Fig. 3: Immunogenicity and protective efficacy of a single-dose immunization with the combination of EDIII-Fc and NS1 circRNAs in ZIKV-infected neonatal mouse model.

a Schematic diagram of maternal immunization and neonatal challenge model. Eight-week-old female C57BL/6 mice were i.m. immunized once with the indicated vaccines at 20 μg each circRNA per mouse or an equal volume of PBS. Immunized mice were mated at 2 weeks after immunization. After birth, 1-day-old pups were s.c. challenged with 1 × 10⁴ FFU of ZIKV. Fifteen days later, pups were sacrificed. b anti-ZIKV nAb titers in C57BL/6 mice at 2 weeks after immunization. LOD is 100 and marked by gray dashed line. c Growth curves of the pups. d Survival curves of the pups. e Neurological scores of the pups at 15 days after challenge. f ZIKV genome copies in neonatal brains at 15 days after challenge or at sacrifice. g H&E staining of brain tissue sections. Representative images of each group are shown. Black arrows, meningeal lymphocyte infiltration. Cyan arrows, necrotic cells in cortex. Scale bar = 50 μm. h Pathological scores of neonatal brains. Meningeal lymphocyte infiltration and cortex laminar necrosis were scored in a single-blind manner. Data points represent mean values of two (b) or three (f) technical replicates for one mouse, or mean values of one group (c), or values for individual mice (e, h). n = 10 for (b). n = 7 (HC, EN(LNP)), 8 (EN(RNA), PBS) and 9 (EDIII-Fc, NS1) for (c–h). Data are representative of two independent experiments and presented as mean ± s.d. Comparisons were performed between each group and HC by Log-rank (Mantel-Cox) test (d). 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.