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. Author manuscript; available in PMC: 2015 Apr 22.
Published in final edited form as: Vaccine. 2013 Oct 5;31(48):5713–5721. doi: 10.1016/j.vaccine.2013.09.040

Figure 6. DVG-derived naked RNA shows strong localized pro-inflammatory activity in mice.

Figure 6

Mice were injected subcutaneously in the footpad with 50 µg of DVG-324 or poly I:C (high molecular weight). (A–B) Footpad tissue was harvested after 36 h and RNA was extracted for the analysis of cytokine expression. The experiment was independently repeated three times. Each assay was performed in triplicates. Data shown is a compilation of all experiments. p=0.0192 (Ifnb), p=0.0201 (Il6), p=0.006 (Il1b), p=0.009 (Tnf) and p=0.0001 (Cxcl10) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test among different conditions is indicated in the graphs as *=p<0.05, **=p<0.05 and ***= p<0.001. Dunn’s denominator = 3. To control for experimental variation, data was also analyzed by two-way ANOVA for the parameters treatment/experiment. Significance of variation among treatments are: p=0.0158 (Ifnb), p=0.0002 (Il6), p=<0.0001 (Il1b), p=<0.0001 (Tnf), and p<0.0001 (Cxcl10). (B) Analysis of cytokine expression in mice footpads collected at 6 h post infection. Data shown is an average of two independent experiments (n=4–5/group). Each assay was performed in triplicates. Data are expressed as copy numbers relative to the housekeeping gene rps11. p=0.0357 (Ifnb), p=0.0374 (Il1b), p=0.0490 (Tnf), and p=0.0979 (Cxcl10) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test among different conditions is indicated in the graphs as *=p<0.05. Dunn’s denominator = 3. (C–D) Single cell suspensions from draining popliteal lymph nodes were analyzed by flow cytometry. (C) Percentage of CD11c+CD11b+ cells from the live cell gate is shown (n = 2 for PBS and 5 for poly I:C and DVG 324). p=0.387 by one-way ANOVA with Bonferroni post-hoc test. Bonferroni denominator = 3. (D) CD11c+CD11b+ cells were further gated for expression of CD103 and B220 to quantify CD11c+CD11b+CD103− DCs and CD11bloCD11cloB220+ plasmacytoid DCs. Data show a representative plot for poly I:C treatment and two representative plots for DVG-324 treatment. (E) Antibodies in the sera of Balb/c mice three weeks after immunization with a single i.m. dose of 180 µg of inactivated respiratory syncytial virus (inRSV) in the presence of 50 µg poly I:C, 50 µg DVG-324, or PBS. Sera pre-immunization (pre-bleed: PB) was also analyzed. n=4/group. p=0.0013 (total IgG), p=0.0011 (IgG1), p=0.0013 (IgG2b), and p=0.0008 (IgG2a) by Kruskal-Wallis test. Significance after Dunn’s post-hoc test among different conditions is indicated in the graphs as *=p<0.05, **p<0.01, and ***p<0.001. Dunn’s denominator = 4