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WO2003059386A2 - Conjuges-vecteurs de proteines prion - Google Patents

Conjuges-vecteurs de proteines prion Download PDF

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Publication number
WO2003059386A2
WO2003059386A2 PCT/EP2003/000460 EP0300460W WO03059386A2 WO 2003059386 A2 WO2003059386 A2 WO 2003059386A2 EP 0300460 W EP0300460 W EP 0300460W WO 03059386 A2 WO03059386 A2 WO 03059386A2
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WIPO (PCT)
Prior art keywords
prion
antigen
virus
peptide
composition
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Ceased
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PCT/EP2003/000460
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English (en)
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WO2003059386A3 (fr
Inventor
Martin Bachmann
Patrick Maurer
Erica Pellicioli
Wolfgang A Renner
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Cytos Biotechnology AG
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Cytos Biotechnology AG
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Priority claimed from US10/050,902 external-priority patent/US7264810B2/en
Application filed by Cytos Biotechnology AG filed Critical Cytos Biotechnology AG
Priority to AU2003205623A priority Critical patent/AU2003205623A1/en
Publication of WO2003059386A2 publication Critical patent/WO2003059386A2/fr
Publication of WO2003059386A3 publication Critical patent/WO2003059386A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0007—Nervous system antigens; Prions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C07K14/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1, LDCF-2
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5409—IL-5
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5437—IL-13
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2730/00—Reverse transcribing DNA viruses
    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention is related to the fields of molecular biology, virology, immunology and medicine.
  • the invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array, and in particular a prion peptide or prion protein-array. More specifically, the invention provides a composition comprising a virus-like particle and at least one prion protein (PrP) or a dimer thereof, or a PrP peptide bound thereto.
  • PrP prion protein
  • the invention also provides a process for producing the conjugates and the ordered and repetitive arrays, respectively.
  • the compositions of the invention are useful in the production of vaccines for the treatment of prion diseases and as a pharmaccine to prevent or cure prion diseases and to efficiently induce immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self- specific immune responses within the indicated context.
  • Prion diseases are an increasing threat for society.
  • prion- induced BSE Bovine Spongiform Encephalopathy
  • CJD Chronic Jakob- Disease
  • CJD Creutzfeldt- Jakob- Disease
  • the number of infected people has been relatively low so far, it seems possible that the disease may become epidemic.
  • long-term prognosis for the development of vCJD may be particular difficult, since incubation times between infection and overt disease are very long and estimated to be 10 years or more.
  • Prions are cellular proteins existing in most mammalian species. Prion proteins (PrP) exist in two forms, a normally folded form that is usually present in healthy individuals (PrP c ) and a misfolded form that is associated with prion diseases (Prp Sc ). The current prion hypotheses postulate that the misfolded prion form Prp Sc can catalyze the refolding of healthy prion PrP c into disease causing Prp Sc (Aguzzi, A and Weissmann, C, Nature 389:795- 798 (1997)). In some rare instances, this transition may also occur spontaneously, causing classical CJD in humans.
  • PrP Sc may also be infectious and may be transmitted by blood transfusion or via the food chain.
  • the latter form of prion mediated disease is known as Kuru and used to occur in human cannibals.
  • Kuru The latter form of prion mediated disease is known as Kuru and used to occur in human cannibals.
  • species that are feeding on their own individuals are not abundant, this form of orally transmitted disease was too rare to be documented for other species.
  • FDCs are a poorly studied cell type but it is now clear that they depend upon the production of lymphotoxin and/or TNF by B cells for their development (Mackay, F., and Browning, J.L., Nature 395:26- 27 (1998)). Indeed, mice deficient for lymphotoxin do not exhibit FDCs (Matsumoto, M.S., et al., Science 264:703-707 (1996)). Moreover, they fail to be productively infected with prions and do not succumb to disease.
  • viruses induce prompt and efficient immune responses in the absence of any adjuvants both with and without T -cell help (Bachmann and Zinkernagel, Ann. Rev. Immunol. 15:235-270 (1991)). Although viruses often consist of few proteins, they are able to trigger much stronger immune responses than their isolated components. For B-cell responses, it is known that one crucial factor for the immunogenicity of viruses is the repetitiveness and order of surface epitopes. Many viruses exhibit a quasi-crystalline surface that displays a regular array of epitopes which efficiently crosslinks epitope- specific immuno globulins on B cells (Bachmann and Zinkernagel, Immunol.
  • the immune system usually fails to produce antibodies against self-derived structures.
  • soluble antigens present at low concentrations this is due to tolerance at the Th cell level.
  • coupling the self-antigen to a carrier that can deliver T help may break tolerance.
  • soluble proteins present at high concentrations or membrane proteins at low concentration B and Th cells may be tolerant.
  • B cell tolerance may be reversible (anergy) and can be broken by administration of the antigen in a highly organized fashion coupled to a foreign carrier (Bachmann and Zinkernagel, Ann. Rev. Immunol 75:235-270 (1997)).
  • prion proteins or dimers thereof, or prion peptides which are bound to a core particle having a structure with an inherent repetitive organization, and hereby in particular to virus-like-particles (VLP's) and subunits of VLP's, respectively, leading to highly ordered and repetitive conjugates represent potent immunogens for the induction of antibodies specific for PrP. Therefore, the present invention provides a therapeutic mean for the treatment of prion diseases, which is based on an ordered and repetitive PrP-core particle array, and in particular a VLP-PrP-conjugate and -array, respectively. This therapeutic is able to induce high titers of anti-PrP antibodies in a vaccinated animal.
  • the present invention provides for a composition
  • a composition comprising: (a) a core particle with at least one first attachment site; and (b) at least one antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is a prion protein (PrP) or a dimer thereof, or a prion peptide, and wherein said second attachment site being selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant, wherein said second attachment site is capable of association to said first attachment site; and wherein said antigen or antigenic determinant and said core particle interact through said association to form an ordered and repetitive antigen array.
  • PrP prion protein
  • Preferred embodiments of core particles suitable for use in the present invention are a virus, a virus-like particle, a bacteriophage, a bacterial pilus or flagella or any other core particle having an inherent repetitive structure capable of forming an ordered and repetitive antigen array in accordance with the present invention.
  • the invention provides a composition comprising an ordered and repetitive antigen or antigenic determinant array, and hereby in particular prion protein or a dimer thereof, or prion peptide VLP conjugates. More specifically, the invention provides a composition comprising a viruslike particle and at least one prion protein (PrP) or a dimer thereof, or a PrP peptide bound thereto. The invention also provides a process for producing the conjugates and the ordered and repetitive arrays, respectively.
  • the compositions of the invention are useful in the production of vaccines for the treatment of prion diseases and as a pharmaccine to prevent or cure prion diseases and to efficiently induce immune responses, in particular antibody responses. Furthermore, the compositions of the invention are particularly useful to efficiently induce self-specific immune responses within the indicated context.
  • a prion protein or a dimer thereof, or a prion peptide is bound to a core particle and VLP, respectively, typically in an oriented manner, yielding an ordered and repetitive prion protein or prion peptide antigen array. Furthermore, the highly repetitive and organized structure of the core particles and VLPs, respectively, mediates the display of the prion protein or a dimer thereof, or prion peptide in a highly ordered and repetitive fashion leading to a highly organized and repetitive antigen array.
  • binding of the prion protein or a dimer thereof, or prion peptide to the core particle and VLP, respectively provides T helper cell epitopes, since the core particle and VLP is foreign to the host immunized with the core particle-prion protein/peptide array and VLP-prion protein/peptide array, respectively.
  • Those arrays differ from prior art conjugates in their highly organized structure, dimensions, and in the repetitiveness of the antigen on the surface of the array.
  • the prion protein or prion peptide is expressed in a suitable expression host compatible with proper folding of the prion protein, or synthesized, while the core particle and the VLP, repespectively, is expressed and purified from an expression host suitable for the folding and assembly of the core particle and the VLP, repespectively.
  • Prion peptides may be chemically synthesized.
  • the prion protein, prion protein domain or prion peptide array is then assembled by binding the prion protein, prion protein domain or prion peptide to the core particle and the VLP, respectively.
  • the present invention provides for a composition
  • a composition comprising (a) a virus-like particle, and (b) at least one antigen or antigenic determinant, wherein said antigen or said antigenic determinant is a prion protein (PrP) or a dimer thereof, or a PrP peptide, and wherein said at least one antigen or antigenic determinant is bound to said virus-like particle.
  • PrP prion protein
  • the present invention provides for a pharmaceutical composition
  • a pharmaceutical composition comprising (a) the composition of claim 1 or claim 22, and (b) an acceptable pharmaceutical carrier.
  • the present invention provides for a vaccine composition
  • a vaccine composition comprising a composition comprising: (a) a core particle with at least one first attachment site; and (b) at least one antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is a prion protein (PrP) or a dimer thereof, or a prion peptide, and wherein said second attachment site being selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant, wherein said second attachment site is capable of association to said first attachment site; and wherein said antigen or antigenic determinant and said core particle interact through said association to form an ordered and repetitive antigen array.
  • PrP prion protein
  • the present invention provides for a vaccine composition
  • a vaccine composition comprising a composition, wherein said composition comprising (a) a virus-like particle; and (b) at least one antigen or antigenic determinant, wherein said antigen or said antigenic determinant is a prion protein (PrP) or a dimer thereof, ⁇ a PrP peptide; and wherein said at least one antigen or antigenic determinant is bound to said virus-like particle.
  • said composition comprising (a) a virus-like particle; and (b) at least one antigen or antigenic determinant, wherein said antigen or said antigenic determinant is a prion protein (PrP) or a dimer thereof, ⁇ a PrP peptide; and wherein said at least one antigen or antigenic determinant is bound to said virus-like particle.
  • PrP prion protein
  • the present invention provides for a process for producing a composition of claim 1 comprising (a) providing a virus-like particle; and (b) providing at least one antigen or antigenic determinant, wherein said antigen or said antigenic determinant is a prion protein (PrP) or a dimer thereof, or a PrP peptide; (c) combining said virus-like particle and said at least one antigen or antigenic determinant so that said at least one antigen or antigenic determinant is bound to said virus-like particle.
  • PrP prion protein
  • the present invention provides a process for producing a composition of claim 22 comprising: (a) providing a core particle with at least one first attachment site; (b) providing at least one antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is a prion protein (PrP) or a dimer thereof, or a prion peptide, and wherein said second attachment site being selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant; and wherein said second attachment site is capable of association to said first attachment site; and (c) combining said core particle and said at least one antigen or antigenic determinant, wherein said antigen or antigenic determinant and said core particle interact through said association to form an ordered and repetitive antigen array.
  • PrP prion protein
  • the present invention provides for a method of immunization comprising administering the composition of claim 1 or claim 22 to an animal or human.
  • the present invention provides for a use of a composition of claim 1 or claim 22 for the manufacture of a medicament for treatment of prion diseases.
  • the present invention provides for a use of a composition of claim 1 or claim 22 for the preparation of a medicament for the therapeutic or prophylactic treatment of prion diseases, preferably of mammalian encephalopathies.
  • the present invention provides for a use of a composition of claim 1 or claim 22, either in isolation or in combination with other agents, for the manufacture of a composition, vaccine, drug or medicament for therapy or prophylaxis of prion diseases, in particular mammalian encephalopathies, and/or for stimulating the mammalian immune system.
  • the invention provides, in particular, vaccine compositions which are suitable for preventing and/or attenuating prion diseases or conditions related thereto.
  • the invention further provides and immunization and vaccination methods, respectively, for preventing and/or attenuating prion diseases or conditions related thereto, in animals, and in particular in cows, sheep and catties as well as in humans.
  • the inventive compositions may be used prophylactically or therapeutically.
  • the invention provides methods for preventing and/or attenuating prion diseases or conditions related thereto which are caused or exacerbated by "self gene products, i.e. "self antigens” as used herein.
  • the invention provides methods for inducing immunological responses in animals and individuals, respectively, which lead to the production of antibodies that prevent and/or attenuate prion diseases or conditions related thereto, which are caused or exacerbated by "self gene products.
  • compositions of the invention when administered to an animal or a human, they may be in a composition which contains salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition.
  • examples of materials suitable for use in preparing pharmaceutical compositions are provided in numerous sources including Remington's Pharmaceutical Sciences (Osol, A, ed., Mack Publishing Co. (1990)).
  • Compositions of the invention are said to be “pharmacologically acceptable” if their administration can be tolerated by a recipient individual. Further, the compositions of the invention will be administered in a "therapeutically effective amount" (i.e., an amount that produces a desired physiological effect).
  • compositions of the present invention may be administered by various methods known in the art, but will normally be administered by injection, infusion, inhalation, oral administration, or other suitable physical methods.
  • the compositions may alternatively be administered intramuscularly, intravenously, or subcutaneously.
  • Components of compositions for administration include sterile aqueous (e.g., physiological saline) or non-aqueous solutions and suspensions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption.
  • FIG. 1 shows the cloning and expression of prion protein (cf.
  • FIG. 2 shows the coupling of prion peptides (mPrPl and mPrPs) to Q ⁇ capsid protein; SDS-Page analysis (cf. Example 14).
  • FIG. 3 shows ELISA of total immunoglobulins (IgG+A+M) recognizing full-length recombinant prion protein detected in serum of mice immunized with mPrPs-Q ⁇ (FIG. 3A) and mPrPl-Q ⁇ (FIG. 3B) (cf. Example 15).
  • Endpoint ELISA titers (log 2 -dilution of 20x prediluted serum) were analysed on day 0, day 13 and day 18.
  • Preimmune serum and serum of mice immunized with murine prion peptide cprpshort, murine prion peptide cprplong and Q ⁇ served as negative controls.
  • Serum of transgenic mice expressing anti-PrP antibody (6H4 ⁇ ) and serum of Pmpolo mice immunized with mPrPs-Q ⁇ and mPrPl-Q ⁇ were used as positive controls.
  • 50 ⁇ g of mPrPs- Q ⁇ and 50 ⁇ g of mPrPl-Q ⁇ were administered in alum, as CFA/IFA-emulsion or without adjuvant on day 0, day 14 and day 28.
  • 50 ⁇ g of Q ⁇ , 100 ⁇ g of cprpshort and 100 ⁇ g of cprplong were applied on day 0, day 14 and day 28.
  • Average anti-PrP titers and standard deviation of four mice (mPrPs-Q ⁇ and mPrPl-Q ⁇ ) and average anti-PrP titers of two mice (controls) are shown.
  • Immunization with mPrPs-Q ⁇ and mPrPl-Q ⁇ resulted in antibodies recognizing full-length recombinant PrP in wild-type and Prnpo/o mice.
  • the highest anti-PrP serum antibody levels were detected in Prnpo/o mice.
  • the anti-PrP titer induced by mPrPs-Q ⁇ was higher than the anti-PrP titer induced by mPrPl-Q ⁇ .
  • FIG. 4 shows long-term anti-PrP antibody response induced by mPrPs-
  • Endpoint ELISA titers (log 2 -dilution of 20x prediluted serum) of total immunoglobulins (IgG+A+M) recognizing recombinant mouse PrP 121-230 are displayed.
  • 50 ⁇ g of mPrPs-Q ⁇ , 50 ⁇ g of Q ⁇ and 100 ⁇ g of murine prion peptide cprpshort were injected subcutaneously on day 0, day 14 and day 28 (indicated by blue arrows).
  • Preimmune serum and serum of mice immunized with murine prion peptide cprpshort and Q ⁇ served as negative controls.
  • Serum of transgenic mice expressing anti-PrP antibody (6H4 ⁇ ) and serum of Prnpo/o mice immunized with mPrPs-Q ⁇ were used as positive controls. Average anti-PrP titers and standard deviation of four mice (mPrPs-Q ⁇ ) and average anti-PrP titers of two mice (controls) are shown.
  • FIG. 5 shows Westernblot-analysis of spleens harvested at 62 dpi. 10%
  • PrP Sc accumulated in scrapie-infected mice that had received no treatment, mPrPs-Q ⁇ or PrPs-peptide. However, the level of deposition of PrP Sc was markedly reduced in mice immunized with mPrPs-Q ⁇ .
  • FIG. 6A-C show the purification of AP205 proteins for use in VLPs, as analysed by SDS PAGE and Western-blotting.
  • FIG. 7A-B ⁇ show electron micrographs comparing AP205 phage particles to AP205 virus like particles spontaneously assembled from recombinant protein expressed in E. coll and purified.
  • Amino acid linker An "amino acid linker”, or also just termed “linker” within this specification, as used herein, either associates the antigen or antigenic determinant with the second attachment site, or more preferably, already comprises or contains the second attachment site, typically - but not necessarily - as one amino acid residue, preferably as a cysteine residue.
  • amino acid linker does not intend to imply that such an amino acid linker consists exclusively of amino acid residues, even if an amino acid linker consisting of amino acid residues is a preferred embodiment of the present invention.
  • amino acid residues of the amino acid linker are, preferably, composed of naturally occuring amino acids or unnatural amino acids known in the art, all-L or all-D or mixtures thereof.
  • an amino acid linker comprising a molecule with a sulfhydryl group or cysteine residue is also encompassed within the invention.
  • Such a molecule comprise preferably a C1-C6 alkyl-, cycloalkyl (C5,C6), aryl or heteroaryl moiety.
  • a linker comprising preferably a C1-C6 alkyl-, cycloalkyl- (C5,C6), aryl- or heteroaryl- moiety and devoid of any amino acid(s) shall also be encompassed within the scope of the invention.
  • Association between the antigen or antigenic determinant or optionally the second attachment site and the amino acid linker is preferably by way of at least one covalent bond, more preferably by way of at least one peptide bond.
