Figure 1.
E16-opsonized WNV enters Vero cells.
(A) Vero cells were incubated with WNV at an MOI of 100 in the presence or absence of 100 μg/ml Alexa 488-E16 or E53 MAbs. Lysotracker red (50 nM) was added to the cells for the last 30 min of the incubation prior to paraformaldehyde fixation. Green staining indicates Alexa 488 conjugated anti-WNV MAbs (panels B, F, J, and N), red staining indicates lysotracker red dye (panels C, G, K, and O), and yellow staining represents co-localization as reflected by the merged images (panels D, H, L, and P). Cells were incubated for 2 h on ice, washed, fixed, and observed by confocal microscopy (panels A–H). Cells were shifted to 37°C following the incubation on ice, fixed after 15 min and observed by confocal microscopy (panels I–P). White arrows indicate examples of co-localization of anti-WNV MAbs and lysotracker red dye. (B) Cells were infected for 3 h at 37°C in the presence of 100 μg/ml E16 (B, D) or E53 (panels F and H) and lysotracker dye (panels C, D, G, and H) fixed, and analyzed by confocal microscopy. The scale bars represent 10 μm.
Figure 2.
Blockade of endosomal acidification with concanamycin A1 mimics treatment with E16.
Vero cells were infected at an MOI of 100 in the presence of 10 nM concanamycin A1 (panels A and D) or 100 μg/ml hE16 (panels B, C, E, and F) for 3 h (panels A–C) or 24 h (panels D–F) and then fixed. Cells were then stained with a pool of Alexa-488 conjugated mouse anti-E MAbs (blue; A-F) and Alexa-647-conjugated goat anti-human IgG (red; C and F) as indicated. Cells were analyzed by confocal microscopy. The white arrows indicate co-localization of hu-E16 and the oligoclonal pool of mouse anti-E MAbs. Representative images are shown from one of two independent experiments. The images were analyzed using the LSM510 confocal microscopy software to assess the overlap in staining. Because of the overlay appearance, we converted the fluorescence images into blue (Alexa-488) and red (Alexa-647) colors for Figure display. The scale bars represent 10 μm.
Figure 3.
E16 scFv or IgG blocks infection in a plasma membrane fusion assay.
WNV (106 PFU) was bound to Vero cells for 2 h on ice after pretreatment with 10 nM concanamycin A1. Subsequently, media, 100 μg/ml E16 IgG, E16 scFv or E60 IgG was added for 30 min on ice, and then the pH shifted at 37°C to pH 7.5 or pH 5.5 for ∼7 min. Cells were washed, the pH normalized, incubated at 37°C for ∼18 h, permeabilized and stained with an oligoclonal pool of anti-E MAbs. The level of infection was assessed by flow cytometry. (A) Representative flow cytometric histogram plots from each condition are shown. The plots are gated to show the percentage of cells that stained positive with an anti-WNV E MAb. The treatment and percentage of positive cells are shown in the top right corner of each plot. (B) The data averaged from three independent experiments is shown with error bars indicating standard deviations. Statistically significant differences between different experimental conditions are described in the text.
Figure 4.
E16 blocks low pH-induced fusion of WNV with liposomes.
Fusion of pyrene-labeled WNV with liposomes at pH 5.4. Fusion was measured in real-time as described in the Materials and Methods. (A) (a) no antibody; (b) 0.1 nM E16; (c) 1 nM E16; and (d) 50 nM E16. Representative viral fusion curves are from at least three independent experiments. (B) Effect of different concentrations of MAbs on WNV-liposome fusion. The WNV-liposome fusion profiles are shown as a percentage of the control (pH 5.4, without MAbs). Black bars, E16; gray bars, E60; and white bars, E111. Data are expressed as the mean of at least three independent experiments and the error bars indicate standard deviations.
Figure 5.
E16 Fab protects WNV from pH-induced inactivation in solution.
WNV (3×103 PFU) was incubated alone in the presence of media, 100 μg/ml E16 Fab, E60 Fab or E9 Fab for 30 min on ice. The reaction was diluted 5-fold in media at pH 7.5 or pH 5.5 and incubated at 37°C for 15 min, and then back-neutralized with a 25-fold excess of media at pH 7.5. This mixture was added to a monolayer of Vero cells prior to an overlay with 2% agarose. Three days later, plaques were fixed and scored. The dotted line represents the lower limit of detection the assay (2×101 PFU). Data is expressed as the mean of three separate experiments performed in duplicate. Statistical significance is indicated in the graph and was calculated using a two-tailed paired t test.
Figure 6.
Model of anti–WNV MAb neutralization.
The neutralization of WNV infection in Vero cells occurs by different mechanisms depending on the epitopes occupied by the MAbs. WNV infection in the absence of antibodies results in attachment, endocytosis, fusion, uncoating and release of the viral RNA into the cytoplasm. Vero cell infection in the presence of neutralizing concentrations of the fusion loop MAbs E53 or E60 results in a blockade in viral attachment. In contrast, infection in the presence of E53 or E60 at sub-neutralizing concentrations allows for efficient attachment, entry, fusion and infection. Infection in the presence of neutralizing concentrations of E16 or E24 (which require lower fractional occupancy for neutralization) results in relatively normal attachment and endocytosis. However, these MAbs inhibit fusion of the viral membrane with the endosomal membrane leading to subsequent targeting of the virus particles to the lysosome.