WO2004007743A2 - Use of cpg nucleic acids in prion-disease - Google Patents

Use of cpg nucleic acids in prion-disease Download PDF

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Publication number
WO2004007743A2
WO2004007743A2 PCT/IB2003/003727 IB0303727W WO2004007743A2 WO 2004007743 A2 WO2004007743 A2 WO 2004007743A2 IB 0303727 W IB0303727 W IB 0303727W WO 2004007743 A2 WO2004007743 A2 WO 2004007743A2
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prion
method according
disease
nucleic acid
cpg
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PCT/IB2003/003727
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French (fr)
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WO2004007743A3 (en
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Hermann Wagner
Hans Kretzschmar
Shneh Sethi
Grayson Lipford
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Coley Pharmaceutical Gmbh
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Priority to US60/396,432 priority
Application filed by Coley Pharmaceutical Gmbh filed Critical Coley Pharmaceutical Gmbh
Publication of WO2004007743A2 publication Critical patent/WO2004007743A2/en
Publication of WO2004007743A3 publication Critical patent/WO2004007743A3/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088Compounds having three or more nucleosides or nucleotides
    • A61K31/7125Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters

Abstract

Methods are provided which are useful in the treatment of prion diseases and other protein deposit diseases, including, for example, post-exposure prophylaxis against the development of iatrogenic Creutzfeldt-Jakob disease. The methods involve the use of immunostimulatory nucleic acids, including CpG nucleic acids.

Description

USE OF CpG NUCLEIC ACIDS IN PRION DISEASE

Field of the Invention

The instant invention pertains to methods useful in the treatment of prion diseases, including, for example, post-exposure prophylaxis against the development of iatrogenic Creutzfeldt- Jakob disease. The methods involve the use of immunostimulatory nucleic acids.

Background of the Invention

Prion diseases include a number of fatal, neurodegenerative diseases believed to be caused by aggregates of normal protein that is present in an abnormal conformation. The normal protein, prion protein, is usually present in the cell membrane of many tissues, particularly neuronal tissue. The abnormally conformed prion protein is believed to be directly involved in converting normally conformed prion protein into more of the abnormally conformed prion protein, which then self-assembles into aggregates that are damaging to neuronal tissue anatomy and function.

At least some of the prion diseases are transmissible. However, unlike bacteria, viruses, fungi, parasites, and other replicating pathogens, transmissible prions are simply proteins; they are transmissible without any accompanying nucleic acid. For reasons that are not yet fully understood, the abnormally conformed prion proteins normally do not induce an immune response. Thus, exposure of a healthy individual to abnormally conformed prion protein can initiate a prion disease that can go unchecked by the immune system.

Exposure to abnormally conformed prion protein thus represents a health risk to susceptible individuals. Such individuals include humans and non-humans, principally cows and sheep. Exposure can come about through contact with prion-diseased animal products through ingestion, iatrogenic or work-related exposure, transplantation, and administration of pharmaceutical preparations.

Summary of the Invention

The instant invention is based in part on the unexpected discovery by the inventors that administration of immunostimulatory nucleic acid to a subject that is exposed to abnormally conformed prion protein is an effective treatment of prion disease. The immunostimulatory nucleic acid effectively delayed and even prevented disease in animals administered very large doses of prion-diseased brain homogenates that uniformly caused fatal disease in all untreated control animals similarly exposed to prion-diseased brain homogenates.

The immunostimulatory nucleic acids are useful in the treatment of prion diseases, including Creutzfeldt- akob disease (CJD), bovine spongiform encephalopathy (BSE), and scrapie. The methods of the invention are also useful for the study of these diseases, for instance, in animal models. The methods will also be useful for developing an understanding of how prion proteins normally fail to elicit an immune response and how an immune response can be elicited and used to treat prion protein disease.

The immunostimulatory nucleic acids are also useful in the treatment of other neurologic diseases involving abnormal protein deposits or aggregates. Such diseases include Alzheimer's disease, which involves deposits of amyloid. The main component of amyloid plaques is amyloid-β peptide (Aβ), a fibrillar 40-42 amino acid peptide that accumulates extracellularly and causes neuronal death.

In one aspect the invention provides a method for treating a prion disease in a subject. The method involves administering to a subject having or at risk of developing a prion disease a CpG nucleic acid in an effective amount to treat the prion disease. In one embodiment the administering follows exposure of the subject to a prion protein that is associated with a prion disease. In one embodiment the prion disease is a transmissible spongiform encephalopathy (TSE). In one embodiment the subject is a human.

In various preferred embodiments, the prion disease is scrapie, BSE, or a form of CJD. The CJD in one embodiment is iatrogenic CJD (iCJD). In another embodiment the CJD is variant CJD (vCJD). In yet another embodiment the CJD is sporadic CJD.

In one aspect the invention provides a method for inducing an immune response to a prion protein. The method according to this aspect of the invention involves the steps of contacting an antigen-presenting cell (APC) with a prion protein and contacting the APC with a CpG nucleic acid in an effective amount to induce an immune response to the prion protein. In one embodiment the immune response occurs in vivo. In one embodiment the immune response occurs in vitro. In all embodiments according to this aspect of the invention the APC is preferably chosen from a B cell, a dendritic cell, a macrophage, and a monocyte. In one preferred embodiment the APC is a dendritic cell. Also according to this aspect of the invention, in one embodiment the APC expresses a Toll-like receptor (TLR) that signals in response to the CpG nucleic acid. It has recently been reported that CpG nucleic acid can specifically induce a particular TLR, designated TLR9, to signal. Accordingly, in one embodiment the TLR is TLR9.

In one embodiment the prion protein includes prion proteimscrapie form (PrPSc). In another embodiment the prion protein includes a fragment of PrPSc lacking at least the amino terminus of full-length PrP c. In yet another embodiment the prion protein includes a derivative of PrPSc or a derivative of a fragment of PrPSc lacking at least the amino terminus of full-length PrPSc.

In one embodiment the prion protein is prion protein: scrapie form (PrP c). In another embodiment the prion protein is a fragment of PrPSc lacking at least the amino terminus of full-length PrPSc. In yet another embodiment the prion protein is a derivative of PrPSc or a derivative of a fragment of PrPSc lacking at least the amino terminus of full-length PrPSc.

It has been reported that certain CpG nucleic acids are more effective in one species than in another. Accordingly, in preferred embodiments the CpG nucleic acid is optimized for use in a species of the subject.

It has also been reported that CpG nucleic acids appear to fall into different classes based on certain structural features as well as their function. At least three classes are believed to exist, denoted Class A, Class B, and Class C. In one embodiment the CpG nucleic acid is a class B CpG nucleic acid. In one embodiment the CpG nucleic acid is a class A CpG nucleic acid. In one embodiment the CpG nucleic acid is a class C CpG nucleic acid.

These and other features of the invention are described in greater detail in connection with the detailed description of the invention.

Detailed Description of the Invention

A major step in the study of prions and the diseases that they cause was the discovery and purification of a protein designated prion protein (PrP). Bolton et al. (1982) Science 218: 1309-11; Prusiner SB et al. (1982) Biochemistry 21 :6942-50; McKinley MP et al. (1983) Cell 35:57-62. Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrPc is encoded by a single-copy host gene (Basler K et al. (1986) Cell 46:417-28) and is normally found at the outer surface of neurons. Prion diseases are accompanied by the conversion of PrP into a modified form called PrP c.

The scrapie isoform of the prion protein (PrP c) is thought necessary for both the transmission and pathogenesis of the transmissible neurodegenerative diseases of animals and humans. See Prusiner SB (1991) Science 252:1515-22. The most common prion diseases of animals are scrapie of sheep and goats and bovine spongiform encephalopathy (BSE; "mad cow disease") of cattle. Wilesmith J et al. (1991) Microbiol Immunol 172:21-38. Four prion diseases of humans have been identified: (1) kuru, (2) Creutzfeldt-Jakob disease (CJD), (3) Gerstmann-Straussler-Scheinker (GSS) syndrome, and (4) fatal familial insomnia. Gajdusek DC (1977) Science 197:943-60; Medori et al. (1992) NEnglJMed 326:444-9.

Most CJD cases are sporadic, but about 10-15% are inherited as autosomal dominant disorders that are caused by mutations in the human PrP gene. Hsiao et al. (1990) Neurology 40:1820-7; Goldfarb et al. (1992) Science 258:806-8. Iatrogenic CJD has been caused by human growth hormone derived from cadaveric pituitaries as well as dura mater grafts. Brown et al. (1992) Lancet 340:24-7. Despite numerous attempts to link CJD to an infectious source such as the consumption of meat from scrapie-infected sheep, none has been identified to date (Harries- Jones et al. (1988) J Neurol Neurosurg Psychiatry 51 :1113-9) except in cases of iatrogenically induced disease. On the other hand, kuru, which for many decades devastated the Fore and neighboring tribes of the New Guinea highlands, is believed to have been spread by infection during ritualistic cannibalism.

The major component of purified infectious prions, designated PrP 27-30, is the proteinase K resistant core of a larger native protein PrP c which is the disease causing form of the ubiquitous cellular protein PrPc. PrPSc is found only in scrapie infected cells, whereas PrPc is present in both infected and uninfected cells implicating PrPSc as the major, if not the sole, component of infectious prion particles. Since both PrPc and PrPSc are encoded by the same single copy gene, great effort has been directed toward unraveling the mechanism by which PrPSc is derived from PrPc. Central to this goal has been the characterization of physical and chemical differences between these two molecules. Properties distinguishing PrPSc from PrPc include low solubility (Meyer RK et al. (1986) Proc Natl Acad Sci USA 83:2310-4), poor antigenicity (Kascsak RJ et al. (1987) J Virol 61 :3688-93; Serban D et al. (1990) Neurology 40:110-7), protease resistance (Oesch B et al. (1985) Cell 40:735-46), and polymerization of PrP 27-30 into rod-shaped aggregates which are very similar, on the ultrastructural and histochemical levels, to the PrP amyloid plaques seen in scrapie-diseased brains (Prusiner SB et al. (1983) Cell 35(2 Pt l):349-58). By using proteinase K it is possible to denature PrPc but not PrPSc. To date, attempts to identify any post-translational chemical modifications in PrPc that lead to its conversion to PrPSc have proven fruitless. Stahl N et al. (1993) Biochemistry 31:5043-53. Consequently, it has been proposed that PrPc and PrPSc are in fact conformational isomers of the same molecule.

Conformational description of PrP using conventional techniques has been hindered by problems of solubility and the difficulty in producing sufficient quantities of pure protein. However, PrPc and PrPSc are conformationally distinct. Theoretical calculations based upon the amino acid sequences of PrPs from several species have predicted four putative helical motifs in the molecule. Experimental spectroscopic data would indicate that in PrP these regions adopt alpha-helical arrangements, with virtually no beta-sheet. Pan et al. (1993) Proc Natl Acad Sci USA 90:10962-6). In dramatic contrast, in the same study it was found that PrPSc and PrP 27-30 possess significant beta-sheet content, which is typical of amyloid proteins. Moreover, studies with extended synthetic peptides, corresponding to PrP amino acid residues 90-145, have demonstrated that these truncated molecules may be converted to either alpha-helical or beta-sheet structures by altering their solution conditions. The transition of PrPc to PrPSc requires the adoption of beta-sheet structure by regions that were previously alpha-helical.

