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

Pseudotyped viruses are molecular tools that can be used to study host-virus interactions and to test neutralizing activity of serum samples. In the work we present today, a general methodology to produce pseudotype viruses based on three plasmid will be presented. For neutralization assays, Y type viruses are generally used, but they are highly pathogenic and cannot be handled in BSL-2 laboratories, rendering this system poorly applicable on large scale.

The technology of pseudoviruses can be a safer alternative compared to live virus to allow the study of pathogen to host interaction and to test neutralizing activity of antibodies. Future experiments will investigate the role of specific antibodies in the maintenance of immunity after vaccination, using the technology of pseudoviruses. To begin, in a 6-well plate, seed one milliliter of human embryonic kidney 293T cells.

Add two milliliters of complete DMEM. The next day, replace the medium with fresh antibiotic-free medium to prepare the cells for transfection. To transfect the adherent cells, first, prepare mix A by adding plasmid DNA to 100 microliters of Opti-MEM.

Next, add 17.5 microliters of polyethylenimine to 100 microliters of Opti-MEM. Mix the contents of the mix A and B, then incubate. During incubation, gently flick the tube every three to four minutes to enhance mixing.

Add the whole volume of the mixture to the plate with the HEK 293T cells. After 16 to 20 hours of transfection, replace the culture medium with fresh complete DMEM. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide to produce the pseudotype viruses.

After 72 hours of transfection, harvest the supernatant into a collection tube. Centrifuge the supernatant at 1600 G for seven minutes at room temperature. Then filter the supernatant through a 0.45 micrometer cellulose acetate filter.

Distribute 50 microliters of complete DMEM in the wells of a 96-well plate. Leave row A empty. Add 100 microliters of the pseudovirus stock to the wells in row A.Next pipette 50 microliters of the solution from row A to B.Repeat this process up to row G to obtain serial dilutions.

Remove the exhausted medium from a plate with cultured susceptible HEK 293T ACE2 cells, and wash the cells with four milliliters of DPBS. Then detach the cells with one milliliter of trypsin-EDTA in DPBS saline. Now add 50 microliters of the susceptible cell suspension into each well containing the pseudotype viruses.

Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for 48 hours. Add 100 microliters of the luciferase reagent to each well. After incubation, transfer the contents of each well into a black 96-well plate, then read the plate in a plate reader.

In a 96-well plate, add 50 microliters of fresh complete DMEM to columns one to ten from row B to H.Add 95 microliters of fresh complete DMEM to the corresponding wells of row A.Now add 50 microliters of complete DMEM to the wells in column 11 and 100 microliters of DMEM to column 12. Pipette five microliters of heat-inactivated serum samples to row A.With a multi-channel pipette, mix the samples in the first row and transfer 50 microliters of medium-containing serum from row A to B, up to row H.Distribute 50 microliters of dilute pseudovirus-containing medium to each well from columns 1 to 11. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide for one hour.

Prepare a suspension of susceptible HEK 293T ACE2 cells in five milliliters. Add 50 microliters of the cell suspension to each well, then incubate before performing the luciferase assay. Lower serum dilution resulted in decreased viral entry into the cells.

This is confirmed by the increased percentage of neutralization.