Materials and Reagents

Filtered Pipette tips 1-10, 1-20, 1-200, 100-1,000 μl (Starlab, catalog numbers: S1120-3810 [P10]; S1120-1810 [P20]; S1120-8810 [P200]; S1122-1830 [P1000]) or equivalent
10 cm plates (tissue-culture treated 100 mm dish; Corning, catalog number: 430167) or equivalent
24-well plates (TC-treated 24-well plate; Corning^® Costar^®, catalog number: 3527) or equivalent
Microcentrifuge tube with screw caps write-on graduated with lid latch 1.5 ml
15 ml conical centrifuge tubes (Merck, Corning^®, catalog number: CLS430791) or equivalent
Nunc^TM EasYFlask^TM 75 cm² cell culture flasks (ThermoFisher, catalog number: 156472) or equivalent
Sterile syringes (20 ml), generic
Millex-HV syringe filter unit, 0.45 µm, PVDF, 33 mm, gamma sterilized (Merck, catalog number: SLHV033RB) or equivalent
96-well V-bottom plate (96 Well TC-treated microplates, Corning, catalog number: 3894) or equivalent
Dry ice
Ethanol absolute ≥99.8% (Avantor, VWR Chemicals, catalog number: 20821.467DP) or equivalent
HEK 293T/17 cells (ATCC, catalog number: CRL-11268).
Engineered HEK 293T/17 cells with human ACE2 and TMPRSS2 (NIBSC code CFAR#101008)
Plasmids:
1. Transfer vector: pCCL.SFFV.eGFP
  Lentiviral transfer vector encoding eGFP downstream of an SFFV promoter in a pCCL backbone.
2. Second-generation lentiviral packaging plasmid: pCMVR8.74 (Addgene, catalog number: 22036)
3. Glycoprotein expression plasmid: pCDNA-SARS-CoV-2-S^(D614G)ΔERR
  SARS-CoV-2 Spike glycoprotein downstream a CMV promoter in a pCDNA backbone. The glycoprotein contains the D614G substitution and deletion of the last 19 amino acids on its carboxyl-terminus to remove its ER retention signal (ΔERR).
Iscove’s Modified Dulbecco’s Medium (Merck, Sigma-Aldrich, catalog number: I3390) supplemented with 2mM L-glutamine and 10% fetal bovine serum (FBS) (Biosera, catalog number: FB-1001/500)
Puromycin (InvivoGen, catalog number: ant-pr-1) or equivalent
Reduced Serum media Opti-MEM^TM (Thermo Fisher Scientific, Gibco^TM catalog number: 31985047)
GeneJuice^® Transfection Reagent (Merck Millipore, catalog number: 70967)
Phosphate-buffered saline (PBS) (Sigma, catalog number: D8537) or equivalent
Cell dissociation buffer Enzyme-free PBS-based (Thermo Fisher Scientific, Gibco^TM catalog number: 13151014) to avoid surface cleavage of both hACE2 and TMPRSS2
Polybrene transfection reagent 10 mg/ml (Merk, catalog number: TR-1003-G)
Viability dye (Thermo Fisher Scientific, eBioscience^TM Fixable Viability Dye eFluor^TM 780, catalog number: 65086514) or equivalent

Equipment

Class II biosafety cabinet (Thermo Fisher Scientific, Thermo Scientific^TM, model: HeraSafe^TM 2030i)
Water bath (Avantor, VWB2 series, VWB2 2, catalog number: 462-0554) or equivalent
Incubator (Eppendorf, model: CellXpert^® C170) or equivalent
Pipettes (Eppendorf Research^® plus, P2, P20, P200, and P1000) or equivalent
Cell counter (Chemometec, Automated cell Analyzer NucleoCounter^® NC-250^TM) with Solution 18 AO.DAPI (Chemometec, catalog number: 910-3018) or equivalent
Plate centrifuge (Thermo Fisher Scientific, model: Heraeus Multifuge X3R) or equivalent
Flow cytometer equipped with a blue and red laser
This protocol utilized a BD LSRFortessa^TM X-20 cell analyser but is not restricted to this equipment. In the absence of a flow cytometer, a fluorescent microscope fitted with a 488 nm excitation laser can be used.

Software

FlowJo^TM Software, version 10.7.1 (BD Bioscience)
An alternative software for Flow Cytometry analysis can be used.

Procedure

Spike LV production by transient transfection
Day 1: Cell seeding
Seed 2.0 × 10⁶ cells HEK293T/17 cells per 10 cm plate in 10 ml of culturing media supplemented with 2 mM L-glutamine and 10% FCS aiming for 80% confluence on day of transfection.

Day 3: Transfection
1. Prepare two sterile 1.5 ml microcentrifuge tubes labeled Tube 1 and Tube 2.
2. Add 5.56 µg of pCMVR8.74, 2.77 µg of pCDNA-SARS-CoV-2-S^(D614G)ΔERR, and 4.17 µg of pCCL.SFFV.eGFP, for a total of 12.5 µg of DNA, to Tube 1 (equivalent to 2:1:1.5 plasmid ratios for scalable production).
3. Prepare the transfection mixture in Tube 2, as shown in Table 1.
  
