Scientists develop suitable human cell line for anti-SARS-CoV-2 drug screening
In a recent article published in the journal Virusscientists described the development of a human cell line suitable for high-throughput screening of antiviral drugs targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
Scientists engineered the A549 human lung carcinoma cell line to express high levels of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), two vital host proteins required for viral entry .
The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 has placed an immense burden on the global health system, with more than 539 million confirmed cases of COVID-19 and more than 6.3 million deaths worldwide. With the progression of the pandemic, many viral variants with new mutational landscapes have emerged around the world. Some of them have been designated as the variants of concern (COVs) due to their greatly enhanced transmissibility, infectivity, virulence and immune evasion. Given the growing threat of the pandemic to global public health, the identification and development of SARS-CoV-2 specific antiviral drugs is urgently needed.
In the current study, scientists described the development of a modified A549 cell line that shows robust expression of ACE2 and TMPRSS2 and high susceptibility to SARS-CoV-2 infection. ACE2 and TMPRSS2 are essential for harboring proteins necessary for SARS-CoV-2 entry into host cells.
Modification of the A549 cell line
Modification of the A549 cell line was performed by transducing the cells with a human lentivirus expressing ACE2, followed by puromycin-based clone selection. Then, the selected clones were transduced with a lentivirus expressing TMPRSS2.
More than 50 clones were selected. Of which, one clone showed robust expression of ACE2 and TMPRSS2 and high SARS-CoV-2 infection rate (60%). The infection rate in the modified cell line is similar to that of the Vero E6 cell line, which is a well-established and widely used cell line to characterize SARS-CoV-2 infection.
Single cell sorting of these clones was performed to generate single cell derived subclones. The susceptibility of these subclones (modified cells) to viral infection was determined by infecting them with wild-type SARS-CoV-2 and its delta and omicron variants. Overall, the modified cells showed high susceptibility to infection by all viral variants tested. The infection rate of engineered cells was higher than that of a commercially available human A549 cell line expressing ACE2 and TMPRSS2.
Characterization of the modified A549 cell line
Reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry were conducted to determine mRNA expressions of ACE2 and TMPRSS2 and cell surface expression of ACE2, respectively.
The results revealed that the engineered cells express significantly higher levels of ACE2 and TMPRSS2 than commercially available ACE2 and TMPRSS2-expressing A549 cells. Additionally, the engineered cells showed higher infectivity for SARS-CoV-2 containing the D614G mutation compared to the wild-type virus. D614G is known to interfere with ACE2 receptor binding and viral entry.
Application of modified A549 cells
The utility of modified A549 cells as a screening platform for antiviral drugs was tested in the study. Specifically, engineered cells infected with SARS-CoV-2 were used to compare the antiviral efficacy of nine antiviral drugs (repurposed anti-COVID 19 drugs Nirmatrelvir, EIDD-1931, remdesivir; anti-HIV drug Nelfinavir; and candidate widely used antiviral drugs camostat mesylate, naphthofluorescein, E64d and decanoyl-RVKR-CMK).
Dose-response analysis revealed that all repurposed drugs as well as the anti-HIV drug strongly inhibited SARS-CoV-2 infection in engineered cells. Among the drug candidates, only camostat mesylate significantly inhibited viral infection. These observations indicate that the modified cells can be used for high throughput screening of antiviral drugs.
Additionally, the modified cells were used to test the antiviral efficacy of a panel of drugs against delta and omicron infections. All antiviral drugs tested (EIDD-1931, remdesivir, nirmatrelvir, and nelfinavir) showed dose-dependent inhibition of infection caused by wild-type SARS-CoV-2 and delta and omicron variants. In engineered cells, the omicron variant showed higher susceptibility to drug treatment compared to wild-type SARS-CoV-2 and the delta variant.
Significance of the study
A human cell model suitable for SARS-CoV-2 infection was developed in the study. The lentiviral transduction method was used to stably and robustly express human ACE2 and TMPRSS2 in A549 cells. As suggested by the scientists, these modified A549 cells could potentially be used to study emerging SARS-CoV-2 variants and screen for antiviral drugs.