Study shows expression of viral proteases may be used to quantitate SARS-CoV-2 infected cells

By Dr. Priyom Bose, Ph.D.Reviewed by Danielle Ellis, B.Sc.Feb 14 2022

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the coronavirus disease 2019 (COVID-19) pandemic. Different laboratories use different approaches to measure SARS-CoV-2 infection, and these methods are laborious and time-consuming. There is no consensus on how to quantify neutralizing antibodies against SARS-CoV-2, which are key to blocking infection and protecting individuals from COVID-19.

A new study published in PLoS Pathogens describes a new method to quantify SARS-CoV-2 infection, which is much simpler and faster compared to existing methods.

Background

SARS-CoV-2 infection or vaccination induces neutralizing antibodies and prevents COVID-19. Novel emerging variants have brought about the ability to escape antibody neutralization in vitro and protective immunity in vivo. The variability in the assays used to measure antibody titers has made it difficult to define serological correlates of protection. Plaque Reduction Neutralisation Tests (PRNTs) are considered to be the gold standard, but low throughput and long turnaround times make it difficult to be used for large-scale screening of sera.

The reverse genetic modification of SARS-CoV-2 is laborious, and existing approaches are restricted to re-engineered reference strains. Lentiviral particles pseudotyped with SARS-CoV-2 spike proteins have been commonly deployed as surrogates. Still, there are heterogeneities across laboratories, and these variations mean that these results correlate imperfectly with results for wild-type virus. There is an urgent need for a simple, high-throughput method to quantify infection and neutralize authentic SARS-CoV-2 isolates. The new method should also account for new variants of concern.

A new study

Previously, in the case of the human immunodeficiency virus-1 (HIV-1), cell-based optical reporter systems have been instrumental in the pandemic response. To gain a deeper understanding of the disease mechanics, luciferase-based neutralization assays using TZM-bl reporter cells are employed worldwide. These assays have also been crucial in the development of antiviral therapeutics. This study develops an equivalent luminescent reporter cell line for SARS-CoV-2.

Scientists showed that the expression of viral proteases could be used to quantify infected cells. The cleavage of specific oligopeptide linkers, which led to cell-based optical biosensors, was utilized for these assays. The biosensors using recombinant SARS-CoV-2 proteases were characterized first. Subsequently, the ability to detect viral protease expression was confirmed.

Lastly, researchers generated reporter cells stably expressing an optimized luciferase-based biosensor. This enabled them to measure viral infection with a 24-96-hour window, including variants of concern. This led to the development of a luminescent SARS-CoV-2 reporter cell line. Researchers showed its usefulness for quantifying the infectious virus and titration of neutralizing antibodies.

Key findings

In the current study, scientists demonstrated the usability of protease-activatable biosensors for the quantification of authentic SARS-CoV-2 infection. They further showed the practical utility of a luciferase-based reporter cell line to measure neutralizing antibody activity. Luminescent assays are capable of being adapted to high-throughput platforms, and the potential applications are similar to TZM-bl reporter cells for HIV infection.

Before the current pandemic, protease-activatable reporters were used to detect recombinant proteases from a range of viruses. However, it was not used to identify viral proteases expressed during authentic infection. The main objective of this study was to develop a simple optical biosensor for authentic COVID-19 infection. The luminescent reporter cell line did not require any sophisticated high-content microscopy and allowed assays to be conducted in 24 hours, in a 96-well or 384-well plate format.

Scientists stated that the window for luciferase-based biosensor activation was greatly elevated compared to the FlipGFP biosensors. Additionally, the 30F-PLP2/PLPro biosensor performed markedly better, during SARS-CoV-2 infection, than the 30F-Opt3/MPro biosensor. This result could be reflective of the inversion of the relative levels of expression of MPro and PLPro. It could also be that the reporters (substrates) have differential accessibility to viral proteases in the context of infection.

Researchers also suspected that PLPro of SARS-CoV-2 could readily access various cellular proteins, including cytosolic reporters. The reporter cells were able to detect different clinical SARS-CoV-2 isolates. This was brought about without any modification of the viral genome. The results suggested that Papain-like proteases of these viruses would also be able to activate 30F-PLP2.

Conclusion

In this study, scientists developed a protease-activatable luminescent biosensor and reporter cell line, which provides an “off the shelf” solution for quantifying authentic neutralizing antibody activity and betacoronavirus infection. The method should be applicable in the current COVID-19 pandemic as well as potential future pandemics.