Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes

Cecilia Di Genova; Alex Sampson; Simon Scott; Diego Cantoni; Martin Mayora-Neto; Emma Bentley; Giada Mattiuzzo; Edward Wright; Mariliza Derveni; Bethany Auld; Bill T Ferrara; Dale Harrison; Mohamed Said; Arwa Selim; Elinor Thompson; Craig Thompson; George Carnell; Nigel Temperton

doi:10.21769/BioProtoc.4236

Production, Titration, Neutralisation, Storage and Lyophilisation of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lentiviral Pseudotypes

Bio Protoc. 2021 Nov 5;11(21):e4236. doi: 10.21769/BioProtoc.4236.

Authors

Cecilia Di Genova¹, Alex Sampson², Simon Scott¹, Diego Cantoni¹, Martin Mayora-Neto¹, Emma Bentley³, Giada Mattiuzzo³, Edward Wright⁴, Mariliza Derveni⁴, Bethany Auld⁴, Bill T Ferrara⁵, Dale Harrison⁵, Mohamed Said⁵, Arwa Selim⁵, Elinor Thompson⁵, Craig Thompson⁶, George Carnell², Nigel Temperton¹

Affiliations

¹ Viral Pseudotype Unit (VPU Kent), Medway School of Pharmacy, University of Kent and Greenwich at Medway, Chatham Maritime, Kent, UK.
² Laboratory of Viral Zoonotics, Department of Veterinary Medicine, Cambridge University, Madingley Road, Cambridge, UK.
³ Division of Virology, National Institute for Biological Standards and Control (NIBSC), Potters Bar, Hertfordshire, UK.
⁴ Viral Pseudotype Unit (VPU Sussex), School of Life Sciences, University of Sussex, Brighton, UK.
⁵ School of Science, University of Greenwich, Chatham Maritime, Kent, UK.
⁶ Department of Zoology, University of Oxford, Oxford, UK.

Abstract

This protocol details a rapid and reliable method for the production and titration of high-titre viral pseudotype particles with the SARS-CoV-2 spike protein (and D614G or other variants of concern, VOC) on a lentiviral vector core, and use for neutralisation assays in target cells expressing angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2). It additionally provides detailed instructions on substituting in new spike variants via gene cloning, lyophilisation and storage/shipping considerations for wide deployment potential. Results obtained with this protocol show that SARS-CoV-2 pseudotypes can be produced at equivalent titres to SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) pseudotypes, neutralised by human convalescent plasma and monoclonal antibodies, and stored at a range of laboratory temperatures and lyophilised for distribution and subsequent application.

Keywords: COVID-19; Lentiviral pseudotype; Pseudotype lyophilisation; SARS-CoV-2 coronavirus; Spike variants; Virus neutralisation.

Abstract

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