Fig. 3

In vitro expression of LNP mRNAs and in vivo immunogenicity A mRNA from two different antigens (E80 dengue virus serotype 3 and LinKAP from Leishmania) were in vitro synthesized and HEK293T cells were transfected with both LNP-mRNAs respectively. Forty-eight hours after transfection, cells were collected, and a Western blot was performed with B culture supernatant from cells transfected with E80 or C total protein extract from cells transfected with LinKAP. D Female C57BL/6 mice were immunized IM with 10 µg of LNP-E80 or LNP-LinKAP, naked mRNA or empty-LNP, with a prime-boost or prime-boost-boost regimen at a 3-week interval. Serum was collected on days 20, 41 and 62 to analyze specific total IgG. E Total IgG titers after mice immunization with LNP-E80 after the boost (p < 0.0001). Thirty days after the last immunization, mice spleens were collected and used in splenocyte cultures with the appropriate stimuli. F IFN-γ production in mice immunized with LNP-E80 (****p < 0.0001, ***p < 0.001). G Total IgG titers after mice immunization with LNP-LinKAP (p < 0.0001). H IFN-γ production in mice immunized with LNP-LinKAP (**p = 0.0001, *p = 0.0001). Statistical analysis was performed with 2-way ANOVA with Turkey’s multiple comparisons test