In general, scrapie infection fails to produce an immune response, with host organisms being tolerant to PrPSc from the same species. Polyclonal anti-PrP antibodies have been raised in rabbits following immunization with large amounts of Syrian hamster PrP 27- 30. Bendheim PE et al. (1985) Proc Natl Acad Sci USA 82:997-1001; Bode L et al. (1985) J Gen Virol 66:2471-8. Similarly, a handful of anti-PrP monoclonal antibodies have been produced in mice. Kascsak Rj et al. (1987) J Virol 61 :3688-93; Barry RA et al. (1986) J Infect Dis 154:518-21. These antibodies are able to recognize native PrP and denatured PrPSc from both Syrian hamsters and humans equally well, but do not bind to murine PrP. Unsurprisingly, the epitopes of these antibodies were mapped to regions of sequence containing amino acid differences between Syrian hamster and murine PrP. Rogers et al. (1991) J Immunol 147:3568-74.

The DNA sequence of the human, sheep and cow PrP genes have been determined, allowing, in each case, the prediction of the complete amino acid sequence of their respective PrP proteins. The normal amino acid sequence which occurs in the vast majority of individuals is refened to as the wild-type PrP sequence. This wild-type sequence is subject to certain characteristic polymorphic variations. In the case of human PrP, two polymorphic amino acids occur at residues 129 (Met/Nal) and 219 (Glu/Lys). Sheep PrP has two amino acid polymorphisms at residues 136 and 171, while bovine PrP has either five or six repeats of an eight amino acid motif sequence (octarepeats) in the amino terminal region of the mature prion protein. While none of these polymorphisms are of themselves pathogenic, they appear to influence prion diseases. Distinct from these normal variations of the wild-type PrP proteins, certain mutations of the human PrP gene which alter either specific amino acid residues of PrP or the number of octarepeats have been identified which segregate with inherited human prion diseases.

For example, sequences of chicken, bovine, sheep, rat, and mouse PrP genes are disclosed and published within Gabriel JM et al. (1992) Proc Natl Acad Sci USA 89:9097- 101. A sequence for a PrP gene of Syrian hamster is published in Basler K et al. (1986) Cell 46:417-28. A PrP gene of sheep is published by Goldmann W et al. (1990) Proc Natl Acad Sci USA 87:2476-80. A gene sequence for bovine PrP is published in Goldmann W et al. (1991) J Gen Virol 72:201-4. A sequence for chicken PrP gene is published in Harris DA et al. (1991) Proc Natl Acad Sci USA 88:7664-8. A PrP gene sequence for mink is published in Kretzschmar HA et al. (1992) J Gen Virol 73:2757-61. A human PrP gene sequence is published in Kretzschmar HA et al. (1986) DΝA 5:315-24. A PrP gene sequence for mouse is published in Locht C et al. (1986) Proc Natl Acad Sci USA 83:6372-6. A PrP gene sequence for sheep is published in Westaway D et al. (1994) Genes Dev 8:959-69. These publications are all incorporated herein by reference to disclose and describe the PrP gene and PrP amino acid sequences.

Human PrP cDΝA (SEQ ID ΝO:l; GenBank Accession No. Ml 3899) cggcgccgcg agcttctcct ctcctcacga ccgaggcaga gcagtcatta tggcgaacct 60 tggctgctgg atgctggttc tctttgtggc cacatggagt gacctgggcc tctgcaagaa 120 gcgcccgaag cctggaggat ggaacactgg gggcagccga tacccggggc agggcagccc 180 tggaggcaac cgctacccac ctcagggcgg tggtggctgg gggcagcctc atggtggtgg 240 ctgggggcag cctcatggtg gtggctgggg gcagccccat ggtggtggct ggggacagcc 300 tcatggtggt ggctggggtc aaggaggtgg cacccacagt cagtggaaca agccgagtaa 360 gccaaaaacc aacatgaagc acatggctgg tgctgcagca gctggggcag tggtgggggg 420 ccttggcggc tacatgctgg gaagtgccat gagcaggccc atcatacatt tcggcagtga 480 ctatgaggac cgttactatc gtgaaaacat gcaccgttac cccaaccaag tgtactacag 540 gcccatggat gagtacagca accagaacaa ctttgtgcac gactgcgtca atatcacaat 600 caagcagcac acggtcacca caaccaccaa gggggagaac ttcaccgaga ccgacgttaa 660 gatgatggag cgcgtggttg agcagatgtg tatcacccag tacgagaggg aatctcaggc 720 ctattaccag agaggatcga gcatggtcct cttctcctct ccacctgtga tcctcctgat 780 ctctttcctc atcttcctga tagtgggatg aggaaggtct tcctgttttc accatctttc 840 taatcttttt ccagcttgag ggaggcggta tccacctgca gcccttttag tggtggtgtc 900 tcactctttc ttctctcttt gtcccggata ggctaatcaa tacccttggc actgatgggc 960 actggaaaac atagagtaga cctgagatgc tggtcaagcc ccctttgatt gagttcatca 1020 tgagccgttg ctaatgccag gccagtaaaa gtataacagc aaataaccat tggttaatct 1080 ggacttattt ttggacttag tgcaacaggt tgaggctaaa acaaatctca gaacagtctg 1140 aaataccttt gcctggatac ctctggctcc ttcagcagct agagctcagt atactaatgc 1200 cctatcttag tagagatttc atagctattt agagatattt tccattttaa gaaaacccga 1260 caacatttct gccaggtttg ttaggaggcc acatgatact tattcaaaaa aatcctagag 1320 attcttagct cttgggatgc aggctcagcc cgctggagca tgagctctgt gtgtaccgag 1380 aactggggtg atgttttact tttcacagta tgggctacac agcagctgtt caacaagagt 1440 aaatattgtc acaacactga acctctggct agaggacata ttcacagtga acataactgt 1500 aacatatatg aaaggcttct gggacttgaa atcaaatgtt tgggaatggt gcccttggag 1560 gcaacctccc attttagatg tttaaaggac cctatatgtg gcattccttt ctttaaacta 1620 taggtaatta aggcagctga aaagtaaatt gccttctaga cactgaaggc aaatctcctt 1680 tgtccattta cctggaaacc agaatgattt tgacatacag gagagctgca gttgtgaaag 1740 caccatcatc atagaggatg atgtaattaa aaaatggtca gtgtgcaaag aaaagaactg 1800 cttgcatttc tttatttctg tctcataatt gtcaaaaacc agaattaggt caagttcata 1860 gtttctgtaa ttggcttttg aatcaaagaa tagggagaca atctaaaaaa tatcttaggt 1920 tggagatgac agaaatatga ttgatttgaa gtggaaaaag aaattctgtt aatgttaatt 1980 aaagtaaaat tattccctga attgtttgat attgtcacct agcagatatg tattactttt 2040 ctgcaatgtt attattggct tgcactttgt gagtatctat gtaaaaatat atatgtatat 2100 aaaatatata ttgcatagga cagacttagg agttttgttt agagcagtta acatctgaag 2160 tgtctaatgc attaactttt gtaaggtact gaatacttaa tatgtgggaa acccttttgc 2220 gtggtcctta ggcttacaat gtgcactgaa tcgtttcatg taagaatcca aagtggacac 2280 cattaacagg tctttgaaat atgcatgtac tttatatttt ctatatttgt aactttgcat 2340 gttcttgttt tgttatataa aaaaattgta aatgtttaat atctgactga aattaaacga 2400 gcgaagatga gcacc 2415

Mink PrP genomic DNA (SEQ ID NO:2; GenBank Accession No. S46825) tcattttgtt ttgttttgtt ttgtttgcag ataagccatc atggtgaaaa gccacatagg 60 cagctggctc ctggttctct ttgtggccac atggagtgac attggcttct gcaagaagcg 120 gccaaagcct ggaggaggct ggaacactgg ggggagccga tacccagggc agggcagtcc 180 tggaggcaac cgctacccac cccagggtgg tggcggctgg ggccagcccc acgggggtgg 240 ctggggacag ccccacgggg gtggctgggg tcagccccac gggggtggct ggggacagcc 300 gcatggtggc ggtggctggg gtcaaggtgg tgggagccac ggtcagtggg gcaagcccag 360 taagcccaaa accaacatga agcatgtggc gggagccgca gcagccgggg cggtcgtggg 420 gggcctgggc ggctacatgc tggggagcgc catgagcagg cccctcattc attttggcaa 480 cgactatgag gaccgctact accgtgagaa catgtaccgc taccccaacc aagtgtacta 540 caagccggtg gatcagtaca gcaaccagaa caacttcgtg catgactgcg tcaacatcac 600 ggtcaagcag cacacggtga ccaccaccac caagggcgag aacttcacgg agaccgacat 660 gaagatcatg gagcgcgtgg tggagcagat gtgtgtcacc cagtaccagc gagagtccga 720 ggcttactac cagagggggg cgagcgccat cctcttctcg ccccctcccg tgatcctcct 780 catctcactg ctcattctcc tgatagtggg atgaggatgg ccttcccatt ctctccatcg 840 tcttcacctt ttacaggttg ggggaggggg tgtctaccta cagccctgta gtggtggtgt 900 ctcattcctg cttctcttta tcacccatag gctaatcccc ttggccctga tggccctggg 960 aaatgtagag cagacccagg atgctattta ttcaagcccc catgtgttgg agtccttcag 1020 gggccaatgc tagtgcaggg ctgagaataa cagcaaatca tcattggttg acctagggct 1080 gcttttttgt tgttgttgtc tagtgcagct gaccgaggct aaaacaattc tcaaaacagt 1140 tttcaaatac ctttgcctgg aaacctctgg ctcctgctgc agctagagct cagtacatta 1200 atgtcccatc ttagccgtgt cttcatagca acttggggaa gtttttctcc ccactctaaa 1260 agaacgcgat tgcacttccc tgtgcaaaga acatttctgc caaatttgaa aggaggccac 1320 atgatattca ttcaaaaagc aaaactagaa accctttgct cttggacgca agcccggcct 1380 gctaggagca ccaaactggg gcgatggttt gcattctgcg gcgtgggcta tgcggcagcc 1440 gaggtgtcca gcgtaaatat tgatgcgacg ctagacctag gcagaggatg tttgcacagg 1500 gaatgaacat aatcaacagt gcgaaaatgc tacaaaaaat cccacactgg ggagcagtgt 1560 ccttggaggc aagttttttt ccttttggga catttaaagc ccctatatgt ggcattcctt 1620 tctttcgtaa cctaaactat agataattaa ggcagttaaa aattgaactt ccttccaggc 1680 cccaagagca aatctttgtt cacttacctg gaaaccagaa tgattttgac acagaggaag 1740 gtgcagctgt taaaataacc ctcatcctag aagattgcat catggagaaa acgatccgta 1800 gacaaaaatg atcgcatttc ttcattgctg tctcgtaatt gacagaaacc agaattatgt 1860 caagtcctag tttctataat cagcttttga atcaaagaat ggaagtccat ccaaaaaaaa 1920 aaaagaaata ccttaggtca cccatgacag aaatacccat tcaggttaga aaaaaggaat 1980 tctgttaact gttatttaag taaggcaaaa ttattgtccg gattgttcga tatcatcagc 2040 tagcagataa attagcattc tgcaatgttc ccggcttgca ctgtgcgggt atttgatgtt 2100 aaaaaaaatt attatatata ttgtgtatga caaacttaga agtttttgct agaggagtta 2160 acatctgata tatctaatgc accaccagtt ttggaaggta ctaaatactt aatatgtaga 2220 aatccttttg cgtggtcctc aggcttacac gtgcactgaa tagttttgta tgatagagcc 2280 catgtggtct tcgaaatatg catgtacttt atattttcta tatttgtaac tgggcatgta 2340 cttgtataaa aaatgtataa acattcgaac tcttgactag aattaaacag gaactgagtg 2400 tgtcccatgt gtttgcagtg acattcacca ccgcaccctg tgttgg 2446

