Fig. 2: In vitro activity of DNA-VLPs. | Nature Communications

Fig. 2: In vitro activity of DNA-VLPs.

From: Enhancing antibody responses by multivalent antigen display on thymus-independent DNA origami scaffolds

Fig. 2

a An overview schematic of the in vitro activity assays and corresponding DNA-VLPs tested. b, c ACE2-expressing HEK 293 cells were incubated with 200 nM RBD. Binding was detected in flow cytometry experiments using PE-labeled CR3022 and a PE-labeled secondary antibody, demonstrating preserved binding activity for chemically modified monomeric RBD-Cy5 compared to monomeric RBD. Representative histograms are shown for ACE2 binding assays from n = 3 biological replicates, and median fluorescent intensity (MFI) values were determined from n = 3 biological replicates. d, e Incubation with Cy5-labeled DNA-VLP-Cy5-30x at 100 nM RBD revealed enhanced binding compared to monomeric RBD-Cy5, likely due to multivalency effects. No unspecific binding for non-functionalized DNA-VLP-Cy5 was observed. The brightness of Cy5-labeled DNA-VLP-Cy5-30x (5 Cy5 per 30 RBDs) and RBD-Cy5 (1 Cy5 per 1 RBD) were quantified experimentally and MFI values were corrected accordingly. Representative histograms are shown for ACE2 binding assays from n = 3 biological replicates, and MFI values were determined from n = 3 biological replicates. f, g Ramos B cells expressing the BCRs CR3022 and B38 were incubated with α-IgM, monomeric RBD, or DNA-VLPs at 30 nM RBD. Ca2+ flux in response to RBD incubation was assayed using Fura Red. Representative fluorescence intensity (FI) curves are shown from n = 3 biological replicates (top). Total Ca2+ flux was quantified via the normalized area under the curve (AUC) (bottom). Normalized AUC values were determined from n = 3 biological replicates. Error bars represent the standard error of the mean. Two-sided Welch’s t-test was performed at α = 0.05 for (c). One-way ANOVA was performed followed by Dunnett’s T3 multiple comparison test at α = 0.05 for (e, f, and g). For (e), Left: p = 0.0006 for Monomer-Cy5:DNA-VLP-Cy5-0x, p = 0.0161 for Monomer-Cy5:DNA-VLP-Cy5-30x, p = 0.0042 for DNA-VLP-Cy5-0x:DNA-VLP-Cy5-30x; Right: p < 0.0001 for Monomer-Cy5:DNA-VLP-Cy5-0x, p = 0.0036 for Monomer-Cy5:DNA-VLP-Cy5-30x, p = 0.0029 for DNA-VLP-Cy5-0x:DNA-VLP-Cy5-30x. For f, p = 0.0170 for Monomer:DNA-VLP-6x, p = 0.0020 for DNA-VLP-0x:DNA-VLP-6x, p = 0.0038 for DNA-VLP-1x:DNA-VLP-6x, p = 0.0085 for Monomer:DNA-VLP-30x, p = 0.0016 for DNA-VLP-0x:DNA-VLP-30x, p = 0.0019 for DNA-VLP-1x:DNA-VLP-30x, p = 0.0080 for DNA-VLP-6x:DNA-VLP-30x. For (g), p = 0.0448 for Monomer:DNA-VLP-6x, p = 0.0368 for DNA-VLP-0x:DNA-VLP-6x, p = 0.0156 for Monomer:DNA-VLP-30x, p = 0.0213 for DNA-VLP-0x:DNA-VLP-30x, p = 0.0264 for DNA-VLP-1x:DNA-VLP-30x, p = 0.0131 for DNA-VLP-6x:DNA-VLP-30x. *p < 0.05; **p < 0.01; ***p < 0.001.

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