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

1. Recombinant SARS-CoV RBD Protein Preparation

1. Prepare calcium phosphate transfection reagent
   1. 2X HBS buffer preparation: Mix together 16 g of NaCl, 0.4 g of Na₂HPO₄*7H₂O, and 13.0 g of HEPES. Adjust pH to 7.00 and bring up total volume to 1000 mL in distilled water. After filtering the solution for sterilization, aliquot and store it at -20°C.
      Hint: Any variation of the pH value would affect the transfection results. Thus, it is advisable to test several pH values around 7.00 (for example, 6.99, 7.00, or 7.01) and find the best one for the transfection using the method introduced below.
   2. 2.5 M CaCl₂ preparation: Add 73.5 g of CaCl₂*2H₂O to distilled water in a final volume of 200 mL. Filter the solution and store at -20°C.
2. Recombinant plasmid transfection and protein purification
   1. Split 293T cells at 50-70% confluency 24 h before transfection. Grow cells in T-175 cm² tissue culture flasks in 40 mL DMEM containing 10% heat-inactivated (HI) FBS and 1% Penicillin/Streptomycin (P/S) at 37°C in 5% CO₂.
   2. All transfection reagents should be brought up to room temperature before transfection. These reagents include 2X HBS, 2.5M CaCl₂, DiH₂O, and rRBD plasmid ⁶.
      Hint: The final recombinant plasmid construct used for protein expression should contain a signal peptide to ensure secretion of expressed recombinant proteins to the culture supernatant. A 6x His tag may be added to the C-terminal of the expressed protein for easy purification.
   3. Prepare one 50 mL (A) and one 15 mL (B) BD Falcon tube, and add reagents to the tubes as indicated in Table 1. Add 2X HBS buffer to tube A. In tube B, add 2.5M CaCl₂ and rRBD plasmid, and bring the volume to the requirement in DiH₂O.
      

      A. One T-175 cm2 tissue culture flask (4000 μL/flask): prepare for rRBD expression
      Tube A		Tube B
      2X HBS	2000 μL	2.5M CaCl2	200 μL
      		rRBD plasmid DNA	40 μg
      		DiH2O to	2000 μL
      B. One 100-mm petri dish (1000 μL/dish): prepare for SARS pseudovirus production
      Tube A		Tube B
      2X HBS	500 μL	2.5M CaCl2	50 μL
      		SARS-CoV S plasmid DNA	5 μg
      		HIV-1 plasmid (pNL4-3.luc.RE)	5 μg
      		DiH2O to	500 μL

      Table 1. Transfection mixture preparation and volumes
      The volumes listed in Table 1 are for one transfection unit. If more flasks or dishes are used for the transfection, adjust volumes accordingly.
   4. Add the DNA-calcium solution in tube B into tube A in a dropwise manner, while maintaining constant and gentle mixing in a vortex. Let the mixture sit at room temperature for 20-30 min. The key for successful calcium phosphate transfection depends on the size and shape of the precipitate formed. Thus, the mixture should be vortexed constantly and slowly to meet this requirement and, as a result, improve transfection efficacy.
   5. Add mixture in a dropwise and even manner into 293T cells (4000 μL/flask). Culture cells in incubator at 37°C in 5% CO₂.
   6. Replace the culture medium with fresh serum-free OPTI-MEM I Reduced-Serum Medium (50 mL/flask) 8-10 h post-transfection. Continue to culture cells for two more days in the same condition.
   7. Collect supernatant containing expressed rRBD protein 72 h post-transfection. Centrifuge at 6000 rpm for 15 min to remove cell debris. Add protease inhibitor cocktail to the collected supernatant and store at 4°C overnight.
   8. On the next day, purify rRBD recombinant protein from the culture supernatant using Ni-NTA Superflow following manufacturer's instructions.
   9. Concentrate purified protein using Amicon Ultra -15 concentration tubes. After protein concentration, add PBS to the concentration tubes and centrifuge again to remove imidazole in elution buffer. Calculate protein concentration and store purified protein at -80°C until use.

