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Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays

Katharine H.D. Crawford, Rachel Eguia, View ORCID ProfileAdam S. Dingens, View ORCID ProfileAndrea N. Loes, Keara D. Malone, Caitlin R. Wolf, View ORCID ProfileHelen Y. Chu, View ORCID ProfileM. Alejandra Tortorici, View ORCID ProfileDavid Veesler, Michael Murphy, Deleah Pettie, Neil P. King, View ORCID ProfileAlejandro B. Balazs, View ORCID ProfileJesse D. Bloom
doi: https://doi.org/10.1101/2020.04.20.051219
Katharine H.D. Crawford
1Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; (K.D.C.), (R.E.), (A.S.D.), (K.M.), (A.N.L.)
2Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
3Medical Scientist Training Program, University of Washington, Seattle, WA 98195, USA
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  • For correspondence: kdusenbu@fredhutch.org reguia@fredhutch.org adingens@fredhutch.org kmalone2@fredhutch.org aloes@fredhutch.org
Rachel Eguia
1Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; (K.D.C.), (R.E.), (A.S.D.), (K.M.), (A.N.L.)
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  • For correspondence: kdusenbu@fredhutch.org reguia@fredhutch.org adingens@fredhutch.org kmalone2@fredhutch.org aloes@fredhutch.org
Adam S. Dingens
1Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; (K.D.C.), (R.E.), (A.S.D.), (K.M.), (A.N.L.)
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  • For correspondence: kdusenbu@fredhutch.org reguia@fredhutch.org adingens@fredhutch.org kmalone2@fredhutch.org aloes@fredhutch.org
Andrea N. Loes
1Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; (K.D.C.), (R.E.), (A.S.D.), (K.M.), (A.N.L.)
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  • For correspondence: kdusenbu@fredhutch.org reguia@fredhutch.org adingens@fredhutch.org kmalone2@fredhutch.org aloes@fredhutch.org
Keara D. Malone
1Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; (K.D.C.), (R.E.), (A.S.D.), (K.M.), (A.N.L.)
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  • For correspondence: kdusenbu@fredhutch.org reguia@fredhutch.org adingens@fredhutch.org kmalone2@fredhutch.org aloes@fredhutch.org
Caitlin R. Wolf
4Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195, USA; (C.R.W.), (H.Y.C.)
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  • For correspondence: crwolf@uw.edu helenchu@uw.edu
Helen Y. Chu
4Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195, USA; (C.R.W.), (H.Y.C.)
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  • For correspondence: crwolf@uw.edu helenchu@uw.edu
M. Alejandra Tortorici
5Department of Biochemistry, University of Washington, Seattle, WA 98109, USA; (M.A.T.), (D.V.), (M.M.), (D.P.), (N.P.K.)
6Institute Pasteur & CNRS UMR 3569, Unité de Virologie Structurale, Paris 75015, France
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  • For correspondence: tortoric@uw.edu dveesler@uw.edu murphymp@uw.edu ddpettie@gmail.com neil@ipd.uw.edu
David Veesler
5Department of Biochemistry, University of Washington, Seattle, WA 98109, USA; (M.A.T.), (D.V.), (M.M.), (D.P.), (N.P.K.)
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  • For correspondence: tortoric@uw.edu dveesler@uw.edu murphymp@uw.edu ddpettie@gmail.com neil@ipd.uw.edu
Michael Murphy
7Institute for Protein Design, University of Washington, Seattle, WA 98195, USA
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Deleah Pettie
7Institute for Protein Design, University of Washington, Seattle, WA 98195, USA
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Neil P. King
5Department of Biochemistry, University of Washington, Seattle, WA 98109, USA; (M.A.T.), (D.V.), (M.M.), (D.P.), (N.P.K.)
7Institute for Protein Design, University of Washington, Seattle, WA 98195, USA
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  • For correspondence: tortoric@uw.edu dveesler@uw.edu murphymp@uw.edu ddpettie@gmail.com neil@ipd.uw.edu
Alejandro B. Balazs
8The Ragon Institute of Massachusetts General Hospital, the Massachusetts Institute Technology, and Harvard University, Cambridge 02139, USA; (A.B.B.)
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  • For correspondence: abalazs@mgh.harvard.edu
Jesse D. Bloom
1Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA; (K.D.C.), (R.E.), (A.S.D.), (K.M.), (A.N.L.)
2Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA
9Howard Hughes Medical Institute, Seattle, WA 98103, USA
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  • For correspondence: jbloom@fredhutch.org kdusenbu@fredhutch.org reguia@fredhutch.org adingens@fredhutch.org kmalone2@fredhutch.org aloes@fredhutch.org
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Abstract

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 VSV particles, but the reagents and protocols are not widely available. Here we detail 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 make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can 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.

Competing Interest Statement

H.Y.C. is a consultant for Merck and Glaxo Smith Kline and receives research funding from Sanofi Pasteur. The other authors declare no conflicts of interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted April 20, 2020.
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Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays
Katharine H.D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Keara D. Malone, Caitlin R. Wolf, Helen Y. Chu, M. Alejandra Tortorici, David Veesler, Michael Murphy, Deleah Pettie, Neil P. King, Alejandro B. Balazs, Jesse D. Bloom
bioRxiv 2020.04.20.051219; doi: https://doi.org/10.1101/2020.04.20.051219
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Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays
Katharine H.D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Keara D. Malone, Caitlin R. Wolf, Helen Y. Chu, M. Alejandra Tortorici, David Veesler, Michael Murphy, Deleah Pettie, Neil P. King, Alejandro B. Balazs, Jesse D. Bloom
bioRxiv 2020.04.20.051219; doi: https://doi.org/10.1101/2020.04.20.051219

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