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. 2016 Dec 20:4:96.
doi: 10.1186/s40425-016-0189-y. eCollection 2016.

Fusion of the dendritic cell-targeting chemokine MIP3α to melanoma antigen Gp100 in a therapeutic DNA vaccine significantly enhances immunogenicity and survival in a mouse melanoma model

James T Gordy  1 , Kun Luo  1 , Hong Zhang  1 , Arya Biragyn  2 , Richard B Markham  1
Affiliations

Fusion of the dendritic cell-targeting chemokine MIP3α to melanoma antigen Gp100 in a therapeutic DNA vaccine significantly enhances immunogenicity and survival in a mouse melanoma model

James T Gordy et al. J Immunother Cancer. .

Abstract

Background: Although therapeutic cancer vaccines have been mostly disappointing in the clinic, the advent of novel immunotherapies and the future promise of neoantigen-based therapies have created the need for new vaccine modalities that can easily adapt to current and future developments in cancer immunotherapy. One such novel platform is a DNA vaccine fusing the chemokine Macrophage Inflammatory Protein-3α (MIP-3α) to an antigen, here melanoma antigen gp100. Previous published work has indicated that MIP-3α targets nascent peptides to immature dendritic cells, leading to processing by class I and II MHC pathways. This platform has shown enhanced efficacy in prophylactic melanoma and therapeutic lymphoma model systems.

Methods: The B16F10 melanoma syngeneic mouse model system was utilized, with a standard therapeutic protocol: challenge with lethal dose of B16F10 cells (5 × 104) on day 0 and then vaccinate by intramuscular electroporation with 50 μg plasmid on days three, 10, and 17. Efficacy was assessed by analysis of tumor burden, tumor growth, and mouse survival, using the statistical tests ANOVA, mixed effects regression, and log-rank, respectively. Immunogenicity was assessed by ELISA and flow cytometric methods, including intracellular cytokine staining to assess vaccine-specific T-cell responses, all tested by ANOVA.

Results: We demonstrate that the addition of MIP3α to gp100 significantly enhances systemic anti-gp100 immunological parameters. Further, chemokine-fusion vaccine therapy significantly reduces tumor burden, slows tumor growth, and enhances mouse overall survival compared to antigen-only, irrelevant-antigen, and mock vaccines, with efficacy mediated by both CD4+ and CD8+ effector T cells. Antigen-only, irrelevant-antigen, and chemokine-fusion vaccines elicit significantly higher and similar CD4+ and CD8+ tumor-infiltrating lymphocyte (TIL) levels compared to mock vaccine. However, vaccine-specific CD8+ TILs are significantly higher in the chemokine-fusion vaccine group, indicating that the critical step induced by the fusion vaccine construct is the enhancement of vaccine-specific T-cell effectors.

Conclusions: The current study shows that fusion of MIP3α to melanoma antigen gp100 enhances the immunogenicity and efficacy of a DNA vaccine in a therapeutic B16F10 mouse melanoma model. This study analyzes an adaptable and easily produced MIP3α-antigen modular vaccine platform that could lend itself to a variety of functionalities, including combination treatments and neoantigen vaccination in the pursuit of personalized cancer therapy.

Keywords: B16 Melanoma; Chemokine-antigen fusion; DNA Vaccine; Gp100; In vivo electroporation; MIP3α, MIP3alpha, or CCL20; Therapeutic cancer vaccine.