  • Animal As used herein, the term "animal” is meant to include, for example, humans, sheep, elks, deer, mule deer, minks, mammals, monkeys, horses, cattle, pigs, goats, dogs, cats, rats, mice, birds, chicken, reptiles, fish, insects and arachnids.
  • Antibody refers to molecules which are capable of binding an epitope or antigenic determinant.
  • the term is meant to include whole antibodies and antigen-binding fragments thereof, including single-chain antibodies.
  • the antibodies are human antigen binding antibody fragments and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a V or VH domain.
  • the antibodies can be from any animal origin including birds and mammals.
  • the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse or chicken.
  • human antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulins and that do not express endogenous immunoglobulins, as described, for example, in U.S. Patent No. 5,939,598 by Kucherlapati et al.
  • Antigen refers to a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by MHC molecules.
  • TCR T cell receptor
  • An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T- lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a Th cell epitope and is given in adjuvant.
  • An antigen can have one or more epitopes (B- and T- epitopes).
  • the specific reaction referred to above is meant to indicate that the antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens.
  • Antigens as used herein may also be mixtures of several individual antigens.
  • Antigenic determinant As used herein, the term “antigenic determinant” is meant to refer to that portion of an antigen that is specifically recognized by either B- or T-lymphocytes. B-lymphocytes responding to antigenic determinants produce antibodies, whereas T-lymphocytes respond to antigenic determinants by proliferation and establishment of effector functions critical for the mediation of cellular and/or humoral immunity. [0045] Association: As used herein, the term “association” as it applies to the first and second attachment sites, refers to the binding of the first and second attachment sites that is preferably by way of at least one non-peptide bond. The nature of the association may be covalent, ionic, hydrophobic, polar or any combination thereof, preferably the nature of the association is covalent.
  • first attachment site refers to an element of non-natural or natural origin, to which the second attachment site located on the antigen or antigenic determinant may associate.
  • the first attachment site may be a protein, a polypeptide, an amino acid, a peptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combination thereof, or a chemically reactive group thereof.
  • the first attachment site is located, typically and preferably on the surface, of the core particle such as, preferably the virus-like particle. Multiple first attachment sites are present on the surface of the core and virus-like particle, respectively, typically in a repetitive configuration.
  • Attachment Site refers to an element associated with the antigen or antigenic determinant to which the first attachment site located on the surface of the core particle and virus-like particle, respectively, may associate.
  • the second attachment site of the antigen or antigenic determinant may be a protein, a polypeptide, a peptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a combination thereof, or a chemically reactive group thereof.
  • At least one second attachment site is present on the antigen or antigenic determinant.
  • the term "antigen or antigenic determinant with at least one second attachment site” refers, therefore, to an antigen or antigenic construct comprising at least the antigen or antigenic determinant and the second attachment site.
  • these antigen or antigenic constructs comprise an "amino acid linker".
  • bound refers to binding or attachment that may be covalent, e.g., by chemically coupling, or noncovalent, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc. Covalent bonds can be, for example, ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, carbon-phosphorus bonds, and the like.
  • bound is broader than and includes terms such as “coupled,” “fused” and “attached”.
  • Coat protein(s) refers to the protein(s) of a bacteriophage or a RNA-phage capable of being incorporated within the capsid assembly of the bacteriophage or the RNA-phage.
  • the term "CP” is used.
  • the specific gene product of the coat protein gene of RNA-phage Q ⁇ is referred to as "Q ⁇ CP”
  • the "coat proteins” of bacteriophage Q ⁇ comprise the "Q ⁇ CP” as well as the Al protein.
  • the capsid of Bacteriophage Q ⁇ is composed mainly of the Q ⁇ CP, with a minor content of the Al protein.
  • the VLP Q ⁇ coat protein contains mainly Q ⁇ CP, with a minor content of Al protein.
  • Core particle refers to a rigid structure with an inherent repetitive organization.
  • a core particle as used herein may be the product of a synthetic process or the product of a biological process.
  • Coupled refers to attachment by covalent bonds or by strong non-covalent interactions, typically and preferably to attachment by covalent bonds. Any method normally used by those skilled in the art for the coupling of biologically active materials can be used in the present invention.
  • Effective Amount refers to an amount necessary or sufficient to realize a desired biologic effect.
  • An effective amount of the composition would be the amount that achieves this selected result, and such an amount could be determined as a matter of routine by a person skilled in the art.
  • an effective amount for treating an immune system deficiency could be that amount necessary to cause activation of the immune system, resulting in the development of an antigen specific immune response upon exposure to antigen.
  • the term is also synonymous with "sufficient amount.”
  • the effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and/or the severity of the disease or condition.
  • One of ordinary skill in the art can empirically determine the effective amount of a particular composition of the present invention without necessitating undue experimentation.
  • Epitope refers to continuous or discontinuous portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
  • An epitope is recognized by an antibody or a T cell through its T cell receptor in the context of an MHC molecule.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a polypeptide that elicits an antibody response or induces a T-cell response in an animal, as determined by any method known in the art. (See, for example, Geysen et al, Proc. Natl Acad. Sci. USA S/:3998-4002 (1983)).
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic. Antigenic epitopes can also be T-cell epitopes, in which case they can be bound immunospecifically by a T-cell receptor within the context of an MHC molecule.
  • An epitope can comprise 3 amino acids in a spatial conformation which is unique to the epitope. Generally, an epitope consists of at least about 5 such amino acids, and more usually, consists of at least about 8-10 such amino acids. If the epitope is an organic molecule, it may be as small as Nitrophenyl.
  • Fusion refers to the combination of amino acid sequences of different origin in one polypeptide chain by in-frame combination of their coding nucleotide sequences.
  • the term “fusion” explicitly encompasses internal fusions, i.e., insertion of sequences of different origin within a polypeptide chain, in addition to fusion to one of its termini.
  • Immune response refers to a humoral immune response and/or cellular immune response leading to the activation or proliferation of B- and/or T-lymphocytes and/or and antigen presenting cells. In some instances, however, the immune responses may be of low intensity and become detectable only when using at least one substance in accordance with the invention. "Immunogenic” refers to an agent used to stimulate the immune system of a living organism, so that one or more functions of the immune system are increased and directed towards the immunogenic agent.
  • immunogenic polypeptide is a polypeptide that elicits a cellular and/or humoral immune response, whether alone or linked to a carrier in the presence or absence of an adjuvant.
  • antigen presenting cell may be activated.
  • a substance which "enhances" an immune response refers to a substance in which an immune response is observed that is greater or intensified or deviated in any way with the addition of the substance when compared to the same immune response measured without the addition of the substance.
  • the lytic activity of cytotoxic T cells can be measured, e.g. using a 51 Cr release assay, in samples obtained with and without the use of the substance during immunization.
  • the amount of the substance at which the CTL lytic activity is enhanced as compared to the CTL lytic activity without the substance is said to be an amount sufficient to enhance the immune response of the animal to the antigen.
  • the immune response in enhanced by a factor of at least about 2, more preferably by a factor of about 3 or more.
  • the amount or type of cytokines secreted may also be altered.
  • the amount of antibodies induced or their subclasses may be altered.
  • Immunization As used herein, the terms “immunize” or
  • immunosenization or related terms refer to conferring the ability to mount a substantial immune response (comprising antibodies and/or cellular immunity such as effector CTL) against a target antigen or epitope. These terms do not require that complete immunity be created, but rather that an immune response be produced which is substantially greater than baseline. For example, a mammal may be considered to be immunized against a target antigen if the cellular and/or humoral immune response to the target antigen occurs following the application of methods of the invention.
  • Natural origin As used herein, the term “natural origin” means that the whole or parts thereof are not synthetic and exist or are produced in nature.
  • Non-natural As used herein, the term generally means not from nature, more specifically, the term means from the hand of man.
  • Non-natural origin As used herein, the term “non-natural origin” generally means synthetic or not from nature; more specifically, the term means from the hand of man.
  • Ordered and repetitive antigen or antigenic determinant array generally refers to a repeating pattern of antigen or antigenic determinant, characterized by a typically and preferably uniform spacial arrangement of the antigens or antigenic determinants with respect to the core particle and virus-like particle, respectively.
  • the repeating pattern may be a geometric pattern.
  • suitable ordered and repetitive antigen or antigenic determinant arrays are those which possess strictly repetitive paracrystalline orders of antigens or antigenic determinants, preferably with spacings of 1 to 30 nanometers, preferably 5 to 15 nanometers.
  • Pili As used herein, the term “pili” (singular being “pilus”) refers to extracellular structures of bacterial cells composed of protein monomers (e.g., pilin monomers) which are organized into ordered and repetitive patterns. Further, pili are structures which are involved in processes such as the attachment of bacterial cells to host cell surface receptors, inter-cellular genetic exchanges, and cell-cell recognition. Examples of pili include Type-1 pili, P-pili, FlC pili, S-pili, and 987P-pili. Additional examples of pili are set out below.
  • Pilus-like structure refers to structures having characteristics similar to that of pili and composed of protein monomers.
  • One example of a "pilus-like structure” is a structure formed by a bacterial cell which expresses modified pilin proteins that do not form ordered and repetitive arrays that are identical to those of natural pili.
  • Polypeptide refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). It indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide. This term is also intended to refer to post- expression modifications of the polypeptide, for example, glycosolations, acetylations, phosphorylations, and the like. A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence. It may also be generated in any manner, including chemical synthesis.
  • Prion Protein refers to a protein encoded by a PrP gene. Prions are distinct from bacteria, viruses and viroids. Known prions include those which infect animals to cause scrapie, a transmissible, degenerative disease of the nervous system of sheep and goats as well as bovine spongiform encephalopathies (BSE) or mad cow disease and feline spongiform encephalopathies of cats.
  • BSE bovine spongiform encephalopathies
  • prion diseases known at present to affect humans are (1) kuru, (2) Creutzfeldt- Jakob Disease (CJD), (3) Gerstmann-Straussler-Scheinker Disease (GSS), (4) fatal familial insomnia (FFI) and (5) new variant of CJD (nvCJD).
  • CJD Creutzfeldt- Jakob Disease
  • GSS Gerstmann-Straussler-Scheinker Disease
  • FFI fatal familial insomnia
  • nvCJD new variant of CJD
  • prion includes all forms of prions causing all or any of these diseases or others in any animals used - and in particular in humans, deer, elk and in domesticated farm animals including but not limited to cows and sheep.
  • PrP gene and the PrP gene of other species There are a number of at present known variants to the human PrP gene and the PrP gene of other species.
  • prion protein PrP
  • prion protein variants including but not limiting to the above indicated preferred examples.
  • PrP peptide or “prion peptide” are broadly defined as any peptide which represents a fraction of a PrP protein and containing at least two, preferably at least three, more prefereably at least four, more prefereably at least five, more prefereably at least six consecutive aminoacids of the original PrP protein.
  • PrP peptide and “PrP fragment” are used interchangeably.
  • PrP peptide and fragments thereof or “prion peptide and fragments thereof, as used herein shall encompass beside the PrP peptide and prion peptide, respectively, any fraction of said PrP peptide and prion peptide, respectively, wherein said fraction may be, preferably, derived by deletion of one or more amino acids at the N and/or C terminus.
  • the PrP peptide can be obtained by recombinant expression in eukaryotic or prokaryotic expression systems as PrP peptide alone or as a fusion with other amino acids or proteins, e.g. to facilitate folding, expression or solubility of the PrP peptide or to facilitate purification of the PrP peptide.
  • one or more amino acids may be added N- or C-terminally to PrP petides.
  • at least one second attachment site may be added to the PrP peptide.
  • PrP peptides may be synthesized using methods known to the art.
  • PrP peptide as used herein shall also encompass a peptide which simulates the three dimensional surface structure of PrP c or PrP sc .
  • Such PrP peptide is not necessarily derived from a continuous amino acid sequence of PrP, but may be formed by discontinuous amino acid residues from PrP. Such peptides may even contain amino acids which are not present in the corresponding PrP protein.
  • PrP c refers to the cellular PrP protein, which is the PrP form that does not typically correlate with the induction of prion disease and which is experimentally defined to be sensitive to Proteinase K digestion. Spectroscopic studies of PrPc showed that PrPc contains -40% alpha-helix and is devoid of beta-sheet.
  • PrP sc refers to a post-translationally altered form of PrP c .
  • PrP sc is formed during a process in which PrP c forms a complex with PrP sc and is transformed into a second PrP sc .
  • the amino acid sequence of PrP c before transformation and after transformation to PrP sc is identical.
  • Spectroscopic studies of PrP sc showed that PrP sc contains a high degree of beta-sheet, is at least partially resistant to Proteinase K and correlates with prion disease.
  • PrP2 refers to a mammalian prion-like polypeptide and fragments thereof and allelic variants thereof. PrP2 proteins from human and mouse were identified in part on the basis of homology of the cDNA encoding for PrP2 to PrP of bovine, chicken, human hamster, mouse and sheep as described in WO 01/46419.
  • Residue As used herein, the term “residue” is meant to mean a specific amino acid in a polypeptide backbone or side chain.
  • Self antigen refers to proteins encoded by the hosf s DNA and products generated by proteins or RNA encoded by the host's DNA are defined as self.
  • proteins that result from a combination of two or several self-molecules or that represent a fraction of a self-molecule and proteins that have a high homology two self- molecules as defined above may also be considered self.
  • Treatment As used herein, the terms “treatment”, “treat”, “treated” or
  • treating refers to prophylaxis and/or therapy.
  • the term refers to a prophylactic treatment which increases the resistance of a subject to infection with a pathogen or, in other words, decreases the likelihood that the subject will become infected with the pathogen or will show signs of illness attributable to the infection, as well as a treatment after the subject has become infected in order to fight the infection, e.g., reduce or eliminate the infection or prevent it from becoming worse.
  • treatment refers to a prophylactic or therapeutic treatment which increases the resistance of a subject against, and/or which reverts formation and/or aggregation of the misfolded form of prion protein (Prp Sc ) that is associated to prion diseases.
  • Prp Sc misfolded form of prion protein
  • Vaccine refers to a formulation which contains the composition of the present invention and which is in a form that is capable of being administered to an animal.
  • the vaccine comprises a conventional saline or buffered aqueous solution medium in which the composition of the present invention is suspended or dissolved.
  • the composition of the present invention can be used conveniently to prevent, ameliorate, or otherwise treat a condition.
  • the vaccine Upon introduction into a host, the vaccine is able to provoke an immune response including, but not limited to, the production of antibodies and/or cytokines and/or the activation of cytotoxic T cells, antigen presenting cells, helper T cells, dendritic cells and/or other cellular responses.
  • the vaccine of the present invention additionally includes an adjuvant which can be present in either a minor or major proportion relative to the compound of the present invention.
  • adjuvant refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine of the present invention provide for an even more enhanced immune response.
  • adjuvants can be used. Examples include complete and incomplete Freund's adjuvant, aluminum hydroxide and modified muramyldipeptide.
  • virus-like particle refers to a structure resembling a virus particle.
  • a virus-like particle in accordance with the invention is non replicative and noninfectious since it lacks all or part of the viral genome, in particular the replicative and infectious components of the viral genome.
  • a virus-like particle in accordance with the invention may contain nucleic acid distinct from their genome.
  • a typical and preferred embodiment of a virus-like particle in accordance with the present invention is a viral capsid such as the viral capsid of the corresponding virus, bacteriophage, or RNA-phage.
  • viral capsid refers to a macromolecular assembly composed of viral protein subunits.
  • the viral protein subunits assemble into a viral capsid and capsid, respectively, having a structure with an inherent repetitive organization, wherein said structure is, typically, spherical or tubular.
  • the capsids of RNA-phages or HBcAg's have a spherical form of icosahedral symmetry.
  • capsid-like structure refers to a macromolecular assembly composed of viral protein subunits reproducing the capsid morphology in the above defined sense but deviating from the typical symmetrical assembly while maintaining ,a sufficient degree of order and repetitiveness.
  • virus-like particle of a bacteriophage refers to a virus-like particle resembling the structure of a bacteriophage, being non replicative and noninfectious, and lacking at least the gene or genes encoding for the replication machinery of the bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.
  • This definition should, however, also encompass virus-like particles of bacteriophages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and noninfectious virus-like particles of a bacteriophage.
  • VLP of RNA phage coat protein The capsid structure formed from the self-assembly of 180 subunits of RNA phage coat protein and optionally containing host RNA is referred to as a "VLP of RNA phage coat protein".
  • VLP of Q ⁇ coat protein A specific example is the VLP of Q ⁇ coat protein.
  • the VLP of Q ⁇ coat protein may either be assembled exclusively from Q ⁇ CP subunits (generated by expression of a Q ⁇ CP gene containing, for example, a TAA stop codon precluding any expression of the longer Al protein through suppression, see Kozlovska, T.M., et al, Intervirology 39: 9-15 (1996)), or additionally contain Al protein subunits in the capsid assembly.
  • virus particle refers to the morphological form of a virus. In some virus types it comprises a genome surrounded by a protein capsid; others have additional structures (e.g., envelopes, tails, etc.).
  • certain embodiments of the invention involve the use of recombinant nucleic acid technologies such as cloning, polymerase chain reaction, the purification of DNA and RNA, the expression of recombinant proteins in prokaryotic and eukaryotic cells, etc.
  • recombinant nucleic acid technologies such as cloning, polymerase chain reaction, the purification of DNA and RNA, the expression of recombinant proteins in prokaryotic and eukaryotic cells, etc.
  • Such methodologies are well known to those skilled in the art and can be conveniently found in published laboratory methods manuals (e.g., Sambrook, J. et al, eds., Molecular Cloning, A Laboratory Manual, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al, eds., Current Protocols in Molecular Biology, John H.
