Herein we show that a DNA vaccine encoding PrPCcan break tolerance to this host protein, inducing both humoral and PrP-specific T-cell responses and a degree of protection against PrPScchallenge. human health (16). Transformation of the ubiquitous cellular prion protein (PrPC) into a pathological conformer (PrPSc) seems to be the key event in pathogenesis (5,11). Emerging data indicate that this devastating group of diseases may be amenable to immunotherapy and immunoprophylaxis, raising the prospect that the spread of these diseases may be countered by vaccination (23). Several reports have shown that antibodies to the normal cellular protein, PrPC, when introduced by injection (27) or by transgenic means (9,25), can arrest the accumulation of the pathological protein conformer PrPSc, thereby interfering with disease progression. However, active immunization strategies against this infectious disease are seriously compromised by host tolerance to PrPC(2); the feasibility of inducing humoral (7,14,24) and T-cell (22) responses to prion protein in wild-type (WT) mice has previously been demonstrated, but Pamabrom to our knowledge, no reports have described the concomitant induction of PrP-specific B-cell, CD4+T-cell, and CD8+T-cell responses in healthy wild-type mice after DNA immunization. Nucleic acid immunization has previously been shown to break tolerance to host proteins (6,12), and we have exploited this phenomenon to confer protection against a murine melanoma (29). Herein we show that a DNA vaccine encoding Rabbit Polyclonal to XRCC2 PrPCcan break tolerance to this host protein, inducing both humoral and PrP-specific T-cell responses and a degree of protection against PrPScchallenge. Several different plasmid constructs were evaluated, each encoding a different version Pamabrom of the mouse PrP. The first plasmid (pCMV-PrP) expressed an unmodified protein; the second (pCMV-UbPrP) encoded PrP fused to the cellular protein ubiquitin to enhance antigen presentation via major histocompatibility complex (MHC) class I, thereby improving the induction of antigen-specific CD8+T cells (17,21); and the third (pCMV-PrPLII) encoded PrP fused to the lysosomal integral membrane protein type II (LIMPII) lysosome-targeting signal, which enhances MHC class II antigen presentation and the induction of CD4+T-cell responses (18). We find that the third construct was the most effective at breaking immune tolerance and enhanced both humoral and cellular responses in wild-type animals. Most importantly, this DNA vaccine conferred significant protection against intracerebral prion challenge, dramatically delaying the onset of disease. However, this benefit had an associated cost: once disease appeared, it was rapidly progressive. The advantages and disadvantages of vaccinating against prion diseases and the possible explanations for the clinical effects are discussed. == MATERIALS AND METHODS == == Mice. == The 129/ola strain was purchased from Harlan, and 129/ola PrP knockout (PrPKO) mice were generously provided by Jean Manson, IAH, Edinburgh, United Kingdom. == Construction of recombinant plasmids expressing the mouse prion protein. == The mouse prion open reading frames with or without their termination codon was obtained by PCR using the forward (5-AGATCTATGGCGAACCTTGGCTACTGGC-3) and the stop reverse (5-AGATCTCATCCCACGATCAGGAAGATG-3) or the nonstop reverse (5-AGATCTTCCCACGATCAGGAAGATGAGG) primers. The amplified fragments were cloned either in the pCMV-LII plasmid (18) or in the pCMV-F1/F2Ubiq plasmid (19) to obtain pCMV-PrP, pCMV-PrPLII, and Pamabrom pCMV-UbPrP plasmids. All the products were expressed under the control of the immediate early promoter of human cytomegalovirus using the pCMV expression vector (Clontech). == Protocol for DNA immunization. == DNA purification was carried out using endotoxin-free columns (QIAGEN). DNA was dissolved at 1 mg/ml in 1 N saline, and mice were immunized into each anterior tibial muscle with 100 g of DNA per mouse and dose. == Inoculation of mice with prions. == The prion inoculum used in these experiments was derived by twofold serial passage of the BSE1inoculum (4) in mice. Twenty microliters of a 10% brain homogenate was delivered by intracerebral injection into the right parietal lobes of mice. Mice were monitored weekly until the onset of terminal disease, at which time daily examination was performed. Early clinical signs included a waddling gait, ruffled coat, and kyphosis for at least three consecutive weeks. Severe clinical signs included tremors, a blank stare, lethargy, and immobility. Mice were sacrificed at.