Neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection

Abstract

Background: Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the principal etiologic agent of SARS. We analyzed serum samples obtained from 623 patients with SARS in Beijing, to determine whether infection with SARS-CoV can elicit neutralizing antibodies (NAbs).

Methods: We developed a highly sensitive and safe neutralization assay using the SARS-CoV pseudotyped virus and used this assay to determine the titers of the NAbs in serum samples from patients with SARS.

Results: We found that 85.9% of serum samples contained NAbs against SARS-CoV and that most of the NAb activities could be attributed to immunoglobulin G. The NAbs became detectable first at 5-10 days after the onset of symptoms, and their levels peaked at 20-30 days and then were sustained for >150 days. The serum samples could neutralize the pseudotype particles bearing the spike glycoproteins from different SARS-CoV strains, suggesting that the NAbs to SARS-CoV were broadly reactive.

Conclusions: NAbs to SARS-CoV are broadly elicited in patients with SARS and, according to their kinetics, may correlate with viral load during the early stages of the disease. These results suggest that it is possible to develop effective vaccines against SARS and that NAbs provide a potential strategy for treating patients with SARS.

Figure 1

Detection of neutralizing antibodies in serum samples from a patient with severe acute respiratory syndrome (SARS) that can block the HIV-luc/SARS pseudovirus from entering and infecting Huh7 cells. Note the ability of the serum sample from the patient with SARS (PS) to neutralize and block the infectivity of HIV-luc/SARS (SARS); the absence of such blocking activity in normal serum (NS); and the lack of neutralizing activity of the SARS serum sample against the pseudotyped virus bearing the G protein of the vesicular stomatitis virus (VSV-G), which could infect the target cells at a level similar to that of HIV-luc/SARS. Data are the average of triplicate determinations. Bar, SD.

Figure 2

Detection of spike (S) protein-specific antibodies in serum samples, with flow cytometric analysis. C50 and C81 are 2 normal serum samples, and D73-D158 are 10 randomly selected serum samples from patients with severe acute respiratory syndrome. Data of flow cytometric analysis were analyzed with the Summit V3.1 program (Dako Cytomation). FITC, fluorescein isothiocyanate; HeLa, normal HeLa cells used as a control; S-HeLa, S protein-expressing HeLa cells.

Figure 3

Kinetics of generation of neutralizing antibodies (NAbs) in serum samples obtained from patients with severe acute respiratory syndrome (SARS). A, Analysis of the NAbs in sequential serum samples obtained from 4 patients (P1, P2, P3, and P4). B, Analysis of NAbs in serum samples obtained from 623 patients with SARS. Each point on the curve contains 18 samples, and the entire curve contains data derived from the 623 serum samples. The titers of the NAbs, expressed in terms of ID₅₀, are plotted against days after the onset of symptoms. The titer at day 0 is the mean ID₅₀ (88) from 153 normal human serum samples.

Figure 4

Neutralizing abilities of 4 serum samples from patients with severe acute respiratory syndrome (SARS) (PS1, PS2, PS3, and PS4) to the pseudotype viruses bearing the spike protein from 4 different SARS-associated coronavirus strains (BJ01, Frankfurt, TOR2, and TW1). Similar results were obtained in 3 independent experiments.

Figure 5

Neutralization caused by IgG in serum samples from patients with severe acute respiratory syndrome (SARS). A, Comparison of the neutralizing abilities of serum samples from patients with SARS, with or without IgG, using the neutralization assay. Serum samples from 3 patients (PS5, PS6, and PS7) were tested; “PS5+,” “PS6+,” and “PS7+ ”denote the same serum sample after IgG depletion. B, Neutralization assay of purified IgG from pooled positive serum samples. Similar results were obtained in 3 independent experiments

Table 1

Titers of neutralizing antibodies in 12 serum samples analyzed with pseudovirus and live severe acute respiratory syndrome-associated coronavirus (SARS-CoV) neutralization assays.