Application of Ha-CoV-2 Pseudovirus for Rapid Quantification of SARS-CoV-2 Variants and Neutralizing Antibodies

1. Virus and viral particle assembly

1. Vectors: Purchase expression vectors for SARS-CoV-2 M, E, or N as well as the SARS-CoV-2 S (Wild-type,Wt), Delta (B.1.617.2), and Omicron (B.1.1.529) commercially.
   NOTE: Protein sequences of the expression vectors are provided in Supplementary File 1. The vendor of these expression vectors can also be found under the Table of Materials.
2. Cells and cell culture: Maintain HEK293T cells in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), 50 units/mL of penicillin, and 50 µg/mL of streptomycin.
   ​NOTE: All cell culture work needs to be done in a laminar airflow biosafety cabinet. The polyethyleneimine (PEI) transfection typically requires 5-6 h incubation. Start times need to be considered carefully beforehand.
3. Virus assembly and PEI-based cotransfection: Assemble Ha-CoV-2 particles by cotransfection of HEK293T cells. Seed cells in a 10 cm cell culture Petri dish (4-5 x 10⁶ cells per dish) in 10 mL of complete DMEM medium the day prior to cotransfection. For this study, three Petri dishes are used to seed cells for the assembly of Ha-CoV-2 wild-type, Delta, and Omicron, respectively.
4. Incubate Petri dishes overnight in a CO₂ incubator at 37 °C. Check the dishes next morning to ensure that cells are 80% confluent. Remove complete medium and replace it with 9 mL of DMEM serum-free media.
5. For each dish, prepare a cotransfection mixture with 2.5 µg of each of the SARS-CoV-2 structural protein expression vectors (N, E, M), 10 µg pAlphaPro-Luc-GFP-PreΨ (HaCoV2 Genome), and 2.5 µg of the S protein expression vector, either the Delta or the Omicron S variants and 45 µL of PEI-based transfection reagent. Allow the cotransfection mixture to form complexes by incubating for 13 min (do not incubate for longer than 30 min).
6. After incubation, add the cotransfection mixture to each Petri dish slowly, drop by drop. Place the dishes inside a CO₂ incubator at 37 °C for 6 h. After 6 h, remove serum-free DMEM and replace it with complete DMEM medium. Harvest virus at 48-60 h post-cotransfection.
7. Virus harvesting and storage: Harvest particles at 48 h post cotransfection. Detach cells by pipetting repeatedly over the surface of the monolayer and collect cells from each dish, put into 15 mL centrifuge tubes, and centrifuge at 400 x g for 5 min. Collect the supernatant and pass it through a 0.22 µM filter. Store the Ha-CoV-2 pseudovirus at -80 °C.

2. Viral infectivity assay

1. Cells and cell culture: Maintain HEK293T(ACE2/TMPRSS2) cells in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated FBS, 50 units/mL of penicillin and 50 µg/mL of streptomycin.
2. Seeding HEK293T(ACE2/TMPRSS2) cells: The day before viral infectivity assay, seed HEK293T(ACE2/TMPRSS2) cells in a 96-well plate in 50 µL of complete DMEM media. For each 96-well plate, seed 2.5 x 10⁴ cells into each well, and a total of 2.5 x 10⁶ cells are needed for one plate. Place the 96 well plate in a CO₂ incubator at 37 °C overnight.
3. Infection of HEK293T(ACE2/TMPRSS2): Use Ha-CoV-2 variants particles to infect HEK293T(ACE2/TMPRSS2) cells. On the morning of infection, remove 50 µL of DMEM from pre-seeded 96 well plate. Replace media with 50 µL of either Ha-CoV-2 Wild-type, Delta, or Omicron for 18 h at 37 °C.
   NOTE: The protocol can be stopped here until the infection has reached 18 h of incubation and the plate is ready to be analyzed via Luciferase Assay. The success of the infection is determined by the Luciferase assay resulting from the Luciferase reporter gene expressed in the infected cells, therefore the more signal is produced, the more successful the infection of the Ha-CoV-2 variant is.
4. Luciferase assay: After 18 h of incubation, add 7.5 µL of cell lysis buffer directly to each well and mix by orbital shaking for 2 min. Lyse cells in lysis buffer for at least 5 min at room temperature.
5. Prepare the Firefly luciferase assay solution by mixing the D-luciferin solution with the Firefly luciferase assay solution in a 1:50 ratio. For a whole 96-well plate, combine 3 mL of the luciferase substrate solution with 60 µL of the D-luciferin solution with 2940 µL of the Firefly luciferase buffer solution.
6. Add 25 μL of the Firefly luciferase assay solution to the cell lysates and mix the plate by orbital shaking for 1 min. Analyze the luciferase activity by using a commercial luciferase microplate reader.

