Identify neutralizing antibodies that inhibit SARS-CoV-2 host cell entry
Montana Molecular’s SARS-CoV-2 pseudovirus neutralization assays provide safe and cost effective solutions for testing the effects of Spike antibodies in live cells. Pseudo-SARS-CoV-2 is a BSL-1 BacMam pseudo typed with Spike protein. BacMam does not infect or replicate in mammals so it is safe to use without risk of infection.
The SARS-CoV-2 Spike protein (S-protein) is a focus of COVID-19 research and drug discovery because Spike interacts with ACE2 on the host cell to mediate viral entry into the host cell. To safely screen for neutralizing antibodies or compounds that inhibit viral entry, cells expressing ACE2 can be used as a “pseudo host” and treated with Pseudo-SARS-CoV-2. When the pseudovirus enters pseudo host cells, the cell nuclei turn bright green.
In the example below, A549 cells expressing ACE2 were exposed to Pseudo-SARS-CoV-2 pseudovirus and treated with a neutralizing antibody. Host cell fluorescence decreases in a dose-dependent manner relative to untreated cells.
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Pseudo SARS-CoV-2 D614G
This pseudovirus is based on an early isolate that first appeared in Europe that encoded a Spike protein with a single conservative mutation D614G. The Korber group was tracking the spread of this mutation, and it soon became clear to them that it was spreading far more rapidly than the original virus (Korber et al. 2020). They posited both structural and immunological explanations for how the mutation affected viral spread. Since the initial report, biochemical approaches in the Choe laboratory have demonstrated that the mutation causes a decrease in S1 shedding and an increase in infectivity (Zhang et al. 2020).
Mpro Inhibitor Assay
The 3CLpro, or Main protease, Mpro is crucial to the processing of the replicase polyprotein of SARS-CoV-2. Because the protease is unique, and required for viral replication, it is a promising drug target. Indeed, it has recently been shown that the protease inhibitor GC376 can block SARS-CoV-2 replication (Hung et al. 2020). The Mpro Inhibitor assay is a cost effective BacMam solution that will make it possible to rapidly screen, in living cells, for Mpro inhibitors. The Mpro assay kit includes an optimized Mpro and a fluorescent sensor for Mpro activity. This assay will also enable investigators working with isolates of the SARS-CoV-2 virus to identify infected cells in culture without the need for immunohistochemistry or the long waiting period to identify cytopathic effects.
Tools to express and purify SARS-CoV-2 Spike protein
The SARS-CoV-2 Spike protein in baculovirus is pseudotyped with VSVG for entry into most mammalian cell types. The Spike protein is truncated just before the transmembrane domain and a convenient His tag is appended for easy purification. Transduction of suspension cultures produces large amounts of secreted Spike protein that can be purified from the media.
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Hung, Hui-Chen, Yi-Yu Ke, Sheng Yu Huang, Peng-Nien Huang, Yu-An Kung, Teng-Yuan Chang, Kuei-Jung Yen, et al. 2020. “Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.” Antimicrobial Agents and Chemotherapy, July. https://doi.org/10.1128/AAC.00872-20.
Korber, B., W. M. Fischer, S. Gnanakaran, H. Yoon, J. Theiler, W. Abfalterer, B. Foley, et al. 2020. “Spike Mutation Pipeline Reveals the Emergence of a More Transmissible Form of SARS-CoV-2.” bioRxiv. https://doi.org/10.1101/2020.04.29.069054.
Zhang, Lizhou, Cody B. Jackson, Huihui Mou, Amrita Ojha, Erumbi S. Rangarajan, Tina Izard, Michael Farzan, and Hyeryun Choe. 2020. “The D614G Mutation in the SARS-CoV-2 Spike Protein Reduces S1 Shedding and Increases Infectivity.” bioRxiv. https://doi.org/10.1101/2020.06.12.148726.