Development of a safe neutralization assay for SARS-CoV and characterization of S-glycoprotein

Abstract

The etiological agent of severe acute respiratory syndrome (SARS) has been identified as a novel coronavirus SARS-CoV. Similar to other coronaviruses, spike (S)-glycoprotein of the virus interacts with a cellular receptor and mediates membrane fusion to allow viral entry into susceptible target cells. Accordingly, S-protein plays an important role in virus infection cycle and is the primary target of neutralizing antibodies. To begin to understand its biochemical and immunological properties, we expressed both full-length and ectodomain of the protein in various primate cells. Our results show that the protein has an electrophoretic mobility of about 160-170 kDa. The protein is glycosylated with high mannose and/or hybrid oligosaccharides, which account for approximately 30 kDa of the apparent protein mass. The detection of S-protein by immunoassays was difficult using human convalescent sera, suggesting that the protein may not elicit strong humoral immune response in virus-infected patients. We were able to pseudotype murine leukemia virus particles with S-protein and produce SARS pseudoviruses. Pseudoviruses infected Vero E6 cells in a pH-independent manner and the infection could be specifically inhibited by convalescent sera. Consistent with low levels of antibodies against S-protein, neutralizing activity was weak with 50% neutralization titers ranging between 1:15 to 1:25. To facilitate quantifying pseudovirus-infected cells, which are stained blue with X-Gal, we devised an automated procedure using an ELISPOT analyzer. The high-throughput capacity of this procedure and the safety of using SARS pseudoviruses should make possible large-scale analyses of neutralizing antibody responses against SARS-CoV.

Fig. 1

Construction of SARS-CoV S glycoprotein expression vectors. (A) Genomic organization of SARS-CoV. Only the major nonstructural (replicase ORF1a and 1b) and structural genes (S, E, M, and N) are illustrated. The signal peptide (SP) and transmembrane (TM) domains of the S-protein, and the locations of 23 potential N-linked glycosylation sites, are indicated. Histidine-tagged ectodomain of S-protein is indicated as eS-His. (B) A cloning strategy for expressing S glycoprotein. See Materials and methods for details.

Fig. 2

Analyses of SARS-CoV S-protein glycosylation. (A) HeLa cells transfected with either pcDNA (empty vector), pcDNA-S, or pTM-S were infected with vTF7-3. S-protein (triangle) was detected by Western blot using convalescent sera. Vaccinia-virus-specific bands are indicated by VV. Ten percent acrylamide gel was used. (B) Histidine-tagged ectodomain of S-protein expressed in HeLa cells was either untreated (lane 2) or treated with Endo-H or PNGaseF glycosidases (lanes 3 and 4, respectively). The protein was detected by Western blot with anti-6×His antibody. No band was detected from cells transfected with an empty vector (lane 1). Acrylamide gradient gel (4–12%) was used.

Fig. 3

SARS-CoV pseudovirus infection. (A) Infectivity of SARS pseudoviruses in Vero E6 cells. Infected cells are stained with X-Gal. Mock-infected cells are shown on the top. Infectivity of SARS pseudoviruses was unaffected by lysosomotropic agents chloroquine (B) and by NIH₄Cl (C). In contrast, VSV-G pseudovirus infection was inhibited by both agents.

Fig. 4

Pseudovirus neutralization assay. (A) Three different pseudoviruses (HIV 1, VSV-G, and SARS-S) were incubated with either normal serum, or two convalescent sera from patients 703497 and 56053 (1:5 dilution). Neutralizing activity was observed only from convalescent sera from SARS-CoV-infected patients against SARS pseudovirus. HIV-1 pseudovirus infection was done with HOS-CCR5-CD4 cells while VSV-G and SARS-S pseudovirus infections were done with Vero E6 cells. (B) Titration of neutralizing antibodies. Convalescent sera from seven different SARS-CoV patients were used to titer pseudovirus-neutralizing activity. Approximately 70 infectious units were used.

Fig. 5

Automation of counting pseudovirus-infected cells using an ELISPOT reader. HOS cells were infected with twofold serially diluted pseudovirus (10–0.625 μl inoculum) in duplicate, in 96-well plate. MuLV pseudotyped with VSV-G was used. (A) Images of wells infected with five different dilutions of pseudovirus, in duplicate. (B) A magnified image of the boxed well. (C) The number of infectious foci is plotted as a function of virus inoculum.