2. Mouse Immunization and Sample Collection

1. Pre-warm Sigma adjuvant system (SAS) to 40-45°C according to manufacturer's instructions. Add 1 mL PBS per vial and mix thoroughly.
2. Prepare protein-adjuvant emulsion according to the protocol in Table 2. Pipette calculated rRBD protein into a 1.5 mL tube. Add equal volume of SAS adjuvant to the tube and vortex vigorously for 2-3 min to form emulsion.
   Hint: The volumes listed in Table 2 are for one mouse. Adjust volumes according to the actual mouse numbers used. For each group, mix together the proteins and adjuvant required for each vaccine. Always prepare one extra sample for the vaccination to ensure accuracy.
   

   Group	1st vaccine (200 μL/mouse)	2nd vaccine (200 μL/mouse)	3rd vaccine (200 μL/mouse)
   rRBD protein	20 μg protein in PBS (100 μL) + 100 μL SAS	10 μg protein in PBS (100 μL) + 100 μL SAS	10 μg protein in PBS (100 μL) + 100 μL SAS
   PBS control	100 μL PBS + 100 μL SAS	100 μL PBS + 100 μL SAS	100 μL PBS + 100 μL SAS

   Table 2. Mouse immunization protocol
3. Subcutaneously prime-immunize female BALB/c mice (4-6 weeks old, 5 mice/group), and boost twice with rRBD and SAS as indicated in Table 2. Use PBS group as the control. The site for subcutaneous injections usually chosen is the loose skin between the shoulder blades. Alternatively, the ventral abdomen is commonly used, because it is easier to inject, and may observe any leakage from the injection sites. When repeated doses of vaccines are used, it is easy to select various injection sites, preventing potential local skin reactions.
4. Bleed mice via retro-orbital with anesthetic before immunization and 10 days after each vaccination, and heat sera at 56°C for 30 min to inactivate complement. Store mouse sera at -20°C until use.

3. Neutralization Detection using Pseudovirus Neutralization Assay

1. Prepare SARS pseudovirus
   1. Split 293T cells in 100 mm tissue culture petri dishes (2x10⁶ cells/dish) 16 h before transfection, and grow cells as above.
   2. Prepare transfection reagents as indicated in Table 1. Co-transfect a plasmid encoding SARS-CoV S protein and a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) using calcium phosphate transfection reagent.
   3. Replace medium with 10 mL fresh DMEM containing 10% FBS and 1% P/S 8-10 h post-transfection. Collect supernatant containing SARS pseudovirus 72 h post-transfection.
   4. Filter pseudovirus through a 0.45 μm filter. Aliquot and store at -80°C until use.
2. SARS pseudovirus neutralization assay
   1. Split 293T cells expressing SARS-CoV receptor ACE2 (ACE2/293T) at 10⁴ cells/100 μL/well in 96-well tissue culture plates 16 h before infection.
   2. Dilute SARS pseudovirus by 2-fold to detect the virus titer in ACE2/293T cells.
   3. Serially dilute mouse sera in 96-well tissue culture plates, and add equal volume of titrated SARS pseudovirus. Preincubate the plates at 37°C for 1 h.
   4. After incubation, add 100 μL of the sera-pseudovirus mixture to ACE2/293T cells, and continue to grow cells at 37°C in 5% CO₂. Add fresh DMEM 24 h later.
   5. Completely remove culture supernatants from the plates 72 h post-infection. Add 1X luciferase cell culture lysis reagent (60 μL/well), and promote cell lysis with constant shaking of plates for 1-2 h at room temperature.
   6. Transfer cell lysates (50 μL/well) into luminometer plates (Microfluor 96-well plates). Add luciferase substrate (50 μL/well) included in luciferase assay system, and detect relative luciferase activity in Ultra 384 luminometer.
   7. Calculate SARS pseudovirus neutralization titer and present as 50% neutralizing antibody titer (NT₅₀) ⁶.

Cell density is an important factor affecting the efficacy of calcium phosphate-based transfection. In our experience, less than 70% confluency of the cells brings the best results. Thus, in order to improve transfection efficiency, it is recommended that cell density be kept at around 50-70% of confluency. The calcium phosphate transfection method is generally thought to be less efficient compared with other transfection methods, such as lipid transfection ¹⁶. However, in this protocol, we used a modified calcium phosphate transfection method whereby constant and slow mixing of the transfection solution in a vortex ensures the formation of a precipitate with the appropriate size and shape, thus improving transfection efficiency.