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Open AccessProtocol

Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays

1
Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
2
Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
3
Medical Scientist Training Program, University of Washington, Seattle, WA 98195, USA
4
Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195, USA
5
Department of Biochemistry, University of Washington, Seattle, WA 98109, USA
6
Institute Pasteur & CNRS UMR 3569, Unité de Virologie Structurale, Paris 75015, France
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Institute for Protein Design, University of Washington, Seattle, WA 98195, USA
8
The Ragon Institute of Massachusetts General Hospital, the Massachusetts Institute Technology, and Harvard University, Cambridge, MA 02139, USA
9
Howard Hughes Medical Institute, Seattle, WA 98103, USA
*
Author to whom correspondence should be addressed.
Viruses 2020, 12(5), 513; https://doi.org/10.3390/v12050513
Received: 20 April 2020 / Revised: 2 May 2020 / Accepted: 3 May 2020 / Published: 6 May 2020
(This article belongs to the Special Issue Pathogenesis of Human and Animal Coronaviruses)
SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral, and vesicular stomatitis virus (VSV) particles, but the reagents and protocols are not widely available. Here, we detailed how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also made all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrated how these pseudotyped lentiviral particles could be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization. View Full-Text
Keywords: SARS-CoV-2; COVID-19; coronavirus; neutralization assay; lentiviral pseudotype; Spike; cytoplasmic tail; ACE2; 293T-ACE2; luciferase; ALAYT SARS-CoV-2; COVID-19; coronavirus; neutralization assay; lentiviral pseudotype; Spike; cytoplasmic tail; ACE2; 293T-ACE2; luciferase; ALAYT
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Crawford, K.H.D.; Eguia, R.; Dingens, A.S.; Loes, A.N.; Malone, K.D.; Wolf, C.R.; Chu, H.Y.; Tortorici, M.A.; Veesler, D.; Murphy, M.; Pettie, D.; King, N.P.; Balazs, A.B.; Bloom, J.D. Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays. Viruses 2020, 12, 513.

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