Figure 1. Vaccination-induced immune responses.

(A) Putative B16 tumor neoantigen (TNA) identified by using the NAP-CNB platform ranked by scores of peptide sequences for a complete 12mer sequence. The TNA sequence, the mutation exclusive to tumor cells (in red), and gene name are shown. The gene expression is quantified as fragments per kilobase million (FPKM). The TNA score is also indicated. (B) Scheme of immunization. Two doses of peptides emulsified in Freund’s adjuvant were subcutaneously (s.c.) injected, separated by 14 days. 14 days after the last dose the efficacy of the vaccine was analyzed by enzyme-linked immunosorbent spot (ELISpot) and intracellular cytokine staining (ICS) assays. (C) ELISpot analysis of interferon-gamma (IFNγ)-producing T-cell effectors from mice vaccinated with the indicated mutated peptides. The upper images show the response of non-vaccinated (phosphate-buffered saline [PBS]) animals after stimulation with the indicated peptides, the bottom images show the response of the animals vaccinated (vaccin) with the indicated peptides after restimulation with the same peptides. It is shown duplicates from representative animals. (D) As in C but showing the mean and sd of 5 mice/group. Asterisks indicate statistically significant differences analyzed by one-way ANOVA (* represent p<0.05, **p<0.005, ***p<0.0005). (E) ICS analysis of CD8⁺ T-cells expressing tumor necrosis factor alpha (TNFα), perforin, or CD107a from mice vaccinated with the indicated mutated peptides: *Pnp (black triangle), *Adar (black circle), *Lrrc28 (black square), *Wiz (black rhombus), *Herc6 (gray circle), and Trp2 (green circle). The mean of the ratio of vaccinated divided by unvaccinated is shown, as the 95% confidence intervals. Intervals that do not include 1 and are therefore statistically significant are marked with *. 5 mice/group were used.