Syrian Hamster PrP cDNA (SEQ ID NO:3; GenBank Accession No. M14054) tcgaaaatct ccctctttag caatttcttg ctcctagagt ttcagcaatt gctttctcgc 60 tccattaggc aacctttcat tttctcacct tccccattat gtaacgggag caatgggttc 120 tggaccagtc ttccattaaa gatgattttt atagtcggtg agcgccgtca gggagtgatg 180 acacctgggg gcggtttaaa ccgtacaatc ccttaaacca gtctggagcg gtgactcatt 240 tccccaggga gaagtggcgc ggccattggt gagcacgacg caagccccgc cccacccagc 300 ccggccccgc cctgctaccc ctcctgactc actgccccgc ccgctccccc gcggcgtccg 360 agcagcagac cgagaaggca catcgagtcc actcgtcgcg tcggtggcag gtaagcggct 420 tctgaagcct ggccccggga agggtgctgg agccaggcct cggtaagcct tcggcttccc 480 agagccaagc ccggcttact ccggctctcg gggcgctgag gccgcggggc tgaggttgag 540 tctggctggg aggtgaccgc gcacccgcag ccgcgcgtct ccttgaggga ccgaacccca 600 ggagaggcca ggagccatcc cttcctcccg agcccggctc acccccagag tcgctcgggg 660 atgggggatg ggggatgggg tggcatcttt tgactgtcgt ttgctgtttt cttctctctt 720 tgtaatagct acagcgaaca taattttacc cagggttcca ccgtggtctc gtccgtcctc 780 ggcatctctc agtccagtac atacccaagg 810

Sheep PrP cDNA (SEQ ID NO:4; GenBank Accession No. AJ223072) atggtgaaaa gccacatagg cagttggatc ctggttctct ttgtggccat gtggagtgac 60 gtgggcctct gcaagaagcg accaaaacct ggcggaggat ggaacactgg ggggagccga 120 tacccgggac agggcagtcc tggaggcaac cgctatccac ctcagggagg gggtggctgg 180 ggtcagcccc atggaggtgg ctggggccaa cctcatggag gtggctgggg tcagccccat 240 ggtggtggct ggggacagcc acatggtggt ggaggctggg gtcaaggtgg tagccacagt 300 cagtggaaca agcccagtaa gccaaaaacc aacatgaagc atgtggcagg agctgctgca 360 gctggagcag tggtaggggg ccttggtggc tacatgctgg gaagtgccat gagcaggcct 420 cttatacatt ttggcaatga ctatgaggac cgttactatc gtgaaaacat gtaccgttac 480 cccaaccaag tgtactacag accagtggat cagtatagta accagaacaa ctttgtgcat 540 gactgtgtca acatcacagt caagcaacac acagtcacca ccaccaccaa gggggagaac 600 ttcaccgaaa ctgacatcaa gataatggag cgagtggtgg agcaaatgtg catcacccag 660 taccagagag aatcccaggc ttattaccaa aggggggcaa gtgtgatcct cttttcttcc 720 cctcctgtga tcctcctcat ctctttcctc atttttctca tagtaggata ggggcaacct 780 tcctgttttc attatcttct taatctttgc caggttgggg gagggagtgt ctacctgcag 840 ccctgtagtg gtggtgtctc atttcttgct tctctcttgt tacctgtata ataataccct 900 tggcgcttac agcactggga aatgacaagc agacatgaga tgctgtttat tcaagtccca 960 ttagctcagt attctaatgt cccatcttag cagtgatttt gtagcaattt tctcatttgt 1020 ttcaagaaca cctgactaca tttccctttg ggaatagcat ttctgccaag tctggaagga 1080 ggccacataa tattcattca aaaaaacaaa actggaaatc cttagttcat agacccaggg 1140 tccaccctgt tgagagcatg tgtcctgtgt ctgcagagaa ctataaagga tattctgcat 1200 tttgcaggtt acatttgcag gtaacacagc catctattgc atcaagaatg gatattcatg 1260 caacctttga cttatgggca gaggacatct tcacaaggaa tgaacataat acaaaggctt 1320 ctgagactaa aaaattccaa catatggaag aggtgccctt ggtggcagcc ttccattttg 1380 tatgtttaag caccttcaag tgatattcct ttctttagta acataaagta tagataatta 1440 aggtacctta attaaactac cttctagaca ctgagagcaa atctgttgtt tatctggaac 1500 ccaggatgat tttgacattg cttagggatg tgagagttgg actgtaaaga aagctgagtg 1560 ctgaagagtt catgcttttg aactatagtg ttggagaaaa ctcttgagag tcccttggac 1620 tgaaaggaga tcagtcctga atattcattg gaaggactga tgctgaagct gaaactccaa 1680 tactttggtc acctgatggg aagaactgaa ggcaggaggg atgctaggaa agactgaagg 1740 caggaggaga aggggacgac agaggatgag atggctagat ggcatcatgg actcaatgga 1800 catgagctta agtaaactcc aggagttggc aatggacagg gagacctggc gtcctgcagt 1860 ccatggtgtc gcagagtcgg acacgattga gtgactaaat tgaggtgacc cagatttaac 1920 atagagaatg cagatacaaa actcatattc atttgattga atcttttcct gaaccagtgc 1980 tagtgttgga ctggtaaggg tataacagca tatataggtt atgtgatgaa gagatagtgt 2040 acatgaaata tgtgcatttc tttattgctg tcttataatt gtcaaaaaag aaaattaggt 2100 ccttggtttc tgtaaaattg acttgaatca aaagggaggc atttaaagaa ataaattaga 2160 gatgatagaa atctgatcca ttcagagtag aaaaagaaat tccattactg ttattaaaga 2220 aggtaaaatt attccctgaa ttgttcaata ttgtcaccta gcagatagac actattctgt 2280 actgttttta ctagcttgca ccttgtggta tcctatgtaa aaacatattt gcatatgaca 2340 aactttttct gttagagcaa ttaacatctg aaccacctaa tgcattacct gtttttgtaa 2400 ggtacttttt gtaaggtact aaggagatgt gggtttaatc cctaggtcag gtaaatcccc 2460 tagaggaaga aatggcaacc cactccagta ttcttgccag gaaaatccag tgggcagagg 2520 agcctggcag ggtacagtct aagagcatgg ggttgcaaag agtgagacaa gacttgagct 2580 actgaacaat aaggacaata aatgctgggt cggctaaaag gttcattagg ttttttttct 2640 gtaagatggc tctagtagta cttgtcttta tcttcattcg aaacaatttt gttagattgt 2700 atgtgacagc tcttgtatca gcatgcattt gaaaaaaaca tcacaattgg taaatttttg 2760 tatagccatc ttactattga agatggaaga aaagaagcaa aattttcagc atatcatgct 2820 gtacttattt caagaaagat aaccaaaatg caaaaatgta tttgtgaagt gtatggagaa 2880 ggggctgcaa ctgatcaagc ttgtcaaagt agtttgtgaa gtttcgtgct ggagatttct 2940 tattggacga tgctccacag ttggatatac cagttgaagt tgatagtgat caaattgaga 3000 tattgagaat aatcgatgtt ataccacgcg ggagatagct gacatactca aaatatccaa 3060 atagaacctt gaaaaccatt tgcaccatct cagttatgtt aatcactttg atgtttgagt 3120 tccacataag caaaaaaaca acaacaaaaa aaaatacaac cttgaccata tttgcgcatg 3180 cagttctcta ctgaaatgat tgaaaacact ttgtttttaa aaacagattt tgattaacag 3240 tgggtacgat acaataacgt agatggaaga aattgtaggg tgagcaaaat gaaccacacc 3300 accaaaggcc agtcttcctc taaagaagat gtgtgtatgg tgggattgga aagtaatcct 3360 ctattatgga ttcttctgga aaacactgct cctaattaga ccaactgaaa acagcactca 3420 acgaaaagca tccagaatta gtcaatagaa aacataatct tccatcagga taacgcaaga 3480 ctacatattt ctttgatgac ccagcatggc tggagtttct gattcatctg ttgtattcag 3540 acgttgcatc tttggatttt ttccatttat ttcagtctac aaaattatca taatggaaaa 3600 aatttccatt ccctggaaga tgtaaagtgc atctggaaaa tttctttgct caaaaagata 3660 aaaagttttg tgaacacaga attatgacgt tgcctgaaaa atggcagaag gtagtggaac 3720 aaaagagtga ctatgttgtt tggtaaagtt cttagtgaaa atgaaaaatg tgtcttttat 3780 ttttatttaa acaccaaagg cacattttag caacccaata ctgaatctaa aggaaactct 3840 tctgtgtgtt gtccttacag tgtgcactga tagtttgtat aagaatccag agtgatattt 3900 gaaatacgca tgtgcttata ttttttatat ttgtaacttt gcatgtactt gttttgtgtt 3960 aaaagtttat aaatatttaa tatctgacta aaattaaaca ggagctaaaa ggagtatctt 4020

