More than 600,000 people worldwide have fallen victim to the lung disease COVID-19 so far, which is caused by the SARS coronavirus-2 (SARS-CoV-2).
In order to obtain an effective therapy for COVID-19 as quickly as possible, drugs that are being used to treat other diseases are currently being repurposed for COVID-19 treatment.
The Infection Biology Unit of the German Primate Center (DPZ) – Leibniz Institute for Primate Research in Göttingen, together with colleagues at the Charité in Berlin, was able to show that the malaria drug chloroquine, which has been demonstrated to inhibit the SARS-CoV-2 infection of African green monkey kidney cells, is not able to prevent infection of human lung cells with the novel coronavirus.
Chloroquine is therefore unlikely to prevent the spread of the virus in the lung and should not be used for the treatment of COVID-19.
It is known that SARS-CoV-2 is able to use two different routes to enter cells. First, after attaching to the cells, the virus can fuse directly with the plasma membrane and introduce its genetic material into the host cell.
Second, it can enter the interior of the cells upon uptake via transport structures, called endosomes. In both cases, the attachment of the virus to the cells and subsequent entry is mediated by the viral spike protein.
For this purpose, the spike protein must be activated either by the enzyme cathepsin L (in endosomes) or by the enzyme TMPRSS2 (on the cell surface). Depending on the cell type, both enzymes or only one of them can be available for activation.
Chloroquine is a drug that is used to treat malaria. Since chloroquine inhibits the infection of monkey kidney cells with SARS-CoV-2, chloroquine has been tested in clinical trials as a possible candidate for the treatment of COVID-19.
However, how chloroquine inhibits the infection of monkey kidneycells was not clear. The current study shows that chloroquine inhibits viral entry into these cells, most likely by blocking cathepsin L activity.
This raised the question whether chloroquine also inhibits the infection of lung cells that are known to produce TMPRSS2 but only a small amount of cathepsin L.
The study shows that chloroquine does not prevent SARS-CoV-2 entry into human lung cells and subsequent spread of the virus in these cells. “In this study, we show that the antiviral activity of chloroquine is cell type-specific and that chloroquine does not block the infection of lung cells.
This means that in future tests of potential COVID-19 drugs, care should be taken that relevant cell lines are used for the investigations in order not to waste unnecessary time and resources in our search for effective COVID-19 therapeutics,” says Stefan Pöhlmann, head of the Infection Biology Unit at DPZ, adding: “COVID-19 is primarily caused by the infection of lung cells, for this reason these cells should be given priority in efficacy tests.”
The COVID-19 pandemic, which is caused by the novel coronavirus SARS-CoV-2, has been associated with more than 600,000 fatal cases worldwide.
In order to develop antiviral interventions quickly, drugs used for treatment of unrelated diseases are currently being repurposed to combat COVID-19.
Chloroquine is a anti-malaria drug that is frequently employed for COVID-19 treatment since it inhibits SARS-CoV-2 spread in the kidney-derived cell line Vero1–3.
Here, we show that engineered expression of TMPRSS2, a cellular protease that activates SARS-CoV-2 for entry into lung cells4, renders SARS-CoV-2 infection of Vero cells insensitive to chloroquine.
Moreover, we report that chloroquine does not block SARS-CoV-2 infection of the TMPRSS2-positive lung cell line Calu-3.
These results indicate that chloroquine targets a pathway for viral activation that is not operative in lung cells and is unlikely to protect against SARS-CoV-2 spread in and between patients.
Chloroquine and hydroxychloroquine are used for Malaria treatment and have been widely employed to treat COVID-19 patients, with
more than 80 registered clinical trials worldwide2,3.
Chloroquine and hydroxychloroquine inhibit SARS-CoV-2 infection of Vero cells1,5,6, providing a rational for using these drugs for COVID-19 treatment. However, it is unknown whether these drugs inhibit infection of lung cells and it is poorly understood how they inhibit SARS-CoV-2 infection.