  • compositions of the invention comprise, or alternatively consist of (a) a core particle with at least one first attachment site; and (b) at least one antigen or antigenic determinant with at least one second attachment site, wherein said antigen or antigenic determinant is a prion protein (PrP) or a prion peptide, and wherein said second attachment site being selected from the group consisting of (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant, wherein said second attachment site is capable of association to said first attachment site; and wherein said antigen or antigenic determinant and said core particle interact through said association to form an ordered and repetitive antigen array.
  • compositions of the invention comprise, or alternatively consist of, a virus-like particle and at least one antigen or antigenic determinant, wherein the antigen or antigenic determinant is a prion protein (PrP) or a PrP peptide, and wherein the at least one antigen or antigenic determinant is bound to the virus-like particle so as to form an ordered and repetitive antigen- VLP- array.
  • the antigen or antigenic determinant is a prion protein (PrP) or a PrP peptide
  • the invention conveniently enables the practitioner to construct such a composition, inter alia, for treatment and/or prophylactic prevention of prion diseases such as iatrogenic CJD, new variant CJD, familial CJD, sporadic CJD, Kuru, GSS, fatal familial insomnia, fatal sporadic insomnia, scrapie, BSE, TME, chronic wasting disease, FSE and exotic ungulate encephalopathy (F. Heppner, A. Aguzzi, Encyclopedia of Life Sciences, 2001, Nature Publishing Group; "Prion Diseases”).
  • prion diseases such as iatrogenic CJD, new variant CJD, familial CJD, sporadic CJD, Kuru, GSS, fatal familial insomnia, fatal sporadic insomnia, scrapie, BSE, TME, chronic wasting disease, FSE and exotic ungulate encephalopathy (F. Heppner, A. Aguzzi, Encyclopedia of Life Sciences, 2001, Nature Publishing Group; "Prion Diseases").
  • the core particle comprises a virus, a bacterial pilus, a structure formed from bacterial pilin, a bacteriophage, a virus-like particle, a viral capsid particle or a recombinant form thereof.
  • Any virus known in the art having an ordered and repetitive coat and/or core protein structure may be selected as a core particle of the invention; examples of suitable viruses include Sindbis and other alphaviruses, rhabdoviruses (e.g.
  • vesicular stomatitis virus picornaviruses (e.g., human rhino virus, Aichi virus), togaviruses (e.g., rubella virus), orthomyxoviruses (e.g., Thogoto virus, Batken virus, fowl plague virus), polyomaviruses (e.g., polyomavirus BK, polyomavirus JC, avian polyomavirus BFDV), parvoviruses, rotaviruses, Norwalk virus, foot and mouth disease virus, a retrovirus, Hepatitis B virus, Tobacco mosaic virus, Flock House Virus, and human Papilomavirus, and preferably a RNA phage, bacteriophage Q ⁇ , bacteriophage R17, bacteriophage Mi l, bacteriophage MX1, bacteriophage NL95, bacteriophage fr, bacteriophage GA, bacteriophage SP, bacteriophage
  • the invention utilizes genetic engineering of a virus to create a fusion between an ordered and repetitive viral envelope protein and a first attachment site comprising a heterologous protein, peptide, antigenic determinant or a reactive amino acid residue of choice.
  • a virus used for the present invention is replication incompetent due to chemical or physical inactivation or, as indicated, due to lack of a replication competent genome.
  • the viral protein selected for fusion to the first attachment site should have an organized and repetitive structure.
  • Such an organized and repetitive structure includes paracrystalline organizations with a spacing of 5-30 nm, preferably 5-15 nm, on the surface of the virus.
  • the creation of this type of fusion protein will result in multiple, ordered and repetitive first attachment sites on the surface of the virus and reflect the normal organization of the native viral protein.
  • the first attachment site may be or be a part of any suitable protein, polypeptide, sugar, polynucleotide, peptide (amino acid), natural or synthetic polymer, a secondary metabolite or combination thereof that may serve to specifically attach the antigen or antigenic determinant leading an ordered and repetitive antigen array.
  • the core particle is a recombinant alphavirus, and more specifically, a recombinant Sinbis virus.
  • Alphaviruses are positive stranded RNA viruses that replicate their genomic RNA entirely in the cytoplasm of the infected cell and without a DNA intermediate (Strauss, J. and Strauss, E., Microbiol. Rev. 55:491-562 (1994)).
  • Sindbis Xiong, C. et al, Science 243:1188-1191 (1989); Schlesinger, S., Trends Biotechnol 11:18-22 (1993)
  • Semliki Forest Virus SFV
  • alphaviral core particles of the invention may be done by means generally known in the art of recombinant DNA technology, as described by the aforementioned articles, which are incorporated herein by reference.
  • a variety of different recombinant host cells can be utilized to produce a viral-based core particle for antigen or antigenic determinant attachment.
  • alphaviruses are known to have a wide host range; Sindbis virus infects cultured mammalian, reptilian, and amphibian cells, as well as some insect cells (Clark, H., J. Natl Cancer Inst. 5.7:645 (1973); Leake, C, J. Gen. Virol 35:335 (1977); Stollar, V. in THE TOGAVIRUSES, R.W. Schlesinger, Ed., Academic Press, (1980), pp.583-621).
  • Sindbis virus infects cultured mammalian, reptilian, and amphibian cells, as well as some insect cells (Clark, H., J. Natl Cancer Inst. 5.7:645 (1973); Leake, C, J. Gen. Virol 35:335 (1977); Stollar, V. in THE TOGAVIRUSES
  • BHK, COS, Vero, HeLa and CHO cells are particularly suitable for the production of heterologous proteins because they have the potential to glycosylate heterologous proteins in a manner similar to human cells (Watson, E. et al, Glycobiology 4:227, (1994)) and can be selected (Zang, M. et al, Bio/Technology 13:389 (1995)) or genetically engineered (Renner W. et al, Biotech. Bioeng. 4:476 (1995); Lee K. et al. Biotech. Bioeng. 50:336 (1996)) to grow in serum-free medium, as well as in suspension.
  • Packaged RNA sequences can also be used to infect host cells. These packaged RNA sequences can be introduced to host cells by adding them to the culture medium. For example, the preparation of non- infective alpahviral particles is described in a number of sources, including "Sindbis Expression System", Version C (Invitrogen Catalog No. K750-1).
  • mammalian cells When mammalian cells are used as recombinant host cells for the production of viral-based core particles, these cells will generally be grown in tissue culture. Methods for growing cells in culture are well known in the art (see, e.g., Celis, J., ed., CELL BIOLOGY, Academic Press, 2 nd edition, (1998); Sambrook, J. et al, eds., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel, F. et al, eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John H. Wiley & Sons, Inc.
  • RNA viruses suitable for use as core particle in the present invention include, but are not limited to, the following: members of the family Reoviridae, including the genus Orthoreovirus (multiple serotypes of both mammalian and avian retroviruses), the genus Orbivirus (Bluetongue virus, Eugenangee virus, Kemerovo virus, African horse sickness virus, and Colorado Tick Fever virus), the genus Rotavirus (human rotavirus, Kansas calf diarrhea virus, murine rotavirus, simian rotavirus, bovine or ovine rotavirus, avian rotavirus); the family Picomaviridae, including the genus Enterovirus (poliovirus, Coxsackie virus A and B, enteric cytopathic human orphan (ECHO) viruses, hepatitis A, C, D
  • members of the family Reoviridae including the genus Orthoreovirus (multiple serotypes of both mammalian and avi
  • the family Bunyaviridae including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean-Congo hemorrhagic fever virus, Kenya sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtype
  • the family Bunyaviridae including the genus Bunyvirus (Bunyamwera and related viruses, California encephalitis group viruses), the genus Phlebovirus (Sandfly fever Sicilian virus, Rift Valley fever virus), the genus Nairovirus (Crimean- Congo hemorrhagic fever virus, Kenya sheep disease virus), and the genus Uukuvirus (Uukuniemi and related viruses); the family Orthomyxoviridae, including the genus Influenza virus (Influenza virus type A, many human subtypes
  • Illustrative DNA viruses that may be used as core particles include, but are not limited to: the family Poxviridae, including the genus Orthopoxvirus (Variola major, Variola minor, Monkey pox Vaccinia, Cowpox, Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus (Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avian poxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genus Suipoxvirus (Swinepox), the genus Parapoxvirus (contagious postular dermatitis virus, pseudocowpox, bovine papular stomatitis virus); the family Iridoviridae (African swine fever virus, Frog viruses 2 and 3, Lymphocystis virus of fish); the family Herpesviridae, including the
  • a bacterial pilin, a subportion of a bacterial pilin, or a fusion protein which contains either a bacterial pilin or subportion thereof is used to prepare compositions and vaccine compositions, respectively, of the invention.
  • pilin proteins include pilins produced by Escherichia coli, Haemophilus influenzae, Neisseria meningitidis, Neisseria gonorrhoeae, Caulobacter crescentus, Pseudomonas stutzeri, and Pseudomonas aeruginosa.
  • amino acid sequences of pilin proteins suitable for use with the present invention include those set out in GenBank reports AJ000636 (SEQ ID NO:l), AJ132364 (SEQ ID NO:2), AF229646 (SEQ ID NO:3), AF051814 (SEQ ID NO:4), AF051815 (SEQ ID NO:5), and X00981 (SEQ ID NO:6), the entire disclosures of which are incorporated herein by reference.
  • Bacterial pilin proteins are generally processed to remove N-terminal leader sequences prior to export of the proteins into the bacterial periplasm. Further, as one skilled in the art would recognize, bacterial pilin proteins used to prepare compositions and vaccine compositions, respectively, of the invention will generally not have the naturally present leader sequence.
  • pilin protein suitable for use in the present invention is the P-pilin of E. coli (GenBank report AF237482 (SEQ ID NO:7)).
  • An example of a Type-1 E. coli pilin suitable for use with the invention is a pilin having the amino acid sequence set out in GenBank report P04128 (SEQ ID NO:8), which is encoded by nucleic acid having the nucleotide sequence set out in GenBank report M27603 (SEQ ID NO:9).
  • GenBank report P04128 SEQ ID NO:8
  • SEQ ID NO:9 The entire disclosures of these GenBank reports are incorporated herein by reference. Again, the mature form of the above referenced protein would generally be used to prepare compositions and vaccine compositions, respectively, of the invention.
  • Bacterial pilins or pilin subportions suitable for use in the practice of the present invention will generally be able to associate to form ordered and repetitive antigen arrays.
  • Pilin proteins are then purified by chromatography, after which pilin dimers are formed by dialysis against 5 mM tris(hydroxymethyl) aminomethane hydrochloride (pH 8.0). Eshdat et al. also found that pilin dimers reassemble to form pili upon dialysis against the 5 mM tris(hydroxymethyl) aminomethane (pH 8.0) containing 5 mM MgCl 2 .
  • pilin proteins may be modified to contain a first attachment site to which an antigen or antigenic determinant is linked through a second attachment site.
  • antigens or antigenic determinants can be directly linked through a second attachment site to amino acid residues which are naturally resident in these proteins.
  • These modified pilin proteins may then be used in vaccine compositions of the invention.
  • Bacterial pilin proteins used to prepare compositions and vaccine compositions, respectively, of the invention may be modified in a manner similar to that described herein for HBcAg.
  • cysteine and lysine residues may be either deleted or substituted with other amino acid residues and first attachment sites may be added to these proteins.
  • pilin proteins may either be expressed in modified form or may be chemically modified after expression.
  • intact pili may be harvested from bacteria and then modified chemically.
  • pili or pilus-like structures are harvested from bacteria (e.g., E. coli) and used to form compositions and vaccine compositions of the invention.
  • bacteria e.g., E. coli
  • pili suitable for preparing compositions and vaccine compositions is the Type-1 pilus of E. coli, which is formed from pilin monomers having the amino acid sequence set out in SEQ ID NO:8.
  • pili or pilus-like structures may be modified in a variety of ways. For example, a first attachment site can be added to the pili to which antigens or antigen determinants may be attached through a second attachment site. In other words, bacterial pili or pilus-like structures can be harvested and modified to lead to ordered and repetitive antigen arrays.
  • Antigens or antigenic determinants could be linked to naturally occurring cysteine resides or lysine residues present in Pili or pilus-like structures. In such instances, the high order and repetitiveness of a naturally occurring amino acid residue would guide the coupling of the antigens or antigenic determinants to the pili or pilus-like structures.
  • the pili or pilus-like structures could be linked to the second attachment sites of the antigens or antigenic determinants using a heterobifunctional cross-linking agent.
  • compositions and vaccine compositions of the invention When structures which are naturally synthesized by organisms (e.g., pili) are used to prepare compositions and vaccine compositions of the invention, it will often be advantageous to genetically engineer these organisms so that they produce structures having desirable characteristics. For example, when Type-1 pili of E. coli are used, the E. coli from which these pili are harvested may be modified so as to produce structures with specific characteristics. Examples of possible modifications of pilin proteins include the insertion of one or more lysine residues, the deletion or substitution of one or more of the naturally resident lysine residues, and the deletion or substitution of one or more naturally resident cysteine residues (e.g., the cysteine residues at positions 44 and 84 in SEQ ID NO:8).
  • pilin genes which result in the expression products containing a first attachment site other than a lysine residue (e.g., a FOS or JUN domain).
  • suitable first attachment sites will generally be limited to those which do not prevent pilin proteins from forming pili or pilus-like structures suitable for use in vaccine compositions of the invention.
  • Pilin genes which naturally reside in bacterial cells can be modified in vivo (e.g., by homologous recombination) or pilin genes with particular characteristics can be inserted into these cells.
  • pilin genes could be introduced into bacterial cells as a component of either a replicable cloning vector or a vector which inserts into the bacterial chromosome.
  • the inserted pilin genes may also be linked to expression regulatory control sequences (e.g., a lac operator).
  • pili or pilus-like structures used in compositions and vaccine compositions, respectively, of the invention will be composed of single type of a pilin subunit.
  • Pili or pilus-like structures composed of identical subunits will generally be used because they are expected to form structures which present highly ordered and repetitive antigen arrays.
  • compositions of the invention also include compositions and vaccines comprising pili or pilus-like structures formed from heterogenous pilin subunits.
  • the pilin subunits which form these pili or pilus-like structures can be expressed from genes naturally resident in the bacterial cell or may be introduced into the cells.
  • a naturally resident pilin gene and an introduced gene are both expressed in a cell which forms pili or pilus-like structures, the result will generally be structures formed from a mixture of these pilin proteins.
  • the relative expression of each pilin gene will typically be the factor which determines the ratio of the different pilin subunits in the pili or pilus-like structures.
  • the expression of at least one of the pilin genes can be regulated by a heterologous, inducible promoter.
  • promoters as well as other genetic elements, can be used to regulate the relative amounts of different pilin subunits produced in the bacterial cell and, hence, the composition of the pili or pilus-like structures.
  • the antigen or antigenic determinant can be linked to bacterial pili or pilus-like structures by a bond which is not a peptide bond
  • bacterial cells which produce pili or pilus-like structures used in the compositions of the invention can be genetically engineered to generate pilin proteins which are fused to an antigen or antigenic determinant.
  • Such fusion proteins which form pili or pilus-like structures are suitable for use in vaccine compositions of the invention.
  • virus-like particles in the context of the present application refer to structures resembling a virus particle but which are not pathogenic. In general, virus-like particles lack the viral genome and, therefore, are noninfectious. Also, virus-like particles can be produced in large quantities by heterologous expression and can be easily purified. [0112] In a preferred embodiment, the virus-like particle is a recombinant virus-like particle. The skilled artisan can produce VLPs using recombinant DNA technology and virus coding sequences which are readily available to the public.
  • the coding sequence of a virus envelope or core protein can be engineered for expression in a baculovirus expression vector using a commercially available baculovirus vector, under the regulatory control of a virus promoter, with appropriate modifications of the sequence to allow functional linkage of the coding sequence to the regulatory sequence.
  • the coding sequence of a virus envelope or core protein can also be engineered for expression in a bacterial expression vector, for example.
  • VLPs include, but are not limited to, the capsid proteins of Hepatitis B virus (Ulrich, et al, Virus Res. 50:141-182 (1998)), measles virus (Warnes, et al, Gene 160:173-178 (1995)), Sindbis virus, rotavirus (US 5,071,651 and US 5,374,426), foot-and-mouth-disease virus (Twomey, et al, Vaccine 73:1603-1610, (1995)), Norwalk virus (Jiang, X., et al, Science 250:1580-1583 (1990); Matsui, S.M., et al, J. Clin. Invest.
  • the retroviral GAG protein (WO 96/30523), the retrotransposon Ty protein pi, the surface protein of Hepatitis B virus (WO 92/11291), human papilloma virus (WO 98/15631), RNA phages, Ty, fr-phage, GA-phage and Q ⁇ -phage.
  • the VLP of the invention is not limited to any specific form.
  • the particle can be synthesized chemically or through a biological process, which can be natural or non- natural.
  • this type of embodiment includes a virus-like particle or a recombinant form thereof.
  • the VLP can comprise, or alternatively essentially consist of, or alternatively consist of recombinant polypeptides, or fragments thereof, being selected from recombinant polypeptides of Rotavirus, recombinant polypeptides of Norwalk virus, recombinant polypeptides of Alphavirus, recombinant polypeptides of Foot and Mouth Disease virus, recombinant polypeptides of measles virus, recombinant polypeptides of Sindbis virus, recombinant polypeptides of Polyoma virus, recombinant polypeptides of Retrovirus, recombinant polypeptides of Hepatitis B virus (e.g., a HBcAg), recombinant polypeptides of Tobacco mosaic virus, recombinant polypeptides of Flock House Virus, recombinant polypeptides of human Papillomavirus, recombinant polypeptides
  • the virus-like particle can further comprise, or alternatively essentially consist of, or alternatively consist of, one or more fragments of such polypeptides, as well as variants of such polypeptides.
