Figure 1.

Anti-DC-SIGN antibodies and soluble DC-SIGN inhibit the ability of dengue virus type 1 to infect DC-SIGN-expressing monocyte-derived dendritic cells. (A) The expression levels of the cell-surface markers CD1a, CD14, langerin and DC-SIGN on immature human monocyte-derived dendritic cells (MDDCs) were assessed by direct fluorescence. DC-SIGN-expressing cells were detected using the anti-DC-SIGN monoclonal antibody, 1B10. The relative fluorescence intensity was measured by FACScan analysis and the histograms show the binding of the specific antibody (grey) and isotype-matched control antibody (black). (B) MDDCs were infected for 40 h with dengue virus (DV)-1 at a multiplicity of infection of 5 and were immunostained with anti-DV-1-specific hyperimmune mouse ascites fluids (HMAF) or anti-DV-1 NS1 monoclonal antibody (anti-NS1), and were observed using a fluorescent microscope. (C) Before infection, MDDCs were mock-treated (control) or incubated with 20 µg ml⁻¹ monoclonal antibody 1B10 (DC-SIGN) or a 1:50 dilution of anti-DV E monoclonal antibody 9D12 (E) in RPMI, 0.2% BSA, for 20 min at 25 °C. Treated MDDCs were infected for 2 h at 37 °C in the continuous presence of antibodies. DV antigens were visualized by an immunofluorescence assay using anti-DV-1-specific HMAF. (D) Before infection, MDDCs were mock-treated (control) or were incubated with 20 µg ml⁻¹ anti-LCMV (lymphocytic choriomeningitis virus) monoclonal antibody (BD12.5), anti-DC-SIGN monoclonal antibody (8A5) or anti-DC-SIGN monoclonal antibody (1B10). FGA/NA d1d virus (1 × 10⁵ AP61 focus-forming units (FFU)) was mixed with 10 µg ml⁻¹ sDC-SIGN in RPMI, 0.2% BSA, for 20 min at 25 °C. MDDCs were infected for 2 h at 37 °C in the continuous presence of inhibitors. Infectious virus particles produced in the supernatants were titred. Each experimental point represents the mean ± s.d. of results obtained from three chambers. Magnification in (B), ×100. DC-SIGN, dendritic-cell-specific ICAM3-grabbing non-integrin; sDC-SIGN, the soluble, tetrameric ectodomain of DC-SIGN.