Journal: bioRxiv
Article Title: Human antibodies against West Nile and related orthoflaviviruses
doi: 10.64898/2026.04.02.715800
Figure Lengend Snippet: (A) IgG binding to WNV EDIII by serial serum dilution. ELISA curves for samples from the 72 WND/WNF-suspected individuals and 3 orthoflavivirus naïve controls (black) are shown. The average of two independent experiments is shown. (B) Serum neutralization screening with WNV RVP . Shown is the rank-ordered NanoLuc activity relative to no-serum control for the 72 WND/WNF-suspected individuals and 1 orthoflavivirus naïve control (black); lower values correspond to higher neutralization. Samples with relative NanoLuc signal below 0.3 (dotted line) were selected for neutralization curves in (C). Each bar represents and individual participant sample analyzed at 1:100 dilution. Average signal of triplicate wells from a single experiment. (C) Neutralization of WNV RVP by serial serum dilution. Shown is the NanoLuc activity relative to no-serum control for 36 WND/WNF cases and 1 orthoflavivirus naïve control (black). Mean ± SD of triplicates. Representative of two independent experiments. (D-F) Identification of serum autoantibodies to IFN-α2 and IFN-ω. Plots compare the ability of serum IgG to bind, and of serum to neutralize, IFN-α2 (D) and IFN-ω (E) (n=39; only samples for which sufficient serum was available were assayed). The comparison of IFN-α2 and IFN-ω neutralization is shown in (F). Binding is shown as relative Mean Fluorescence Intensity (MFI) and neutralization as relative ISG15-promoter driven luciferase signal compared to no serum control. The dotted lines indicate the threshold for positivity of the assay. , Representative of 2 independent experiments. (G) Age and gender distribution of the study participants with or without IFN-α2 and/or IFN-ω neutralizing autoantibodies. Welch’s test. (H and I) Serum IgG binding to WNV EDIII and serum neutralization of WNV RVP in study participants with or without IFN-α2 and/or IFN-ω neutralizing autoantibodies. The two groups are compared with respect to (H) IgG binding to WNV EDIII (Area Under the Curve (AUC) of ELISA and (I) neutralization of WNV RVP (NT 50 values). Horizontal lines indicate the mean. Mann-Whitney test. In (A to C), green, blue and red indicate samples from WNV-infected individuals from which antibodies were derived (Figure S2B and C). In (D to I), female is circle, male is square, dark blue neutralizes both IFN-α2 and IFN-ω, light blue neutralizes IFN-α2 only, grey does not neutralize either.
Article Snippet: Serum samples were diluted 1:20 in OptiMEM (Optimized Minimal Essential Medium; Gibco, 31985070) containing 0.01 ng/mL IFN-α2 (Novus Biologicals, NBP2-34971) or 0.02 ng/mL IFN-ω (Novus Biologicals, NBP2-35893), together with a live-cell Renilla luciferase substrate (EnduRen, Promega, E6481; 1:10,000), and incubated for 1 hour with constant shaking at 600 rpm.
Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Activity Assay, Control, Comparison, Fluorescence, Luciferase, MANN-WHITNEY, Infection, Derivative Assay
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