Vaccination-induced immune responses.

A. Putative B16 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. 2 doses of peptides emulsified in Freund’s adjuvant were s.c. injected separated by 14 days. 14 days after the last dose the efficacy of the vaccine was analyzed by ELISPOT and ICS assays. C, ELISPOT analysis of IFNψ-producing T-cell effectors from mice vaccinated with the indicated mutated peptides. The upper images show the response of non-vaccinated (non-vac) animals after stimulation with the indicated peptides, the bottom images show the response of the animals vaccinated (vac) 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 per group. Asterisks indicate statistically significant differences analyzed by one-way ANOVA (* represent P< 0.05, ** P< 0.005, *** P< 0.0005). E, Intracellular Cytokine Staining (ICS) analysis of CD8+ T cells expressing 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 (grey 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 *.

Antitumor activity of vaccination with predicted TNA.

A. Scheme of immunization. 2 doses of peptides emulsified in Freund’s adjuvant were s.c. injected separated by 14 days. 14 days after the last dose the B16 F10 melanoma cells were injected s.c. in the mid-right flank of C57BL/6J host mice and the tumor size over time was analyzed using a dial caliper. B-I. Tumor growth on non-vaccinated (B) or vaccinated mice with the indicated peptide (C-I) (or peptide mix (H)), monitored every 1–3 days. Each line corresponds to the tumor size in one animal. The median of the tumors in non-vaccinated (PBS) and vaccinated with TRP2 (positive control) are shown as dashed lines in grey and green color respectively. Mice with tumors ≥ 900 mm3 were sacrificed. Following the rules of our ethical committee animals presenting ulcers were also sacrificed. J. Percentage of animals showing tumors with a volume ≤ 100 mm3 15 days after implantation.

A,B Kaplan-Meier survival curves of mice vaccinated with the indicated TNA peptides and challenged with B16 F10 melanoma. Comparison of Survival Curves has been performed using the Log-rank (Mantel-Cox) test. The significant P values comparing the control (PBS) group with each other group are indicated and * represents P< 0.05.