3. RNA extraction of Ha-CoV-2 and quantitative reverse transcriptase PCR (RT-qPCR)

1. Viral RNA extraction: Extract the viral RNA from Ha-CoV-2 Wild-type and the Ha-CoV-2 Delta and Omicron variant particles using a commercial viral RNA extraction kit, following the manufacturer's instructions. Store the extracted viral RNA at -80 °C or use it immediately for RT-qPCR.
2. RT-qPCR: Perform RT-qPCR on viral RNA using a one-step master mix. Perform the reaction in a commercial PCR machine. Please note that the target of amplification is the genomic RNA of Ha-CoV-2. Use Ha-CoV-2 vector DNA as a standard for creating a standard curve and calculating the RNA copy number of each variant.

4. Neutralizing antibody assay

1. Seeding HEK293T(ACE2/TMPRSS2) cells: The day before the assay, seed HEK293T(ACE2/TMPRSS2) cells in a 96 well plate in 50 µL of complete DMEM media. HEK293T(ACE2/TMPRSS2) cells are commercially purchased.
2. For counting cells, obtain 20 µL cells from a T75 flask containing HEK293T(ACE2/TMPRSS2) cells, and mix with 20 µL of trypan blue solution. Add 20 µL of this mixture to cell counting chamber and count the number of cells per mL. To seed a 96 well plate for infection, use 2.5 x 10⁴ cells per well, and 2.5  x 10⁶ cells will be needed in total. Place 96 well plate in a CO₂ incubator at 37 °C overnight.
3. Neutralizing antibody assay: In a sterile polypropylene 96-well plate, prepare the standard 27BV neutralizing antibody and Ha-CoV-2 mixture. Add 8 µL of 27BV (45 mg/mL) to the plate and perform serial dilutions of the antibody with 6 µL of serum-free DMEM.
   NOTE: Be sure to exchange pipette tips between well transfers and ensure the antibody and serum-free medium are mixed thoroughly to produce accurate results.
4. To the serially diluted antibody, add 54 µL of Ha-CoV-2 particle and mix the virus and the antibody. Pre-incubate Ha-CoV-2 particles with serially diluted 27BV for 1 h at 37 °C at 5% CO₂. After the 1 h incubation, apply 50 µL of the antibody and Ha-CoV-2 mixture to the 96-well plate containing the HEK293T(ACE2/TMPRSS2) cells (2.5 x 10⁴ cells per well) seeded the day before.
5. For controls, leave at least three wells containing only the HEK293T(ACE2/TMPRSS2) cells. Add 50 µL of complete medium to these wells to serve as non-infected wells for background signal of the luciferase assay readings.
   ​NOTE: The protocol can be stopped here until the infection has reached 18 h of incubation at 37 °C, and then the plate is ready to be analyzed via luciferase assay.
6. Luciferase assay: After 18 h of incubation, add 7.5 µL of lysis buffer directly to each well and mix by orbital shaking for 2 min. Lyse cells in lysis buffer for at least 5 min at room temperature.
7. Prepare the Firefly luciferase assay solution by mixing the D-luciferin solution with the Firefly luciferase assay solution in a 1:50 ratio. For a whole 96-well plate, combine 3 mL of the luciferase substrate solution with 60 µL of the D-luciferin solution with 2940 µL of the firefly luciferase buffer solution.
8. Add 25 μL of the Firefly luciferase assay solution to the cell lysates and mix the plate by orbital shaking for 1 min. Analyze the luciferase activity by using a commercial luciferase microplate reader.

5. Quantification and statistical analysis

1. Data collection: Perform infection and luciferase assays in triplicate as indicated (Figure 1). Quantify luciferase expression with luciferase assay readings. The mean is the average value of the three luciferase assay readings. Background signal readings from non-infected wells are subtracted from this mean value. Standard deviation (SD) is determined from the average value of the luciferase assay readings.
2. Data analysis: Plot antibody neutralization activity and calculate the ID₅₀ (50% inhibition dosage) values using commercial graphing software. The ID₅₀ value is defined as the antibody inhibitory dilutions at which a 50% reduction in the infection of HEK293T(ACE2/TMPRSS2) cells (based on luciferase assay readings) is attained.