Bovine PrP cDNA (SEQ ID NO:5; GenBank AccessionNo. X55882) atggtgaaaa gccacatagg cagttggatc ctggttctct ttgtggccat gtggagtgac 60 gtgggcctct gcaagaagcg accaaaacct ggaggaggat ggaacactgg ggggagccga 120 tacccaggac agggcagtcc tggaggcaac cgttatccac ctcagggagg gggtggctgg 180 ggtcagcccc atggaggtgg ctggggccag cctcatggag gtggctgggg ccagcctcat 240 ggaggtggct ggggtcagcc ccatggtggt ggctggggac agccacatgg tggtggaggc 300 tggggtcaag gtggtaccca cggtcaatgg aacaaaccca gtaagccaaa aaccaacatg 360 aagcatgtgg caggagctgc tgcagctgga gcagtggtag ggggccttgg tggctacatg 420 ctgggaagtg ccatgagcag gcctcttata cattttggca gtgactatga ggaccgttac 480 tatcgtgaaa acatgcaccg ttaccccaac caagtgtact acaggccagt ggatcagtat 540 agtaaccaga acaactttgt gcatgactgt gtcaacatca cagtcaagga acacacagtc 600 accaccacca ccaaggggga gaacttcacc gaaactgaca tcaagatgat ggagcgagtg 660 gtggagcaaa tgtgcattac ccagtaccag agagaatccc aggcttatta ccaacgaggg 720 gcaagtgtga tcctcttctc ttcccctcct gtgatcctcc tcatctcttt cctcattttt 780 ctcatagtag gatag 795 Chicken PrP cDNA (SEQ ID NO:6; GenBank Accession No. M61145) gaattccctc ggcagccagc tcctccctct cgctatttat tcctttctcc cccccctacg 60 ctggatctgg atcatctcaa gccgagcggt gacggcttct tggatcgctc atacataaat 120 atctgtgagt cagaggaagc aaccaccgac cccaagacct caccccgagc catggctagg 180 ctcctcacca cctgctgcct gctggccctg ctgctcgccg cctgcaccga cgtcgccctc 240 tccaagaagg gcaaaggcaa acccagtggt gggggttggg gcgccgggag ccatcgccag 300 cccagctacc cccgccagcc gggctaccct cataacccag ggtaccccca taacccaggg 360 tacccccaca accctggcta tccccataac cccggctacc cccagaaccc tggctacccc 420 cataacccag gttacccagg ctggggtcaa ggctacaacc catccagcgg aggaagttac 480 cacaaccaga agccatggaa accccccaaa accaacttca agcacgtggc gggggcagca 540 gcggcgggtg ctgtggtggg gggcttgggg ggctacgcca tggggcgcgt tatgtcaggg 600 atgaactacc acttcgatag acccgatgag taccgatggt ggagtgagaa ctcggcgcgt 660 tatcccaacc gggtttacta ccgggattac agcagccccg tgccacagga cgtcttcgtg 720 gccgattgct ttaacatcac agtgactgag tacagcattg gccctgctgc caagaagaac 780 acctccgagg ctgtggcggc agcaaaccaa acggaggtgg agatggagaa caaagtggtg 840 acgaaggtga tccgcgagat gtgcgtgcag cagtaccgcg agtaccgcct ggcctcgggc 900 atccagctgc accctgctga cacctggctc gccgtcctcc tcctcctcct caccaccctt 960 tttgccatgc actgatggga tgccgtgccc cggccctgtg gcagtgagat gacatcgtgt 1020 ccccgtgccc acccatgggg tgttccttgt cctcgctttt gtccatcttt ggtgaagatg 1080 tccccccgct gcctccccgc aggctctgat ttgggcaaat gggaggggat tttgtcctgt 1140 cctggtcgtg gcaggacggc tgctggtggt ggagtgggat gcccaaaaaa tggccttcac 1200 cacttcctcc tcctcttcct ttctggggcg gagatatggg ctcgtccagc ccttattgtc 1260 cctgcaagag cgtatctgaa aatcctcttt gctaacaagc agggttttac ctaatctgct 1320 tagccccagt gacagcagag cgcctttccc cagggcacac caaccccaag ctgaggtgct 1380 tggcagccac acgtcccatg gaggctgatg ggttttgggg cgtcccaagc aacaccctgg 1440 gctactgagg tgcaattgta gctctttaat ctgccaatcc caaccctacc gtgtagatag 1500 gaactgcctg ctctgcattt tgcatgctgc aaacacctcc tgccgcagcg cccccaaaat 1560 agagtgattt gggaatagtg aggctgaagc cacagcagct tgggattggg ctcatcatat 1620 caatccatga tgctttgctt ccagctgagc ctcactgccc ttttatagcc tgcccagagg 1680 aagggagcgc tgctaaatgc ccaaaaaggt aacactgagc aaaagcttat ttcaatgtat 1740 gatagagaac gagtgcatct cgcacagatc agccatggga gcatcgtttg ccatcagccc 1800 caaaacccaa aggatgctaa aatgcagcca aaggggaatc aagcacgcag ggaaggactt 1860 gaatcagctc aactggattg aaatggcaaa aggcatgagt agaacgaacg gcaaggggat 1920 gctggagatc cacctcctgt gagcaaattg ttcgatgcag ccaatggaac tattgcttct 1980 tgtgcttcag ttgctgctga tgtgtacata ggctgtagca tatgtaaagt tacacgtgtc 2040 aagctgctcg caccgcgtag agctaatatg tatcatgtat gtgggcactg aatgccaccg 2100 ttggccatac ccaaccgtcc taaacgattt tcacgtcgct gtaacttaag tggagataca 2160 ctttcagtat attcagcaaa aggaattc 2188

Mouse PrP cDNA (SEQ ID NO:7; GenBank Accession No. M13685) aattccttca gaactgaacc atttcaaccg agctgaagca ttctgccttc ctagtggtac 60 cagtccaatt taggagagcc aagcagacta tcagtcatca tggcgaacct tggctactgg 120 ctgctggccc tctttgtgac tatgtggact gatgtcggcc tctgcaaaaa gcggccaaag 180 cctggagggt ggaacaccgg tggaagccgg tatcccgggc agggaagccc tggaggcaac 240 cgttacccac ctcagggtgg cacctggggg cagccccacg gtggtggctg gggacaaccc 300 catgggggca gctggggaca acctcatggt ggtagttggg gtcagcccca tggcggtgga 360 tggggccaag gagggggtac ccataatcag tggaacaagc ccagcaaacc aaaaaccaac 420 ctcaagcatg tggcaggggc tgcggcagct ggggcagtag tggggggcct tggtggctac 480 atgctgggga gcgccgtgag caggcccatg atccattttg gcaacgactg ggaggaccgc 540 tactaccgtg aaaacatgta ccgctaccct aaccaagtgt actacaggcc agtggatcag 600 tacagcaacc agaacaactt cgtgcacgac tgcgtcaata tcaccatcaa gcagcacacg 660 gtcaccacca ccaccaaggg ggagaacttc accgagaccg atgtgaagat gatggagcgc 720 gtggtggagc agatgtgcgt cacccagtac cagaaggagt cccaggccta ttacgacggg 780 agaagatcca gcagcaccgt gcttttctcc tcccctcctg tcatcctcct catctccttc 840 ctcatcttcc tgatcgtggg atgagggagg ccttcctgct tgttccttcg cattctcgtg 900 gtctaggctg ggggaggggt tatccacctg tagctctttc aattgaggtg gttctcattc 960 ttgcttctct gtgtccccca taggctaata cccctggcac tgatgggccc tgggaaatgt 1020 acagtagacc agttgctctt tgcttcaggt ccctttgatg gagtctgtca tcagccagtg 1080 ctaacaccgg gccaataaga atataacacc aaataactgc tggctagttg gggctttgtt 1140 ttggtctagt gaataaatac tggtgtatcc cctgacttgt acccagagta caaggtgaca 1200 gtgacacatg taacttagca taggcaaagg gttctacaac caaagaagcc actgtttggg 1260 gatggcgccc tggaaaacag cctcccacct gggatagcta gagcatccac acgtggaatt 1320 ctttctttac taacaaacga tagctgattg aaggcaacaa aaaaaaaaaa atcaaattgt 1380 cctactgacg ttgaaagcaa acctttgttc attcccaggg cactaga'atg atctttagcc 1440 ttgcttggat tgaactagga gatcttgact ctgaggagag ccagccctgt aaaaagcttg 1500 gtcctcctgt gacgggaggg atggttaagg tacaaaggct agaaacttga gtttcttcat 1560 ttctgtctca caattatcaa aagctagaat tagcttctgc cctatgtttc tgtacttcta 1620 tttgaactgg ataacagaga gacaatctaa acattctctt aggctgcaga taagagaagt 1680 aggctccatt ccaaagtggg aaagaaattc tgctagcatt gtttaaatca ggcaaaattt 1740 gttcctgaag ttgcttttta ccccagcaga cataaactgc gatagcttca gcttgcactg 1800 tggattttct gtatagaata tataaaacat aacttcaagc ttatgtcttc tttttaaaac 1860 atctgaagta tgggacgccc tggccgttcc atccagtact aaatgcttac cgtgtgaccc 1920 ttgggctttc agcgtgcact cagttccgta ggattccaaa gcagacccct agctggtctt 1980 tgaatctgca tgtacttcac gttttctata tttgtaactt tgcatgtatt ttgttttgtc 2040 atataaaaag tttataaatg tttgctatca gactgacatt aaatagaagc tatgatg 2097

Sheep PrP cDNA (SEQ ID NO:8; GenBank Accession No. X79912) gcagagaagt catcatggtg aaaagccaca taggcagttg gatcctggtt ctctttgtgg 60 ccatgtggag tgacgtgggc ctctgcaaga agcgaccaaa acctggcgga ggatggaaca 120 ctggggggag ccgatacccg ggacagggca gtcctggagg caaccgctat ccacctcagg 180 gagggggtgg ctggggtcag ccccatggag gtggctgggg ccaacctcat ggaggtggct 240 ggggtcagcc ccatggtggt ggctggggac agccacatgg tggtggaggc tggggtcaag 300 gtggtagcca cagtcagtgg aacaagccca gtaagccaaa aaccaacatg aagcatgtgg 360 caggagctgc tgcagctgga gcagtggtag ggggccttgg tggctacatg ctgggaagtg 420 ccatgagcag gcctcttata cattttggca atgactatga ggaccgttac tatcgtgaaa 480 acatgtaccg ttaccccaac caagtgtact acagaccagt ggatcagtat agtaaccaga 540 acaactttgt gcatgactgt gtcaacatca cagtcaagca acacacagtc accaccacca 600 ccaaggggga gaacttcacc gaaactgaca tcaagataat ggagcgagtg gtggagcaaa 660 tgtgcatcac ccagtaccag agagaatccc aggcttatta ccaaaggggg gcaagtgtga 720 tcctcttttc ttcccctcct gtgatcctcc tcatctcttt cctcattttt ctcatagtag 780 gataggggca accttcctgt ttt 803

Rat PrP cDNA (SEQ ID NO:9; GenBank Accession No. NM_012631) atggcgaacc ttggctactg gctgctggcc ctctttgtga ctacatgtac tgatgttggc 60 ctctgcaaaa agcggccaaa gcctggaggg tggaacactg gtggaagccg gtaccctggg 120 cagggaagcc ctggaggcaa ccgttaccca cctcagagtg gtggtacctg ggggcagccc 180 catggtggtg gctggggaca acctcatggt ggtggctggg gacaacctca tggtggtggc 240 tggggtcagc cccatggcgg gggctggagt caaggagggg gtacccataa tcagtggaac 300 aagcccagca agccaaaaac caacctcaag catgtggcag gggctgccgc agctggggca 360 gtagtggggg gccttggtgg ctacatgttg gggagtgcca tgagcaggcc catgctccat 420 tttggcaacg actgggagga ccgctactac cgagaaaaca tgtaccgtta ccctaaccaa 480 gtgtactaca ggccggtgga tcagtacagc aaccagaaca acttcgtgca cgactgtgtc 540 aatatcacca tcaagcagca tacagtcacc accaccacca agggggagaa cttcacggag 600 accgacgtga agatgatgga gcgtgtggtg gagcagatgt gcgtcaccca gtatcagaag 660 gagtcccagg cctattacga cgggagaaga tctagcgccg tgcttttctc ctcccctcct 720 gtgatcctcc tcatctcctt cctcatcttc ctgatcgtgg gatga 765

As used herein, "prion disease" refers to any disease or condition in a subject, the pathogenesis ofwhich involves a prion protein other than PrPc ofthe species ofthe subject. A prion disease will typically but not necessarily be a transmissible spongiform encephalopathy.

