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).
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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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.