WO2008124646A2 - Utilisation de protéines amyloïdes en tant qu'échafaudages pour des vaccins - Google Patents

Utilisation de protéines amyloïdes en tant qu'échafaudages pour des vaccins Download PDF

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WO2008124646A2
WO2008124646A2 PCT/US2008/059499 US2008059499W WO2008124646A2 WO 2008124646 A2 WO2008124646 A2 WO 2008124646A2 US 2008059499 W US2008059499 W US 2008059499W WO 2008124646 A2 WO2008124646 A2 WO 2008124646A2
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seq
protein
amyloid
mycobacterium tuberculosis
amyloid protein
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WO2008124646A3 (fr
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Amy Rosenberg
Erich James Keller
Robert Tycko
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/164—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
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    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
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    • A61K39/02—Bacterial antigens
    • 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
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    • A61K39/0208—Specific bacteria not otherwise provided for
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    • A61K39/025—Enterobacteriales, e.g. Enterobacter
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    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/05—Actinobacteria, e.g. Actinomyces, Streptomyces, Nocardia, Bifidobacterium, Gardnerella, Corynebacterium; Propionibacterium
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    • A61K39/07—Bacillus
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    • A61K39/08—Clostridium, e.g. Clostridium tetani
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/09—Lactobacillales, e.g. aerococcus, enterococcus, lactobacillus, lactococcus, streptococcus
    • A61K39/092—Streptococcus
    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
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    • A61K39/104—Pseudomonadales, e.g. Pseudomonas
    • A61K39/1045—Moraxella
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/107—Vibrio
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • 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
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker
    • 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
    • A61K2039/64—Medicinal preparations containing antigens or antibodies characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Embodiments in the present disclosure relate to the use of amyloid proteins as scaffolds for the display of heterologous peptides. More specifically, some embodiments relate to amyloid proteins, which present at least one heterologous target peptide containing an antigenic sequence or at least one epitope and the use of these compositions to induce an immune response to said heterologous target peptide, antigenic sequence, or epitope. Methods of making and using these compositions to prevent and/or treat human disease are also embodied. Description of the Related Art
  • Amyloid proteins are composed of peptides whose chemical properties are such that they spontaneously aggregate in vitro or in vivo, assuming parallel or antiparallel beta sheet configurations (Tycko, R. 2006 Quarterly Reviews of Biophysics 39:1-55). Amyloid proteins can arise from peptides that may differ in primary amino acid sequences but assume similar tertiary and quaternary structures.
  • an immunogen or vaccine as disclosed herein is stable at room temperature.
  • immunogens or vaccines are relatively inexpensive and safe.
  • Some embodiments comprise an immunogenic composition comprising an amyloid protein and a heterologous peptide that comprises at least one antigen.
  • the heterologous peptide is chemically linked to the amyloid protein.
  • amyloid protein has been modified to contain a chemically-reactive amino acid and said heterologous peptide is chemically linked to the amyloid protein at the chemically reactive amino acid.
  • heterologous peptide and the amyloid protein are joined to form a fusion protein.
  • the heterologous peptide is joined to the N-terminus or the C-terminus of the amyloid protein.
  • amyloid protein is CspA (E. coli cold shock protein A) or retro-CspA (E. coli cold shock protein A in backbone reversed form).
  • the heterologous peptide comprises a sequence selected from the group consisting of: Influenza B: PAKLLKERGFFGAIAGFIE (SEQ ID NO: 44);
  • GNAEVHASFFDIG SEQ ID NO: 45
  • Vibrio cholera VEVPGSQHIDSQKKA
  • Foot and mouth disease VPNLRGDLQVLAQKVARTLP
  • Listeria GYKDGNEYI (SEQ ID NO: 48); Shistosomiasis: GFTTNEERYNVFAE (SEQ ID NO: 49);
  • Tetanus toxin CAGSSRSALVAGC (SEQ ID NO: 50); Streptococcus pneumoniae PspA - KLEELSDKIDELDAE (SEQ ID NO: 51); Streptococcus pneumoniae PspA- SQKKYDEDQKKTEEKAALEKA- ASEEMDKAVAAVQQA (SEQ ID NO: 52); Cryptosporidium P23 parvum - QDKPADAP AAEAP AAEP A AQQ DKPADA
  • HIV P24 - GPKEPFRDYVDRFYKC (SEQ ID NO: 54); HIV GP 120 - RKRIHIGPGRAFYITKN (SEQ ID NO: 55); Foot and Mouth Disease Virus VPl - YNGECR YNRN A VPNLRGDLQV LAQKVARTLP (SEQ ID NO: 56);
  • Corynebacterium toxin - FQVVHNSYNRPAYSPGC (SEQ ID NO: 57); diphtheriae Borrelia burgdorferi OspA - VEIKEGT VTLKREIDKN GKVT (SEQ ID NO: 58);
  • Influenza A8/PR8 - SSVSSFERFEC (SEQ ID NO: 60);
  • Influenza A8/PR8 - YRNLLWLTEK (SEQ ID NO: 61); Yersinia pestis
  • Haemophilus pBOMP -CSSSNNDAAGNGAAQFGGY (SEQ ID NO: 63); influenzae Haemophilus pBOMP - NKLGTVSYGEE (SEQ ID NO: 64); influenzae Haemophilus pBOMP - NDEAAYSKNRRAVLAY (SEQ ID NO: 65); influenzae Moraxella catarrhalis copB - LDIEKDKKKRTDEQLQAELDD KYAGKGY (SEQ ID NO: 66);
  • Porphyromonas gingivalis - GVSPKVCKDVTVEGSNEFAPVQNLT SEQ ID NO: 69
  • Porphyromonas gingivalis - RIQSTWRQKTVDLP AGTKYV SEQ ID NO: 70
  • Trypanosoma cruzi - KAAIAPAKAAAAPAKAATAPA (SEQ ID NO: 72);
  • Plasmodium falciparum MSPl - SVQIPKVPYPNGIVYC (SEQ ID NO: 73);
  • Plasmodium falciparum MSPl - DFNHYYTLKTGLEADC (SEQ ID NO: 74); Streptococcus sobrinus Agl/II - KPRPIYEAKLAQNQKC (SEQ ID NO: 75);
  • Streptococcus sobrinus Agl/II - AKADYEAKLAQYEKDLC SEQ ID NO: 76
  • lymphocytic Choriomeningitis virus - RPQASGVYMGNLTAQC SEQ ID NO: 77
  • Shigella flexneri Invasin - KDRTLIEQK (SEQ ID NO: 78); Respiratory Synctial Virus G - CSICSNNPTCWAICK (SEQ ID NO: 79); Plasmodium vivax CS - GDRADGQP AGDRADGQP AG (SEQ ID NO: 80); Clostridium tetani tox - QYIKANSKFIGITELC (SEQ ID NO: 81); Entamoeba histolytica - VECASTVCQNDNSCPIIADVE KCNQ (SEQ ID NO: 82); Schistosoma japonicum - DLQSEISLSLENGELIRRAKSAESLASELQRRVD
  • Schistosoma mansoni - DLQSEISLSLENSELIRRAKAAESLASDLQRRVD SEQ. ID. No. 84
  • Plasmodium vivax - DRAAGQPAGDRADGQPAG (SEQ ID NO: 85); Influenza virus - CNNPHRIL (SEQ ID NO: 86);
  • Influenza virus - CPKYVKQNTLKLATGMRNVPE KQTR (SEQ ID NO: 87); Influenza virus - SIMRSDAPIGTCSSECITPNGSIPNDKPFQNVNKITY (SEQ ID NO: 88);
  • Influenza virus - RGIFGAIAGFIENGWEGMIDGWYGFRHQN (SEQ ID NO: 89); Influenza virus - EKQTRGIFGA (SEQ ID NO: 90);
  • Mycobacterium tuberculosis - ARRGLERGLNALADAVKV (SEQ ID NO: 96); Mycobacterium tuberculosis - EKIGAELVKEVAKK (SEQ ID NO: 97); Mycobacterium tuberculosis - GLKRGIEKAVEKVTETL (SEQ ID NO: 98); Mycobacterium tuberculosis - IED AVRNAKAA VEEG (SEQ ID NO: 99); Plasmodium falciparum - NANPNANPNANP (SEQ ID NO: 100); and
  • Circumsporozoite - QAQGDGANAGQP (SEQ ID NO: 101).
  • the heterologous peptide is fused to the amyloid protein through a linker.
  • the linker comprises (gly)n-, wherein n>2.
  • Some aspects encompass an immunogenic composition comprising a nucleic acid encoding an amyloid protein and a heterologous peptide that comprises at least one antigen.
  • the heterologous peptide is joined to the N- terminus or the C-terminus of said amyloid protein.
  • the amyloid protein is CspA (E. coli cold shock protein A) or retro-CspA (E. coli cold shock protein A in backbone reversed form).
  • Some embodiments encompass a method of inducing an immune response in a subject comprising providing to the subject an immunogenic composition described herein and measuring the immune response.
  • the method of inducing an immune response in a subject further comprises identifying the subject as an individual in need of an immune response to a pathogen.
  • Some embodiments entail using of any one of the immunogenic compositions of described herein to prepare a medicament for the purpose of inducing an immune response to a pathogen.
  • Some aspects encompass a method of making an immunogenic composition as described herein, comprising: providing an amyloid protein that comprises a chemically reactive amino acid; providing a heterologous peptide that comprises at least one antigen and a chemically reactive amino acid; and coupling said heterologous peptide to said amyloid protein.
  • amyloid protein has been modified to incorporate a chemically reactive amino acid.
  • heterologous peptide has been modified to incorporate at least one chemically reactive amino acid.
  • the amyloid protein is derived from a bacterial source.
  • amyloid protein is selected from the group consisting of E. coli CsgA curlin protein, AgfA Tafi protein, and a Chaplin protein.
  • the amyloid protein is derived from a fungal source.
  • amyloid protein is a hydrophobin.
  • amyloid protein is derived from bacteriophage T7. In some embodiments, the amyloid protein is bacteriophage T7 endonuclease.
  • amyloid protein is designed de novo.
  • amyloid protein is A ⁇ l-40.
  • Figure 1 Transmission electron micrograph showing amyloid fibrils with aggregated A ⁇ 1 -40 peptide. Scale bar: 100 nm.
  • FIG. 1 Transmission electron micrograph showing amyloid fibrils with aggregated ⁇ -amyloid 1-40 peptide containing a tetanus toxin peptide (CAGSSRSALVAGC, SEQ ID NO: 1) at the N-terminus. Scale bar: 500 nm.
  • Figure 3 Schematic diagram of amyloid structure showing regular array of N- Terminal molecules (gray ends without arrows).
  • Figure 4 Schematic diagram of amyloid structure with N-terminal ends modified by addition of peptide of interest (black ends without arrows).
  • Figure 5 Schematic diagram of amyloid structure modified with a linker to which a peptide of interest is attached (crosses).
  • FIG. 6 Transmission electron microscope images (left side) of amyloid fibrils formed by A ⁇ l ⁇ K) (amino-acid sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGA IIGLMVGGVV; SEQ ID NO: 2), amylin (amino acid sequence KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO: 3), with a disulfide bond between C2 and C7), and A ⁇ l 1-25 (amino-acid sequence EVHHQKL VFF AEDVG; SEQ ID NO: 4). Fibrils are negatively stained with uranyl acetate. Scale bars are 100 nm.
  • ChpAl (SEQ ID NO: 5); ChpA2 (SEQ ID NO: 6); ChpBl (SEQ ID NO: 7); ChpB2 (SEQ ID NO: 8); ChpCl (SEQ ID NO: 9); ChpC2 (SEQ ID NO: 10); ChpD (SEQ ID NO: 11); ChpE (SEQ ID NO: 12); ChpF (SEQ ID NO: 13); ChpG (SEQ ID NO: 14); ChpH (SEQ ID NO: 15); Consensus sequence (SEQ ID NO: 16).