As used herein, "transmissible spongiform encephalopathy" and, equivalently, "(TSE)" shall mean any prion disease that is associated with spongiform encephalopathy and is communicable from one individual to another. As prion diseases can include entities other than TSE, this term refers to at least a subset of all prion diseases. At present TSE includes Creutzfeldt- Jakob disease, kuru, Gerstmann-Straussler-Scheinker syndrome, fatal familial insomnia, bovine spongiform encephalopathy, and scrapie.

As used herein, "Creutzfeldt-Jakob disease" and, equivalently, "(CJD)" refers to the TSE that naturally occurs in humans. CJD includes sporadic, genetic (familial), and infectious (i.e., variant and iatrogenic) forms. CJD is a well described entity in the medical literature, and until now has been widely believed to be a uniformly fatal neurodegenerative disease for which there is no effective form of treatment.

As used herein, "variant Creutzfeldt-Jakob disease" and, equivalently, "(vCJD)", also referred to in the literature as new variant Creutzfeldt-Jakob disease (nvCJD), refers to CJD attributable to the BSE prion. It can be distinguished from sporadic CJD not only by the prion involved but also by certain clinical and preclinical features. See Aguzzi A (2000) Haematologica 85:3-10; Hill AF et al. (1997) Nature 389:448-50; Bruce ME et al. (1997) Nature 389:498-501; Will R et al. (1996) Lancet 347:921-5.

As used herein, "iatrogenic Creutzfeldt-Jakob disease" and, equivalently, "(iCJD)" refers to any form of CJD that is attributable to work- or treatment-related exposure to prion protein that is associated with CJD.

As used herein, "bovine spongiform encephalopathy" and, equivalently, "(BSE)" shall refer to the TSE that occurs naturally in cows and cattle.

As used herein, "scrapie" refers to the TSE that occurs naturally in sheep and goats, as well as to experimental models of scrapie. Scrapie in sheep has been recognized and described in the literature for over 300 years. For a review, see O'Rourke Kl (2001) Vet Clin North Am Food Anim Pract 17:283-300.

As used herein, a "prion protein that is associated with a prion disease" refers to any prion protein involved in the pathogenesis of a prion disease. A prion protein that is associated with a prion disease can be a prion found in nature. Alternatively, prion protein that is associated with a prion disease can be a prion protein made de novo or modified from its natural form through human activity, e.g., by in vitro synthesis, chemical synthesis, chemical derivativization, or genetic alteration. Chemical alteration includes, without limitation, altered glycosylation. In a preferred embodiment, a prion protein that is associated with a prion disease is a prion protein found in nature, e.g., PrPSc. In one embodiment, a prion protein that is associated with a prion disease is a truncated or genetically modified form of a prion protein found in nature. A genetically modified form of a prion protein found in nature includes a fusion protein involving at least a substantial portion of a prion protein as one component, a prion protein that differs from a prion protein found in nature by one or more conservative amino acid substitutions, and allelic variants of the prion protein found in nature.

Naturally occurring residues can be divided into the following classes based on common side chain properties: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr; (3) acidic: Asp, Glu; (4) basic: Asn, Gin, His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. Thus, for example, conservative amino acid substitutions can involve the exchange of a member from one of these classes for another member from the same class. Non-conservative amino acid substitutions can involve the exchange of a member of one of these classes for a member from another class.

A conservative amino acid substitution can involve a substitution of a native amino acid residue with another residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties.

As used herein, "prion proteimscrapie form (PrPSc)" refers to any of a number of naturally occurring, species-specific, proteinase K-resistant forms of prion protein associated with prion disease. Specific examples include, but are not limited to, the following: human PrPSc having an amino acid sequence provided by SEQ ID NO: 10; bovine PrPSc having an amino acid sequence provided by SEQ ID NO:l 1; bovine PrPSc having an amino acid sequence provided by SEQ ID NO: 12; ovine PrPSc having an amino acid sequence provided by SEQ ID NO:13; ovine PrPSc having an amino acid sequence provided by SEQ ID NO:14; and murine PrPSc having an amino acid sequence provided by SEQ ID NO: 15. Human PrPSc (SEQ ID NO: 10; GenBank Accession No. AAE81600)

MANLGCW V LFVAT SD G LCKKRPKPGG NTGGSRYPG QGSPGGNRYP PQGGGGWGQP 60 HGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWN KPSKPKTNMK HMAGAAAAGA 120

WGGLGGYML GSAMSRPIIH FGSDYEDRYY RENMHRYPNQ VYYRPMDEYS NQNNFVHDCV 180

NITIKQHTVT TTTKGENFTE TDVKMMERW EQMCITQYER ESQAYYQRGS SMVLFSSPPV 240

ILLISFLIFL IVG 253

Bovine PrPSc (SEQ ID NO:l l; GenGank Accession No. AAE81601)

MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGWNTGGSRY PGQGSPGGNR YPPQGGGGWG 60

QPHGGGWGQP HGGGWGQPHG GGWGQPHGGG WGQPHGGGGW GQGGTHGQWN KPSKPKTNMK 120

HVAGAAAAGA WGGLGGYML GSAMSRPLIH FGSDYEDRYY RENMHRYPNQ VYYRPVDQYS 180 NQNNFVHDCV NITVKEHTVT TTTKGENFTE TDIKMMERW EQMCVTQYQK ESQAYYDQGA 240

SVILFSSPPV ILLISFLIFL IVG 263

Bovine PrPSc (SEQ ID NO: 12; GenGank Accession No. CAA39368)

MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGGWNTGGSR YPGQGSPGGN RYPPQGGGGW 60

GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GWGQPHGGGG WGQGGTHGQW NKPSKPKTNM 120

KHVAGAAAAG AWGGLGGYM LGSAMSRPLI HFGSDYEDRY YRENMHRYPN QVYYRPVDQY 180

SNQNNFVHDC VNITVKEHTV TTTTKGENFT ETDIKMMERV VEQMCITQYQ RESQAYYQRG 240

ASVILFSSPP VILLISFLIF LIVG 264

Ovine PrPSc (SEQ ID NO: 13; GenBank Accession No. AAE81602)

MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGWNTGGSRY PGQGSPGGNR YPPQGGGGWG 60

QPHGGGWGQP HGGGWGQPHG GSWGQPHGGG GWGQGGSHSQ WNKPΞKPKTN MKHVAGAAAA 120

GAWGGLGGY MLGSAMSRPL IHFGNDYEDR YYRENMYRYP NQVYYRPVDQ YSNQNNFVHD 180

CVNITVKQHT VTTTTKGENF TETDIKIMER WEQMCITQY QRESQAYYQR GASVILFSSP 240

PVILLISFLI FLIVG 255

Ovine PrPSc (SEQ ID NO: 14; GenBank Accession No. CAA56283)

MVKSHIGSWI LVLFVAMWSD VGLCKKRPKP GGGWNTGGSR YPGQGSPGGN RYPPQGGGGW 60

GQPHGGGWGQ PHGGGWGQPH GGGWGQPHGG GGWGQGGSHS QWNKPSKPKT NMKHVAGAAA 120

AGAWGGLGG YMLGSAMSRP LIHFGNDYED RYYRENMYRY PNQVYYRPVD QYSNQNNFVH 180

DCVNITVKQH TVTTTTKGEN FTETDIKIME RWEQMCITQ YQRESQAYYQ RGASVILFSS 240

PPVILLISFL IFLIVG 256

Murine PrPSc (SEQ ED NO: 15; GenBank Accession No. AAE81599) MANLGYWLLA LFVTMWTDVG LCKKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGTWGQPH 60

GGGWGQPHGG SWGQPHGGSW GQPHGGGWGQ GGGTHNQWNK PSKPKTNLKH VAGAAAAGAV 120

VGGLGGYMLG SAMSRPMIHF GNDWEDRYYR ENMYRYPNQV YYRPVDQYSN QNNFVHDCVN 180

ITIKQHTVTT TTKGENFTET DVKMMERWE QMCVTQYQKE SQAYYDGRRS SSTVLFSSPP 240

VILLISFLIF LIVG 254

As used herein, "full-length PrPSc" refers to a form of PrPSc that includes all its amino acids as it occurs in nature. It is to be distinguished, for example, from a truncated form of PrPSc, described elsewhere herein. A full-length PrPSc can, however, be incorporated into a PrPSc conjugate or PrPSc fusion protein.

As used herein, a "derivative of PrPSc" refers to a chemical or genetic derivative of a naturally occurring form of PrPSc, including PrPSc with non-native glycosylation, covalent or non-covalent conjugates formed between PrPSc and another compound, PrPSc fusion proteins, and any combination thereof. A "derivative of a fragment of PrPSc lacking at least the amino terminus of full-length PrPSc" refers to a chemical or genetic derivative of an N-terminally truncated form of PrPSc, including such truncated forms of PrPSc: (i) with non-native glycosylation, (ii) as part of a covalent or non-covalent conjugate formed with another compound, (iii) as part of a fusion protein, or (iv) any combination thereof. In preferred embodiments the fragment of PrPSc lacking at least the amino terminus of full-length PrPSc refers to a fragment lacking one or more, up to and including all, of the octarepeats (e.g., GGGWGQPH (SEQ ID NO: 16) and GGSWGQPH (SEQ ID NO: 17)). See Flechsig E et al. (2000) Neuron 27:399-408.

A truncated form of a prion protein found in nature is identical in primary sequence to the prion protein found in nature except for the absence of one or more amino acid residues from the N-terminal end, the C-terminal end, or both the N-terminal and the C-terminal ends. Truncated forms can also include deletion mutants, in which an internal sequence is omitted without changing either the N-terminal end or the C-terminal end. In a preferred embodiment, a truncated prion protein lacks one or more, up to and including all, N-terminal octarepeats (e.g., GGGWGQPH and GGSWGQPH). See Flechsig E et al. (2000) Neuron 27:399-408.

As used herein, a "fragment of PrPSc lacking at least the amino terminus of full-length PrPSc" shall refer to a truncated form of full-length PrPSc lacking one or more N-terminal amino acids normally present in full-length PrPSc. In a prefened embodiment, the fragment lacks one or more, up to and including all, of the octarepeats (e.g., GGGWGQPH and GGSWGQPH). See Flechsig E et al. (2000) Neuron 27:399-408.

The methods of the instant invention employ immunostimulatory nucleic acids. In the prefened embodiment, the immunostimulatory nucleic acid is a CpG nucleic acid. The terms "nucleic acid" and "oligonucleotide" are used interchangeably to mean multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the terms "nucleic acid" and "oligonucleotide" refer to oligoribonucleotides as well as oligodeoxyribonucleotides. The terms "nucleic acid" and "oligonucleotide" shall also include polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by nucleic acid synthesis).

The terms "nucleic acid" and "oligonucleotide" also encompass nucleic acids or oligonucleotides with substitutions or modifications, such as in the bases and/or sugars. For example, they include nucleic acids having backbone sugars that are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 2' position and other than a phosphate group at the 5' position. Thus modified nucleic acids may include a 2'-O- alkylated ribose group. In addition, modified nucleic acids may include sugars such as arabinose instead of ribose. Thus the nucleic acids may be heterogeneous in backbone composition thereby containing any possible combination of polymer units linked together such as peptide-nucleic acids (which have an amino acid backbone with nucleic acid bases).

