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. 2016 Apr:86:83-91.
doi: 10.1016/j.biomaterials.2016.01.056. Epub 2016 Feb 1.

Viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses

Nicholas M Molino  1 , Medea Neek  1 , Jo Anne Tucker  2 , Edward L Nelson  3 , Szu-Wen Wang  4
Affiliations

Viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses

Nicholas M Molino et al. Biomaterials. 2016 Apr.

Abstract

The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8(+) T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimetic platform for cancer vaccine delivery. Simultaneous conjugation of a melanoma-associated gp100 epitope and CpG to the E2 nanoparticle (CpG-gp-E2) yielded an antigen-specific increase in the CD8(+) T cell proliferation index and IFN-γ secretion by 1.5-fold and 5-fold, respectively, compared to an unbound peptide and CpG formulation. Remarkably, a single nanoparticle immunization resulted in a 120-fold increase in the frequency of melanoma epitope-specific CD8(+) T cells in draining lymph nodes and a 30-fold increase in the spleen, relative to free peptide with free CpG. Furthermore, in the very aggressive B16 melanoma murine tumor model, prophylactic immunization with CpG-gp-E2 delayed the onset of tumor growth by approximately 5.5 days and increased animal survival time by approximately 40%, compared to PBS-treated animals. These results show that by combining optimal particle size and simultaneous co-delivery of molecular vaccine components, antigen-specific anti-tumor immune responses can be significantly increased.

Keywords: Biomimetic; CD8; Protein nanoparticle; T cells; Tumor-associated antigen; Vaccine.

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Figures

Fig. 1
Fig. 1
Physicochemical characterization of functionalized nanoparticles. A) Functionalization of the E2 nanoparticle (E2; 28105 Da monomer) with the CKVPRNQDWL peptide (gp-E2) shows a broad band in the 30–35 kDa range, supporting heterogeneous conjugation of the gp100 peptide to the external E2 lysines. Simultaneous conjugation of gp100 peptide and CpG (lane CpG-gp-E2) shows two distinct broad signals in the 30–35 kDa and 35–40 kDa range. B) Representative DLS data reveal nanoparticle sizes within the optimal reported vaccine size range. C) Transmission electron micrograph of CpG-gp-E2 stained with 2% uranyl acetate confirms monodisperse, intact nanoparticles. Scale bar is 100 nm.
Fig. 2
Fig. 2
Pmel-1 CD8+ T cells show increased antigen-specific IFN-γ secretion when stimulated by BMDCs loaded with the CpG-gp-E2 nanoparticle, compared to other formulations. IFN-γ levels measured with ELISA were normalized to the free gp100 peptide formulation (gp100) as baseline. Data are presented as mean ± S.E.M. (n = 3) and were analyzed using a one-way ANOVA followed by Dunnett's test comparing all means to CpG-gp-E2 within each concentration (*p < 0.05; **p < 0.01; ***p < 0.001).
Fig. 3
Fig. 3
Pmel-1 CD8+ T cells exhibit increased proliferative capacity when cultured in the presence of BMDCs loaded with the CpG-gp-E2 nanoparticle, compared to other gp100 formulations (100 nM gp100 peptide, either free or E2-bound). (A) Representative flow cytometry histograms of CFSE-labeled CD8+ T cells show increased proliferation in the CpG-gp-E2 group. (B) The CpG-gp-E2 nanoparticle induced the greatest CTL proliferative capacity. Data represents mean proliferation index (PI) ± S.E.M. (n = 3) and is normalized to the free gp100 peptide formulation. Statistical analysis used a one-way ANOVA followed by Dunnett's test comparing all groups to CpG-gp-E2 (**p < 0.01).
Fig. 4
Fig. 4
Mice immunized with CpG-gp-E2 and gp100 + CpG-E2 formulations exhibited increased secondary lymphoid organ antigen presenting cell numbers in the A) draining lymph nodes and B) spleens. Vaccine formulations with antigen contained 5 µg each of gp100 peptide and CpG ODN (either free or E2-bound). Cells measured in the secondary lymphoid organs included natural killer cells (NK), dendritic cells (DC), B cells, macrophages (Mac), T cells, CD8+ T cells, and CD4+ T cells. Data is presented as average ± S.E.M. total cell numbers relative to the PBS control of at least 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by a post hoc Tukey's test, with a pairwise comparison of all statistical means. *p < 0.05; **p < 0.01 compared to the PBS background control.
Fig. 5
Fig. 5
Immunization with the CpG-gp-E2 nanoparticle increased the gp100-specific CTL response. Cells were isolated from the A) draining lymph nodes and B) spleens of mice immunized with different formulations (5 µg gp100 peptide and 5 µg CpG; unbound or bound to E2) and were cultured ex vivo in the presence of KVPRNQDWL peptide (gp100) or irrelevant SIINFEKL peptide (OVA) and analyzed for IFN-γ-secreting cells by ELISpot. The lower panels show representative wells from the immunization groups for negative control irrelevant peptide (OVA) and tumor antigen peptide (gp100). Data is presented as average ± S.E.M. spots per million cells from at least 3 independent experiments. Statistical significance was determined by ANOVA followed by Dunnett's test, comparing all means to CpG-gp-E2 (*p < 0.05; **p < 0.01).
Fig. 6
Fig. 6
Splenocytes from mice receiving a single immunization of the CpG-gp-E2 (50 µg) nanoparticle formulation exhibited enhanced lytic ability toward B16-F10 melanoma cells (measured by release of lactate dehydrogenase). Data is presented as average ± S.E.M. % lysis of at least 3 independent experiments. Statistical significance was determined by ANOVA followed by Dunnett's test, comparing all means to CpG-gp-E2 (*p < 0.05).
Fig. 7
Fig. 7
Immunization with the CpG-gp-E2 nanoparticle delayed B16-F10 tumor growth and increased animal survival time. Data is representative of a duplicate set of independent experiments. Mice (n = 5 per group) were immunized subcutaneously with CpG-gp-E2 (50 µg per injection; equivalent to 5 µg each of gp100 peptide and CpG ODN) or PBS at Days −28 and −14, followed by tumor challenge at Day 0. A) Immunization with CpG-gp-E2 exhibited a delayed tumor growth, compared to PBS-treated control. Each line represents the tumor growth of a single animal. B) Immunization with CpG-gp-E2 significantly prolonged animal survival, compared to PBS-treated controls (p < 0.002, log-rank test).
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