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

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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.

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Acknowledgments

This work was supported in part by the Virginia-Maryland Regional College of Veterinary Medicine (to I.B.) and NIH grant P01 CA073743 (to O.J.F.). SPG was supported by The Sass Foundation for Medical Research Postdoctoral Fellowship. We would like to thank Hamp Edwards and Kristen Gambles from the University of Maryland for help with electron microscopy and insect cell culture work.

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Authors and Affiliations

  1. Department of Veterinary Medicine, University of Maryland, 8075 Greenmead Drive, College Park, MD, 20742, USA

    Yogendra Rajawat, Zoe Hilioti & Ioannis Bossis

  2. Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA, 15261, USA

    Sharmila Pejawar-Gaddy, Jia Xue, Daniel F. Gaddy & Olivera J. Finn

  3. Stanley Division of Developmental Neurovirology, Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, 21287, USA

    Raphael P. Viscidi

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  1. Sharmila Pejawar-Gaddy
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Correspondence to Ioannis Bossis.

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Supplemental Figure 1

Bovine Papillomavirus type-1 L1 protein 3-D structure with prediction of virion surface-exposed areas. The L1 monomer contains 6 loops (BC, CD, DE, EF, FG, HI), and 5 helices (H1-H5). In red are the 3 loops and the one helix that we replaced one at a time with a polyglutamic-cysteine epitope, and generated four chimeric constructs. (DOC 130 kb)

Supplemental Figure 2

Effect of reaction conditions on conjugation of MUC1 polycationic peptides on chimeri VLPs. a) Effect of ionic strength on conjugation efficiency of BPV-HI-E8c, and BPV-H4-E8c with the polyarginine MUC1 peptide R8c-MUC1. The conjugation efficiency is presented as the amount of MUC1 reactivity (OD) in Elisa assays where the plates were coated with conjugated particle. b) Effect of the ratio of oxidized (GSSG) to reduced glutathione (GSH) on conjugation efficiency of BPV-HI-E8c, and BPV-H4-E8c with the polyarginine MUC1 peptide R8c-MUC1. c) Quantitative assessment of conjugation efficiency in the BPV-HI-E8c, and BPV-H4-E8c with the polyarginine MUC1 peptide R8c-MUC1. The MUC1 reactivity of conjugated particles in ELISA assays were compared to the reactivity of various amounts of free R8c-MUC1 peptide. (DOC 87 kb)

Supplemental Figure 3 BMDC activation following uptake of BPV and BPV-MUC1

. 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 24h were stained for standard DC maturation markers CD40, CD80, CD86 and MHC class II and analyzed by flow cytometry. Shown here are representative histograms for each of the costimulatory molecules analyzed. DC alone (untreated - UT), MUC1 peptide (250ng-GVTSAPDTRPAPGSTAPPAH)(peptide). (DOC 147 kb)

Supplemental Figure 4 MUC1-speific IgG in MUC1 transgenic mice.

Mice were vaccinated three times, two weeks apart, with vector control (BPV-HI-E8c), vaccine (BPV-HI-E8c-MUC1) or left untreated (UT). Blood was collected from the mice prior to vaccination (pre vaccine) and following final treatment (post vaccine). Serum (1:40 dilution) was then analyzed to determine the presence of antibodies to MUC1. 4 mice were analyzed in the untreated group and 9 mice in the vector and vaccine groups. (DOC 51 kb)

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Pejawar-Gaddy, S., Rajawat, Y., Hilioti, Z. et al. Generation of a tumor vaccine candidate based on conjugation of a MUC1 peptide to polyionic papillomavirus virus-like particles. Cancer Immunol Immunother 59, 1685–1696 (2010). https://doi.org/10.1007/s00262-010-0895-0

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