Nucleic acids also include substituted purines and pyrimidines such as C-5 propyne modified bases. Wagner RW et al. (1996) Nat Biotechnol 14:840-4. Purines and pyrimidines include but are not limited to adenine, cytosine, guanine, thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other naturally and non-naturally occurring nucleobases, substituted and unsubstituted aromatic moieties. Other such modifications are well known to those of skill in the art.

The immunostimulatory oligonucleotides of the instant invention can encompass various chemical modifications and substitutions, in comparison to natural RNA and DNA, involving a phosphodiester internucleoside bridge, a β-D-ribose unit and/or a natural nucleoside base (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to the skilled person and are described, for example, in Uhlmann E et al. (1990) Chem Rev 90:543; "Protocols for Oligonucleotides and Analogs" Synthesis and Properties & Synthesis and Analytical Techniques, S. Agrawal, Ed, Humana Press, Totowa, USA 1993; Crooke ST et al. (1996) Annu Rev Pharmacol Toxicol 36:107-129; and Hunziker J et al. (1995) Mod Synth Methods 7:331-417. An oligonucleotide according to the invention can have one or more modifications, wherein each modification is located at a particular phosphodiester internucleoside bridge and/or at a particular β-D-ribose unit and/or at a particular natural nucleoside base position in comparison to an oligonucleotide of the same sequence which is composed of natural DNA or RNA.

For example, the invention relates to an oligonucleotide which comprises one or more modifications and wherein each modification is independently selected from: a) the replacement of a phosphodiester internucleoside bridge located at the 3' and/or the 5' end of a nucleoside by a modified internucleoside bridge, b) the replacement of phosphodiester bridge located at the 3' and/or the 5' end of a nucleoside by a dephospho bridge, c) the replacement of a sugar phosphate unit from the sugar phosphate backbone by another unit, d) the replacement of a β-D-ribose unit by a modified sugar unit, and e) the replacement of a natural nucleoside base by a modified nucleoside base. More detailed examples for the chemical modification of an oligonucleotide are as follows.

A phosphodiester internucleoside bridge located at the 3' and/or the 5' end of a nucleoside can be replaced by a modified internucleoside bridge, wherein the modified internucleoside bridge is for example selected from phosphorothioate, phosphorodithioate, NR1R2-phosphoramidate, boranophosphate, α-hydroxybenzyl phosphonate, phosphate-(Cι- C ι)-O-alkyl ester, phosphate-[(C6-Cι2)aryl-(Cι-C ι)-O-alkyl]ester, (Cι-C8)alkyl-phosphonate and/or (C6-Cι2)-arylphosphonate bridges, (C7-Cι2)-α-hydroxymethyl-aryl (e.g., disclosed in WO 95/01363), wherein (C6-C]2)aryl, (C6-C20)aryl and (C6-C14)aryl are optionally substituted

1 ") by halogen, alkyl, alkoxy, nitro, cyano, and where R and R are, independently of each other, hydrogen, (Cι-Cιs)-alkyl, (C6-C20)-aryl, (C6-Cι )-aryl-(Cι-C8)-alkyl, preferably hydrogen,

1

(Ci-C8)-alkyl, preferably (C]-C4)-alkyl and/or methoxyethyl, or R and R form, together with the nitrogen atom canying them, a 5-6-membered heterocyclic ring which can additionally contain a further heteroatom from the group O, S and N.

The replacement of a phosphodiester bridge located at the 3' and/or the 5' end of a nucleoside by a dephospho bridge (dephospho bridges are described, for example, in Uhlmann E and Peyman A in "Methods in Molecular Biology", Vol. 20, "Protocols for Oligonucleotides and Analogs", S. Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp. 355ff), wherein a dephospho bridge is for example selected from the dephospho bridges formacetal, 3'-thioformacetal, methylhydroxylamine, oxime, methylenedimethyl-hydrazo, dimethylenesulfone and/or silyl groups.

A sugar phosphate unit (i.e., a β-D-ribose and phosphodiester internucleoside bridge together forming a sugar phosphate unit) from the sugar phosphate backbone (i.e., a sugar phosphate backbone is composed of sugar phosphate units) can be replaced by another unit, wherein the other unit is for example suitable to build up a "morpholino-derivative" oligomer (as described, for example, in Stirchak EP et al. (1989) Nucleic Acids Res 17:6129-41), that is, e.g., the replacement by a morpholino-derivative unit; or to build up a polyamide nucleic acid ("PNA"; as described for example, in Nielsen PE et al. (1994) Bioconjug Chem 5:3-7), that is, e.g., the replacement by a PNA backbone unit, e.g., by 2-aminoethylglycine.

A β-ribose unit or a β-D-2'-deoxyribose unit can be replaced by a modified sugar unit, wherein the modified sugar unit is for example selected from β-D-ribose, α-D-2'- deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-O-(C1-C6)alkyl-ribose, preferably 2'- O-(CrC6)alkyl-ribose is 2'-O-methylribose, 2'-O-(C2-C6)alkenyl-ribose, 2'-[O-(C|-C6)alkyl- O-(C!-C6)alkyl]-ribose, 2'-NH2-2'-deoxyribose, β-D-xylo-furanose, α-arabinofuranose, 2,4-dideoxy-β-D-erythro-hexo-pyranose, and carbocyclic (described, for example, in Froehler J (1992) Am Chem Soc 114:8320) and/or open-chain sugar analogs (described, for example, in Vandendriessche et al. (1993) Tetrahedron 49:7223) and/or bicyclosugar analogs (described, for example, in Tarkov M et al. (1993) Helv Chim Ada 76:481).

A natural nucleoside base can be replaced by a modified nucleoside base, wherein the modified nucleoside base is for example selected from hypoxanthine, uracil, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkyluracil, 5-(C2-C6)- alkenyluracil, 5-(C2-C6)-alkynyluracil, 5-(hydroxymethyl)uracil, 5-chlorouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytosine, 5-(Cι-C6)-alkylcytosine, 5-(C2-C6)- alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5-chlorocytosine, 5-fluorocytosine, 5-bromocytosine, N2-dimethylguanosine, 2,4-diamino-purine, 8-azapurine, a substituted 7-deazapurine, preferably 7-deaza-7-substituted and/or 7-deaza-8-substituted purine or other modifications of a natural nucleoside bases. This list is meant to be exemplary and is not to be interpreted to be limiting.

The oligonucleotides of the present invention are nucleic acids that contain specific sequences found to elicit an immune response. These specific sequences that elicit an immune response are refened to as "immunostimulatory motifs", and the oligonucleotides that contain immunostimulatory motifs are refened to as "immunostimulatory nucleic acid molecules" and, equivalently, "immunostimulatory nucleic acids" or "immunostimulatory oligonucleotides". The immunostimulatory oligonucleotides of the invention thus include at least one immunostimulatory motif.

In one embodiment of the invention the immunostimulatory oligonucleotides include immunostimulatory motifs which are "CpG dinucleotides". A CpG dinucleotide can be methylated or unmethylated. An immunostimulatory nucleic acid containing at least one unmethylated CpG dinucleotide is a nucleic acid molecule which contains an unmethylated cytosine-guanine dinucleotide sequence (i.e., an unmethylated 5' cytosine followed by 3' guanosine and linked by a phosphate bond) and which activates the immune system; such an immunostimulatory nucleic acid is a CpG nucleic acid. CpG nucleic acids have been described in a number of issued patents, published patent applications, and other publications, including U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068.

An immunostimulatory nucleic acid containing at least one methylated CpG dinucleotide is a nucleic acid which contains a methylated cytosine-guanine dinucleotide sequence (i.e., a methylated 5' cytosine followed by a 3' guanosine and linked by a phosphate bond) and which activates the immune system. In other embodiments the immunostimulatory oligonucleotides are free of CpG dinucleotides. These oligonucleotides which are free of CpG dinucleotides are refened to as non-CpG oligonucleotides, and they have non-CpG immunostimulatory motifs. The invention, therefore, also encompasses nucleic acids with other types of immunostimulatory motifs, which can be methylated or unmethylated. The immunostimulatory oligonucleotides of the invention, further, can include any combination of methylated and unmethylated CpG and non-CpG immunostimulatory motifs. In some embodiments the immunostimulatory oligonucleotide is not an antisense oligonucleotide.

As used herein, a "Toll-like receptor (TLR) that signals in response to the CpG nucleic acid" refers to any TLR that engages or initiates an intracellular signaling pathway associated with the development of an immune response, as a result of contacting the TLR with CpG nucleic acid. The pathway typically involves the adaptor protein MyD88 and subsequent downstream molecules including TRAF, IRAK, Jun, Erk, p38 MAPK, and NF-κB. The TLRs are a family of at least ten highly conserved receptors that share as a common feature a cytoplasmic Toll homology IL-1 receptor (TIR) domain believed to be involved in such signaling. TLR9 is reported to be the natural receptor for CpG nucleic acid.

As to CpG nucleic acids, it has recently been described that there are different classes of CpG nucleic acids. One class is potent for activating B cells but is relatively weak in inducing IFN-α and NK cell activation; this class has been termed the B class. The B class CpG nucleic acids typically are fully stabilized and include an unmethylated CpG dinucleotide within certain prefened base contexts. See, e.g., U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; and 6,339,068. Another class is potent for inducing IFN-α and NK cell activation but is relatively weak at stimulating B cells; this class has been termed the A class. The A class CpG nucleic acids typically have stabilized poly-G sequences at 5' and 3' ends and a palindromic phosphodiester CpG dinucleotide-containing sequence of at least 6 nucleotides. See, for example, published patent application PCT US00/26527 (WO 01/22990). Yet another class of CpG nucleic acids activates B cells and NK cells and induces IFN-α; this class has been termed the C class. The C class CpG nucleic acids typically are fully stabilized, include a B class-type sequence and a GC-rich palindrome or near-palindrome. This class has been described in published patent application PCT/US02/26468 (WO 03/015711), the entire content of which is incorporated herein by reference.

Immunostimulatory oligonucleotides are effective in vertebrates. Different immunostimulatory oligonucleotides can cause optimal immune stimulation depending on the type of subject and the sequence of the immunostimulatory oligonucleotide. Many vertebrates have been found according to the invention to be responsive to the same class of immunostimulatory oligonucleotides, sometimes refened to as human specific immunostimulatory oligonucleotides. Rodents, however, respond to different nucleic acids. Immunostimulatory oligonucleotides causing optimal stimulation in humans may not generally cause optimal stimulation in a mouse and vice versa. An immunostimulatory oligonucleotide causing optimal stimulation in humans often does, however, cause optimal stimulation in other animals such as cow, horses, sheep, etc. For example, within Class B CpG ODN, prefened immunostimulatory sequences have been identified for use in mice (ODN 1826, 5'- TCCATGACGTTCCTGACGTT -3', SEQ ID NO: 18) and for use in humans (ODN 2006, 5'- TCGTCGTTTTGTCGTTTTGTCGTT -3', SEQ ID NO: 19). One of skill in the art can identify the optimal immunostimulatory nucleic acid sequences useful for a particular species of interest using routine assays described herein and/or known in the art, using the guidance supplied herein.

