Testing the Possibility to Protect Bovine PrPC Transgenic Swiss Mice Against Bovine PrPSc Infection by DNA Vaccination Using Recombinant Plasmid Vectors Harboring and Expressing the Complete or Partial cDNA Sequences of Bovine PrPc

Abstract

The objective of this study was to investigate the molecular mechanisms of neurobiological processes involved in the degeneration of the central nervous system. The bovine spongiform encephalopathy (BSE) was used as experimental model system for investigation of transmissible spongiform encephalopathy (TSE). The experimental strategy was to evaluate the possibility for protection of bovine PrP(C) transgenic mice against a bovine PrP(Sc) infection by DNA vaccination using the complete or partial cDNA sequences of the bovine prion protein. Three recombinant plasmids pCR3.1-EX-PrP-BSE-C20 (C20), pCR3.1-EX-PrP-BSE-90-235-C4 (C4), and pCR3.1-EX-PrP-BSE-106-131-C14 (C14) were constructed. These mammalian expression vectors harbor complete (C20) or partial (C4 and C14) cDNA sequences of the Bos taurus PrP(C) (BTPrP(C)) encoding for amino acid residues 1-264 (C20), 90-235 (C4), and 106-131 (C14) of the BTPrP(C). Transgenic mice harboring and expressing BTPrP(C) were generated using the donor strain C57/CBA, receptor strain Swiss mouse, and recombinant plasmid MoPrPXho-boPrP. Crossing of positive transgenic mice to bovine PrP and negative to murine PrP with 129/OLA (murine PrP-/-) and C57BL6x129/OLA (murine PrP+/-) mice was carried out to amplify the colony of transgenic mice termed bovine PrP(C) transgenic Swiss mice (BTPrP-TgM). The capabilities of C20, C4, and C14 to express the corresponding cDNA sequence of BTPrP(C) in vitro and in vivo were confirmed prior to DNA vaccination of the BTPrP-TgM using NIH 3T3 cells and BALB/c mice, respectively. In order to prove the capability of the constructed expression vectors to protect BTPrP-TgM in vivo against a BSE infection 80 female BTPrP-TgM were vaccinated intramuscularly and subcutaneously with DNA of the plasmids C20, C4, C14, and parental vector pCR3.1 (100 microg DNA corresponding to about 26-30 pmol DNA/animal and application) in four groups (each consists of 20 animals). DNA vaccination was followed by three additional boosters. The vaccinated animals (15 animals of each group) were challenged twice per oral with homogenates of brain material obtained from BSE cattle containing the infectious PrP(Sc) (100 microl/animal which corresponds to 15 mg of a 15% brain homogenate). The first and second challenge experiments were performed 76-83 and 181 days post DNA vaccination, respectively. A part of the vaccinated animals (3-5 animals of each group) that served as internal negative control were mock infected using the brain homogenate of healthy cattle or Phosphate saline buffer (PBS). A variety of symptoms and clinical pictures were observed during the monitoring of DNA vaccinated animals. However, the observed diseases seem to be similar in all experimental animal groups. After an observation period of 14 months post the second challenge experiment the remaining animals (some animals died or were sacrificed when moribund during the study) were sacrificed after expiration of the experimental schedule. The right hemisphere of the brain and a half of the spleen tissue of the individual animals were used for detection of PrP(Sc) by Western blot analysis. The misfolded bovine PrP(Sc) was not detected in the brain or spleen tissues of those animals that were vaccinated with DNA of C20, which was able to express the complete bovine PrP(C) protein in vitro and in vivo. In contrast, the bovine PrP(Sc) was detected in the brain or spleen tissues of animals that were DNA vaccinated with DNA of the parental vector pCR3.1, with DNA of C4, or with DNA of C14. The results of these studies underline that the constructed expression vector C20 possesses the protective capacity to inhibit the formation of misfolded bovine PrP(Sc) in BTPrP-TgM under the conditions used. A delay of occurrence of TSE-specific symptoms in the majority of the vaccinated animals seems to be due to the prolonged incubation time of BSE infection.