This method provides a way of answering one of the more important questions concerning the SARS-coronavirus-2 vaccines under development. Is the vaccine able to elicit a neutralizing antibody response? The main advantage of this technique is that it can be done in a containment level two facility.
It is also relatively fast and inexpensive, making it well-suited to analyzing many samples at once. Demonstrating the neutralizing acid procedure, we have Ricardo Marius, a senior technician in the lab and Taylor Jamieson, an MD PhD student. Begin by culturing Vero E6 cells in DMEM supplemented with 10%FBS at 37 degrees Celsius and 5%carbon dioxide.
To passage the cells, first wash them by adding 10 milliliters of PBS and gently rocking the dish four to five times. After aspirating the PBS, add three milliliters of trypsin EDTA and rock the dish to ensure that the entire surface is covered before incubating it at 37 degrees Celsius. Once the cells have detached, deactivate the trypsin by adding seven milliliters of DMEM supplemented with 10%FBS, then resuspend the cells by pipetting up and down several times.
Remove the trypsin by centrifugation and aspirate the supernatant without disturbing the cell pellet before resuspending the cells in 10 milliliters of fresh DMEM. After counting, add approximately one time 10 to the seventh cells to each 15 centimeter plate and incubate the culture for one to two days until the cells are 100%confluent. To prepare the VSV spike EGFP pseudovirus for titering, infect the cells at 0.01 multiplicity of infection with the stock virus diluted in 12 milliliters of serum-free DMEM.
After adding the virus, incubate the cells for one hour at 37 degrees Celsius and 5%carbon dioxide with occasional rocking. At the end of the incubation, replace the inoculum with fresh DMEM supplemented with 2%FBS and 20 millimolar heaps, then incubate the cells at 34 degrees Celsius and 5%carbon dioxide for 24 hours. The next day, use a fluorescent microscope to visualize the EGFP expression of the infected cells.
Once an extensive cytopathic effect and cell detachment are observed, collect the culture supernatant and remove the debris by centrifugation. To avoid multiple freeze-thaw cycles, aliquot the supernatant before storage at minus 80 degrees Celsius. To determine the viral titer, plate Vero E6 cells in six well plates at a seeding density of six times 10 to the fifth cells per well and incubate overnight.
The next day, set up a tenfold serial dilution series of the viral stock by adding 900 microliters of medium to seven micro-centrifuge tubes and adding 100 microliters of viral stock to the first tube. After brief vortexing, transfer 100 microliters of diluted virus to each subsequent tube, vortexing between each tube. Then replace the supernatant in each well of the Vero E6 culture plate with 500 microliters of the 10 to the minus second to 10 to the minus seventh viral dilutions.
After a 45 minute incubation in the cell culture incubator with gently rocking every 15 minutes, replace the inoculum with an overlay solution and incubate the cells at 34 degrees Celsius with 5%carbon dioxide for 48 hours. At the end of the incubation, to visualize the plaques by crystal violet staining, wash the cells one time with PBS before adding two milliliters of 0.1%crystal violet to each well. Place the plate on a rocker for approximately 20 minutes at room temperature before gently washing each well two times with PBS.
After the last wash, allow the plates to air dry for at least one hour before using the formula as indicated to calculate the titer of the virus in each well in plaque forming units per milliliter. To plate cells for a neutralization assay, use a multi-channel pipette to add 100 microliters of Vero E6 cells to each well of a 96 well plate at a density of two times 10 to the fifth cells per milliliter, and incubate the plate for 24 hours at 37 degrees Celsius and 5%carbon dioxide. The following day, heat inactivate the patient serum samples to be tested in a 56 degrees Celsius water bath for 30 minutes.
Then set up a tenfold dilution series of the patient's serum samples in an empty 96 well tissue culture plate by adding eight microliters of the serum to 72 microliters of serum-free DMEM with antibiotics in each well of row A.Then add 40 microliters of serum-free DMEM to each well of rows B to G and 80 microliters to each well of row H.Using a 12 well multichannel pipette, mix the samples in row A, then transfer 40 microliters of the sample from each well of row A to each well of row B.After mixing, repeat the dilution for each subsequent row of wells until row F.Next, add 40 microliters of VSV spike EGFP at 0.05 multiplicity of infection to each well in rows A to G, and mix by pipetting four to five times before incubating the plate for one hour at 37 degrees Celsius and 5%carbon dioxide. At the end of the incubation, carefully aspirate the supernatant from each well of the Vero E6 culture plate and transfer 60 microliters of antibody virus mixture from each well of the sample dilution plate to the corresponding well of the cell culture plate. When all the samples have been transferred, place the cell culture plate at 37 degrees Celsius and 5%carbon dioxide for one hour with rocking every 20 minutes.
After the incubation is complete, add 140 microliters of carboxymethylcellulose overlay solution to each well of the cell culture plate and transfer the plate to a 34 degrees Celsius and 5%carbon dioxide incubator. After 24 hours, image the plates on an automated fluorescent imager at 488 nanometer wavelength, and use the automated counting feature of the imager to quantify the individual EGFP foci. In this representative example, a commercially available neutralizing antibody against the SARS-coronavirus-2 spike receptor binding domain was used as a positive control, alongside IgG as a negative control.
The percent inhibition was calculated based on the number of EGFP foci detected via fluorescent imaging. Pseudovirus neutralization by convalescent patient samples collected approximately three months post SARS-coronavirus-2 infection showed that hospitalized patients demonstrated an increased neutralizing capacity compared to those who did not require hospitalization. This procedure can also be used to evaluate other COVID-19 prevention and therapeutic agents that aim to block viral infection.
