The C1-mRNA nanovaccine induced a TLR4-dependent immune response in vivo. (A) Cytokine and chemokine profiles in serum samples from WT and Tlr4−/− mice after C1-mRNA vaccination. Upper: The schedule of vaccination and peripheral blood collection. Lower: Multiplex immunoassay measuring 26 cytokines/chemokines from serum samples. Control: PBS injection group. C1-OVA mRNA dose per mouse: mRNA encoding OVA257–264,10 μg; C1, 1.6 mg; n = 5 mice per group. (B and C) Antitumor efficacy of the C1-mRNA nanovaccine on WT and Tlr4−/− mice. Irrelevant mRNA that does not encode antigen serve as a negative control. Control: PBS injection group. The mRNA nanovaccine dose per mouse: mRNA, 10 μg; C1, 1.6 mg; n = 5 mice per group. Data represent the mean ± SEM ****P < 0.001; ns, not significant.