the currently circulating VoC, and reinforce the concept of repurposing drugs in clinical trials against COVID-19.

The causative agent of COVID-19, SARS-CoV-2 rapidly evolves in the human population at high circulation frequency despite increasing natural and vaccine-induced immunity.

Variants of concern (VoC) emerge and continue to turn over. For example, the Alpha and Beta VoC were first reported in the UK and South Africa in Summer 2020, and were replaced by the Delta VoC, first reported in India October 2020. Delta soon became the dominant VoC by mid 2021 (3) (4) (5) . Lately, the Omicron variant emerged, as first reported in South Africa in November 2021, and became abundant worldwide from December 2021 onward, replacing Delta by the beginning of 2022 (6) . Omicron variants feature up to at least 30 amino acid substitutions, several deletions and also insertions in the spike protein (S) open reading frame (ORF). Remarkably, many of the substitutions are directly localized within the receptor-binding domain (RBD) (7) , possibly the result of immune pressure upon infection and vaccination (8) (9) (10) (11) . Accordingly, neutralizing antibody titers against Omicron are found to be lower than against earlier VoC, as suggested in a preprint study from 23 laboratories (12) . Nonetheless, Omicron appears to cause less severe disease than Delta, possibly because the immune status of many people still partially protects against Omicron and its VoC. However, Omicron continues to cause death notably affecting also non-vaccinated or incompletely vaccinated individuals (11, 13) .

A growing body of evidence suggests that the multiple amino acid substitutions in the Sprotein reduce the dependency of the virus on the serine protease TMPRSS2, compared to the Delta VoC (14) . The altered usage of TMPRSS2 by Omicron appears to make the virion more dependent on low-pH endosomes and the cathepsin entry pathway (14) (15) (16) .

This highlights a considerable genetic flexibility of SARS-CoV-2 to adapt to different cell entry pathways, and may render the S-protein dependent entry a rather difficult target for powerful pan-interference strategies against SARS-CoV-2. 4 By engaging a multicycle drug repurposingscreen against coronaviruses we previously identified and validated several broadly acting compounds against SARS-CoV-2 infection of human nasal and bronchial airway epithelial explant cultures (HAEEC) grown at air liquid interface, namely, Methylene blue (MB), Mycophenolic acid (MPA), and Posaconazole (POS) (2) .These compounds have been used in the clinics for unrelated applications, and can be considered for repurposing against SARS-CoV-2. Here we report that these compounds strongly inhibit SARS-CoV-2 Omicron, along with the antihelminthic drug niclosamide (Niclo). Finally, we provide evidence for SARS-CoV-2 persistent infection of HAEEC.

The partial immunity against circulating Omicron subvariants BA.1 and BA. is not known, these compounds do not inhibit SARS-CoV-2 cell entry, and have little effects on viral genome replication, but strongly inhibit the release of infectious progeny to the apical medium, indicating that they affect one or several post replication steps, such as particle formation or egress (2). 5 Nasal HAEEC (Epithelix, MucilAir™) grown at air-liquid interface (ALI) were apically inoculated with Omicron BA.1 (1,000 TCID 50 per tissue) and treated with compounds added to the basolateral medium one day post infection (pi). Following five consecutive days of daily treatment and apical sampling, infectious virus titer was determined with 50% tissue culture infectious dose (TCID 50 ) assays. Treatments with MB, MPA, and POS strongly inhibited the release of Omicron progeny (Fig. 1A) , similarly as previously observed withthe Alpha, Beta, Gamma, and Delta VoC (2). The basolateral medium had no detectable virus titer, akin to the earlier study (2) . The Omicron titers on the apical side, however, were in the range of 4.5 to 4.9-log 10 TCID 50 /ml as early as one day pi Fig. 1B and 1C ). Importantly, we could not detect evidence that MB and MPA induced phospholipidosis (Suppl. Fig. 2 ). Phospholipidosis manifests itself by a foamy appearance of internal cellular membranes likely owing to disregulated membrane signalling, sorting or transport (18) . It was reported that cationic amphiphilic drugsmay have unspecific antiviral activity correlated with phospholipidosis, for example compounds such as chloroquine, that had been discussed for repurposing in the early days of COVID-19 (19) .

We next compared the susceptibility of human primary epithelial explant cells of nasal and bronchial origin to Omicron BA.1 infection. Our results indicate that Omicron similarly infected both nasal and bronchial lung epithelial cells yielding apical titers in the range of 10 4 to 10 5 TCID 50 /ml (Fig.2) . These results suggest that bronchial cells can be used as a model for drug assessment against SARS-CoV-2 Omicron, and are in agreement with a 6 report using ex vivo cultures, where Omicron exhibited higher replication in bronchial cells than in lung parenchymal cells mimicking alveoli of the lower respiratory tract (16) .

