Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications

ABSTRACT Clinical trials of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics often include virological secondary endpoints to compare viral clearance and viral load reduction between treatment and placebo arms. This is typically achieved using quantitative reverse-transcript...

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Main Authors: Dominic Wooding, Kate Buist, Alessandra Romero-Ramirez, Helen Savage, Rachel Watkins, Daisy Bengey, Caitlin Greenland-Bews, Caitlin R. Thompson, Nadia Kontogianni, Richard Body, Gail Hayward, Rachel L. Byrne, Susan Gould, Christopher Myerscough, Barry Atkinson, Victoria Shaw, Bill Greenhalf, Emily Adams, Ana Cubas-Atienzar, Saye Khoo, Tom Fletcher, Thomas Edwards
Format: Article
Language:English
Published: American Society for Microbiology 2024-11-01
Series:mSphere
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Online Access:https://journals.asm.org/doi/10.1128/msphere.00304-24
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author Dominic Wooding
Kate Buist
Alessandra Romero-Ramirez
Helen Savage
Rachel Watkins
Daisy Bengey
Caitlin Greenland-Bews
Caitlin R. Thompson
Nadia Kontogianni
Richard Body
Gail Hayward
Rachel L. Byrne
Susan Gould
Christopher Myerscough
Barry Atkinson
Victoria Shaw
Bill Greenhalf
Emily Adams
Ana Cubas-Atienzar
Saye Khoo
Tom Fletcher
Thomas Edwards
author_facet Dominic Wooding
Kate Buist
Alessandra Romero-Ramirez
Helen Savage
Rachel Watkins
Daisy Bengey
Caitlin Greenland-Bews
Caitlin R. Thompson
Nadia Kontogianni
Richard Body
Gail Hayward
Rachel L. Byrne
Susan Gould
Christopher Myerscough
Barry Atkinson
Victoria Shaw
Bill Greenhalf
Emily Adams
Ana Cubas-Atienzar
Saye Khoo
Tom Fletcher
Thomas Edwards
author_sort Dominic Wooding
collection DOAJ
description ABSTRACT Clinical trials of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics often include virological secondary endpoints to compare viral clearance and viral load reduction between treatment and placebo arms. This is typically achieved using quantitative reverse-transcriptase PCR (RT-qPCR), which cannot differentiate replicant competent virus from non-viable virus or free RNA, limiting its utility as an endpoint. Culture-based methods for SARS-CoV-2 exist; however, these are often insensitive and poorly standardized for use as clinical trial endpoints. We report optimization of a culture-based approach evaluating three cell lines, three detection methods, and key culture parameters. We show that Vero-angiotensin-converting enzyme 2-transmembrane serine protease 2 cells in combination with RT-qPCR of culture supernatants from the first passage provides the greatest overall detection of Delta viral replication (22 of 32, 68.8%), being able to identify viable virus in 83.3% (20 of 24) of clinical samples with initial Ct values of <30. Likewise, we demonstrate that RT-qPCR using culture supernatants from the first passage of Vero human signaling lymphocytic activation molecule cells provides the highest overall detection of Omicron viral replication (9 of 31, 29%), detecting live virus in 39.1% (9 of 23) of clinical samples with initial Ct values of <25. This assessment demonstrates that combining RT-qPCR with virological endpoint analysis has utility in clinical trials of therapeutics for SARS-CoV-2; however, techniques may require optimization based on dominant circulating strain.IMPORTANCERT-qPCR is commonly used for virological endpoints during clinical trials for antiviral therapy to determine the quantity and presence of virus in a sample. However, RT-qPCR identifies viral RNA and cannot determine if viable virus is present. Existing culture-based techniques for SARS-CoV-2 are insensitive and not sufficiently standardized to be employed as clinical study endpoints. The use of a culture system to monitor replicating viruses could mitigate the possibility of molecular techniques identifying viral RNA from inactive or lysed viral particles. The methodology optimized in this study for detecting infectious viruses may have application as a secondary virological endpoint in clinical trials of therapeutics for SARS-CoV-2 in addition to numerous research processes.