As used herein, the term "treat" as used in reference to a disease or condition shall mean to intervene in such disease or condition so as to prevent or slow the development of, slow the progression of, halt the progression of, or eliminate the disease or condition. Thus the phrase "to treat the prion disease" as used herein means to prevent or slow the development of, slow the progression of, halt the progression of, or eliminate the prion disease.

As used herein, a "subject" refers to a human or non-human vertebrate. Prefened non-human vertebrates include feed livestock susceptible to TSE, including cows and cattle, sheep, goats, and pigs. Non-human subjects also specifically include non-human primates as well as rodents. Non-human subjects also include, without limitation, chickens, horses, dogs, cats, guinea pigs, hamsters, mink, and rabbits.

As used herein, a "subject having a prion disease" is a subject known or diagnosed to have a prion disease as disclosed herein. Generally a subject having a prion disease will have some objective manifestation of the prion disease, such as a sign, symptom, or result of a suitable diagnostic test that indicates the presence of a prion disease. In the transmissible spongiform encephalopathies, such objective manifestations can include dementia, ataxia, myoclonus, tremor, presence of protease-resistant prion protein in brain extract, and typical or characteristic abnormalities on brain CT, brain MRI, and/or EEG. This list is not meant to be limiting in any way, and those of skill in the art will recognize what criteria are suitable for making a diagnosis of prion disease in a given species in question. A subject having a prion disease shall also include any subject having a test result which specifically indicates the presence in that subject of any amount of prion protein that is associated with a prion disease, since it is believed that prion protein that is associated with a prion disease is not present in a subject without a prion disease.

A subject having a prion disease can but need not necessarily have an identifiable risk factor for having a prion disease. An identifiable risk factor for having a prion disease can include a family history of prion disease, a history of consuming known or suspected prion- diseased tissue, or a history of exposure to a prion protein that is associated with a prion disease or to a product derived from a known or suspected prion-diseased tissue (e.g., through administration of pituitary extract).

As used herein, a "subject at risk of developing a prion disease" is a subject with a known or suspected exposure to prion-diseased tissue, a known or suspected exposure to prion protein that is associated with a prion disease, or a known or suspected predisposition to develop a prion disease (e.g., family history of prion disease). In one embodiment the subject at risk of developing a prion disease is a subject residing in or traveling to an area in which TSE is endemic. In one embodiment the subject at risk of developing a prion disease is a subject residing in or traveling to an area in which food or water contains or is likely to contain prion protein that is associated with prion disease. In one embodiment, a subject at risk of developing a prion disease is a subject with a known or suspected iatrogenic exposure to prion-diseased tissue, e.g., neurosurgeons, neuropathologists, pathologists, nurses, morticians, histology technicians and laboratory workers at special risk of contracting iCJD. In one embodiment, a subject at risk of developing a prion disease is a subject with a known or suspected iatrogenic exposure to prion-diseased tissue through receiving a tissue or organ allograft from a subject having a prion disease. Such tissues can include, without limitation, corneas and dural grafts.

As used herein, an "effective amount" of a substance generally refers to that amount of the substance that is sufficient to bring about a desired effect. With reference to CpG nucleic acid, an "effective amount to induce an immune response to the prion protein" shall refer to that amount of CpG nucleic acid that is sufficient to induce an immune response to a particular prion protein. The immune response can occur in vitro, in vivo, ex vivo, and any combination thereof. An immune response to a prion protein can be measured using any suitable means to determine that an immune response occurs in association with exposure of an immune cell to the prion protein. The immune response can be antigen-specific, including any of the following: production of prion protein- specific antibody, proliferation of prion protein-specific lymphocytes, and cell-mediated immunity against cells expressing prion protein. The immune response can alternatively or additionally be antigen-nonspecific, including any of the following: induction of a Toll-like receptor signaling pathway, inflammation, and production of a cytokine and/or chemokine. Because prion proteins are generally believed not to evoke an immune response, any prion protein-specific immune response which occurs in association with exposure of an immune cell to a prion protein will generally indicate an immune response to the prion protein.

Also with reference to CpG nucleic acid, an "effective amount to treat the prion disease" shall refer to that amount of CpG nucleic acid that is sufficient to treat a particular prion disease. In one embodiment, an effective amount to treat the prion disease is that amount that is sufficient to slow the development of prion disease, compared to the rate of development of prion disease that would occur without CpG administration according to the instant invention. In one embodiment, an effective amount to treat the prion disease is that amount that is sufficient to prevent the development of prion disease, compared to development of prion disease that would occur without CpG administration according to the instant invention. In one embodiment an effective amount to treat the prion disease is that amount that is sufficient to slow the progression of prion disease, compared to the rate of progression of prion disease that would occur without CpG administration according to the instant invention. In one embodiment an effective amount to treat the prion disease is that amount that is sufficient to stop the progression of prion disease, compared to the progression of prion disease that would occur without CpG administration according to the instant invention. In one embodiment an effective amount to treat the prion disease is that amount that is sufficient to resolve prion disease, compared to the prion disease that would occur without CpG administration according to the instant invention.

Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular immunostimulatory oligonucleotide being administered, the antigen, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular immunostimulatory oligonucleotide and/or other therapeutic agent without necessitating undue experimentation.

Subject doses of the immunostimulatory oligonucleotides for mucosal or local delivery typically range from about 0.1 μg to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time therebetween. More typically doses range from about 10 μg to 5 mg per administration, and most typically from about 100 μg to 1 mg, with repeated administrations being spaced days or weeks apart. Subject doses of immunostimulatory oligonucleotides for parenteral delivery for the purpose of inducing an antigen-specific immune response, wherein the compounds are delivered with an antigen but not another therapeutic agent are typically 5 to 10,000 times higher than the effective mucosal dose for vaccine adjuvant or immune stimulant applications, and more typically 10 to 1,000 times higher, and most typically 20 to 100 times higher. Doses of the immunostimulatory oligonucleotides for parenteral delivery for the purpose of inducing an innate immune response or for inducing an antigen-specific immune response when the immunostimulatory nucleic acids are administered in combination with other therapeutic agents or in specialized delivery vehicles typically range from about 0.1 μg to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time therebetween. More typically parenteral doses for these purposes range from about 10 μg to 5 mg per administration, and most typically from about 100 μg to 1 mg, with repeated administrations being spaced days or weeks apart. In some embodiments, however, parenteral doses for these purposes may be used in a range of 5 to 10,000 times higher than the typical doses described above.

For any immunostimulatory oligonucleotide the therapeutically effective amount can be initially determined from animal models. A therapeutically effective dose can also be determined from human data for CpG oligonucleotides which have been tested in humans (human clinical trials have been initiated) and for compounds which are known to exhibit similar pharmacological activities, such as other mucosal adjuvants, e.g., LT and other antigens for vaccination purposes, for mucosal or local administration. Higher doses are required for parenteral administration. The applied dose can be adjusted based on the relative bioavailability and potency of the administered immunostimulatory oligonucleotide. Adjusting the dose to achieve maximal efficacy based on the methods described herein and other methods as are well-known in the art is well within the capabilities of the ordinarily skilled artisan.

The immunostimulatory oligonucleotide can be administered alone or with antigen or other therapeutic agent. In this context, "antigen" refers to any biological molecule capable of eliciting specific immunity. Antigens specifically include peptides (oligopeptides, polypeptides, proteins, and glycosylated derivatives thereof), and polysaccharides. Peptide antigen can be administered preformed or as a polynucleotide encoding the peptide. Also in this context, "other therapeutic agent" includes any suitable composition useful in treating prion disease, including an antibody capable of binding a prion protein. When the immunostimulatory oligonucleotide is administered with antigen or other therapeutic agent, the immunostimulatory oligonucleotide can be administered before, concunently with, or following administration of the antigen or other therapeutic agent. The immunostimulatory oligonucleotide and the antigen or other therapeutic agent can be formulated together or separately when the immunostimulatory oligonucleotide is administered concunently with the antigen or other therapeutic agent. When the immunostimulatory oligonucleotide is administered before or following administration of antigen or other therapeutic agent, the immunostimulatory oligonucleotide and the antigen or other therapeutic agent can be administered by the same route of administration or by different routes of administration. In addition, when the immunostimulatory oligonucleotide is administered before or following administration of antigen or other therapeutic agent, the immunostimulatory oligonucleotide and the antigen or other therapeutic agent can be administered to the same site or to different sites.

The immunostimulatory oligonucleotides may be directly administered to the subject or may be administered in conjunction with a nucleic acid delivery complex. A nucleic acid delivery complex shall mean a nucleic acid molecule associated with (e.g., ionically or covalently bound to; or encapsulated within) a targeting means (e.g., a molecule that results in higher affinity binding to target cell (e.g., B cell surfaces and or increased cellular uptake by target cells). Examples of nucleic acid delivery complexes include nucleic acids associated with a sterol (e.g., cholesterol), a lipid (e.g., a cationic lipid, virosome or liposome), or a target cell specific binding agent (e.g., a ligand recognized by target cell specific receptor). Preferred complexes may be sufficiently stable in vivo to prevent significant uncoupling prior to internalization by the target cell. However, the complex can be cleavable under appropriate conditions within the cell so that the nucleic acid is released in a functional form.

Delivery vehicles or delivery devices for delivering antigen and nucleic acids to surfaces have been described. The immunostimulatory oligonucleotide and/or the antigen and/or other therapeutics may be administered alone (e.g., in saline or buffer) or using any delivery vehicles known in the art. For instance the following delivery vehicles have been described: cochleates (Gould-Fogerite et al., 1994, 1996); emulsomes (Vancott et al., 1998, Lowell et al., 1997); ISCOMs (Mowat et al., 1993, Carlsson et al., 1991, Hu et., 1998, Morein et al., 1999); liposomes (Childers et al., 1999, Michalek et al., 1989, 1992, de Haan 1995a, 1995b); live bacterial vectors (e.g., Salmonella, Escherichia coli, bacillus Calmette- Guerin, Shigella, Lactobacillus) (Hone et al., 1996, Pouwels et al., 1998, Chatfield et al., 1993, Stover et al., 1991, Nugent et al., 1998); live viral vectors (e.g., Vaccinia, adenovirus, Herpes Simplex) (Gallichan et al., 1993, 1995, Moss et al., 1996, Nugent et al., 1998, Flexner et al., 1988, Monow et al., 1999); microspheres (Gupta et al., 1998, Jones et al., 1996, Maloy et al., 1994, Moore et al., 1995, O'Hagan et al., 1994, Eldridge et al., 1989); nucleic acid vaccines (Fynan et al., 1993, Kuklin et al., 1997, Sasaki et al., 1998, Okada et al., 1997, Ishii et al., 1997); polymers (e.g., carboxymethylcellulose, chitosan) (Hamajima et al., 1998, Jabbal-Gill et al., 1998); polymer rings (Wyatt et al., 1998); proteosomes (Vancott et al., 1998, Lowell et al., 1988, 1996, 1997); sodium fluoride (Hashi et al., 1998); transgenic plants (Tacket et al., 1998, Mason et al., 1998, Haq et al., 1995); virosomes (Gluck et al., 1992, Mengiardi et al., 1995, Cryz et al., 1998); virus-like particles (Jiang et al., 1999, Leibl et al., 1998).

