1 Laboratory of Structural Biology, Tsinghua University, Beijing 100084, China
2 National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China
3 Harbin Veterinary Research Institute, Chinese Academy of Agriculture, 427 Maduan Street, Harbin 150001, China
Correspondence
Zihe Rao
raozh{at}xtal.tsinghua.edu.cn
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ABSTRACT |
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MAIN TEXT |
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Eight truncated fragments covering the S protein (Fig. 1a) and the full-length N protein were expressed in Escherichia coli, using genes of the BJ01 strain (GenBank accession no. AY278488) of SARS-CoV isolated in China, while the M protein was expressed in Pichia pastoris as it could not be easily expressed in E. coli. All fragments of the S protein containing a six-histidine tag were expressed in E. coli as inclusion bodies. The proteins were purified by sonication and repeated washing, and then dissolved in Tris/HCl buffer with 8 M urea and further purified using Ni-NTA agarose (Invitrogen). Finally, the denatured proteins were refolded by dialysis in Tris/HCl buffer (pH 7·4). Soluble N and M proteins containing six-histidine tags were also purified using Ni-NTA agarose (Fig. 1b
). It should be noted that, unlike our expression products, the native S protein is highly glycosylated and that oligosaccharides may affect the generation of neutralization antibodies. Consequently, we cannot rule out the possibility that the lack of glycosylation in E. coli has influenced the outcome of our experiments. Nevertheless, expression of the proteins in E. coli could provide a simple and cheap method for large-scale production of vaccines protecting against SARS-CoV infection.
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Notably, the neutralizing capacity of antiserum to SD (1 : 128), which overlapped both the SG1 and SG3 fragments, was higher than that of either SG1 or SG3 individually (Fig. 1a and 2b). To confirm this synergistic effect of neutralizing capacity between antibodies to epitopes in SG1 and SG3, we mixed the two antisera in equal volumes to measure the neutralizing titre. The neutralizing capacity of the mixed antisera against SG1 and SG3 was equal to that of SD (Fig. 2b and c
). Neutralization of coronaviruses is a specific event that requires the presentation of the epitope involved in neutralization in the appropriate structural context. Work with transmissible gastroenteritis coronavirus (TGEV) has shown that the S glycoprotein domain recognized by the receptor on ST cells is located in the globular domain of the protein close to the antigenic sites A and D (Sune et al., 1990
). In TGEV neutralization experiments using binary combinations of mAbs specific for different antigenic sites, a synergistic effect was also observed (Sune et al., 1990
).
To investigate synergistic effects in more detail, we measured the neutralizing capacity of a mixture of antisera against SA, SB and SD, which covered residues 13877 of the S protein. As expected, the mixed sera showed a higher neutralizing titre of 1 : 342 (Fig. 2c). Similarly, the mixed sera to SA, SB and SC, covering residues 13730 of the S protein, had a titre of 1 : 297. The mixture of antisera against SA, SB, SC and SG3, covering residues 13882 of the S protein, had a titre of 1 : 342, which was the same as for the mixture of sera to SA, SB and SD (Fig. 2c
). Generally, the humoral response against a virus is directed towards many different envelope protein epitopes. The results suggest that the region from residues 13877 of the S protein, including an important epitope in fragment SG3 (residues 735882), is essential for the development of an effective protein-based vaccine preventing SARS-CoV infection.
It has been reported that the M protein of TGEV is required for virus assembly and budding, that it is immunodominant and that M-specific antibodies both neutralize TGEV and mediate the complement-dependent lysis of TGEV-infected cells (Delmas et al., 1986; Risco et al., 1995
; Woods et al., 1987
). To investigate the neutralizing capacity of antiserum to the M protein of SARS-CoV, we examined its neutralizing titre using serum of heat-inactivated complement. The neutralization titre of the M protein antiserum was higher than 1 : 128 (Fig. 2b
), suggesting that the M protein is important in development of an effective protein-based vaccine preventing SARS-CoV infection. To evaluate the synergistic effect between antisera to different epitopes, the M antiserum was mixed with antisera against the SA, SB and SD fragments (covering residues 13877 of the S protein) and a neutralization assay was performed. The neutralizing capacity of the mixed SA+SB+SD+M antisera showed a considerable synergistic effect, with a neutralizing titre of 1 : 396 (Fig. 2c
). This suggested that sera to the M and S proteins can effectively enhance each other's activity.
We also assayed the neutralizing capacity of antiserum to the N protein, but no neutralizing ability was detected in vitro (Fig. 2c). However, recent evidence indicates that the N protein can induce a T-cell response to SARS-CoV infection (Gao et al., 2003
). An effective vaccine may also aim to elicit a vigorous cellular immunity. Therefore, the N protein is also important in development of an effective vaccine preventing SARS-CoV infection.
Neutralizing antibodies that bind efficiently to the envelope spikes of SARS-CoV can offer protection or may be beneficial if present at appropriate concentrations before exposure to the virus. To define the neutralizing capacity of convalescent antisera from SARS-CoV patients, we determined the neutralization titres for sera from three convalescent patients under the same experimental conditions, by pooling together equal volumes of antisera from each patient. The resulting neutralization titre was found to be 1 : 342. Clearly, the neutralization titre of mixed antisera to fragments SA, SB and SD (covering residues 13877 of the S protein) and the M protein was higher than that of antisera from convalescent patients (Fig. 2c).
Our results have confirmed that, as major antigens, the expressed unglycosylated structural proteins can induce a strong, SARS-CoV-specific, neutralizing antibody response in rabbits. These results provide crucial information for a rational approach towards maximizing antibody responses elicited by potential vaccines. It will also be essential to determine whether humans and animals generate similar responses to SARS-CoV (Cyranoski, 2004; Fouchier et al., 2003
; Guan et al., 2003
; Martina et al., 2003
). Virus-challenge tests in animal models will be required for different SARS-CoV isolates to assess the protective capacity of the expressed proteins as vaccines. Furthermore, before any potential vaccine can be tested in humans, a non-human primate challenge model must be used to evaluate its effectiveness against SARS-CoV infection and to exclude any potential side effects (Fouchier et al., 2003
), such as those observed for candidate vaccines against feline infectious peritonitis virus (Vennema et al., 1990
). In addition, the development of a vaccine against SARS-CoV infection in animals also needs to prevent the virus being carried and transmitted to humans by animals, such as masked palm civet, ferret and cat, and causing infection in humans (Cyranoski, 2004
; Guan et al., 2003
; Martina et al., 2003
).
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ACKNOWLEDGEMENTS |
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Received 15 March 2004;
accepted 2 July 2004.