  • Figure 9 Sequence of the rodA gene (nucleotide, (SEQ ID NO: 18); amino acid, (SEQ ID NO: 19). Putative TATA and CAAT boxes in the 5' region of the gene are double underlined. Two introns (49 and 46 bp) were identified by comparison with the rodA sequence of A. nidulans and are shown in lower case; the consensus splice signals are underlined. The putative translation open reading frame is given in one-letter code below the DNA sequence.
  • Figure 10 DNA sequence (SEQ ID NO: 20) of the transcriptional unit of MPGl and the predicted amino acid sequence of the translation product (SEQ ID NO: 21).
  • FIG. 11 Comparison of the polypeptide sequence predicted by MPGl with the fungal hydrophobins.
  • the predicted MPGl polypeptide sequence (SEQ ID NO: 162) was aligned with the known hydrophobins.
  • ScI, (SEQ ID NO: 157); Sc3, (SEQ ID NO: 159); and Sc4, (SEQ ID NO: 158) are proteins from S. commune; ssgA, (SEQ ID NO: 160); rodA, (SEQ ID NO: 161); and Eas, (SEQ ID NO: 164) are translated from genes of M. anisopilae, A. nidulans and N. crassa; cerato-ulmin, (SEQ ID NO: 163); is a peptide from O. ulmi.
  • the sequences were aligned based on the conserved cysteine residues (shown in bold) using the GCG PILEUP program. Highly conserved amino acid residues are shown in uppercase and as a consensus sequence.
  • FIG. 12 Amino acid sequence of T7 endonuclease I (SEQ ID NO: 22) expressed in E. coli. Numbering used is that of the wild type protein.
  • the sequence MGHHHHHHHHHHSSGHDDDDKHM (SEQ ID NO: 23) is the His-tag plus enterokinase site.
  • the first residue of the wild type protein (M) is at position 1.
  • FIG. 13 (A) Schematic illustration of the design of a combinatorial library of de novo proteins containing six ⁇ -strands (arrows) punctuated by turns. (B) Designed binary sequence pattern. Alternating pattern in the ⁇ -strands is indicated with polar residues (E) as white font in black background and nonpolar residues (F) as black font in gray background. Combinatorial diversity is incorporated at positions marked E, F, and t (turn). Fixed residues are incorporated at the termini and in some of the turns.
  • FIG. 14 Construction of a combinatorial library of genes.
  • Each single-stranded DNA oligonucleotide encodes degenerate sequences in the ⁇ -strands and in turns 1 and 4.
  • Degenerate codons encoding polar residues are shown as (E)
  • those encoding nonpolar residues are shown as (F)
  • those encoding turns are shown as (t).
  • Fixed sequences occur in turns 2, 3, and 5 and also in the gene termini. These are shown in shadow font.
  • the fixed sequences at the 39 ends of each oligonucleotide allow annealing and priming for synthesis of complementary DNA. Elongation of complementary DNA by polymerase is indicated by arrows.
  • the entire procedure was carried out twice: once to construct the first three ⁇ -strands (and associated turns) and again to construct the last three ⁇ -strands (and associated turns).
  • the two libraries of double-stranded pieces were then used as PCR templates to construct a library of full length genes.
  • the non-degenerate sequence of turn no. 3 served as an internal annealing sequence, and oligonucleotides complementary to the N- and C- terminal sequences served as external primers.
  • Figure 15 Design of the cc ⁇ model system.
  • A Amino acid sequence of cc ⁇ (SEQ ID NO: 41). PositionsX 7 and Xi 4 are occupied by Ala and Leu residues, respectively, in cc ⁇ -p and by Met residues in cc ⁇ -Met. The heptad repeats (abcdefg), capping residues (N cap -acetyl and C cap -amide), and the sequence patterning of polar (p) and hydrophobic (h) residues are indicated.
  • B Helical wheel representation of cc ⁇ as seen along the helix axis from the N terminus.
  • C Schematic representation of cc ⁇ in its ⁇ -strand conformation as seen along the ⁇ -sheet plane.
  • FIG. 1 Cyclized B cell epitope. The amino acid sequence and strategy for selective oxidation of a chimeric measles virus fusion protein (MVF) epitope-HER-2/neu epitope is shown.
  • VMF virus fusion protein
  • amyloid proteins can be used as scaffolds or platforms on to which peptide determinants, antigenic sequences, or epitopes (e.g., an antigenic peptide containing a plurality of epitopes of a pathogen, such as bacteria, fungi, mold, virus, or a cancer cell) can be presented or displayed so as to induce an immune response to said peptide determinants, antigenic sequences, or epitopes.
  • a pathogen such as bacteria, fungi, mold, virus, or a cancer cell
  • the diversity of amyloid proteins that can be used with aspects of the invention is quite large.
  • an amyloid protein is selected because it is non-toxic in humans.
  • amyloid proteins that can be used with aspects of the invention described herein include one of bacterial origin, which should array and display peptides in a similar fashion as that of the human ⁇ -amyloid protein.
  • CspA is. coli cold shock protein A, 73 amino acids
  • retro-CspA, 92 or 93 residues have N-termini which may be modified with peptides of at least 14 amino acids and still retain amyloidogenic properties (Shukla A et al. 2003 Protein Engineering 16:875-879). Sequences of several amyloid proteins are also readily available on Genbank, for example.
  • the amyloid proteins used in the embodiments described herein can be made recombinantly or chemically synthesized by conventional techniques.
  • heterologous peptides, protein fragments, antigenic peptide sequences, or epitopes, which can be displayed on the amyloid proteins described herein can also be diverse and, preferably, have a length of at least, 5-10, 10-15, 20-25, 30-40, or 40-50 amino acids. That is, some embodiments include an amyloid protein described herein comprising a heterologous peptide that is at least or equal to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 , 27, 28, 29, 30, 35, 40, 45, or 50 amino acids, wherein said heterologous peptide comprises, consists of or consists essentially of at least one antigen or epitope to which an immune response can be raised.
  • antigenic peptides for incorporation into amyloid proteins include immunogenic sequences from influenza hemagluttinin, tetanus or botulinum toxin or pseudomonas pili peptide sequences.
  • Antigenic peptides can include any one or more of the following sequences, for example:
  • Influenza B PAKLLKERGFFGAIAGFIE (SEQ ID NO: 44) is the consensus sequence of influenza B virus in HA1/HA2 joining region. It is poorly immunogenic when injected as an unaltered peptide but produces good titers and protection when coupled to adjuvant (Bianchi E et al. 2005 J Virol 79:7380-7388.
  • GNAEVHASFFDIG SEQ ID NO: 45
  • PA anthrax Protective Antigen
  • Vibrio Cholera VEVPGSQHIDSQKKA (SEQ ID NO: 46): immunization of rabbits with this sequence conjugated to a protein carrier generates neutralizing antibody that neutralizes E. coli heat labile toxin (LT), inhibits adenylate cyclase activity mediated by LT and neutralized LT activity in a rat ileal loop model (Jacob CO et al. 1984 EMBO 3 :2889-2893).
  • LT E. coli heat labile toxin
  • Foot and mouth disease the peptide sequence of amino acids 141-160 of the VPl protein of foot and mouth disease virus (FMDV) elicits virus neutralizing antibodies that protect a variety of animals when administered in a variety of adjuvant settings, but the most potent is linkage to the hepatitis B core protein, which arrays them at high density: VPNLRGDLQVLAQKVARTLP (SEQ ID NO: 47) Clarke BE et all 987 Nature 330: 381-384. 5) Listeria; Immunity to Listeria is mediated principally by CTL to the listeriolysin O toxin, essential for promoting escape of organisms from the phagolysosome to the cytosol and thence to infection of other cells.
  • FMDV foot and mouth disease virus
  • a peptide sequence from LLO, residues 91-99 has been found to be the dominant epitope in LLO for generation of CTL and protection against lethal challenge: GYKDGNEYI (SEQ ID NO: 48).
  • the 9 residues in the N-terminus of amyloid- ⁇ with LLO91-99 have been substituted by the present inventors and was found to generate amyloid fibrils, which will be sonicated and tested in a protection model (Pamer E. 2004. Nature Rev Immunol 4: 812-823).
  • Shistosomiasis administration of the 9B peptide-1 adjuvanted to flagellin induced serum antibodies that recognized schistosomula and cercariae and reduced infection by 30-53%.
  • GFTTNEERYNVFAE SEQ ID NO: 49
  • Tetanus toxin Tetanus toxin.
  • a tetanus toxin peptide CAGS SRSALVAGC, SEQ ID NO: 50
  • Fig. 2 aggregated protein
  • Such fibrils were sonicated prior to injection. 2ug of sonicated fibrils in lOOul of PBS were injected, which can be sonicated and injected into mice. Such immunization was safe.
  • Streptococcus pneumoniae PspA - KLEELSDKIDELDAE SEQ ID NO: 51
  • Streptococcus pneumoniae PspA - KLEELSDKIDELDAE SEQ ID NO: 51
  • Streptococcus pneumoniae PspA- SQKKYDEDQKKTEEKAALEKA- ASEEMDKAVAAVQQA (SEQ ID NO: 52);
  • Foot and Mouth Disease Virus VPl - YNGECRYNRNAVPNLRGDLQV LAQKVARTLP (SEQ ID NO: 56); Corynebacterium toxin - FQVVHNSYNRP AYSPGC (SEQ ID NO: 57); diphtheriae Borrelia burgdorferi OspA - VEIKEGTVTLKREIDKNGKVT (SEQ ID NO: 58);
  • Influenza A8/PR8 - YRNLLWLTEK (SEQ ID NO: 61);
  • Porphyromonas gingivalis - GVSPKVCKDVTVEGSNEF APVQNLT SEQ ID NO: 69
  • Trypanosoma cruzi - SHNFTLVASVIIEEAPSGNTC SEQ ID NO: 71
  • Trypanosoma cruzi - KAAIAP AKAAAAP AKAATAPA SEQ ID NO: 72;
  • Plasmodium falciparum MSPl - SVQIPKVPYPNGIVYC (SEQ ID NO: 73);
  • Plasmodium falciparum MSPl - DFNHYYTLKTGLEADC (SEQ ID NO: 74);
  • Streptococcus sobrinus Agl/II - KPRPIYEAKLAQNQKC SEQ ID NO: 75;
  • Streptococcus sobrinus Agl/II - AKADYEAKLAQYEKDLC SEQ ID NO: 76
  • lymphocytic Choriomeningitis virus - RPQASGVYMGNLTAQC SEQ ID NO: 76
  • Shigella flexneri Invasin - KDRTLIEQK (SEQ ID NO: 78);
  • Respiratory Synctial Virus G - CSICSNNPTCWAICK (SEQ ID NO: 79); Plasmodium vivax CS - GDRADGQPAGDRADGQPAG (SEQ ID NO: 80);
  • Clostridium tetani tox - QYIKANSKFIGITELC (SEQ ID NO: 81);
  • Schistosoma mansoni - DLQSEISLSLENSELIRRAKAAESLASDLQRRVD SEQ. ID. No. 84
  • Plasmodium vivax - DRAAGQP AGDRADGQP AG (SEQ ID NO: 85); Influenza virus - CNNPHRIL (SEQ ID NO: 86);
  • Influenza virus - CPKYVKQNTLKLATGMRNVPE KQTR (SEQ ID NO: 87);
  • Influenza virus - SIMRSDAPIGTCSSECITPNGSIPNDKPFQNVNKITY SEQ ID NO: 88;
  • Influenza virus - RGIFGAIAGFIENGWEGMIDGWYGFRHQN (SEQ ID NO: 89);
  • Influenza virus - EKQTRGIFGA (SEQ ID NO: 90);
  • Mycobacterium tuberculosis - AVLEDPYILLVSSKV (SEQ ID NO: 91);
  • Mycobacterium tuberculosis - LLVS SKVST VKDLLP (SEQ ID NO: 92);
  • Mycobacterium tuberculosis - LLPLLEKVIGAGKPL (SEQ ID NO: 93); Mycobacterium tuberculosis - AILTGGQVISEEVGL (SEQ ID NO: 94);
  • Mycobacterium tuberculosis - IAFNSGLEPGWAEK (SEQ ID NO: 95);
  • Mycobacterium tuberculosis - ARRGLERGLNALADAVKV (SEQ ID NO: 96);
  • Mycobacterium tuberculosis - EKIGAELVKEVAKK (SEQ ID NO: 97);
  • Mycobacterium tuberculosis - GLKRGIEKA VEKVTETL (SEQ ID NO: 98); Mycobacterium tuberculosis - IEDA VRNAKAA VEEG (SEQ ID NO: 99);
  • Plasmodium falciparum - NANPNANPNANP (SEQ ID NO: 100).
