Recombinant receptor-binding domain of SARS-CoV spike protein expressed in mammalian, insect and E. coli cells elicits potent neutralizing antibody and protective immunity

Abstract

Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease. The potential recurrence of the disease from animal reservoirs highlights the significance of development of safe and efficient vaccines to prevent a future SARS epidemic. In this study, we expressed the recombinant receptor-binding domain (rRBD) in mammalian (293T) cells, insect (Sf9) cells, and E. coli, respectively, and compared their immunogenicity and protection against SARS-CoV infection in an established mouse model. Our results show that all rRBD proteins expressed in the above systems maintained intact conformation, being able to induce highly potent neutralizing antibody responses and complete protective immunity against SARS-CoV challenge in mice, albeit the rRBD expressed in 293T cells elicited stronger humoral immune responses with significantly higher neutralizing activity (P<0.05) than those expressed in Sf9 and E. coli cells. These results suggest that all three rRBDs are effective in eliciting immune responses and protection against SARS-CoV and any of the above expression systems can be used for production of rRBD-based SARS subunit vaccines. Preference will be given to rRBD expressed in mammalian cells for future evaluation of the vaccine efficacy in a non-human primate model of SARS because of its ability to refold into a native conformation more readily and to induce higher level of neutralizing antibody responses than those expressed in E. coli and insect cells.

Fig. 1

Expressed rRBD proteins (RBD-293T, RBD-Sf9 and RBD-Ec) were detected by Western blot for their reactivity with a panel of mAbs that recognize conformational and linear epitopes in the SARS-CoV RBD region.

Fig. 2

Humoral immune responses were detected using sera of mice vaccinated with rRBD proteins. PBS was used as the negative control. (A) Reactivity of serum antibodies with rRBDs. RBD-specific IgG was detected by ELISA using sera (1:3000 dilution) from mice before (pre-immune) and 10 days after each vaccination. The data are presented as mean A450 ± standard error (SE) of five mice per group. ⁎ indicates significant difference (P < 0.05) between RBD-293T or RBD-Sf9, respectively, and RBD-Ec group. (B) The ability of antibody binding to rRBD was detected by ELISA using serial dilutions of sera collected from mice 10 days post-last vaccination. The data are presented as mean A450 ± SE of five mice per group at various dilution points. RBD-293T, RBD-Sf9 and RBD-Ec indicate rRBD proteins expressed in 293T, Sf9 and E. coli cells, respectively.

Fig. 3

Neutralizing antibody activity was detected using sera of mice vaccinated with rRBD proteins. PBS was used as the negative control. Sera collected at 10 days post-last vaccination were used for the detection. (A) neutralizing antibody titers against SARS pseudovirus infection. The data are presented as mean ± SE of 50% neutralizing antibody titers (NT₅₀) from five mice per group. ⁎ indicates significant difference (P < 0.05) between RBD-293T and other groups, or RBD-Sf9 and RBD-Ec groups. (B) neutralizing antibody titers against live SARS-CoV infection. The titers were determined as the highest dilutions of sera that could completely prevent CPE in at least 50% of the wells (NT₅₀) and are presented as mean ± SE of five mice per group. ⁎ indicates significant difference (P < 0.05) between the RBD-293T vaccination group and other groups.

Fig. 4

Protective immunity was detected in mice vaccinated with rRBD proteins. Mice respectively vaccinated with RBD-293T, RBD-Sf9 and RBD-Ec were challenged with SARS-CoV GZ50. The SARS-CoV replication in the challenged mouse lung tissues was detected and expressed as Log₁₀TCID₅₀/g of tissues. M1–M5 indicates five mice per group. The limit of detection was 1.5 Log₁₀TCID₅₀/g of lung tissues.