Fig. 2

Characterization of hybrid flagellin filaments. a The top panel represents molecular structure of the parent FliC filament13 (left panel: XY view and middle panel: Z view). Negative stain TE micrograph of WT FliC filament generated by recombinantly produced FliC (right panel); FliC filament diameter was calculated to be 20 nm. The bottom panel represents a hypothetical molecular model of hFliC, where DENV2 + GS linkers were modeled into native FliC filament to replace D3 domain. The diameter of hFliC was computationally determined to be 35 nm. Negative stain TE micrographs confirmed resulting hFliC filaments had increased diameter compared to FliC filament. b Binding ELISA was performed using DENV2 specific monoclonal IgA antibody. hFliC showed binding to monoclonal IgA, compared to FliC alone, which showed no binding. Experiments were performed in triplicate (n = 3), and error bars represent mean +/- SEM, Student’s t-test was used to determine statistics, and a p < 0.05 were considered statistically significant. c Immunogold particle labeling against DENV2 E specific IgG (primary antibody, isolated from 4G2 culture supernatant), was used to determine the placement of the DENV2 E portion relative to the rest of the filament. Negative stain transmission electron micrograph of immunogold labeling of WT FliC filaments (left panel), confirming that no immunogold particles bound to WT filaments. Right panel is a negative stain transmission electron micrograph of immunogold labeling of hFliC filaments, which indicated that the immunogold particles primarily associate with the outside of the hFliC filament