2021 Volume 3 Issue 3 Pages 112-114
The development of highly active anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) agents is of great importance. However, the insufficient access of researchers to SARS-CoV-2 infection experiments that require the facilities and regulations for Bio Safety Level 3 (BSL3) is a bottleneck in expanding the activity for drug development against SARS-CoV-2. To overcome this limitation, we constructed a system that supports SARS-CoV-2 infection experiments in cell culture and is widely open to both academia and industries under collaboration. Our institute, the National Institute of Infectious Diseases (NIID), Japan, not only possesses well-organized BSL3 facilities but also has established original cell models that are highly susceptible to the SARS-CoV-2 infection, including the VeroE6/TMPRSS2 cells [1], and developed cell-based high-throughput systems to evaluate the antiviral activity of compounds [2], as well as isolated a series of SARS-CoV-2 clinical strains, including variants of concern such as alpha, beta, gamma, and delta [3]. These advantages can allow us to serve as a collaborative hub by providing SARS-CoV-2 infection cell culture systems for drug evaluations.
Our infection cell culture system (shown below in detail) can be applied to the evaluation of any modalities aimed at inhibiting the virus life cycle, including small molecules, natural products, nucleic acids, peptides, antibodies, and sera derived from infected or vaccinated animals. Candidate substances with high priority would be, but not limited to, those already selected from screening in in silico (e.g., compounds virtually bound to a SARS-CoV-2 protein), in vitro (e.g., compounds shown to inhibit the enzymatic activity of a SARS-CoV-2 protein or to inhibit a protein-protein interaction essential for virus infection), or virus-free cell culture systems (e.g., compounds inhibiting a protein-protein interaction or cell fusion mimicking virus-cell membrane fusion), and sera derived from animals treated with a vaccine candidate. Those rationally designed to target SARS-CoV-2 (e.g., nucleic acids or molecules designed to target viral genome RNA) are also potential candidates. Structurally unique compounds and libraries can be the sources for new structures possessing anti-SARS-CoV-2 activity.
Our proposed scheme for drug evaluation is as follows (Fig. 1): 1) Primary screening using VeroE6/TMPRSS2 cells as a target cell line and the Wk521 strain (Wuhan type) as SARS-CoV-2 inoculum [2]. As the infection exhibits a robust cytopathic effect within 2 days post-infection with high reproducibility, survival cell numbers serve as an initial marker for antiviral activity that can be quantified in a high-throughput manner using a high-content imaging analyzer in 96- or 384-well plates. Representatively, SARS-CoV-2 infection reduces the viability of VeroE6/TMPRSS2 cells to below 1%, but treatment with remdesivir (RDV), a SARS-CoV-2 polymerase inhibitor, at 10 µM rescues the cell viability to more than 30%, as a positive control [4]. The primary screening selects compounds that significantly increase the cell viability compared to that of DMSO-treated cells. 2) The second screening is to quantify viral RNA in the supernatant of infected VeroE6/TMPRSS2 cells before showing cytopathic effects (e.g., at 18–24 hr post-infection). Compounds that reduce the SARS-CoV-2 RNA level, examined by real-time RT-PCR are then selected. Hit compounds of processes 1) and 2) are those that reduce viral RNA and protect SARS-CoV-2-induced cytopathology without cytotoxic effects. 3) Validation of hit compounds can then be further examined by detecting SARS-CoV-2 proteins by either immunoblotting or immunofluorescence analysis and by quantifying the infectivity titer of the supernatant of infected cells, as well as by examining the reproducibility and dose-dependency of the observed effects. Dose-response curves for antiviral activity and cytotoxic effect examined with varying compound concentrations can be used to calculate the 50% and 90% maximal inhibitory concentrations (IC50 and IC90, respectively), 50% cytotoxic concentrations (CC50), and Hill coefficient, which are useful for prioritizing and determining the focused compounds in the following assay. 4) The antiviral activity of compounds, especially those targeting host factors, may depend on the cellular environment. As the VeroE6/TMPRSS2 cells is a monkey kidney-derived cell line, the examination in human lung-derived cells is desirable. Calu-3 cells are frequently used as a model originating from human lung epithelial cells that are susceptible to SARS-CoV-2 infection, although the virus propagation is far less than that in VeroE6/TMPRSS2 cells [4]. In our assay, some compounds (e.g., chloroquine) show very diverse antiviral activities among these cell types, partly because the viral entry process follows different mechanisms depending on the cell type [4]. As more physiologically relevant models, air-liquid interface culture and human lung organoids are reported to be permissive to SARS-CoV-2 infection. 5) The mode of action of compounds, especially the target step within the virus life cycle (virus entry into cells, virus genome replication, or assembly/release) can be examined by virological assays, including time of addition analysis, where the compound is added at different times after virus inoculation to observe the antiviral activity [2, 4]. Alternatively, for the evaluation of entry inhibitors, including antibodies and vaccinated sera, pseudovirus carrying the SARS-CoV-2 spike protein and mimicking the SARS-CoV-2 spike-mediated cell entry is a useful model as the pseudovirus can be handled in BSL2 and can easily evaluate viral infection by monitoring the luciferase activity driven by pseudovirus infection [4] (Fig. 1).
Schematic representation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection assays. Upper, SARS-CoV-2 infection is evaluated by measuring cytopathic effect (1), SARS-CoV-2 RNA (2), and protein (3) at 48 or 24 hr post-inoculation with SARS-CoV-2 Wk521 strain to VeroE6/TMPRSS2 cells. Lower, pseudovirus infection to VeroE6/TMPRSS2 cells can also be used to evaluate the anti-SARS-CoV-2 activity of entry inhibitors, antibodies, and vaccinated sera.
Our infection assays evaluate the anti-SARS-CoV-2 activity of the candidate compounds. Information on the structure-activity relationship obtained in the assays provide a strategy for further chemical synthesis of compounds and enable the optimization of compounds through chemical synthesis-infection assay cycles. In combination with the pharmacokinetics of compounds in animals, the antiviral profile obtained through the above assay also provides information to predict the appropriate dosage and treatment frequency that helps to design the appropriate protocol for further infection assays using animal models. Thus, providing such infection cell culture assays to researchers in academia and industries will accelerate drug development against COVID-19.
The authors have nothing to disclose.
This work was supported by the Agency for Medical Research and Development (AMED) (Grant No. JP20fk0108411 and JP20fk0108511).
