Production of Recombinant Replication-defective Lentiviruses Bearing the SARS-CoV or SARS-CoV-2 Attachment Spike Glycoprotein and Their Application in Receptor Tropism and Neutralisation Assays

For enveloped viruses, such as SARS-CoV-2, transmission relies on the binding of viral glycoproteins to cellular receptors. Conventionally, this process is recapitulated in the lab by infection of cells with isolated live virus. However, such studies can be restricted due to the availability of high quantities of replication-competent virus, biosafety precautions and associated trained staff. Here, we present a protocol based on pseudotyping to produce recombinant replication-defective lentiviruses bearing the SARS-CoV or SARS-CoV-2 attachment Spike glycoprotein, allowing the investigation of viral entry in a lower-containment facility. Pseudoparticles are produced by cells transiently transfected with plasmids encoding retroviral RNA packaging signals and Gag-Pol proteins, for the reconstitution of lentiviral particles, and a plasmid coding for the viral attachment protein of interest. This approach allows the investigation of different aspects of viral entry, such as the identification of receptor tropism, the prediction of virus host range, and zoonotic transmission potential, as well as the characterisation of antibodies (sera or monoclonal antibodies) and pharmacological inhibitors that can block entry. Graphic abstract: SARS-CoV and SARS-CoV-2 pseudoparticle generation and applications.

6. For example, the titre of 256 for serum sample 1 and 512 for serum sample 3 indicates that IC50 was calculated at a dilution of 1:256 and 1:512, respectively. The conclusion that could be drawn from this is that serum sample 3 is able to neutralise SARS-CoV-2 S-mediated entry more efficiently than serum sample 1 and therefore has higher antibody titres ( Figure 3B).
7. For serum sample 4, this value has been plotted as 1,024, which is the upper limit of detection (ULoD) for this assay. This means that this sample was able to inhibit 50% of the luciferase signal in all wells and at the lowest dilution that was tested. This serum would have to be retitrated with a broader dilution series to determine the neutralisation titre. For serum sample 2, none of the wells in the dilution range yielded a recordable IC50. The neutralisation titre is therefore plotted as an arbitrary value below the lower limit of detection (LLoD), which in this case is 40, but would be reported as <40 as the true titre is unknown (Figure 3B). Examples of IC50 neutralisation titres using this method can be found in Figure 4F in  and in Figure 2D and 2E in Graham et al. (2020). used. For example, if the cell types used are larger (e.g., BHK-21 cells are larger than HEK293T cells) or have a high doubling rate, we recommend starting at a lower seeding density. On the other hand, if the cells are smaller, or have a slower growth rate, and are difficult to reach confluency (e.g., Calu3 cells) or indeed are suspension cells, you may want to start with a higher seeding density. In both instances, we recommend testing different seeding densities to find the optimal for any given experiment.
3. An example of optimal confluency of HEK293T cells prior to infection (60-80%) is shown in   8. Neutralisation titres do not always need to be recorded as IC50 values. Other cut-off points can be chosen dependent on the level of neutralisation expected in a given assay, and to provide a more stringent measure of neutralisation (e.g., 80% neutralisation, IC80).
9. The surface expression of different ACE2 receptors may differ, which may affect the level of Spike-ACE2 interaction leading to misinterpretation of results. Therefore, it is important to investigate and normalise the cell surface expression of the ACE2 receptors used. The mammalian ACE2 receptors described and used herein were HA-tagged at the C-terminus, which allowed detection of surface expression by flow cytometry. Additionally, protein expression was assessed by Western blotting (Conceicao et al., 2020).
2. Working solution of 1 mg/ml PEI a. Weigh the viscous liquid to get 50 mg/ml in water (e.g., 0.42 g PEI + 8.4 ml water) and transfer to a sterile 50 ml Falcon.
b. Place Falcon in a water bath set to 50°C and gently pipette up and down using a 1 ml pipette until fully dissolved.
c. Dilute to 1 mg/ml with water (e.g., take 0.5 ml of your 50 mg/ml stock and add 24.5 ml water) d. The solution in its current state will be very basic. Adjust pH to 7 using diluted hydrochloric acid.
e. Filter through a 0.22 µm filter and aliquot into 1.5 ml Eppendorf tubes.
f. Store at -20°C long-term and at 4°C for up to one month while in use.