Design of Peptide-Based Nanovaccines Targeting Leading Antigens From Gynecological Cancers to Induce HLA-A2.1 Restricted CD8+ T Cell Responses

Abstract

Gynecological cancers are a leading cause of mortality in women. CD8⁺ T cell immunity largely correlates with enhanced survival, whereas inflammation is associated with poor prognosis. Previous studies have shown polystyrene nanoparticles (PSNPs) are biocompatible, do not induce inflammation and when used as vaccine carriers for model peptides induce CD8⁺ T cell responses. Herein we test the immunogenicity of 24 different peptides, from three leading vaccine target proteins in gynecological cancers: the E7 protein of human papilloma virus (HPV); Wilms Tumor antigen 1 (WT1) and survivin (SV), in PSNP conjugate vaccines. Of relevance to vaccine development was the finding that a minimal CD8⁺ T cell peptide epitope from HPV was not able to induce HLA-A2.1 specific CD8⁺ T cell responses in transgenic humanized mice using conventional adjuvants such as CpG, but was nevertheless able to generate strong immunity when delivered as part of a specific longer peptide conjugated to PSNPs vaccines. Conversely, in most cases, when the minimal CD8⁺ T cell epitopes were able to induce immune responses (with WT1 or SV super agonists) in CpG, they also induced responses when conjugated to PSNPs. In this case, extending the sequence around the CD8⁺ T cell epitope, using the natural protein context, or engineering linker sequences proposed to enhance antigen processing, had minimal effects in enhancing or changing the cross-reactivity pattern induced by the super agonists. Nanoparticle approaches, such as PSNPs, therefore may offer an alternative vaccination strategy when conventional adjuvants are unable to elicit the desired CD8⁺ T cell specificity. The findings herein also offer sequence specific insights into peptide vaccine design for nanoparticle-based vaccine carriers.

Keywords: CD8 T cell epitopes; HLA-A2.1; HPV; WT1; immunogenicity; nanoparticles; survivin; vaccine.

Figure 1

Optimization of conjugation conditions to covalently conjugate HPV peptides to PSNPs to produce uniform HPV(peptide)-PSNPs nanovaccine formulations. PSNPs (1% solid final) were pre-activated following the standard procedure (detailed in Materials and Methods), and then re-conditioned in different buffer and pH solutions before mixing with each peptide antigen (0.5 mg/ml final) for conjugation. After conjugation, the final particle sizes for each peptide-PSNPs formulation was assessed using a Zetasizer. Data presented as peptide-PSNPs conjugate size (nm) ± SD (3 repeated measurements) under each conjugation conditions for each peptide. The dotted lines indicated the acceptable nanovaccine formulation size range at 40–60 nm.

Figure 2

Antigen-specific T cell responses in HLA-A2.1/Kb mice induced by HPV peptides with CpG or PSNPs. HPV01, -HPV05 and –HPV08 peptides were either mixed with CpG or covalently conjugated to PSNPs forming nanovaccine formulations. Each formulation was injected with matching amount of target peptide antigen (all contained 0.5 μg/peptide antigen/injection in 100–200 μl volume). Matching amount of HPV01, HPV05 and HPV08 peptides were also mixed with CpG (20 μg/injection) as comparison. Mice were immunized once intradermally. 15 days after the immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with different concentration of antigen specific peptides (5, 10, 20, and 50 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from pooled cells within each group of mice (n = 3). Results were expressed as Stimulation Index (SI) of the antigen-induced IFN-γ responses (measured by SFU) over the background levels (media alone responses) (± SD triplicated in assay) upon stimulation with HPV05, HPV08 and HPV01 peptide at 20 μg/ml. ^***p < 0.001 (A): HPV05-PSNPs formulation vs. HPV05+CpG ± HPV12 formulations (representative 1 of 3 experiments); (B): HPV01-, HPV05-, and HPV08-PSNPs formulations vs. each peptide adjuvanted by CpG formulations (summarized from multiple experiments) in comparison.

Figure 3

Impact of immunization schedules and time interval on HPV08-PSNPs immunogenicity. HPV08 peptides were covalently conjugated to PSNPs forming HPV08-PSNPs nanovaccine formulation (final containing 0.37 mg/ml of HPV08 conjugated to PSNPs, 100 μl (or 37 μg)/injection). Mice were immunized following the schedules listed in the figure. Twelve days after the last immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with antigen specific peptides (HPV05 and HPV08, all at 25 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from individual mouse (n = 4). Results are expressed as net spot-forming-unit (SFU)/million splenocytes/mouse upon each peptide recall ± SD (n = 4 individual mice). Two-way ANOVA analysis indicated the significance of HPV05 and HPV08 peptides induced specific responses in the HPV08-PSNPs formulations ^*p < 0.05, ^**p < 0.01.

