Optimised Method for the Production and Titration of Lentiviral Vectors Pseudotyped with the SARS-CoV-2 Spike
Leila Mekkaoui,
Emma Bentley,
Mathieu Ferrari,
Katarina Lamb,
Katarzyna Ward,
Rajeev Karattil,
Zulaikha Akbar,
Reyisa Bughda,
James Sillibourne,
Shimobi Onuoha ,
Giada Mattiuzzo,
Yasu Takeuchi,
Martin Pule
Affiliations
Leila Mekkaoui
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Emma Bentley
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QC, United Kingdom
Mathieu Ferrari
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Katarina Lamb
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Katarzyna Ward
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Rajeev Karattil
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Zulaikha Akbar
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Reyisa Bughda
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
James Sillibourne
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Shimobi Onuoha
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
Giada Mattiuzzo
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QC, United Kingdom
Yasu Takeuchi
National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, EN6 3QC, United KingdomDivision of Infection and Immunity, University College London, Cruciform Building, Gower Street, WC1E 6BT, United Kingdom
Martin Pule
Autolus Limited, The MediaWorks, 191 Wood Lane, London, W12 7FP, United Kingdom
The use of recombinant lentivirus pseudotyped with the coronavirus Spike protein of SARS-CoV-2 would circumvent the requirement of biosafety-level 3 (BSL-3) containment facilities for the handling of SARS-CoV-2 viruses. Herein, we describe a fast and reliable protocol for the transient production of lentiviruses pseudotyped with SARS-CoV-2 Spike (CoV-2 S) proteins and green fluorescent protein (GFP) reporters. The virus titer is determined by the GFP reporter (fluorescent) expression with a flow cytometer. High titers (>1.00 E+06 infectious units/ml) are produced using codon-optimized CoV-2 S, harbouring the prevalent D614G mutation and lacking its ER retention signal. Enhanced and consistent cell entry is achieved by using permissive HEK293T/17 cells that were genetically engineered to stably express the SARS-CoV-2 human receptor ACE2 along with the cell surface protease TMPRSS2 required for efficient fusion. For the widespread use of this protocol, its reagents have been made publicly available.Graphic abstract:Production and quantification of lentiviral vectors pseudotyped with the SARS-CoV-2 Spike glycoprotein
