Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis.

Keywords: Anti-spike antibody; Antibody-dependent enhancement; Fc receptors; HL-CZ; SARS-CoV.

Fig. 1

HL-CZ cells are susceptible to SARS-CoV infection. After infecting cells with SARS-CoV, culture supernatant and cell lysates were collected for (A) SARS-CoV detection and (B) cellular cytokine mRNA expression. (C) Results from HL-CZ cytopathic effect observation and immunofluorescence staining 2 d post-infection. (D) Supernatants from mock and SARS-CoV-infected HL-CZ cells were used to infect Vero-E6 cells for 2 d. Results are from one representative experiment of the three performed.

Fig. 2

HL-CZ cells express ACE2 and Fc receptors, and display antibody-dependent enhancement. (A) Results from flow cytometry analyses of Fc receptors and ACE2 expression in HL-CZ cells. (B) SARS-CoV or mock infections in the presence of various dilutions of anti-sera drawn from SARS-CoV patients. (C) Transmission electron microscopy images of HL-CZ cells infected with SARS-CoV in the presence of various dilutions of anti-sera against SARS-CoV. Images 1 and 3 indicate 10-, and 2000-fold dilutions of anti-sera against SARS-CoV, respectively. Image 2 indicates 10-fold dilutions of normal sera control treatment.

Fig. 3

Antibody-dependent enhancement of SARS-CoV infection is mediated by anti-spike protein antibodies. (A) Results from infectivity assays using HL-CZ cells infected with SARS-CoV and treated with various dilutions of anti-sera collected from SARS-CoV patients. (B) Apoptosis analyses of HL-CZ cells infected with SARS-CoV in the presence of 1000-fold diluted normal sera and anti-SARS-CoV sera (Annexin V staining). (C) HL-CZ cells infected with SARS-CoV in the presence of various dilutions (10-, 100-, 1000- or 2000-fold) of mouse anti-spike protein serum and isotype control mouse serum. (D) HL-CZ cells infected with SARS-CoV in the presence of different dilutions (10-, 100-, 1000- or 2000-fold) of mouse anti-nucleocapsid serum and isotype control mouse serum. C, normal sera (control); S, anti-SARS sera (sample). Shown are results from one representative experiment of three performed (^∗p < 0.05; ^∗∗p < 0.01).

Fig. 4

Monoclonal antibodies against the SIa regions of spike proteins show antibody-dependent enhancement capability. (A) Isotype and epitope mapping of monoclonal antibodies against SARS-CoV spike proteins (NI^∗, not identified; bold, conservative amino acid residues). Lower part, fragment scheme of spike proteins used to generate monoclonal antibodies. (B) Evaluations of ADE effects of SARS-CoV-pseudotyped virus infections of HL-CZ cells in the presence of the monoclonal antibodies listed in (A). (C) Infectivity assays were performed using SARS-CoV-infected HL-CZ cells treated with SIb4, SIb2, or isotype control antibodies. (D) Cytopathic effects observed in HL-CZ cells treated with SIb4, SIb2, or isotype control antibodies. Presented results are from one representative experiment of three performed (^∗p < 0.05; ^∗∗p < 0.01).