The adjuvant effect of melanin is superior to incomplete Freund's adjuvant in subunit/peptide vaccines in mice

Abstract

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

Keywords: Cancer vaccine; Immunotherapy; Melanin; Neoepitope; PD1.

Fig. 1

pOVA30-melanin vaccine is superior to IFA in terms of CTL response and anti-tumor effect. a Outline of the in vivo experiments in E.G7-OVA model. b ELISpot analysis of splenocytes on day 21, after immunizations on days 0 and 14. Each point represents an individual mouse (n = 8 mice/group with pooled data from 2 different experiments). c Expression of CD62L, CD44, CCR7, T-bet and PD1 in the CD8⁺dextramer⁺ population of [pOVA30-Mel + CpG] and [pOVA30-IFA + CpG] immunized mice (n = 8 mice/group, with pooled data from two different experiments). d Tumor growth curve of C57BL/6 mice bearing subcutaneous tumors and treated with different protocols. Bars = mean values ± standard error of the mean (SEM). *p < 0.5, **p < 0.01, ***p < 0.001

Fig. 2

Tumor escape mechanisms in the E.G7-OVA model. Peripheral CTL response in tumor-bearing and tumor-free mice after immunization on days 4 and 18 with [pOVA30-Mel + CpG]: a SIINFELKL-specific T cells within the splenic T CD8+ cells by flow cytometry; b PD1 expression within the CD8⁺dextramer⁺ population (SIINFELKL-specific T cells). c–g Cytometry analysis of CD8 + and CD4+ TILs in tumor-bearing mice immunized on days 4 and 18 with [melanin + CpG], [pOVA30-Mel + CpG] or [pOVA30-Mel + CpG] and sacrificed on day 24. h Number of RNA OVA copies (normalized to β actin) by qRT-PCR analysis in tumors that have grown in [melanin + CpG]- and [pOVA30-Mel + CpG]-treated mice, at day 28. Non-transfected-E.G7 and E.G7-OVA tumors cells, cultured in vitro, were used as controls. Each point represents an individual mouse, pooled data from two different experiments. Bars = mean values ± SEM. ns: not statistically significant; *p < 0.5, **p < 0.01, *** p < 0.001

Fig. 3

mAdpgk-melanin vaccine significantly increases CTL response in the MC38 tumor model but does not inhibit tumor growth. a Outline of the in vivo experiments in MC38 model. b ELIspot analysis of splenocytes on day 21, after immunizations on days 0 and 14. Each point represents an individual mouse (n = 8 mice/group with pooled data from 2 different experiments). c Immune phenotype of mAdpgk-specific CD8+ T splenic cells (dextramer⁺ cells) in the different groups of mice. d Tumor growth in MC38-bearing mice, treated with the different vaccine formulations. Results are expressed as the mean ± SEM of tumor volumes. e Correlation between tumor volume at day 24 and number of IFNγ-SFCs (Spot forming cells) after stimulation in vitro of splenocytes with the MHC class I-epitope (CD8). Pearson coefficient = 0.68. f Percentage of CD8+ and CD4+ cells of CD45+ total cells in cytometry analysis of TILs (tumor infiltrating lymphocytes) of the [mAdpgk-Mel + CpG] immunized mice and [Mel + CpG] mice. g, h Phenotype of tumor infiltrating lymphocytes: percentage of PD1+ cells, TIM3+ cells and LAG3+ cells within CD4+ and CD8+ TILs in [mAdpgk-Mel + CpG] immunized mice. i Percentage of FoxP3+ cells in CD4+ TILs in [mAdpgk-Mel + CpG] and [Mel + CpG] group. Each point represents an individual mouse (pooled data from 2 different experiments). Bars = mean values ± SEM. *p < 0.5, **p < 0.01, ***p < 0.001

Fig. 4

MC38 tumor cells are poorly recognized by anti-mAdpgk CD8 T cells. a Percentage of MHC class I (H2 Kb/Db) positive cells within MC38 cells in cytometry analysis of in vitro MC38 line cells. b CD8 splenic T cells isolated from mice vaccinated with [mAdpgk-Mel + CpG] were co-cultured for 24 h with medium + mAdpgk, MC38 cells, MC38 cells with mAdpgk peptide, ex vivo tumor cells or ex vivo tumor cells with mAdpgk peptide. Each point represents the mean value of IFNγ-SFC in triplicate wells (pooled data from three different experiments). Bars = mean values ± SEM. *p < 0.05, **p < 0.01