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spelling doaj-art-b9fee6f82ea04a7885599c6002c3fe272024-11-21T14:00:48ZengAmerican Society for MicrobiologymSphere2379-50422024-11-0191110.1128/msphere.00304-24Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applicationsDominic Wooding0Kate Buist1Alessandra Romero-Ramirez2Helen Savage3Rachel Watkins4Daisy Bengey5Caitlin Greenland-Bews6Caitlin R. Thompson7Nadia Kontogianni8Richard Body9Gail Hayward10Rachel L. Byrne11Susan Gould12Christopher Myerscough13Barry Atkinson14Victoria Shaw15Bill Greenhalf16Emily Adams17Ana Cubas-Atienzar18Saye Khoo19Tom Fletcher20Thomas Edwards21Centre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomDepartment of Clinical Sciences, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomManchester University NHS Foundation Trust, Research and Innovation, Manchester, United KingdomNuffield Department of Primary Care Health Sciences, University of Oxford, Oxford, United KingdomDepartment of Clinical Sciences, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomResearch and Evaluation, UK Health Security Agency, Porton Down, Salisbury, United KingdomUniversity of Liverpool, Liverpool, United KingdomUniversity of Liverpool, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomUniversity of Liverpool, Liverpool, United KingdomDepartment of Clinical Sciences, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomCentre for Drugs and Diagnostics, Liverpool School of Tropical Medicine and Hygiene, Liverpool, United KingdomABSTRACT Clinical trials of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics often include virological secondary endpoints to compare viral clearance and viral load reduction between treatment and placebo arms. This is typically achieved using quantitative reverse-transcriptase PCR (RT-qPCR), which cannot differentiate replicant competent virus from non-viable virus or free RNA, limiting its utility as an endpoint. Culture-based methods for SARS-CoV-2 exist; however, these are often insensitive and poorly standardized for use as clinical trial endpoints. We report optimization of a culture-based approach evaluating three cell lines, three detection methods, and key culture parameters. We show that Vero-angiotensin-converting enzyme 2-transmembrane serine protease 2 cells in combination with RT-qPCR of culture supernatants from the first passage provides the greatest overall detection of Delta viral replication (22 of 32, 68.8%), being able to identify viable virus in 83.3% (20 of 24) of clinical samples with initial Ct values of <30. Likewise, we demonstrate that RT-qPCR using culture supernatants from the first passage of Vero human signaling lymphocytic activation molecule cells provides the highest overall detection of Omicron viral replication (9 of 31, 29%), detecting live virus in 39.1% (9 of 23) of clinical samples with initial Ct values of <25. This assessment demonstrates that combining RT-qPCR with virological endpoint analysis has utility in clinical trials of therapeutics for SARS-CoV-2; however, techniques may require optimization based on dominant circulating strain.IMPORTANCERT-qPCR is commonly used for virological endpoints during clinical trials for antiviral therapy to determine the quantity and presence of virus in a sample. However, RT-qPCR identifies viral RNA and cannot determine if viable virus is present. Existing culture-based techniques for SARS-CoV-2 are insensitive and not sufficiently standardized to be employed as clinical study endpoints. The use of a culture system to monitor replicating viruses could mitigate the possibility of molecular techniques identifying viral RNA from inactive or lysed viral particles. The methodology optimized in this study for detecting infectious viruses may have application as a secondary virological endpoint in clinical trials of therapeutics for SARS-CoV-2 in addition to numerous research processes.https://journals.asm.org/doi/10.1128/msphere.00304-24molecular diagnosticsclinical trialsCOVID-19
spellingShingle Dominic Wooding
Kate Buist
Alessandra Romero-Ramirez
Helen Savage
Rachel Watkins
Daisy Bengey
Caitlin Greenland-Bews
Caitlin R. Thompson
Nadia Kontogianni
Richard Body
Gail Hayward
Rachel L. Byrne
Susan Gould
Christopher Myerscough
Barry Atkinson
Victoria Shaw
Bill Greenhalf
Emily Adams
Ana Cubas-Atienzar
Saye Khoo
Tom Fletcher
Thomas Edwards
Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications
mSphere
molecular diagnostics
clinical trials
COVID-19
title Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications
title_full Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications
title_fullStr Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications
title_full_unstemmed Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications
title_short Optimization of SARS-CoV-2 culture from clinical samples for clinical trial applications
title_sort optimization of sars cov 2 culture from clinical samples for clinical trial applications
topic molecular diagnostics
clinical trials
COVID-19
url https://journals.asm.org/doi/10.1128/msphere.00304-24
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