  • Variants of polypeptides can share, for example, at least 80%, 85%, 90%, 95%, 97%, or 99% identity at the amino acid level with their wild-type counterparts.
  • the virus-like particle comprises, consists essentially of, or alternatively consists of recombinant proteins, or fragments thereof, of a RNA-phage.
  • the RNA-phage is selected from the group consisting of a) bacteriophage Q ⁇ ; b) bacteriophage R17; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage Mi l; h) bacteriophage MX1; i) bacteriophage NL95; j) bacteriophage £; and k) bacteriophage PP7.
  • the viruslike particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant proteins, or fragments thereof, of the RNA- bacteriophage Q ⁇ or of the RNA-bacteriophage fr.
  • the recombinant proteins comprise, or alternatively consist essentially of, or alternatively consist of coat proteins of RNA phages.
  • RNA-phage coat proteins forming capsids or VLP's, or fragments of the bacteriophage coat proteins compatible with self-assembly into a capsid or a VLP are, therefore, further preferred embodiments of the present invention.
  • Bacteriophage Q ⁇ coat proteins for example, can be expressed recombinantly in E. coli. Further, upon such expression these proteins spontaneously form capsids. Additionally, these capsids form a structure with an inherent repetitive organization.
  • bacteriophage coat proteins which can be used to prepare compositions of the invention include the coat proteins of RNA bacteriophages such as bacteriophage Q ⁇ (SEQ ID NO: 10; PIR Database, Accession No. VCBPQ ⁇ referring to Q ⁇ CP and SEQ ID NO: 11; Accession No. AAA16663 referring to Q ⁇ Al protein), bacteriophage R17 (SEQ ID NO: 12; PIR Accession No. VCBPR7), bacteriophage fr (SEQ ID NO: 13; PIR Accession No. VCBPFR), bacteriophage GA (SEQ ID NO: 14 GenBank Accession No.
  • bacteriophage SP SEQ ID NO: 15 GenBank Accession No. CAA30374 referring to SP CP and SEQ ID NO: 16 Accession No. referring to SP Al protein
  • bacteriophage MS2 SEQ ID NO: 17; PIR Accession No. VCBPM2
  • bacteriophage Mi l SEQ ID NO: 18 GenBank Accession No. AAC06250
  • bacteriophage MX1 SEQ ID NO: 19 GenBank Accession No. AAC 14699
  • bacteriophage NL95 SEQ ID NO:20 GenBank Accession No. AAC 14704
  • bacteriophage £ SEQ ID NO: 21 GenBank Accession No.
  • bacteriophage PP7 SEQ ID NO: 22
  • Al protein of bacteriophage Q ⁇ or C-terminal truncated forms missing as much as 100, 150 or 180 amino acids from its C-terminus may be incorporated in a capsid assembly of Q ⁇ coat proteins.
  • the percentage of Q ⁇ Al protein relative to Q ⁇ CP in the capsid assembly will be limited, in order to ensure capsid formation.
  • Q ⁇ coat protein has also been found to self-assemble into capsids when expressed in E. coli (Kozlovska TM. et al, GENE 137: 133-137 (1993)).
  • the capsid contains 180 copies of the coat protein, which are linked in covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R.
  • Capsids or VLP's made from recombinant Q ⁇ coat protein may contain, however, subunits not linked via disulfide links to other subunits within the capsid, or incompletely linked.
  • bands corresponding to monomeric Q ⁇ coat protein as well as bands corresponding to the hexamer or pentamer of Q ⁇ coat protein are visible. Incompletely disulfide-linked subunits could appear as dimer, trimer or even tetramer band in non-reducing SDS-PAGE.
  • Q ⁇ capsid protein also shows unusual resistance to organic solvents and denaturing agents. Surprisingly, we have observed that DMSO and acetonitrile concentrations as high as 30%, and Guanidinium concentrations as high as 1 M do not affect the stability of the capsid.
  • the high stability of the capsid of Q ⁇ coat protein is an advantageous feature, in particular, for its use in immunization and vaccination of mammals and humans in accordance of the present invention.
  • N-terminal methionine of Q ⁇ coat protein is usually removed, as we observed by N-terminal Edman sequencing as described in Stoll, E, et al, J. Biol. Chem. 252:990-993 (1977).
  • VLP composed from Q ⁇ coat proteins where the N-terminal methionine has not been removed, or VLPs comprising a mixture of Q ⁇ coat proteins where the N-terminal methionine is either cleaved or present are also within the scope of the present invention.
  • RNA phage coat proteins have also been shown to self- assemble upon expression in a bacterial host (Kastelein, RA. et al, Gene 23: 245-254 (1983), Kozlovskaya, TM. et al, Dokl Akad. Nauk SSSR 287: 452- 455 (1986), Adhin, MR. et al, Virology 170: 238-242 (1989), Ni, CZ., et al, Protein Sci. 5: 2485-2493 (1996), Priano, C, et al, J. Mol. Biol. 249: 283-297 (1995)).
  • the Q ⁇ phage capsid contains, in addition to the coat protein, the so called read-through protein Al and the maturation protein A2. Al is generated by suppression at the UGA stop codon and has a length of 329 aa.
  • the capsid of phage Q ⁇ recombinant coat protein used in the invention is devoid of the A2 lysis protein, and contains RNA from the host.
  • the coat protein of RNA phages is an RNA binding protein, and interacts with the stem loop of the ribosomal binding site of the replicase gene acting as a translational repressor during the life cycle of the virus. The sequence and structural elements of the interaction are known (Witherell, GW. & Uhlenbeck, OC.
  • the viruslike particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant proteins, or fragments thereof, of a RNA-phage, wherein the recombinant proteins comprise, consist essentially of or alternatively consist of mutant coat proteins of a RNA phage, preferably of mutant coat proteins of the RNA phages mentioned above.
  • mutant coat proteins of the RNA phage have been modified by removal of at least one lysine residue by way of substitution, or by addition of at least one lysine residue by way of substitution; alternatively, the mutant coat proteins of the RNA phage have been modified by deletion of at least one lysine residue, or by addition of at least one lysine residue by way of insertion.
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant proteins, or fragments thereof, of the RNA-bacteriophage Q ⁇ , wherein the recombinant proteins comprise, or alternatively consist essentially of, or alternatively consist of coat proteins having an amino acid sequence of SEQ ID NO: 10, or a mixture of coat proteins having amino acid sequences of SEQ ID NO: 10 and of SEQ ID NO: 11 or mutants of SEQ ID NO: 11 and wherein the N-terminal methionine is preferably cleaved.
  • the viruslike particle comprises, consists essentially of or alternatively consists of recombinant proteins of Q ⁇ , or fragments thereof, wherein the recombinant proteins comprise, or alternatively consist essentially of, or alternatively consist of mutant Q ⁇ coat proteins.
  • these mutant coat proteins have been modified by removal of at least one lysine residue by way of substitution, or by addition of at least one lysine residue by way of substitution.
  • these mutant coat proteins have been modified by deletion of at least one lysine residue, or by addition of at least one lysine residue by way of insertion.
  • Q ⁇ mutants for which exposed lysine residues are replaced by arginines can also be used for the present invention.
  • the following Q ⁇ coat protein mutants and mutant Q ⁇ VLP's can, thus, be used in the practice of the invention: "Q ⁇ -240" (Lysl3-Arg; SEQ ID NO:23), "Q ⁇ -243" (Asn 10-Lys; SEQ ID NO:24), "Q ⁇ -250" (Lys 2-Arg, Lysl3-Arg; SEQ ID NO:25), “Q ⁇ - 251” (SEQ ID NO:26) and "Q ⁇ -259” (Lys 2-Arg, Lysl6-Arg; SEQ ID NO:27).
  • the virus-like particle comprises, consists essentially of or alternatively consists of recombinant proteins of mutant Q ⁇ coat proteins, which comprise proteins having an amino acid sequence selected from the group of a) the amino acid sequence of SEQ ID NO:23; b) the amino acid sequence of SEQ ID NO:24; c) the amino acid sequence of SEQ ID NO:25; d) the amino acid sequence of SEQ ID NO:26; and e) the amino acid sequence of SEQ ID NO:27.
  • mutant Q ⁇ coat protein VLP's and capsids are disclosed in pending U.S. Application No. 10/050,902 filed by the present assignee on January 18, 2002. In particular is hereby referred to Example 18 of above mentioned application.
  • the viruslike particle comprises, or alternatively consists essentially of, or alternatively consists of recombinant proteins of Q ⁇ , or fragments thereof, wherein the recombinant proteins comprise, consist essentially of or alternatively consist W 03
  • the viruslike particle comprises, or alternatively essentially consists of, or alternatively consists of recombinant proteins, or fragments thereof, of RNA-phage AP205.
  • the AP205 genome consists of a maturation protein, a coat protein, a replicase and two open reading frames not present in related phages; a lysis gene and an open reading frame playing a role in the translation of the maturation gene (Klovins, J., et al, J. Gen. Virol. 83: 1523-33 (2002)).
  • AP205 coat protein can be expressed from plasmid pAP283-58 (SEQ ID NO: 94), which is a derivative of pQblO (Kozlovska, T. M.. et al, Gene 737:133- 37 (1993)), and which contains an AP205 ribosomal binding site.
  • AP205 coat protein may be cloned into pQbl85, downstream of the ribosomal binding site present in the vector. Both approaches lead to expression of the protein and formation of capsids as described in the co- pending US provisional patent application with the title "Molecular Antigen Arrays” and having filed by the present assignee on July 16, 2002, which is incorporated by reference in its entirety.
  • Vectors pQblO and pQbl85 are vectors derived from pGEM vector, and expression of the cloned genes in these vectors is controlled by the trp promoter (Kozlovska, T. M. et al, Gene 137:133-37 (1993)).
  • Plasmid pAP283-58 (SEQ ID NO:94) comprises a putative AP205 ribosomal binding site in the following sequence, which is downstream of the Xbal site, and immediately upstream of the ATG start codon of the AP205 coat protein: tet ⁇ g ⁇ ATTTTCTGCGCACCCAT CCCGGGTGGCGCCCAAAGTGAGGAAAATCAC ⁇ tg.
  • the vector pQbl85 comprises a Shine Delagarno sequence downstream from the Xbal site and upstream of the start codon (tct ⁇ g ⁇ TTAACCCAACGCGTAGGAG TCAGGCC tg, Shine Delagarno sequence underlined).
  • the vims- like particle comprises, or alternatively essentially consists of, or alternatively consists of recombinant coat proteins, or fragments thereof, of the RNA-phage AP205..
  • This preferred embodiment of the present invention thus, comprises
  • AP205 coat proteins that form capsids. Such proteins are recombinantly expressed, or prepared from natural sources. AP205 coat proteins produced in bacteria spontaneously form capsids, as evidenced by Electron Microscopy (EM) and immunodiffusion. The structural properties of the capsid formed by the AP205 coat protein (SEQ ID NO: 95) and those formed by the coat protein of the AP205 RNA phage are nearly indistinguishable when seen in EM.
  • AP205 VLPs are highly immunogenic, and can be linked with antigens and/or antigenic determinants to generate vaccine constructs displaying the antigens and/or antigenic determinants oriented in a repetitive manner. High titers are elicited against the so displayed antigens showing that bound antigens and/or antigenic determinants are accessible for interacting with antibody molecules and are immunogenic.
  • the viruslike particle comprises, or alternatively essentially consists of, or alternatively consists of recombinant mutant coat proteins, or fragments thereof, of the RNA-phage AP205.
  • Assembly-competent mutant forms of AP205 VLPs including AP205 coat protein with the subsitution of proline at amino acid 5 to threonine (SEQ ID NO: 96), may also be used in the practice of the invention and leads to a further preferred embodiment of the invention.
  • AP205 P5-T mutant coat protein can be expressed from plasmid pAP281-32 (SEQ ID No. 97), which is derived directly from pQbl85, and which contains the mutant AP205 coat protein gene instead of the Q ⁇ coat protein gene.
  • Vectors for expression of the AP205 coat protein are transfected into E. coli for expression of the AP205 coat protein.
  • Methods for expression of the coat protein and the mutant coat protein, respectively, leading to self-assembly into VLPs are described in co-pending US provisional patent application with the title "Molecular Antigen Arrays" and having filed by the present assignee on July 16' 2002, which is incorporated by reference in its entirety. Suitable E.
  • coli strains include, but are not limited to, E. coli K802, JM 109, RR1.
  • Suitable vectors and strains and combinations thereof can be identified by testing expression of the coat protein and mutant coat protein, respectively, by SDS-PAGE and capsid formation and assembly by optionally first purifying the capsids by gel filtration and subsequently testing them in an immunodiffusion assay (Ouchterlony test) or Electron Microscopy (Kozlovska, T. M., et al, Gene 137:133-37 (1993)).
  • AP205 coat proteins expressed from the vectors pAP283-58 and pAP281-32 may be devoid of the initial Methionine amino-acid, due to processing in the cytoplasm of E. coli. Cleaved, uncleaved forms of AP205 VLP, or mixtures thereof are further preferred embodiments of the invention.
  • the viruslike particle comprises, or alternatively essentially consists of, or alternatively consists of a mixture of recombinant coat proteins, or fragments thereof, of the RNA-phage AP205 and of recombinant mutant coat proteins, or fragments thereof, of the RNA-phage AP205.
  • the viruslike particle comprises, or alternatively essentially consists of, or alternatively consists of fragments of recombinant coat proteins or recombinant mutant coat proteins of the RNA-phage AP205.
  • Recombinant AP205 coat protein fragments capable of assembling into a VLP and a capsid, respectively are also useful in the practice of the invention. These fragments may be generated by deletion, either internally or at the termini of the coat protein and mutant coat protein, respectively. Insertions in the coat protein and mutant coat protein sequence or fusions of antigen sequences to the coat protein and mutant coat protein sequence, and compatible with assembly into a VLP, are further embodiments of the invention and lead to chimeric AP205 coat proteins, and particles, respectively. The outcome of insertions, deletions and fusions to the coat protein sequence and whether it is compatible with assembly into a VLP can be determined by electron microscopy.
  • the particles formed by the AP205 coat protein, coat protein fragments and chimeric coat proteins described above can be isolated in pure form by a combination of fractionation steps by precipitation and of purification steps by gel filtration using e.g. Sepharose CL-4B, Sepharose CL-2B, Sepharose CL- 6B columns and combinations thereof as described in the co-pending US provisional patent application with the title "Molecular Antigen Arrays" and having filed by the present assignee on July 16, 2002, which is incorporated by reference in its entirety.
  • Other methods of isolating virus-like particles are known in the art, and may be used to isolate the virus-like particles (VLPs) of bacteriophage AP205.
  • VLPs virus-like particles
  • the use of ultracentrifugation to isolate VLPs of the yeast retrotransposon Ty is described in U.S. Patent No. 4,918,166, which is incorporated by reference herein in its entirety.
  • RNA- phage coat proteins can be modified such that one or more reactive amino acid residues can be inserted by way of insertion or substitution.
  • those modified forms of bacteriophage coat proteins can also be used for the present invention.
  • variants of proteins which form capsids or capsid-like structures e.g., coat proteins of bacteriophage Q ⁇ , bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS2
  • coat proteins of bacteriophage Q ⁇ , bacteriophage R17, bacteriophage fr, bacteriophage GA, bacteriophage SP, and bacteriophage MS2 can also be used to prepare compositions of the present invention.
  • the invention further includes compositions and vaccine compositions, respectively, which further includes variants of proteins which form capsids or capsid-like structures, as well as methods for preparing such compositions and vaccine compositions, respectively, individual protein subunits used to prepare such compositions, and nucleic acid molecules which encode these protein subunits.
  • variant forms of wild-type proteins which form capsids or capsid-like structures and retain the ability to associate and form capsids or capsid-like structures.
  • compositions and vaccine compositions comprising proteins, which comprise, or alternatively consist essentially of, or alternatively consist of amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identical to wild-type proteins which form ordered arrays and having an inherent repetitive structure, respectively.
  • nucleic acid molecules which encode proteins used to prepare compositions of the present invention.
  • the invention further includes compositions comprising proteins, which comprise, or alternatively consist essentially of, or alternatively consist of amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identical to any of the amino acid sequences shown in SEQ ID NOs: 10-27.
  • Proteins suitable for use in the present invention also include
  • C-terminal truncation mutants of proteins which form capsids or capsid-like structures, or VLP's include proteins having an amino acid sequence shown in any of SEQ ID NOs: 10-27 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the C-terminus. Typically, theses C-terminal truncation mutants will retain the ability to form capsids or capsid-like structures.
  • proteins suitable for use in the present invention also include
  • N-terminal truncation mutants of proteins which form capsids or capsid-like structures include proteins having an amino acid sequence shown in any of SEQ ID NOs: 10-27 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the N-terminus.
  • these N-terminal truncation mutants will retain the ability to form capsids or capsid-like structures.
  • Additional proteins suitable for use in the present invention include N- and C-terminal truncation mutants which form capsids or capsid-like structures.
  • Suitable truncation mutants include proteins having an amino acid sequence shown in any of SEQ ID NOs: 10-27 where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the N-terminus and 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the C-terminus.
  • these N-terminal and C-terminal truncation mutants will retain the ability to form capsids or capsid-like structures.
  • compositions comprising proteins which comprise, or alternatively consist essentially of, or alternatively consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the above described truncation mutants.
