Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid - PubMed (original) (raw)

. 2004 Jul 15;190(2):379-86.

doi: 10.1086/422040. Epub 2004 Jun 16.

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Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid

Danny Tze Ming Leung et al. J Infect Dis. 2004.

Abstract

The recent outbreak of severe acute respiratory syndrome (SARS) provided an opportunity to study the antibody response of infected individuals to the causative virus, SARS coronavirus. We examined serum samples obtained from 46 patients with SARS, 40 patients with non-SARS pneumonia, and 38 healthy individuals, by use of Western blotting (WB), enzyme-linked immunoassay (ELISA), and immunofluorescence assay, using both native and bacterially produced antigens of the virus. We found a highly restricted, immunoglobulin G-dominated antibody response in patients with SARS, directed most frequently (89% by ELISA) and predominantly at the nucleocapsid. Almost all of the subjects without SARS had no antinucleocapsid antibodies. The spike protein was the next most frequently targeted, but only 63% of the patients (by ELISA) responded. Other targets of the response identified by use of WB included antigens of 80 and 60 kDa. Several nonstructural proteins cloned were not antigenic, and the culture-derived nucleocapsid appeared to be specifically degraded.

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Figures

Figure 1.

Figure 1.

Identification of viral antigens that are reactive with severe acute respiratory syndrome (SARS) serum. A, Drawing of the SARS coronavirus structure showing the main antigens. B, Crude viral antigens obtained from culture cells were separated on gel and stained with Coomassie blue (CB). U, uninfected control cells. Lanes 1 and 2, Different antigen preparations showing slight variation in protein intensities at 36–48 kDa. Shown are results of Western blotting (WB) of preparation no. 2 or preparation U reacted with a serum sample from a patient with SARS showing the highly reactive antigens—nucleocapsid (N) 1, N2, and N3—in the former. MW, molecular weight markers (in kDa). C, WB resultsof 6 patients with SARS (S) and 4 patients with non-SARS pneumonia(P), showing strong reactivities of the N1–N3 antigens and lesser reactivities of the spike and the 80- and 60-kDa proteins or the partial reactivity (N1) or total lack of reactivity.

Figure 2.

Figure 2.

Comparison of the efficiency of various detection assays for severe acute respiratory syndrome (SARS). Individual serum samples from each group (46 patients with SARS, 40 patients with non-SARS pneumonia, and 38 healthy individuals) were examined by use of immunofluore scenceassay (IFA), Western blotting (WB), or ELISA. Antigens used included native spike (S), native nucleocapsid (N), crude antigen, crude viral extract, recombinant nucleocapsid (rNa), gel-purified native N2 and N3 antigens (pN2, 3), gel-purified native spike (pS), and recombinant spike (rSc). Except where marked “IgM”, all tests are based on detection of IgG. In each test, the cut off for positivity is shown by the shaded bar; for ELISA, this is based on the mean+1 SD value of the combined cohorts of patients with non-SARS pneumonia and healthy individuals. In WB, the intensity of the reaction was arbitrarily scored by eye (3, strongest). Serum samples that were not examined because of a lack of antigen or serum or because the results were not readable are shown by a dot.

Figure 3.

Figure 3.

Demonstration that nucleocapsid (N) 1-N4 are all N antigens. A, Western blotting (WB) of 2 serum samples from patients with severe acute respiratory syndrome (SARS) (nos. S35 and S44) against the crude viral extract in the presence of various antigens used as inhibitor. B, WB of serum samples obtained from 3 mice immunized with recombinant N (rNa)-GST (lanes 1, 2, and 3) or from a representative mouse immunized with recombinant spike (rSa)-GST or recombinant spike (rSc)-GST (lane S), against the crude viral extract. MW, molecular weight (in kDa); pN2,3, gel-purified native N2 and N3 antigens; rNSP, recombinant nonstructural protein; U, unimmunized mouse.

Figure 4.

Figure 4.

Performance of the recombinant nucleocapsid (rNa)—ELISA using serum samples from patients with severe acute respiratory syndrome (SARS). A, Comparison with the crude ELISA by regression analysis. B, Comparison with immunofluorescence assay (IFA). C, Two examples where both of the serum samples have similar ELISA but vastly different IFA titers. D, Relationship between stage of disease and test sensitivity.

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