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


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.

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Antigen-presenting cell


C–C chemokine receptor type 7


Cytotoxic T lymphocyte


Cytotoxic T-lymphocyte-associated protein 4


Dulbecco modified Eagle medium


Ethylenediaminetetraacetic acid


Immune check-point inhibitor


Incomplete Freund’s adjuvant


Interferon gamma


Lymphocyte-activation gene 3


Major histocompatibility complex


Phosphate-buffered saline


Programmed cell death 1


Quantitative real-time reverse transcriptase–polymerase chain reaction


Ribonucleic acid


Standard error of the mean


Spot-forming cell


Tumor-associated antigens


Tumor-infiltrating lymphocyte


T-cell immunoglobulin mucin-3


Toll-like receptor 9


Regulatory T cells


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The authors thank the Association Oligocyte and the Association pour le development des neurosciences a Avicenne (ADNA) for supporting our research.


No funding sources were used for this report.

Author information




SC, CT, ET and AFC contributed to the study concept and design of experiments. SC, CB, MSR and TT performed the experiments. CB and TT helped in methodology and software analysis. SC and CB were involved in data analysis and interpretation of results. SC and AFC wrote the preliminary version of the paper. All authors participated in critical review and revision of the final manuscript.

Corresponding author

Correspondence to Stefania Cuzzubbo.

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Conflict of interest

The AP/HP (Assistance Publique de Hopitaux de Paris) filed a provisional patent application on this method. AF Carpentier & C Banissi are listed as inventors. AF Carpentier holds shares in Altevax inc. and is consultant for BMS. The authors declare that there are no other conflicts of interest.

Ethical approval and ethical standards.

Experiments were conducted on female C57BL/6 mice. All animal experiments were approved by the ethics committee of Paris Descartes University (Project APAFIS #5337 N° 2016021517305775) and performed in accordance with European Union guidelines for animal experiments.

Human and animal rights

Animal source: Female C57BL/6 aged 5 to 6 week old were purchased from Janvier Labs (Le Genest-Saint-Isle, France) and kept under specific-pathogen-free conditions.

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Published as a poster abstract at the 5th Immunotherapy of Cancer Conference: [1]

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Cuzzubbo, S., Banissi, C., Rouchon, M.S. et al. The adjuvant effect of melanin is superior to incomplete Freund’s adjuvant in subunit/peptide vaccines in mice. Cancer Immunol Immunother (2020).

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  • Cancer vaccine
  • Immunotherapy
  • Melanin
  • Neoepitope
  • PD1