Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles

Abstract

Virus-like particles (VLPs) are promising vaccine technology due to their safety and ability to elicit strong immune responses. Chimeric VLPs can extend this technology to low immunogenicity foreign antigens. However, insertion of foreign epitopes into the sequence of self-assembling proteins can have unpredictable effects on the assembly process. We aimed to generate chimeric bovine papillomavirus (BPV) VLPs displaying a repetitive array of polyanionic docking sites on their surface. These VLPs can serve as platform for covalent coupling of polycationic fusion proteins. We generated baculoviruses expressing chimeric BPV L1 protein with insertion of a polyglutamic-cysteine residue in the BC, DE, HI loops and the H4 helix. Expression in insect cells yielded assembled VLPs only from insertion in HI loop. Insertion in DE loop and H4 helix resulted in partially formed VLPs and capsomeres, respectively. The polyanionic sites on the surface of VLPs and capsomeres were decorated with a polycationic MUC1 peptide containing a polyarginine-cysteine residue fused to 20 amino acids of the MUC1 tandem repeat through electrostatic interactions and redox-induced disulfide bond formation. MUC1-conjugated fully assembled VLPs induced robust activation of bone marrow-derived dendritic cells, which could then present MUC1 antigen to MUC1-specific T cell hybridomas and primary naïve MUC1-specific T cells obtained from a MUC1-specific TCR transgenic mice. Immunization of human MUC1 transgenic mice, where MUC1 is a self-antigen, with the VLP vaccine induced MUC1-specific CTL, delayed the growth of MUC1 transplanted tumors and elicited complete tumor rejection in some animals.

Fig. 1

Expression profile of chimeric L1 constructs and electron micrographs of purified chimeric particles. a Western blot analysis using a monoclonal anti-BPV L1 in lysates of Hi5 cells infected with the four recombinant baculoviruses BPV-BC-E8c, BPV-DE-E8c, BPV-HI-E8c, BPV-H4-E8c, and BPV-WT expressing the chimeric and native L1 proteins. b Western blot analysis of purified BPV-DE-E8c, BPV-HI-E8c, and BPV-H4-E8c. The BPV-BC-E8c did not result in any particle formation. c BPV-HI-E8c VLPs, magnification 30 K, the scale bar is 100 nm; d BPV-H4-E8c capsomeres, magnification at 70 K, scale bar is 50 nm; e BPV-DE-E8c partially assembled VLPs, magnification at 30 K, the scale bar is 100 nm. For electron microscopy, the purified particles were loaded on carbon-coated copper grids, negatively stained with 2% potassium phosphotungstate (pH = 7), and visualized under a JEOL 1200 TEM

Fig. 2

Quantitative and qualitative assessment of MUC1 peptide conjugation on chimeric VLPs. a SDS-PAGE and Coomassie brilliant blue staining of conjugated BPV-HI-E8c VLPs. As much as 10, 20, and 30 μg of conjugated VLPs and 100 ng of R8c-MUC1 (mass standard) were loaded on separate lanes. Due to overloading of the gel (at 30 μg), delayed band migration is observed. The L1 runs according to the theoretical MW of 56 kDa. The R8c-MUC1 runs as ~6 kDa. b Immunogold labeling of BPV-HI-E8c VLPs conjugated with the R8c-MUC1 peptide. The conjugated VLPs were adsorbed on formvar/carbon-coated nickel grids. The primary antibody was monoclonal anti-MUC1 IgG, and the secondary was colloidal-gold-conjugated (6 nm) goat anti-mouse-IgG. Negative staining was performed with 1% sodium silicotungstate (pH = 6.5). Magnification is at 40 K and the scale bar is 100 nm

Fig. 3

BMDC activation following uptake of BPV and BPV-MUC1. a Bone marrow DC were loaded with various BPV constructs (WT BPV, BPV-HI-E8c-MUC1, BPV-HI-E8c; BPV-H4-E8c-MUC1, BPV-H4-E8c) for 24 h, and subsequently were stained for standard DC maturation markers CD40, CD80, CD86, and MHC class II and analyzed by flow cytometry (unconjugated vs. mock: p = 0.000195; conjugated vs. mock: p = 0.0000035). b Supernatants harvested from DC cultures, 24 h post-treatment with various constructs, were used to assess IL-12 secretion using IL-12 ELISA (unconjugated vs. mock: p = 0.0236; conjugated vs. mock: p = 0.00346684). DC alone (untreated UT), MUC1 peptide (250 ng-GVTSAPDTRPAPGSTAPPAH)(pep) *p < 0.05; **p < 0.01

Fig. 4

MUC1 conjugated to chimeric BPV VLPs can be cross-presented to primary, naïve MUC1-specific T cells. IFNγ production following mock-treatment (untreated), addition of MUC1 peptide (10, 50, and 250 ng), or treatment with unconjugated (BPV-HI-E8c) and MUC1-conjugated (BPV-HI-E8c-MUC1) chimeric VLPs (1, 5, and 25 μg). p values were calculated against peptide alone for each concentration shown. **p < 0.01

Fig. 5

T cell activation in MUC1-Tg mice. Proliferation of MUC1-specific CD8⁺ T cells (a) and CD4⁺ T cells (b) following in vitro culture of CFSE-labeled splenocytes for 5 days with the immunizing antigens as indicated. c On day 5, supernatants were harvested and measured for the presence of IFNγ using ELISA. The results shown are for individual mice. Values of *p < 0.05 were considered significant. n = 6–7 mice per group

Fig. 6

Tumor progression following vaccinations. Mice were injected with 5 × 10⁴ RMA-MUC1 tumor cells 2 weeks following the final vaccine boost (5 μg per dose). Tumor progression in individual MUC1-Tg treated with PBS (a), vector alone BPV-HI-E8c (b), and vaccine BPV-HI-E8c-MUC1 (c) was followed for 60 days. On day 21 (day before the first mouse in the PBS negative control groups was sacrificed), tumors in all mice in all groups were measured (d). Values of *p < 0.05 were considered significant; **p < 0.01. n = 21 per group for vaccine and vector group and n = 9 mice per group for PBS group