The expression and antigenicity of a truncated spike-nucleocapsid fusion protein of severe acute respiratory syndrome-associated coronavirus

Abstract

BackgroundIn 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.ResultsGenes 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.ConclusionThe truncated S-N fusion protein is a suitable immunodiagnostic antigen and vaccine candidate.

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“…MERS-CoV patients were observed to have low-level crossreactivity to SARS-CoV 88 and in one study HCoV-HKU1 N protein 76 . Because of this cross-reactivity, researchers have developed diagnostic assays with truncated SARS-CoV S, N, and M proteins [91][92][93][94] . A study with longitudinal follow-up suggests that the titers may indeed reflect stimulation of preexisting antibodies from past infections as avidity of IgG Fig.…”

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“…MERS-CoV patients were observed to have low-level cross-reactivity to SARS-CoV-1 77 and in one study HCoV-HKU1 N protein 79 . Because of this cross-reactivity, researchers have developed diagnostic assays with truncated SARS-CoV-1 S, N, and M proteins [81][82][83][84] . See Figure 5 for associations between homologous and heterologous titers.…”

Unravelling Antigenic Cross-Reactions toward the World of Coronaviruses: Extent of the Stability of Shared Epitopes and SARS-CoV-2 Anti-Spike Cross-Neutralizing Antibodies

“…The spike protein of HCoV-HKU1, HCoV-NL63, HCoV-229E, and SARS-CoV-2, analyzed by a pairwise similarity plot, showed that HCoV-HKU1 had the highest similarity in all spike regions to the SARS-CoV-2 spike, compared with HCoV-229E and HCoV-NL63 [ 43 ]. SARS-CoV-1 was reported to share epitopes with HCoV-OC43, and weak cross-reactions were also found with HCoV-229E [ 44 , 45 , 46 ]. Che and colleagues [ 45 ] found that sera from SARS-CoV-1 showed a several fold increase in antibody titers against HCoV-229E and HCoV-OC43 compared to controls.…”