Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

The present protocol has been approved by and follows the guidelines of the Ethical Committee of the University of Verona (approval protocol number 1538). Informed written consent was obtained from the human subjects participating in the study. Whole blood samples were collected from healthcare worker (HCW) volunteers who were in the process of receiving anti-SARS-CoV-2 vaccines. These samples were collected in plastic tubes containing anticoagulants for the subsequent isolation of serum¹⁵.

All the following processes must be performed in a Class-2 biological hood, working under sterile conditions. Virus handling must be performed with care, and all waste products must be neutralized in a diluted bleach solution. An overview of the protocol is displayed in Figure 1.

Figure 1: Graphical representation of a neutralization assay. (A) PV production, (B) PV titration, and (C) neutralization assay. All the procedures are performed in a class-2 biological hood under sterile conditions. Titration step (B) needs to be performed to standardize the infectivity levels of PVs before use in the neutralization assay (C). This figure was created with BioRender. Please click here to view a larger version of this figure.

1. SARS-CoV-2 PVs production and infectivity test

1. Seed 5 x 10⁵ HEK293T cells in complete Dulbecco's Modified Eagle Medium (DMEM, high-glucose, 10% foetal bovine serum (FBS), 1% L-glutamine, 1% penicillin/streptomycin) in a 6-well plate (6WP) to reach a suitable cell density compatible with the transfection reagent used. In the case of performing transfection with polyehtylenimine (PEI) (prepare the reagent following the manufacturer instructions), ensure that the cells reach 40-60% density on the day of transfection (step 1.3). Keep the cells in a humidified incubator at 37 °C and 5% CO₂.
2. Prior to transfection, replace the spent cell medium with fresh medium without antibiotics (DMEM, high-glucose, 10% FBS, 1% L-glutamine) to achieve higher transfection efficiency.
   NOTE: The day after seeding, HEK293T cells are ready to be transfected.
3. Transfect adherent HEK293T cells with a suitable transfection reagent according to the manufacturer's instructions. If using PEI, prepare two mixes and follow the steps below.
   1. To prepare mix A, add 500 ng of pCMV-dR8.91 packaging plasmid¹⁶, 750 ng of pCSFLW reporter plasmid¹⁶, and 450 ng of SARS-CoV-2 Spike expressing plasmid in 100 µL of reduced serum medium.
   2. To prepare mix B, add 17.5 µL of PEI (concentration: 1 mg/mL) to 100 µL of the reduced serum medium.
   3. Allow both mixes to incubate at room temperature (RT) for 5 min. Next, mix the contents of both tubes together by adding the PEI mix B to DNA mix A.
   4. Incubate the tube for 20-30 min at RT. Flick the tube gently every 3-4 min to enhance the mixing. Finally, add the mixture to the HEK293T cells.
4. 16-20 h after the transfection, replace the culture medium with fresh, complete DMEM. Incubate at 37 °C and 5% CO₂, to allow for the production of PVs by transfected cells.
5. 72 h after the transfection, harvest the supernatant containing PVs. Then centrifuge at 1600 x g for 7 min at room temperature to remove cell debris and dead cells and filter it through a 0.45 µm cellulose acetate filter.
6. OPTIONAL STEP: To increase the final yield of PV titer, perform multiple transfections, pool the cell media containing PVs, and concentrate it using concentrating tubes.
7. Proceed directly with the next steps ("PVs titration", section 2) or aliquot the PV-containing medium in suitable tubes to store at -80 °C until use. Prepare an additional aliquot (400-500 µL) to be used for titration.
   NOTE: Making multiple aliquots will guarantee reproducibility between experiments by avoiding excessive thaw-freeze cycles.

2. PVs titration

1. Use the fresh PV-containing medium for the next steps or thaw the testing aliquot (step 1.7) to perform the titration of the new viral stock. Freezing aliquots of the same PV stock will guarantee reproducibility.
2. Add 50 µL of complete DMEM (or complete medium compatible with the target cell line in usage) in all the wells of a 96 well-plate (96WP) necessary to test in duplicate the PV stock, leaving row "A" empty. Add 100 µL of PVs stock to row "A". Based on the number of preparations to be tested, leave one column without the virus as a "cell only" control (Figure 2).
3. Pipette 50 µL from row A to row B and repeat this process up to row G to obtain serial dilutions of the initial stock. Discard the excess volume from the last row.
4. Detach cellsusing trypsin/ethylenediaminetetraacetic acid 1x (EDTA) in Dulbecco's phosphate buffer saline 1x (DPBS 1x), after removing the spent medium and washing cells with DPBS 1x twice. Prepare cells to a density of 4 x 10⁵ cells/mL.
   NOTE: In this protocol, PVs infection was tested on the susceptible cell line HEK293T/ACE2; such cells were derived from HEK293T, transduced using a lentiviral vector to express ACE2 receptor.
5. Add 50 µL of the cell suspension into each well to ensure a cell count of 2 x 10⁴ cells per well.
6. Incubate at 37 °C and 5% CO₂, for 48 h.
7. After the incubation, perform the Luciferase assay to obtain the reading as per the manufacturer's instructions. Add 100 µL of the luciferase reagent to the wells and incubate in the dark at RT for 2 min. Move the content of each well to a black 96 well plate (compatible with the available plate reader) and read the plates in a 96 well plate reader.
   NOTE: The luminometer used for the luciferase readout will produce a spreadsheet file with the raw, unprocessed data that will be used for downstream analysis (in this case, an Excel file). The virus' infectivity will be expressed as relative luminescence units (RLU) (described in paragraph 4.1).

