Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2
Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an H...
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| Veröffentlicht in: | PloS one 2021-03, Vol.16 (3), p.e0248348-e0248348 |
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| creator | Neerukonda, Sabari Nath Vassell, Russell Herrup, Rachel Liu, Shufeng Wang, Tony Takeda, Kazuyo Yang, Ye Lin, Tsai-Lien Wang, Wei Weiss, Carol D |
| description | Pseudoviruses are useful surrogates for highly pathogenic viruses because of their safety, genetic stability, and scalability for screening assays. Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays. |
| doi_str_mv | 10.1371/journal.pone.0248348 |
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Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0248348</identifier><identifier>PMID: 33690649</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>ACE inhibitors ; ACE2 ; Acquired immune deficiency syndrome ; AIDS ; Angiotensin-converting enzyme 2 ; Angiotensin-Converting Enzyme 2 - genetics ; Angiotensin-Converting Enzyme 2 - immunology ; Antibodies, Neutralizing - immunology ; Antibodies, Viral - immunology ; Biology and life sciences ; Coronaviruses ; COVID-19 ; COVID-19 - metabolism ; Diagnosis ; Drug Evaluation, Preclinical - methods ; Editing ; Enzymatic activity ; Epidemics ; Epidemiology ; Evaluation ; Fireflies ; Fluorescence ; Gag protein ; Genetic aspects ; Glycoproteins ; HEK293 Cells ; HIV ; Human immunodeficiency virus ; Humans ; Lentivirus - metabolism ; Leukemia ; Medicine and health sciences ; Methodology ; Neon ; Neutralization ; Neutralization Tests - methods ; Plasmids ; Prevention ; Proteases ; Regulatory agencies ; Research and analysis methods ; Research facilities ; SARS-CoV-2 - metabolism ; SARS-CoV-2 - pathogenicity ; Severe acute respiratory syndrome ; Severe acute respiratory syndrome coronavirus 2 ; Spike Glycoprotein, Coronavirus - genetics ; Stomatitis ; Testing ; United States ; Vaccines ; Viruses</subject><ispartof>PloS one, 2021-03, Vol.16 (3), p.e0248348-e0248348</ispartof><rights>COPYRIGHT 2021 Public Library of Science</rights><rights>This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication: https://creativecommons.org/publicdomain/zero/1.0/ (the “License”). 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Many different pseudovirus platforms exist, each with different advantages and limitations. Here we report our efforts to optimize and characterize an HIV-based lentiviral pseudovirus assay for screening neutralizing antibodies for SARS-CoV-2 using a stable 293T cell line expressing human angiotensin converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). We assessed different target cells, established conditions that generate readouts over at least a two-log range, and confirmed consistent neutralization titers over a range of pseudovirus input. Using reference sera and plasma panels, we evaluated assay precision and showed that our neutralization titers correlate well with results reported in other assays. Overall, our lentiviral assay is relatively simple, scalable, and suitable for a variety of SARS-CoV-2 entry and neutralization screening assays.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>33690649</pmid><doi>10.1371/journal.pone.0248348</doi><tpages>e0248348</tpages><orcidid>https://orcid.org/0000-0002-9965-1289</orcidid><oa>free_for_read</oa></addata></record> |
| fulltext | fulltext |
| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2021-03, Vol.16 (3), p.e0248348-e0248348 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_2499869242 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Public Library of Science (PLoS); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | ACE inhibitors ACE2 Acquired immune deficiency syndrome AIDS Angiotensin-converting enzyme 2 Angiotensin-Converting Enzyme 2 - genetics Angiotensin-Converting Enzyme 2 - immunology Antibodies, Neutralizing - immunology Antibodies, Viral - immunology Biology and life sciences Coronaviruses COVID-19 COVID-19 - metabolism Diagnosis Drug Evaluation, Preclinical - methods Editing Enzymatic activity Epidemics Epidemiology Evaluation Fireflies Fluorescence Gag protein Genetic aspects Glycoproteins HEK293 Cells HIV Human immunodeficiency virus Humans Lentivirus - metabolism Leukemia Medicine and health sciences Methodology Neon Neutralization Neutralization Tests - methods Plasmids Prevention Proteases Regulatory agencies Research and analysis methods Research facilities SARS-CoV-2 - metabolism SARS-CoV-2 - pathogenicity Severe acute respiratory syndrome Severe acute respiratory syndrome coronavirus 2 Spike Glycoprotein, Coronavirus - genetics Stomatitis Testing United States Vaccines Viruses |
| title | Establishment of a well-characterized SARS-CoV-2 lentiviral pseudovirus neutralization assay using 293T cells with stable expression of ACE2 and TMPRSS2 |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-01-15T04%3A58%3A36IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Establishment%20of%20a%20well-characterized%20SARS-CoV-2%20lentiviral%20pseudovirus%20neutralization%20assay%20using%20293T%20cells%20with%20stable%20expression%20of%20ACE2%20and%20TMPRSS2&rft.jtitle=PloS%20one&rft.au=Neerukonda,%20Sabari%20Nath&rft.date=2021-03-10&rft.volume=16&rft.issue=3&rft.spage=e0248348&rft.epage=e0248348&rft.pages=e0248348-e0248348&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0248348&rft_dat=%3Cgale_plos_%3EA654518929%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=2499869242&rft_id=info:pmid/33690649&rft_galeid=A654518929&rft_doaj_id=oai_doaj_org_article_0523c5026ddf407ea47ec70af1314726&rfr_iscdi=true |