Effective inhibition of melanoma tumorigenesis and growth via a new complex vaccine based on NY-ESO-1-alum-polysaccharide-HH2

Abstract

Background: A safe and effective adjuvant plays an important role in the development of a vaccine. However, adjuvants licensed for administration in humans remain limited. Here, for the first time, we developed a novel combination adjuvant alum-polysaccharide-HH2 (APH) with potent immunomodulating activities, consisting of alum, polysaccharide of Escherichia coli and the synthetic cationic innate defense regulator peptide HH2.

Methods: The adjuvant effects of APH were examined using NY-ESO-1 protein-based vaccines in prophylactic and therapeutic models. We further determined the immunogenicity and anti-tumor effect of NY-ESO-1-APH (NAPH) vaccine using adoptive cellular/serum therapy in C57/B6 and nude mice. Cell-mediated and antibody-mediated immune responses were evaluated.

Results: The APH complex significantly promoted antigen uptake, maturation and cross-presentation of dendritic cells and enhanced the secretion of TNF-α, MCP-1 and IFN-γ by human peripheral blood mononuclear cells compared with individual components. Vaccination of NAPH resulted in significant tumor regression or delayed tumor progression in prophylactic and therapeutic models. In addition, passive serum/cellular therapy potently inhibited tumor growth of NY-ESO-1-B16. Mice treated with NAPH vaccine produced higher antibody titers and greater antibody-dependent/independent cellular cytotoxicity. Therefore, NAPH vaccination effectively stimulated innate immunity, and boosted both arms of the adaptive humoral and cellular immune responses to suppress tumorigenesis and growth of melanoma.

Conclusions: Our study revealed the potential application of APH complex as a novel immunomodulatory agent for vaccines against tumor refractory and growth.

Figure 1

NAPH vaccine inhibits the melanoma tumorigenesis and growth. A, C57/B6 mice (n = 12) were treated with different vaccines (on Days 0, 14 and 28) and challenged with 2 × 10⁵ NY-ESO-1-B16 cells (on Day 42) in a prophylactic model. B, NY-ESO-1-B16-bearing mice (n = 12) were treated with vaccines (on Days 5, 12 and 19) in a therapeutic model. C, D, Adoptive transfer of cells or serum from NAPH-treated mice induced tumor regression in recipient mice. Donor splenocytes (2 × 10⁷) were injected i.v. into recipient mice for 2 consecutive days (on Days 1 and 2). In serum adoptive therapy, tumor-bearing mice were injected i.v. with 0.1 ml of the sera from Days 1 to 10. E, Adoptive transfer of serum from immunized-Balb/c mice inhibited A375 melanoma growth in a nude mouse model. * P < 0.05; ** P < 0.005.

Figure 2

NAPH vaccine stimulates the innate immune response. 48 hours after NAPH injection, the mice were sacrificed. A standard 4 h NK assay against YAC-1 targets revealed that the NK cytotoxicity in the NAH group was higher than that in the NS, NA and NAP groups; in particular, NAPH-treated mice had the greatest cytotoxic activity against YAC-1 cells among all groups (* P < 0.05; ** P < 0.005).

Figure 3

NAPH increases NY-ESO-1-specific antibody immune response. A, The titers of anti-NY-ESO-1 total IgG were measured using ELISA. Dot, titer of each mouse. B, C, The titers of anti-NY-ESO-1 IgG2a/IgG1 titers in the treated groups were measured. D, FACS analysis using NY-ESO-1-B16 cells to detect NY-ESO-1-specific IgG antibodies (n = 3 mice, pooled composite results are shown). E, Antibody-dependent cellular cytotoxicity was determined using the ⁵¹Cr-release assay. NY-ESO-1-B16 cells were labeled with ⁵¹Cr and coated with sera from control or treated mice. Splenocytes from untreated or treated mice were used as effectors. The assay was run in three replicates.

Figure 4

NAPH vaccination elicits robust NY-ESO-1-specific cellular response in vitro. A, The cytotoxicity of splenocytes against NY-ESO-1-B16 cells was examined in a 4-h ⁵¹Cr-release assay. Points, mean of triplicate samples; bars, SD. B, IL-4/IFN-γ ELISPOT responses were performed at one week post-immunization (n = 3, three replicates). C, Intracellular staining of IFN-γ in CD4⁺ and CD8⁺ T cells. Similar results were obtained from three independent experiments (*P < 0.05; ** P < 0.005).

Figure 5

Immunohistochemistry analyses were performed to study infiltrating lymphocytes (TILs) in tumors. A. Tumor tissue sections were separately incubated with anti-CD4, anti-CD8 and anti-CD57 mAbs overnight at 4°C. Sections were then incubated with biotinylated anti-mouse antibody and streptavidin-biotinylated peroxidase complex. B. Intensity of lympocyte infiltration in the tumors was determined by the mean positive cell counts in the dermis around the B16-NY-ESO-1 tumor per field (10 randomly selected high power fields/slide). For each site, 3 pathologists performed a blind read of the glass slides.

Figure 6

PS-HH2 complex adjuvant promotes the antigen uptake and cross -presentation and maturation of DCs. A, DC were incubated with NHS-conjugated native NY-ESO-1 or protein adjuvant complex for 3 h, fixed, stained with DAPI and analyzed via confocal laser microscopy. B, DCs were incubated with Alexa Fluor-labeled NY-ESO-1 or NY-ESO-1/complex adjuvant for 3 h, fixed, permeabilized, blocked and stained with mouse FITC-anti-LAMP1 or FITC-anti-LMP2 antibody, respectively. DNA was stained with DAPI and analyzed via confocal laser microscopy. C, Analysis of surface maturation markers of DCs stimulated with PS-HH2 for 24 h.

Figure 7

Alum and PS-HH2 synergistically triggers activation of the NF-κB signal pathway. Immature DCs derived from bone marrow were incubated and harvested on Day 5 for Western blotting. After treatment with alum (125 μg/ml), PS-HH2 (PS: 20 μg/ml and HH2: 40 μg/ml), or alum-PS-HH2 complex for 0, 5, 10 and 30 min, respectively. DCs were washed, lysed and used for Western blot analysis using antibodies to phospho-Syk, phospho-Akt, phospho-IkBα and phospho-NF-κB, respectively. GAPDH was detected on the same membrane and used as the loading control.