Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays
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 simpl...
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| Format: | Article |
| Language: | English |
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MDPI AG
2020-05-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/12/5/513 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Katharine H. D. Crawford |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-10T20:00:25Z |
| format | Article |
| id | doaj.art-88a2dcbba5204bbe892d526add0ceb42 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-10T20:00:25Z |
| publishDate | 2020-05-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-88a2dcbba5204bbe892d526add0ceb422023-11-19T23:39:03ZengMDPI AGViruses1999-49152020-05-0112551310.3390/v12050513Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization AssaysKatharine H. D. Crawford0Rachel Eguia1Adam S. Dingens2Andrea N. Loes3Keara D. Malone4Caitlin R. Wolf5Helen Y. Chu6M. Alejandra Tortorici7David Veesler8Michael Murphy9Deleah Pettie10Neil P. King11Alejandro B. Balazs12Jesse D. Bloom13Division of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USADivision of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USADivision of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USADivision of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USADivision of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USADivision of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195, USADivision of Allergy and Infectious Diseases, University of Washington, Seattle, WA 98195, USADepartment of Biochemistry, University of Washington, Seattle, WA 98109, USADepartment of Biochemistry, University of Washington, Seattle, WA 98109, USAInstitute for Protein Design, University of Washington, Seattle, WA 98195, USAInstitute for Protein Design, University of Washington, Seattle, WA 98195, USADepartment of Biochemistry, University of Washington, Seattle, WA 98109, USAThe Ragon Institute of Massachusetts General Hospital, the Massachusetts Institute Technology, and Harvard University, Cambridge, MA 02139, USADivision of Basic Sciences and Computational Biology Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USASARS-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.https://www.mdpi.com/1999-4915/12/5/513SARS-CoV-2COVID-19coronavirusneutralization assaylentiviral pseudotypeSpike |
| spellingShingle | 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 Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays Viruses SARS-CoV-2 COVID-19 coronavirus neutralization assay lentiviral pseudotype Spike |
| title | Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays |
| title_full | Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays |
| title_fullStr | Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays |
| title_full_unstemmed | Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays |
| title_short | Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays |
| title_sort | protocol and reagents for pseudotyping lentiviral particles with sars cov 2 spike protein for neutralization assays |
| topic | SARS-CoV-2 COVID-19 coronavirus neutralization assay lentiviral pseudotype Spike |
| url | https://www.mdpi.com/1999-4915/12/5/513 |
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