  • the invention thus includes compositions and vaccine compositions prepared from proteins which form capsids or VLP's, methods for preparing these compositions from individual protein subunits and VLP's or capsids, methods for preparing these individual protein subunits, nucleic acid molecules which encode these subunits, and methods for vaccinating and/or eliciting immunological responses in individuals using these compositions of the present invention.
  • the invention includes virus-like particles or recombinant forms thereof.
  • the particles used in compositions of the invention are composed of a Hepatitis B core protein (HBcAg) or a fragment of a HBcAg.
  • the particles used in compositions of the invention are composed of a Hepatitis B core protein (HBcAg) or a fragment of a HBcAg protein, which has been modified to either eliminate or reduce the number of free cysteine residues. Zhou et al. (J. Virol.
  • VLP's suitable for use in compositions of the invention include those comprising modified HBcAgs, or fragments thereof, in which one or more of the naturally resident cysteine residues have been either deleted or substituted with another amino acid residue (e.g., a serine residue).
  • the HBcAg is a protein generated by the processing of a Hepatitis B core antigen precursor protein.
  • a number of isotypes of the HBcAg have been identified and their amino acids sequences are readily available to those skilled in the art.
  • compositions and vaccine compositions, respectively, of the invention will be prepared using the processed form of a HBcAg (i.e., a HBcAg from which the N-terminal leader sequence of the Hepatitis B core antigen precursor protein have been removed).
  • the HBcAgs when HBcAgs are produced under conditions where processing will not occur, the HBcAgs will generally be expressed in "processed" form.
  • these proteins when an E. coli expression system directing expression of the protein to the cytoplasm is used to produce HBcAgs of the invention, these proteins will generally be expressed such that the N-terminal leader sequence of the Hepatitis B core antigen precursor protein is not present.
  • Hepatitis B virus-like particles which can be used for the present invention, is disclosed, for example, in WO 00/32227, and hereby in particular in Examples 17 to 19 and 21 to 24, as well as in WO 01/85208, and hereby in particular in Examples 17 to 19, 21 to 24, 31 and 41, and in pending U.S. Application No. 10/050,902 filed by the present assignee on January 18, 2002. For the latter application, it is in particular referred to Example 23, 24, 31 and 51. All three documents are explicitly incorporated herein by reference.
  • the present invention also includes HBcAg variants which have been modified to delete or substitute one or more additional cysteine residues. It is known in the art that free cysteine residues can be involved in a number of chemical side reactions. These side reactions include disulfide exchanges, reaction with chemical substances or metabolites that are, for example, injected or formed in a combination therapy with other substances, or direct oxidation and reaction with nucleotides upon exposure to UV light. Toxic adducts could thus be generated, especially considering the fact that HBcAgs have a strong tendency to bind nucleic acids. The toxic adducts would thus be distributed between a multiplicity of species, which individually may each be present at low concentration, but reach toxic levels when together.
  • HBcAg variants suitable for use in the practice of the present invention have been identified. Yuan et al, (J. Virol. 73:10122-10128 (1999)), for example, describe variants in which the isoleucine residue at position corresponding to position 97 in SEQ ID NO:28 is replaced with either a leucine residue or a phenylalanine residue.
  • HBcAg variants differ in amino acid sequence at a number of positions, including amino acid residues which corresponds to the amino acid residues located at positions 12, 13, 21, 22, 24, 29, 32, 33, 35, 38, 40, 42, 44, 45, 49, 51, 57, 58, 59, 64, 66, 67, 69, 74, 77, 80, 81, 87, 92, 93, 97, 98, 100, 103, 105, 106, 109, 113, 116, 121, 126, 130, 133, 135, 141, 147, 149, 157, 176, 178, 182 and 183 in SEQ ID NO:77.
  • Further HBcAg variants suitable for use in the compositions of the invention, and which may be further modified according to the disclosure of this specification are described in WO 00/198333, WO 00/177158 and WO 00/214478.
  • HBcAgs As noted above, generally processed HBcAgs (i.e., those which lack leader sequences) will be used in the compositions and vaccine compositions, respectively, of the invention.
  • the present invention includes vaccine compositions, as well as methods for using these compositions, which employ the above described variant HBcAgs.
  • amino acid sequence of a polypeptide has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to one of the above wild-type amino acid sequences, or a subportion thereof, can be determined conventionally using known computer programs such the Bestfit program.
  • Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference amino acid sequence, the parameters are set such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the HBcAg variants and precursors having the amino acid sequences set out in SEQ ID NOs: 29-72 and 73-76 are relatively similar to each other.
  • reference to an amino acid residue of a HBcAg variant located at a position which corresponds to a particular position in SEQ ID NO: 77 refers to the amino acid residue which is present at that position in the amino acid sequence shown in SEQ ID NO:77.
  • the homology between these HBcAg variants is for the most part high enough among Hepatitis B viruses that infect mammals so that one skilled in the art would have little difficulty reviewing both the amino acid sequence shown in SEQ ID NO:77 and that of a particular HBcAg variant and identifying "corresponding" amino acid residues.
  • the HBcAg amino acid sequence shown in SEQ ID NO:73 which shows the amino acid sequence of a HBcAg derived from a virus which infect woodchucks, has enough homology to the HBcAg having the amino acid sequence shown in SEQ ID NO:77 that it is readily apparent that a three amino acid residue insert is present in SEQ ID NO:64 between amino acid residues 155 and 156 of SEQ ID NO:77.
  • the invention also includes vaccine compositions which comprise
  • HBcAg variants of Hepatitis B viruses which infect birds, as wells as vaccine compositions which comprise fragments of these HBcAg variants.
  • these HBcAg variants one, two, three or more of the cysteine residues naturally present in these polypeptides could be either substituted with another amino acid residue or deleted prior to their inclusion in vaccine compositions of the invention.
  • cysteine residues of the Hepatitis B virus capsid protein have been either deleted or substituted with another amino acid residue.
  • compositions and vaccine compositions, respectively, of the invention will contain HBcAgs from which the C-terminal region (e.g., amino acid residues 145-185 or 150-185 of SEQ ID NO:77) has been removed.
  • additional modified HBcAgs suitable for use in the practice of the present invention include C-terminal truncation mutants. Suitable truncation mutants include HBcAgs where 1, 5, 10, 15, 20, 25, 30, 34, 35, amino acids have been removed from the C-terminus.
  • HBcAgs suitable for use in the practice of the present invention also include N-terminal truncation mutants.
  • Suitable truncation mutants include modified HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, or 17 amino acids have been removed from the N-terminus.
  • HBcAgs suitable for use in the practice of the present invention include N- and C-terminal truncation mutants. Suitable truncation mutants include HBcAgs where 1, 2, 5, 7, 9, 10, 12, 14, 15, and 17 amino acids have been removed from the N-terminus and 1, 5, 10, 15, 20, 25, 30, 34, 35 amino acids have been removed from the C-terminus.
  • compositions and vaccine compositions comprising HBcAg polypeptides comprising, or alternatively essentially consisting of, or alternatively consisting of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97%, or 99% identical to the above described truncation mutants.
  • a lysine residue is introduced into a HBcAg polypeptide, to mediate the binding of the prion protein, prion protein domain or PrP peptide to the VLP of HBcAg.
  • compositions of the invention are prepared using a HBcAg comprising, or alternatively consisting of, amino acids 1-144, or 1-149, 1-185 of SEQ ID NO:77, which is modified so that the amino acids corresponding to positions 79 and 80 are replaced with a peptide having the amino acid sequence of Gly-Gly-Lys-Gly-Gly (SEQ ID NO:78).
  • cysteine residues at positions 48 and 107 of SEQ ID NO:77 are mutated to serine.
  • the invention further includes compositions comprising the corresponding polypeptides having amino acid sequences shown in any of SEQ ID NOs:29-74, which also have above noted amino acid alterations. Further included within the scope of the invention are additional HBcAg variants which are capable of associating to form a capsid or VLP and have the above noted amino acid alterations.
  • compositions and vaccine compositions comprising HBcAg polypeptides which comprise, or alternatively consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or 99% identical to any of the wild-type amino acid sequences, and forms of these proteins which have been processed, where appropriate, to remove the N-terminal leader sequence and modified with above noted alterations.
  • compositions or vaccine compositions of the invention may comprise mixtures of different HBcAgs.
  • these vaccine compositions may be composed of HBcAgs which differ in amino acid sequence.
  • vaccine compositions could be prepared comprising a "wild-type" HBcAg and a modified HBcAg in which one or more amino acid residues have been altered (e.g., deleted, inserted or substituted).
  • preferred vaccine compositions of the invention are those which present highly ordered and repetitive antigen array, wherein the antigen is a prion protein, prion protein domain or prion peptide.
  • the at least one prion protein (PrP), PrP peptide or PrP domain is bound to said core particle and virus-like particle, respectively, by at least one covalent bond.
  • the least one prion protein (PrP), PrP peptide or PrP domain is bound to the core particle and virus-like particle, respectively, by at least one covalent bond, said covalent bond being a non-peptide bond leading to a core particle-PrP ordered and repetitive array and a PrP-VLP-array or -conjugate, respectively.
  • This PrP-VLP array and conjugate has typically and preferably a repetitive and ordered structure since the at least one, but usually more than one, prion protein (PrP), PrP peptide or PrP domain is bound to the VLP in an oriented manner. Preferably, more than 10, 20, 40, 80, 120 PrP peptides or proteins are bound to the VLP.
  • the formation of a repetitive and ordered PrP-VLP array and conjugate, respectively, is ensured by an oriented and directed as well as defined binding and attachment, respectively, of the at least one prion protein (PrP), PrP peptide or PrP domain to the VLP as will become apparent in the following.
  • the typical inherent highly repetitive and organized structure of the VLP's advantageously contributes to the display of the prion protein (PrP), PrP peptide or PrP domain in a highly ordered and repetitive fashion leading to a highly organized and repetitive PrP-VLP array and conjugate, respectively.
  • PrP prion protein
  • the preferred inventive conjugates and arrays differ from prior art conjugates in their highly organized structure, dimensions, and in the repetitiveness of the antigen on the surface of the array.
  • the preferred embodiment of this invention furthermore, allows expression of both the particle and the antigen in an expression host guaranteeing proper folding of the antigen, i.e. the at least one prion protein (PrP), PrP peptide or PrP domain, and proper folding and assembly of the VLP.
  • PrP prion protein
  • the present invention discloses methods of binding of prion protein, prion protein domain or prion peptide to core particles and VLPs, repectively.
  • the prion protein, prion protein domain or prion peptide is bound to the core particle and VLP, respectively, by way of chemical cross-linking, typically and preferably by using a heterobifunctional cross-linker.
  • a heterobifunctional cross-linker typically and preferably by using a heterobifunctional cross-linker.
  • the hetero-bifunctional cross- linker contains a functional group which can react with preferred first attachment sites, i.e.
  • the first step of the procedure is the reaction of the core particle or the VLP with the cross-linker.
  • the product of this reaction is an activated core particle or activated VLP, also called activated carrier.
  • unreacted cross-linker is removed using usual methods such as gel filtration or dialysis.
  • the prion protein, prion protein domain or prion peptide is reacted with the activated carrier, and this step is typically called the coupling step. Unreacted prion protein, prion protein domain or prion peptide may be optionally removed in a fourth step, for example by dialysis. Several hetero-bifunctional cross-linkers are known to the art.
  • cross-linkers include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers available for example from the Pierce Chemical Company (Rockford, IL, USA) , and having one functional group reactive towards amino groups and one functional group reactive towards cysteine residues.
  • the above mentioned cross-linkers all lead to formation of a thioether linkage.
  • cross-linkers suitable in the practice of the invention is characterized by the introduction of a disulfide linkage between the prion protein, prion protein domain or prion peptide and the core particle or VLP upon coupling.
  • Preferred cross-linkers belonging to this class include for example SPDP and Sulfo-LC-SPDP (Pierce).
  • SPDP Sulfo-LC-SPDP
  • the extent of derivatization of the core particle and VLP, respectively, with cross-linker can be influenced by varying experimental conditions such as the concentration of each of the reaction partners, the excess of one reagent over the other, the pH, the temperature and the ionic strength.
  • the degree of coupling i.e.
  • the amount of prion protein, prion protein domain or prion peptides per subunits of the core particle and VLP, respectively, can be adjusted by varying the experimental conditions described above to match the requirements of the vaccine. Solubility of the prion protein, prion protein domain or prion peptide may impose a limitation on the amount of prion protein, prion protein domain or prion peptide that can be coupled on each subunit, and in those cases where the obtained vaccine would be insoluble, reducing the amount of prion protein, prion protein domain or prion peptides per subunit is beneficial.
  • a particularly favored method of binding of prion protein, prion protein domain or prion peptides to the core particle and the VLP, respectively, is the linking of a lysine residue on the surface of the core particle and the VLP, respectively, with a cysteine residue on the prion protein, prion protein domain or prion peptide.
  • the first attachment site is a lysine residue and the second attachment site is a cysteine residue.
  • an amino acid linker containing a cysteine residue as a second attachment site or as a part thereof, to the prion protein, prion protein domain or prion peptide for coupling to the core particle and VLP, respectively, may be required.
  • a cysteine may be introduced either by insertion or mutation within the prion protein, prion protein domain or prion peptide.
  • the cysteine residue may be introduced by chemical coupling.
  • amino acid linker will be dependent on the nature of the antigen and self-antigen, respectively, i.e. on the nature of the PrP protein, PrP peptide or PrP domain, on its biochemical properties, such as pi, charge distribution and glycosylation. In general, flexible amino acid linkers are favored.
  • amino acid linkers are the hinge region of Immunoglobulins, glycine serine linkers (GGGGS) n , and glycine linkers (G) n all further containing a cysteine residue as second attachment site and optionally further glycine residues.
  • amino acid linkers are N-terminal gammal: CGDKTHTSPP; C-terminal gamma 1: DKTHTSPPCG; N-terminal gamma 3: CGGPKPSTPPGSSGGAP; C-terminal gamma 3: PKPSTPPGSSGGAPGGCG; N-terminal glycine linker: GCGGGG; C-terminal glycine linker: GGGGCG; C-terminal glycine-lysine linker: GGKKGC; N-terminal glycine-lysine linker: CGKKGG.
  • the antigen is a prion peptide, GGCG, GGC or GGC-NH2 ("NH2" stands for amidation) linkers at the C-terminus of the peptide or CGG at its N- terminus are preferred as amino acid linkers.
  • NH2 stands for amidation
  • linkers at the C-terminus of the peptide or CGG at its N- terminus are preferred as amino acid linkers.
  • glycine residues will be inserted between bulky amino acids and the cysteine to be used as second attachment site, to avoid potential steric hindrance of the bulkier amino acid in the coupling reaction.
  • the cysteine residue present on the prion protein, prion protein domain or prion peptide has to be in its reduced state to react with the hetero- bifunctional cross-linker on the activated VLP, that is a free cysteine or a cysteine residue with a free sulfhydryl group has to be available.
  • the cysteine residue to function as binding site is in an oxidized form, for example if it is forming a disulfide bridge, reduction of this disulfide bridge with e.g. DTT, TCEP or ⁇ -mercaptoethanol is required.
  • Other methods of binding the prion protein, prion protein domain or prion peptide to the core particle and the VLP, respectively include methods wherein the prion protein, prion protein domain or prion peptide is cross-linked to the core particle and the VLP, respectively, using the carbodiimide EDC, and NHS.
  • the prion protein, prion protein domain or prion peptide may also be first thiolated through reaction, for example with SATA, SATP or iminothiolane.
  • the prion protein, prion protein domain or prion peptide, after deprotection if required, may then be coupled to the core particle and the VLP, respectively, as follows.
  • the prion protein, prion protein domain or prion peptide is reacted with the core particle and the VLP, respectively, previously activated with a hetero-bifunctional cross-linker comprising a cysteine reactive moiety, and therefore displaying at least one or several functional groups reactive towards cysteine residues, to which the thiolated prion protein, prion protein domain or prion peptide can react, such as described above.
  • a reducing agent are included in the reaction mixture.
  • the prion protein, prion protein domain or prion peptide is attached to the core particle and the VLP, respectively, using a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO] 4 , BS 3 , (Pierce Chemical Company, Rockford, IL, USA) or other known homo-bifunctional cross-linkers whith functional groups reactive towards amine groups or carboxyl groups of the core particle and the VLP, respectively,.
  • a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO] 4 , BS 3 , (Pierce Chemical Company, Rockford, IL, USA) or other known homo-bifunctional cross-linkers whith functional groups reactive towards amine groups or carboxyl groups of the core particle and the VLP, respectively,.
  • the prion protein, or prion protein domain is bound to the core particle and the VLP, respectively, through modification of the carbohydrate moieties present on glycosylated prion protein or prion domain and subsequent reaction with the core particle and the VLP, respectively.
  • the glycosylated prion protein or prion protein domain is reacted with sodium periodate in a mild oxidation reaction of the carbohydrate moiety, to yield an activated prion protein or prion protein domain with one or more aldehyde functional groups.
  • the so activated prion protein or prion protein domain is separated from excess sodium periodate, and further reacted with the core particle and the VLP, respectively, wherein lysine residues of the core particle and the VLP, respectively, or of at least one VLP subunit are reacting with the previously formed aldehyde functional group on the prion protein or prion protein domain, for example as described by Hermanson, G.T. in Bioconjugate Techniques, Academic Press Inc., San Diego, CA, USA.
  • Self polymerization of the activated prion protein or prion protein domain may be controlled by adjusting the pH as described in the aforementioned publication.
  • the formed Schiff base is preferably further reduced with sodium cyanoborohydride, which is subsequently removed by gel filtration or dialysis.
  • the core particle and the VLP may be reacted with EDC at carboxyl groups of the core particle and the VLP, respectively, or at least one VLP subunit and a dihydrazyde, such as adipic acid dihydrazyde, to yield a hydrazide moiety available for reaction with the one or more aldehyde functional groups present on the activated prion protein or prion protein domain.
  • the so formed hydrazone may be further reduced with sodium cyanoborohydride.
  • the activated prion protein or prion protein domain with one or more aldehyde functional groups is reacted with cysteamine, resulting in the introduction of a cysteine group in the prion protein or prion protein domain.
  • Additional cross-linking methods and cross-linkers, suitable for binding a prion protein, prion protein domain or prion peptide to a core particle and a VLP, respectively, as well as guidance on performing the coupling reactions and on the use of chemical cross-linkers and chemical cross-linking procedures can be found in Hermanson, G.T. in Bioconjugate Techniques, Academic Press Inc., San Diego, CA, USA.
  • Other methods of binding the VLP to a prion protein, prion protein domain or prion peptide include methods where the core particle and the VLP, respectively, is biotinylated, and the prion protein, prion protein domain or prion peptide expressed as a streptavidin-fusion protein, or methods wherein both the prion protein, prion protein domain or prion peptides and the core particle and the VLP, respectively, are biotinylated, for example as described in WO 00/23955.
  • the prion protein, prion protein domain or prion peptide may be first bound to streptavidin or avidin by adjusting the ratio of prion protein, prion protein domain or prion peptide to streptavidin such that free binding sites are still available for binding of the core particle and the VLP, respectively, which is added in the next step.
  • all components may be mixed in a "one pot" reaction.
  • ligand-receptor pairs where a soluble form of the receptor and of the ligand is available, and are capable of being cross-linked to the core particle and the VLP, respectively, or the prion protein, prion protein domain or prion peptide, may be used as binding agents for binding the prion protein, prion protein domain or prion peptide to the core particle and the VLP, respectively,.
  • either the ligand or the receptor may be fused to the prion protein, prion protein domain or prion peptide, and so mediate binding to the core particle and the VLP, respectively, chemically bound or fused either to the receptor, or the ligand respectively. Fusion may also be effected by insertion or substitution.
  • the VLP is the VLP of a RNA phage, and in a more preferred embodiment, the VLP is the VLP of RNA phage Q ⁇ coat protein.
  • One or several antigen molecules i.e. a prion peptide, a prion protein or a prion protein domain, can be attached to one subunit of the capsid or VLP of RNA phages coat proteins, preferably through the exposed lysine residues of the VLP of RNA phages, if sterically allowable.
  • a specific feature of the VLP of the coat protein of RNA phages and in particular of the Q ⁇ coat protein VLP is thus the possibility to couple several antigens per subunit. This allows for the generation of a dense antigen array.
  • the binding and attachment, respectively, of the at least prion protein (PrP) or PrP peptide or PrP domain to the core particle and the virus-like particle, respectively, is by way of interaction and association, respectively, between at least one first attachment site of the virus-like particle and at least one second attachment of the antigen or antigenic determinant.
  • VLPs or capsids of Q ⁇ coat protein display a defined number of lysine residues on their surface, with a defined topology with three lysine residues pointing towards the interior of the capsid and interacting with the RNA, and four other lysine residues exposed to the exterior of the capsid. These defined properties favor the attachment of antigens to the exterior of the particle, rather than to the interior of the particle where the lysine residues interact with RNA.
  • VLPs of other RNA phage coat proteins also have a defined number of lysine residues on their surface and a defined topology of these lysine residues.
  • the first attachment site is a lysine residue and/or the second attachment comprises sulfhydryl group or a cysteine residue.
  • the first attachment site is a lysine residue and the second attachment is a cysteine residue.
  • the prion protein, prion protein domain or prion peptide is bound via a cysteine residue, either naturally present on the prion protein, prion protein domain or prion peptide or engineered, to lysine residues of the VLP of RNA phage coat protein, and in particular to the VLP of Q ⁇ coat protein.
  • VLPs derived from RNA phages are their high expression yield in bacteria that allows production of large quantities of material at affordable cost.
  • the inventive conjugates and arrays differ from prior art conjugates in their highly organized structure, dimensions, and in the repetitiveness of the antigen on the surface of the array.
  • the use of the VLPs as carriers allow the formation of robust antigen arrays and conjugates, respectively, with variable antigen density.
  • the use of VLP's of RNA phages, and hereby in particular the use of the VLP of RNA phage Q ⁇ coat protein allows to achieve very high epitope density.
  • the preparation of compositions of VLPs of RNA phage coat proteins with a high epitope density can be effected by using the teaching of this application.
  • the second attachment site may be either naturally or non-naturally present with the antigen or the antigenic determinant. In the case of the absence of a suitable natural occurring second attachment site on the antigen or antigenic determinant, such a, then non-natural second attachment has to be engineered to the antigen.
  • lysine residues are exposed on the surface of the VLP of Q ⁇ coat protein. Typically these residues are derivatized upon reaction with a cross-linker molecule. In the instance where not all of the exposed lysine residues can be coupled to an antigen, the lysine residues which have reacted with the cross-linker are left with a cross-linker molecule attached to the ⁇ -amino group after the derivatization step. This leads to disappearance of one or several positive charges, which may be detrimental to the solubility and stability of the VLP.
  • a Q ⁇ mutant coat protein with one additional lysine residue suitable for obtaining even higher density arrays of antigens.
  • This mutant Q ⁇ coat protein, Q ⁇ -243 (Asn 10-Lys; SEQ ID NO:24), was cloned, the protein expressed, and the capsid or VLP isolated and purified, showing that introduction of the additional lysine residue is compatible with self-assembly of the subunits to a capsid or VLP.
  • prion protein, prion protein domain or prion petide arrays and conjugates, respectively may be prepared using VLP of Q ⁇ coat protein mutants.
  • a particularly favored method of attachment of antigens to VLPs, and in particular to VLPs of RNA phage coat proteins is the linking of a lysine residue present on the surface of the VLP of RNA phage coat proteins with a cysteine residue naturally present or engineered on the antigen, i.e. the prion protein or prion peptide.
  • a cysteine residue In order for a cysteine residue to be effective as second attachment site, a sulfhydryl group must be available for coupling. Thus, a cysteine residue has to be in its reduced state, that is, a free cysteine or a cysteine residue with a free sulfhydryl group has to be available.
  • the cysteine residue to function as second attachment site is in an oxidized form, for example if it is forming a disulfide bridge
  • reduction of this disulfide bridge with e.g. DTT, TCEP or ⁇ -mercaptoethanol is required.
  • concentration of reductand, and the molar excess of reductand over antigen has to be adjusted for each antigen.
  • a titration range, starting from concentrations as low as 10 ⁇ M or lower, up to 10 to 20 mM or higher reductand if required is tested, and coupling of the antigen to the carrier assessed. Although low concentrations of reductand are compatible with the coupling reaction as described in pending U.S. Application No.
  • the pH of the dialysis or equilibration buffer is lower than 7, preferably 6.
  • the compatibility of the low pH buffer with antigen activity or stability has to be tested.
  • Epitope density on the VLP of RNA phage coat proteins can be modulated by the choice of cross-linker and other reaction conditions.
  • the cross-linkers Sulfo-GMBS and SMPH typically allow reaching high epitope density.
  • Derivatization is positively influenced by high concentration of reactands, and manipulation of the reaction conditions can be used to control the number of antigens coupled to VLPs of RNA phage coat proteins, and in particular to VLPs of Q ⁇ coat protein.
  • the position at which it should be fused, inserted or generally engineered has to be chosen.
  • the selection of the position of the second attachment site may, by way of example, be based on a crystal structure of the antigen.
  • Such a crystal structure of the antigen may provide information on the availability of the C- or N- termini of the molecule (determined for example from their accessibility to solvent), or on the exposure to solvent of residues suitable for use as second attachment sites, such as cysteine residues.
  • Exposed disulfide bridges as is the case for Fab fragments, may also be a source of a second attachment site, since they can be generally converted to single cysteine residues through mild reduction.
  • the second attachment site will be added such that it allows generation of antibodies against the site of interaction with the natural ligands.
  • the location of the second attachment site will be selected such that steric hindrance from the second attachment site or any amino acid linker containing the same is avoided.
  • an antibody response directed at a site distinct from the interaction site of the self-antigen with its natural ligand is desired.
  • the second attachment site may be selected such that it prevents generation of antibodies against the interaction site of the self-antigen with its natural ligands.
  • the prion protein or prion peptide comprises a single second attachment site or a single reactive attachment site capable of association with the first attachment sites on the core particle and the VLPs or VLP subunits, respectively.
  • the provision of a single second attachment site or a single reactive attachment site on the antigen thus, ensures a single and uniform type of binding and association, respectively leading to a very highly ordered and repetitive array.
  • the binding and association, respectively is effected by way of a lysine- (as the first attachment site) and cysteine- (as a second attachment site) interaction, it is ensured, in accordance with this preferred embodiment of the invention, that only one cysteine residue per antigen, independent whether this cysteine residue is naturally or non- naturally present on the antigen, is capable of binding and associating, respectively, with the VLP and the first attachment site of the core particle, respectively.
  • an amino acid linker is bound to the antigen or the antigenic determinant by way of at least one covalent bond.
  • the amino acid linker comprises, or alternatively consists of, the second attachment site.
  • the amino acid linker comprises a sulfhydryl group or a cysteine residue.
  • the amino acid linker is cysteine.
  • the at least one antigen or antigenic determinant i.e. the PrP protein, PrP peptide or the PrP domain is fused to the core particle and the virus-like particle, respectively.
  • a VLP is typically composed of at least one subunit assembling into a VLP.
  • the antigen or antigenic determinant preferably the at least one prion peptide or PrP domain, is fused to at least one subunit of the virus-like particle or of a protein capable of being incorporated into a VLP generating a chimeric VLP-subunit-prion peptide protein fusion.
  • Fusion of the prion peptides can be effected by insertion into the VLP subunit sequence, or by fusion to either the N- or C-terminus of the VLP- subunit or protein capable of being incorporated into a VLP.
  • fusion proteins of a peptide to a VLP subunit the fusion to either ends of the subunit sequence or internal insertion of the peptide within the subunit sequence are encompassed.
  • Fusion may also be effected by inserting the prion peptide sequences into a variant of a VLP subunit where part of the subunit sequence has been deleted, that are further referred to as truncation mutants.
  • Truncation mutants may have N- or C-terminal, or internal deletions of part of the sequence of the VLP subunit.
  • the specific VLP HBcAg with, for example, deletion of amino acid residues 79 to 81 is a truncation mutant with an internal deletion. Fusion of prion peptides to either the N- or C-terminus of the truncation mutants VLP-subunits also lead to embodiments of the invention.
  • fusion of an epitope into the sequence of the VLP subunit may also be effected by substitution, where for example for the specific VLP HBcAg, amino acids 79-81 are replaced with a foreign epitope.
  • fusion as referred to hereinafter, may be effected by insertion of the prion peptide sequence in the sequence of a VLP subunit, by substitution of part of the sequence of the VLP subunit with the prion peptide sequence, or by a combination of deletion, substitution or insertions.
  • the chimeric prion peptide- VLP subunit will be in general capable of self-assembly into a VLP.
  • VLP displaying epitopes fused to their subunits are also herein referred to as chimeric VLPs.
  • the virus-like particle comprises or alternatively is composed of at least one VLP subunit.
  • the virus-like particle comprises or alternatively is composed of a mixture of chimeric VLP subunits and non- chimeric VLP subunits, i.e. VLP subunits not having an antigen fused thereto, leading to so called mosaic particles. This may be advantageous to ensure formation of and assembly to a VLP.
  • the proportion of chimeric VLP-subunits may be 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95% or higher.
  • Flanking amino acid residues may be added to either end of the sequence of the peptide or epitope to be fused to either end of the sequence of the subunit of a VLP, or for internal insertion of such peptidic sequence into the sequence of the subunit of a VLP.
  • Glycine and serine residues are particularly favored amino acids to be used in the flanking sequences added to the prion peptide to be fused.
  • Glycine residues confer additional flexibility, which may diminish the potentially destabilizing effect of fusing a foreign sequence into the the sequence of a VLP subunit.
  • the VLP is a Hepatitis B core antigen VLP.
  • Fusion proteins to either the N-terminus of a HBcAg (Neyrinck, S. et al, Nature Med. 5:1157-1163 (1999)) or insertions in the so called major immunodominant region (MIR) have been described (Pumpens, P. and Grens, E., Intervirology 44:98-114 (2001)), WO 01/98333), and are preferred embodiments of the invention.
  • MIR major immunodominant region
  • Example 6 We also describe by way of example (Example 6) the insertion of an epitope into the MIR of HBcAg, resulting in a chimeric self-assembling HBcAg.
  • An important factor for the optimization of the efficiency of self-assembly and of the display of the epitope to be inserted in the MIR of HBcAg is the choice of the insertion site, as well as the number of amino acids to be deleted from the HBcAg sequence within the MIR (Pumpens, P.
  • HBcAg contains a long arginine tail (Pumpens, P.
  • the VLP is a VLP of a RNA phage.
  • the major coat proteins of RNA phages spontaneously assemble into VLPs upon expression in bacteria, and in particular in E. coli.
  • bacteriophage coat proteins which can be used to prepare compositions of the invention include the coat proteins of RNA bacteriophages such as bacteriophage Q ⁇ (SEQ ID NO: 10; PIR Database, Accession No. VCBPQ ⁇ referring to Q ⁇ CP and SEQ ID NO: 11; Accession No. AAA16663 referring to Q ⁇ Al protein) and bacteriophage fr (SEQ ID NO:4; PIR Accession No. VCBPFR).
  • the at least one prion peptide is fused to a Q ⁇ coat protein.
  • Fusion protein constructs wherein epitopes have been fused to the C-terminus of a truncated form of the Al protein of Q ⁇ , or inserted within the Al protein have been described (Kozlovska, T. M., et al, Intervirology, 39:9-15 (1996)).
  • the Al protein is generated by suppression at the UGA stop codon and has a length of 329 aa, or 328 aa, if the cleavage of the N-terminal methionine is taken into account. Cleavage of the N-terminal methionine before an alanine (the second amino acid encoded by the Q ⁇ CP gene) usually takes place in E.
  • the production of mosaic particles may be effected in a number of ways.
  • Kozlovska et al, Intervirolog, 39:9-15 (1996) describe two methods, which both can be used in the practice of the invention.
  • efficient display of the fused epitope on the VLPs is mediated by the expression of the plasmid encoding the Q ⁇ Al protein fusion having a UGA stop codong between CP and CP extension in a E.
  • coli strain harboring a plasmid encoding a cloned UGA suppressor tRNA which leads to translation of the UGA codon into Trp (pISM3001 plasmid (Smiley B.K., et al, Gene 734:33-40 (1993))).
  • the CP gene stop codon is modified into UAA, and a second plasmid expressing the Al protein-prion peptide fusion is cotransformed.
  • the second plasmid encodes a different antibiotic resistance and the origin of replication is compatible with the first plasmid (Kozlovska, T. M., et al, Intervirology 39:9-15 (1996)).
  • CP and the Al protein-prion peptide fusion are encoded in a bicistronic manner, operatively linked to a promoter such as the Trp promoter, as described in FIG. 1 of Kozlovska et al, Intervirology, 39:9-15 (1996).
  • the prion peptide is inserted between amino acid 2 and 3 (numbering of the cleaved CP, that is wherein the N-terminal methionine is cleaved) of the fr CP, thus leading to a prion peptide-fr CP fusion protein.
  • Vectors and expression systems for construction and expression of fr CP fusion proteins self-assembling to VLP and useful in the practice of the invention have been described (Pushko P. et al, Prot. Eng. (5:883-891 (1993)).
  • the prion peptide sequence is inserted into a deletion variant of the fr CP after amino acid 2, wherein residues 3 and 4 of the fr CP have been deleted (Pushko P. et al, Prot. Eng. 5:883-891 (1993)).
  • the prion peptides are fused to a capsid protein of papillomavirus.
  • the prion peptides are fused to the major capsid protein LI of bovine papillomavirus type 1 (BPV-1).
  • BPV-1 bovine papillomavirus type 1
  • Vectors and expression systems for construction and expression of BPV-1 fusion proteins in a baculovirus/insect cells systems have been described (Chackerian, B. et al, Proc. Natl. Acad. Sci. USA 96:2373- 2378 (1999), WO 00/23955).
  • BPV-1 LI- prion peptide fusion protein which is a preferred embodiment of the invention.
  • Cloning in a baculovirus vector and expression in baculovirus infected Sf9 cells has been described, and can be used in the practice of the invention (Chackerian, B. et al, Proc. Natl. Acad. Sci. USA 95:2373-2378 (1999), WO 00/23955).
  • Purification of the assembled particles displaying the fused prion peptides can be performed in a number of ways, such as for example gel filtration or sucrose gradient ultracentrifugation (Chackerian, B. et al, Proc. Natl. Acad. Sci. USA 95:2373- 2378 (1999), WO 00/23955).
  • the prion peptides are fused to a Ty protein capable of being incorporated into a Ty VLP.
  • the prion peptides are fused to the pi or capsid protein encoded by the TYA gene (Roth, J.F., Yeast 75:785-795 (2000)).
  • the yeast retrotransposons Tyl, 2, 3 and 4 have been isolated from Saccharomyces Serevisiae, while the retrotransposon Tfl has been isolated from Schizosaccharomyces Pombae (Boeke, J.D.
  • the retrotransposons Tyl and 2 are related to the copia class of plant and animal elements, while Ty3 belongs to the gypsy family of retrotransposons, which is related to plants and animal retroviruses.
  • the pi protein also referred to as Gag or capsid protein, has a length of 440 amino acids.
