Fig. 1: Immunogenicity of circRNAs encoding EDIII, EDIII-Fc, or EDIII-Fd. | Nature Communications

Fig. 1: Immunogenicity of circRNAs encoding EDIII, EDIII-Fc, or EDIII-Fd.

From: A single-dose circular RNA vaccine prevents Zika virus infection without enhancing dengue severity in mice

Fig. 1

a Schematic diagram of PIE (Ana)-mediated RNA circularization. Coding sequences for EDIII, EDIII-Fc and EDIII-Fd were fused with tPA signal sequence at the 5’ termini and cloned into circRNA plasmids (Supplementary Fig. 1a). circRNAs were produced by IVT followed by circularization in the presence of GTP and Mg2+. b Schematic diagram of immunization assay. c, d Anti-E IgG titers at 7 (c) or 14 (d) days after immunization were measured by ELISA. Titers were calculated as the reciprocals of the highest dilutions at which the optical density values at 450 nm (OD450) were equal to or higher than cut-off values. Limits of detection (LODs) are 100 and marked by gray dashed lines. e anti-ZIKV nAb titers at 14 days after immunization were measured by flow cytometry-based neutralization test (FNT) and calculated as the dilutions at which the percentages of ZIKV-positive cells were reduced to 50% of negative controls. LOD is 50. f–h Frequencies of Tfh cells in CD4+ T cells (f), activated GL7+ GC B cells in CD38- B cells (g) and plasma cells in lymphocytes (h) in the ILNs. At 14 days after immunization, ILN lymphocytes were isolated, labeled with surface marker antibodies, and measured by flow cytometry. i, j Frequencies of CD4+ T (i) and CD8+ T (j) cells secreting IFN-γ, TNFα, or IL-2. At 14 days after immunization, splenic lymphocytes were isolated, stimulated with ZIKV E peptide pools, and measured by intracellular cytokine staining assays. Box plots (c–e) indicate median (middle line), 25-75 percentile (box), 5-95 percentile (whiskers) and outliers (single points). Data points represent values for individual mice (f–h) or three technical replicates of pooled lymphocytes (i, j). n = 12 for (c–e). n = 8 (LNP) and 6 (EDIII, EDIII-Fc, EDIII-Fd) for (f–h). n = 3 for (i, j). Data are representative of two independent experiments and presented as mean ± standard derivation (s.d.). Comparisons are performed by one-way analysis of variation (ANOVA) and Tukey’s multiple comparison tests. p values are shown on the graphs. Source data are provided as a Source Data file.

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