  • Circumsporozoite - QAQGDGANAGQP (SEQ ID NO: 101).
  • IEDB Immune Epitope Database and Analysis Resource
  • NIAID Infectious Diseases
  • NHIH National Institutes of Health
  • HHS Department of Health and Human Services
  • the IEDB contains data related to antibody and T cell epitopes for humans, non-human primates, rodents, and other animal species. Curation of data relating to NIAID Category A, B, and C priority pathogens, NIAID Emerging and Re-emerging infectious diseases, Malaria, Hepatitis B, Clostridium tetani, Leishmania, and Candida albicans is current through September 2007.
  • the IEDB Analysis Resource for predicting linear epitopes is available on the internet.
  • the epitope prediction tool uses a semi-empirical method for prediction of antigenic determinants on protein antigens (Kolaskar AS, Tongaonkar PC. 1990 FEBS Lett 276:172-174) or an alternative method for predicting linear B-cell epitopes described by Larsen, J.E. et al. 2006 Immunome Res 2:2.
  • Another tool for predicting epitopes can be found on the world wide web at epitope-informatics. Cancer Peptides Expressed in the Context of Amyloid Proteins
  • Peptides from proteins expressed as tumor associated antigens may be incorporated into the amyloid scaffold for elicitation of cellular and humoral immunity to cancer cells.
  • Peptides may be attached to amyloid scaffold proteins via linker, by "native chemical ligation" in which a peptide with a C-terminal thioester reacts with one with an N-terminal cysteine, by incorporation into the sequence via chemical synthesis, or by cloning the genetic sequence into a plasmid containing the amyloid sequences in frame.
  • linker by "native chemical ligation" in which a peptide with a C-terminal thioester reacts with one with an N-terminal cysteine, by incorporation into the sequence via chemical synthesis, or by cloning the genetic sequence into a plasmid containing the amyloid sequences in frame.
  • Non-limiting examples of such peptides are as follows: Breast Cancer Peptides:
  • HER-2/neu family The amino acid sequence of HER-2/neu (Homo sapiens ERBB2) is shown in Fig. 16. a) Thelper/CTL epitopes: p98-114 (RLRIVRGTQLFEDNYAL; SEQ ID NO: 102) and p369-386 (KIFGSLAFLPESFDGDPA; SEQ ID NO: 103) from extracellular domain of HER-2; p776-790 (GVGSPYVSRLLGICL; SEQ ID NO: 104) and p927-941 (PAREIPDLLEKGERL; SEQ ID NO: 105) from intracellular domain of HER-2 (Disis, M.L. 2003 Clinical Cancer Research 9:5559-5565.
  • MVF HER-2 p628-647 combines a measles virus fusion protein (MVF) epitope (amino acids 288-302) with p628-647 (INCTHSCVDLDDKGCPAEQR; SEQ ID NO :106) to engage T helper cells (Dakappagari N. 2000 Cancer Research 60:3782-89.
  • MVF measles virus fusion protein
  • Cyclized B cell epitope Peptides can be engineered to include a disulfide bond to form a conformationally correct HER-2/neu epitope (Dakappagari N. et al. 2005 J Biol Chem 280:54-63). Cyclization according to this method is shown in Fig. 17.
  • a peptide epitope from Mamaglobin-A comprises the MAM3 peptide sequence: FMQLIYDSSL (SEQ ID NO: 107), Goedegebuure PS et al. 2003 Surgery 133:74-80.
  • melanoma peptide epitopes include MAGE-Al %-i 04 (SLFRAVITK; SEQ ID NO: 108), MAGE-IO 254-262 (GLYDGMEHL; SEQ lD NO: 109), and GpIOO 614-622 (LIYRRRLMK; SEQ ID NO: 110) (Chianese-Bullock K et al. 2005 J Immunol 174:3080-3086).
  • tyrosinase peptide sequences including KCDICTDEY (SEQ ID NO: 1]
  • antigenic peptides or haptens as described herein may also contain one or more chemically-reactive amino acids that allow-for chemical coupling to an amyloid protein.
  • heterologous peptides, protein fragments or antigens can be chemically synthesized or recombinantly produced by available techniques in peptide chemistry and/or recombinant techniques.
  • heterologous peptides, antigens, or epitopes can be fused to the amyloid protein through chemical linkage or incorporated into the amyloid protein sequence itself (e.g., by recombinant techniques such that the peptide is expressed along with the amyloid protein as a fusion protein).
  • antigenic peptides may be readily chemically-linked to amyloid proteins by a number of techniques, as described below; depending on the amyloid protein used and the amount of antigenic peptide incorporation desired, the insertion of chemically- reactive amino acids into the amyloid protein at sites that preferably do not abolish aggregation or do not perturb folding or aggregation of the amyloid protein may be desired. Insertion of the chemically-reactive amino acids can be accomplished by PCR, site directed mutagenesis, or other techniques known in the art.
  • An introduced chemically-reactive amino acid residue has a chemically-reactive side chain that provides a functional group for derivatizing the amyloid protein (e.g., conjugating a hapten or antigenic peptide to the amyloid protein).
  • useful side chain functional groups include epsilon-amino groups, beta-or gamma-carboxyl groups, thiol (- SH) groups and aromatic rings (e.g. tyrosine and histidine).
  • the chemically-reactive amino acid residue is typically a lysine, cysteine, or histidine residue or a carboxyl-containing residue, such as aspartic acid or glutamic acid. Lysine is a particularly preferred chemically- reactive amino acid residue.
  • That activated carrier is then reacted with a polypeptide that either contains a terminal cysteine or to which an additional amino- or carboxy-terminal cysteine residue has been added to form a covalently bonded amyloid protein.
  • the amino group of a polypeptide hapten can be first reacted with N-succinimidyl 3-(2-pyridylthio)propionate (SPDP, Pharmacia), and that thiol-containing polypeptide can be reacted with the activated carrier after reduction.
  • SPDP N-succinimidyl 3-(2-pyridylthio)propionate
  • the sulfur-containing moiety and double bond-containing Michael acceptor can be reversed.
  • a large number of heterobifunctional agents that form a disulfide link at one functional group end and a peptide link at the other, including N- succidimidyl-3-(2-pyridyldithio) propionate (SPDP).
  • SPDP N- succidimidyl-3-(2-pyridyldithio) propionate
  • This reagent creates a disulfide linkage between itself and a thiol in either the amyloid protein or the hapten, for example a cysteine residue in a polypeptide hapten, and an amide linkage on the coupling partner, for example the amino on a lysine or other free amino group in the carrier protein.
  • a variety of such disulfide/amide forming agents are known. (See e.g., Immun. Rev. (1982) 62:185).
  • bifunctional coupling agents form a thioether rather than a disulfide linkage.
  • Many of these thioether-forming agents are commercially available and include reactive esters of 6- maleimidocaproic acid, 2-bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido- methyl)cyclohexane-l-carboxylic acid and the like.
  • the carboxyl groups can be activated by combining them with succinimide or 1 -hydroxy-2-nitro-4-sulfonic acid, sodium salt.
  • the particularly preferred coupling agent is succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC) obtained from Pierce Company, Rockford, 111.
  • SMCC succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate
  • the "native chemical ligation" reaction can be used to link the antigenic target peptide directly into the amyloid-forming peptide backbone (either before or after fibril formation).
  • the NCL reaction is a spontaneous reaction between a peptide with a C- terminal thioester and another peptide with an N-terminal cysteine (Kent, S. 2003 J Peptide Sci 9:574-593).
  • the immune response induced by the compositions described herein can be humoral and/or cell mediated.
  • a variety of assays are known to evaluate the type and strength of the immune response mounted to a composition described herein (e.g., production of antibodies, such as IgG, IgA, or IgM titer, neutralization, protection, and ELISPOT).
  • the type and/or strength of an immune response is measured before, during, or after providing a composition as described herein to a subject, preferably a human.
  • the amyloid structure presents a regular array of accessible N- terminal molecules (ends without arrows).
  • the N-terminal ends of the amyloid peptides can be directly modified with the peptide of interest. As shown in Fig. 4, the N-terminal peptide ends are replaced by the target peptide of interest (Black). By another approach, as shown in Fig. 5, the N-terminal peptides are modified with a linker to which the peptides of interest (crosses) are attached. This can be accomplished by introducing a cysteine near the N-terminus of the amyloid peptide, and cross-linking the sulfhydryl group of the cysteine side chain to a maleimide group on the antigen of interest.
  • amyloid protein may be modified to create a chimeric molecule, in essence "grafting" antigens of interest that already exist in a ⁇ -sheet configuration onto the amyloid scaffold.
  • a chimeric molecule is created by incorporating the functional central ⁇ -hairpin loop of a snake toxin, sequence 26-37 is onto the structurally similar ⁇ -hairpin of a scorpion toxin.
  • amyloidogenic peptides that produce different amyloid proteins is contemplated.
  • the A ⁇ peptide's C-terminal encephalitogenic residues 15-40 will be modified and replaced with amino acids favoring T cell responses to the immunogen/vaccine antigen (Brown, M.E. et al. 2005 Expert Opin Biol Ther 5:809-816).
  • amyloid proteins including those derived from microbial sources, may be used.
  • amyloid proteins generated de novo from first principles such as the 17 residue model system known as cc ⁇ (Kammerer, R. et al. 2004 Proc Natl Acad Sci USA 101:4435-4440) will be constructed. Such modifications are expected to enhance immunogenicity of the scaffold-target peptide compositions, by including foreign peptide sequences.
  • Some embodiments incorporate or attach a bacterial or viral protein or peptide, whose neutralization confers clinical benefit (such as influenza hemagluttinin, tetanus or botulinum toxin or pseudomonas pili peptide sequences) into the amyloid protein.
  • a target peptide sequence from anthrax toxin, to which an antibody response is predicted to be neutralizing is fused with an amyloid protein scaffold.
  • the amyloid fibrils are generated in vitro, subjected to sonication and injected subcutaneously into the host.
  • amyloid proteins assemble into fibrils that have repetitive organization, thereby providing a scaffold structure to which peptide epitopes can be linked. Accordingly, the amyloid protein/target peptide fusion proteins disclosed herein are designed to elicit mammalian immune responses.
  • Amyloid fibrils are filamentous structures resulting from the spontaneous aggregation of peptides and proteins (Tycko, R. 2006 Quarterly Review of Biophysics 39:1- 55).
  • Fig. 6 shows examples of amyloid fibrils as they appear in transmission electron microscope (TEM) images with negative staining. The fibrils are typically straight and unbranched, with diameters on the order of 10 nm and lengths ranging from 100 nm to many microns. Referring to Fig. 6, the amyloid fibril morphologies are similar, particularly for A ⁇ l-40 and amylin, despite the differences in sequence. X-ray fiber diffraction data show that amyloid fibrils contain cross- ⁇ structural motifs (right side).
