Protocol for Recombinant RBD-based SARS Vaccines: Protein Preparation, Animal Vaccination and Neutralization Detection

In this procedure a recombinant vaccine expressing SARS COV RBD protein is prepared and its ability to induce neutralizing activity against a SARS pseudo virus is evaluated.First. The RBD protein is expressed in purified from the culture supernatant of transfected 2 9 3 T cells. The purified protein is then used to immunize.

Mice and sera are collected from the vaccinated mice at various time points following immunization to quantify the neutralization potential of the immune sera. A pseudo virus neutralization assay is performed, recombinant produced luciferase expressing sars pseudo virus is produced and incubated with a titrated mouth sra. Next, the ability of these neutralized viruses to infect SARS co-receptor ACE expressing cells is assessed by measuring the relative luciferase activity in infected cells.

50%neutralizing antibody titer can then be calculated. My name is tu. I'm currently a postal in the laboratory.

The main advantage of the pseudo virus neutralization asay over existing techniques such as live virus based neutralization assays is that this technique is relatively safe. Does not require an infectious SAR virus and can be performed in a non by safety three laboratory. My name is demonstrating procedure will be laying due and DU is a research post research fellow in my laboratory who has focused on the development of vaccine against emerging infectious disease such as SARS and infl Thaw aliquots of sterile two XHBS buffer pH seven 2.5 molar calcium chloride, and RBD plasmid at room temperature.

Once the solutions are thawed, transfer two milliliters of two XHBS to a 50 milliliter conical tube in a 15 milliliter tube mix 200 microliters of calcium chloride with 40 micrograms of RBD plasmid. Adjust the volume to two milliliters using room temperature sterile distilled water using a sterile five milliliter pipette. Add the plasmid solution to the two XHBS in a dropwise manner while maintaining the mix under a constant but gentle vortex.

Keeping this vortex slow and continuous is crucial for obtaining a precipitant of adequate consistency for high transfection efficiency. Store for 20 minutes at room temperature. Following this incubation, add the mixture drop by drop to a flask containing 50 to 70%confluent 2 9 3 T cells.

In a volume of 40 milliliters of cell culture medium, it is important to distribute the transfection solution evenly. Mix the medium immediately after adding the transfection mix. Place the cells at 37 degrees Celsius in a 5%carbon dioxide incubator.

After eight to 10 hours of incubation, remove the complete cell medium and replace it with 50 milliliters of fresh serum free optimum, one medium to avoid any interference of serum on the subsequent protein purification and then return the cells to the 37 degree Celsius incubator. Two days later collect the supernatant, which will contain expressed RBD protein and centrifuge at 15 minutes at 6, 000 RPM to remove cell debris. Once the cells have been pelleted, transfer the supernat into a fresh tube and add protease inhibitor cocktail.

To prevent protein degradation, use nickel NTA super flow following the manufacturer's instructions to purify the RBD recombinant protein from the harvested supernat ELUTE RBD protein with buffer containing 250 millimolar ole. Next using Emon Ultra 15 tubes, concentrate the purified protein containing OLE by centrifugation at 3000 RPM four degrees Celsius for around 15 minutes. Discard the flow through.

ADD PBS to the RBD concentration tubes and centrifuge again at 3000 RPM four degrees Celsius for 20 minutes. Repeat the wash twice. To ensure complete removal of ole the purified protein will now dissolve in PBS pipette protein to a clean 1.5 milliliter EOR tube.

Finally, calculate the concentration of the purified protein using a NanoDrop with PBS as the blank and store the sample at minus 80 degrees Celsius. First, add one milliliter of sterile PBS to one vial of Sigma adjuvant system. Prewarm in a water bath at 40 to 45 degrees Celsius and vortex the vial thoroughly for two to three minutes.

Next, prepare the protein adjuvant emulsion in a 1.5 milliliter einor tube. Mix the desired amount of thawed purified protein diluted in PBS with an equal volume of adjuvant system and vortex vigorously for two to three minutes. Also, prepare control emulsions by mixing the adjuvant with PBS alone.Subcutaneously.

Inject at least five four to six week old BC mice with 200 microliters each of the initial immunization mixture containing 20 micrograms of protein per dose or with the control emulsion at 21 days following the initial inoculation subcutaneously. Inject booster doses containing 10 micrograms of protein in a final volume of 200 microliters each. Collect blood samples from each mouse before the initial immunization and 10 days after each vaccination and store the isolated serum at minus 20 degrees Celsius after heat.