Chloroquine and hydroxychloroquine elevate endosomal pH and inhibit viruses that depend on low pH for cell entry7.
We asked whether they also block SARS-CoV-2 cell entry and whether entry inhibition accounts for blockade of SARS-CoV-2 infection. Moreover, we investigated whether entry inhibition is cell type-dependent, since the virus can employ pH-dependent and pH-independent pathways for entry into cells.
Thus, SARS-CoV-2 spike protein (SARS-2-S), which mediates viral entry, is activated by the endosomal pH-dependent cysteine protease cathepsin L (CatL) in certain cell lines4.
In contrast, entry into airway epithelial cells, which express low levels of CatL8, depends on the pH-independent, plasma membrane resident serine protease TMPRSS24. Importantly, CatL usage by coronaviruses is restricted to cell lines8–10 while TMPRSS2 activity is essential for viral spread and pathogenesis in the infected host11,12.
We compared chloroquine and hydroxychloroquine-mediated inhibition of SARS-2-S-mediated entry into Vero (kidney), Vero-TMPRSS2 and Calu-3 (lung) cells. Calu-3 cells, like airway epithelium, express low amounts of CatL8 and SARS-CoV-2 entry into these cells is TMPRSS2-dependent4.
In contrast, Vero cell entry of SARS-CoV-2 is CatL-dependent while both CatL and TMPRSS2 support entry into Vero-TMPRSS2 cells4.
As control, we used camostat mesylate, which inhibits TMPRSS2-dependent entry4.
Camostat mesylate treatment did not interfere with cell viability while chloroquine and hydroxychloroquine slightly reduced viability when applied at the highest concentration (Fig. 1a). Inhibition of SARS-2-S-driven entry by camostat mesylate was only observed with TMPRSS2+ cell lines, as expected (Fig. 1a, Table 1).
Moreover, chloroquine and hydroxychloroquine inhibited SARS-2-S-driven entry into Vero cells (TMPRSS2-) with high efficiency while inhibition of entry into Calu-3 and Vero-TMPRSS2 (both TMPRSS2+) cells was inefficient or absent, respectively (Fig. 1a, Table 1).
Thus, chloroquine and hydroxychloroquine can block SARS-2-S-driven entry but inhibition is cell line-dependent and efficient inhibition is not observed with TMPRSS2+ lung cells.
We next investigated whether the cell type-dependent differences in entry inhibition translated into differential inhibition of authentic
SARS-CoV-2. Indeed, chloroquine efficiently blocked SARS-CoV-2 Infection of Vero kidney cells, as expected1, but failed to efficiently inhibit SARS-CoV-2 infection of Calu-3 lung cells (Fig. 1b, c).
A subtle reduction in SARS-CoV-2 infection was seen in the presence of 100 μM chloroquine, in keeping with the modest inhibition of pseudotype entry under those conditions (Fig. 1a), but this effect was not statistically significant. In sum, chloroquine failed to efficiently block Calu-3 cell infection with SARS-2-S-bearing pseudotypes and authentic SARS-CoV-2, indicating that in these cells chloroquine does not appreciably interfere with viral entry or the subsequent steps of the viral replication cycle.
Confirmation of our results with primary respiratory epithelium is pending. Moreover, we note that virus production in Calu-3 relative to Vero E6 cells was more robust in the present study as compared to a published one13, potentially due to use of the Calu-3 subclone 2B4 in the previous but not the present study.
Nevertheless, our results suggest that chloroquine and hydroxychloroquine will exert no antiviral activity in human lung tissue and will not be effective against COVID-19, in keeping with the results of recent clinical trials14,15. Moreover, they highlight that cell lines mimicking important aspects of respiratory epithelial cells should be used when analyzing the antiviral activity of drugs targeting host cell functions.
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More information: Markus Hoffmann et al. Chloroquine does not inhibit infection of human lung cells with SARS-CoV-2, Nature (2020). DOI: 10.1038/s41586-020-2575-3
