Fig. 6: Immunogenicity and protective efficacy of optimized EN(LNP) in mice.

a Schematic diagram of optimized circRNA. Detailed elements have been depicted in Supplementary Fig. 9a. b Western blot analysis of EDIII-Fc and NS1 in the culture media of HEK293T cells transfected with prototype (P) or optimized (Opt) circRNAs (without β-mercaptoethanol). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in cell lysates was also examined. Analysis was independently repeated twice with similar results. c Vaccine doses and schedule for maternal immunization and neonatal challenge model. One-day-old pups were s.c. challenged with 1 × 10⁴ FFU of ZIKV. Fifteen days later, pups were sacrificed. d anti-ZIKV nAb titers in C57BL/6 mice at 2 weeks after immunization. LOD is 100 and marked by gray dashed line. e Survival curves of the pups. f Neurological scores of the pups at 15 days after challenge. g ZIKV genome copies in neonatal brains at 15 days after challenge or at sacrifice. Box plots (f, g) indicate median (middle line), 25–75 percentile (box), 5-95 percentile (whiskers) and outliers (single points). h H&E staining of brain tissue sections. Representative images are shown. Black arrows, meningeal lymphocyte infiltration. Cyan arrows, necrotic cells in cortex. Scale bar = 100 μm. i Vaccine doses and schedule for immunization and challenge assay in Ifnar^-/- mice. Fifteen days after challenge, mice were sacrificed. j anti-ZIKV nAb titers in Ifnar^-/- mice at 2 weeks after immunization. k Survival curves. l, ZIKV genome copies in the sera at 1, 4, and 7 days after challenge. Data points represent mean values of two (d, j) or three (l) technical replicates for one mouse. n = 6 for (d). n = 10 (HC), 15 (P-5), 12 (P-20) and 14 (Opt-5, Opt-20, PBS) for (e–h). n = 6 for (j–l). Data are representative of two independent experiments and presented as mean ± s.d. Comparisons of survival rates between each group and HC were performed by Log-rank (Mantel-Cox) test (e, k). Other comparisons were performed by one-way ANOVA and Tukey’s multiple comparison tests. p values are shown on the graphs. Source data are provided as a Source Data file.