Inactivation of complement at 56 degrees Celsius for 30 minutes 16 hours before transfection plate 2 9 3 T cells in 100 millimeter tissue culture dishes at a density of 2 million cells per dish. Prepare the transfection reagent as described earlier and cot transfect the cells with a plasmid encoding the SARS cove S protein and another encoding the ENV defective luciferase expressing HIV one genome. Eight to 10 hours post transfection.

Replaced the medium with 10 milliliters of fresh DMEM medium containing 10%serum and then return the cells to the incubator. Two days later, harvest the snat containing the SARS pseudo virus. Pass the supernatant through a 0.45 micron syringe filter to remove cell debris aliquot the virus.

Use immediately or freeze at minus 80 degrees Celsius for future use using a multi-channel pipette plate. 2 9 3 T cells expressing SARS COV receptor ACE two in the 96 well tissue culture plate at a concentration of 10, 000 cells per a hundred microliters per well and incubate the cells at 37 degrees Celsius for 16 hours. Use a separate 96 well plate to titrate the recombinant luciferase expressing SARS pseudo virus and determine the appropriate virus concentration for the neutralization assay in rows A and B first plate.

150 microliters per well of 10%F-B-S-D-M-E-M. Next, add 150 microliters of undiluted virus to rows A one and B one and perform a duplicate serial twofold dilution by carrying over 150 microliters from the wells in row one to the wells in the next row and so forth until row 11. Discard the last 150 microliters and do not add virus to row 12.

Once the serial dilution has been performed and the ACE 2 93 T cells have been incubated for 16 hours, infect the cells by transferring 100 microliters from each well of the virus plate into the corresponding well of the plate containing the cells. Incubate the cells for two days at 37 degrees Celsius to allow viruses to infect the cells. Determine the level of infection in each well by reading the plate in an ultra 3 84 luminometer.

The amount of infection is proportional to the intensity of luciferase activity. The virus concentration at which the relative luciferase unit is about a thousand times greater than the background RLU of the cells alone in column 12 will be used in the neutralization assay. Set up the neutralization in a separate 96 well plate first using a multi-channel pipette at 150 microliters of 10%F-B-S-D-M-E-M to the wells in column one and 120 microliters to the wells in the other columns.

Next, add three microliters of each mouse serum to be tested to duplicate wells in column one. In this example, the first two wells have 50 fold diluted serum from an RBD immunized mouse. The next two have serum from a second RBD immunized mouse and the last four wells have serum from two control PBS immunized animals using a multi-channel pipette, perform fivefold serial dilution of the serum by transferring 30 microliters from all the wells in the first column into wells of the second column, and repeating this until column 11.

Discard the remaining 30 microliters and leave the wells in column 12 without serum. Next, add 120 microliters per well of a diluted SAR pseudo virus to each. Well incubate the plate at 37 degrees Celsius for one hour to allow any anti RBD antibodies in the mouse serum to bind and neutralize the SARS pseudo virus.

Following this incubation, transfer 100 microliters of neutralized virus from each well into the corresponding wells. The 96 well plate containing ACE two expressing 2 9 3 T cells. Incubate the cells at 37 degrees Celsius for 24 hours to allow infection once the cells have been infected, remove the sate and using a multi-channel pipette and replace it with 100 microliters per well of fresh medium.

Return the plate to the incubator after 72 hours at 37 degrees Celsius. Once again, remove the supra natin At this point, any pseudo virus that has not been neutralized by the mouse serum should have been able to infect cells using a multichannel pipette. Add 60 microliters per well of one luciferase cell culture lysis reagent.

Place the plate on a shaker at room temperature and allow the cells to lyse for one to two hours. Once the cells have been lyed, transfer 50 microliters of the cell lysates into a 96 well luminometer plate and add 50 microliters per well of luciferase substrate. Use an ultra 3 84 luminometer set in luminescence in 96 well plates mode to read the plate and assess relatively luciferase activity in the different wells from which the percentage of virus neutralized can be derived.

These data show that the percentage of virus neutralization achieved is proportional to the concentration of serum from RBD immunized mice. In contrast, serum from PBS injected animals fail to neutralize the virus at any concentration from these data. The NT 50 or serum titer at which 50%of the virus is neutralized can be easily derived.

While attempting this procedure is important to to remember that confidence and patience are essential to the success of experiments. After watching these videos, you should have a better understanding how to express recombinant SARS vaccine in maman cell sub, and to determine ability induce neutralized antibody response in mouse models.