  • Solid-state NMR data show that the cross- ⁇ motifs in A ⁇ 1 — 40 and amylin fibrils are comprised of parallel ⁇ - sheets (right side of schematic diagram), while those in A ⁇ l 1-25 fibrils are comprised of antiparallel ⁇ -sheets (left side of schematic diagram).
  • Amyloid fibrils are also of current interest because of the relatively recent realization that the ability to form amyloid fibrils is not a property restricted to peptides and proteins that are associated with amyloid diseases (Jimenez et al. 1999; Pertinhez et al. 2001 ; Fandrich & Dobson, 2002).
  • amyloid fibril appears to be a genetically stable structural state of a polypeptide chain, competing thermodynamically and kinetically with globular monomelic states and unfolded monomelic states.
  • Peptides and proteins that are known to form amyloid fibrils have widely diverse amino-acid sequences and molecular weights. Although sequences with long hydrophobic segments and sequences rich in glutamine and asparagine residues are two classes of amyloid-forming sequences that are discussed in detail below, the only feature of an amino-acid sequence that is strongly prohibitive of amyloid formation may be a high net charge.
  • the principal structural motif in amyloid fibrils is the cross- ⁇ motif (Fig. 6).
  • the cross- ⁇ structure is a ribbon-like ⁇ -sheet that extends over the length of the fibril and is comprised of ⁇ -strands that run approximately perpendicular to the long axis of the fibril. Backbone hydrogen bonds that link the ⁇ -strands are nearly parallel to the long axis.
  • ⁇ -helices have not been detected in the core of amyloid structures.
  • Secondary structure in amyloid fibrils therefore refers to the identification of ⁇ -strand segments, non- ⁇ -strand segments (e.g. bends, loops, or turns), and structurally disordered segments.
  • Tertiary structure in amyloid fibrils refers to the organization of ⁇ -strands into parallel or antiparallel ⁇ -sheets, and to the precise registry of interstrand hydrogen bonds. Both parallel and antiparallel ⁇ -sheets have been identified in amyloid fibrils by solid-state NMR, although parallel ⁇ -sheets appear to be more common.
  • Quaternary structure in amyloid fibrils refers to the relative orientation of and contacts between ⁇ -sheets.
  • the ⁇ -sheets that make contact with one another can be formed by different sets of molecules or, when each peptide or protein contains more than one ⁇ - strand segment, by different segments of the same set of molecules.
  • quaternary contacts can be either intermolecular or intramolecular.
  • tertiary, and quaternary structure are properties of the 'protofilamenf in an amyloid fibril, defined as the filamentous unit with minimal width and minimal mass- per-length (MPL) that exists in isolation (Antzutkin et al. 2002; Petkova et al. 2005).
  • MPL mass- per-length
  • protofilaments may associate laterally into thicker fibrils with greater MPL values (i.e., form 'bundles'). Lateral association of amyloid protofilaments into thicker fibrils may be considered a higher level of structural organization, beyond quaternary structure.
  • MPL mass- per-length
  • the TEM image of A ⁇ l-40 fibrils in Fig. 6 shows fibrils with several distinct morphologies. Fibrils grown under quiescent conditions exhibit a periodic modulation in their apparent diameters, indicating a periodic twist, have maximum diameters of 9 ⁇ 1 nm, and do not tend to adhere to one another under TEM or AFM conditions. Fibrils grown with gentle agitation have diameters of 5 ⁇ 1 nm, lack a resolvable twist in TEM images (or a resolvable height modulation in AFM images), and tend to adhere to one another laterally, forming flat bundles of protofilaments.
  • Sonication of either quiescent or agitated 'parent' fibrils produces fragments with lengths of 50-100 nm that can be used as seeds for subsequent fibril growths.
  • 'Daughter' fibrils grown from these seeds i.e., from peptide solutions to which seeds have been added at the beginning of incubation
  • Sonicated daughter fibrils can be used as seeds for the growth of 'granddaughter' fibrils, again with preservation of the parent morphology.
  • fibril morphology and molecular structure are attributable to the existence of multiple, distinct fibril nucleation events.
  • the term 'nucleation event' means the formation of a peptide aggregate (called the critical nucleus), whose structure probably resembles the structure of a short segment of the amyloid protofilament, that is equally likely to dissociate or to extend into a long protofilament by the binding of additional peptide molecules.
  • fibril morphologies and molecular structures are determined by the structural details of the nucleation events. Hence, subtle variations in experimental conditions, such as the presence or absence of gentle agitation, can favor one nucleation event over others.
  • a ⁇ l ⁇ 2 differs from A ⁇ l-40 by the addition of isoleucine and alanine residues at positions 41 and 42, respectively, and normally accounts for -30% of the ⁇ -amyloid peptides in blood plasma.
  • investigators have developed a detailed model for fibrils formed by A ⁇ l-42(M35ox), based on existing evidence for in-register, parallel ⁇ - sheets in A ⁇ l ⁇ -2 fibrils and on H/D exchange, mutagenesis, and cryo-EM experiments.
  • This model differs from the A ⁇ l ⁇ 40 model in that the protofilament consists of a single layer of A ⁇ l-42(M35ox) molecules, the N-terminal ⁇ -strand is comprised of residues 17- 26, and the internal quaternary contacts are shifted so that side-chains of Fl 9 residues lie between side-chains of V36 and V40 residues. These differences may be attributable to disruption of the external quaternary interface by oxidation of the M35 side-chain and to extension of the C-terminal hydrophobic segment by 141 and A42.
  • the internal quaternary contacts in A ⁇ l ⁇ 42 fibrils may be intermolecular in nature). Structure of Fibrils formed by short ⁇ - Amyloid Fragments
  • a ⁇ lO-35 forms fibrils containing in-register, parallel ⁇ -sheets, as originally demonstrated by the solid-state NMR measurements.
  • TEM images and MPL data from STEM images of A ⁇ lO-35 fibrils closely resemble those of A ⁇ l-42 fibrils and A ⁇ l ⁇ 40 fibrils. It appears that the molecular structures of A ⁇ lO-35 fibrils are quite similar to the structures of full-length ⁇ -amyloid fibrils, supporting the notion that A ⁇ 10-35 fibrils are appropriate models for full-length ⁇ -amyloid fibrils.
  • Shorter fragments including A ⁇ l6-22 (N-acetyl-KLVFFAE-amide) (SEQ ID NO: 114), A ⁇ l l-25, and A ⁇ 34-42, have been shown by solid-state NMR measurements to contain antiparallel ⁇ -sheets.
  • All three of these peptides contain a single hydrophobic segment (residues 17-21 in A ⁇ l6-22 and A ⁇ l 1-25; residues 34-42 in A ⁇ 34-42). Maximal alignment of hydrophobic segments can therefore be achieved in either parallel or antiparallel ⁇ -sheets.
  • Near neutral pH, A ⁇ l 6-22 and A ⁇ 34-42 have positive charges at their N-termini and negative charges at their C-termini. Antiparallel ⁇ - sheets may then be favored by the electrostatic interactions within each ⁇ -sheet layer.
  • N-octanoyl-KLVFFAE-amide (SEQ ID NO: 115) forms amyloid fibrils that contain in-register, parallel ⁇ -sheets (Gordon et al. 2004).
  • interactions of the 7-carbon alkyl chains at the N-termini favor the parallel alignment of peptide molecules, either kinetically (e.g., by 'pre-organizing' the parallel alignment in an oligomeric state that precedes fibril formation) or thermodynamically.
  • Ure2plO-39 and full-length Ure2p fibrils Ure2p is a 354-residue protein that regulates the utilization of particular nitrogen sources by yeast cells.
  • Ure2p is a yeast prion protein.
  • Residues 1-89 of Ure2p constitute its prion, or amyloid- forming, domain.
  • the remainder of the Ure2p sequence forms a globular structure resembling glutathione-S-transferase that is apparently retained in the amyloid form.
  • the prion domain of Ure2p does not contain contiguous hydrophobic segments longer than two residues, unlike the ⁇ -amyloid peptides discussed above.
  • the prion domain of Ure2p contains a high percentage of glutamine (11%) and asparagine (37%) residues. Similar glutamine- and asparagine-rich sequences are found in other yeast prion proteins, such as the Sup35 protein that produces the [PSI+] phenotype upon aggregation into amyloid fibrils, and in amyloid-forming proteins associated with neurodegenerative disorders such as Huntingdon's disease and spinocerebellar ataxias.
  • TTRl 05-115 conformation is an untwisted ⁇ -strand with a very high level of structural order in the central hydrophobic segment (1107 through Ll 11).
  • Studies of TTRl 05-115 fibrils demonstrate that modern solid-state NMR techniques are capable of determining all aspects of peptide conformations in non-crystalline systems. HET-sfibrils
  • the HET-s protein is the product of one of the heterokaryon incompatibility genes of the filamentous fungus Podospera anserina, which control the viability of mixed cells resulting from fusion of two parent cells. Fusion of cells containing het-s and het-S alleles results in programmed cell death, but only if the het-s parent is in the [Het-s] (rather than the [Het-s*]) phenotypic state, corresponding to an aggregated, amyloid state of the HET-s protein.
  • the [Het-s] phenotype is transmissible among het-s cells, either by exchange of cytoplasm or by mitotic division.
  • HET-s is a fungal prion protein. Moreover, unlike the vast majority of amyloid fibrils, HET-s fibrils have a well-characterized and presumably evolved biological function. It has been established that residues 218-289 of HET-s (HET- s218-289) constitute the prion domain, i.e. make up the core structure of HET-s fibrils. Unlike the amyloid-forming peptides discussed above, the amino-acid sequence of HET- s218-289 contains neither hydrophobic segments longer than four residues nor a high percentage of glutamine and asparagine residues. In addition, unlike the [URE3] and [PSI+] prions, distinct strains of the [HET-s] prion have not been found. Amylin fibrils
  • Amylin a 37-residue peptide co-secreted with insulin by ⁇ -cells in the islets of Langerhans of the pancreas, forms amyloid deposits in the pancreas of patients with type 2 diabetes. These amyloid deposits may contribute to the destruction of islet cells.
  • amylin fibrils prepared in vitro are polymorphic and essentially indistinguishable from A ⁇ l-40 fibrils in TEM images, despite the fact that amylin contains an intramolecular disulfide bridge between cysteines at residues 2 and 7, only one 5-residue hydrophobic segment, and a total of seven glutamine and asparagine residues. EPR measurements on spin-labeled amylin fibrils suggest an in-register, parallel ⁇ -sheet structure, with a disordered N-terminal segment. PrP fibrils
  • PrP the mammalian prion protein associated with TSE
  • TSE is a 230-residue protein with a primarily helical globular domain (residues 125-228) and a disordered N-terminal domain in the monomelic, soluble form characterized by solution NMR.
  • the TSE infectious agent is believed to be an aggregated form of PrP, but the precise nature of infectious PrP aggregates has not been firmly established.
  • the 17-residue cc ⁇ peptide was designed to form a trimeric helical coiled-coil structure, but was also found by investigators to form amyloid fibrils at elevated temperatures in an apparently irreversible structural conversion. According to the
  • Amyloid fibrils are formed by the 32-residue peptide hormone calcitonin.
  • Amyloid Proteins of Microbial or de novo Design Origin Microbial amyloid proteins are also known (Gebbink et al. 2005 Nature Reviews
  • E. coli CsgA curlin A 131 amino acid, 15.3kDa protein, encoded by the csgA gene. Fusion proteins have been constructed including his tagged protein. Purified CsgA- his forms amyloid fibers in vitro on prolonged incubation 4°C for 4-12 hours. Use of this amyloid will require creation of a fusion protein and production in E. coli or other bacterial cell substrates (Chapman MR et al 2002 Science 295:851-855). 2) AgfA Tafi protein is highly homologous to the E. coli curlin protein CsgA (74% amino acid identity), produced in Salmonella species.
  • Chaplin coelicolor hydrophobic aeriel proteins amyloids: minimum size of 52 to maximum size of 259 amino acids.
  • Table 1 The coding sequences of Chaplins have been identified and a consensus sequence developed (Fig. 7). These chaplin proteins assume amyloid properties when vortexed in aqueous solution.
  • Fusion proteins can be generated using any of the above sequences and peptide or protein sequences and production of such proteins in a bacterial cell substrate (e.g., Enter obacter, Streptomyces) followed by purification using standard methods. Amyloid formation will require vortexing (Claessen D. et al. 2003 Genes and Development 17:1714-1726). Amyloids of Fungal Origin 4) Hydrophobins: These are small secreted proteins whose structure depends on environment: the ⁇ -sheet (amyloid) state termed "rodlets” occurs after self-assembly at air- water interfaces. They are low molecular weight (7-9kD) and distinguished by the conserved pattern of cysteines in the following consensus sequence:
  • EAS Hydrophobin from Neurospora crassa has the amino acid sequence shown in Fig. 8. This protein forms amyloid rodlets on evaporation of water. The residues involved in amyloid formation have been identified.
  • SC3 Hydrophobin of Schizophyllum commune. This hydrophobin is secreted in a monomelic state that is soluble in water. It assumes an ⁇ -helical state on contact with a hydrophobic surface and a ⁇ -sheet amyloid configuration at the air-water interface. A plasmid containing the SC3 sequence can be modified to create a fusion protein containing a target peptide or protein. A fungal cell substrate would be required to produce the fusion protein which may then be purified by standard methods. Conversion of the monomer or a-helical form into amyloid structures may be accomplished by either evaporation from a hydrophobic surface, or by addition of Tween-20 at 85°C (Schuurs T et al. 1997 Genetics
  • Rod A Conidial hydrophobin of Aspergillus species. A 16kDa protein encoded by the rodA gene. The DNA and amino acid sequence are shown in Fig. 9 (Thau N et al. 1994 Infection and Immunity 62:4380-4388). A fusion protein consisting of Rod A and the peptide or protein of interest is created and expressed in a fungal cell substrate such as Aspergillus fumigatus. Purification is by standard methods and amyloid formation by air/water interface or Tween 20 exposure as for SC3 (Paris S et al. 2003 Applied and Environmental Microbiology 69:1581-1588).
  • MPGl hydrophobin from rice blast fungus is a 15kD protein that forms rodlets at hydrophobic surfaces. Its DNA and amino acid sequence are shown in Fig. 10 (Talbot N et al 1993 The Plant Cell
  • fusion proteins are generated that incorporating the target peptide or protein genetic sequence into that of the hydrophobin sequence and the plasmid used to transfect a fungal cell substrate.
  • the hydrophobins are purified by standard methods and amyloid formation is generated as above by evaporative means or by Tween 20 exposure on a hydrophobic surface.
  • the bacteriophage T7 endonuclease forms amyloid like fibrils. Fibril formation occurs upon incubation at 37 0 C for 3-7 days and is pH dependent such that fibrils form at pH 6.8 but not at pH 6.0 or 8.0 (Guo, Z. and Eisenberg, D. 2007 J Biol Chem 282:14968- 14974).
  • the amino acid sequence of bacteriophage T7 endonuclease is shown in Fig. 12 (Hadden et al. 2001 Nature Structural Biology 8:62-67).
  • the protein can be expressed and purified in E. coli.
  • a fusion protein consisting of the target peptide or protein of interest can be constructed and amyloid formation tested by incubation under ideal amyloid formation conditions. Amyloids via de novo Design:
  • This construct facilitates rapid screening of the library for genes that assemble in the correct reading frame and this could be adapted to our invention by using a peptide specific antibody rather than an epitope tag as was used here.
  • Proteins were purified by standard methods and found at moderate concentrations to have assembled into an oligomeric state with high MW (>1, 000,000 Daltons), which were found to be fibrils by EM. The amyloid fibrils were found to be stable at physiologic temperatures.
  • This type of high throughput system would allow us to insert target peptides in the N- and C-terminal regions, clone into the T7/E. coli expression system and discover peptides that are optimal for amyloid formation with each target peptide.
  • cc ⁇ 17 residue peptide termed cc ⁇ was generated that forms a native coiled coil structure under ambient conditions, but which is converted into amyloid fibrils by raising the temperature to 37-50 0 C.
  • this peptide's propensity to form amyloid fibrils is diminished by small sequence changes, it may be that modifications, including lengthening of the peptide chain, would render the peptide less susceptible to disrupted amyloid formation by an introduced peptide.
  • the amino acid sequence is shown in Fig. 15 (Kammerer, R. et al. 2004 Proc Natl Acad Sd USA 101:4435-4440).
  • the amino acid sequence of the target peptide of interest may be placed at non-critical areas of the cc ⁇ peptide and amyloid generated by raising the temperature to 50 0 C.
  • Some embodiments concern an immunogen comprising a chimeric amyloid protein that comprises at least one heterologous epitope.
  • Additional immunogens include polynucleotides comprising a nucleotide sequence encoding a chimeric amyloid protein or a variant thereof, wherein the nucleotide sequence further encodes a heterologous epitope.
  • heterologous epitope comprises a domain that is not present in the native amyloid protein or encoded by the polynucleotide encoding therefore.
  • Polypeptides comprising such heterologous epitopes or polynucleotides encoding therefor are referred to herein as “chimeric polypeptides” or “chimeric polynucleotides”, respectively.
  • chimeric polypeptides or chimeric polynucleotides
  • the chimeric polypeptides or polynucleotides encoding therefor are isolated or substantially purified polynucleotide or polypeptide compositions.
  • an "isolated” or “purified” polynucleotide or polypeptide, or biologically active portion thereof is substantially or essentially free from components that normally accompany or interact with the polynucleotide or polypeptide as found in its naturally occurring environment.
  • an isolated or purified polynucleotide or polypeptide is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an "isolated" polynucleotide is free of sequences (optimally protein encoding sequences) that naturally flank the polynucleotide (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the polynucleotide is derived.
  • the isolated polynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank the polynucleotide in genomic DNA of the cell from which the polynucleotide is derived.
  • a protein that is substantially free of cellular material includes preparations of protein having less than about 30%, 20%, 10%, 5%, or
  • optimally culture medium represents less than about 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or non-protein- of-interest chemicals.
  • Some embodiments provide fusion nucleic acids encoding the fusion polypeptides described herein.
  • nucleic acids may be made, all of which encode the fusion proteins of the present invention.
  • those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the fusion protein.
  • heterologous epitope can be placed anywhere in the amyloid protein sequence, as long as the chimeric polypeptide retains the structural integrity of the native scaffold polypeptide so that it presents the epitope in an ordered fashion as described herein.
  • the nucleotide sequence encoding the heterologous epitope or the domain it is contained in can be of any length including about 15 to about 30 nucleotides, about 31 to about 60 nucleotides, about 61 to about 90 nucleotides, about 91 to about 120 nucleotides, about 121 to about 150 nucleotides, about 151 to about 180 nucleotides, about 181 to about 210 nucleotides, about 210 to about 240 nucleotides, about 241 to about 270, about 271 to about 300, about 301 to about 330 nucleotides, or longer.
  • the expression vectors may be either self- replicating extrachromosomal vectors or vectors which integrate into a host genome.
  • these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the fusion protein.
  • control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism.
  • the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
  • Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
  • Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • DNA for a pre-sequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
  • a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
  • "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
  • transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the fusion protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the fusion protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • Promoter sequences encode either constitutive or inducible promoters.
  • the promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the promoters are strong promoters, allowing high expression in cells, particularly mammalian cells, such as the CMV promoter, particularly in combination with a Tet regulatory element.
  • the expression vector may comprise additional elements.
  • the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a prokaryotic host for cloning and amplification.
  • the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
  • the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.
  • the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
  • Selection genes are well known in the art and will vary with the host cell used.
  • a retroviral vector system is used.
  • the candidate nucleic acids are introduced into the cells for screening, as is more fully outlined below.
  • introduction into or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid.
  • the method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaPO 4 precipitation, liposome fusion, lipofectin®, electroporation, viral infection, etc.
  • the candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e., through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.). As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfecting such targets are preferred. Variant Polypeptides and Polynucleotides encoding therefor
  • compositions disclosed herein can employ variant amyloid proteins, polynucleotides encoding therefor, as well as variants of heterologous epitopes.
  • the heterologous epitopes may be recognized by previously identified neutralizing antibodies.
  • variants is intended to mean substantially similar sequences.
  • a “variant” protein is intended to mean a protein derived from the native protein by deletion (so-called truncation) of one or more amino acids at the N-terminal and/or C-terminal end of the native protein; deletion and/or addition of one or more amino acids at one or more internal sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein activity as described herein for scaffold. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Variants of a native amyloid protein employed in the methods of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs and parameters described elsewhere herein.
  • a variant of a protein of the invention may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • a fragment of an amyloid protein of the invention will encode at least 15, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 800 contiguous amino acids, or up to the total number of amino acids present in a full-length amyloid protein.
  • a variant comprises a polynucleotide having deletions (i.e., truncations) at the 5' and/or 3' end; deletion and/or addition of one or more nucleotides at one or more internal sites in the native polynucleotide; and/or substitution of one or more nucleotides at one or more sites in the native polynucleotide.
  • a "native" polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode an amino acid sequence comprising an amyloid protein or a functional variant thereof, wherein the amino acid sequences further comprise a heterologous target peptide epitope.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, such as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined below.
  • Variant polynucleotides also include synthetically derived polynucleotides, such as those generated, for example, by using site- directed mutagenesis but that still encode an amyloid protein of the invention.
  • variants of a particular polynucleotide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by sequence alignment programs and parameters as described elsewhere herein.
  • Variants of a particular polynucleotide can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described elsewhere herein.
  • the percent sequence identity between the two encoded polypeptides has at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • a fragment of an amyloid protein-encoding polynucleotide may encode the cross- ⁇ structural motif that is commonly found in amyloid proteins.
  • Polynucleotides that are fragments of an amyloid protein-encoding nucleotide sequence comprise at least 15, 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400 or more contiguous nucleotides, or up to the number of nucleotides present in a full-length amyloid protein polynucleotide of the invention.
  • variant amyloid proteins as well as polynucleotides encoding these variants, are known in the art and are discussed in further detail elsewhere herein.
  • the polypeptide employed in the methods of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art.
  • variant polypeptides or polynucleotides of the invention can comprise heterologous target peptide epitopes.
  • amino acid sequence variants and fragments of the amyloid proteins can be prepared by mutations in the DNA. Methods for mutagenesis and polynucleotide alterations are well known in the art. See, for example, Kunkel (1985) Proc. Natl.
  • polypeptides and polynucleotides employed in the methods of the invention encompass naturally occurring sequences as well as variations and modified forms thereof. Such variants will continue to possess the desired activity for scaffold as discussed elsewhere herein. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and optimally will not create complementary regions that could produce disadvantageous secondary mRNA structure.
  • the deletions, insertions, and substitutions of the protein sequences encompassed herein are not expected to produce radical changes in the characteristics of the protein. However, when it is difficult to predict the exact effect of the substitution, deletion, or insertion in advance of doing so, one skilled in the art will appreciate that the effect will be evaluated by routine screening assays. That is, the activity can be evaluated for functional variants of the amyloid proteins by the ability to behave as scaffolds.
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequence identity or “identity” in the context of two polynucleotides or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • sequences differ in conservative substitutions the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have "sequence similarity" or “similarity”. Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, California).
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 using the following parameters: % identity and % similarity for a nucleotide sequence using GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmp scoring matrix; % identity and % similarity for an amino acid sequence using GAP Weight of 8 and Length Weight of 2, and the BLOSUM62 scoring matrix; or any equivalent program thereof.
  • equivalent program is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by GAP Version 10.
  • GAP uses the algorithm of Needleman and Wunsch (1970) J MoI. Biol. 48.443-453 to find the alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty.
  • gap creation penalty values and gap extension penalty values in Version 10 of the GCG Wisconsin Genetics Software Package for protein sequences are 8 and 2, respectively.
  • the default gap creation penalty is 50 while the default gap extension penalty is 3.
  • the gap creation and gap extension penalties can be expressed as an integer selected from the group of integers consisting of from 0 to 200.
  • the gap creation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.
  • the structural bias of the target peptide may be taken into account.
  • target peptides with a helical bias or extended structure bias may be made through fusion to the N- terminus and/or C-terminus of the amyloid proteins.
  • peptide targets with coiled coil bias may be made via fusion to the N- and/or C- terminus of the amyloid proteins.
  • the target peptides are inserted within the amyloid scaffold protein. Some embodiments may utilize loop formations via insertion into loops in the amyloid scaffold proteins; amino acid residues within the respective loop structures may be replaced by the target peptide or the target peptide may be inserted in between two amino acid residues located within a loop structure.
  • aspects of the present invention provide fusion proteins of amyloid scaffold proteins and targeted peptides.
  • fusion protein or “fusion polypeptide” or grammatical equivalents herein is meant a protein composed of a plurality of protein components that, while typically not joined in their native state, typically are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide.
  • Protein in this context includes proteins, polypeptides and peptides. Plurality in this context means at least two, and preferred embodiments generally utilize two components. It will be appreciated that the protein components can be joined directly or joined through a peptide linker/spacer as outlined below. In addition, other components such as fusion partners including presentation structures, targeting sequences, etc. may be used.
  • aspects of the present invention provide fusion proteins of amyloid scaffold proteins and target peptides.
  • amyloid scaffold protein amyloid scaffold polypeptide
  • amyloid scaffold or grammatical equivalents thereof, herein is meant a protein to which amino acid sequences, such as the target peptides described herein, can be fused.
  • the peptides are exogeneous to the scaffold; that is, they are not usually present in the amyloid protein.
  • the amyloid scaffold protein Upon fusion, the amyloid scaffold protein usually allows the display of the target peptides in a way that they are accessible to other molecules.
  • a target peptide is fused to an amyloid scaffold protein to form a fusion polypeptide.
  • fused or operably linked” herein is meant that the target peptide and the amyloid scaffold protein, are linked together, in such a manner as to minimize the disruption to the stability of the scaffold structure.
  • the fusion polypeptide (or fusion polynucleotide encoding the fusion polypeptide) can comprise further components as well, including multiple peptides at multiple loops, fusion partners, etc.
  • the fusion polypeptide includes additional components, including, but not limited to, fusion partners and linkers.
  • the target peptide is fused to the N-terminus of the amyloid protein.
  • the fusion can be direct, i.e. with no additional residues between the C- terminus of the peptide and the N-terminus of the amyloid protein, or indirect; that is, intervening amino acids are used, such as one or more fusion partners, including a linker.
  • intervening amino acids such as one or more fusion partners, including a linker.
  • a presentation structure may be used, to confer some conformational stability to the target peptide.
  • some embodiments include the use of dimerization sequences.
  • N-terminal residues of the amyloid protein are deleted, i.e., one or more amino acids of the amyloid protein can be deleted and replaced with the target peptide.
  • the fusion is directly to the first amino acid of the amyloid protein.
  • the target peptide is fused to the C-terminus of the amyloid protein.
  • the fusion can be direct or indirect, and C- terminal residues may be deleted.
  • target peptides and fusion partners are added to both the N- and the C- terminus of the amyloid protein.
  • the target peptide is fused to an internal position of the amyloid protein. While the target peptide can be inserted at virtually any position, preferred positions include insertion at the tips of "loops" on the surface of the amyloid protein, to minimize disruption of the amyloid protein structure.
  • the random peptide is inserted, without any deletion of amyloid protein residues. That is, the insertion point is between two amino acids in the loop, adding the new amino acids of the peptide, optionally including fusion partners and/or linkers. Generally, when linkers are used, the linkers are directly fused to the amyloid protein, with additional fusion partners, if present, being fused to the linkers and the peptides.
  • the target peptide is inserted into the amyloid protein, with one or more amyloid protein residues being deleted; that is, the random peptide (optionally including fusion partners and/or linkers) replaces one or more residues.
  • the linkers are attached directly to the amyloid protein.
  • linker residues that replace the amyloid protein residues, again generally at the tip of the loop.
  • residues from one to five residues of amyloid protein are deleted, with deletions of one, two, three, four and five amino acids all possible.
  • fusion polypeptides comprising amyloid protein and target peptides are provided.
  • one embodiment provides a polynucleotide encoding an amyloid protein with a multisite cloning site inserted into the region that corresponds to at least one loop outlined above.
  • the amyloid scaffold protein is a ⁇ -amyloid protein ( ⁇ AP).
  • the amyloid scaffold protein is derived from a bacterial source.
  • the amyloid protein is an E. coli CsgA curlin protein.
  • the amyloid protein is AgfA Taf ⁇ protein.
  • the amyloid protein is a Chaplin protein.
  • amyloid scaffold protein is derived from a fungal source. In one embodiment, the amyloid protein is a hydrophobin.
  • amyloid scaffold protein is derived from bacteriophage T7. In one embodiment, the amyloid protein is bacteriophage T7 endonuclease.
  • the amyloid scaffold protein is designed de novo.
  • linker amino acids on one or both sides may comprise 2, 3, 4, or more glycines, in order to provide a flexible region between the random library and the rest of the protein.
  • the linker may not need any glycines.
  • the presence of multiple glycines at least partly conformational ⁇ decouples the inserted peptide from the rest of the protein. In some cases, this enhances the ability of the target peptide to fold properly.
  • amyloid scaffold protein folds well and independently of the sequence of the target peptide.
  • the target peptide is fused to the N- or C-terminus of an amyloid protein.
  • the target peptide is attached to the amyloid protein via one or more linkers.
  • the fusion proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a fusion protein, under the appropriate conditions to induce or cause expression of the fusion protein.
  • the conditions appropriate for fusion protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.
  • the timing of the harvest is important.
  • the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
  • Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells.
  • yeast Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E coli, Bacillus subtilis, SF9 cells, C 129 cells, 293 cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoid cell lines, Jurkat cells, mast cells and other endocrine and exocrine cells, and neuronal cells.
  • the fusion proteins are expressed in mammalian cells.
  • Mammalian expression systems are also known in the art, and include retroviral systems.
  • a mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3 ! ) transcription of a coding sequence for the fusion protein into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box located 25- 30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
  • transcription terminator and polyadenylation signals include those derived form SV40.
  • mammalian cells used in the present invention can vary widely. Basically, any mammalian cells may be used, with hamster, mouse, rat, primate and human cells being particularly preferred, although as will be appreciated by those in the art, modifications of the system by pseudotyping allows all eukaryotic cells to be used, preferably higher eukaryotes.
  • Suitable cells include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos, etc.
  • the ATCC cell line catalog which can be found on the world wide web at atcc.org provides a list of commonly used cell lines.
  • the cells may be additionally genetically engineered, that is, contain exogeneous nucleic acid other than the fusion nucleic acid.
  • the fusion proteins are expressed in bacterial systems.
  • Bacterial expression systems are well known in the art.
  • a suitable bacterial promoter is any nucleic acid sequence capable of binding bacterial RNA polymerase and initiating the downstream (3') transcription of the coding sequence of the fusion protein into mRNA.
  • a bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerase binding site and a transcription initiation site. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences.
  • promoter sequences derived from sugar metabolizing enzymes such as galactose, lactose and maltose
  • sequences derived from biosynthetic enzymes such as tryptophan.
  • Promoters from bacteriophage may also be used and are known in the art.
  • synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences.
  • a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
  • the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation codon and a sequence 3-9 nucleotides in length located 3-1 1 nucleotides upstream of the initiation codon.
  • SD Shine-Delgarno
  • the expression vector may also include a signal peptide sequence that provides for secretion of the fusion protein in bacteria.
  • the signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art.
  • the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
  • the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.
  • bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
  • fusion proteins are produced in insect cells.
  • Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
  • fusion protein is produced in yeast cells.
  • Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
  • Promoter sequences for expression in yeast include the inducible GALl-IO promoter, the promoters from alcohol dehydrogenase, enolase, glucokinase, glucose-6- phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acid phosphatase gene.
  • Yeast selectable markers include ADE2, HIS4, LEU2, TRPl, and
  • ALG7 which confers resistance to tunicamycin
  • the neomycin phosphotransferase gene which confers resistance to G418
  • the CUPl gene which allows yeast to grow in the presence of copper ions.
  • the fusion polypeptides of the invention may be further fused to other proteins, if desired, for example to increase expression.
  • the fusion protein is isolated from a cell.
  • fusion protein may be isolated using a rescue sequence.
  • rescue sequences comprising epitope tags or purification sequences may be used to pull out the fusion protein using immunoprecipitation or affinity columns.
  • the peptide may be detected using mass spectroscopy.
  • Embodiments described herein include various immunogenic compositions.
  • an "immunogenic composition” refers to any composition that is capable of eliciting an immune response.
  • the term “vaccine” refers to an immunogenic composition that reduces the risk of, or prevents, infection by an infectious agent (a "prophylactic vaccine”) or that ameliorates, to any extent, an existing infection (a "therapeutic vaccine”).
  • a vaccine protects an organism from subsequent challenge with the infectious agent, the vaccines is said to be "protective.”
  • Immunogenic compositions can include an isolated chimeric polypeptide or active variant thereof or an isolated polynucleotide encoding the chimeric amyloid protein of the invention or an active variant thereof.
  • An isolated chimeric amyloid protein is present in an immunogenic composition in an amount sufficient to elicit an immune response against the heterologous epitope upon administration of a suitable dose to a subject.
  • An isolated chimeric polynucleotide encoding a chimeric amyloid protein can also be present in the immunogenic composition in an amount sufficient such that administration of a suitable dose to a subject results in the expression of the encoded chimeric amyloid protein polypeptide, which stimulates an immune response against the heterologous epitope.
  • a "subject" is defined as any animal including any mammal, such as, rodents, rabbits, goats, sheep, non-human primates, humans etc.
  • an immunogenic composition comprising a chimeric amyloid protein of the invention or a variant or fragment thereof.
  • an immunogenic composition includes cells expressing a chimeric amyloid protein of the invention, a cell lysate, or a fraction thereof, containing the chimeric polypeptide, such as, e.g., a membrane fraction.
  • the immunogenic composition comprises an isolated chimeric amyloid protein or variant thereof.
  • the immunogenic chimeric amyloid protein or active variant thereof can be provided as a virus-derived vaccine.
  • virus-derived vaccine refers to a vaccine containing a recombinantly engineered virus that either does not cause disease in human or has been deliberately weakened so that it cannot cause disease.
  • weakened (attenuated) viruses are used as vectors, or vehicles, to deliver polynucleotide encoding the chimeric amyloid protein into the cells of the body. Once inside cells, the cells produce the chimeric amyloid proteins. These chimeric proteins can stimulate an immune response.
  • Virus-derived vaccines have been prepared using a canarypox virus, a vaccinia virus, the alphavirus VEE, and a replication-defective adenovirus or adenovirus.
  • Other viruses that can be engineered to produce recombinant viruses useful in vaccines include retroviruses that are packaged in cells with amphotropic host range, and attenuated or defective DNA viruses, such as, but not limited to, herpes simplex virus (HSV), papillomavirus, Epstein Barr virus (EBV), adeno-associated virus (AAV), poxvirus, and the like.
