Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol
The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutraliz...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2021-01-01
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| Series: | Frontiers in Cardiovascular Medicine |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcvm.2020.618651/full |
| _version_ | 1819298001346101248 |
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| author | Jose Manuel Condor Capcha Jose Manuel Condor Capcha Guerline Lambert Derek M. Dykxhoorn Alessandro G. Salerno Alessandro G. Salerno Joshua M. Hare Joshua M. Hare Michael A. Whitt Savita Pahwa Dushyantha T. Jayaweera Dushyantha T. Jayaweera Lina A. Shehadeh Lina A. Shehadeh Lina A. Shehadeh |
| author_facet | Jose Manuel Condor Capcha Jose Manuel Condor Capcha Guerline Lambert Derek M. Dykxhoorn Alessandro G. Salerno Alessandro G. Salerno Joshua M. Hare Joshua M. Hare Michael A. Whitt Savita Pahwa Dushyantha T. Jayaweera Dushyantha T. Jayaweera Lina A. Shehadeh Lina A. Shehadeh Lina A. Shehadeh |
| author_sort | Jose Manuel Condor Capcha |
| collection | DOAJ |
| description | The COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. This is particularly important for patients who are at high risk for severe outcomes related to COVID-19. The production of pseudotyped viral particles may seem like a daunting task for a non-virology laboratory without experience in the two most commonly used pseudotyping systems, namely retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVΔG). By incorporating the most up-to-date knowledge, we have developed a detailed, easy-to-follow novel protocol for producing SARS-CoV-2 spike-bearing pseudovirus using the VSV-ΔG system. We describe the infection assay which uses GFP fluorescence as a measure of infection in a 24-well live imaging system. We present results of our optimization of the system to enhance viral infection levels through the over-expression of human ACE2 receptor and the overexpression of at least one of two proteases - TMPRSS2 or Furin, as well as, supplementation with Poloxamer 407 (P407) and Prostaglandin E2 (PGE2) as adjuvants. We show that the system works efficiently in three unrelated, clinically relevant cell lines: human 293T (renal epithelial) cells, human Calu-3 (lung epithelial) cells, and the non-human primate (African Green Monkey) cell line, Vero-E6 (renal epithelial) cells. In addition, we have used this system to show infection of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). This system is efficient (virus generation, titration, and infection assays can be performed in 1 week), quantitative, inexpensive, and readily scalable for application in drug development and therapeutic screening approaches. |
| first_indexed | 2024-12-24T05:22:57Z |
| format | Article |
| id | doaj.art-1f2f57c7537c4768944f2e70a6238ceb |
| institution | Directory Open Access Journal |
| issn | 2297-055X |
| language | English |
| last_indexed | 2024-12-24T05:22:57Z |
| publishDate | 2021-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cardiovascular Medicine |
| spelling | doaj.art-1f2f57c7537c4768944f2e70a6238ceb2022-12-21T17:13:24ZengFrontiers Media S.A.Frontiers in Cardiovascular Medicine2297-055X2021-01-01710.3389/fcvm.2020.618651618651Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week ProtocolJose Manuel Condor Capcha0Jose Manuel Condor Capcha1Guerline Lambert2Derek M. Dykxhoorn3Alessandro G. Salerno4Alessandro G. Salerno5Joshua M. Hare6Joshua M. Hare7Michael A. Whitt8Savita Pahwa9Dushyantha T. Jayaweera10Dushyantha T. Jayaweera11Lina A. Shehadeh12Lina A. Shehadeh13Lina A. Shehadeh14Division of Cardiology, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesInterdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesDivision of Cardiology, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesDr. John T. Macdonald Foundation Department of Human Genetics, John P. Hussman Institute for Human Genomics, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesDivision of Cardiology, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesInterdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesDivision of Cardiology, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesInterdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesDepartment of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN, United StatesDepartment of Microbiology and Immunology, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesInterdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesDivision of Infectious Disease, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesDivision of Cardiology, Department of Medicine, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesInterdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesPeggy and Harold Katz Family Drug Discovery Center, University of Miami Leonard M. Miller School of Medicine, Miami, FL, United StatesThe COVID-19 pandemic caused by the SARS-CoV-2 coronavirus requires reliable assays for studying viral entry mechanisms which remains poorly understood. This knowledge is important for the development of therapeutic approaches to control SARS-CoV-2 infection by permitting the screening for neutralizing antibodies and other agents that can block infection. This is particularly important for patients who are at high risk for severe outcomes related to COVID-19. The production of pseudotyped viral particles may seem like a daunting task for a non-virology laboratory without experience in the two most commonly used pseudotyping systems, namely retro/lentiviruses and vesicular stomatitis virus (VSV) which lacks the VSV envelope glycoprotein (VSVΔG). By incorporating the most up-to-date knowledge, we have developed a detailed, easy-to-follow novel protocol for producing SARS-CoV-2 spike-bearing pseudovirus using the VSV-ΔG system. We describe the infection assay which uses GFP fluorescence as a measure of infection in a 24-well live imaging system. We present results of our optimization of the system to enhance viral infection levels through the over-expression of human ACE2 receptor and the overexpression of at least one of two proteases - TMPRSS2 or Furin, as well as, supplementation with Poloxamer 407 (P407) and Prostaglandin E2 (PGE2) as adjuvants. We show that the system works efficiently in three unrelated, clinically relevant cell lines: human 293T (renal epithelial) cells, human Calu-3 (lung epithelial) cells, and the non-human primate (African Green Monkey) cell line, Vero-E6 (renal epithelial) cells. In addition, we have used this system to show infection of human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). This system is efficient (virus generation, titration, and infection assays can be performed in 1 week), quantitative, inexpensive, and readily scalable for application in drug development and therapeutic screening approaches.https://www.frontiersin.org/articles/10.3389/fcvm.2020.618651/fullSARS-CoV-2pseudoviruslentivirusACE2TMPRSS2furin |
| spellingShingle | Jose Manuel Condor Capcha Jose Manuel Condor Capcha Guerline Lambert Derek M. Dykxhoorn Alessandro G. Salerno Alessandro G. Salerno Joshua M. Hare Joshua M. Hare Michael A. Whitt Savita Pahwa Dushyantha T. Jayaweera Dushyantha T. Jayaweera Lina A. Shehadeh Lina A. Shehadeh Lina A. Shehadeh Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol Frontiers in Cardiovascular Medicine SARS-CoV-2 pseudovirus lentivirus ACE2 TMPRSS2 furin |
| title | Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol |
| title_full | Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol |
| title_fullStr | Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol |
| title_full_unstemmed | Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol |
| title_short | Generation of SARS-CoV-2 Spike Pseudotyped Virus for Viral Entry and Neutralization Assays: A 1-Week Protocol |
| title_sort | generation of sars cov 2 spike pseudotyped virus for viral entry and neutralization assays a 1 week protocol |
| topic | SARS-CoV-2 pseudovirus lentivirus ACE2 TMPRSS2 furin |
| url | https://www.frontiersin.org/articles/10.3389/fcvm.2020.618651/full |
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