A 219-mer CHO-expressing receptor-binding domain of SARS-CoV S protein induces potent immune responses and protective immunity

Abstract

Development of vaccines is essential for the prevention of future recurrences of severe acute respiratory syndrome (SARS), caused by the SARS coronavirus (SARS-CoV). The spike (S) protein, especially receptor-binding domain (RBD) of SARS-CoV, plays important roles in the prevention of SARS infection, and is thus an important component in SARS vaccine development. In this study, we expressed a 219-mer (residues 318-536) RBD protein in Chinese hamster ovary (CHO)-K1 cells (RBD219-CHO), and tested its immune responses and protective immunity in a mouse model. The results showed that this recombinant protein was correctly folded, being able to maintain intact conformation and authentic antigenicity. It could induce strong humoral and cellular immune responses and high titers of neutralizing antibodies in the vaccinated mice. RBD219-CHO protein elicited potent protective immunity that protected all vaccinated mice from SARS-CoV challenge. These results suggest that the recombinant RBD219-CHO protein has great potential for the development of an effective and safe SARS subunit vaccine.

FIG. 1.

Vaccination protocol for the experiments.

FIG. 2.

Western blot for detecting reactivity of RBD219-CHO protein with a panel of mAbs specific for distinct conformational epitopes in RBD of SARS-CoV S protein. One mAb recognizing a linear epitope in RBD was also selected for detection.

FIG. 3.

Detection of RBD-specific antibodies in sera of mice immunized with RBD219-CHO protein and PBS controls. The data are presented as mean A450 ± standard error (SE) from five mice per group. (a) IgG antibody detected in sera (at 1:3000 dilution) collected before immunization (Pre) and 10 d after each vaccination are shown here. (b) RBD-specific antibody titer detected in sera collected 10 d after the last vaccination are shown here.

FIG. 4.

Detection of neutralizing antibodies in sera of mice vaccinated with RBD219-CHO or PBS controls. Sera collected 10 d after the last immunization were tested for neutralizing activity against infection by SARS pseudovirus in ACE2/293T cells, and live SARS-CoV in Vero E6 cells. The data are presented as mean NT₅₀ ± SE of five mice per group.

FIG. 5.

Detection of protective immunity in mice immunized with RBD219-CHO protein or PBS controls. Mice vaccinated with RBD219-CHO protein were challenged with live SARS-CoV (GZ50). (a) SARS-CoV replication in the lung tissues of challenged mice was detected and expressed as log₁₀TCID₅₀/g of tissue. The detection limit was 1.5 log₁₀TCID₅₀/g of lung tissue. M1–M5 indicate the five mice used per group. (b) The number of SARS-CoV RNA copies in the lung tissues of mice challenged with live SARS-CoV GZ50 were measured by qRT-PCR, and expressed as RNA copies/μg of lung tissue. The neutralizing antibody titers (NT₅₀) in the sera of the mice are also shown. The data are presented as mean ± SE of five mice per group. The experiment was performed three times and similar results were obtained.