Fig. 5: ADE risks of prototype circRNA vaccines in mice.
a Cross-reactivity of immune sera to ZIKV and DENV. Mice sera were serially diluted and tested by ELISA using EDIII of ZIKV and DENV1, or E of DENV2, DENV3, and DENV4. LOD is 100 and marked by gray dashed line. Box plots indicate median (middle line), 25-75 percentile (box), 5-95 percentile (whiskers) and outliers (single points). b In vitro ADE activities of immune sera. Equally pooled mice sera in each group were 5-fold serially diluted (starting at 1:100) and incubated with ZIKV or DENV. The mixtures were used to infect K562 cells. Infected cells were examined by flow cytometry using anti-ZIKV mAb 8D10 or a cross-reactive mAb ZK8-4. c In vivo ADE activities of immune sera. Equally pooled mice sera in each group were diluted tenfold with PBS. Twelve-week-old Ifnar-/- mice were i.p. transferred with 200 μl diluted sera 1 day before i.p. challenge with 1 × 106 FFU of mouse-adapted DENV2. d Survival curves. e DENV2 genome copies in the sera at 1, 4, and 7 days after challenge. f In vivo ADE activities of circRNA immunization. Twelve-week-old Ifnar-/- mice were i.m. immunized with EN(LNP) or empty LNPs 2 weeks before challenge. Mice receiving 200 μl diluted ZIKV sera 1 day before challenge were used as controls. g Survival curves. h DENV2 genome copies in the sera at 1, 4, and 7 days after challenge. Data points represent mean values of three technical replicates for one mouse (e, h). n = 10 (ZIKV sera), 16 (EDIII-Fc) and 6 (EN(LNP), EN(RNA), NS1, PBS) for (a). n = 5 for (c–e). n = 6 for (f–h). Data are representative of at least two independent experiments and presented as mean ± s.d. Comparisons were performed by unpaired two-tailed Student’s t-test between each group and ZIKV sera group (a), Log-rank (Mantel-Cox) tests between PBS (d) or LNP group (g) and the rest groups, or by one-way ANOVA and Tukey’s multiple comparison tests (e, h). p values are shown on the graphs. Source data are provided as a Source Data file.