  Table 1. Transient transfection mixture.
  Reagents μl/10 cm plate
  Gene Juice 30
  OptiMEM^® 470
4. Mix GeneJuice/OptiMEM^® transfection mixture using a pipette and incubate for 5 min at room temperature.
5. Pipette 500 µl of transfection mixture into tube containing the plasmid constructs (Tube 1) and gently mix solution five times using the same pipette, avoiding the formation bubbles.
6. Incubate solution for 15 min at room temperature.
7. Pipette the DNA/GeneJuice/OptiMEM solution onto HEK293T/17 cells in a dropwise manner throughout the total surface of the plate.
8. Place plate in an incubator at 37 °C with 5% CO₂for 16-18 h.
Day 4: Media change
1. Carefully remove the culture media from plate 16-18 h post-transfection.
2. Slowly add 7 ml of fresh culturing media supplemented with 2 mM L-glutamine and 10% FCS by pipetting media to one side of the plate to avoid detachment of cells.
3. Place plate back in the incubator at 37 °C with 5% CO₂for 16-18 h.
Day 5: Viral supernatant harvesting
1. Harvest the viral supernatant containing the lentiviral vector pseudotyped with SARS-CoV-2-S^(D614G)ΔERR into a 15 ml falcon previously chilled by either placing it in the fridge or on wet ice for 10 min.
2. Centrifuge at 400 × g for 5 min to remove cellular debris.
3. Clarify viral supernatant with 0.45 µm microfiltration to remove viral aggregates.
4. To avoid multiple freeze-thaw cycles, aliquot viral supernatants into 0.2-0.5 ml aliquots in sterile 1.5 ml microcentrifuge tubes with screw caps.
5. For later use, snap-freeze vials using dry ice and ethanol before storing them at -80 °C.
6. For immediate use, store vials on ice until needed.
Spike-LV titration by infectivity assay
Day 1:
1. Seeding permissive cells in a 24-well plate:
  1. Pre-warm IMDM containing 10% FCS, 1% GlutaMAX, and 1 µg/ml Puromycin. This will be referred to as culturing media.
  2. Harvest stable HEK293T/17-hACE2+TMPRSS2+ cells previously thawed and cultured for at least two passages (with the addition of 1 µg/ml Puromycin from the first passage after thawing) (Figure S1) as follows:
    1. Remove and discard cellular supernatant.
    2. Gently wash cells by pipetting 5 ml of PBS to remove any excess culturing media.
    3. Add 3 ml of Cell dissociation buffer Enzyme-free PBS-based to the flask and incubate for 5 min.
    4. Gently tap the sides of the flask to prompt cell detachment.
    5. Add 7 ml of culturing media to flask, collect cells, and pipette into a new falcon.
    6. Centrifuge at 400 × g for 5 min.
    7. Discard supernatant and flick falcon with finger to gently dislodge cellular pellet.
    8. Resuspend the cells by adding 5 ml of culturing media.
    9. Determine cell density:
      1. Passage cells by platting 1:6-1:8 of resuspended cells in a new T75 flask with 9 ml of culturing media, with a recommended minimum seeding of 3.3 × 10⁴ cells/cm².
      2. Proceed to Step 1c for titration cell seeding.
  3. Determine cell density using an Automated cell Analyzer NucleoCounter^® NC-250^TM.
  4. Resuspend cells at 5.0 × 10⁴cells/ml in 8 ml per viral vector supernatant to be quantified by titration.
  5. Add 0.88 µl/ml of polybrene to resuspended cells; the final volume in each well will be 1.1 ml, leading to a final concentration of 8 µg/ml of polybrene.
  6. Mix the resuspended cells and plate 1 ml/well in a total of seven wells (six wells for vector infection and one well for a no-vector control).
  7. Add 1 ml of PBS to remaining unoccupied wells.
2. Preparing Spike LV dilutions for infection of permissive cells:
  1. If using frozen Spike-LV, thaw in a water bath set at 37 °C and immediately use. If using fresh Spike-LV placed on ice, mix viral vectors by pipetting a few times, avoiding the formation of bubbles.
  2. In a V-bottom 96-well plate, prepare the following 1:4 serial dilutions of Spike-LV in pre-chilled culture media for six points:
    1. Pipette 200 µl of Spike-LV into one well in the V-bottom plate (e.g., well A1).
    2. Pipette 150 µl of pre-chilled complete IMDM into five every other well in V-bottom plate (e.g., wells A3, A5, A7, A9, and A11).
      Tip: Using every other well on the plate allows moving between 96-well plates and 24-well plates using a multichannel pipette (see Figure 1).
    Figure 1. Transfer between 96-well and 24-well plates
  3. Move 50 µl from the first well (A1) containing Spike-LV into the subsequent well (A3).
  4. Gently mix with a pipette ten times, being careful to avoid the formation of bubbles.
  5. Move onto the third well (A5) and continue the serial dilution until reaching the 6^th well (A11).
  6. Using a multichannel with every other tip occupied, pipette 100 μl of vector dilutions to each well of HEK293T/17-hACE2+TMPRSS2+ cells already plated in a 24-well plate (Figure 1).
  7. Centrifuge the plate at 1,000 × g for 20 min to enhance transduction by spinoculation.
  8. Place plate back in the incubator at 37 °C with 5% CO₂ for 72 h.
Day 3: Determining transduction efficiency and titer calculation
1. Discard culture media by flicking 24-well plate into a laboratory box that can be subsequently disinfected or a laboratory sink if the correct risk assessments are in place. Blot the plate on paper to remove excess liquid.
2. Add 200 μl/well of cell dissociation buffer and incubate for 10 min.
3. Gently tap the plate to detach cells.
4. Add 400 μl of PBS/well, resuspend cells by pipetting, and harvest 200 μl into a new V-bottom 96-well plate.
  Tip: Using every other well on the plate allows moving between 96-well plates and 24-well plates using a multichannel pipette (Figure 1).
5. Centrifuge the plate at 400 × g for 5 min.
6. Discard supernatant by quickly flicking plate in an appropriate manner (see step 1) and blot on paper to remove excess liquid.
7. Add 250 μl PBS/well and centrifuge plate at 400 × g for 5 min.
8. In the meantime, prepare a master mix for Fixable Viability Dye eFluor^TM 780 (0.1 µl + 99.9 µl PBS/well).
9. Discard supernatant by quickly flicking plate in an appropriate manner (see step 1) and blot on paper to remove excess liquid.
10. Resuspend cells in 100 µl /well staining master mix.
11. Incubate at room temperature for 30 min in the dark.
12. Add 150 μl PBS/well and centrifuge plate at 400 × g for 5 min.
13. Discard PBS, blot plate, and resuspend cells in 50 μl of PBS using P200 tips.
14. Acquire plate on flow cytometer with the following bandpass filters:
  1. RL2 780/60 for Fixable Viability Dye eFluor^TM 780.
  2. BL1 530/60 for fluorescent marker of transduction, eGFP.
    Note: If the user is unfamiliar with flow cytometric analysis of cells, it is advisable that a trained flow cytometry operator assists from this step onward.

