Background

Studies on SARS-CoV-2 viruses are hampered by the difficulty to produce and manipulate the live viruses that require biosafety level 3 (BSL-3) labs. An alternative to using live virus is to use recombinant lentivirus pseudotyped with the SARS-CoV-2 Spike protein. Pseudotyped viral vectors are very powerful tools for studying biological processes related to enveloped viruses, such as viral entry and immunological response. Pseudotyped viral particles consist of the envelope glycoprotein of one virus with a replication-deficient core of another virus. This allows the deficient core to be dependent on the pseudotyping envelope for target cell entry, thus allowing the investigation of SARS-CoV-2 infection and related serological responses.

Spike is a type-I fusion transmembrane protein expressed on the surface of viral particles as a crown-shaped trimer of heterodimers. In host cells, precursor glycoproteins are proteolytically cleaved by furin at the multibasic S1/S2 site, resulting in dimers composed of an extracellular subunit (S1) containing the receptor-binding domain, which is non-covalently attached to a transmembrane subunit (S2) responsible for viral fusion and subsequent cell entry (Hoffmann et al., 2020a; Peacock et al., 2020). Spike is both necessary and sufficient to induce membrane fusion and cell entry by first binding to its human receptor, ACE2 (hACE2), followed by its proteolytic cleavage by target cell proteases such as the transmembrane protease serine 2 (TMPRSS2) (Hoffmann et al., 2020b; Walls et al., 2020). Thus, Spike has been shown to be the primary target for neutralizing antibodies in COVID-19 convalescent patient sera (Chen et al., 2020; Ju et al., 2020; Pinto et al., 2020).

Spike from different coronaviruses have been successfully pseudotyped on different non-replication competent viruses (Carnell et al., 2017; Grehan et al., 2015; Yan et al., 2007; Fukushi et al., 2005). Accordingly, different viral vectors have been pseudotyped with Spike from SARS-CoV-2, including VSV (Letko et al., 2020; Nie et al., 2020), HIV (Ou et al., 2020; Wu, 2020), and MLV (Pinto et al., 2020; Quinlan et al., 2020) based vectors for neutralization assays, which have been reported to correlate with the live strain. However, a detailed protocol of pseudovector production has not been described for widespread application.

Herein, we describe a fast and reliable protocol for the production of a self-inactivating lentiviral vector pseudotyped with SARS-CoV-2’s Spike glycoprotein and expressing enhanced green fluorescent protein (eGFP) as a marker of infection, which has recently been used to determine neutralisation efficiency of COVID-19 therapeutics on four SARS-CoV-2 variants (Ferrari et al., 2021). The protocol employs a three-plasmid transfection in HEK293T/17 cells with the following plasmids: (i) plasmid expressing the HIV-1 lentiviral genes gag, pol, rev, and tat: (ii) a self-inactivating transfer vector encoding eGFP under an internal viral promoter derived from the spleen focus-forming virus (SFFV); and (iii) a plasmid encoding codon-optimized Spike, with or without the prevalent D614G mutation (Korber et al., 2020), under the cytomegalovirus (CMV) promoter with its ER retention signal (amino acid residues 1255 to 1273) deleted as it has been shown to enhance surface expression (Ou et al., 2020). Produced vectors are then quantified by an infectivity assay on genetically engineered HEK293T/17 cells that stably express hACE2 and TMPRSS2 (Supplementary Figure 1). This protocol has been recently used to produce lentiviral vectors pseudotyped with different SARS-CoV-2's Spike variants. These cells have been deposited in the repository of the National Institute for Biological Standards and Control’s Covid-19-related research reagents (CFAR #101008) for widespread use.