Induction of insert-specific immune response in mice by hamster polyomavirus VP1 derived virus-like particles carrying LCMV GP33 CTL epitope

Abstract

Hamster polyomavirus (HaPyV) major capsid protein VP1 based chimeric virus-like particles (VLPs) carrying model GP33 CTL epitope derived from Lymphocytic choriomeningitis virus (LCMV) were generated in yeast and examined for their capability to induce CTL response in mice. Chimeric VP1-GP33 VLPs were effectively processed in antigen presenting cells in vitro and in vivo and induced antigen-specific CD8+ T cell proliferation. Mice immunized only once with VP1-GP33 VLPs without adjuvant developed an effective GP33-specific memory T cell response: 70% were fully and 30% partially protected from LCMV infection. Moreover, aggressive growth of tumors expressing GP33 was significantly delayed in these mice in vivo. Therefore, HaPyV VP1-derived VLP harboring CTL epitopes are attractive vaccine candidates for the induction of insert-specific CTL immune response.

Fig. 1

Generation and purification of HaPyV VP1 based VLPs. (A) Nucleotide and amino acid sequences of LCMV GP33 epitope. (B) Schematic figure of VP1, VP1-GP33-1 and VP1-GP33-4 proteins (aa positions of insertion sites #1 and #4 are indicated) and their identification in SDS-PAGE and Western blot analysis (WB) with anti-HaPyV VP1 mAbs 6D11 (C). In lanes: 1 – purified VP1 protein; 2 – lysate of yeast cells expressing VP1-GP33-1 VLPs; 3 – lysate of yeast cells expressing VP1-GP33-4 VLPs; 4 – purified VP1-GP33-1 protein; 5 – purified VP1-GP33-4 protein; 6 – PageRuler prestained protein ladder (#SM0671 Fermentas, Lithuania). (D) Electron microscopy pictures of VLPs, stained with 2% aqueous uranyl acetate solution and examined by Morgagni electron microscope.

Fig. 2

Proliferation of GP33-specific CD8+ T cells in vitro. CFSE-labeled splenocytes were labeled with PE conjugated anti-mouse CD8 antibodies after 4–5 days of stimulation and analyzed by flow cytometry. Living cells were gated on CD8+ T cells and percentage of proliferating CD8+ cells (identified by low CFSE level) was determined. In (A) flow cytometry data from one representative experiment where CFSE-labeled P14 mouse splenocytes were stimulated with GP33 peptide, PBS or VLPs (VP1, VP1-GP33-1, and VP1-GP33-4 VLPs) were shown. For better visualization and for evaluation of statistical significance data from 4 independent experiments (2 P14 mice per group were used in each experiment) were summarized in diagram (B). Values were shown as mean ± SD. All the differences were statistically significant (p < 0.02) if not labeled n.s. – not significant (t-test). (C) CFSE-labeled splenocytes from P14 mouse and from B6 mouse (for negative control) were stimulated with DCs treated with 10 μg/mL VP1, VP1-GP33-1, VP1-GP33-4 VLPs (10⁶ splenocytes + 10⁵ DCs/mL). PBS and untreated DCs were used as negative controls. GP33 peptide and DCs pulsed with GP33 peptide were used as positive controls. The percentage of proliferating CD8+ T cells for better visualization and for evaluation of statistical significance was shown in diagrams. Values from two independent experiments (3 B6 and P14 mice per group were used in both experiments) were shown as mean ± SD. All the differences were statistically significant (p < 0.05) if not labeled n.s. (t-test).

Fig. 3

Detection of chimeric VLPs-induced GP33-specific memory CTL response in mice by CD8+ T cell restimulation in vitro (A) and LCMV challenge in vivo (B). Adult B6 mice were immunized i.v. with 50 μg of VLPs (VP1, VP1-GP33-1 and VP1-GP33-4), or 200 pfu LCMV, or PBS for positive and negative controls respectively. 8 weeks later CFSE-labeled splenocytes from immunized mice were restimulated with GP33 peptide for 3 days (A) or mice were challenged with LCMV (B). (A) Splenocytes from 3 B6 mice per group were restimulated and the percentage of proliferating CD8+ T cells for better visualization and for evaluation of statistical significance was shown in diagrams. Values from three independent experiments (3 B6 mice per group) were shown as mean ± SD. All the differences were statistically significant (p < 0.05) if not labeled n.s. (t-test). (B): Mice were challenged i.v. with 200 pfu of LCMV; viral titer in the spleen was determined 4 days after the challenge and expressed as pfu/g of spleen tissue. Each circle represents an individual mouse, horizontal dash – mean titer value in the group. The dotted line represents the assay cutoff. Data from two representative experiments (3 and 2 B6 mice per group) are shown. Differences between groups are statistically significant, p < 0.05 according Wilcoxon Mann–Whitney test (except VP1-GP33-1 vs. VP1-GP33-4).

Fig. 4

Protective and therapeutical efficacy of HaPyV VP1 based chimeric VLPs in vivo using tumor model system. (A and B) Adult B6 mice were immunized i.v. with 50 μg of VLPs (VP1, VP1-GP33-1 and VP1-GP33-4), or 200 pfu LCMV, or PBS for positive and negative controls respectively. 8 weeks later mice were challenged by injecting s.c. into the flank (A) 10⁶ of MCA102 tumor cells not expressing GP33 (3 B6 mice per group) or (B) MCA102-GP33 tumor cells expressing GP33 (7 B6 mice per group). Progression in time of tumor size of individual mice calculated as the product of bisecting tumor diameters is displayed. The horizontal dotted line marks tumor size 50 mm². (D) B6 mice were injected with 10⁶ of MCA102-GP33 tumor cells s.c. into the flank. Therapeutical effect of HaPyV VP1 and VP1-GP33 VLPs injections on MCA102-GP33 tumor growth was examined when tumors reached ∼25 mm² and mice were treated i.v. with 10 μg VLPs or with PBS (6 B6 mice per group). Progression in time of tumor size of individual mice calculated as the product of bisecting tumor diameters is displayed. The horizontal dotted line marks tumor size 50 mm². Schedule of immunization on days 9, 11 and 13 when the treatments were administrated is shown by arrows.

Fig. 5

Detection of antiVP1-GP33 antibodies in serum of B6 mice immunized with VP1-GP33 chimeric VLPs. Four B6 mice were immunized with VP1-GP33-1 or VP1-GP33-4 VLPs three times at 28-day intervals. (A) Antibodies against VP1-GP33 VLPs were detected by Western blot analysis with after third immunization from VP1-GP33-1 VLPs (middle panel) and VP1-GP33-4 VLPs (right panel) immunized mice. The SDS-PAGE of same proteins shown in left panel. In lines: 1 – VP1 protein; 2 – VP1-GP33-1; 3 – VP1-GP33-4; 4 – negative control, Bocavirus VP2 protein; 5 – PageRuler prestained protein ladder (#SM0671 Fermentas, Lithuania). (B) GP33 specific antibodies were detected by flow cytometry of MCA102-GP33 cells after incubation with serum samples and staining. Parental MCA102 cells without GP33 antigen were used as negative control.