  • PI is cleaved during maturation of the VLP at position 408, leading to the p2 protein, the essential component of the VLP.
  • prion protein peptide may be fused to pi by inserting a sequence coding for the prion protein peptide into the BamHl site of the pMA5620 plasmid (Adams, S.E., et al, Nature 329:68-70 (1987)).
  • fusion proteins comprising amino acids 1-381 of pi of Tyl-15, fused C-terminally to the N-terminus of the foreign epitope.
  • N-terminal fusion of prion peptides, or internal insertion into the pi sequence, or substitution of part of the pi sequence is also meant to fall within the scope of the invention.
  • insertion of prion peptides into the Ty sequence between amino acids 30-31, 67-68, 113-114 and 132-133 of the Ty protein pi leads to preferred embodiments of the invention.
  • VLPs suitable for fusion of prion peptides are, for example,
  • Retrovirus-like-particles (WO9630523), HIV2 Gag (Kang, Y.C., et al, Biol. Chem. 350:353-364 (1999)), Cowpea Mosaic Virus (Taylor, K.M.et al, Biol. Chem. 380:387-392 (1999)), parvovirus VP2 VLP (Rueda, P. et al, Virology 263:89-99 (1999)), HBsAg (US 4,722,840, EP0020416B1).
  • VLPs suitable for the practice of the invention are also those described in Intervirology 39:1 (1996). Further examples of VLPs contemplated for use in the invention are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, COPV, HIV GAG, Tobacco Mosaic Virus. Virus-like particles of SV-40, Polyomavirus, Adenovirus, Herpes Simplex Virus, Rotavirus and Norwalk virus have also been made, and chimeric VLPs of those VLPs are also within the scope of the present invention.
  • the antigen or antigenic determinant is a prion protein.
  • the antigen or antigenic determinant comprises or is a prion protein dimer.
  • Two prion protein monomers may be brought together by linkage of the C-terminus of one prion protein monomer with the N-terminus of another prion protein monomer via an amino acid linker.
  • Amino acid linkers suitable for dimerizing two prion protein monomers have been described in the Art, such for example by Dolezal O., et al, Protein Eng. 13: 565-74 (2000), or Perisic O., et al, Structure 2: 1217-26 (1994)).
  • the more preferred length of the described Glycine-Serine amino acid linkers is of 1 to 10 amino acids, preferably 3-10 amino acids, and even more preferably 3-5 amino acids.
  • Dimerization of two prion protein monomers may also be carried out by attaching a dimerization domain to the prion protein monomer, for example the human IgGl CH3 domain (Hu S., et al, Cancer Res. 56: 3055-61 (1996)) at the C-terminus of said prion protein monomer. Attachment of the dimerization domain to the C-terminus of the prion protein monomer is preferably mediated by an amino acid linker or connecting peptide, as described (Hu S., et al, Cancer Res. 56: 3055-61 (1996)).
  • dimerization domains which may be attached at the N- or C- terminus of the prion protein monomer, via an amino acid linker or connecting peptide have been described by De Kruif et al, J. Biol. Chem. 271: 7630-34 (1996). The authors describe the use of Fos and Jun Leucine zippers for the dimerization of a scFv antibody fragment.
  • the dimerization domain is attached to the C-terminus of the scFv fragment, via an amino acid linker of sequence: PKPSTPPGSSCGG.
  • the sequence GGC is added at the C-terminus of both the FOS and Jun helix for improved dimerization.
  • one Jun helix and one Fos helix is attached at the C-terminus of each prion protein monomer to ensure dimer formation, with the sequence GGC at the C- terminus of each helix.
  • the amino acid linker is attached at the C- terminus of both Jun and fos helices, immediately at the N-terminus of each of the prion protein monomers.
  • the sequence CGG is attached to the N-terminus of both Jun and Fos helices.
  • GCN4 leucine zipper of Saccharomyces cerevisiae as homo-dimerization domain The GCN4 leucine zipper is fused to the N- or C-terminus of the prion protein monomer according to the guidance provided above.
  • an amino acid linker containing a cysteine residue as second attachment site is fused to the C-terminus of the dimerization domain, or to the N-terminus of the prion protein monomer, for binding of the prion protein to the VLP.
  • an amino acid linker containing a cysteine residue as second attachment site is attached to the N-terminus of the dimerization domain, or to the C-terminus of the prion protein monomer for binding to the VLP.
  • the antigen or antigenic determinant is selected form the group consisting of a) human prion protein; b) bovine prion protein; c) sheep prion protein; d) elk prion protein; e) mule deer prion protein; f) white-tailed deer prion protein; g) pig prion protein; h) chicken prion protein; i) mouse prion protein; j) goat prion protein; and k) a peptide or a fragment thereof of any prion protein of a)-j).
  • the antigen or antigenic determinant comprises, alternatively essentially consists of, or alternatively consists of an amino acid sequence selected from the group consisting a) the amino acid sequence of SEQ ID NO: 79; b) the amino acid sequence of SEQ ID NO: 80; c) the amino acid sequence of SEQ ID NO: 81; d) the amino acid sequence of SEQ ID NO: 82; e) the amino acid sequence of SEQ ID NO: 83; f) the amino acid sequence of SEQ ID NO: 84; g) the amino acid sequence of SEQ ID NO: 85; h) the amino acid sequence of SEQ ID NO: 86; i) the amino acid sequence of SEQ ID NO: 87; j) the amino acid sequence of SEQ ID NO: 88; and k) the amino acid sequence of any fragment of any of SEQ ID NO:79-88.
  • the antigen or antigenic determinant is a PrP protein variant, e.g. containing amino acid substitutions or peptide insertions or polymorphisms.
  • compositions and vaccine compositions, respectively, comprising PrP protein variants are included within the scope of the present invention.
  • a list of preferred examples including but not limited to such PrP variants, in particular to human PrP variants is given in Table 1.
  • the antigen or antigenic determinant is the human, sheep or bovine prion protein.
  • Prion proteins can be produced by expression of the PrP cDNA in procaryotic or eucaryotic expression systems.
  • Various examples hereto have been described in the literature and can be used, possibly after modifications, to express any prion protein of any desired species. For example as described by Volkel et al. (Volkel, D., Eur J Biochem 257:462-471 (1998)) for E.
  • sheep PrP was produced in large quantities in Escherichia coli after fusion with a carboxy-terminal hexahistidine sequence as described by Baron et.al (Baron., T., et al, FEMS Immunol Med Microbiol 25:379-384 (1999)).
  • Other tags such as the FLAG tag, the HA tag or the constant region of an antibody (Fc region) may also be used for purification.
  • Fc region constant region of an antibody
  • a tetracycline-inducible expression system was established for high level expression of mouse PrP in murine N2a neuroblastoma cells using standard molecular and cell biological methods.
  • N2a clones were fully controllable with respect to expression of PrP, as over-expression could be switched on and off as desired using the tetracycline-derivative doxycycline as an effector substance (Windl, O., et al, J. Gen. Virol 80:15-21, (1999)).
  • the antigen or antigenic determinant is a PrP peptide or a fragment thereof.
  • the prion peptide is selected from the group consisting: a) human prion peptide; b) bovine prion peptide; c) sheep prion peptide; d) elk prion peptide; e) mule deer prion peptide; f) white-tailed deer prion peptide; g) pig prion peptide; h) chicken prion peptide; i) mouse prion peptide; j) goat prion peptide; and k) a fragment of any prion peptide of a)-j).
  • the prion protein fragment is a construct comprising the globular domain extending from residues 125 to 228 of the human prion protein or other mammalian homologues, described by Zahn et al, Proc. Natl. Acad. Sci. USA 97: 145-150 (2000).
  • Such constructs include the fragments 23-230, 121-230 and 90-230, all modified with a C-terminal or N-terminal amino acid linker containing a cysteine residue as second attachment site.
  • the fragments may be expressed and purified as described (Zahn et al, Proc. Natl. Acad. Sci.
  • the cDNA encoding these fragments may be cloned as set out in Example 13 in the pCEP-SP-EK-Fc* vector and expressed and purified as described in Example 13.
  • the constructs may be cloned in pMod vectors as described in WO/02056905 filed by the present assignee.
  • PrP peptides or fragments thereof can be produced using standard molecular biological technologies where the nucleotide sequence coding for the fragment of interest is amplified by PCR and is cloned as a fusion to a polypeptide tag, such as the histdine tag, the Flag tag, myc tag or the constant region of an antibody (Fc region).
  • a polypeptide tag such as the histdine tag, the Flag tag, myc tag or the constant region of an antibody (Fc region).
  • enterokinase cleavage site between the PrP fragment and the tag the PrP fragment can be separated from the tag after purification by digestion with enterokinase.
  • the PrP fragment can be syntesized in vitro using standard peptide synthesis reactions known to a person skilled in the art.
  • PrP fragments can be produced by protease digestion or chemical cleavage of the full length prion protein, both methods which are well known to people trained in the art.
  • Corsaro et al. describe a procedure, which allows the expression and purification of the ' native form of a human prion protein fragment 90-231, corresponding to the protease resistant core of PrPsc.
  • GST glutathione S-transferase
  • PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin (Corsaro, A., et al, Neurochem. Int. 47:55-63 (2002)).
  • the prion peptide comprises at least one antigenic site of a prion protein.
  • the skilled person in the art knows how to identify the corresponding peptides and amino acid sequences, respectively.
  • Several prion peptides having at least one antigenic site of a prion protein and being suitable for use in the present invention are described in WO 93/11155, the entirety of which is explicitly incorporated by reference.
  • six regions defined by general formulas providing amino acid sequences of synthetic peptides are reported. One of these regions was shown to harbor two additional frame shift mutations resulting in additional amino acid sequences.
  • Typical and preferred examples for human PrP peptides useful for the present invention include but are not limited to the following human PrP peptides as well as fragments thereof: HU PRP 109-131: MKHMAGAAAAGAVVGGLGGYMLGSAMSRPII HU PRP 132-160: SAMSRPIIHFGSDYEDRYYRENMHRYPNQ HU PRP 153-181: NMHRYPNQVYYRPMDEYSNQNNFVHDCVN HU PRP 225-253: YYQRGSSMVLFSSPPVILLISFLIFLIVG HU PRP 54-69: GGGWGQPHGGGWGQPH HU PRP 174-204: NFVHDCVNITIKQHTVTTTTKGENFTETDVK HU PRP 211-232: EQMCITQYERESQAYYQRGSSM Guidance on how to modify human prion protein, prion peptide or a fragment thereof, in particular, for binding to the virus-like particle is given throughout the
  • Mouse prion protein constructs are disclosed, and a preferred human prion protein constructs, i.e. a human prion peptide, can also be generated and have, for example, the sequence of SEQ ID NO: 89.
  • Further constructs comprise the whole human prion protein sequence and fragments thereof.
  • Immunization against prion protein using the inventive compositions comprising, preferably a human prion protein or human prion peptide bound to a core particle and VLP, respectively, may provide a way of treatment or prevention of Creutzfeldt- Jakob (variant form) or other prion diseases. In the former case, this corresponds to an immunization against a self-antigen.
  • compositions of the invention comprising the prion protein or prion peptide may provide a way of treatment against prion mediated diseases in other animals as well.
  • Preferred embodiments thus, include the corresponding sequences of bovine and sheep prion protein constructs, which are given in SEQ ID NO:90 and SEQ ID NO:91.
  • the corresponding amino acid sequences of the elk prion peptide, mule and white-tailed deer are also preferred embodiments of the invention.
  • the antigen or antigenic determinant is a prion peptide comprising, or alternatively essentially consisting of, or alternatively consisiting of an amino acid sequence selected from the group consisting of:
  • the antigen or antigenic determinant further comprise at least one second attachment site being selected from the group consisting of: (i) an attachment site not naturally occurring with said antigen or antigenic determinant; and (ii) an attachment site naturally occurring with said antigen or antigenic determinant.
  • the antigen or antigenic determinant with said at least second attachment site comprises, or alternatively consists of, or alternatively consists of an amino acid sequence selected from the group consisting of:
  • These peptides comprise an N-terminal cysteine residue as a second attachment added for coupling to VLPs and Pili.
  • PrP peptides suitable for usein the present invention can be experimentally determined by their intrinsic property to induce a T cell or an antibody response. This is generally achieved by immunizing an experimental animal separately with selected peptides in an immunologically suitable formulation and by measuring T cell and B cell, i.e. antibody responses, using methods known to a person trained in the art. Suoan et al. immunized mice with a panel of overlapping mouse PrP peptides, in toto covering the full length mouse PrP protein and measured T cell and B cell responses (Souan L., et al, Eur. J. Immunol. 37:2338-2346, (2001)).
  • mouse PrP peptides comprising, or alternatively essentially consisting of, or alternatively consisting of an amino acid sequence selected from (a) SAMSRPMIHFGNDWEDRYYR (Mouse PrP 131-150), (b)
  • QMCVTQYQKESQAYYDGRRS (Mouse PrP 211-230), and the corresponding amino acid sequences of (a) or (b) of human, bovine, sheep, elk, mule deer, white-tailed deer, pig, chicken or goat as well as the amino acid sequence of any fragment of any indicated species and animal, respectively, of (a) or (b).
  • prion infectivity resides in PrP sc which is a conformational variant of a host encoded cellular protein designated PrP c .
  • Further prion peptides suitable for the present invention are peptides which contain at least one antibody epitope of PrP c or PrP sc .
  • the antigen is a protein, a polypeptide or a peptide, this region can be formed by a continuous aminoacid sequence.
  • the antibody epitope can be formed by a discontinuous aminoacid sequence in which, after three dimensional folding of the protein, polypeptide or peptide, the aminoacids are arranged in such a manner that they spatially come close together and form the epitope. Continuous peptide fragments of interest can identified by immunization experiments as described above.
  • PrP peptides suitable for use for the present invention have an amino acid sequence, which comprises, or alternatively essentially consists of, or alternatively consists of an amino acid sequence selected from CYYR, CYYRRYYRRYY, AYYQ, and AYYQYYQYYYQ.
  • Peptide fragments suitable for use as antigen or antigenic determinant for the present invention forming an epitope on the surface of PrP S0 and capable of binding PrP-specific antibodies were identified in mapping experiments using immobilized peptide banks (WO/9915651). These peptide banks (available from Jerini Biotools, Berlin) cover the full length PrP protein sequence and were screened against a monoclonal antibody named 15B3 specifically binding PrP sc . Again, this systematic approach defined general formulae defining PrP petides suitable for use for the present invention.
  • PrP peptides suitable for use for the present invention have an amino acid sequence, which comprises, or alternatively essentially consists of, or alternatively consists of an amino acid sequence selected from (a) GSDYEDRYY (Human PrP 142-150), (b) GNDYEDRYY (Sheep PrP 145- 153), and the corresponding amino acid sequences of (a) or (b) of human, bovine, sheep, elk, mule deer, white-tailed deer, pig, chicken or goat as well as the amino acid sequence of any fragment of any indicated species and animal, respectively, of (a) or (b).
  • prion peptides suitable for use for the present invention can be identified by using existing or future monoclonal or polyclonal antibodies.
  • Such antibodies may include for example the monoclonal antibodies
  • PrP peptides suitable for use for the present invention can be defined by other antibodies including monoclonal antibodies mAb 3-11 or mAb 2-40, both recognizing human PrP peptide 106-126, or related antibodies which inhibit PrP peptide neurotoxicity or fibril formation (Forloni, G., et. al. Nature 352:543-546, (1993); Brown, D.R., Glla 75:59-67 (1996)).
  • PrP peptides suitable for use for the present invention are defined by antibodies including monoclonal antibody CNCM 1-2476 which is capable of selectively binding the three dimensional conformation provided by the C- terminal part of the PrP Sc isoform of the prion protein or part thereof, while not binding to the PrP c form (WO 01/90191).
  • the antigen or antigenic determinant is a prion peptide having an amino acid sequence, which comprises, or alternatively essentially consists of, or alternatively consists of an amino acid sequence selected from the human PrP sequence 214-226 CITQYERESQAYY or variants thereof or corresponding sequences in other species for example bovine PrP peptide 225-237 CITQYQ RESQAYY or variants thereof.
  • PrP peptides suitable for use for the present invention may be identified by screening phage display peptide libraries with antibodies specific for PrP sc or PrP c , a method well known to a person trained in the art. Furthermore, PrP peptides which have fusogenic and fibril formation properties and therefore are neurotoxic (Pillot, T.J., Mol. Biol. 5:381-393 (1997)) are further preferred embodiments suitable for use for the present invention.
  • the antigen or antigenic determinant is isolated or recombinant PrP2 of any animal as well as any antigenic fragments of PrP2 of any animal.
  • PrP2 represents mammalian prion-like compositions identified in part on the basis of homology of the cDNA encoding for PrP2 to PrP of various species as outlined in WO 01/46419.
  • the plasmid pQ ⁇ lO (Kozlovska, TM, et al, Gene 737: 133-137) was used as an initial plasmid for the construction of pQ ⁇ -240.
  • the mutation Lysl3— >Arg was created by inverse PCR.
  • the inverse primers were designed in inverted tail-to-tail directions:
  • the plasmid pQ ⁇ lO was used as an initial plasmid for the construction of pQ ⁇ -243.
  • the mutation AsnlO- Lys was created by inverse PCR.
  • the inverse primers were designed in inverted tail-to-tail directions:
  • the plasmid pQ ⁇ -240 was used as an initial plasmid for the construction of pQ ⁇ -250.
  • the mutation Lys2— »Arg was created by site- directed mutagenesis.
  • 5'-GATTTAGGTGACACTATAG-3 ' were used for the synthesis of the mutant PCR-fragment, which was introduced into the pQ ⁇ -185 expression vector at the unique restriction sites Ncol and Hindlll.
