Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity

Abstract

Both viral effect and immune-mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type-2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non-SARS-pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti-epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross-reactive epitopes on SARS-CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti-S2 Abs and IFN-gamma resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti-S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS-CoV infection may be implicated in SARS pathogenesis.

Fig. 2

Epithelial cell cytotoxicity caused by SARS patient sera. Culture supernatants were obtained from A549 cells incubated with a 1 : 20 dilution of patient sera for 72 h. The levels of A549 cell cytotoxicity were determined by the lactate dehydrogenase (LDH) activity, and (a) the relative OD and (b) the percentage of cytotoxicity are shown. Relationship between anti-A549 cell IgG (c), IgM (d), or IgA (e) levels of SARS patients as determined by percentage cell binding and cell cytotoxicity as determined by LDH activity.

Fig. 3

(a) The locations of four synthetic peptides on SARS-CoV spike protein sharing sequence homology with human proteins are indicated. (b) Binding ability of SARS patient sera to spike peptides. SARS patient sera (from a pool of five patients) or healthy control sera (from a pool of five healthy controls) were 1 : 20 diluted and added to the wells coated with spike protein S2 domain, D02, D03, D07, or D08 peptides. ELISA was performed for the detection of the binding activity of patient or healthy control sera to these peptides. Experiments were carried out in duplicate, and the averages are shown. (c) Blockage of A549 cell binding ability by spike peptides. SARS patient sera or healthy control sera were preabsorbed with 10 µg S2, D02, D03, D07, or D08 peptides overnight at 4 °C before incubated with A549 cells. The binding activity of patient or healthy control sera to A549 cells was determined by flow cytometry. Experiments were carried out in duplicate, and the averages are shown. *P < 0·05, **P < 0·01, ***P < 0·001.

Fig. 5

(a) Effect of anti-S2 Abs and IFN-γ on PBMC adherence to epithelial cells. A549 cells were cultured in 8-well glass chamber slides and treated with 25 ng of IFN-γ for 24 h. Cells were washed with PBS, and then cocultured with fresh human PBMC for 1 h in the presence or absence of 1 : 50 dilution of anti-S2 hyperimmune sera. Cell adhesion was observed using Liu's stain. The numbers of adherent cells were quantified in each tested culture. (b) Blockage of PBMC adherence to epithelial cells by spike peptides. Anti-S2 hyperimmune sera were preabsorbed with 10 µg S2, D02, D03, D07, or D08 peptides at room temperature for 2 h before incubated with A549 cells. After 1 h at 37 °C, fresh human PBMC were added and the rest of procedures were followed as described in (a). Experiments were carried out in triplicate, and the averages are shown. *P < 0·05, ***P < 0·001.

Fig. 1

Anti-A549 cell autoAbs in SARS patient sera. Human A549 epithelial cells were incubated with a 1 : 20 dilution of sera from SARS patients with different days of fever onset (n = 140), non-SARS-pneumonia patients (n = 8), or healthy controls (n = 10), followed by FITC-conjugated anti-human IgG, IgM, or IgA, and then observed by fluorescent microscopy (a) or analysed by flow cytometry (b). The percentages of A549 cells that reacted with patient or control sera are shown.

Fig. 4

The epithelial cell cross-reactivity of anti-spike Abs. A549 cells (a, b) and HL cells (c) were incubated with a 1 : 50 dilution of anti-S, S1, or S2 mouse hyperimmune sera, followed by FITC-conjugated anti-mouse IgG, and then analysed by flow cytometry (a, c) or viewed with confocal microscopy (b). The normal mouse sera were used as the negative control. MFI: mean fluorescence intensity.