Data analysis

FlowJo analysis
The gating strategy to identify the percentage of eGFP-positive population is depicted in Figure 2A, starting with the live cell gate, followed by the forward versus side scatter gate, and the singlets identification. Singlets are then plotted as histograms to identify the percentage of eGFP-positive cells (Figure 2B).
Determine the percent of transduced cells in each well
When calculating titers, only transduction efficiencies of less than 20% of transduced cells should be considered. This method assumes one integration per cell. Using >20% risks counting cells with multiple integration sites which leads to an underestimation of the titer. Transduction efficiencies between 0.5% and 20% were used to compare vector production between a control LV incorporating a Vesicular Stomatitis virus glycoprotein (VSV-G) and LV pseudotyped with CoV-2 S^ΔERR, with or without the prevalent D614G mutation. The D614G mutation was found to increase the titer by approximately 60% (Figure 2C). Titration calculations are presented in Table 1 as an example.

Figure 2. Flow cytometric analysis of pseudotyped lentiviral vectors on stable HEK293T/17-hACE2+TMPRSS2+. (A) Gating strategy starting with live (eF780) vs. FSC-A to identify live cells, followed by SSC-A vs. FSC-A to identify HEK293T/17 cells, and by SSC-W vs. FSC-A for singlets identification. (B) Transiently produced VSV-G LV, S^ΔERRCoV-2 LV, and S^(D614G)ΔERRCoV-2 LV were serially diluted from 100 µl in a 4-fold dilution for six points and added on control HEKT293T/17 cells (blue population) and CoV-2 S permissive HEK293T/17-hACE2+TMPRSS2+ cells (red population) in the presence of 8 µg/ml of polybrene. Staggered histograms of transduced populations are presented. (C) Graph representing viral titers of the three pseudotyping glycoproteins quantified by an infectivity assay on HEK293T/17 and HEK293T/17-hACE2+TMPRSS2 cells. Data presented are ±SD of duplicate determinations.
Calculate the functional titer as infectious units per ml (IU/ml) using the following equation:

Note: For a more accurate titer, take the average of multiple dilutions.

Table 2. Calculation of lentiviral vector titers. Viral titer calculation of lentiviral vector pseudotyped with VSV-G, CoV-2 S^ΔERRLV, and CoV-2 S^(D614G)ΔERRinfected on both HEK293T/17 cells and HEK293T/17-hACE2+TMPRSS2+ cells. Transduction efficiencies higher than 20% and lower than 0.5% are highlighted in grey font and omitted from titer calculation. Average titers are presented from duplicate determinations.

Acknowledgments

The authors acknowledge Dr Nigel Temperton (Medway School of Pharmacy, The University of Kent) for the pCDNA-SARS-CoV-2-Spike plasmid expressing the full-length codon-optimised Spike glycoprotein. E. M. B was funded by the Coalition for Epidemic Preparedness Innovations (CEPI).

Competing interests

The authors declare no competing interests.

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