Figure 3.

Intramuscular injection of repRNA/LION induces minimal systemic cytokine response in mice

(A) Mx1-GFP Tg mice received i.m. injection of repRNA/LION or repRNA/LNP encoding SEAP (10 μg). Twenty-four hours later, mice were sacrificed, and the indicated tissues were isolated. GFP signaling (reporting type I/III IFN responses) in the extracted tissues was analyzed by IVIS (representative data of two biological replicates are shown). The bar graphs indicate means +SD (n = 2) with individual values. (B) C57BL/6 mice received i.m. injection of repRNA/LION or repRNA/LNP encoding SEAP (10 μg). Serum IFN-α2 levels at the indicated time points were determined by ELISA and shown as boxplots (n = 5). (C) Cytokine profiling of sera isolated from 10 μg of repRNA/LION- and repRNA/LNP-injected mice at 14 h was determined by cytokine array. The results from the calculation of the values of repRNA/LION divided by the values of repRNA/LNP are shown (n = 3). (D) C57BL/6 mice were i.m. injected with 10 μg of repRNA/LION or repRNA/LNP encoding SEAP. Serum levels of cardiac troponin-I (cTNI) at indicated time points were determined by ELISA and shown as boxplots (n = 5). (E) C57BL/6 mice were i.m. injected with 10 μg total of repRNA/LION or repRNA/LNP encoding mixed antigens derived from EV-D68 and RSV. Their body weight changes were monitored over 3 days after the immunization, and the percentages of the body weight loss are shown as bar graphs (mean) ± SD (error bars) with individual values (square dots). All the boxplots show min to max values with all data points. Statistical comparison of mean values among groups was performed by ordinary two-way ANOVA and Tukey’s multiple comparisons test between each group. Statistically significant results are shown as asterisks: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant.