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WO2005034995A1 - Chimeres de prions et ses utilisations - Google Patents

Chimeres de prions et ses utilisations Download PDF

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Publication number
WO2005034995A1
WO2005034995A1 PCT/US2003/031057 US0331057W WO2005034995A1 WO 2005034995 A1 WO2005034995 A1 WO 2005034995A1 US 0331057 W US0331057 W US 0331057W WO 2005034995 A1 WO2005034995 A1 WO 2005034995A1
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WIPO (PCT)
Prior art keywords
prion
seq
chimera
antibody
protein
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Application number
PCT/US2003/031057
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English (en)
Inventor
Sergio Abrignani
Fred Cohen
Melissa D. Michelitsch
Celine Yuan-Hwei Hu
Bruce Phelps
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Chiron Corporation
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Priority to AU2003279088A priority Critical patent/AU2003279088A1/en
Publication of WO2005034995A1 publication Critical patent/WO2005034995A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2872—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against prion molecules, e.g. CD230
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2828—Prion diseases

Definitions

  • the invention relates to prion chimeras, polynucleotides encoding these chimeras, methods of generating antibodies using such chimeras and polynucleotides, and to antibodies generated using these methods.
  • the invention further relates to methods of using such chimeras, polynucleotides and/or antibodies to detect the presence of pathogenic prions in a biological sample and to methods of using such antibodies, polynucleotides and/or prion protein chimeras to raise an immune response and/or as components in a therapeutic or prophylactic vaccine.
  • BACKGROUND Conformational diseases include a variety of unrelated diseases, including transmissible spongiform encephalopathies (e.g., prion diseases) and Alzheimer disease, arising from aberrant conformational transition of a protein which in turn leads to self-association of the aberrant protein forms, with consequent tissue deposition and damage.
  • transmissible spongiform encephalopathies e.g., prion diseases
  • Alzheimer disease arising from aberrant conformational transition of a protein which in turn leads to self-association of the aberrant protein forms, with consequent tissue deposition and damage.
  • prion diseases include Creutzfeldt- Jakob disease (CJD), Gerstmann-Straussler-Scheinker syndrome, Fatal Familial Insomnia, and Kuru (see, e.g., Harrison's Principles of Internal Medicine, Isselbacher et al., eds., McGraw-Hill, Inc. New York, (1994); Medori et al. 1992 N. Engl. J. Med., 326: 444-9.).
  • CJD Creutzfeldt- Jakob disease
  • Gerstmann-Straussler-Scheinker syndrome Fatal Familial Insomnia
  • Kuru see, e.g., Harrison's Principles of Internal Medicine, Isselbacher et al., eds., McGraw-Hill, Inc. New York, (1994); Medori et al. 1992 N. Engl. J. Med., 326: 444-9.
  • TSEs transmissible spongiform encephalopathies
  • BSE bovine spongiform encephalopathy
  • DSE transmissible mink encephalopathy
  • chronic wasting disease of captive mule deer and elk Gajdusek, (1990)
  • Subacute Spongiform Encephalopathies Transmissible Cerebral Anr loidoses Caused by Unconventional Viruses. Pp. 2289-2324 In: Virology, Fields, ed. New YOrk: Raven Press, Ltd.).
  • Transmissible spongiform encephalopathies are characterized by the same hallmarks: a spongiform degeneration, reactive gliosis in the cortical and subcortical gray matters of the brain, and transmission when experimentally inoculated into laboratory animals including primates, rodents, and transgenic mice.
  • the term "prion" was originally coined as a reference to a proteinaceous infectious agent that lacks nucleic acid. (See, e.g., Bolton, McKinley et al. (1982) Science 218:1309-1311; Prusiner, Bolton et al. (1982) Biochemistry 21:6942-6950; McKinley, Bolton et al.
  • PrP Complete prion protein-encoding genes have since been cloned, sequenced and expressed in transgenic animals. PrP is encoded by a single-copy host gene. See, e.g., Basler, Oesch et al. (1986) Cell 46:417-428. At least 20 different mutations in the human PrP gene have been found to be associated with inherited prion diseases. It is thought likely that these inherited forms of PrP Sc are also pathogenic should they be transmitted to another host.
  • PrP c generally contains about 40% ⁇ -helix and little ⁇ -sheet, while PrP sc contains about 30% ⁇ -helix and about 45% ⁇ -sheet.
  • the proposed PrP sc parallel ⁇ -helix structure is thought to be an unusually stable conformation, similar to ⁇ -helical structures generally found in proteins subjected to harsh, denaturing environments such as bacterial or viral virulence factors or plant pollens.
  • the proposed ⁇ -helical fold may be formed in a two-state manner analogous to ⁇ -helix formation. Based on this predicted structure, it has been suggested that the conversion of PrP c to PrP sc may result in the stabilization of a proto- ⁇ -helical motif by a neighboring PrP molecule and subsequent extension to form the complete ⁇ -helical structure.
  • PrP c is soluble in non-denaturing detergents, PrP sc is insoluble; PrP c is readily digested by proteases, while PrP sc is partially resistant, resulting in the formation of an
  • PrPres N-terminally truncated fragment known as "PrPres” (Baldwin et al. (1995); Cohen & Pruisner (1995)), "PrP 27-30” (27-30 kDa) or “PK-resistant” (proteinase K resistant) form.
  • PrP sc acts as a template for the conversion of PrP c to PrP sc . Only a minute amount of PrP sc is believed to be needed to start this process and eventually lead to irreversible neurodegenerative damage.
  • Pathogenic prions have also been transmitted through improperly sterilized depth electrodes, transplanted corneas, human growth hormone (HGH) and gonadotrophin derived from cadaveric pituitaries, and dura matter grafts.
  • HGH human growth hormone
  • gonadotrophin derived from cadaveric pituitaries and dura matter grafts.
  • transmission of pathogenic prions is also blamed in the BSE epidemic in Great Britain, where cattle were purportedly infected with prions through a meat and bone meal (MBM) fed primarily to dairy cows.
  • MBM meat and bone meal
  • the tragic consequences of accidental transmission of these diseases see, e.g., Gajdusek, Infectious Amyloids, and Prusiner Prions In Fields Virology. Fields, et al., eds. Lippincott-Ravin, Pub. Philadelphia (1996); Brown et al.
  • PrP is resistant to many proteases, and it is generally thought that this characteristic prevents pathogenic prion conformations from being processed and presented to antigen presenting cells (APC).
  • APC antigen presenting cells
  • the amino acid sequence similarity between non-pathogenic prions and pathogenic prions makes it difficult for a host's immune system to generate antibodies specific to PrP sc .
  • prion-related diseases typically kill the host without any sign of an immune response to the pathogenic prion.
  • U.S. Patent No. 6,562,341 is directed to a device including a solid support and an antibody that binds to native PrP sc in situ.
  • the present invention is related to prion chimeras that can be used to raise antibodies o specific to PrP .
  • the antibodies and chimeras of the invention can be used in a wide range of applications, including as diagnostic tools to detect the presence of pathogenic prions in a biological sample, in methods of generating immune response, and/or as components of a therapeutic or prophylactic vaccine.
  • the invention comprises a prion chimera comprising a prion polypeptide and a non-prion polypeptide.
  • the prion polypeptide comprises an amino acid sequence of a prion protein that can adapt the ⁇ -helical conformation of pathogenic prion, for example, the sequences depicted in FIG.
  • the non-prion polypeptide may be derived from a beta-helical protein.
  • the beta- helical protein may be left- or right- handed, for example, pertactin or GCA (or fragments thereof), as depicted in FIGs. 2 and 7 (SEQ ID NO:3, SEQ ID NO: 183, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:4, SEQ ID NO:184, SEQ ID NO:10 and SEQ ID NO: 11)).
  • the prion chimeras are those depicted in FIGs.
  • the non-prion polypeptide of the chimera does not contain one or more of the amino acids of the non-prion protein that adapt the ⁇ -helix conformation.
  • the prion chimeras are typically not, themselves, pathogenic and/or infectious.
  • a polynucleotide e.g., a DNA construct
  • encodes a prion chimera for example the constructs shown in FIGs. 20-23 (SEQ ID NO: 155-182).
  • the non-prion protein segment preferably allows the prion protein segment to adapt its ⁇ -helical structure in the prion chimera.
  • the component amino acid sequences e.g., non-prion sequences and prion sequences
  • the prion sequences may be co-linear (e.g., the prion sequences are N- and/or C- terminal to the non-prion sequences).
  • one component may be inserted into the other component.
  • the prion chimera may further comprise a tag sequence, for example, a histidine tag sequence as depicted in SEQ ID NO. 5 or SEQ ID NO:6.
  • the invention includes a method of generating antibodies specific to a pathogenic prion, the method comprising the step of administering to an animal a prion chimera, wherein the chimera comprises a prion protein or a fragment or derivative thereof; and a non-prion, ⁇ -helical protein or a fragment or derivative thereof.
  • the method may also include the step of isolating the antibodies from the animal.
  • the prion protein or fragment or derivative thereof has a ⁇ -helical conformation of a pathogenic prion.
  • any of the chimeras described herein may include prion proteins containing the amino acid sequences set forth in FIG 1 (SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:6, SEQ LO NO: 12, SEQ ID NO:7 or SEQ ID NO: 13). Further, in any of the chimeras described herein, one or more amino acids of the ⁇ -helix of the ⁇ -helical protein may be replaced with the prion protein or fragment or derivative thereof. Any of the chimeras described herein may include non-prion proteins (e.g., left handed or right handed ⁇ -helical proteins such as pertactin (P69) or ⁇ carbonic anhydrase), for example, containing the amino acid sequences set forth in FIGs.
  • P69 pertactin
  • the prion chimera may further comprise a tag sequence, for example, a histidine tag sequence as depicted in SEQ ID NO. 5 or SEQ LD NO:6.
  • specific antibodies may be from the animal.
  • Prion chimeras include, for example, the chimeras depicted in Figures 3-6, 8-23 and 25-28 (SEQ ID NO: 14-153 and 185-208).
  • any of the prion chimeras used to generate antibodies may also be encoded by polynucleotides, for example polynucleotides operably linked to control elements that are expressed to produce the prion chimera.
  • the polynucleotide constructs encoding the prion chimeras may also include a signal sequence, for example a leader sequence derived from tpa. Exemplary polynucleotide constructs are depicted in Figures 20-23 (SEQ ID NO:155-182).
  • the invention includes antibodies specific a prion chimera, for example an antibody that is specific for the ⁇ -helical region of a pathogenic prion.
  • the antibodies are generated by any of the methods described herein, for example, by administering one or more of the prion chimeras (or polynucleotides encoding these prion chimeras) to an animal (e.g., non-human or human mammal) subject, by phage display techniques, or the like.
  • the antibodies of the present invention may be monoclonal and polyclonal antibodies. Any of the antibodies described herein may also be encoded by one or more polynucleotides.
  • the invention includes a method for detecting the presence of a pathogenic prion in a biological sample, comprising (a) exposing the biological sample to any of the prion chimera-specific antibodies described herein (e.g., an antibody generated by the methods described herein); and (b) detecting the presence or absence of the antibody bound to a pathogenic prion.
  • the antibody is directed against a ⁇ -helical region of a pathogenic prion.
  • the invention includes a method for detecting the presence of a pathogenic prion in a biological sample comprising: (a) exposing the biological sample suspected of containing a pathogenic prion to a prion chimera; and (b) detecting the presence or absence of the prion chimera bound to the pathogenic prion, if any, in the sample.
  • the invention includes a method for detecting a pathogenic prion in a biological sample, comprising: (a) providing a solid support comprising a first antibody (or prion chimera) bound thereto, wherein the first antibody specific to a prion chimera (e.g., an antibody generated by the methods described herein); (b) exposing the solid support to a biological sample under conditions which allow pathogenic prions, when present in the biological sample, to bind to the first antibody or prion chimera; (c) exposing the solid support to a detectably labeled second antibody specific to pathogenic prions or detectably labeled prion chimera under conditions which allow the second antibody or detectably labeled prion chimera to bind to pathogenic prions bound by the first antibody or prion chimera; and (d) detecting complexes formed between the first antibody or prion chimera, a pathogenic prion from the biological sample and the second antibody or detectably labeled prion chimera
  • the invention includes a method for detecting the presence of a pathogenic prion in a biological sample comprising: (a) providing a solid support comprising a first antibody or prion chimera bound thereto, wherein the first antibody or prion chimera recognizes pathogenic and non-pathogenic prions; (b) exposing the solid support to a biological sample under conditions which allow prion proteins, when present in the biological sample, to bind to the first antibody or prion chimera; (c) exposing the solid support to a detectably labeled second antibody specific to a prion chimera (e.g., labeled antibody generated according to the methods described herein) or a detectably labeled prion chimera that binds to pathogenic prions; and (d) detecting complexes formed between the first antibody, a pathogenic prion from the biological sample, and the second antibody or the detectably labeled prion chimera.
  • the invention includes a method for detecting the presence of a pathogenic prion in a biological sample comprising: (a) providing a solid support comprising a first antibody specific for a prion chimera (e.g., an antibody generated according to the methods described herein) or prion chimera bound thereto, wherein the first antibody or prion chimera is specific to pathogenic prions; (b) combining the solid support with a detectably labeled first ligand or antigen, wherein the first antibody's or prion chimera's binding affinity to the detectably labeled first ligand or antigen is weaker than the first antibody's (or prion chimera's) binding affinity to a pathogenic prion; (c) combining a biological sample with the solid support under conditions which allow a pathogenic prion, when present in the biological sample, to bind to the first antibody (or prion chimera's) and replace the first ligand or antigen; and (d)
  • the biological sample can be organs, whole blood, blood fractions, plasma, cerebrospinal fluid (CSF), urine, tears, tissue, organs, and/or biopsies.
  • the biological sample is blood.
  • the solid support can be, for example, nitrocellulose, polystyrene latex, polyvinyl fluoride, diazotized paper, nylon membranes, activated beads, and/or magnetically responsive beads.
  • the invention includes a solid support comprising at least one prion chimera or at least one antibody specific to pathogenic and/or non-pathogenic prions bound thereto, for example, an antibody generated according to the methods described herein that is specific for pathogenic prions (e.g., directed against a ⁇ -helical region of a pathogenic prion).
  • the solid supports as described herein are used in an immunoassay (e.g., where the antibody bound the solid support is specific to pathogenic prions or where the antibody bound to the solid s support is specific to pathogenic and nonpathogenic prions).
  • the solid support can comprise, for example, nitrocellulose, polystyrene latex, polyvinyl fluoride, diazotized paper, nylon membranes, activated beads, and magnetically responsive beads.
  • the invention includes a kit for detecting the presence of a pathogenic prion in a biological sample comprising: (a) a solid support comprising at least one antibody (e.g., an antibody generated according to the methods described herein) or prion chimera specific to pathogenic prions bound thereto; and (b) ancillary reagents and, optionally, positive and negative controls.
  • the invention includes an immunogenic composition comprising a prion chimera and, optionally, an adjuvant, wherein the prion chimera comprises a prion protein or a fragment or derivative thereof and a non-prion ⁇ -helical protein or a fragment or derivative thereof.
  • the prion protein may comprise, for example, a prion protein (or fragment thereof) that can adopt a ⁇ -helical conformation of a pathogenic prion protein (e.g., the amino acid sequences set forth in SEQ ID NO:l; SEQ ID NO:2; SEQ LD NO:6, SEQ LD NO: 12, SEQ ID NO:7 and/or SEQ ID NO: 13).
  • the non-prion ⁇ -helical protein can be a left handed helical protein or a right handed helical protein or a fragment thereof, for example, pertactin (e.g., P69 pertactin) or ⁇ carbonic anhydrase or fragments thereof (e.g., SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:183; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ JD NO:240 and/or SEQ JD NO: 11).
  • pertactin e.g., P69 pertactin
  • ⁇ carbonic anhydrase or fragments thereof e.g., SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:183; SEQ ID NO:8; SEQ ID NO:9; SEQ ID NO:10; SEQ JD NO:240 and/or SEQ JD NO: 11).
  • the immunogenic composition comprises one or more prion chimeras depicted in FIGs. 3-6, 8-23 and 25-28 (SEQ ID NO: 14-153 and SEQ ID NO: 185-208).
  • the immunogenic composition comprises one or more polynucleotides encoding prion chimeras depicted in FIGs. 20-23 (SEQ ID NO: 155- 182).
  • the adjuvant component may be encoded by a polynucleotide.
  • the immunogenic compositions may also comprise combinations and/or mixtures of polypeptides and polynucleotides.
  • the invention includes a method of raising an immune response to an infectious prion comprising administering to an animal an immunologically effective amount of any of the immunogenic compositions described herein (e.g., prion protein chimeras and/or polynucleotides encoding prion chimeras).
  • Antibodies (or polynucleotide encoding these antibodies) as described herein may also be administered to a subject.
  • one or more components of the immunogenic composition is encoded by a polynucleotide that is incorporated into a gene delivery vehicle, for example a viral vector (e.g., alphaviral vector), a non-viral vector, a particular carrier (e.g., PLG), and/or a liposome preparation.
  • a viral vector e.g., alphaviral vector
  • a non-viral vector e.g., PLG
  • PLG particular carrier
  • the invention includes "prime-boost" type methods of inducing an immune response in a subject, for example methods comprising (a) administering a first composition comprising an immunogenic composition comprising a polynucleotide (e.g., a polynucleotide encoding a prion chimera or a polynucleotide an antibody specific for pathogenic prions) in a priming step and (b) administering a second composition comprising a polypeptide (e.g., a prion chimera or an antibody specific for pathogenic prions), as a booster, in an amount sufficient to induce an immune response in the subject.
  • a first composition comprising an immunogenic composition comprising a polynucleotide (e.g., a polynucleotide encoding a prion chimera or a polynucleotide an antibody specific for pathogenic prions) in a priming step
  • a second composition comprising a polypeptide (
  • one or more adjuvants may be included.
  • the subject may be a mammal, for example, a human.
  • one or more components may be administered intramuscularly, intramucosally, intranasally, subcutaneously, intradermally, transdermally, intravaginally, intrarectally, orally and or intravenously.
  • the immune response may be prophylactic and/or therapeutic.
  • FIG 1 A depicts a full-length human sequence (SEQ JD NO:l) and fragments extending from amino acid residues 135-155 (SEQ JD NO:6) and amino acid residues 126-154 (SEQ ID NO: 12).
  • FIG IB depicts full-length mouse sequence (SEQ ID NO: 2) and fragments extending from amino acid residues 135-155 (SEQ JD NO:7) and amino acid residues 126-154 (SEQ JD NO:13).
  • FIGs. 2A - 2B depict amino acid sequences of an exemplary non-prion sequence designated "P69" pertactin and derived from Pertussis toxin.
  • FIG 2A depicts a full-length amino acid sequence of P69 (SEQ JD NO:3).
  • FIG. 2B depicts fragments of SEQ JD NO:3 designated "P69 Control A” (SEQ JD NO: 8) and "P69 Control B” (SEQ JD NO:9).
  • FIGs. 3A - 3D depict exemplary prion chimeras including "P69 control A" non-prion amino acid sequences and human PrP prion amino acid sequences (135-155).
  • FIG. 3A depicts sequences of prion chimeras designated P69 A/human Chimera No. 1 (SEQ JD NO: 14) and P69A/human Chimera No. 2 (SEQ LD NO: 15).
  • FIG. 3B depicts sequences of prion chimeras designated P69 A/human Chimera No.
  • FIG. 3 depicts sequences of prion chimeras designated P69 A/human Chimera No. 5 (SEQ JD NO: 18) and P69A/human Chimera No. 6 (SEQ ID NO: 19).
  • FIG. 3D depicts sequences of prion chimeras designated P69 A/human Chimera No. 7 (SEQ JD NO:20) and P69A/human Chimera No. 8 (SEQ ID NO:21).
  • FIGs. 1-8 include sequences derived from P69 Control A (SEQ ID NO:8); sequences derived from human PrP (135-155) (SEQ JD NO:6); and a His tag.
  • FIG. 4A depicts sequences of prion chimeras designated P69Amouse Chimera No. 1 (SEQ ID NO:22) and P69A/mouse Chimera No. 2 (SEQ ID NO:23).
  • FIG. 4B depicts sequences of prion chimeras designated P69 A/mouse Chimera No.
  • FIG. 4C depicts sequences of prion chimeras designated P69A/mouse Chimera No. 5 (SEQ ID NO:26) and P69A/mouse Chimera No. 6 (SEQ ID NO:
  • FIG. 4D depicts sequences of prion chimeras designated P69 A/mouse Chimera No. 7 (SEQ ID NO:28) and P69A/mouse Chimera No. 8 (SEQ ID NO:29).
  • Chimeras designated P69A/mouse (135-155) Chimera No. 1-8 include sequences derived from P69 Control A (SEQ JD NO:8); sequences derived from mouse PrP (135-155) (SEQ JD NO:7); and a His tag.
  • FIGs. 5A - 5D depict exemplary prion chimeras including "P69 control B" non-prion amino acid sequences and human PrP prion amino acid sequences (135-155).
  • FIG. 5A depicts sequences of prion chimeras designated P69B/human Chimera No. 1 (SEQ JD NO:30) and P69B/human Chimera No. 2 (SEQ JD NO:31).
  • FIG. 5B depicts sequences of prion chimeras designated P69B/human Chimera No. 3 (SEQ JD NO: 32) and P69B/human Chimera No. 4 (SEQ JD NO:33).
  • FIG. 5C depicts sequences of prion chimeras designated P69B/human Chimera No. 5 (SEQ JD NO:34) and P69B/human Chimera No. 6 (SEQ JD NO: 35).
  • FIG. 5D depicts sequences of prion chimeras designated P69B/human Chimera No. 7 (SEQ ID NO:36) and P69B/human Chimera No. 8 (SEQ JD NO:37).
  • Chimeras designated P69B/human (135-155) Chimera No. 1-8 include sequences derived from P69 Control B (SEQ JD NO:9); sequences derived from human PrP (135-155) (SEQ JD NO:6); and a His tag.
  • FIGs. 6A - 6D depict exemplary prion chimeras including "P69 Control B" non-prion amino acid sequences and mouse PrP prion amino acid sequences (135-155).
  • FIG. 6A depicts sequences of prion chimeras designated P69B/mouse Chimera No. 1 (SEQ JD NO: 38) and P69B/mouse Chimera No. 2 (SEQ JD NO:39).
  • FIG. 6B depicts sequences of prion chimeras designated P69B/mouse Chimera No. 3 (SEQ ID NO:40) and P69B/mouse Chimera No. 4 (SEQ JD NO:41).
  • FIG. 6C depicts sequences of prion chimeras designated
  • FIG. 6D depicts sequences of prion chimeras designated P69B/mouse Chimera No. 7 (SEQ JD NO:44) and P69B/mouse Chimera No. 8 (SEQ JD NO:45).
  • Chimeras designated P69B/mouse (135-155) Chimera No. 1-8 include sequences derived from P69 Control B (SEQ JD NO:9); sequences derived from mouse PrP (135-155) (SEQ ID NO:7); and a His tag.
  • FIG. 7 depicts an exemplary non-prion amino acid sequences derived from left- handed non-prion ⁇ -helical protein Methanosarcina thermophila ⁇ carbonic anhydrase ("GCA").
  • GCA left- handed non-prion ⁇ -helical protein Methanosarcina thermophila ⁇ carbonic anhydrase
  • SEQ ID NO:4 depicts the full length sequence of GCA
  • SEQ ID NO: 183 depicts full length GCA with the leader sequence removed
  • SEQ JD NO: 10 depicts a fragment of GCA designated "GCA Control A (GCAA);”
  • SEQ JD NO: 11 depicts a fragment of GCA designated as "GCA Control B (GCAB);
  • SEQ JD NO: 184 depicts a fragment of GCA with the leader sequence removed designated "GCABnoL.”
  • FIG. 8A - 8B depict exemplary prion chimeras including "GCA Contol A" non- prion sequences and human PrP 135-155 sequences.
  • FIG. 8A depicts sequences of prion chimeras designated GCAA human Chimera No. 1 (SEQ ID NO:46); GCAA human Chimera No. 2 (SEQ JD NO:47); and GCAA/human Chimera No. 3 (SEQ ID NO:48).
  • FIG. 8B depicts sequences of prion chimeras designated GCAA/human Chimera No. 4 (SEQ JD
  • FIG. 9A - 9B depict exemplary prion chimeras including "GCA Contol A" non- prion sequences and mouse PrP 135-155 sequences.
  • FIG. 9A depicts sequences of prion chimeras designated GCAA/mouse Chimera No.
  • FIG. 9B depicts sequences of prion chimeras designated GCAA/mouse Chimera No. 4 (SEQ ID NO:55); GCAA/mouse Chimera No. 5 (SEQ ID NO:56); and GCAA/mouse Chimera No. 6 (SEQ JD NO:57).
  • FIGs. 10A - 10B depict exemplary prion chimeras including "GCA Control B" non- prion sequences and human PrP 135-155 sequences.
  • FIG. 10A depicts sequences of prion chimeras designated GCAB/human Chimera No. 1 (SEQ JD NO:58); GCAB/human Chimera No. 2 (SEQ ID NO:59); and GCAB/human Chimera No. 3 (SEQ ID NO:60).
  • FIG. 10A depicts sequences of prion chimeras designated GCAB/human Chimera No. 1 (SEQ JD NO:58); GCAB/human Chimera No. 2 (SEQ ID NO:59); and GCAB/human Chimera No. 3 (SEQ ID NO:60).
  • FIG. 10B depicts sequences of prion chimeras designated GCAB/human Chimera No. 4 (SEQ ID NO:61); GCAB/human Chimera No. 5 (SEQ JD NO:62); and GCAB/human Chimera No. 6 (SEQ ID NO:63).
  • Chimeras designated GCAB/human (135-155) Chimera No. 1-6 include sequences derived from GCAB (SEQ JD NO: 11); sequences derived from human PrP (135- 155) (SEQ ID NO:6); and a His tag.
  • FIGs. 11A - 11B depict exemplary prion chimeras including "GCA Control B" non- prion sequences and mouse PrP 135-155 sequences.
  • FIG. 11A depicts sequences of prion chimeras designated GCAB/mouse Chimera No. 1 (SEQ ID NO:64); GCAB/mouse Chimera No. 2 (SEQ ID NO:65); and GCAB/mouse Chimera No. 3 (SEQ ID NO:66).
  • FIG. 11B depicts sequences of prion chimeras designated GCAB/mouse Chimera No. 4 (SEQ ID NO:67); GCAB/mouse Chimera No. 5 (SEQ JD NO:68); and GCAB/mouse Chimera No. 6 (SEQ ID NO:69).
  • FIGs. 12A - 12D depict exemplary prion chimeras including "P69 control A" non- prion amino acid sequences and human PrP prion amino acid sequences (126-154).
  • FIG. 12A depicts sequences of prion chimeras designated P69A/human Chimera No. 1 (SEQ JD
  • FIG. 12B depicts sequences of prion chimeras designated P69A/human Chimera No. 3 (SEQ JD NO:72) and P69A human Chimera No. 4 (SEQ ID NO:73).
  • FIG. 12C depicts sequences of prion chimeras designated P69A/human Chimera No. 5 (SEQ JD NO:74) and P69 A/human Chimera No. 6 (SEQ JD NO:75).
  • FIG. 12D depicts sequences of prion chimeras designated P69A/human Chimera No. 7 (SEQ ID NO:76) and P69A/human Chimera No.
  • FIG. 13 A - 13D depict exemplary prion chimeras including "P69 Control A" non- prion amino acid sequences and mouse PrP prion amino acid sequences (126-154).
  • FIG. 13A depicts sequences of prion chimeras designated P69 A/mouse Chimera No. 1 (SEQ ID NO:78) and P69 A/mouse Chimera No. 2 (SEQ ID NO:79).
  • FIG. 13B depicts sequences of prion chimeras designated P69A/mouse Chimera No. 3 (SEQ JD NO: 80) and P69 A/mouse Chimera No. 4 (SEQ JD NO:81).
  • FIG. 13C depicts sequences of prion chimeras designated P69A/mouse Chimera No. 5 (SEQ ID NO:82) and P69 A/mouse Chimera No. 6 (SEQ JD NO:83).
  • FIG. 13D depicts sequences of prion chimeras designated P69A/mouse Chimera No. 7 (SEQ ID NO: 84) and P69A/mouse Chimera No. 8 (SEQ ID NO: 85).
  • Chimeras designated P69A mouse 126-154 Chimera No. 1-8 include sequences derived from P69 Control A (SEQ ID NO:8); sequences derived from mouse PrP (126-154) (SEQ ID NO:13); and a His tag.
  • FIG. 14A depicts sequences of prion chimeras designated P69B/human Chimera No. 1 (SEQ ID NO: 86) and P69B/human Chimera No. 2 (SEQ JD NO: 87).
  • FIG. 14B depicts sequences of prion chimeras designated P69B/human Chimera No. 3 (SEQ ID NO: 88) and P69B/human Chimera No. 4 (SEQ JD NO:89).
  • FIG. 14C depicts sequences of prion chimeras designated P69B/human Chimera No. 5 (SEQ ID NO:90) and P69B/human Chimera No. 6 (SEQ ID NO:91).
  • FIG. 14D depicts sequences of prion chimeras designated P69B/human Chimera No. 7 (SEQ ID NO:92) and P69B/human Chimera No. 8 (SEQ ID NO:93). Chimeras designated P69B/human 126-154 Chimera No.
  • FIGs. 15A - 15D depict exemplary prion chimeras including "P69 Control B" non- prion amino acid sequences and mouse PrP prion amino acid sequences (126-154).
  • FIG. 15A depicts sequences of prion chimeras designated P69B/mouse Chimera No. 1 (SEQ JD NO:94) and P69B/mouse Chimera No. 2 (SEQ ID NO:95).
  • FIG. 15B depicts sequences of prion chimeras designated P69B/mouse Chimera No. 3 (SEQ ID NO:96) and P69B/mouse Chimera No. 4 (SEQ ID NO:97).
  • FIG. 15C depicts sequences of prion chimeras designated
  • FIG. 15D depicts sequences of prion chimeras designated P69B/mouse Chimera No. 7 (SEQ ID NO: 100) and P69B/mouse Chimera No. 8 (SEQ ID NO: 101).
  • Chimeras designated P69B/mouse 126-154 Chimera No. 1-8 include sequences derived from P69 Control B (SEQ ID NO:9); sequences derived from mouse PrP (126-154) (SEQ JD NO: 13); and a His tag.
  • FIG. 16A - 16B depict exemplary prion chimeras including "GCA Control A" non- prion sequences and human PrP 126-154 sequences.
  • FIG. 16 A depicts sequences of prion chimeras designated GCAA/human Chimera No. 1 (SEQ ID NO: 102); GCAA/human Chimera No. 2 (SEQ ID NO: 103); and GCAA/human Chimera No. 3 (SEQ JD NO: 104).
  • FIG. 16B depicts sequences of prion chimeras designated GCAA/human Chimera No. 4 (SEQ ID NO: 105); GCAA/human Chimera No. 5 (SEQ ID NO: 106); and GCAA/human Chimera No.
  • FIG. 17A - 17B depict exemplary prion chimeras including "GCA Control A" non- prion sequences and mouse PrP 126-154 sequences.
  • FIG. 17A depicts sequences of prion chimeras designated GCAA/mouse Chimera No. 1 (SEQ JD NO: 108); GCAA/mouse Chimera No.
  • FIG. 17B depicts sequences of prion chimeras designated GCAA/mouse Chimera No. 4 (SEQ JD NO: 111); GCAA/mouse Chimera No. 5 (SEQ JD NO: 112); and GCAA/mouse Chimera No. 6 (SEQ ID NO: 113).
  • Chimeras designated GCAA/mouse 126-154 Chimera No. 1-6 include sequences derived from GCAA (SEQ JD NO: 10); sequences derived from mouse PrP (126-154) (SEQ ID NO: 8); and a His tag.
  • FIG. 18A - 18B depict exemplary prion chimeras including "GCA Control B" non- prion sequences and human PrP 126-154 sequences.
  • FIG. 18A depicts sequences of prion chimeras designated GCAB/human Chimera No. 1 (SEQ ID NO: 114); GCAB/human Chimera No. 2 (SEQ ID NO: 115); and GCAB/human Chimera No. 3 (SEQ JD NO: 116).
  • FIG. 18B depicts sequences of prion chimeras designated GCAB/human Chimera No. 4 (SEQ JD NO: 117); GCAB/human Chimera No. 5 (SEQ JD NO: 118); and GCAB/human Chimera No.
  • Chimeras designated GCAB/human (126-154) Chimera No. 1-6 include sequences derived from GCAB (SEQ ID NO: 11); sequences derived from human PrP (126-154) (SEQ ID NO: 12); and a His tag.
  • FIG. 19 A depicts sequences of prion chimeras designated GCAB/mouse Chimera No. 1 (SEQ JD NO: 120); GCAB/mouse Chimera No.
  • FIG. 19B depicts sequences of prion chimeras designated GCAB/mouse Chimera No. 4 (SEQ JD NO: 123); GCAB/mouse Chimera No. 5 (SEQ JD NO: 124); and GCAB/mouse Chimera No. 6 (SEQ JD NO: 125).
  • Chimeras designated GCAB/mouse (126-154) Chimera No. 1-6 include sequences derived from GCAB (SEQ ID NO: 11); sequences derived from mouse PrP (126-154) (SEQ ID NO: 13); and a His tag.
  • FIGs. 19B depicts sequences of prion chimeras designated GCAB/mouse Chimera No. 4 (SEQ JD NO: 123); GCAB/mouse Chimera No. 5 (SEQ JD NO: 124); and GCAB/mouse Chimera No. 6 (SEQ JD NO: 125).
  • FIG. 20A depicts the DNA construct designated Tpa.p69.mprpfl (SEQ ID NO: 155) and the prion chimera encoded by Tpa.p69.mprpfl (SEQ ID NO: 126).
  • FIG. 20B depicts the DNA construct designated T ⁇ a.p69.mprpf2 (SEQ ID NO: 156) and the prion chimera encoded by Tpa.p69.mprpf2 (SEQ ID NO: 127).
  • FIG. 20A depicts the DNA construct designated Tpa.p69.mprpfl (SEQ ID NO: 155) and the prion chimera encoded by Tpa.p69.mprpfl (SEQ ID NO: 126).
  • FIG. 20B depicts the DNA construct designated T ⁇ a.p69.mprpf2 (SEQ ID NO: 156) and the prion chimera encoded by Tpa.p69.mprpf2 (SEQ ID NO: 127).
  • FIG. 20C depicts the DNA construct designated Tpa.p69.mprpf3 (SEQ ID NO: 157) and the prion chimera encoded by Tpa.p69.mprpf3 (SEQ ID NO: 128).
  • FIG. 20D depicts the DNA construct designated Tpa.p69.mprpf4 (SEQ JD NO: 158) and the prion chimera encoded by Tpa.p69.mprpf4 (SEQ ID NO: 129).
  • FIG. 20E depicts the DNA construct designated Tpa.p69.mprpf5 (SEQ ID NO: 159) and the prion chimera encoded by Tpa.p69.mprpf5 (SEQ ID NO: 130).
  • FIG. 20F depicts the DNA construct designated and Tpa.p69.mprpf6 (SEQ ID NO: 160) and the prion chimera encoded by Tpa.p69.mprpf6 (SEQ ID NO: 131).
  • FIG. 20G depicts the DNA construct designated Tpa.p69.mprpf7 (SEQ ID NO: 161) and the prion chimera encoded by Tpa.p69.mprpf7 (SEQ JD NO: 132).
  • FIG. 20H depicts the DNA construct designated Tpa.p69.mprpf8 (SEQ ID NO: 162) and the prion chimera encoded by Tpa.p69.mprpf8 (SEQ ID NO: 133).
  • the constructs designated t ⁇ a.p69.mprpfl through tpa.p69.mprpf8 include sequences encoding a tpa leader peptide sequence (SEQ ID NO: 154); P69 non-prion sequences and prion sequences (135-155, SEQ ID NO:7).
  • the prion sequences are 3 ' to the P69-encoding sequences in the construct and C-terminal to the P69 sequences in the chimera.
  • FIGs. 21 A - 21H depict exemplary DNA constructs and prion chimeras encoded by these constructs, including a tpa leader sequence and mouse prion sequences. Human prion sequences may be used in these molecules.
  • FIG. 21A depicts the DNA construct designated Tpa.m ⁇ rpf 1.69 (SEQ ID NO: 163) and the chimera encoded by Tpa.mprpf 1.69 (SEQ JD
  • FIG. 21B depicts the DNA construct designated T ⁇ a.mprpf2.69 (SEQ JD NO:164) and the prion chimera encoded by Tpa.mprpf2.69 (SEQ JD NO.T35).
  • FIG. 21C depicts the DNA construct designated Tpa.mprpf3.69 (SEQ ID NO:165) and the prion chimera encoded by Tpa.mprpf3.69 (SEQ JD NO: 136).
  • FIG. 21D depicts the DNA construct designated T ⁇ a.mprpf4.69 (SEQ ID NO: 166) and the prion chimera encoded by Tpa.mprpf4.69 (SEQ ID NO: 137).
  • FIG. 21E depicts the DNA construct designated Tpa.mpr ⁇ f5.69 (SEQ JD NO.T67) and the prion chimera encoded by Tpa.mprpf5.69 (SEQ ID NO: 138).
  • FIG. 21F depicts the DNA construct designated Tpa.mprpf6.69 (SEQ ID NO.T68) and the prion chimera encoded by and T ⁇ a.mpr ⁇ f6.69 (SEQ JD NO: 139).
  • FIG. 21G depicts the DNA construct designated T ⁇ a.mprpf7.69 (SEQ ID NO: 169) and the prion chimera encoded by Tpa.mprpf7.69 (SEQ JD NO: 140).
  • 21H depicts the DNA construct designated Tpa.mprpf 8.69 (SEQ ID NO: 170) and the prion chimera encoded by and Tpa.mprpf869 (SEQ JD NO: 141).
  • the constructs designated tpa.mprpf 1.69 through tpa.mprpf 8.69 include sequences encoding a tpa leader peptide sequence (SEQ ID NO: 154); P69 non-prion sequences and prion sequences (135-155, SEQ ID NO:7).
  • the prion sequences are 5' to the P69-encoding sequences in the construct and N-terminal to the P69 sequences in the chimera.
  • FIG. 22 A depicts DNA constructs designated T ⁇ a.mprpf 1.gca (SEQ ID NO: 171); the prion chimera encoded by Tpa.mprpf 1.gca (SEQ JD NO: 142); Tpa.mprpf2.gca (SEQ ID NO: 172); and the prion chimera encoded by T ⁇ a.mprpf2.gca (SEQ ID NO: 143).
  • FIG. 22 A depicts DNA constructs designated T ⁇ a.mprpf 1.gca (SEQ ID NO: 171); the prion chimera encoded by Tpa.mprpf 1.gca (SEQ JD NO: 142); Tpa.mprpf2.gca (SEQ ID NO: 172); and the prion chimera encoded by T ⁇ a.mprpf2.gca (SEQ ID NO: 143).
  • FIG. 22B depicts DNA constructs designated Tpa.mprpf3.gca (SEQ ID NO: 173); the prion chimera encoded by Tpa.mprpf3.gca (SEQ ID NO:144); Tpa.mprpf4.gca (SEQ ID NO: 174); and the prion chimera encoded by Tpa.mprpf4.gca (SEQ ID NO: 145).
  • Tpa.mprpf5.gca SEQ ID NO:175); the prion chimera encoded by Tpa.mprpf5.gca (SEQ ID NO: 146); Tpa.mprpf6.gca (SEQ JD NO: 176); and the prion chimera encoded by Tpa.mprpf6.gca (SEQ JD NO: 147).
  • the constructs designated Tpa.mprpf 1. gca through Tpa.mprpf6.gca include sequences encoding a tpa leader peptide sequence (SEQ ID NO:175); the prion chimera encoded by Tpa.mprpf5.gca (SEQ ID NO: 146); Tpa.mprpf6.gca (SEQ JD NO: 176); and the prion chimera encoded by Tpa.mprpf6.gca (SEQ JD NO: 147).
  • FIGS. 23 A - 23C depict exemplary DNA constructs and prion chimeras encoded by these constructs, including a tpa leader sequence and mouse prion sequences. Human prion sequences may be used in these molecules.
  • FIG. 23A depicts exemplary DNA constructs and prion chimeras encoded by these constructs, including a tpa leader sequence and mouse prion sequences. Human prion sequences may be used in these molecules.
  • FIG.23A depicts the DNA construct designated Tpa.gca.mprpfl (SEQ ID NO: 177); the chimera encoded by Tpa.gca.m ⁇ rpfl (SEQ JD NO: 148); the DNA construct designated T ⁇ a.gca.mprpf2 (SEQ ID NO: 178); and the chimera encoded by Tpa.gca.mprpf2 (SEQ JD NO: 149).
  • FIG.23B depicts the DNA construct designated T ⁇ a.gac.mprpf3 (SEQ ID NO: 179); the chimera encoded by Tpa.gca.mprpf3
  • FIG. 23C depicts the DNA construct designated Tpa.gca.mprp5 (SEQ JD NO:181); the chimera encoded by Tpa.gca.mprpf5 (SEQ JD NO: 152); the DNA construct designated Tpa.gca.mprp ⁇ (SEQ ID NO:182); and the chimera encoded by Tpa.gca.mprpf6 (SEQ ID NO:153).
  • the constructs designated Tpa.gca.mprpfl through Tpa.gca.mprpf ⁇ include sequences encoding a tpa leader peptide sequence (SEQ ID NO: 154); GCA non-prion sequences and mouse prion sequences (135-155, SEQ ID NO:7).
  • the prion sequences are 3' to the GCA-encoding sequences in the constructs and C-terminal to the GCA non-prion sequences in the chimeras.
  • FIG. 24 depicts an exemplary tpa leader amino acid sequence of 25 amino acids in length (SEQ ID NO: 154) and an exemplarly GCA leader sequence M M F N K (SEQ ID NO:209) that was removed from GCA full length (SEQ ID NO:4) and GCA Control B (SEQ ID NO: 11) to make GCA full length no leader (SEQ ID NO: 183) and GCA Control B No Leader (SEQ JD NO: 184), respectively.
  • FIGs. 25A - 25B depict exemplary prion chimeras including "GCA Control B no leader” non-prion sequences (SEQ JD NO:184) and human PrP 135-155 sequences.
  • FIG. 25A depicts sequences of prion chimeras designated GCABnoL/human Chimera No. 1 (SEQ JD NO: 185); GCABnoL/human Chimera No. 2 (SEQ ID NO: 186); and GCABnoL/human Chimera No. 3 (SEQ ID NO:187).
  • FIG. 25B depicts sequences of prion chimeras designated GCABnoL/human Chimera No. 4 (SEQ JD NO: 188); GCABnoL/human Chimera No. 5 (SEQ 3D NO: 189); and GCABnoL/human Chimera No. 6 (SEQ JD NO: 190).
  • Chimeras designated GCABnoL/human (135-155) Chimera No. 1-6 include sequences derived from GCABnoL (SEQ ID NO: 184); sequences derived from human PrP (135-155) (SEQ JD NO:6); and a His tag.
  • FIG. 26A depicts sequences of prion chimeras designated GCABnoL/mouse Chimera No. 1 (SEQ JD NO: 191); GCABnoL/mouse Chimera No. 2 (SEQ JD NO: 192); and GCABnoL/mouse Chimera No. 3 (SEQ JD NO: 193).
  • GCABnoL/mouse Chimera No. 4 SEQ ID NO: 194
  • GCABnoL/mouse Chimera No. 5 SEQ JD NO: 195
  • GCABnoL/mouse Chimera No. 6 SEQ ID NO: 196
  • Chimera No. 1-6 include sequences derived from GCABnoL (SEQ JD NO:184); sequences derived from mouse PrP (135-155) (SEQ JD NO:7); and a His tag.
  • FIG. 27A - 27B depict exemplary prion chimeras including "GCA Control B no leader" non-prion sequences and human PrP 126-154 sequences.
  • FIG. 27 A depicts sequences of prion chimeras designated GCABnoL/human Chimera No. 1 (SEQ ID NO: 197); GCABnoL/human Chimera No. 2 (SEQ ID NO: 198); and GCABnoL/human Chimera No. 3 (SEQ JD NO: 199).
  • FIG. 27B depicts sequences of prion chimeras designated
  • GCABnoL/human Chimera No. 4 SEQ ID NO:200
  • GCABnoL/human Chimera No. 5 SEQ ID NO:201
  • GCABnoL/human Chimera No. 6 SEQ JD NO:202
  • Chimeras designated GCABnoL/human (126-154) Chimera No. 1-6 include sequences derived from GCABnoL (SEQ JD NO:184); sequences derived from human PrP (126-154) (SEQ JD NO: 12); and a His tag.
  • FIGs. 28A - 28B depict exemplary prion chimeras including "GCA Control B no leader" non-prion sequences and mouse PrP 126-154 sequences.
  • FIG. 28A depicts sequences of prion chimeras designated GCABnoL/mouse Chimera No. 1 (SEQ JD NO:203); GCABnoL/mouse Chimera No. 2 (SEQ ID NO:204); and GCABnoL/mouse Chimera No. 3 (SEQ JD NO:205).
  • FIG. 28B depicts sequences of prion chimeras designated GCABnoL/mouse Chimera No. 1 (SEQ JD NO:203); GCABnoL/mouse Chimera No. 2 (SEQ ID NO:204); and GCABnoL/mouse Chimera No. 3 (SEQ JD NO:205).
  • FIG. 28B depicts sequences of prion chimeras designated GCABnoL/mouse Chimera No. 1 (SEQ JD NO:203); GCABnoL/mouse Chimera No. 2 (SEQ ID NO:204);
  • Chimeras designated GCABnoL/mouse (126-154) Chimera No. 1-6 include sequences derived from GCABnoL (SEQ 3D NO:184); sequences derived from mouse PrP (126-154) (SEQ JD NO:13); and a His tag.
  • SEQ JD NO. 1 represents a full-length amino acid sequence of the human prion protein.
  • SEQ ID NO. 2 represents a full-length amino acid sequence of the mouse prion protein.
  • SEQ JD NO. 3 represents an amino acid sequence of P69.
  • SEQ ID NO. 4 represents an amino acid sequence of GCA.
  • SEQ JD NO. 5 is the following amino acid sequence that represents a histidine tag: G GHHHHHH.
  • SEQ JD NO. 6 represents an amino acid sequence of a human PrP fragment (135 - 155).
  • SEQ JD NO. 7 represents an amino acid sequence of a mouse PrP fragment (135 - 155).
  • SEQ ID NO. 8 represents an amino acid sequence of P69 Control A.
  • SEQ 3D NO. 9 represents an amino acid sequence of P69 Control B.
  • SEQ JD NO. 10 represents an amino acid sequence of GCA Control A.
  • SEQ 3D NO. 11 represents an amino acid sequence of GCA Control B.
  • SEQ 3D NO. 12 represents an amino acid sequence of a human PrP fragment (126 - 154).
  • SEQ 3D NO. 13 represents an amino acid sequence of a mouse PrP fragment (126 - 154).
  • SEQ 3D NOS. 14 - 21 represent examples of prion chimeras comprising human PrP (135 - 155) - P69 Control A - His Tag.
  • SEQ 3D NOS. 30 - 37 represent examples of prion chimeras comprising a His Tag -
  • SEQ ID NOS. 38 - 45 represent examples of prion chimeras comprising a His Tag - P69 Control B - Mo PrP (135 - 155).
  • SEQ 3D NOS. 46 - 51 represent examples of prion chimeras comprising Hu PrP (135
  • SEQ 3D NOS. 52 - 57 represent examples of prion chimeras comprising Mo PrP (135
  • SEQ JD NOS. 58 - 63 represent examples of prion chimeras comprising His Tag -
  • SEQ JD NOS. 64 - 69 represent examples of prion chimeras comprising His Tag - GCA Control B - Mo PrP (135 - 155).
  • SEQ ID NOS. 70 - 77 represent examples of prion chimeras comprising human PrP (126 - 154) - P69 Control A - His Tag.
  • SEQ ID NOS. 78 - 85 represent examples of prion chimeras comprising mouse PrP (126 - 154) - P69 Control A - His Tag.
  • SEQ ID NOS. 94 - 101 represent examples of prion chimeras comprising a His Tag -
  • SEQ 3D NOS. 102 - 107 represent examples of prion chimeras comprising Hu PrP (126 - 154) - GCA Control A - His Tag.
  • SEQ 3D NOS. 108 - 113 represent examples of prion chimeras comprising Mo PrP (126 - 154) - GCA Control A - His Tag.
  • SEQ 3D NOS. 114 - 119 represent examples of prion chimeras comprising His Tag - GCA Control B - Hu PrP (126 - 154).
  • SEQ ID NOS. 126 - 133 represent examples of prion chimeras encoded by DNA constructs where the chimeras include a tpa signal sequence and P69 non-prion sequences.
  • SEQ ID NOS. 134 - 141 represent examples of prion chimeras encoded by DNA constructs where the chimeras include a tpa signal sequence, prion sequences and P69 non- prion sequences.
  • SEQ ID NOS. 148 - 153 represent examples of prion chimeras encoded by DNA constructs where the chimeras include a tpa signal sequence, prion sequences and GCA non- prion sequences.
  • SEQ 3D NO: 154 is an exemplary tpa leader sequence.
  • SEQ 3D NOs: 155 - 170 represent examples of DNA constructs encoding prion chimera polypeptides that include a tpa signal sequence, prion sequences and P69 non-prion sequences.
  • SEQ ID NOs: 171 - 182 represent examples of DNA constructs encoding prion chimera polypeptides that include a tpa signal sequence, prion sequences and GCA non-prion sequences.
  • SEQ 3D NO: 183 represents an amino acid sequence of GCA without the leader sequence.
  • SEQ 3D NO: 184 represents an amino acid sequence of GCA Control B without the leader sequence.
  • SEQ 3D NOS. 185 - 190 represent examples of prion chimeras comprising His Tag -
  • SEQ 3D NOS. 191 - 196 represent examples of prion chimeras comprising His Tag - GCA Control B no leader - Mo PrP (135 - 155).
  • SEQ 3D NOS. 197 - 202 represent examples of prion chimeras comprising His Tag - GCA Control B no leader - Hu PrP (126 - 154).
  • SEQ ID NOS. 203 - 208 represent examples of prion chimeras comprising His Tag - GCA Control B no leader - Mo PrP (126 - 154).
  • SEQ ID NO. 209 is the following amino acid sequence that represents a GCA leader sequence: M M F N K.
  • the invention relates to prion chimeras, polynucleotides encoding prion chimeras, methods of generating antibodies using prion chimeras and polynucleotides encoding prion chimeras, as well as to antibodies specific for prion chimeras and polynucleotides encoding these antibodies.
  • the invention further relates to methods of using such antibodies, chimeras and/or polynucleotides to detect the presence of pathogenic prions, for example in a biological sample.
  • the invention further relates to methods of using such antibodies, chimeras and/or polynucleotides as a component in a therapeutic or prophylactic vaccine.
  • the prion chimeras (and polynucleotides encoding these chimeras) used in the invention typically comprise a polypeptide comprising a first segment and a second segment.
  • the first segment comprises a prion polypeptide (or fragment thereof) and the second segment comprises a non-prion protein (or fragment thereof).
  • the prion polypeptide comprises an amino acid sequence of a prion protein that can adapt the ⁇ -helical conformation of a pathogenic prion protein.
  • the prion component of the chimera adapts a ⁇ -helix conformation within the chimera.
  • the non-prion protein typically comprises at least a fragment of a non-prion ⁇ -helical protein, for example pertactin or GCA.
  • the non-prion protein used in the chimera generally does not contain a portion of the amino acids of the non-prion protein that adapt the ⁇ -helix conformation.
  • the practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Methods In Enzymology (S. Colowick and N.
  • PrP protein and PrP
  • Prions are naturally produced in a wide variety of mammalian species, including human, sheep, cattle, and mice.
  • a representative amino acid sequence of a human prion protein is set forth as SEQ ID NO:l.
  • a representative amino acid sequence of a mouse prion protein is set forth as SEQ ID. NO:2.
  • the pathogenic conformation of the prion protein typically includes at least one region that can adapt a ⁇ -helical conformation (referred to as a " ⁇ -helical region").
  • a ⁇ -helical region For example, in Figure 1A (SEQ 3D NO:l), helical regions occur at approximately amino acid residues 144 to 153 and at approximately residues 173 to 194, numbered relative to the human PrP shown in SEQ 3D NO: 1.
  • Beta-strand regions occur at approximately amino acid 129 to 133 and at approximately amino acid residues 160 to 163, numbered relative to the human PrP shown in SEQ ID NO: 1.
  • Pathogenic forms may or may not be infectious.
  • pathogenic may mean that the protein actually causes the disease or it may simply mean that the protein is associated with the disease and therefore is present when the disease is present.
  • a pathogenic protein as used in connection with this disclosure is not necessarily a protein that is the specific causative agent of a disease.
  • a "prion protein” or “conformational disease protein” as defined herein is not limited to a polypeptide having the exact sequence to those described herein. It is readily apparent that the terms encompass conformational disease proteins (e.g., prion proteins) from any of the identified or unidentified species or disease. Descriptions of structural features are given herein with reference to a human and/or mouse sequence, presented in the Figures.
  • PrP gene is used herein to describe any genetic material that expresses proteins including known polymorphisms and pathogenic mutations.
  • PrP gene refers generally to any gene of any species that encodes any form of a PrP protein.
  • the PrP gene can be from any animal, including the "host” and “test” animals described herein and any and all polymorphisms and mutations thereof, it being recognized that the terms include other such PrP genes that are yet to be discovered.
  • the protein expressed by such a gene can assume either a PrP c (non-disease) or PrP sc (disease) form.
  • Gyometry or "tertiary structure" of a polypeptide or protein is meant the overall 3-D configuration of the protein.
  • ⁇ -helix refers to a protein conformation comprising a helical structure that contains highly ordered, stacked side chains on both the inside and outside of the helix. This side-chain stacking adds to the rigid geometry of the ⁇ -helices, which tend to have planar sides and essentially no interstrand twist, ⁇ -helices include both left- and right- handed structures. The number of residues per ⁇ -helical turn for left-handed helices averages about 18 residues per helical turn.
  • the number of residues per ⁇ -helical turn for right-handed helices can vary substantially, but averages about 24 residues per helical turn. See Wille, et al., "Structural Studies of the Scrapie Prion Protein by Electron Crystallography", Proc. Natl. Acad. Sci. USA, 99 (6): 3563-3568 (2002) and T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); and A.L. Lehninger, Biochemistry (Worth Publishers, Inc., 1975).
  • Primary-related disease refers to a disease caused in whole or in part by a pathogenic prion particle (PrP Sc ).
  • Prion-related diseases include scrapie, bovine spongiform encephalopathies (BSE), mad cow disease, feline spongiform encephalopathies, kuru, Creutzfeldt- Jakob Disease (CJD), Gerstmann-Strassler-Scheinker Disease (GSS), and fatal familial insomnia (FFI).
  • BSE bovine spongiform encephalopathies
  • CJD Creutzfeldt- Jakob Disease
  • GSS Gerstmann-Strassler-Scheinker Disease
  • FFI fatal familial insomnia
  • Minimum fragments of polypeptides useful in the invention can be at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25 or even longer amino acids.
  • the polypeptide comprises a ⁇ -helical region of a prion protein
  • the polypeptide preferably comprises enough amino acids to adapt a ⁇ -helical conformation.
  • polypeptides useful in this invention can have a maximum length suitable for the intended application. Generally, the maximum length is not critical and can easily be selected by one skilled in the art. Reference to polypeptides and the like also includes derivatives of the amino acid sequences of the invention.
  • a first polypeptide is "derived from" a second polypeptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide, or (ii) displays sequence identity to the second polypeptides as described herein.
  • Such derivatives can include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like.
  • Amino acid derivatives can also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the proteins or errors due to PCR amplification.
  • a "fragment” as used herein refers to a polypeptide consisting of only a part of the intact full-length polypeptide sequence and structure as found in nature. For instance, a fragment can include a C-terminal deletion and/or an N-termmal deletion of a protein. Typically, the fragment retains one, some or all of the functions of the full-length polypeptide sequence from which it is derived.
  • a “recombinant” protein is a protein that has been prepared by recombinant DNA techniques as described herein.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below.
  • the host organism expressed the foreign gene to produce the protein under expression conditions.
  • the phrase "prion chimera” as used herein generally refers to a polypeptide comprising at least two different segments (e.g., amino acid sequences obtained from different sources/proteins).
  • At least one segment comprises a prion polypeptide (or fragment thereof) or a sequence derived from a prion, while the other segment comprises a non-prion polypeptide or a sequence derived from a non-prion polypeptide (e.g., a sequence obtained from a ⁇ -helical protein).
  • Prion chimeras may be the form of a fusion protein and may include additional elements, for example a tagging sequence (e.g., a His tagging sequence that may serve to facilitate purification and increase solubility of the chimera).
  • the different segments may be co-linear (N- and/or C-terminal to each other) or, alternatively, one segment may be inserted into another segment so that it is flanked by the sequences of the other segment.
  • a “non-prion protein” as used herein refers to any protein that is not a prion polypeptide.
  • a “non-prion ⁇ -helical protein” as used herein refers to a non-prion protein that contains an amino acid sequence that can adapt a ⁇ -helical conformation. Such ⁇ -helical proteins include proteins that contain right-handed and/or left-handed ⁇ -helices.
  • Non- limiting examples of right handed beta-helix proteins include pectate lyase, pectin lyase, galacturonase, chondroitinase B, pectin methylesterase, P22 tailspike protein, virulence factor P69 pertactin, insect cysteine-rich antifreeze protein, cell-division inhibitor MinC, alpha subunit of glutamate synthase (e.g., C-terminal domain) and C-terminal domain of adenylylcyclase associated protein.
  • Non-limiting examples of left handed beta-helix proteins include UDP N-acetylglucosamine acyltransferase, galactoside acetyltransferase, xenobiotic acetyltransferase, tetrahydrodipicolinate-N-succinlytransf erase, THDP-succinlytransferase, DapD N-acetylglucosamine 1-phosphate uridyltransferase GlmU, and C-terminal domain of carbonic anhydrase.
  • the non-prion protein comprises sequence derived from ⁇ carbonic anhydrase (left handed beta-helical protein) while in other embodiments, the non-prion polypeptide comprises sequence derived from pertactin (right handed beta-helical protein).
  • polynucleotide generally refers to a nucleic acid molecule.
  • a "polynucleotide” can include both double- and single-stranded sequences and refers to, but is not limited to, cDNA from viral, prokaryotic or eukaryotic MRNA, genomic RNA and DNA sequences from viral (e.g. RNA and DNA viruses and retroviruses) or prokaryotic DNA, and especially synthetic DNA sequences.
  • the term also captures sequences that include any of the known base analogs of DNA and RNA, and includes modifications such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the nucleic acid molecule encodes a therapeutic or antigenic protein. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts that produce the antigens. Modifications of polynucleotides may have any number of effects including, for example, facilitating expression of the polypeptide product in a host cell.
  • a polynucleotide can encode a biologically active (e.g., immunogenic or therapeutic) protein or polypeptide.
  • a polynucleotide can include as little as 10 nucleotides, e.g., where the polynucleotide encodes an antigen.
  • the polynucleotide encodes a prion or fragment thereof
  • the polynucleotide is long enough to encode for a ⁇ -helical region of the prion.
  • the polynucleotide includes at least 18, 19, 20, 21, 22, 23, 24, 25, 30 or even more amino acids.
  • a "polynucleotide coding sequence” or a sequence that "encodes” a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • Exemplary coding sequences are the modified viral polypeptide-coding sequences of the present invention.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • control elements include, but are not limited to, transcription regulators, such as promoters, transcription enhancer elements, transcription termination signals, and polyadenylation sequences; and translation regulators, such as sequences for optimization of initiation of translation, e.g., Shine-Dalgarno (ribosome binding site) sequences, Kozak sequences (i.e., sequences for the optimization of translation, located, for example, 5' to the coding sequence), leader sequences (heterologous or native), translation initiation codon (e.g., ATG), and translation termination sequences.
  • transcription regulators such as promoters, transcription enhancer elements, transcription termination signals, and polyadenylation sequences
  • translation regulators such as sequences for optimization of initiation of translation, e.g., Shine-Dalgarno (ribosome binding site) sequences, Kozak sequences (i.e., sequences for the optimization of translation, located, for example, 5' to the coding sequence), leader sequences (heterologous or
  • one or more translation regulation or initiation sequences are derived from wild-type translation initiation sequences, i.e., sequences that regulate translation of the coding region in their native state. Wild-type leader sequences that have been modified, using the methods described herein, also find use in the present invention. Native or modified leader sequences can be from any source, for example other sources (e.g., wild-type or modified tpa leader sequence exemplified herein and/or leader sequences derived from prion-encoding polynucleotides).
  • Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters.
  • a "nucleic acid” molecule can include, but is not limited to, prokaryotic sequences, eukaryotic mRNA, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences.
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • a "recombinant" nucleic acid molecule as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation: (1) is not associated with all or a portion of the polynucleotide with which it is associated in nature; and/or (2) is linked to a polynucleotide other than that to which it is linked in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • Recombinant host cells refer to cells which can be, or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell which has been transfected. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation.
  • Progeny of the parental cell which are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a desired peptide, are included in the progeny intended by this definition, and are covered by the above terms.
  • isolated is meant, when referring to a polynucleotide or a polypeptide, that the indicated molecule is separate and discrete from the whole organism with which the molecule is found in nature or, when the polynucleotide or polypeptide is not found in nature, is sufficiently free of other biological macromolecules so that the polynucleotide or polypeptide can be used for its intended purpose.
  • Antibody as known in the art includes one or more biological moieties that, through chemical or physical means, can bind to or associate with an epitope of a polypeptide of interest.
  • the antibodies of the invention specifically bind to pathogenic prion conformations.
  • the term “antibody” includes antibodies obtained from both polyclonal and monoclonal preparations, as well as the following: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991) Nature 349: 293-299; and U.S. Patent No. 4,816,567; F(ab') 2 and F(ab) fragments; F v molecules (non-covalent heterodimers, see, for example, Inbar et al.
  • antibody further includes antibodies obtained through non-conventional processes, such as phage display.
  • monoclonal antibody refers to an antibody composition having a homogeneous antibody population. The term is not limited regarding the species or source of the antibody, nor is it intended to be limited by the manner in which it is made. Thus, the term encompasses antibodies obtained from murine hybridomas, as well as human monoclonal antibodies obtained using human rather than murine hybridomas.
  • polyclonal antibodies are desired, a selected mammal (e.g., mouse, rabbit, goat, horse, etc.) is generally immunized with an immunogenic composition (e.g., including a prion chimera or polynucleotide encoding a prion chimera that are expressed in the animal). Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the prion chimera contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography.
  • an immunogenic composition e.g., including a prion chimera or polynucleotide encoding a prion chimera that are expressed in the animal.
  • Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies to the prion chimera contains antibodies to other antigens, the polyclonal antibodies can be purified by immunoaffinity chromatography.
  • a "single domain antibody” is an antibody that is comprised of an HL domain, which binds specifically with a designated antigen.
  • a dAb does not contain a VL domain, but may contain other antigen binding domains known to exist to antibodies, for example, the kappa and lambda domains. Methods for preparing dabs are known in the art. See, for example, Ward et al, Nature 341: 544 (1989). Antibodies can also be comprised of VH and VL domains, as well as other known antigen binding domains. Examples of these types of antibodies and methods for their preparation and known in the art (see, e.g., U.S. Pat. No. 4,816,467, which is incorporated herein by reference), and include the following.
  • vertebrate antibodies refers to antibodies that are tetramers or aggregates thereof, comprising light and heavy chains which are usually aggregated in a " Y" configuration and which may or may not have covalent linkages between the chains.
  • the amino acid sequences of the chains are homologous with those sequences found in antibodies produced in vertebrates, whether in situ or in vitro (for example, in hybridomas).
  • Vertebrate antibodies include, for example, purified polyclonal antibodies and monoclonal antibodies, methods for the preparation of which are described infra.
  • Hybrid antibodies are antibodies where chains are separately homologous with reference to mammalian antibody chains and represent novel assemblies of them, so that two different antigens are precipitable by the tetramer or aggregate.
  • hybrid antibodies one pair of heavy and light chains are homologous to those found in an antibody raised against a first antigen, while a second pair of chains are homologous to those found in an antibody raised against a second antibody. This results in the property of "divalence”, i.e., the ability to bind two antigens simultaneously.
  • Such hybrids can also be formed using chimeric chains, as set forth below.
  • Chimeric antibodies refers to antibodies in which the heavy and/or light chains are fusion proteins.
  • one portion of the amino acid sequences of the chain is homologous to corresponding sequences in an antibody derived from a particular species or a particular class, while the remaining segment of the chain is homologous to the sequences derived from another species and/or class.
  • the variable region of both light and heavy chains mimics the variable regions or antibodies derived from one species of vertebrates, while the constant portions are homologous to the sequences in the antibodies derived from another species of vertebrates.
  • the definition is not limited to this particular example.
  • Another example is "altered antibodies", which refers to antibodies in which the naturally occurring amino acid sequence in a vertebrate antibody has been varies.
  • antibodies can be redesigned to obtain desired characteristics.
  • the possible variations are many, and range from the changing of one or more amino acids to the complete redesign of a region, for example, the constant region.
  • Changes in the constant region in general, to attain desired cellular process characteristics, e.g., changes in complement fixation, interaction with membranes, and other effector functions. Changes in the variable region can be made to alter antigen-binding characteristics.
  • the antibody can also be engineered to aid the specific delivery of a molecule or substance to a specific cell or tissue site.
  • the desired alterations can be made by known techniques in molecular biology, e.g., recombinant techniques, site-directed mutagenesis, etc.
  • antibodies are aggregates comprised of a heavy-chain/light-chain dimer bound to the Fc (i.e., stem) region of a second heavy chain. This type of antibody escapes antigenic modulation. See, e.g., Glennie et al. Nature 295: 712 (1982). Included also within the definition of antibodies are “Fab” fragments of antibodies.
  • the “Fab” region refers to those portions of the heavy and light chains which are roughly equivalent, or analogous, to the sequences which comprise the branch portion of the heavy and light chains, and which have been shown to exhibit immunological binding to a specified antigen, but which lack the effector Fc portion.
  • Fab includes aggregates of one heavy and one light chain (commonly known as Fab'), as well as tetramers containing the 2H and 2L chains (referred to as F(ab)2), which are capable of selectively reacting with a designated antigen or antigen family.
  • Fab antibodies can be divided into subsets analogous to those described above, i.e., “vertebrate Fab”, “hybrid Fab”, “chimeric Fab”, and “altered Fab”. Methods of producing Fab fragments of antibodies are known within the art and include, for example, proteolysis, and synthesis by recombinant techniques.
  • Antigen-antibody complex refers to the complex formed by an antibody that is specifically bound to an epitope on an antigen.
  • binds specifically is meant high avidity and/or high affinity binding of an antibody to a specific polypeptide i.e., epitope of a PrP protein.
  • Antibody binding to its epitope on this specific polypeptide is preferably stronger than binding of the same antibody to any other epitope, particularly those which may be present in molecules in association with, or in the same sample, as the specific polypeptide of interest e.g., binds more strongly to PrP sc than denatured fragments of PrP c so that by adjusting binding conditions the antibody binds almost exclusively to PrP sc and not denatured fragments of PrP c .
  • Antibodies that bind specifically to a polypeptide of interest may be capable of binding other polypeptides at a weak, yet detectable, level (e.g., 10% or less of the binding shown to the polypeptide of interest). Such weak binding, or background binding, is readily discernible from the specific antibody binding to the compound or polypeptide of interest, e.g., by use of appropriate controls.
  • antibodies of the invention bind pathogenic prions with a binding affinity of greater than 10 6 mole/1, more preferably greater than 10 7 mole/1 or even more preferably 10 8 mole/liters or more. Techniques for determining amino acid sequence "similarity" or “percent identity” are well known in the art.
  • similarity means the amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed “percent similarity” then can be determined between the compared polypeptide sequences.
  • Techniques for determining nucleic acid and amino acid sequence identity also are well known in the art and include determining the nucleotide sequence of the mRNA for that gene (usually via a cDNA intermediate) and determining the amino acid sequence encoded thereby, and comparing this to a second amino acid sequence.
  • identity refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively.
  • Two or more polynucleotide sequences can be compared by determining their "percent identity.”
  • Two or more amino acid sequences likewise can be compared by determining their "percent identity.”
  • the percent identity of two sequences, whether nucleic acid or peptide sequences is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981).
  • percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated, the "Match" value reflects
  • sequence identity Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, such as the alignment program BLAST, which can also be used with default parameters.
  • search parameters may vary based on the size of the sequence in question.
  • Computer programs are also available to determine the likelihood of certain polypeptides forming structures such as ⁇ -helices.
  • One such program, described herein, is the "ALB" program for protein and polypeptide secondary structure calculation and predication.
  • secondary protein structure can be predicted from the primary amino acid sequence, for example using protein crystal structure and aligning the protein sequence related to the crystal structure (e.g., using Molecular Operating Environment (MOE) programs available from the Chemical Computing Group Inc., Montreal, P.Q., Canada). Other methods of predicting secondary structures are described, for example, in Gamier et al.
  • MOE Molecular Operating Environment
  • Homology can also be determined by hybridization of polynucleotides under conditions that form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • Two DNA, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 80%-85%, preferably at least about 90%, and most preferably at least about 95%-98% sequence identity over a defined length of the molecules, as determined using the methods above.
  • substantially homologous also refers to sequences showing complete identity to the specified DNA or polypeptide sequence.
  • DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
  • immunogenic composition refers to any composition (e.g., polypeptides and/or polynucleotides) where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response.
  • the immunogenic composition can be introduced directly into a recipient subject, such as by injection, inhalation, oral, intranasal or any other parenteral or mucosal (e.g., intra-rectally or intra-vaginally) route of administration.
  • epitope is meant a site on an antigen to which specific B cells and/or T cells respond, rendering the molecule including such an epitope capable of eliciting an immunological reaction or capable of reacting with antibodies present in a biological sample.
  • the term is also used interchangeably with "antigenic determinant” or "antigenic determinant site.”
  • An epitope can comprise 3 or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and, more usually, consists of at least 8-10 such amino acids. Methods of determining spatial conformation of amino acids are known in the art and include, for example, x-ray crystallography and 2- dimensional nuclear magnetic resonance.
  • Gene transfer refers to methods or systems for reliably inserting DNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • Gene delivery expression vectors include, but are not limited to, vectors derived from alphaviruses, pox viruses and vaccinia viruses.
  • sample includes biological samples derived from an animal (living or dead) such as organs (e.g., brain, liver, kidney, etc), whole blood, blood fractions, plasma, cerebrospinal fluid (CSF), urine, tears, tissue, organs, biopsies and the like as well as pharmaceuticals, foods, cosmetics and the like.
  • organs e.g., brain, liver, kidney, etc
  • CSF cerebrospinal fluid
  • urine tears, tissue, organs, biopsies and the like as well as pharmaceuticals, foods, cosmetics and the like.
  • label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.
  • fluorescer refers to a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range.
  • labels include, but are not limited to fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, NADPH, beta-galactosidase, horseradish peroxidase, glucose oxidase, alkaline phosphatase and urease.
  • compositions comprising a prion chimera (and/or polynucleotides encoding prion chimeras), antibodies specific to pathogenic prions, and methods of making and using these prion chimeras and/or antibodies.
  • the prion chimeras comprise a prion polypeptide and a non-prion polypeptide. Without being bound by one theory, it appears that the non-prion polypeptide serves as a backbone (or scaffold) for the prion polypeptide.
  • the prion polypeptide is either incorporated into the chimera in a disease conformation or can be induced into a disease conformation after incorporation into the chimera (e.g., by culture conditions and/or exposure to disease conformations).
  • the chimeras allow for ready detection (diagnosis) of prion diseases and for the generation of antibodies (e.g., therapeutic antibodies) against the disease conformation.
  • the prion portion of the chimera may be involved in formation of a beta-helices that can be characteristic of disease-form proteins.
  • the disease-form protein(s) present in the sample can be ordered, aggregated or otherwise induced to a state that can then be detected.
  • the antibodies and/or chimera (or one or more of their component parts) can be labeled or marked to facilitate detection.
  • Conformational Disease (Prion) Proteins The following is a non-limiting list of diseases with associated insoluble proteins that assume two or more different conformations.
  • the conformational disease protein is a prion and the chimera is a prion chimera.
  • Alzheimer's Disease APP A* peptide, *l-antichymotrypsin, tan, non-A* component
  • insoluble proteins listed above each include a number of variants or mutations that result in different strains that are all encompassed by the present invention.
  • Known pathogenic mutations and polymorphisms in the PrP gene related to prion diseases are given below and the sequences of human, sheep and bovine are given in U.S. Pat. No. 5,565,186, issued Oct. 15, 1996.
  • prion proteins and other conformational disease proteins
  • prion proteins have two different 3-dimensional conformations with the same amino acid sequence. One conformation is associated with disease characteristics and is generally insoluble whereas the other conformation is not associated with disease characteristics and is soluble.
  • the present invention is not limited to the diseases, proteins and strains listed. As noted above, a conformational conversion of the non-pathogenic "cellular" forms into pathogenic isoforms is believed to be fundamental event in the onset of conformational diseases.
  • the prion component used in the invention comprises an amino acid sequence of a prion protein that can adapt the ⁇ -helical conformation of a pathogenic prion protein. Discussion of the proposed ⁇ -helical structure of pathogenic prion proteins can be found in Wille, et al., "Structural Studies of the Scrapie Prion Protein by Electron Crystallography", Proc. Natl. Acad. Sci. USA, 99 (6): 3563-3568 (2002).
  • ⁇ -helices are known to contain highly ordered, stacked side chains on both the inside and outside of the helix. This side-chain stacking adds to the rigid geometry of the ⁇ -helices, which tend to have planar sides and essentially no interstrand twist, ⁇ -helices include both left- and right-handed structures.
  • the number of residues per ⁇ -helical turn for left-handed helices averages about 18 residues per helical turn.
  • the number of residues per ⁇ -helical turn for right-handed helices can vary substantially, but averages about 24 residues per helical turn. See Wille, et al., "Structural Studies of the Scrapie Prion Protein by Electron Crystallography", Proc. Natl. Acad. Sci.
  • Non-limiting examples of conformation (prion) proteins suitable for use in the chimeras described herein include peptides that interact with helix HI and beta-strand S2 (residues 142-166, as numbered relative to human PrP (SEQ 3D NO:l)).
  • polypeptides derived from or designed to interact with other regions of disease-form proteins can also be used, including hydrophobic regions, metal binding domains and the like.
  • the amino acid sequence AGAAAAGA comprising residues 112-119 of a hydrophobic region of murine PrP (SEQ 3D NO:2) has been shown to be amyloidogenic and evolutionarily conserved.
  • Prion polypeptides components of the chimeras described herein may also be designed to include metal binding domains, including, but not limited to, the highly conserved octarepeat sequence PHGGGWGQ or HGGGW spanning residues 60-91 of human PrP (SEQ 3D NO:l). Jackson et al. Proc Natl Acad Sci U S A 2001 Jul 17;98(15):8531-5; see, also, Burns et al. (2002) Biochemistry 41(12):3991-4001.
  • the prion polypeptide comprises amino acid residues from about amino acid 135 to about amino acid 155 of the prion amino acid sequence (e.g., human (SEQ ID NO:6) or mouse (SEQ 3D NO:7).
  • the prion polypeptide comprises amino acid residues from about amino acid 126 to about 154 of a prion protein (e.g., human (SEQ ID NO: 12) or mouse (SEQ 3D NO: 13).
  • the prion polypeptide of the prion chimera adapts a ⁇ -helix conformation within the prion chimera.
  • the prion polypeptide segment of the chimeras described herein can include one or more amino acid replacements, additions, and deletions thereof.
  • the conformational (prion) protein of the chimera can be anywhere from 5 to about 75 amino acids long (or any value therebetween) or even longer, preferably from about 20 to 50 amino acids (or any value therebetween), and even more preferably between about 20 and 30 amino acids (or any value therebetween) in length.
  • the polypeptides can be identical to naturally-occurring beta-helical forming regions of these conformation proteins.
  • one or more residues may be different from wild-type, for example introducing polar, non-polar residues or aromatic residues in certain positions to favor beta-helix formation and/or pi -stacking.
  • polar, non-polar residues or aromatic residues in certain positions to favor beta-helix formation and/or pi -stacking.
  • the polynucleotide and amino acid sequence for prion proteins produced by many different species are known, including human, mouse, sheep and cattle. Variants to these sequences exist within each species, including variants that are more likely to adapt the ⁇ - helical conformation of pathogenic prions.
  • the prion polypeptides (or fragments thereof) used in the invention can comprise fragments or derivatives of the amino acid sequences of any species that produces prion proteins. Derivatives of such prion proteins, including amino acid replacements, deletions, additions and other mutations to these sequences can also be used in the prion sequences. Preferably, any amino acid replacements, additions, and deletions of the prion protein sequence will not affect the ability of the prion polypeptide to adapt a ⁇ -helical conformation (e.g., within the prion chimera). In one embodiment, conservative amino acid replacements are preferred. Conservative amino acid replacements are those that take place within a family of amino acids that are related in their side chains.
  • the prion proteins used in the invention may further comprise amino acids from portions of the prion that do not adapt a ⁇ -helical conformation. Preferably, these additional amino acids do not interfere with the ability of the prion polypeptide to adapt a ⁇ -helical conformation.
  • Non-Conformational Disease (Non-Prion) Proteins The chimeras described herein will also comprise a sequence derived from a non- conformational disease, for example a fragment of a non-prion ⁇ -helical protein. Virtually any protein can serve as the non-prion portion of the chimera, although it is prefe ⁇ ed that the non-prion portion of the chimera be capable of adapting a ⁇ -helical conformation, for example to facilitate and/or maintain that pathogenic conformation of the prion portion.
  • the non-prion protein used in the chimera acts as a carrier (or scaffold or backbone) protein and allows the prion polypeptide (or fragment thereof) to adapt its ⁇ - helical conformation in the prion chimera.
  • Non-prion ⁇ -helical protein suitable for use in the invention include proteins with right handed ⁇ -helices, such as the Bordetella pertussis pertactin protein, and proteins with left-handed ⁇ -helices, such as Methanosarcina thermophila ⁇ carbonic anhydrase. (See, e.g., Emsley et al. (1996) Nature May 2;381(6577):90-2 for a description of the structure of pertactin and Smith et al.
  • beta-helix proteins suitable for use as described herein will be known to those of skill in the art, including, but not limited to, right handed beta-helix proteins such as pectate lyase, pectin lyase, galacturonase, chondroitinase B, pectin methylesterase, P22 tailspike protein, insect cysteine-rich antifreeze protein, cell- division inhibitor MinC, alpha subunit of glutamate synthase (e.g., C-terminal domain) and C-terminal domain of adenylylcyclase associated protein as well as left handed beta-helix proteins such as UDP N-acetylglucosamine acyltransferase, galactoside acetyltransferase, xenobiotic acetyltransferase
  • One or more amino acids of the non-prion protein of the chimera may be replaced with a prion polypeptide.
  • the prion polypeptide can adapt a ⁇ -helical conformation of a pathogenic prion.
  • the prion portion may be located at either the C- terminal or the N-terminal region of the non-prion polypeptides.
  • the prion polypeptide is placed after the N-terminal methionine.
  • the prion polypeptide may be inserted into (or embedded in) the non-prion portion. Non-limiting examples of prion chimeras are shown in FIGs.
  • the non-prion ⁇ -helical sequences of the prion chimera can further comprise fragments or derivatives of a non-prion ⁇ -helical proteins. For instance, amino acids can be removed from either the N terminal or the C terminal regions.
  • the prion chimeras of the invention may further comprise additional segments.
  • a non-limiting example of an additional segment is a tagging sequence. This tagging sequence can facilitate purification and solubilization of the chimera. Suitable tagging sequences are known in the art and include, for instance, sequences comprising histidine residues (e.g., SEQ 3D NO:5).
  • the non-prion polypeptide is derived from a Bordetella pertussis pertactin protein. Many variants of Bordetella pertussis pertactin protein are known in the art. (See, e.g., FIG. 8 (SEQ 3D NO:3)).
  • P69 refers to the pertactin of a specific strain of pertussis.
  • SEQ 3D NO:3 represents the full-length sequence of the P69 protein, including all of the amino acids that are known to adapt the ⁇ -helical structure.
  • P69 fragments suitable for use as the non-prion ⁇ -helical protein include the amino acid sequences selected from the group consisting of SEQ 3D NO:8 and SEQ ID NO:9.
  • SEQ JD NO:8 and 9 represent fragments of P69 where a portion of the amino acid sequence that adapts the ⁇ -helical conformation has been removed.
  • SEQ 3D NO: 8 is referred to in the figures as P69 Control A.
  • SEQ ID NO: 9 is referred to in the figures as P69 Control B.
  • the prion chimera comprises SEQ ID NO:8 and a prion protein (or fragment thereof).
  • the prion chimera comprises SEQ ID NO: 9 and a prion portion.
  • Non-limiting examples of prion chimeras using both human and mouse prion sequences and P69 non-prion ⁇ -helical protein sequences are shown in FIGs 3 - 6, 12 - 15, 20 - 23, 20 and 21.
  • Left-handed non-prion ⁇ -helical proteins can also be used in the invention.
  • One example of a left handed non-prion ⁇ -helical protein suitable for use in the invention is Methanosarcina thermophila ⁇ carbonic anhydrase (GCA).
  • GCA Methanosarcina thermophila ⁇ carbonic anhydrase
  • SEQ ID NO:4 represents the full-length sequence of GCA, including all of the amino acids that adapt the ⁇ -helical structure.
  • a prion chimera comprising a polypeptide comprising SEQ 3D NO:4 and a prion portion is used.
  • GCA non-prion ⁇ -helical protein fragments suitable for use in the invention include the amino acid sequences selected from the group consisting of SEQ ID NO: 10 and SEQ 3D NO: 11.
  • SEQ 3D NOs: 10 and 11 depicts fragments of GCA in which some of the amino acids that adapt the ⁇ -helical conformation have been removed.
  • SEQ ID NO: 10 is referred to in the figures as GCA Control A.
  • SEQ 3D NO. 11 is refe ⁇ ed to in the figures as GCA Control B.
  • the prion chimera comprises a polypeptide comprising SEQ ID NO. 10 and a prion sequence.
  • the prion chimera comprises a polypeptide comprising SEQ 3D NO. 11 and prion sequences.
  • Non-limiting examples of prion chimeras using both human and mouse sequences and GCA non-prion ⁇ -helical proteins are shown in FIGs 8 - 11, 16 - 19, 22, 23 and 25-28.
  • the prion chimeras used in the invention are not, themselves, pathogenic and/or infectious.
  • the prion chimeras used in the invention can be used to generate antibodies that specifically bind to prions in a pathogenic prion conformation.
  • prion chimeras themselves can be used to specifically bind to prions in a pathogenic prion conformation. Such specific binding generally refers to a greater affinity of the antibody or chimera to the pathogenic conformation relative to the nonpathogenic conformation, as can be determined by numerous suitable assays known in the art.
  • III.C. Polypeptide Production The chimeras (and components thereof) of the present invention can be produced in any number of ways all of which are well known in the art. In one embodiment, the polypeptides are generated using recombinant techniques, well known in the art. One of skill in the art could readily determining nucleotide sequences that encode the desired chimera using standard methodology and the teachings herein.
  • Non- limiting examples of polynucleotide constructs encoding prion chimeras are depicted in SEQ 3D NO:211-238 and the constructs depicted in Table 1.
  • Oligonucleotide probes can be devised based on the known sequences of the prion- encoding and non-prion-encoding polynucleotides and used to probe genomic or cDNA libraries. The sequences can then be further isolated using standard techniques and, e.g., restriction enzymes employed to truncate the gene at desired portions of the full-length sequence. Similarly, sequences of interest can be isolated directly from cells and tissues containing the same, using known techniques, such as phenol extraction and the sequence further manipulated to produce the desired truncations.
  • the sequences encoding the chimera (or components thereof) can also be produced synthetically, for example, based on the known sequences.
  • the nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired.
  • the complete sequence is generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) /. Biol. Chem. 259:6311; Siemmer et al.
  • the primer can be made specific by keeping primer length and base composition within relatively narrow limits and by keeping the mutant base centrally located. See, e.g., Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is effected using DNA polymerase, the product cloned and clones containing the mutated DNA, derived by segregation of the primer extended strand, selected. Selection can be accomplished using the mutant primer as a hybridization probe. The technique is also applicable for generating multiple point mutations. See, e.g., Dalbie-McFarland et al. Proc. Natl. Acad.
  • chimera proteins Once coding sequences for the desired components of chimera proteins have been isolated and/or synthesized, they can be cloned into any suitable vector or replicon for expression. (See, also, Examples).
  • a wide variety of vectors encoding modified polypeptides can be generated by creating expression constructs which operably link, in various combinations, polynucleotides encoding prion and non-prion polypeptides having deletions or mutations therein. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • Examples of recombinant DNA vectors for cloning and host cells which they can transform include the bacteriophage ⁇ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pM ⁇ 290 (non-E. coli gram-negative bacteria), pHV14 (E.
  • baculo virus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego CA ("MaxBac” kit). Plant expression systems can also be used to produce the prion chimeras. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; and Ffackland et al., Arch. Virol. (1994) 139:1-22. Viral systems, such as a vaccinia based infection/transfection system, as described in Tomei et al., J. Virol.
  • cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the DNA of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA that is then translated into protein by the host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).
  • the gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence. With the present invention, both the naturally occurring signal peptides or heterologous sequences can be used. Leader sequences can be removed by the host in post-translational processing. See, e.g., U.S. Patent Nos.
  • Such sequences include, but are not limited to, the TPA leader, as well as the honey bee mellitin signal sequence.
  • Other regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell.
  • Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • the control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
  • the coding sequence can be cloned directly into an expression vector that already contains the control sequences and an appropriate restriction site. In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols.
  • the expression vector is then used to transform an appropriate host cell.
  • mammalian cell lines include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, as well as others.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G293 cells
  • bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs.
  • Yeast hosts useful in the present invention include inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.
  • Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa c ⁇ lifornica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
  • the proteins of the present invention are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The selection of the appropriate growth conditions is within the skill of the art.
  • the transformed cells secrete the polypeptide product into the surrounding media.
  • Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example using a tissue plasminogen activator (TPA) leader sequence, an interferon ( ⁇ or ⁇ ) signal sequence or other signal peptide sequences from known secretory proteins.
  • TPA tissue plasminogen activator
  • ⁇ or ⁇ interferon
  • the secreted polypeptide product can then be isolated by various techniques described herein, for example, using standard purification techniques such as but not limited to, hydroxyapatite resins, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.
  • the transformed cells are disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the recombinant polypeptides substantially intact.
  • Jntracellular proteins can also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the polypeptides occurs.
  • methods of disrupting cells for use with the present invention include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecylsulphate, Triton, NP40 and CHAPS.
  • the particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pre-treatment used.
  • cellular debris is removed, generally by centrifugation, and the intracellularly produced polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion- exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoadsorbent techniques, affinity chromatography, immunoprecipitation, and the like.
  • one method for obtaining the intracellular polypeptides of the present invention involves affinity purification, such as by immunoaffinity chromatography using antibodies (e.g., previously generated prion chimera specific antibodies), or by lectin affinity chromatography.
  • Particularly preferred lectin resins are those that recognize mannose moieties such as but not limited to resins derived from Galanthus nivalis agglutinin (GNA), Lens culinaris agglutinin (LCA or lentil lectin), Pisum sativum agglutinin (PSA or pea lectin), Narcissus pseudonarcissus agglutinin (NPA) and Allium ursinum agglutinin (AUA).
  • GAA Galanthus nivalis agglutinin
  • LCA Lens culinaris agglutinin
  • PSA Pisum sativum agglutinin
  • NPA Narcissus pseudonarcissus agglutinin
  • polypeptides can be further purified using conventional techniques well known in the art, such as by any of the techniques described above.
  • Polypeptides can be conveniently synthesized chemically, for example by any of several techniques that are known to those skilled in the peptide art. In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group.
  • the protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage.
  • the protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth.
  • any remaining protecting groups and any solid support, if solid phase synthesis techniques are used) are removed sequentially or concurrently, to render the final polypeptide.
  • Typical protecting groups include t-butyloxycarbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4- dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o- nitrophenylsulfonyl and the like.
  • Typical solid supports are cross-linked polymeric supports.
  • polypeptide analogs of the present invention can also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis. See, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131-5135; U.S. Patent No. 4,631,211.
  • the antibodies of the invention are preferably specific and selective for pathogenic prions and can be used to distinguish between pathogenic and non-pathogenic prions.
  • the antibodies of the invention recognize a ⁇ -helical region of a pathogenic prion.
  • the antibodies of the invention are generated by administering a prion chimera or polynucleotide encoding a prion chimera to an animal. The methods may also include isolating the antibodies from the animal.
  • the antibodies of the invention may be polyclonal or monoclonal antibody preparations, monospecific antisera, human antibodies, or may be hybrid or chimeric antibodies, such as humanized antibodies, altered antibodies (Fab') 2 fragments, F(ab) fragments, Fv fragments, single-domain antibodies, dimeric or trimeric antibody fragments or constructs, minibodies, or functional fragments thereof which bind to the antigen in question.
  • Antibodies are produced using techniques well known to those of skill in the art and disclosed in, for example, U.S. Patent Nos. 4,011,308; 4,722,890; 4,016,043; 3,876,504; 3,770,380; and 4,372,745.
  • polyclonal antibodies are generated by immunizing a suitable animal, such as a mouse, rat, rabbit, sheep, or goat, with an antigen of interest (e.g., prion chimera).
  • an antigen of interest e.g., prion chimera
  • the antigen can be linked to a carrier prior to immunization.
  • a carrier are well known to those of ordinary skill in the art.
  • Immunization is generally performed by mixing or emulsifying the antigen in saline, preferably in an adjuvant such as Freund's complete adjuvant, and injecting the mixture or emulsion parenterally (generally subcutaneously or intramuscularly).
  • the animal is generally boosted 2 - 6 weeks later with one or more injections of the antigen in saline, preferably using Freund's incomplete adjuvant.
  • Antibodies may also be generated by in vitro immunization, using methods known in the art. Polyclonal antiserum is then obtained from the immunized animal. Monoclonal antibodies are generally prepared using the method of Kohler and Milstein (1975) Nature 256:495-497, or a modification thereof. Typically, a mouse or rat is immunized as described above. However, rather than bleeding the animal to extract serum, the spleen (and optionally several large lymph nodes) is removed and dissociated into single cells.
  • the spleen cells may be screened (after removal of nonspecifically adherent cells) by applying a cell suspension to a plate or well coated with the antigen.
  • B-cells expressing membrane-bound immunoglobulin specific for the antigen, will bind to the plate, and are not rinsed away with the rest of the suspension.
  • Resulting B-cells, or all dissociated spleen cells are then induced to fuse with myeloma cells for form hybridomas, and are cultured in a selective medium (e.g., hypoxanthine, aminopterin, thymidine medium,
  • a selective medium e.g., hypoxanthine, aminopterin, thymidine medium
  • hybridomas are plated by limiting dilution, and are assayed for the production of antibodies that bind specifically to the immunizing antigen (and which do not bind to unrelated antigens).
  • the selected monoclonal antibody-secreting hybridomas are then cultured either in vitro (e.g., in tissue culture bottles or hollow fiber reactors), or in vivo (e.g., as ascites in mice).
  • Humanized and chimeric antibodies are also useful in the invention.
  • Hybrid (chimeric) antibody molecules are generally discussed in Winter et al. (1991) Nature 349: 293-299 and U.S. Patent No. 4,816,567. Humanized antibody molecules are generally discussed in Riechmann et al.
  • One approach to engineering a humanized antibody involves cloning recombinant DNA containing the promoter, leader, and variable-region sequences from a mouse antibody gene and the constant-region exons from a human antibody gene to create a mouse-human chimera, a humanized antibody. See generally, Kuby, "Immunology, 3 rd Edition", W.H. Freeman and Company, New York (1998) at page 136.
  • Antibodies both monoclonal and polyclonal, which are directed against chimeras as described herein are particularly useful in diagnosis and therapeutic applications, for example, those antibodies that are neutralizing are useful in passive immunotherapy.
  • Monoclonal antibodies in particular, may be used to raise anti-idiotype antibodies.
  • Anti-idiotype antibodies are immunoglobulins that carry an "internal image" of the antigen of the agent against which protection is desired. Techniques for raising anti-idiotype antibodies are known in the art. See, e.g., Grzych (1985), Nature 316:74; MacNamara et al. (1984), Science 226:1325, Uytdehaag et al (1985), J. Immunol. 134:1225.
  • Antibody fragments that retain the ability to recognize the pathogenic prion conformation are also included within the scope of the invention.
  • a number of antibody fragments are known in the art that comprise antigen-binding sites capable of exhibiting immunological binding properties of an intact antibody molecule.
  • functional antibody fragments can be produced by cleaving a constant region, not responsible for antigen binding, from the antibody molecule, using e.g., pepsin, to produce F(ab') fragments. These fragments will contain two antigen binding sites, but lack a portion of the constant region from each of the heavy chains.
  • Fab fragments comprising a single antigen binding site, can be produced, e.g., by digestion of polyclonal or monoclonal antibodies with papain.
  • Functional fragments including only the variable regions of the heavy and light chains, can also be produced, using standard techniques such as recombinant production or preferential proteolytic cleavage of immunoglobulin molecules. These fragments are known as F v . See, e.g., Inbar et al. (1972) Proc. Nat. Acad. Sci USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; and Ehrlich et al. (1980) Biochem 19:4091-4096.
  • a single-chain Fv (“sFv” or scFv”) polypeptide is a covalently linked V H - V heterodimer that is expressed from a gene fusion including VH - and V - encoding genes linked by a peptide-encoding linker.
  • a number of methods have been described to discern and develop chemical structures (linkers) for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an sFv molecule which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S.
  • Patent Nos. 5,091,513; 5,132,405; and 4,946,778 The sFv molecules may be produced using methods described in the art. See, e.g., Huston et al. (1988) Proc. Nat. Acad. Sci USA 85:5879-5338; U.S. Patent Nos. 5,091,513; 5,132,405 and 4,946,778. Design criteria include determining the appropriate length to span the distance between the C- terminus of one chain and the N-terminus of the other, wherein the linker is generally formed from small hydrophilic amino acid residues that do not coil or form secondary structures. Such methods have been described in the art. See, e.g., U.S. Patent Nos.
  • Suitable linkers generally comprise polypeptide chains of alternating sets of glycine and serine residues, and may include glutamic acid and lysine residues inserted to enhance solubility.
  • "Mini-antibodies” or “minibodies” will also find use with the present invention. Minibodies are sFv polypeptide chains that include oligomerization domains at their C- termini, separated from the sFv by a hinge region. Pack et al., (1992) Biochem 31:1579- 1584.
  • the oligomerization domain comprises self-associating ⁇ -helices, e.g., leucine zippers, that can be further stabilized by additional disulfide bonds.
  • the oligomerization domain is designed to be compatible with vectorial folding across a membrane, a process thought to facilitate in vivo folding of the polypeptide into a functional binding protein.
  • minibodies are produced using recombinant methods well known in the art. See, e.g., Pack et al., (1992) Biochem 31:1579-1584; Cumber et al. (1992) J. Immunology 149B: 120-126.
  • Non-conventional means can also be used to generate and identify the antibodies of the invention.
  • a phage display library can be screened for antibodies that bind to the prion chimeras of the invention.
  • the antibody will be specific to the ⁇ - helical conformation of a pathogenic prion. See generally, Siegel, "Recombinant Monoclonal Antibody Technology", Transfus. Clin. Biol. (2002) 9(1): 15-22; Sidhu, "Phage Display in Pharmaceutical Biotechnology", Curr. Opin. Biotechnol. (2000) 11(6):610 - 616; Sharon, et al., "Recombinant Polyclonal Antibody Libraries", Comb. Chem.
  • the antibodies of the invention may also be generated by administering a polynucleotide sequence encoding a prion chimera into an animal.
  • a polynucleotide sequence encoding a prion chimera into an animal.
  • the prion chimera is expressed in vivo, antibodies specific to the prion chimera are generated in vivo.
  • Methods for polynucleotide delivery of the prion chimeras of the invention are discussed in section VI.A., below. The specificity of the antibodies of the invention can be tested in a variety of ways.
  • prions having a pathogenic conformation are generally resistant to certain proteases, such as proteinase K.
  • the same proteases are able to degrade prions in a nonpathogenic conformation.
  • One method of testing the specificity of the antibodies of the present invention is to select a biological sample containing both pathogenic and non-pathogenic prions. The sample can be separated into two equal volumes.
  • Antibodies of the invention can be added adsorbed onto a solid support (as further described below) and used to obtain a quantitative value directly related to the number of antibody- prion binding interactions on the solid support.
  • Protease can be added to the second sample and the same test performed.
  • any antibody-prion binding interactions in the second volume can be attributed to pathogenic prions. Variations and other assays known in the art can also be used to demonstrate the specificity of the antibodies of the invention.
  • the prion chimeras, polynucleotides and/or antibodies of the invention can be used in a variety of assays to screen samples (e.g., biological samples) to detect the presence of pathogenic prions.
  • Assays involving antibodies may use, for example, a monoclonal antibody directed towards a single epitope, a combination of monoclonal antibodies directed towards the same epitope(s), monoclonal antibodies directed towards different epitopes, monoclonal antibodies directed to epitopes of different chimeras, polyclonal antibodies directed towards the same antigen, polyclonal antibodies directed towards different antigens and/or a combination of monoclonal and polyclonal antibodies.
  • the antibodies of the invention can be used to capture a pathogenic prion in a biological sample. Any suitable means of detection can then be used to identify binding between the antibody and a pathogenic prion.
  • the invention includes a method of detecting pathogenic forms of prion particles in a sample (e.g., a biological sample), the method comprising (a) exposing the sample suspected of containing a pathogenic prion to a first antibody, wherein the first antibody is specific to pathogenic prions; and (b) detecting the presence or absence of the first antibody binding to a pathogenic prion.
  • the antibody is generated according to any of the methods described herein.
  • the invention includes a method of detecting a pathogenic prion in a biological sample, the method comprising (a) exposing the biological sample suspected of containing a pathogenic prion to a prion chimera; and (b) detecting the presence or absence of the prion chimera bound to the pathogenic prion.
  • assay e.g., immunoassay
  • protocols may be based, for example, upon competition, or direct reaction, or sandwich type assays.
  • Protocols may also, for example, use solid supports, or may be by immunoprecipitation. Most assays involve the use of labeled antibody or polypeptide (e.g., chimera).
  • the labels may be, for example, fluorescent, cherniluminescent, radioactive, or dye molecules.
  • Detectable labels suitable for use in the invention include any molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin, strepavidin or haptens) and the like.
  • Additional labels include, but are not limited to, those which use fluoresce, including those substances or portions thereof which are capable of exhibiting fluorescence in the detectable range.
  • labels that may be used in the invention include, but are not limited to, horse radish peroxidase (HRP), fluorescein, F3TC, rhodamine, dansyl, umbelliferone, dimethyl acridinium ester (DMAE), Texas red, luminol, NADPH and ⁇ -galactosidase.
  • Assays that amplify the signals from the probe are also known. Examples of which are assays that utilize biotin and avidin, and enzyme-labeled and mediated immunoassays, such as ELIS A assays.
  • any of the assays described herein may be conducted in solution (e.g., a liquid medium) or a solid medium.
  • the antibodies and/or prion chimeras are exposed to the biological sample in a liquid medium.
  • the presence or absence of binding between the antibody and/or prion chimera and a pathogenic prion in the biological sample can be detected in several ways.
  • immunoprecipitation is used to separate out antibody and/or prion chimeras that are bound to the pathogenic prion.
  • the immunoprecipitation is facilitated by the addition of a precipitating enhancing agent.
  • a precipitation enhancing agent includes moieties that can enhance or increase the precipitation of the antibodies and/or prion chimeras that are bound to pathogenic prions.
  • Such precipitation enhancing agents include polyethylene glycol (PEG), protein G, protein A and the like.
  • protein G or protein A are used as precipitation enhancing agents, the protein can optionally be attached to a bead, preferably a magnetic bead. Precipitation can be further enhanced by use of centrifugation or with the use of magnetic force. Use of such precipitating enhancing agents are known in the art.
  • the antibodies and the chimeras of the invention can be adsorbed onto solid supports for use in the assays of the invention.
  • a solid support for purposes of the invention, can be any material that is an insoluble matrix and can have a rigid or semi-rigid surface.
  • Exemplary solid supports include, but are not limited to, substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinyl fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • Particular supports include plates, pellets, disks, capillaries, hollow fibers, needles, pins, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally crosslinked with N-N'-bis- acryloylethylenediamine, and glass particles coated with a hydrophobic polymer.
  • the molecules to be added to the solid support can readily be functionalized to create styrene or acrylate moieties, thus enabling the incorporation of the molecules into polystyrene, polyacrylate or other polymers such as polyimide, polyacrylamide, polyethylene, polyvinyl, polydiacetylene, polyphenylene-vinylene, polypeptide, polysaccharide, polysulfone, polypyrrole, polyimidazole, polythiophene, polyether, epoxies, silica glass, silica gel, siloxane, polyphosphate, hydrogel, agarose, cellulose and the like.
  • polyimide polyacrylamide
  • polyethylene polyvinyl, polydiacetylene, polyphenylene-vinylene
  • polypeptide polysaccharide
  • polysulfone polysulfone
  • polypyrrole polyimidazole
  • polythiophene polyether
  • epoxies silica glass
  • the solid support coupled to an antibody or chimera is reacted with a sample (e.g., biological sample).
  • a sample e.g., biological sample.
  • a secondary binder moiety e.g., antibodies specific to pathogenic prions or chimeras
  • suitable binding conditions such that the secondary binder is capable of associating selectively with the bound pathogenic prions, if any, in the sample.
  • the presence of the secondary binder can then be detected using techniques well known in the art.
  • a number of anti-human immunoglobulin (Ig) molecules are known in the art (e.g., commercially available goat anti-human Ig or rabbit anti-human Ig).
  • Ig molecules for use herein will preferably be of the IgG or IgA type, however, Ig may also be appropriate in some instances.
  • the Ig molecules can be readily conjugated to a detectable enzyme label, such as horseradish peroxidase, glucose oxidase, Beta-galactosidase, alkaline phosphatase and urease, among others, using methods known to those of skill in the art.
  • An appropriate enzyme substrate is then used to generate a detectable signal.
  • An exemplary sandwich type assay can be conducted using the antibodies and/or prion chimeras described herein as follows.
  • a sample (e.g., biological sample) suspected of containing a pathogenic prion is exposed to a first antibody specific to pathogenic prions (e.g., an antibody generated using a prion chimera) and/or a prion chimera.
  • a first antibody specific to pathogenic prions e.g., an antibody generated using a prion chimera
  • pathogenic prions e.g., an antibody generated using a prion chimera
  • a prion chimera e.g., an antibody generated using a prion chimera
  • a prion chimera e.g., an antibody generated using a prion chimera
  • the solid support is reacted first with one or more of the antibodies directed against prion chimeras or prion chimeras themselves, washed and then exposed to the test sample.
  • Antibodies or prion chimeras are again added and the reaction visualized using either a direct color reaction or using a labeled second antibody, such as an anti-immunoglobulin labeled with horseradish peroxidase, alkaline phosphatase or urease.
  • a solid support is first reacted with an antibody and/or chimera such that the antibody and/or chimera are sufficiently immobilized to the support.
  • immobilization to the support can be enhanced by first coupling the antibody and/or chimera to a protein with better solid phase-binding properties.
  • Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobuline, ovalbumin, and other proteins well known to those skilled in the art.
  • BSA bovine serum albumin
  • Other reagents that can be used to bind molecules to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules to antigens, are well known to those of ordinary stall in the art.
  • any non-immobilized components may be removed from the support by washing, and the support-bound components are then contacted with a biological sample suspected of containing pathogenic prions under suitable binding conditions.
  • a second antibody and/or a prion chimera may be added under suitable binding conditions.
  • the added second antibody and/or prion chimera can include a detectable label.
  • Suitable controls can also be used in the assays of the invention. For instance, a negative control of PrP can be used in the assays. A positive control of a prion chimera could also be used in the assays. Such controls can optionally be detectably labeled.
  • an antibody specific to pathogenic prions is adsorbed onto (immobilized on) a solid support, for example an antibody generated using a prion chimera.
  • the solid support is combined with a sample (e.g., a biological sample) suspected of containing a pathogenic prion, under conditions that allow a pathogenic prion, when present in the sample, to bind to the first antibody or to the prion chimera.
  • the solid support can then be washed to remove any unbound materials.
  • a detectably labeled second antibody and/or a detectably labeled prion chimera can be added to the solid support under complex forming conditions.
  • the second antibody (or labeled prion chimera) can be specific to pathogenic prions or it may be capable of binding to both pathogenic and non- pathogenic prions. Complexes formed between the first antibody and/or the prion chimera, a pathogenic prion from the biological sample, and the second detectably labeled antibody and/or the detectably labeled prion chimera may then be detected to determine the presence of pathogenic prions in the sample.
  • the invention includes a method of detecting the presence of a pathogenic prion particle in a sample (e.g., a biological sample), the method comprising (a) providing a solid support comprising one or more of a first antibody specific to pathogenic prions (e.g., an antibody generated according to the methods described herein) or a prion chimera bound thereto; (b) combining a sample with the solid support under conditions which allow a pathogenic prion, when present in a biological sample, to bind to the first antibody or to the prion chimera; (c) adding to the solid support, under complex forming conditions, one or more of a detectably labeled second antibody or a detectably labeled prion chimera, wherein the second antibody may optionally recognize both pathogenic and nonpathogenic prions; and (d) detecting complexes formed between the first antibody and/or prion chimera, a pathogenic prion from the biological sample, and the second detectably labeled antibody and/
  • ELIS A methods can be used, wherein the wells of a microtiter plate are coated with the first antibody and/or a prion chimera. A biological sample containing or suspected of containing pathogenic prions is then added to the coated wells. After a period of incubation sufficient to allow any pathogenic prions to bind to the first antibody and/or the prion chimeras, the ⁇ late(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule, such as a second antibody or a prion chimera, added.
  • a detectably labeled secondary binding molecule such as a second antibody or a prion chimera
  • This secondary binding molecular is allowed to react with any captured sample prion, the plate washed and the presence of the labeled antibodies and/or labeled chimeras detected using methods well known in the art.
  • an antibody that can recognize both pathogenic and nonpathogenic prions and/or a prion chimera can be bound to the solid support.
  • the solid support is then combined with a sample (e.g., a biological sample) suspected of containing a pathogenic prion, under conditions which allow any prion proteins, both pathogenic and nonpathogenic, to bind to the first antibody and/or prion chimera.
  • the solid support can then be washed to remove any unbound materials.
  • a detectably labeled second antibody and/or a detectably labeled prion chimera can be added to the solid support under complex forming conditions.
  • the detectably labeled second antibody is specific for pathogenic prions (e.g., is generated using a prion chimera).
  • Complexes formed between the first antibody and/or the prion chimera, a pathogenic prion from the sample, and the second detectably labeled antibody and/or detectably labeled prion chimera can be detected.
  • the antibodies and chimeras of the invention may also be used in competition assays. Means of detection can be used to identify when an antigen weakly binding to an antibody specific to pathogenic prions is displaced by a pathogenic prion.
  • a first antibody specific to pathogenic prions is adsorbed onto a solid support.
  • the antibody is generated according to methods described herein.
  • the solid support is combined with a detectably labeled first antigen or ligand so that the first antigen ligand is bound to the first antibody (or prion chimera).
  • the binding affinity of the first antibody (or prion chimera) and the detectably labeled first antigen or ligand (or detectably labeled prion chimera) is weaker than the binding affinity of the first antibody (prion chimera) and a pathogenic prion.
  • the support is combined with a sample (e.g., a biological sample) suspected of containing a pathogenic prion, under conditions that allow the pathogenic prion, when present in the sample, to bind to the first antibody (or prion chimera) and replace the first detectably labeled antigen or ligand.
  • a sample e.g., a biological sample
  • the first antibody (or prion chimera) and pathogenic prions from the biological sample can be detected.
  • detection can be accomplished by, for instance, comparing the amount of detectably labeled first antigen or ligand present on the support prior to exposure to the biological sample with the amount present after exposure to the biological sample.
  • kits with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above.
  • the kit may additionally or alternatively comprise such antibodies and/or prion chimeras adsorbed onto one or more solid supports.
  • the kit may further contain suitable positive and negative controls, as described above.
  • the kit can also contain, depending on the particular immunoassay used, suitable labels ad other packaged reagents and materials (i.e., wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.
  • the invention is directed to solid supports comprising an antibody specific to pathogenic prions, an antibody capable of binding to both pathogenic and nonpathogenic prions, and/or a prion chimera.
  • Methods of producing these solid supports are also provided, for example by (a) providing a solid support; and (b) binding thereto one or more moieties selected from the group consisting of an antibody specific to pathogenic prions, an antibody capable of binding to both pathogenic and nonpathogenic prions, and a prion chimera.
  • the polyclonal or monoclonal antibodies may further be used to isolate disease form proteins by immunoaffinity columns.
  • the antibodies can be affixed to a solid support by, for example, adsorption or by covalent linkage so that the antibodies retain their immunoselective activity.
  • spacer groups may be included so that the antigen binding site of the antibody remains accessible.
  • the immobilized antibodies can then be used to bind the target from a biological sample, such as blood or plasma.
  • the bound proteins or complexes are recovered from the column matrix by, for example, a change in pH.
  • Biological samples that can be tested according to the invention include any sample amenable to an antibody assay, including blood and tissue samples.
  • the invention further relates to immunogenic compositions comprising the antibodies and prion chimeras (and polynucleotides encoding the chimeras and/or antibodies) and methods of using these compositions in therapeutic and prophylactic vaccines for the treatment or prevention of prion-related diseases.
  • the antibodies, prion chimeras (and polynucleotides encoding these antibodies and/or chimeras) can also be used in immunogenic (e.g., vaccine) compositions, individually or in combination, for prophylactic (i.e., to prevent pathogenesis) or therapeutic (to treat disease following infection) purposes.
  • the immunogenic compositions can comprise mixtures of one or more of the antibodies, chimeras and/or polynucleotides. These molecules may be obtained from a variety of sources, for example, recombinantly produced protein, synthetically produced proteins, etc.
  • the vaccine may also be administered in conjunction with other antigens and immunoregulatory agents, for example, immunoglobulins, cytokines, lymphokines, and chemokines, including but not limited to 3L-2, modified 3L-2 (cysl25- serl25), GM-CSF, IL-12, alpha- or gamma-interferon, IP-10, MIP1 and RANTES.
  • the vaccines may be adrninistered as polypeptides or, alternatively, as naked nucleic acid vaccines (e.g., DNA), using viral vectors (e.g., retroviral vectors, adenoviral vectors, adeno- associated viral vectors, alphaviral vectors) or non-viral vectors (e.g., liposomes, particles coated with nucleic acid or protein).
  • viral vectors e.g., retroviral vectors, adenoviral vectors, adeno- associated viral vectors, alphaviral vectors
  • non-viral vectors e.g., liposomes, particles coated with nucleic acid or protein.
  • the immunogenic compositions may also comprise a mixture of protein and nucleic acid, which in turn may be delivered using the same or different modalities and/or vehicles.
  • the vaccine may be given more than once (e.g., a "prime" administration followed by one or more "boosts”) to achieve
  • the same composition can be administered as the prime and as the one or more boosts.
  • different compositions can be used for priming and boosting.
  • the methods of the invention comprise administering an immunogenic composition comprising a prion chimera, an antibody specific for pathogenic prions and/or polynucleotides encoding these chimeras or antibodies to an animal.
  • the immunogenic compositions used in the invention preferably comprise an immunologically effective amount of these components.
  • An "immunologically effective amount" is an amount sufficient to allow the mammal to raise an immune response to a prion protein, preferably a pathogenic prion. Still more preferably, the immune response is directed against the ⁇ -helical conformation of pathogenic prions.
  • the immune response generally involves the production of antibodies specific to the prion chimera and the pathogenic prion conformation.
  • the amount of antibodies produced will vary depending on several factors including the animal used, the presence of an adjuvant, etc.
  • the immunogenic compositions of the invention may further comprise one or more adjuvants.
  • Adjuvants suitable for use in the invention include one or more of the following: - E.coli heat-labile enterotoxin ("LT”), or detoxified mutants thereof, such as the K63 or R72 mutants; cholera toxin ("CT”), or detoxified mutants thereof; microparticles (i.e., a particle of -lOOnm to ⁇ 150 ⁇ m in diameter, more preferably ⁇ 200nm to ⁇ 30 ⁇ m in diameter, and most preferably ⁇ 500nm to ⁇ 10 ⁇ m in diameter) formed from materials that are biodegradable and non-toxic (e.g.
  • an immunostimulatory oligonucleotide e.g. a CpG oligonucleotide
  • a saponin see International patent application WO 00/62800
  • immunostimulatory double stranded PvNA aluminum compounds (e.g. aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, oxyhydroxide, orthophosphate, sulfate etc. (e.g. see chapters 8 & 9 of Vaccine design: the subunit and adjuvant approach, eds.
  • Vaccine design or mixtures of different aluminum compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous etc.), and with adsorption being prefe ⁇ ed; MF59 (5% Squalene, 0.5% Tween 80, and 0.5% Span 85, formulated into submicron particles using a microfluidizer) (see Chapter 10 of Vaccine design; see also International patent application WO 90/14837); liposomes (see Chapters 13 and 14 of Vaccine design); ISCOMs (see Chapter 23 of Vaccine design); SAF, containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121, and thr-MDP, either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion (see Chapter 12 of Vaccine design); P biTM adjuvant
  • a polyoxyethylene ether or a polyoxyethylene ester International patent application WO 99/52549
  • a polyoxyethylene sorbitan ester surfactant in combination with an octoxynol International patent application WO 01/21207
  • a polyoxyethylene alkyl ether or ester surfactant in combination with at least one additional non-ionic surfactant such as an octoxynol
  • an immunostimulatory oligonucleotide e.g.
  • Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D- isoglutamine (thr-MDP), N-acteyl-normuramyl-L-alanyl-D-isogluatme (nor-MDP), N- acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(l'-2'-dipalmitoyl-5 , n-glycero-3- huydroxyphosphoryloxy)-ethylamine (MTP-PE), etc.
  • Other adjuvants suitable for mucosal or parenteral administration are also available (e.g.
  • Vaccine design the subunit and adjuvant approach, eds. Powell & Newman, Plenum Press 1995 (ISBN 0-306-44867-X).
  • Mutants of LT are preferred adjuvants (e.g., mucosal adjuvants), in particular the "K63" and "R72" mutants (e.g. see International patent application WO 98/18928), as these result in an enhanced immune response.
  • Microparticles are also useful as adjuvants.
  • PHA poly(lactide)
  • PLG poly(D,L-lactide-co-glycolide)
  • PLGA poly(D,L-lactide-co-glycolide)
  • the microparticles may be derived from any of various polymeric starting materials that have a variety of molecular weights and, in the case of the copolymers such as PLG, a variety of lactide:glycolide ratios, the selection of which will be largely a matter of choice, depending in part on the coadministered antigen.
  • the prions, antibodies, polynucleotides and/or adjuvants of the invention may be entrapped within the microparticles, or may be adsorbed to them. Entrapment within PLG microparticles is preferred. PLG microparticles are discussed in further detail in Morris et al., (1994), Vaccine, 12:5 - 11, in chapter 13 of Mucosal Vaccines, eds. Kiyono et al.,
  • the vaccine compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect, as discussed above.
  • compositions of the invention can also be used in compositions of the invention, for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates.
  • mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates
  • organic acids such as acetates, proprionates, malonates, or benzoates.
  • Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art.
  • compositions of the invention can also contain liquids or excipients, such as water, saline, glycerol, dextrose, ethanol, or the like, singly or in combination, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents.
  • a carrier is optionally present which is a molecule that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.
  • one or more polypeptides in the immunogenic composition may be conjugated to a bacterial toxoid, such as toxoid from diphtheria, tetanus, cholera, etc.
  • Vaccine compositions ⁇ vill typically comprise a therapeutically effective amount of the molecules (chimeras) or nucleotide sequences encoding the same, antibodies directed to these molecules and any other of the above-mentioned components, as needed.
  • therapeutically effective amount is meant an amount that will induce a protective and/or therapeutic immunological response in the uninfected, infected or unexposed individual to whom it is administered.
  • Such a response will generally result in the development in the subject of a secretory, cellular and or antibody-mediated immune response to the vaccine.
  • a response includes but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M; the proliferation of B and T lymphocytes; the provision of activation, growth and differentiation signals to immunological cells; expansion of helper T cell, suppressor T cell, and/or cytotoxic T cell.
  • the antibodies of the invention are specific to the pathogenic prion conformation. Still more preferably, the antibodies are specific to the ⁇ -helical region of pathogenic prions.
  • the invention also comprises a composition comprising an effective amount of antibodies specific to pathogenic prions, for example generated using the methods described herein.
  • the invention further comprises a composition comprising an effective amount of antibodies specific to the ⁇ - helical region of a pathogenic prion.
  • a "therapeutically effective amount” will fall in a relatively broad range that can be determined through routine trials. The exact amount necessary will vary depending on the subject being treated; the age and general condition of the individual to be treated; the capacity of the individual's immune system to synthesize antibodies; the degree of protection desired; the severity of the condition being treated; the particular vaccine selected and its mode of administration, among other factors.
  • the immunogenic (e.g., vaccine) compositions of the invention are preferably pharmaceutically acceptable and pharmacologically acceptable.
  • compositions are preferably not biologically or otherwise undesirable, i.e., the material may be administered to an individual in a formulation or composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • the immunogenic compositions of the invention may be administered in a single dose, or as part of an administration regime.
  • Nucleic acids, antibodies and or peptides may be administered may be administered by any suitable modality including, but not limited to intramuscularly, intramucosally, subcutaneously, intradermally, transdermally, intravaginally, intrarectally, orally and/or intravenously.
  • the dosage regime may include priming and boosting doses, which may be administered mucosally, parenterally, or various combinations thereof.
  • one or more components of the immunogenic compositions are administered parenterally or mucosally.
  • Suitable routes of parenteral administration include intramuscular (IM), subcutaneous, intravenous, intraperitoneal, intradermal, transcutaneous, and transdermal (see e.g., International patent application WO 98/20734) routes, as well as delivery to the interstitial space of a tissue.
  • Suitable routes of mucosal administration include oral, intranasal, intragastric, pulmonary, intestinal, rectal, ocular and vaginal routes.
  • the immunogenic composition may be adapted for mucosal administration.
  • composition for oral administration, it may be in the form of tablets or capsules, optionally enteric-coated, liquid, transgenic plants, etc.
  • composition for intranasal administration, it may be in the form of a nasal spray, nasal drops, gel or powder.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the nucleic acid-containing immunogenic compositions e.g., vaccines
  • the nucleic acid-containing immunogenic compositions may be accomplished with or without viral vectors, as described above, by injection using either a conventional syringe or a gene gun, such as the Accell® gene delivery system (PowderJect Technologies, Inc., Oxford, England).
  • DNA into cells of the epidermis is particularly preferred as this mode of administration provides access to skin- associated lymphoid cells and provides for a transient presence of DNA in the recipient.
  • polynucleotide sequences coding for the above-described molecules can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells expressing the gene, or by deriving the gene from a vector known to include the same.
  • the desired gene can be isolated directly from cells and tissues containing the same, using standard techniques, such as phenol extraction and PCR of cDNA or genomic DNA. See, e.g., Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.
  • the gene of interest can also be produced synthetically, rather than cloned.
  • the nucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed.
  • the complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, e.g., Edge, Nature (1981) 292:756; Nambair et al., Science (1984) 223:1299; Jay et al., /. Biol. Chem. (1984) 259:6311; Stemmer, W.P.C., (1995) Gene 164:49-53.
  • the gene sequence encoding the desired chimera or antibody can be inserted into a vector. Insertions can be made within the coding sequence or at either end of the coding sequence.
  • Vectors may include control elements operably linked to the coding sequence, which allow for the expression of the gene in vivo in the subject species.
  • typical promoters for mammalian cell expression include the S V40 early promoter, a CMV promoter such as the CMV immediate early promoter, the mouse mammary tumor virus LTR promoter, the adeno virus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others.
  • transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon.
  • a sequence for optimization of initiation of translation located 5' to the coding sequence, is also present.
  • transcription terminator/polyadenylation signals include those derived from S V40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence. Enhancer elements may also be used herein to increase expression levels of the mammalian constructs.
  • Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBO J. (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence.
  • LTR long terminal repeat
  • the constructs may be uni-cistronic or, alternatively, multi-cistronic cassettes (e.g., bi-cistronic cassettes) can be constructed allowing expression of multiple antigens from a single mRNA using the EMC IRES, or the like.
  • the constructs can be used for nucleic acid immunization using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466.
  • Polynucleotides can be delivered either directly to the vertebrate subject or, alternatively, delivered ex vivo, to cells derived from the subject and the cells reimplanted in the subject.
  • retroviral systems have been developed for gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retro viral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems have been described (U.S. Patent No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A.D., Human Gene Therapy (1990) 1:5-14; Scarpa et al., Virology (1991) 180:849-852; Burns et al., Proc. Natl. Acad.
  • adenovims vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al.,
  • Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B.J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129; Kotin, R.M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.
  • Additional viral vectors that will find use for delivering the nucleic acid molecules encoding the antigens of interest include those derived from the pox family of viruses, including vaccinia virus and avian pox virus.
  • Vaccinia virus recombinants expressing the genes can be constructed as follows. The DNA is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells that are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome.
  • the resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and by picking viral plaques resistant thereto.
  • avipox viruses such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species.
  • an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
  • Picornavirus-derived vectors can also be used. (See, e.g., U.S. Patent Nos. 5,614,413 and 6,063,384).
  • Alphavirus genus such as, but not limited to, vectors derived from the Sindbis, Semliki Forest, and Venezuelan Equine Encephalitis viruses, will also find use as viral vectors for delivering the polynucleotides of the present invention.
  • Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al., J. Virol. (1996) 70:508-519; and International Publication Nos. WO 95/07995 and WO 96/17072; as well as, Dubensky, Jr., T.W., et al., U.S. Patent No.
  • a vaccinia based infection/transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest in a host cell.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays vibrant specificity in that it only transcribes templates bearing T7 promoters.
  • RNA RNA that is then translated into protein by trie host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. US ⁇ (1986) 83:8122-8126.
  • an amplification system can be used that will lead to high level expression following introduction into host cells.
  • a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase that in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene.
  • T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase .
  • Polynucleotides encoding chimeras and/or antibodies as described herein can also be delivered without a viral vector.
  • the construct can be packaged in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation is generally accomplished using liposomes that are able to stably bind or entrap and retain nucleic acid.
  • the ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid.
  • liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.
  • Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred.
  • Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081); and purified transcription factors (Debs et al., /. Biol. Chem. (1990) 265:10189-10192), in functional form. Cationic liposomes are readily available.
  • N[l-2,3-dioleyloxy)pro ⁇ yl]-N,N,N-triethyl-ammonium (DOTMA) liposomes are available under the trademark Lipofectin, from G3J3CO BRL, Grand Island, NY. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416).
  • Other commercially available lipids include (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
  • Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci.
  • DOTAP l,2-bis(oleoyloxy)-3-(trimethylarnmonio)propane liposomes.
  • anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
  • Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
  • the liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).
  • the various liposorne-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in METHODS OF IMMUNOLOGY (1983), Vol. 101, pp. 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Ada (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al, Biochem. Biophys. Res.
  • the DNA, antibodies and/or protein chimera(s) described herein can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta. (1975) 394:483-491. See, also, U.S. Patent Nos. 4,663,161 and 4,871,488.
  • the vaccine compositions (or components thereof) may also be encapsulated, adsorbed to, or associated with, particulate carriers. Such carriers present multiple copies of a selected antigen to the immune system and promote trapping and retention of antigens in local lymph nodes.
  • the particles can be phagocytosed by macrophages and can enhance antigen presentation through cytokine release.
  • particulate earners include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al.,
  • Suitable microparticles may also be manufactured in the presence of charged detergents, such as anionic or cationic detergents, to yield microparticles with a surface having a net negative or a net positive charge.
  • charged detergents such as anionic or cationic detergents
  • microparticles manufactured with anionic detergents such as hexadecyltrimethylammonium bromide (CTAB), i.e. CTAB-PLG microparticles, adsorb negatively charged macromolecules, such as DNA.
  • CTAB hexadecyltrimethylammonium bromide
  • particulate systems and polymers can be used for the in vivo or ex vivo delivery.
  • polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest.
  • DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods.
  • Peptoids Zuckerman, R.N., et al., U.S. Patent No. 5,831,005, issued November 3, 1998, herein incorporated by reference
  • biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering polynucleotides of the present invention.
  • the particles are coated with the polynucleotide(s) and/or polypeptides to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun.”
  • a gun powder discharge from a "gene gun” For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Patent Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744.
  • needle-less injection systems can be used (Davis, H.L., et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, OR).
  • the methods of the invention further comprise treating or preventing a prion-relating disease by administering to an animal a composition comprising an effective amount of the antibodies of the invention.
  • Methods of treatment may combine both immunogenic compositions and antibody compositions.
  • the invention comprises a method for treating or preventing a prion-related disease comprising administering an immunogenic composition comprising an immunologically effective amount of a prion chimera and administering an effective amount of antibodies specific to pathogenic prions.
  • the immunogenic composition and the antibodies may be administered together or separately.
  • the invention further comprises a composition comprising an immunogenic composition comprising an immunologically effective amount of a prion chimera and further comprising an effective amount of antibodies specific to pathogenic prions.
  • the chimeras, polynucleotides and/or antibodies used in the invention can be administered to an animal.
  • Animals suitable for use in the methods of the invention include humans and other primates, including non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses, domestic animals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese and the like.
  • Animals suitable for use in the invention can be of any age, including both adult and newborn. Transgenic animals can also be used in the invention.
  • compositions of the invention can be used to treat or prevent prion-related diseases.
  • prion-related diseases include a disease cause in whole or in part by a pathogenic prion particle (PrP Sc ).
  • Prion-related diseases include scrapie, bovine spongiform encephalopathies (BSE), mad cow disease, feline spongiform encephalopathies, kuru, Creutzfeldt- Jakob Disease (CJD), Gerstmann-Strassler-Scheinker Disease (GSS), and fatal familial insomnia (EFI).
  • BSE bovine spongiform encephalopathies
  • CJD Creutzfeldt- Jakob Disease
  • GSS Gerstmann-Strassler-Scheinker Disease
  • EXAMPLES The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.
  • DNA sequences were amplified directly from Bordatella pertussis strains W28 and SA1 using standard PCR techniques. PrP DNA sequences were also amplified from genomic DNA using standard PCR techniques. The chimeras were then created by taking advantage of unique restriction sites within the natural sequences. All constructs were made in the non- expressing plasmid vector pUCl 8 and later excised and religated into the Stratagene pET3a plasmid vector. Nucleic acids encoding a leader sequence (e.g., a tpa leader sequence) may also be included in the constructs. (See, e.g., FIGs.
  • A. Purification Bacteria were cracked open by 2 rounds of freezing and thawing at -80 degrees Celsius and room temperature respectively. The bacteria were further disrupted with 5 one minute rounds of sonication with a Branson Sonifier Microtip near the energy limit for the microtip. The bacteria were then centrifuged for 45 minutes at 16,000 rpm in a Beckman JA20 rotor. For GCA chimera purification the supernatant was decanted and saved for the next step in the purification. For P.69 purification the supernatant was discarded and the pellet was saved for the next step in the purification.
  • A.l. P.69 Purification P.69 was purified from inclusion bodies. The pellet fraction following centrifugation was resolublized in 6M guanadine-HCl overnight. The solublized material was slowly adjusted to 2M guanadine-HCl and then dialyzed against PBS, changing the buffer every hour for five hours. Precipitate was collected by centrifugation for 30 minutes at 16,000 rpm in a Beckman JA20 rotor. Expression levels and resolublization was evaluated by running SDS-PAGE gels of the supernatant and pellet fractions. In general the protein would be
  • GCA was purified to a high degree of homogeneity (>95%) by passing the supernatant over a Ni-NTA resin (Qiagen) and eluting with a gradient of increasing imidizole concentration. All purifiable constructs eluted between 200 and 400 mM imidizole. Fractions were analyzed using SDS-PAGE. Protein samples were further purified by reducing the pooled GCA containing fractions in lOmM DTT followed by size exclusion chromatography and buffer exchange using a Superdex 75 column (Pharmacia) in PBS. This protein was then used directly for a variety of experiments including CD spectrometery, direct binding assays with PrP Sc , and as an antigen for the generation of polyclonal antibodies in rabbits (Josman, LLC).
  • Example 3 Chimera Binding Assays Chimeras are tested for their ability to directly bind to prion proteins by incubating either prion infected mouse brains (both treated and untreated with proteinase K) or normal mouse brains with fusion protein coated nickel resin over night. The resin is washed extensively, and bound protein is eluted from the resin by incubating it with SDS-PAGE gel loading buffer containing 500 mM imidizole. The eluted protein is detected by Western blotting with an antibody that recognizes denatured PrP.
  • Example 4 Antibody Production The following provides an example of a protocol that can be used to generate antibodies to the prion chimeras of the invention. Mice are immunized with a composition comprising a prion chimera either TM
  • Freund's adjuvant complete, is used as an adjuvant for the first injection followed by Incomplete Freund's adjuvant for the remaining infections, except for the IV infection.
  • the 3N injections are prepared in saline.
  • SEQ 3D ⁇ OS. 14 - 153 are non-limiting examples several prion chimeras which can be used in this procedure.
  • Example 5 Antibody Production The following provides an example of a protocol that can be used to generate antibodies to the prion chimeras of the invention.
  • Mice are immunized with a composition comprising a polynucleotide encoding for a prion chimera either IM (intramuscular) or IP (intraperitoneal) on day 0, followed by 2 - 5 boosts at intervals of not more frequently than every 2 weeks. Blood is collected before the first immunization and then 7 days following each boost to monitor the humoral response to the antigen. 6 orbital eye bleeds are taken from each animal (three from each eye) of approximately 0.2 mis or less per bleed. The final boost is delivered by IV (intravenous) injection.
  • IM intramuscular
  • IP intraperitoneal
  • mice are euthanized by exposure to CO 2 or isofluorane followed by cervical dislocation. Spleens are then harvested for hybridoma production.
  • Adjuvants suitable for polynucleotide administration can optionally be used in the administered compositions.
  • the polynucleotides can be adsorbed onto a microparticle, such as a poly(lactide-co-glycolide) (PLG) microparticle.
  • PLG poly(lactide-co-glycolide)
  • Polynucleotides encoding for SEQ 3D NOS. 14 - 125 are examples of polynucleotides that can be used in this procedure.
  • Table 1 summarizes selected characteristics of exemplary constructs encoding various exemplary prion chimeras. Sequence Identification Numbers are given relative to the chimera encoded by the construct.
  • pAbs refers to generation of polyclonal antibodies.
  • TBD refers to experiments to be done.

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Abstract

L'invention concerne des chimères de prions, des anticorps spécifiques aux prions, des polynucleotides codant pour lesdits chimères et anticorps et des procédés pour générer des anticorps à partir de chimères. L'invention concerne en outre des procédés d'utilisation desdits chimères et anticorps pour détecter la présence de prions pathogènes dans un échantillon biologique et des procédés d'utilisation desdits anticorps, chimères et/ou polynucléotides en tant que composants d'un vaccin thérapeutique ou prophylactique.
PCT/US2003/031057 2003-09-10 2003-09-30 Chimeres de prions et ses utilisations WO2005034995A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8524651B2 (en) 2003-10-24 2013-09-03 Immunaid Pty Ltd. Method of therapy
US9122778B2 (en) 2009-05-27 2015-09-01 Biotempus Limited Methods of treating diseases

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020150571A1 (en) * 1995-09-14 2002-10-17 Prusiner Stanley B. Antibodies specific for native PrPSc
US6602672B1 (en) * 1994-05-13 2003-08-05 The Regents Of The University Of California Recombinant construct encoding epitope tagged PrP protein
US6620629B1 (en) * 1997-02-21 2003-09-16 The Regents Of The University Of California Method for detecting prions

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6602672B1 (en) * 1994-05-13 2003-08-05 The Regents Of The University Of California Recombinant construct encoding epitope tagged PrP protein
US20020150571A1 (en) * 1995-09-14 2002-10-17 Prusiner Stanley B. Antibodies specific for native PrPSc
US6620629B1 (en) * 1997-02-21 2003-09-16 The Regents Of The University Of California Method for detecting prions

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8524651B2 (en) 2003-10-24 2013-09-03 Immunaid Pty Ltd. Method of therapy
US9122778B2 (en) 2009-05-27 2015-09-01 Biotempus Limited Methods of treating diseases
US9239904B2 (en) 2009-05-27 2016-01-19 Biotempus Limited Computer systems for treating diseases
US9268908B2 (en) 2009-05-27 2016-02-23 Biotempus Limited Computer systems for treating diseases
US10714208B2 (en) 2009-05-27 2020-07-14 Biotempus Pty Ltd Computer systems for treating diseases

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