Figure 2: Representative layout of a 96 well plate for PVs titration. A fixed volume of PV-containing supernatant is added to row A, columns 1-11, and serially diluted. The last column is left as the "cell only" control. This figure was created with BioRender. Please click here to view a larger version of this figure.

3. Neutralization assay

1. Thaw patients' sera on ice. Inactivate serum samples by incubating them at 56 °C for 30 min.
2. In a 96 well plate, add 50 µL of the fresh, complete DMEM (or complete medium compatible with the target cell line used)in each of the following wells: from row B (columns 1-10) to row H (columns 1-10). Put 95 µL of the fresh, complete DMEM in row A (columns 1-10). Add 50 µL and 100 µL of complete DMEM into the wells of columns 11 and 12, respectively. These will be the infected (virus control, or VC) and uninfected (cell only, or CC) controls, respectively (Figure 3).
3. Add 5 µL of heat-inactivated serum/plasma samples in row A (columns 1-10). Each sample will be in duplicate. With a multichannel pipette, mix the samples in the first row and move 50 µL of medium containing serum from row A to row B. Repeat this process up to the last row (Figure 3). Discard the remaining 50 µL.
4. Thaw the necessary number of PVs' aliquots and dilute to ≥ 10⁴ RLU/mL. Add 50 µL of the diluted PV-containing medium to each well (from column 1 to column 11) using a multichannel pipette to reach a 1:1 dilution of heat inactivated serum/plasma to virus. Incubate at 37 °C and 5% CO₂, for 1 h to allow the antibodies in the serum samples to bind to the SARS-CoV-2 spike protein on the PVs.
5. Prepare at least 5 mL suspension of susceptible cells (HEK293T/ACE2) at a cell density of 4 x 10⁵ cells/mL. Add 50 µL of the cell suspension to each well and incubate at 37 °C and 5% CO₂, for 48 h.
6. After the incubation, perform the luciferase assay reading according to the manufacturer's instructions, as described in step 2.7.
   NOTE: The luminometer used for luciferase readout will produce a spreadsheet file (in this case, .xlsx) with the raw, unprocessed data that will be used for downstream analysis (the Luciferase assay file).

Figure 3: Plate representation based on serum dilution. Bright red corresponds to a higher quantity of serum, and bright blue lane (column 11) corresponds to infected cell control (VC, virus control). Light blue lane (column 12) corresponds to uninfected cells (CC, cell control). This figure was created with BioRender. Please click here to view a larger version of this figure.

4. Titration analysis

1. On the Luciferase assay file, assign the names/titles to the corresponding samples.
2. Multiply the RLU measure by the dilution factors (from the top to the bottom of the grid: 20x, 40x, 80x, 160x, 320x, 640x, 1,280x, 2,560x) to obtain RLU/mL. If different dilution factors are used, change the multiplication factors accordingly.
3. Calculate the average RLU/mL for each PV preparation.

5. PVs neutralization assay analysis

1. On the Luciferase assay spreadsheet file (in this case, .xlsx), assign the corresponding titles to the tested samples. Enter the dilution factor of the sample (40s, 80x, 160x, 320x, 640x, 1,280x, 2,560x, 5,120x). Calculate the Log10 of the dilution factors.
2. Calculate the average RLU of uninfected and infected control (Figure 3, columns 11 and 12, respectively). These values will be useful for the normalization in step 5.5.
3. Open a new document for data analysis. Select X/Y analysis, input X as Numbers and Y as Enter 2 replicate values in side-by-side sub-columns.
4. Enter Log10 (dilution) values as X numbers. Enter the duplicate RLU of the samples.
5. Go to Analyze > Normalize > Flag all the samples on the same sheet. Input the average VC and CC values in How is 0% defined?, and How is 100% defined?, respectively. Click OK.
6. On the normalized data sheet, go to Analyze > XY analyses > Nonlinear analyses (curve fit). Flag all the samples and click OK. For the Dose-response – Inhibition, select log(inhibitor) vs normalized response – variable slope.
7. Under Constrain, change HillSlope a Must be less than 0.
8. Under Output, flag Create summary table and graph. Click on OK to obtain the final analyses. A working sheet with a template for the analysis is provided in Supplementary File 1.