This protocol describes a general procedure for studying recombinant receptor-binding domain (RBD)-based subunit vaccines against SARS. It includes methods for transfection and expression of RBD protein in 293T cells, immunization of mice with RBD and detection of neutralization activity of mouse sera using an established SARS pseudovirus neutralization assay.
Based on their safety profile and ability to induce potent immune responses against infections, subunit vaccines have been used as candidates for a wide variety of pathogens 1-3. Since the mammalian cell system is capable of post-translational modification, thus forming properly folded and glycosylated proteins, recombinant proteins expressed in mammalian cells have shown the greatest potential to maintain high antigenicity and immunogenicity 4-6.
Although no new cases of SARS have been reported since 2004, future outbreaks are a constant threat; therefore, the development of vaccines against SARS-CoV is a prudent preventive step and should be carried out. The RBD of SARS-CoV S protein plays important roles in receptor binding and induction of specific neutralizing antibodies against virus infection 7-9. Therefore, in this protocol, we describe novel methods for developing a RBD-based subunit vaccine against SARS. Briefly, the recombinant RBD protein (rRBD) was expressed in culture supernatant of mammalian 293T cells to obtain a correctly folded protein with proper conformation and high immunogenicity 6. The transfection of the recombinant plasmid encoding RBD to the cells was then performed using a calcium phosphate transfection method 6,10 with some modifications. Compared with the lipid transfection method 11,12, this modified calcium phosphate transfection method is cheaper, easier to handle, and has the potential to reach high efficacy once a transfection complex with suitable size and shape is formed 13,14. Finally, a SARS pseudovirus neutralization assay was introduced in the protocol and used to detect the neutralizing activity of sera of mice vaccinated with rRBD protein. This assay is relatively safe, does not involve an infectious SARS-CoV, and can be performed without the requirement of a biosafety-3 laboratory 15.
The protocol described here can also be used to design and study recombinant subunit vaccines against other viruses with class I fusion proteins, for example, HIV, respiratory syncytial virus (RSV), Ebola virus, influenza virus, as well as Nipah and Handra viruses. In addition, the methods for generating a pseudovirus and subsequently establishing a pseudovirus neutralization assay can be applied to all these viruses.
1. Recombinant SARS-CoV RBD Protein Preparation
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 |
2. Mouse Immunization and Sample Collection
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 |
3. Neutralization Detection using Pseudovirus Neutralization Assay
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.
The authors have nothing to disclose.
This study was supported by the National Institutes of Health (NIH) of the United States (RO1 AI68002).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
NaCl | Sigma | S7653 | ||
Na2HPO4*7H2O | Sigma | S2429 | ||
HEPES | Sigma | H3375 | ||
CaCl2*2H2O | Sigma | C5080 | ||
DMEM | Invitrogen | 12430 | ||
HI FBS | Invitrogen | 10438 | ||
Penicillin/Streptomycin | Invitrogen | 15140 | ||
OPTI-MEM I Medium | Invitrogen | 31985 | ||
Protease inhibitor cocktail | Roche | 11836170001 | ||
Ni-NTA Superflow | Qiagen | 30450 | ||
Sigma adjuvant system | Sigma | S6322 | ||
Luciferase assay system | Promega | E1501 | ||
Luciferase cell culture lysis reagent (5X) | Promega | E1531 | ||
Amicon Ultra – 15 | Millipore | UFC901024 | ||
Microfluor 96-well plates | Thermo Scientific | 7905 | ||
Ultra 384 luminometer | Tecan Systems |