  • HSV herpes simplex virus
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • a pharmaceutically acceptable carrier suitable for use in the invention is non-toxic to cells, tissues, or subjects at the dosages employed, and can include a buffer (such as a phosphate buffer, citrate buffer, and buffers made from other organic acids), an antioxidant (e.g., ascorbic acid), a low-molecular weight (less than about 10 residues) peptide, a polypeptide (such as serum albumin, gelatin, and an immunoglobulin), a hydrophilic polymer (such as polyvinylpyrrolidone), an amino acid (such as glycine, glutamine, asparagine, arginine, and/or lysine), a monosaccharide, a disaccharide, and/or other carbohydrates (including glucose, mannose, and dextrins), a chelating agent (e.g., ethylenediaminetetratacetic acid [EDTA]), a sugar alcohol (such as mannitol and sorbitol), a salt-forming counterion
  • An exemplary sustained-release composition has a semi permeable matrix of a solid hydrophobic polymer to which the polypeptide is attached or in which the polypeptide is encapsulated.
  • suitable polymers include a polyester, a hydrogel, a polylactide, a copolymer of L- glutamic acid and T-ethyl-L-glutamase, non-degradable ethylene-vinylacetate, a degradable lactic acid-glycolic acid copolymer, and poly-D-(-)-3-hydroxybutyric acid.
  • Such matrices are in the form of shaped articles, such as films, or microcapsules.
  • Exemplary sustained release compositions include polypeptides attached, typically via ⁇ -amino groups, to a polyalkylene glycol (e.g., polyethylene glycol [PEG]). Attachment of PEG to proteins is a well-known means of extending in vivo half-life. Any conventional "pegylation” method can be employed, provided the “pegylated” variant retains the desired function(s).
  • a polyalkylene glycol e.g., polyethylene glycol [PEG]
  • a sustained-release composition in another embodiment, includes a liposomally entrapped polypeptide.
  • Liposomes are small vesicles composed of various types of lipids, phospholipids, and/or surfactants. These components are typically arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing polypeptides are prepared by known methods.
  • Immunogenic compositions can be stored in any standard form, including, e.g., an aqueous solution or a lyophilized cake. Such compositions are typically sterile when administered to subjects. Sterilization of an aqueous solution is readily accomplished by filtration through a sterile filtration membrane. If the composition is stored in lyophilized form, the composition can be filtered before or after lyophilization and reconstitution.
  • polypeptide antigen An alternative to traditional immunization with a polypeptide antigen involves the direct in vivo introduction of a polynucleotide encoding the amyloid protein and antigen into tissues of a subject for expression of the antigen by the cells of the subject's tissue.
  • Polynucleotide-based compositions used to vaccinate a subject are termed "polynucleotide vaccines" or “naked DNA” or DNA vaccines or DNA immunogens.
  • polynucleotide-vaccine or a "naked DNA” is a vaccine or immunogen containing one or more polynucleotides encoding an antigen, wherein administration of the polynucleotide to an organism results in expression of the encoded antigen, followed by an immune response to that antigen.
  • an immunogenic composition comprising a chimeric polynucleotide encoding a chimeric amyloid polypeptide or variant thereof is provided.
  • Such compositions can include other components including, for example, a storage solution, such as a suitable buffer, e.g., a physiological buffer.
  • the other component is a pharmaceutically acceptable carrier as described above.
  • the use of polynucleotide vaccines is well known to those skilled in the art.
  • the composition comprising the polynucleotide encoding the chimeric amyloid protein further includes a component that facilitates entry of the polynucleotide into a cell.
  • a component that facilitates entry of the polynucleotide into a cell include, for example, lipids, liposomes, water-oil emulsions, polyethylene imines and dendrimers, any of which can be used in compositions according to the invention.
  • Lipids are among the most widely used components of this type, and any of the available lipids or lipid formulations can be employed with the polynucleotides of the invention. Typically, cationic lipids are preferred.
  • Preferred cationic lipids include N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dioleoyl phosphotidylethanolamine (DOPE), and/or dioleoyl phosphatidylcholine (DOPC).
  • DOTMA N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • DOPE dioleoyl phosphotidylethanolamine
  • DOPC dioleoyl phosphatidylcholine
  • Polynucleotides can also be entrapped in liposomes, as described above for polypeptides.
  • polynucleotides are complex ed to dendrimers, which can be used to transfect cells.
  • Dendrimer polycations are three dimensional, highly ordered oligomeric and/or polymeric compounds typically formed on a core molecule or designated initiator by reiterative reaction sequences adding the oligomers and/or polymers and providing an outer surface that is positively charged.
  • Suitable dendrimers include, but are not limited to, "starburst" dendrimers and various dendrimer polycations. Methods for the preparation and use of dendrimers to introduce polynucleotides into cells in vivo are well known to those of skill in the art.
  • the chimeric polynucleotide of the invention can be provided in an expression cassette for expression in a cell.
  • the cassette can include 5' and 3' regulatory sequences operably linked to the chimeric polynucleotide of the invention.
  • "Operably linked" is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a chimeric polynucleotide and a regulatory sequence i.e., a promoter
  • Operably linked elements may be contiguous or non-contiguous.
  • the cassette may additionally contain at least one additional gene to be cotransformed into the cell of interest.
  • Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the chimeric polynucleotide to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain selectable marker genes.
  • the expression cassette will include in the 5'-3 T direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), a chimeric polynucleotide of the invention, and a transcriptional and translational termination region (i.e., termination region) functional in the cell type of interest.
  • the regulatory regions i.e., promoters, transcriptional regulatory regions, and translational termination regions
  • the chimeric polynucleotide of the invention may be native/analogous to the host cell or to each other.
  • the regulatory regions and/or the chimeric polynucleotide of the invention may be heterologous to the host cell or to each other.
  • the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • a large number of factors can influence the efficiency of expression of antigen genes and/or the immunogenicity of gene-based vaccines.
  • factors include the vector, the promoter used to drive antigen gene expression, and the stability of the inserted gene in the plasmid.
  • promoters differ in tissue specificity and efficiency in initiating mRNA synthesis.
  • Many DNA vaccines and immunogens in mammalian systems have relied upon viral promoters derived from cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • compositions comprising the polynucleotides or polypeptides can be stored in any standard form, including, e.g., an aqueous solution or a lyophilized cake. Such compositions are typically sterile when administered to cells or subjects. Sterilization of an aqueous solution is readily accomplished by filtration through a sterile filtration membrane. If the composition is stored in lyophilized form, the composition can be filtered before or after lyophilization and reconstitution.
  • the various immunogenic compositions of the invention can include one or more adjuvant.
  • adjuvant refers to a compound or mixture that enhances the immune response to an antigen.
  • Exemplary adjuvants include, but are not limited to, Adju-Phos, AdjumerTM, albumin-heparin microparticles, Algal Glucan, Algammulin, Alum, Antigen Formulation, AS-2 adjuvant, autologous dendritic cells, autologous PBMC, Avridine®, B7- 2, BAK, BAY Rl 005, Bupivacaine, Bupivacaine-HCl, BWZL, Calcitriol, Calcium Phosphate Gel, CCR5 peptides, CFA, Cholera holotoxin (CT) and Cholera toxin B subunit (CTB), Cholera toxin Al-subunit-ProteinA D-fragment fusion protein, CpG, CRLl 005, Cytokine-containing Liposomes, D-Murapalmitine, DDA, DHEA, Diphtheria toxoid, DL- PGL, DMPC, DMPG, DOC/ Alum Complex
  • TM Keyhole Limpet Hemocyanin, Lipid-based Adjuvant, Liposomes, Loxoribine, LT(Rl 92G), LT-OA or LT Oral Adjuvant, LT-Rl 92G, LTK63, LTK72, MF59, MONTANIDE ISA 51, MONTANIDE ISA 720, MPLTM, MPL-SE, MTP- PE, MTP-PE Liposomes, Murametide, Murapalmitine, NAGO, nCT native Cholera Toxin, Non-Ionic Surfactant Vesicles, non-toxic mutant El 12K of Cholera Toxin mCT-El 12K, p- Hydroxybenzoique acid methyl ester, pCIL-10, pCIL12, pCMVmCATl, pCMVN, Peptomer-NP, Pleuran, PLG, PLGA, PGA, and PLA, Pluronic Ll 21, PMMA, PODDSTM, Poly rA
  • T helper effector cells or Cytolytic T cells may be elicited and establish protective memory T and B cells.
  • CTL Cytolytic T cells
  • Such scaffolds may further serve to release peptide in a sustained fashion for display or further processing by dendritic cells or other antigen presenting cells for elicitation of T cell responses including T helper cell and cytolytic T cell responses.
  • the N-terminus of amyloid scaffolds have been targeted for replacement with peptides of various lengths, the immune response to which is expected to confer some degree of protection against infection.
  • the human ⁇ -amyloid 1-40 peptide has been chosen as a model protein scaffold in which to test the immunogenicity of a peptide array, as a proof of principle.
  • CspA E. coli cold shock protein A, 73 amino acids
  • retro-CspA 92 or 93 residues
  • the immunogenic compositions can be used to elicit an immune response in a subject.
  • the method comprises introducing into the subject an effective concentration of an immunogenic composition comprising a chimeric amyloid protein as described herein or an active variant thereof.
  • the method comprises administering an immunogenic composition comprising a polynucleotide that encodes a chimeric amyloid protein or a variant thereof and expressing the chimeric polynucleotide in the subject.
  • the immunogenic compositions can be used as vaccines.
  • the immunogenic composition is administered to individuals who are not infected with a pathogen to reduce the risk of, or prevent, infection.
  • the immunogenic composition can also be administered to individuals who are already infected with a pathogen, but are still able to mount an immune response.
  • a so-called "therapeutic vaccine” can ameliorate the existing infection (for example, by improving the subject's condition or slowing or preventing disease progression) and/or can provide prophylaxis against infection with related pathogens (e.g., other strains of the same virus). Accordingly, methods for inhibiting or preventing infection by pathogen in a subject are provided.
  • This method comprises administering to the subject an effective concentration of an immunogenic composition comprising a chimeric amyloid protein of the invention or variant thereof.
  • the method comprises administering an immunogenic composition comprising a polynucleotide that encodes a chimeric amyloid protein of the invention, and expressing the chimeric polynucleotide in the subject.
  • Polypeptide-based immunogenic compositions are conveniently administered by injection (e.g., subcutaneous, intradermal, intramuscular, intraperitoneal, intravenous, etc.).
  • Alternative routes include oral administration (tablets and the like) and inhalation (e.g., using commercially available nebulizers for liquid formulations or lyophilized or aerosolized formulations).
  • Polypeptide compositions may also be administered via microspheres, liposomes, immune-stimulating complexes (ISCOMs), or other microparticulate delivery systems or sustained release formulations introduced into suitable tissues (such as blood).
  • ISCOMs immune-stimulating complexes
  • polynucleotide-based immunogenic compositions of the invention can be employed to express an encoded polypeptide in vivo, in a subject, thereby eliciting an immune response against the encoded polypeptide.
  • Various methods are available for administering polynucleotides into animals. The selection of a suitable method for introducing a particular polynucleotide into an animal is within the level of skill in the art.
  • Polynucleotides of the invention can also be introduced into a subject by other methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), or a DNA vector transporter (see, e.g., Wu et al. (1992) J. Biol. Chem. 267:963-967).
  • An "effective concentration” is defined herein as an amount of a biologically active agent that produces an intended biological activity.
  • the effective concentration of either the chimeric amyloid protein or the chimeric amyloid protein-encoding polynucleotide administered in the immunogenic composition depends on the properties of the particular composition, e.g., the immunogenicity of a particular formulation, administration route, immunization regimen, condition of the subject and the like, and the determination of a suitable dose for a particular set of circumstances is within the level of skill in the art. Different dosages can be used in a series of sequential inoculations. Thus, the practitioner may administer a relatively large dose in a primary inoculation and then boost with relatively smaller doses of the chimeric amyloid protein.
