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. 2012 Sep 25;21(1):251–259. doi: 10.1038/mt.2012.202

Figure 1.

Figure 1

DOTAP/DOPE complexed antigen-encoding mRNA induces potent antigen-specific immune responses. (a) Mice were immunized s.c. with PBS, 20 µg of Gag-encoding mRNA, either as such (“naked”) or complexed with the indicated carriers. At week 4, mice were boosted with the same formulation. Spleens and lymph nodes were isolated at week 8, and the number of Gag-specific interferon-γ secreting cells was determined by enzyme-linked immunosorbent spot. Means ± SEM are shown of a total of 5 mice per group. PBAE, poly(β-amino ester). (b) Frequencies of CD69+ CD4+ and CD8+ T cells in draining lymph nodes 1 day after footpad injection of 16 µl DOTAP/DOPE (DOTAP), 4ug of naked Gag-encoding mRNA (naked gag) or 4 µg DOTAP/DOPE-complexed Gag-encoding mRNA (DOTAP gag). Means ± SD are shown of a total of 6 mice per group. (c) In vivo killing assay was performed by adoptive transfer of carboxyfluorescein diacetate succinimdyl ester-labeled peptide-loaded splenocytes to mice immunized with 20 µg of naked gag or DOTAP gag. The ratio of Gag-pulsed versus unpulsed cells in 4 mice is shown. ***P < 0.001. (d) Gating strategy for B cells and germinal center (GC) B cells. (e) The frequencies of B cells and GC B cells were determined by flow cytometry 7 days post footpad injection of 16 µl DOTAP, 4 µg naked gag or DOTAP gag. B cells were defined as CD45+ CD19+ CD3− cells, and GC B cells as GL-7+ CD95+ B cells. Means ± SD are shown of a total of 6 mice per group.