Antibody recognition of cell surface-associated NS1 triggers Fc-gamma receptor-mediated phagocytosis and clearance of West Nile Virus-infected cells

Abstract

Previous studies have suggested that monoclonal antibodies (MAbs) to flavivirus nonstructural protein-1 (NS-1) protect against infection in mice through an Fc-gamma receptor-dependent pathway. To identify a specific mechanism, we evaluated the protective activity of anti-NS1 MAbs to WNV using mice and cells with deficiencies of specific Fc-gamma receptors. Our results suggest that only MAbs that recognize cell surface-associated NS1 trigger Fc-gamma receptor I- and/or IV-mediated phagocytosis and clearance of WNV-infected cells. These findings may be relevant for generating novel therapeutic MAbs or vaccines against flaviviruses that target the NS1 protein.

FIG. 1.

Efficacy of 10NS1 MAb in C1q^−/−, Fc-γ receptor I^−/−, III^−/−, and IV^−/−, Fc-γ receptor III^−/−, and NK cell-depleted mice. C1q^−/− (A), Fc-γ receptor I^−/−, III^−/−, and IV^−/− (B), or Fc-γ receptor III^−/− (C) C57BL/6 mice were inoculated via footpad with 10² PFU of WNV on day 0. On the same day, mice were administered PBS or a single dose of 10NS1 MAb (500 μg) by an intraperitoneal route. The difference in survival curves between antibody and PBS treatments was statistically significant for the C1q^−/− (n = 20, P < 0.0001) and Fc-γ receptor III^−/− mice (n = 15, P < 0.0001) but not for Fc-γ receptor I^−/−, III^−/−, and IV^−/− mice (n = 13, P = 0.6). (D) NK cells were depleted from wild-type mice after treatment with 150 μg of anti-NK1.1 MAb 2 days before and after infection. Depletion of NK cells was confirmed as >99% by flow cytometry of peripheral blood lymphocytes. Mice were infected with WNV and treated with 10NS1 or PBS as described above. There was no statistically significant difference in mortality between 10NS1-treated, NK-depleted, and nondepleted mice (n = 30, P = 0.8). The survival curves were constructed from two to three independent experiments.

FIG. 2.

Flow cytometry histograms showing immunoreactivity of NS1 on the surface of yeast, within permeabilized Raji-WNV-Rep cells, and on the surface of Raji-WNV-Rep cells with individual anti-NS1 MAbs. Representative histograms are shown for four NS1 MAbs (8NS1, 15NS1, 10NS1, and 17NS1). The E1 MAb against WNV E protein (19) was used as a negative control. WNV NS1 immunostaining was performed as described previously (6). Intracellular and cell surface-associated NS1 levels on Raji-WNV-Rep were measured in permeabilized or nonpermeabilized cells, respectively. Green and red arrows indicate the absence or presence, respectively, of binding of MAbs to cell surface-associated NS1. The data shown are representative of several independent experiments.

FIG. 3.

Anti-NS1 MAb-dependent phagocytosis by peritoneal macrophages. (A) Thioglycolate-elicited peritoneal macrophages were isolated after peritoneal lavage from wild-type, Fc-γ receptor I^−/−, III^−/−, and IV^−/−, or Fc-γ receptor III^−/− mice and adhered (5 × 10⁵ cells) on poly-d-lysine and laminin-coated coverslips. NS1-expressing Raji-WNV-Rep target cells (2 × 10⁶) labeled with CFSE were opsonized with 10NS1 or 15NS1 and incubated with wild-type or Fc-γ receptor-deficient peritoneal macrophages for 1 h at 37°C. After being washed, cells were fixed with paraformaldehyde, stained with Alexa-555-conjugated cholera toxin subunit B, and imaged by confocal microscopy. The yellow and white arrows indicate CFSE-labeled Raji-WNV-Rep target cells bound to and engulfed by macrophages, respectively. (B) Quantitation of the percentage of bound or phagocytosed target cells by wild-type peritoneal macrophages with different anti-NS1 MAbs. Ten random fields were counted (×63 magnification) per antibody treatment, resulting in more than 600 total cells counted. The difference in number between 10NS1, 16NS1, 17NS1, and 6NS1 or 15NS1 treatment was statistically significant for bound (P ≤ 0.0001) and phagocytosed (P ≤ 0.0001) target cells. (C) Quantitation of the number of bound or engulfed target cells by Fc-γ receptor I^−/−, III^−/−, or IV^−/− (left) or Fc-γ receptor III^−/− (right) peritoneal macrophages with 10NS1 or 15NS1 MAbs. The difference in the percentage of bound or phagocytosed Raji-WNV-Rep target cells between 10NS1 and 15NS1 was not different for Fc-γ receptor I^−/−, III^−/−, and IV^−/− macrophages but was for Fc-γ receptor III^−/− macrophages (P < 0.0001).