Mannose-binding lectin in severe acute respiratory syndrome coronavirus infection

Abstract

Little is known about the innate immune response to severe acute respiratory syndrome (SARS) coronavirus (CoV) infection. Mannose-binding lectin (MBL), a key molecule in innate immunity, functions as an ante-antibody before the specific antibody response. Here, we describe a case-control study that included 569 patients with SARS and 1188 control subjects and used in vitro assays to investigate the role that MBL plays in SARS-CoV infection. The distribution of MBL gene polymorphisms was significantly different between patients with SARS and control subjects, with a higher frequency of haplotypes associated with low or deficient serum levels of MBL in patients with SARS than in control subjects. Serum levels of MBL were also significantly lower in patients with SARS than in control subjects. There was, however, no association between MBL genotypes, which are associated with low or deficient serum levels of MBL, and mortality related to SARS. MBL could bind SARS-CoV in a dose- and calcium-dependent and mannan-inhibitable fashion in vitro, suggesting that binding is through the carbohydrate recognition domains of MBL. Furthermore, deposition of complement C4 on SARS-CoV was enhanced by MBL. Inhibition of the infectivity of SARS-CoV by MBL in fetal rhesus kidney cells (FRhK-4) was also observed. These results suggest that MBL contributes to the first-line host defense against SARS-CoV and that MBL deficiency is a susceptibility factor for acquisition of SARS.

Figures

Figure 1

Serum levels of mannose-binding lectin (MBL), for MBL genotypes with or without the mutant allele B in patients with severe acute respiratory syndrome (SARS), 19–23 days after the onset of disease. The horizontal bars indicate the medians for each genotype subgroup. Individuals with undetectable serum levels of MBL (<0.053 μg/mL) are not shown

Figure 2

Binding of mannose-binding lectin (MBL) to severe acute respiratory syndrome (SARS) coronavirus (CoV)–infected FRhK-4 cells. Binding of MBL to SARS-CoV–infected FRhK-4 cells was observed (A and B). Convalescent serum samples from patients with SARS with confirmed SARS-CoV antibody seroconversion were also tested, and binding of specific IgG to SARS-CoV–infected FRhK-4 cells was detected (C and D). Both MBL and anti–SARS-CoV antibody from serum samples from patients with SARS did not bind uninfected FRhK-4 cells (data not shown). Confocal imaging showed that the binding by MBL mostly occurred on the surface of the infected cell (B) and that the binding by specific IgG from serum samples from patients with SARS occurred on both the surface and in the cytoplasm of the infected cell (D). Original magnifications, ×200 (A and C) and ×600 (B and D)

Figure 3

Binding of mannose-binding lectin (MBL) to severe acute respiratory syndrome (SARS) coronavirus (CoV). Binding of MBL to SARS-CoV was determined by use of a microtiter capture assay. Wells of microtiter plates were coated with SARS-CoV, and MBL was incubated for 2 h in the virus-coated wells. A Amount of MBL. Incubation with increasing amounts of MBL resulted in increasing levels of detected MBL bound to immobilized SARS-CoV (Δ). In contrast, MBL binding did not increase when the wells were coated with bovine serum albumin (▪). B Amount of SARS-CoV. The amount of MBL binding was also dependent on the amount of SARS-CoV used to coat the wells. C Presence of Ca2+, EDTA, and/or mannan. To determine the nature of binding of MBL to SARS-CoV, MBL was incubated in wells coated with SARS-CoV in the presence of either Ca2+ or EDTA. The binding of MBL to SARS-CoV was reduced to background levels in the presence of EDTA, indicating that the interaction of MBL with SARS-CoV was Ca2+ dependent. Preincubation of MBL with 2 mg/mL mannan in the presence of Ca2+ also substantially reduced binding of MBL to SARS-CoV. These data provide evidence that MBL bound to immobilized SARS-CoV via its carbohydrate recognition domains. Results are presented as mean ± SE and are representative of 4 experiments

Figure 4

Mannose-binding lectin (MBL)–mediated deposition of complement C4 on severe acute respiratory syndrome (SARS) coronavirus (CoV). Different concentrations of MBL were incubated overnight at 4°C in microtiter plates coated with SARS-CoV. A consistent amount of C4 protein (4 μg/mL) was then incubated for 1.5 h at 37°C in the wells. Incubation with increasing amounts of MBL resulted in increasing deposition of complement C4 on SARS-CoV (○). In contrast, deposition of complement C4 did not increase when wells were coated with bovine serum albumin (▪). Results are presented as mean ± SE and are representative of 2 experiments

Figure 5

Inhibition of severe acute respiratory syndrome (SARS) coronavirus (CoV) infection. SARS-CoV pretreated with different concentrations of mannose-binding lectin (MBL) was added to confluent layers of FRhK-4 cells. After 1.5 h of absorption, the cells were washed and incubated with fresh medium. The infectivity of SARS-CoV was determined by real-time quantitative polymerase chain reaction specific to the 1b gene region of SARS-CoV, with extraction of viral RNA from the culture supernatant after 24 h of incubation. Increasing amounts of MBL resulted in decreasing the infectivity of SARS-CoV. Results are representative of 4 experiments