Vaccination with Prion Peptide-Displaying Polyomavirus-Like Particles Prolongs Incubation Time in Scrapie-Infected Mice

Figures

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  Figure 1. Immunisation and sample collection regimen with indication of treatment dates (days).

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  Figure 2. Schematic illustration of hamster polyomavirus (HaPyV) VP1 or VP2 fusion proteins with murine prion protein (MPrP) peptides (a) and detection of formation of virus-like particles by negative staining electron microscopy (b). L, linker GSSG.

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  Figure 3. Analyses of purified proteins expressed by constructs K1 to K9 (lanes 1–9), VP1/VP2 protein controls (lane 10) and of lysate of non-transformed yeast cells (lane 11) by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (a) and Western blot assay with VP1 specific (b), VP2 specific (c) and PrP specific antibodies SAF70 (d), 3F4 (e) and 8H4 (f). Pre-stained marker (lane 12) encompassing 25–70 kDa.

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  Figure 4. Optical density (OD) of indirect VP1 IgG enzyme-linked immunosorbent assay (ELISA) displaying relative OD (a) as well as raw data (b) from mice sera after immunisation with corresponding VLP constructs K1–K9 at three different timepoints: 0 (priming), 1 (21 days, first boost) and 2 (42 days, second boost). Indirect ELISA is performed with yeast-expressed VP1-protein as antigen. Controls include non-immunized (Co) and VP1/2 immunized mice. * (p < 0.05); ** (p < 0.005); *** (p < 0.001).

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  Figure 5. (a) Western blot analysis of pooled sera from animals immunized with VP1/2 control (VP1, lane 2), K1 (lane 3), K2 (lane 4), K3 (lane 5), K4 (Lane 6), K5 (lane 7), K6 (lane 8), K7 (lane 9), K8 (lane 10), K9 (lane 11). Each lane contains blotted recombinant murine prion protein separated on an 16% SDS-PAGE and probed with corresponding serum pools. mAb SAF70 (lane 1) was used as a positive control (Co). (b) Semi-quantitative analysis by VersaDoc. Western blot signals were measured with a digital camera and evaluated with the respective software (Quantity One).

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  Figure 6. VLP construct K9 prolongs incubation time in mice challenged with murine RML scrapie prions. Comparison of mean survival times ± the standard deviation for mice immunized with VLP constructs K1-K9, VP1/2 control and non-immunized control (Co) mice (a). Columns are labeled with elongation time [days] compared to untreated control. Kaplan–Meier survival analysis of intraperitoneally (i.p.) infected mice after s.c. treatment with construct K9. Controls included untreated controls (Co) and VP1/2 controls (b).

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  Figure 7. PrPSc accumulation in the brains of selected mice challenged with RML. Immunoblot analysis of RML-infected mouse brain homogenate digested with proteinase K (PK). (a) PrPSc of scrapie infected mice without VLP treatment and (b) PrPSc of scrapie infected mice immunized with VLP construct K9. Detection of PrPSc was carried out with mAb SAF70. (M) molecular weight marker.