Figure 4

Optimization of conjugation conditions to covalently conjugate WT1 peptides to PSNPs to produce uniform WT1(peptide)-PSNPs nanovaccine formulations. PSNPs (1% solid final) were pre-activated following the standard procedure (detailed in Materials and Methods), and then re-conditioned in different buffer and pH solutions before mixing with each peptide antigen (0.5 mg/ml final) for conjugation. After conjugation, the final particle sizes for each peptide-PSNPs formulation was assessed using a Zetasizer. Data presented as peptide-PSNPs conjugate size (nm) ± SD (3 repeated measurements) under each conjugation conditions for each peptide. The dotted lines indicated the acceptable nanovaccine formulation size range at 40–60 nm.

Figure 5

Induction of IFNγ-producing antigen specific CD8⁺ T cells following i.d. administrations of WT1(peptide)-PSNPs candidates in HLA-A2.1/Kb mice. WT1 derived peptides (WT1A, WT1B, WT1C, WT1D, and WT1E) were covalently conjugated to PSNPs to constitute PSNPs vaccine formulations (containing 0.5 mg/ml of each peptide in each of the conjugation mix). Mice were immunized 3 times with each formulation (100 μl or 50 μg (including both conjugated and non-conjugated peptide)/injection) intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN γ ELISpot assay upon stimulations with WT1 peptides (5 μg/ml) or controls (media alone or Con A). Each condition was tested in triplicate on splenocytes from individual mouse (n = 4). Results are expressed as stimulation index (SI) of the SFU over the background (media alone) ± SD (n = 4 individual mice). Two-way ANOVA analysis indicated the significance of WT1A and WT1B peptide processing in the WT1peptide-PSNPs formulations. ^*p < 0.05, ^**p < 0.01; ^***p < 0.001. Figure was summarized from multiple experiments.

Figure 6

Optimization of conjugation conditions to covalently conjugate Survivin peptides to PSNPs to produce uniform SV(peptide)-PSNPs nanovaccine formulations. PSNPs (1% solid final) were pre-activated following the standard procedure (detailed in Materials and Methods), and then re-conditioned in different buffer and pH solutions before mixing with each peptide antigen (0.5 mg/ml final) for conjugation. After conjugation, the final particle sizes for each peptide-PSNPs formulation was assessed using a Zetasizer. Data presented as peptide-PSNPs conjugate size (nm) ± SD (3 repeated measurements) under each conjugation conditions for each peptide. The dotted lines indicated the acceptable nanovaccine formulation size range at 40–60 nm.

Figure 7

Antigen-specific T cell responses in HLA-A2.1/Kb mice induced by SV peptides with CpG or PSNPs. SV-derived peptides: (A) SV01 and SV02, (B) SV10, (C) SV10, SV12, SV13, SV14, and SV16 were covalently conjugated to PSNPs forming PSNPs vaccine formulations. Each formulation contained equal amount of each SV peptide target and PSNPs (all at 0.5 mg/ml per peptide, 1% solid for PSNPs; 100 μl/injection). Equivalent amount of SV01, SV02, and SV10 peptides were also mixed with CpG (20 μg/injection) as comparison. For each immunization group, mice were immunized 3 times intradermally, 10 days apart. 11 days after the last immunization, antigen specific T cell responses were evaluated by IFN-γ ELISpot assay upon stimulations with antigen specific peptides (dosages on the figure (μg/ml) except C all at 25 μg/ml) or controls (media alone, or Con A). Each condition was tested in triplicate on splenocytes from individual mouse (n = 3–4). Results are expressed as the Stimulation Index (SI) of the antigen-induced IFN-γ responses (measured by SFU) over the background levels (media alone responses) ± SD (n = 4 individual mice) upon stimulation for each peptide conditions assayed in triplicated wells. Two-way ANOVA analysis indicated the significance of antigen specific responses induced by specific peptides in the SVpeptide-PSNPs or SVpeptide+CpG formulations. ^*p < 0.05, ^**p < 0.01; ^***p < 0.001, and ^****p < 0.0001. Figure was summarized from multiple experiments.