The expression and antigenicity of a truncated spike-nucleocapsid fusion protein of severe acute respiratory syndrome-associated coronavirus.
BACKGROUND
In the absence of effective drugs, controlling SARS relies on the rapid identification of cases and appropriate management of the close contacts, or effective vaccines for SARS. Therefore, developing specific and sensitive laboratory tests for SARS as well as effective vaccines are necessary for national authorities.


RESULTS
Genes encoding truncated nucleocapsid (N) and spike (S) proteins of SARSCoV were cloned into the expression vector pQE30 and fusionally expressed in Escherichia coli M15. The fusion protein was analyzed for reactivity with SARS patients' sera and with anti-sera against the two human coronaviruses HCoV 229E and HCoV OC43 by ELISA, IFA and immunoblot assays. Furthermore, to evaluate the antigen-specific humoral antibody and T-cell responses in mice, the fusion protein was injected into 6-week-old BALB/c mice and a neutralization test as well as a T-cell analysis was performed. To evaluate the antiviral efficacy of immunization, BALB/c mice were challenged intranasally with SARSCoV at day 33 post injection and viral loads were determined by fluorescent quantitative RT-PCR. Serological results showed that the diagnostic sensitivity and specificity of the truncated S-N fusion protein derived the SARS virus were > 99% (457/460) and 100.00% (650/650), respectively. Furthermore there was no cross-reactivity with other two human coronaviruses. High titers of antibodies to SRASCoV appeared in the immunized mice and the neutralization test showed that antibodies to the fusion protein could inhibit SARSCoV. The T cell proliferation showed that the fusion protein could induce an antigen-specific T-cell response. Fluorescent quantitative RT-PCR showed that BALB/c mice challenged intranasally with SARSCoV at day 33 post injection were completely protected from virus replication.


CONCLUSIONS
The truncated S-N fusion protein is a suitable immunodiagnostic antigen and vaccine candidate.
Mu
Feng
F
Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China. mufeng@genomics.org.cn
Niu
Dongsheng
D
Mu
Jingsong
J
He
Bo
B
Han
Weiguo
W
Fan
Baoxing
B
Huang
Shengyong
S
Qiu
Yan
Y
You
Bo
B
Chen
Weijun
W
D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D000917:Antibody Formation; D000956:Antigens, Viral / Q000276:immunology; D049109:Cell Proliferation / Q000187:drug effects; D000086462:Coronavirus Nucleocapsid Proteins; D004797:Enzyme-Linked Immunosorbent Assay; D015967:Gene Expression Regulation, Viral; D006801:Humans; D007111:Immunity, Cellular; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000276:immunology; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology / Q000502:physiology; D012680:Sensitivity and Specificity; D045169:Severe Acute Respiratory Syndrome / Q000517:prevention & control; D064370:Spike Glycoprotein, Coronavirus; D013154:Spleen / Q000166:cytology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology; D014760:Viral Fusion Proteins / Q000235:genetics* / Q000276:immunology* / Q000494:pharmacology; D014779:Virus Replication
D000914:Antibodies, Viral; D000956:Antigens, Viral; D000086462:Coronavirus Nucleocapsid Proteins; D008562:Membrane Glycoproteins; C000706247:N protein, SARS-CoV; D019590:Nucleocapsid Proteins; D064370:Spike Glycoprotein, Coronavirus; D014759:Viral Envelope Proteins; D014760:Viral Fusion Proteins