  • the immune response against the heterologous epitope of the chimeric polypeptide can be generated by one or more inoculations of a subject with an immunogenic composition of the invention.
  • a first inoculation is termed a "primary inoculation” and subsequent immunizations are termed “booster inoculations”.
  • booster inoculations generally enhance the immune response, and immunization regimens including at least one booster inoculation are preferred. Any type of immunogenic composition described above may be used for a primary or booster immunization.
  • an immunogenic composition comprising polynucleotides (e.g., or a virus-derived vaccine) of the invention can be used for a primary immunization, followed by boosting with an immunogenic composition containing polypeptides of the invention, or vice versa.
  • a primary immunization and one or more booster immunization can provide the same chimeric polypeptide and/or different chimeric polypeptides.
  • a suitable immunization regimen includes at least three separate inoculations with one or more immunogenic compositions of the invention, with a second inoculation being administered more than about two, about three to eight, or about four, weeks following the first inoculation.
  • the third inoculation is administered several months after the second inoculation, and in specific embodiments, more than about five months after the first inoculation, more than about six months to about two years after the first inoculation, or about eight months to about one year after the first inoculation. Periodic inoculations beyond the third are also desirable to enhance the subject's "immune memory.”
  • the adequacy of the vaccination parameters chosen can be determined by taking aliquots of serum from the subject and assaying antibody titers during the course of the immunization program.
  • the T cell populations can by monitored by conventional methods.
  • the clinical condition of the subject can be monitored for the desired effect, e.g., prevention of infection, improvement in disease state ⁇ e.g., reduction in viral load), or reduction in transmission frequency to uninfected subjects. If such monitoring indicates that vaccination is sub-optimal, the subject can be boosted with an additional dose of immunogenic composition, and the vaccination parameters can be modified in a fashion expected to potentiate the immune response.
  • the dose of the chimeric amyloid protein or polynucleotide and/or adjuvant can be increased or the route of administration can be changed.
  • the immune system of the host responds to the vaccine by producing antibodies specific for the target protein.
  • the host immune response e.g., T cell recall or antibody titer
  • Such measurements can be made by conventional assays.
  • Clinical evaluation and observation of an improvement in condition can also be a measurement of immune response in some contexts.
  • the host becomes at least partially or completely immune to a pathogen of interest, or resistant to developing moderate or severe disease. Evaluation of an Immune Response
  • Immuno response broadly refers to the antigen-specific responses of lymphocytes to foreign substances. Any substance that can elicit an immune response is said to be “immunogenic” and is referred to as an "immunogen”. All immunogens are antigens, however, not all antigens are immunogenic.
  • An immune response of this invention can be humoral (via antibody activity) or cell-mediated (via T cell activation).
  • the term "memory immune effector cell” is an immune effector cell as defined above, which has previously encountered an antigen. In contrast to its naive counterpart, activation of a memory, antigen specific immune effector cell may not require a costimulatory signal. Recognition of the peptiderMHC complex may be sufficient. "Activated”, when used in reference to a T cell, implies that the cell is no longer in
  • cytotoxins cytokines and other related membrane-associated proteins characteristic of the cell type (e.g., CD8+ or CD4+), and is capable of recognizing and binding any target cell that displays the particular peptide/MHC complex on its surface, and releasing its effector molecules.
  • the term "inducing an immune response in a subject” is a term understood in the art and refers to an increase of at least about 2- fold, or alternatively at least about 5-fold, or alternatively at least about 10-fold, or alternatively at least about 100- fold, or alternatively at least about 500-fold, or alternatively at least about 1000-fold or more in an immune response to an antigen (or epitope) which can be detected or measured, after introducing the antigen (or epitope) into the subject, relative to the immune response (if any) before introduction of the antigen (or epitope) into the subject.
  • An immune response to an antigen includes but is not limited to, production of an antigen-specific (or epitope-specific) antibody, and production of an immune cell expressing on its surface a molecule which specifically binds to an antigen (or epitope).
  • Methods of determining whether an immune response to a given antigen (or epitope) has been induced are well known in the art.
  • antigen-specific antibody can be detected using any of a variety of immunoassays known in the art, including, but not limited to, ELISA, wherein, for example, binding of an antibody in a sample to an immobilized antigen (or epitope) is detected with a detectably-labeled second antibody (e.g., enzyme-labeled mouse anti-human Ig antibody).
  • ELISA electrospray-activated immunosorbent assay for example, antigen-specific antibody.
  • a detectably-labeled second antibody e.g., enzyme-labeled mouse anti-human Ig antibody.
  • Humoral Response In some embodiments, mice are given recombinant amyloid protein/target peptide fusion protein. The mice receiving recombinant amyloid protein/target peptide fusion proteins are immunized no more than twice.
  • the amyloid protein/target peptide fusion protein is expressed as a fusion protein.
  • the recombinant amyloid protein/target peptide fusion protein comprises target protein that is chemically conjugated to the amyloid protein.
  • the fusion protein is synthetically derived (i.e., peptide synthesis).
  • mice are given a booster vaccination of recombinant amyloid protein/target peptide fusion protein. Mice are bled twice a month.
  • Enzyme immunosorbent assays are used to detect the presence of murine pathogen or tumor-specific antibodies. These assays are performed essentially as described (Chen et al., Hepatology 28(1): 219 (1998). Briefly, recombinant amyloid protein/target peptide fusion protein, or peptide alone, is passively adsorbed overnight at 4°C to 96-well microtiter plates (Nunc, Copenhagen, Denmark) at 1 ⁇ g/ml in 50 mM sodium carbonate buffer (pH 9.6). The plates are then blocked by incubation with dilution buffer containing PBS, 2% goat serum, and 1% bovine serum albumin for one hour at 37°C.
  • an enzyme immunoassay (EIA) is performed. (See e.g., Hultgren et al. 1998 J Gen Virol. 79:2381-2391 and Hultgren et al. 1997 Clin Diagn Lab Immunol 4:630-632 ()). The antibody levels are recorded as the highest serum dilution giving an optical density at 405nm more than twice that of non-immunized mice.
  • the immunogenicity of the antigen presenting cells or educated T cells produced by the methods of the invention can be determined by well known methodologies including, but not limited to the following:
  • 51 Cr-release lysis assay Lysis of peptide-pulsed 51 Cr-labeled targets by antigen- specific T cells can be compared. "More active" compositions will show greater lysis of targets as a function of time. The kinetics of lysis as well as overall target lysis at a fixed time point (e.g., 4 hours) may be used to evaluate performance. Ware et al. 1983 Immunol 131:1312.
  • Cytokine-release assay Analysis of the types and quantities of cytokines secreted by T cells upon contacting modified APCs can be a measure of functional activity.
  • Cytokines can be measured by ELISA or ELISPOT assays to determine the rate and total amount of cytokine production. Fujihashi et al. 1993 J Immunol Meth 160:181 ; Tanquay and Killion 1994 Lymphokine Cytokine Res 13:259.
  • compositions of the invention can be assayed for the ability to elicit reactive T cell populations from normal donor or patient-derived PBMC; in this system, elicited T cells can be tested for lytic activity, cytokine-release, polyclonality, and cross reactivity to the antigenic epitope. Parkhurst et al. 1996 Immunol 157:2539.
  • Immunogenicity can be assessed in vivo by vaccinating HLA transgenic mice with the compositions described herein and determining the nature and magnitude of the induced immune response.
  • the hu-PBL-SCID mouse model allows reconstitution of a human immune system in a mouse by adoptive transfer of human PBL. These animals may be vaccinated with the compositions and analyzed for immune response as previously mentioned in Shirai et al. 1995 J Immunol 154:2733;
  • T cells will proliferate in response to reactive compositions.
  • Proliferation can be monitored quantitatively by measuring, for example, 3H-thymidine uptake. Caruso et al. 1997 Cytometry 27:71.
  • a non-human primate (chimpanzee) model system can be utilized to monitor in vivo immunogenicities of HLA-restricted ligands. It has been demonstrated that chimpanzees share overlapping MHC-ligand specificities with human MHC molecules thus allowing one to test HLA- restricted ligands for relative in vivo immunogenicity.
  • the immunogenic compositions of the invention can be employed to generate antibodies that recognize a chimeric amyloid protein of the invention.
  • the method comprises administering to a subject an immunogenic composition comprising a chimeric amyloid protein of the invention or administering to the subject a polynucleotide encoding a chimeric amyloid protein of the invention.
  • immunogenic compositions of the invention can be administered to the subject by any suitable route of administration. Accordingly, in one embodiment, an immunogenic composition is administered to a subject to generate antibodies that recognize the heterologous target peptide epitope. Such antibodies may have use as a research reagent, for example.
  • the subject employed in this embodiment is one typically employed for antibody production. Mammals, such as, rodents, rabbits, goats, sheep, etc., are preferred.
  • the antibodies generated can be either polyclonal or monoclonal antibodies.
  • Polyclonal antibodies are raised by injecting (e.g., subcutaneous or intramuscular injection) antigenic polypeptides into a suitable animal (e.g., a mouse or a rabbit). The antibodies are then obtained from blood samples taken from the animal. The techniques used to produce polyclonal antibodies are extensively described in the literature. Polyclonal antibodies produced by the subjects can be further purified, for example, by binding to and elution from a matrix that is bound with the polypeptide against which the antibodies were raised.
  • Monoclonal antibodies can also be generated using techniques known in the art.

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Abstract

Les modes de réalisation de la présente invention se rapportent à l'utilisation de protéines amyloïdes en tant qu'échafaudages destinés à porter des peptides hétérologues. Plus précisément, certains modes de réalisation portent sur des protéines amyloïdes qui présentent au moins un peptide cible hétérologue contenant une séquence antigénique ou au moins un épitope, et sur l'utilisation de ces compositions pour induire une réponse immunitaire audit peptide cible hétérologue, à la séquence antigénique ou à l'épitope. L'invention concerne également des procédés de fabrication et d'utilisation de ces compositions pour prévenir et/ou traiter une maladie chez l'être humain.
PCT/US2008/059499 2007-04-06 2008-04-04 Utilisation de protéines amyloïdes en tant qu'échafaudages pour des vaccins WO2008124646A2 (fr)

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US20140004151A1 (en) * 2011-09-30 2014-01-02 La Jolla Institute For Allergy And Immunology Antigens and Epitopes Derived From Mycobacterium Tuberculosis
US10703784B2 (en) * 2011-09-30 2020-07-07 La Jolla Institute For Allergy And Immunology Antigens and epitopes derived from Mycobacterium tuberculosis
US9200082B2 (en) 2013-03-14 2015-12-01 The University Of Chicago Methods and compositions involving fibrillizing polypeptides for nanofibers
WO2015097289A1 (fr) 2013-12-24 2015-07-02 Vib Vzw Sécrétion et présentation fonctionnelle de polypeptides chimériques
US9926352B2 (en) * 2014-03-03 2018-03-27 Serendipity Biotech Inc. Chimeric dystrophin-VSV-G protein to treat dystrophinopathies
US20150246950A1 (en) * 2014-03-03 2015-09-03 Sujata Acharjee Chimeric Dystrophin-VSV-G Protein to Treat Dystrophinopathies
WO2019222840A1 (fr) * 2018-05-22 2019-11-28 The Governors Of The University Of Alberta Échafaudages structurés inoffensifs pour vaccins et antigènes de maladies amyloïdes basés sur une structure
CN112533959A (zh) * 2018-05-22 2021-03-19 阿尔伯塔大学理事会 用于基于结构的淀粉样蛋白疾病疫苗和抗原的无害的、结构化的支架
US20220064233A1 (en) * 2018-05-22 2022-03-03 The Governors Of The University Of Alberta Innocuous, structured scaffolds for structure-based amyloid disease vaccines and antigens

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