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

We have established two main findings in this protocol. First, our Ha-CoV-2 platform demonstrates that the newly emerged Omicron subvariant is more infectious than the original Wuhan strain. Additionally, Omicron subvariants are more resistant to neutralizing antibodies induced by previous strains.

SARS-CoV-2 research requires BSL-3 facilities, making it difficult for common laboratories to monitor SARS-CoV-2 variants and to quantify neutralizing antibodies. Our Ha-CoV-2 system is a pseudo-type virus that can be used in common laboratories and provides a faster way to quantify viral variants and their sensitivity to neutralizing antibodies. Ha-CoV-2 is a virus-like particle that has all the structural proteins of SARS-CoV-2 and resembles SARS-CoV-2.

Also, Ha-CoV-2 uses and alpha viral vector for robust and rapid gene expression, producing fast results and a high signal-to-noise ratio for great reproducibility. Begin by maintaining HEK293T cells and DMEM containing heat inactivated FBS, Penicillin, and Streptomycin in a T75 flask. For cell counting, mix 20 microliters of cell suspension with 20 microliters of Trypan Blue solution.

Add 20 microliters of dye-mixed cell suspension to the cell counting chamber and count the number of cells per milliliter. Seed HEK293T cells in 10 milliliters of complete DMEM in a 10-centimeter cell culture Petri dish. Incubate the cells overnight in a carbon dioxide incubator at 37 degrees Celsius.

The next day, remove the complete medium, and replace it with nine milliliters of DMEM serum-free medium. Using the given compounds of viral particle assembly, prepare a co-transfection mixture. Slowly add the mixture to the Petri dish, and incubate at 37 degrees Celsius for six hours.

After incubation, replace the serum-free DMEM with a complete DMEM medium, and continue incubation for co-transfection for up to 48 hours. Then detach the cells by repeatedly pipetting over the surface of the monolayer, and transfer the suspension into a 15-milliliter tube. Centrifuge the cell suspension at 400 g for five minutes.

Collect the supernatant and pass it through a 0.22 micron filter. Store the filtrate containing Ha-CoV-2 pseudo virus at 80 degrees Celsius. Begin by maintaining HEK293T ACE2/TMPRSS2 cells in DMEM containing heated activated FBS, Penicillin, and Streptomycin in a T75 flask.

For cell counting, mix 20 microliters of cell suspension with 20 microliters of Trypan Blue solution. Add 20 microliters of dye-mixed cell suspension to the cell counting chamber, and count the number of cells per milliliter. Seed 2.5 x 10 to the power of 4 HEK293T ACE2/TMPRSS2 cells in 50 microliters of complete DMEM medium into each well of a 96-well plate.

Incubate the cells in a carbon dioxide incubator overnight at 37 degrees Celsius. The next day, replace the 50 microliters of DMEM with 50 microliters of virus suspension, and incubate for 18 hours at 37 degrees Celsius. After incubation, add 7.5 microliters of cell lysis buffer to each well and mix on an orbital shaker for two minutes.

Once the cells are lysed, add freshly prepared Firefly Luciferase Assay Solution, and mix them on an orbital shaker for one minute. Analyze the luciferase activity by using a commercial luciferase microplate reader. For neutralizing antibody assay, seed HEK293T cells in 50 microliters of complete DMEM, as demonstrated previously.

In a 96-well plate, add eight microliters of 27 BV neutralizing antibody, and perform serial dilutions with six microliters of serum-free DMEM. Add 54 microliters of Ha-CoV2 virus particle to the serially diluted antibody, and mix the suspension. Incubate for one hour at 37 degrees Celsius and 5%carbon dioxide.

Then add 50 microliters of virus suspension to the wells containing HEK293T cells. For controls, add 50 microliters of complete DMEM medium into the wells containing only HEK293T cells. Place the plate in an incubator at 37 degrees Celsius for 18 hours.

After lysing the cells, as demonstrated previously, add freshly prepared Firefly Luciferase Assay Solution and mix by orbital shaking for one minute. Analyze the luciferase activity by using a commercial luciferase microplate reader Ha-CoV2 Omicron and Omicron variants generated four to tenfold higher signal than the Wild Type Ha-CoV2, suggesting higher infectivity. The antibody 27 BV demonstrated neutralizing activity against both Delta and Omicron variants.

The ID50 of 27 BV for Omicron was approximately 10 times less potent than the ID50 for Wild Type and Delta.