The immunostimulatory oligonucleotides may be administered by any means known to the skilled artisan. Routes of administration include but are not limited to oral, mucosal, parenteral, intravenous, intramuscular, intraperitoneal, intranasal, intratracheal, sublingual, subcutaneous, intradermal, inhalation, ocular, vaginal, and rectal.

The immunostimulatory oligonucleotides are administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer R (1990) Science 249:1527-33, which is incorporated herein by reference.

The immunostimulatory oligonucleotides and optionally other therapeutics and/or antigens may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non- pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic, tartaric, citric, methane sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and a salt (0.8-2% w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

For oral administration, the immunostimulatory oligonucleotides can be formulated readily by combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpynolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers for neutralizing internal acid conditions or may be administered without any caniers.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. Microspheres formulated for oral administration may also be used. Such microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to the present invention may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifiuoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. The immunostimulatory oligonucleotides, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The immunostimulatory oligonucleotides may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the immunostimulatory oligonucleotides may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

The term "pharmaceutically-acceptable carrier" means one or more compatible solid or liquid filler, diluents or encapsulating substances which are suitable for administration to a human or other vertebrate animal. The term "canier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being commingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.

The immunostimulatory oligonucleotides useful in the invention may be delivered in mixtures with additional adjuvant(s), other therapeutics, or antigen(s). A mixture may consist of several adjuvants in addition to the immunostimulatory oligonucleotide or several antigens or other therapeutics.

The particular mode selected will depend, of course, upon the particular adjuvants or antigen selected, the particular condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of an immune response without causing clinically unacceptable adverse effects. Prefened modes of administration are discussed above.

The immunostimulatory oligonucleotides may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the immunostimulatory oligonucleotides into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. Liquid dose units are vials or ampoules. Solid dose units are tablets, capsules and suppositories. For treatment of a patient, depending on activity of the compound, manner of administration, purpose of the immunization (i.e., prophylactic or therapeutic), nature and severity of the disorder, age and body weight of the patient, different doses may be necessary. The administration of a given dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units. Multiple administration of doses at specific intervals of weeks or months apart is usual for boosting the antigen-specific responses.

Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compounds, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer base systems such as poly(lactide-glycolide), copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the foregoing polymers containing drugs are described in, for example, U.S. Patent 5,075,109. Delivery systems also include non-polymer systems that are: lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-di-and tri-glycerides; hydrogel release systems; sylastic systems; peptide based systems; wax coatings; compressed tablets using conventional binders and excipients; partially fused implants; and the like. Specific examples include, but are not limited to: (a) erosional systems in which an agent of the invention is contained in a form within a matrix such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems in which an active component permeates at a controlled rate from a polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and 5,407,686. In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.

Examples

Example 1. CpG-ODN Protects Mice from Scrapie Prion.

Groups of 8 mice each were inoculated intraperitoneally with 100 μl of 10% brain homogenates from mice terminally ill with RML scrapie prion strain conesponding to an infectious challenge of approximately 104 LD 0. The mice received 0.15 pmol (30 μl of 5 nM solution) CpG-ODN (oligonucleotide 1826, 5'- TCCATGACGTTCCTGACGTT -3', SEQ ID NO: 18), which has been shown to be a strong inducer of innate immunity and to confer sterile immunity against certain infectious diseases. Sparwasser T et al. (2000) Eur J Immunol 30:3591-7. CpG-ODN was administered intraperitoneally at the time of inoculation (Oh) with scrapie prions as well as 4 times at intervals of 24h (4 x q 24h); a second group received 0.15 pmol (30 μl of 5 nM solution) CpG-ODN 7h after infection (7h) and 4 times at intervals of 24h (4 x q 24h); a third group received CpG-ODN at 7h after infection and subsequently 20 times at intervals of 24h (20 x q 24h). Controls matched for age and sex were given saline instead of CpG-ODN at the identical time intervals. Additional control experiments were also performed in uninfected mice using saline and brain homogenates of uninfected mice. All animals were observed and scored daily for clinical signs of disease. Scrapie in mice is characterized by ataxia of gait, tremor, difficulty righting from a supine position, and tail rigidity. Occunence of two of these four symptoms was used as the end point criterion for establishing a clinical diagnosis of scrapie. Western blots of brain homogenates were performed to confirm the diagnosis. All results were analyzed using the Student's t test (Table 1).

Table 1. Mean incubation time in C57BL/6 mice after inoculation with scrapie strain RML and treatment with CpG-ODN.

Figure imgf000033_0001

Mice infected with the RML strain which received CpG-ODN at the time of inoculation and 7h post-infection as well as 4 times at intervals of 24h showed a dramatic prolongation of survival time compared to control mice with an increase in survival time of 38% in both cases. These differences were highly significant (pO.OOOl). The application of CpG-ODN 7h post-inoculation and 20 times at 24h intervals led to disease- free intervals of more than 330 days. All control groups which were not inoculated with the RML strain remained disease-free, and no harmful effect of CpG-ODN application was observed. These results showed that the application of CpG-ODN at the time of infection and 7h after infection led to a dramatic prolongation of survival time. This effect can be amplified when CpG-ODN are given for a longer period of time. The application of CpG-ODN for 20 times at 24h intervals results in a disease-free interval of >330 days, which indicates the great potential of CpG-ODN for post-exposure prophylaxis of people with exposure to infection. The mechanism of disease prevention remains to be determined, but it seems that the most likely explanation for this effect is a stimulation of TLR-expressing cells of the innate immune system, e.g., macrophages, monocytes, and especially dendritic cells. Sparwasser T et al. (2000) Eur J Immunol 30:3591-7. CpG-ODN has been known to induce resistance against other infectious diseases. Zimmermann S et al. (1998) J Immunol 160:3627-30. The induction of extreme prolongation of the incubation time or even resistance to prion disease was a surprising finding in the context of a completely different infectious agent.

The findings presented here show that administration of CpG-ODN prolongs the incubation time by 38% and may have the potential to prevent infection after repeated administration, even when high doses of infectivity are administered intraperitoneally. It may therefore be possible to prevent disease after inadvertent iatrogenic exposure with much lower infectious doses administered peripherally.

Example 2. Effect of Timing Between Exposure to Prion and Administration of CpG-ODN. Mice are injected with RML scrapie prion or control and treated with CpG-ODN or control essentially as in Example 1, except that the interval between injection with RML scrapie prion or control and administration of CpG-ODN or control is varied. In some groups administration of CpG-ODN is delayed as much as a month following injection with RML scrapie prion or control. In some groups administration of CpG-ODN precedes injection with RML scrapie prion or control. In some groups the number and schedule of repeated administrations of CpG-ODN is varied from Example 1. Results, measured as in Example 1, show that CpG-ODN is effective even when administered more than 7h after injection with RML scrapie prion.

Example 3. Mice Protected from Scrapie Prion by CpG-ODN Develop an Immune Response to Prion. Mice are injected with RML scrapie prion or control and treated with CpG-ODN or control essentially as in Example 1 or Example 2. At various time points following injection with RML scrapie prion or control and treatment with CpG-ODN or control, tissue or blood samples are obtained and analyzed for prion-specific and prion-nonspecific immune response. Presence of an immune response is determined by suitable method or measurement including, without limitation, antibody titer, enzyme-linked immunosorbent assay (ELISA), flow cytometry, cell proliferation assay, cytotoxicity assay, polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR), Western immunoblot, Northern blot, and Southern blot. General methods for these types of measurements are standard and are suitably adapted to the specific antigen or stimulus being assayed. For example, ELISA is used to measure production of various secreted products, including antibodies, cytokines and chemokines. Cytokines and chemokines in this example include interleukin (IL)-4, IL-10, IL-6, IL-12, IL-18, interferon (IFN)-α, IFN-β, IFN-γ, tumor necrosis factor (TNF), and EP-10. Flow cytometry is used to measure cell surface and intracellular proteins, including markers associated with immune cell activation. Markers associated with immune cell activation can vary with cell type but include cluster of differentiation (CD) markers such as CD86, major histocompatibility complex (MHC), inducible cytokine receptors, and certain costimulatory molecules. Results show that CpG- ODN induces an immune response to prion protein.

Example 4. Selection of CpG-ODN.

Mice are injected with RML scrapie prion or control and treated with CpG-ODN or control essentially as in Example 1 or Example 2. Various CpG-ODN are compared against ODN 1826 for their effectiveness. Results, measured as in Example 1, show that protection is related to the use of species-optimized CpG-ODN.

Example 5. Use of CpG-ODN in Alzheimer's Disease Model.

Mice genetically susceptible to developing Alzheimer' s-like disease are administered CpG nucleic acid alone or CpG nucleic acid plus antigen (e.g., amyloid precursor protein or Aβ), either prior to or following onset of Alzheimer' s-like disease. Similar mice are administered appropriate control treatment. Animals are monitored for behavioral and histologic evidence of Alzheimer' s-like disease. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one aspect of the invention and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

All references, patents and patent publications that are recited in this application are incorporated in their entirety herein by reference.

We claim:

Claims

Claims
1. A method for treating a prion disease in a subject, comprising: administering to a subject having or at risk of developing a prion disease a CpG nucleic acid in an effective amount to treat the prion disease.
2. The method according to claim 1, wherein the administering follows exposure of the subject to a prion protein that is associated with a prion disease.
3. The method according to claim 1 , wherein the prion disease is a transmissible spongiform encephalopathy (TSE).
4. The method according to claim 1, wherein the prion disease is scrapie.
5. The method according to claim 1, wherein the prion disease is bovine spongiform encephalopathy (BSE).
6. The method according to claim 1, wherein the prion disease is variant Creutzfeldt- Jakob disease (vCJD).
7. The method according to claim 1, wherein the prion disease is iatrogenic Creutzfeldt- Jakob disease (iCJD).
8. The method according to claim 1, wherein the subject is a human.
9. A method for inducing an immune response to a prion protein, comprising: contacting an antigen-presenting cell (APC) with a prion protein; and contacting the APC with a CpG nucleic acid in an effective amount to induce an immune response to the prion protein.
10. The method according to claim 9, wherein the immune response is in vivo.
11. The method according to claim 9, wherein the APC is selected from the group consisting of: a B cell, a dendritic cell, a macrophage, and a monocyte.
12. The method according to claim 9, wherein the APC is a dendritic cell.
13. The method according to claim 9, wherein the APC expresses a Toll-like receptor (TLR) that signals in response to the CpG nucleic acid.
14. The method according to claim 13, wherein the TLR is TLR9.
15. The method according to claim 9, wherein the prion protein is prion proteimscrapie form (PrPSc).
16. The method according to claim 9, wherein the prion protein is a fragment of PrPSc lacking at least the amino terminus of full-length PrPSc.
17. The method according to claim 9, wherein the prion protein is a derivative of PrPSc or a derivative of a fragment of PrPSc lacking at least the amino terminus of full-length PrPSc.
18. The method according to claim 9, wherein the CpG nucleic acid is a Class B CpG nucleic acid.
19. The method according to claim 9, wherein the CpG nucleic acid is a Class A CpG nucleic acid.
20. The method according to claim 9, wherein the CpG nucleic acid is a Class C CpG nucleic acid.
21. The method according to claim 9, wherein the CpG nucleic acid is optimized for use in a species of the subject.
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