To further address theneed of acute medical treatment options with broadly available, safe and effective antivirals, we tested if Niclo inhibited Omicron infection of bronchial HAEEC. Niclo is FDA-approved for the treatment of tapeworm infections. It acts as a protonophore and has broad anti-helminthic and antiviral activity owing to its ability to neutralize acidic cytoplasmic membrane compartments, as well as its effects on membrane trafficking and cell signaling processes (20) (21) (22) (23) . Recently, Niclo was shown to inhibit infection of primary human bronchial epithelial cells with SARS-CoV-2 Alpha, Beta, and Delta VoC (1).

To test if Niclo affected SARS-CoV-2 Omicron infection, we apically inoculated Omicron onto bronchial HAEEC, and treated the cells with different concentrations of Niclo (20, 10, 5, and 1µM) one day pi for up to day 8 in daily treatments and apical sampling, followed by TCID 50 titration of infectious progeny production. While 1µM of Niclo had no effect on Omicron, and 20µM was toxic for the cells, a daily treatment with intermediate concentrations of 5 or 10µM reduced the infectious titer of Omicron at days 3 to 8 up to 2 log 10 (Fig.3) . Similarly we could not detect evidence that Niclo induced phospholipidosis (Suppl. Fig. 2) . These results were similar to the ones with the Alpha, Beta, and Delta VoC reported earlier (1) . Notably, our effective concentrations of Niclo were slightly We took apical samples from the Omicron infected, DMSO control nasal HAEEC cells (presented in Fig. 1 ) for up to 15 days, and found a continuous titer between about 10 4 and 10 5 TCID50/ml in the apical milieu (Fig. 4A) . The RT-qPCR genome equivalents were between about 10 7 and 10 9 copies / ml, indicating continued production and release of viral components over several weeks. In accordance, infected nasal HAEEC cells fixed at 7d and 21d pi followed by RNA FISH staining demonstrated the presence of intracellular SARS-CoV-2 ORF1ab RNA(+) fluorescent puncta predominantly in the cell layer near the apical side of the pseudo-tissue (Fig. 4B) . Similarly, the 21d infected HAEEC cells also exhibited a clear staining of the SARS-CoV-2 ORF1ab RNA(+), albeit to a lesser extent.

Together, these results support the notion that HAEEC can be infected with SARS-CoV-2 for periods of at least several weeks. This gives rise to a situation that resembles a persistent infection, where virus is steadily released without overt tissue death.

Persistence in vivo may enhance the chance for viral recombination, which requires that two or more different infectious agents are present in the same cell. A survey of SARS-CoV-2 genomes from UK firstly observed mosaic genomes structures of Alpha VoC with other co circulating variants, likely to be from recombinant origin (37) . Then, other potential SARS-CoV-2 recombinant lineage sequences were deposited on genomic databases and currently labelled XA to XL by the PANGO lineage nomenclature (38) . 9 Similarly, as both the Delta and the Omicron VoC have been massively co-circulating in many countries across the world, it can be expected that Delta and Omicron recombinants will appear. In fact, this has recently been suggested in several independent preprints (39) (40) (41) , acknowledged by the WHO with the BA.1 x AY.4 recombinant, nicknamed 'Deltacron', and labelled XD in the PANGO lineage nomenclature (38) . Although it is presently unknown, if these recombinants will outcompete the known SARS-CoV-2 variants, virus recombinants raise concerns about increased transmission and pathogenicity. This issue requires continued monitoring, drug and vaccine development and explorations of repurposable drugs against COVID-19. 

Niclo was purchased to Sigma Aldrich (cat#N3510-50G; lot #BCBD3349V) and solubilized according to manufacturer's instructions. 

Phospolipidosis assay was performed as described by Tummino and colleagues (19) . NEAA, 10µg/mL Hoechst, and 2µM Ethidium homodimer-2 (EthD-2), and imaged with ImageXpress Micro confocal (Molecular Devices) microscope.

This was carried out as published in Murer et al (2) . 

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We acknowledge financial support from the Swiss National Science foundation (SNSF) to UFG (31CA30_196177/1) and a special Coronavirus Research grant from the University of Zurich to UFG. We are grateful to Dr. Adriano Aguzzi and Dr. Simone Hornemann for granting generous access to their BSL3 laboratory, and Dr. Maarit Suomalainen for discussions.