FIG. 3.
Breaking tolerance against PrP by DNA immunization. (A) Serial dilutions of sera from WT 129/ola mice immunized three times with pCMV (dashes), pCMV-PrP (circles), pCMV-UbPrP (triangles), and pCMV-PrPLII (squares) were tested against ELISA plates coated with recombinant prion protein. The standard deviation for each group is shown. Serum from a WT 129/ola mouse immunized with pCMV-PrPLII was diluted 1:200 and tested in a Western blot assay (inset) using as the antigen (lane 1) brain extracts from a WT 129/ola mouse (PrPC), (lane 2) recombinant prion protein, or (lane 3) brain extracts from a PrPSc-infected mouse after proteinase K treatment. The data were identical for all mice in the same group. Molecular weight standards in thousands (K) were used as size markers. (B) Splenocytes from mice immunized with pCMV-PrPLII were harvested, incubated together with the indicated semipurified protein stimulus at 1 μg/ml for 36 h, and subjected to ICCS. Circles and percentages shown (values given after subtraction of the control value) indicate the PrP-specific CD4+ T cells (B, left panel) and CD8+ T cells (B, right panel) induced after DNA immunization with pCMV-PrPLII.