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Figures

Fig. 1
Fig. 1
Systemic immune parameters of vaccine groups in prophylactic vaccination setting. Mice were vaccinated three times at 1 week intervals with endotoxin-free PBS, dMIP3α-gp100, and MIP3α-gp100 fusion vaccine. Analysis occurred 2 weeks post third vaccination. Data represent two independent experiments with 3–5 mice per group per experiment. a Analysis of relative antibody production against B16F10 cells. In-Cell ELISA performed utilizing fixed B16F10 cells as antigens. Experimental data are shown at a 1:2000 serum dilution after 30-min colorimetric development. Absorbance values from pre-immune mice were subtracted from post immune mice to obtain the delta absorbance. All groups were significantly different from each other by ANOVA. b-c Analysis of splenic CD8+ T cells reactive to ex vivo stimulation by gp10025-33 peptide. Activation was signaled by cytoplasmic IFN-γ accumulation as measured by Intracellular Cytokine Staining Flow Cytometry. Panel b shows the data as percentage of CD3+ splenocytes. Panel c estimates the total number of reactive CD3 + CD8+ splenocytes by extrapolating flow cytometric data to measured splenic total cell counts. For both panels, all groups differ significantly from each other, as determined by by ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 2
Fig. 2
Vaccine effects on tumor growth in therapeutic model. Vaccinations occurred on days 3, 10, and 17 post challenge. a Tumor growth rate was assessed between days 10 and 16, day 10 being the point at which tumor growth of the negative control group began accelerating and day 16 being the point at which mice began to be censored due to endpoints being reached. The graph shows one representative experiment of two, five to seven mice per group and includes linear regression lines and slopes. The slope of tumor growth among recipients of MIP3α-gp100 vaccine differed significantly from dMIP3α-gp100, MIP3α-CSP, and mock PBS vaccination, as evaluated using a statistical mixed effects regression model. The groups receiving dMIP3α-gp100 and MIP3α-CSP did not differ significantly compared to each other or to the group receiving mock vaccination. Error bars represent standard error. b Tumor size at day 14 post challenge, the last point before any mice were removed from experiments. The data are representative of two experiments, with 5–8 mice per group per experiment. MIP3α-gp100 vaccine recipients had significantly smaller tumors compared to dMIP3α-gp100, MIP3α-CSP, and mock PBS vaccinated mice, as determined by ANOVA. dMIP3α-gp100 and MIP3α-CSP were not significantly different from each other or from mock. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 3
Fig. 3
Vaccine effects on mouse survival in a therapeutic model. Vaccinations occurred on days 3, 10, and 17 post challenge. Mice were removed from the study at the following endpoints: death, tumor size surpassing 2 cm in any dimension, or excessive tumor bleeding and ulceration. Data representative of two experiments, 5–8 mice per group per experiment. Mice in the MIP3α-gp100 vaccine group exhibit significantly enhanced survival compared to the dMIP3α-gp100, MIP3α-CSP, and mock PBS vaccination groups by the log-rank test. dMIP3α-gp100 and MIP3α-CSP did not differ significantly from each other or from mock. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 4
Fig. 4
Vaccine effector T-cell responses analyzed by subset depletions. Mice were vaccinated three times at 1 week intervals and then challenged with a lethal dose (5 × 104) of B16F10 cells. T-cell subsets were depleted one day prior to challenge, on day of challenge, and 7 days post challenge. Quality of the depletions was assessed by flow cytometric analysis of peripheral blood lymphocytes on days 0 and 8 or 10. a Representative flow cytometry plots shown depicting CD4 and CD8 expression gated on overall lymphocytes from blood collected at day 10 post challenge. b Tumor growth regression plot from day 6 to 13 post challenge, assessed by mixed effects regression. c Tumor size at day 13 post challenge, with significance assessed by ANOVA. All data are from two independent experiments of 4–5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 5
Fig. 5
Vaccine effects on tumor infiltrating lymphocytes (TILs). Vaccinations occurred on days 3 and 10 post challenge. Mice were sacrificed on day 17 and lymphocyte-enriched tumor suspensions were analyzed by flow cytometry. a shows CD8+ TILs and b CD4+ TILs. Data show one representative experiment with 3–5 mice per group. Two independent experiments were performed. All three vaccine formulations have significantly higher CD4 and CD8 TILs compared to mock vaccination but not to each other, as assessed by ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001
Fig. 6
Fig. 6
Vaccine-specific CD8+ T-cell tumor infiltrate analysis. Vaccinations occurred on days 3 and 10 post challenge. Mice were sacrificed on day 17 and lymphocyte-enriched tumor suspensions were collected. CD8+ TILs reactive to ex vivo stimulation by gp10025-33 peptide were delineated by Intracellular Cytokine Staining Flow Cytometry measuring cytoplasmic IFN-γ accumulation post stimulation. a Percentage of CD8+ TILs reactive to antigen. b Estimated total number of reactive CD8+ TILs normalized to tumor size. All groups were significantly different from each other by ANOVA except for the comparison of PBS to MIP3α-CSP. HA irrelevant negative peptide and PMA/ionomycin positive controls confirmed the protocol validity (data not shown). Data are from one of two representative experiments with 3–5 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001
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