Optimised Method for the Production and Titration of Lentiviral Vectors Pseudotyped with the SARS-CoV-2 Spike

Mekkaoui, Leila; Bentley, Emma M; Ferrari, Mathieu; Lamb, Katarina; Ward, Katarzyna; Karattil, Rajeev; Akbar, Zulaikha; Bughda, Reyisa; Sillibourne, James; Onuoha, Shimobi; Mattiuzzo, Giada; Takeuchi, Yasuhiro; Pule, Martin

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.