  • the PCR reactions were performed with PCR kit reagents and according to producer protocol (MBI Fermentas, Vilnius, Lithuania). [0251] Sequencing using the direct label incorporation method verified the desired mutations. E.coli cells harbouring pQ ⁇ -250 supported efficient synthesis of 14-kD protein co migrating upon PAGE with control Q ⁇ coat protein isolated from Q ⁇ phage particles.
  • the plasmid pQ ⁇ lO was used as an initial plasmid for the construction of pQ ⁇ -251.
  • the mutation Lysl6- Arg was created by inverse PCR.
  • the inverse primers were designed in inverted tail-to-tail directions:
  • the plasmid pQ ⁇ -251 was used as an initial plasmid for the construction of pQ ⁇ -259.
  • the mutation Lys2-»Arg was created by site- directed mutagenesis.
  • 5'-GATTTAGGTGACACTATAG-3' were used for the synthesis of the mutant PCR-fragment, which was introduced into the pQ ⁇ -185 expression vector at the unique restriction sites Ncol and Hindlll.
  • the PCR reactions were performed with PCR kit reagents and according to producer protocol (MBI Fermentas, Vilnius, Lithuania).
  • E.coli JM109 was transformed with Q ⁇ coat protein expression plasmids.
  • 5 ml of LB liquid medium containing 20 ⁇ g/ml ampicillin were inoculated with clones transformed with with Q ⁇ coat protein expression plasmids.
  • the inoculated culture was incubated at 37 °C for 16-24 h without shaking.
  • the prepared inoculum was subsequently diluted 1:100 in 100-300 ml of fresh LB medium, containing 20 ⁇ g/ml ampicillin. and incubated at 37 °C overnight without shaking.
  • the resulting second inoculum was diluted 1:50 in M9 medium containing 1 % Casamino acids and 0.2 % glucose in flasks, and incubated at 37 °C overnight under shaking.
  • a saturated ammonium sulphate solution was added dropwise under stirring to the above pooled lysate.
  • the volume of the SAS was adjusted to be one fifth of total volume, to obtain 20% of saturation.
  • the solution was left standing overnight, and was centrifuged the next day at 14 000 rpm, for 20 min.
  • the pellet was washed with a small amount of 20% ammonium sulphate, and centrifuged again .
  • the obtained supernatants were pooled, and SAS was added dropwise to obtain 40% of saturation.
  • the solution was left standing overnight, and was centrifuged the next day at 14 000 rpm, for 20 min.
  • the obtained pellet was solubilised in NET buffer.
  • the resolubilized protein was then purified further on a Sepharose 2B column and finally on a Sepharose 6B column.
  • the capsid peak was finally extensively dialyzed against water and lyophilized as described above.
  • the assembly of the coat protein into a capsid was confirmed by electron microscopy.
  • the protein was purified by gel filtration on a Sepharose CL-4B and finally on a Sepharose CL-2B column, and lyophilized as described above. The assembly of the coat protein into a capsid was confirmed by electron microscopy.
  • HBcAg Hepatitis B virus capsid
  • a part of this region (Proline 79 and Alanine 80) was genetically replaced by the peptide Gly-Gly-Lys-Gly-Gly (HBcAg-Lys construct).
  • the introduced Lysine residue contains a reactive amino group in its side chain that can be used for intermolecular chemical crosslinking of HBcAg particles with any antigen containing a free cysteine group.
  • EcoRIHBcAg(s) and HBcAg(l-149)Hind(as) were used with 100 ng of the two purified PCR fragments in a 50 ml reaction mixture containing 2 units of Pwo polymerase, 0.1 mM dNTPs and 2 mM MgSO 4 .
  • PCR cycling conditions were: 94°C for 2 minutes; 30 cycles of 94°C (1 minute), 50°C (1 minute), 72°C (2 minutes).
  • the assembled PCR product was analyzed by agarose gel electrophoresis, purified and digested for 19 hours in an appropriate buffer with EcoRI and Hindlll restriction enzymes.
  • lysate was then centrifuged for 30 minutes at 12,000 x g in order to remove insoluble cell debris.
  • the supernatant and the pellet were analyzed by Western blotting using a monoclonal antibody against HBcAg (YVS1841, purchased from Accurate Chemical and Scientific Corp., Westbury, NY, USA), indicating that a significant amount of HBcAg-Lys protein was soluble.
  • lysates from E. coli cells expressing HBcAg-Lys and from control cells were centrifuged at 14,000 x g for 30 minutes.
  • the cleared cell lysate was used for step-gradient centrifugation using a sucrose step gradient consisting of a 4 ml 65% sucrose solution overlaid with 3 ml 15% sucrose solution followed by 4 ml of bacterial lysate.
  • the sample was centrifuged for 3 hrs with 100,000 x g at 4°C. After centrifugation, 1 ml fractions from the top of the gradient were collected and analyzed by SDS- PAGE followed by Coomassie staining. The HBcAg-Lys protein was detected by Coomassie staining.
  • the HBcAg-Lys protein was enriched at the interface between 15 and
  • HBcAg-Lys was then purified by first precipitating the protein with ammonium sulphate (30% saturation) from the cleared cell lysate, then loading the resolubilized pellet on a gel filtration column (Sephacryl S-400, Pharmacia). The pooled fractions were precipitated again with ammonium sulphate, the pellet resolubilized and loaded a second time on the same gel filtration column. The fractions were finally pooled and concentrated, and the concentration assessed using a Bradford test (BioRad). EXAMPLE 4
  • HBcAg Hepatitis core Antigen
  • 2cys-Mut devoid of cysteine residues at positions corresponding to 48 and 107 in SEQ ID NO:77 and containing an inserted lysine residue was constructed using the following methods.
  • Primer 1 EcoRIHBcAg(s) CCGGAATTCATGGACATTGACCCTTATAAAG
  • Primer 2 48as GTGCAGTATGGTGAGGTGAGGAATGCTCAGGAGACTC
  • Primer 3 48s GSGTCTCCTGAGCATTCCTCACCTCACCATACTGCAC
  • Primer 4 107as CTTCCAAAAGTGAGGGAAGAAATGTGAAACCAC
  • Example 2 A part of the c/el epitope (residues 72 to 88) region (Proline 79 and Alanine 80) was genetically replaced by the peptide Gly-Gly-Lys-Gly-Gly (HBcAgl-185-Lys construct).
  • the introduced Lysine residue contains a reactive amino group in its side chain that can be used for intermolecular chemical crosslinking of HBcAg particles with any antigen containing a free cysteine group.
  • PCR methods and conventional cloning techniques were used to prepare the HBcAgl-185-Lys gene.
  • the Gly-Gly-Lys-Gly-Gly sequence was inserted by amplifying two separate fragments of the HBcAg gene from pEco63, as described above in Example 2 and subsequently fusing the two fragments by PCR to assemble the full length gene.
  • the following PCR primer combinations were used:
  • Primer 1 EcoRIHBcAg(s) (see example 2)
  • Primer 2 HBcAgwtHindllll
  • the residues 79 and 80 of HBcAgl-185 were substituted with the epitope C ⁇ H3 of sequence VNLTWSRASG .
  • the C ⁇ H3 sequence stems from the sequence of the third constant domain of the heavy chain of human IgE.
  • the epitope was inserted in the HBcAgl-185 sequence using an assembly PCR method.
  • the HBcAgl-185 gene originating from ATCC clone pEco63 and amplified with primers HBcAg-wt EcoRI fwd and HBcAg-wt Hind III rev was used as template in two separate reactions to amplify two fragments containing sequence elements coding for the C ⁇ H3 sequence. These two fragments were then assembled in a second PCR step, in an assembly PCR reaction.
  • the overhangs of the pFrd ⁇ vector are filled in with Klenow after digestion with Bspll91, and oligonucleotides coding for the sequence of the prpshort murine prion peptide and additional nucleotides for in frame cloning are ligated in pFrd8 after the Klenow treatment.
  • Clones with the insert in the right orientation are analysed by sequencing. Expression and purification of the chimeric fusion protein in E. coli JM109 or E. coli K802 is performed as described in Pushko, P. et al, Prot. Eng.
  • Two complementary oligonucleotides coding for the murine prion peptide prpshort, of sequence GNDWEDRYYRENMYR, with ends compatible with the Nhel site of pOGS8111 are synthesized. Additional nucleotides are added to allow for in frame insertion of a sequence coding for the murine prpshort epitope according to the description of EP06777111. The amino acids AS and SS flanking the inserted epitope are encoded by the altered Nhel sites resulting from the insertion of the oligonucleotide in the TyA(d) gene of pOGS ⁇ l l l.
  • a sequence coding for the murine prpshort epitope having the sequence GNDWEDRYYRENMYR is substituted to the sequence coding for amino acids 130-136 of the BPV-1 LI gene cloned in the pFastBacl (GIBCO/BRL) vector as described (Chackerian, B. et al, Proc. Natl. Acad. USA 96: 2373-2378 (1999)).
  • the sequence of the construct is verified by nucleotide sequence analysis.
  • Recombinant baculovirus is generated using the GIBCO/BRL baculovirus system as described by the manufacturer.
  • the chimeric VLPs are purified from baculovirus infected Sf9 cells as described by Kirnbauer, R. et al, Proc. Natl. Acad. Sci. 59:12180-84 (1992) and Greenstone, H.L., et al, Proc. Natl Acad. Sci. 95:1800-05 (1998).
  • GNDWEDRYYRENMYR generated in Examples 7-11 are used for immunization of mice as described in Example 15.
  • the sera obtained from the immunized mice are analysed in a Prion protein-specific ELISA as described in Example 15.
  • the protective effect of the vaccine is examined by immunizing a large group of mice prior to intraperitoneal prion inoculation.
  • Spleens, where PrP Sc is detectable as early as 35 days post inoculation (35dpi) are investigated for the presence of prions, to assess the protective effect of the vaccine in vivo.
  • infectivity of the mentioned organs is investigated by utilizing a bioassay, as described by M. Fischer and colleagues, EMBO J. 75:1255-1264 (1996).
  • the onset of prion disease (approximately 240 days post inoculation of wild-type mice) in immunized vs.
  • mice is assessed as a further readout to confirm the efficacy of the vaccine in protecting mice against prion disease.
  • the protective effect of the antibodies induced by the immunization procedure is further assessed in an experiment where the capability of rescuing chronically prion-infected neuroblastoma cells (ScN2a) according to a protocol published by M. Enari et al. (PNAS 98, 9295-9299, (2001)) is measured.
  • ScN2a chronically prion-infected neuroblastoma cells
  • a truncated form (aa 121-230) of the mouse prion protein was recombinantly expressed with a GGGGCG amino acid linker fused at its C-terminus for coupling to VLPs and Pili.
  • the protein was fused to the N-terminus of a human Fc-fragment for purification.
  • An enterokinase (EK) cleavage-site was introduced behind the GGGGCG amino acid linker to cleave the Fc- part of the fusion protein after purification.
  • Mouse PrPt was amplified by PCR with the primer 5'PrP-BamHI and
  • pBPCMVPrP- Fc contained the wild-type sequence of the mouse prion protein.
  • 5 'PrP -BamHI had an internal BamHI site and contained an ATG and 3'PrP-NheI had an internal Nhel site.
  • DNA was used in the 50 ⁇ l reaction mixture (1 unit of PFX Platinum polymerase, 0.3 mM dNTPs and 2 mM MgSO4).
  • the temperature cycles were as follows: 94°C for 2 minutes, followed by 5 cycles of 94°C (15 seconds), 50°C (30 seconds), 68°C (45 seconds), followed by 20 cycles of 94°C (15 seconds), 64°C (30 seconds), 68°C (45 seconds) and followed by 68°C for 10 minutes.
  • PCR product was digested with BamHI and Nhel and inserted into pCEP-SP-EK-Fc* (WO/02056905, filed by the present assignee) containing the GGGGCG linker sequence at the 5 'end of the EK cleavage sequence.
  • the resulting plasmid was named pCEP-SP-mPrPt-EK-Fc*.
  • Plasmid pCEP-SP-mPrPt-EK-Fc* was transfected into 293-EBNA cells (Invitrogen) and purified on a Protein A-sepharose column.
  • mPrPt after cleavage has the sequence as identified in SEQ ID NO:93 with the GGGGCG linker at its C-terminus.
  • the purified fusion protein mPrPt-EK-Fc* was cleaved with enterokinase and analysed on a 16% SDS-PAGE gel under reducing conditions before and after enterokinase cleavage. The gel was stained with Coomassie Brilliant Blue. The result is shown in FIG. 1. Molecular weights of marker proteins are given on the left margin of the gel in the figure. The mPrPt-EK-Fc* fusion protein could be detected as a 50 kDa band. The cleaved mPrPt protein containing the GGGGCG amino acid linker fused to its C- terminus could be detected as a broad band between 18 and 25 kDa.
  • mPrPt The identity of mPrPt was confirmed by western blotting (data not shown). Thus, mPrPt with a C-terminal amino acid linker containing a cysteine residue, could be expressed and purified to be used for coupling to VLPs and Pili.
  • Lane 1 Molecular weight marker. Lane 2: mPrPt-EK-Fc* before cleavage. Lane 3: mPrPt after cleavage. Coupling of mPrP t to Q ⁇ capsid
  • 150 mM NaCl pH 7.2 is reacted for 30 minutes with a 25 fold molar excess of SMPH (Pierce), diluted from a stock solution in DMSO, at 25 °C on a rocking shaker.
  • the reaction solution is subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 °C.
  • the protein-containing fractions eluating from the column are pooled, and the desalted derivatized pili protein is reacted with the mPrP t solution (end concentrations: 60 ⁇ M pili, 60 ⁇ M mPrP t ) for four hours at 25 °C on a rocking shaker. Coupling products are analysed by SDS-PAGE.
  • prion peptide vaccines Coupling of prion peptides to Q ⁇ capsid protein: prion peptide vaccines.
  • CSAMSRPMIHFGNDWEDRYYRENMYR ("murine cprplong") and CGNDWEDRYYRENMYR (“murine cprpshort”), which comprise an added N-terminal cysteine residue for coupling to VLPs and Pili, and used for chemical coupling to Q ⁇ as described in the following.
  • 100 ⁇ l of the dialyzed reaction mixture was then reacted either with 1.35 ⁇ l of a 2 mM stock solution (in DMSO) of the peptide cprpshort (1:2 peptide/Q ⁇ capsid protein ratio) or with 2.7 ⁇ l of the same stock solution (1 :1 peptide/Q ⁇ ratio).
  • 1 ⁇ l of a 10 mM stock solution (in DMSO) of the peptide cprplong was reacted with 100 ⁇ l of the dialyzed reaction mixture.
  • the coupling reactions were performed over night at 15 °C in a water bath.
  • the reaction mixtures were subsequently dialyzed 24 h against 2x 5 L of 20 mM Hepes, 150 mM NaCl, pH 7.4 at 4°C.
  • the coupled products were centrifuged and supernatants and pellets were analysed on 16% SDS-PAGE gels under reducing conditions. Gels were stained with Coomassie Brilliant Blue. The results are shown in FIG. 2. Molecular weights of marker proteins are given on the left margin of the gel in the figure. The bands at a molecular weight between 16.5 and 25 kDa clearly demonstrated the covalent coupling of the peptides cprpshort and cprplong to Q ⁇ capsid protein. [0314]
  • the samples loaded on the gel of FIG. 2 A were the following:
  • Lane 1 Molecular weight marker. Lane 2: derivatized Q ⁇ capsid protein before coupling. Lane 3 and 4: Q ⁇ . capsid protein-cprplong coupling reactions. Soluble fraction (lane 3) and insoluble fraction (lane 4) are shown.
  • NaCl pH 7.2 is reacted for 30 minutes with a 10 fold molar excess of SMPH (Pierce) ), diluted from a stock solution in DMSO, at 25 °C on a rocking shaker.
  • the reaction solution is subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 °C.
  • the dialyzed fr reaction mixture is then reacted with equimolar concentration of peptide cprpshort or a ration of 1:2 cprplong / fr over night at 16 °C on a rocking shaker. Coupling products are analysed by SDS-PAGE.
  • a solution of 120 ⁇ M HBcAg-Lys-2cys-Mut in 20 mM Hepes, 150 mM NaCl pH 7.2 is reacted for 30 minutes with a 10 fold molar excess of SMPH (Pierce), diluted from a stock solution in DMSO, at 25 °C on a rocking shaker.
  • the reaction solution is subsequently dialyzed twice for 2 hours against 1 L of 20 mM Hepes, 150 mM NaCl, pH 7.2 at 4 °C.
  • the dialyzed HBcAg-Lys-2cys-Mut reaction mixture is then reacted with equimolar concentration of peptide cprpshort or a ration of 1 :2 cprplong / HBcAg-Lys- 2cys-Mut over night at 16 °C on a rocking shaker. Coupling products are analysed by SDS-PAGE.
  • the reaction mixture is desalted on a PD-10 column (Amersham-Pharmacia Biotech).
  • the protein-containing fractions eluating from the column are pooled, and the desalted derivatized pili protein is reacted with the prion peptides in equimolar or in a ratio of 1:2 peptide pili over night at 16 °C on a rocking shaker. Coupling products are analysed by SDS-PAGE.
  • mice Female C57BL/6 mice were immunized with the murine prion peptide cprpshort coupled to Q ⁇ (in the example termed "mPrPs-Q ⁇ ") or the peptide cprplong coupled to Q ⁇ (in the example termed "mPrPl-Q ⁇ ”), administered in alum, as CFA/IFA-emulsion, or with no adjuvant.
  • the murine prion peptides cprpshort, cprplong (in the example just referred to cprpshort or cprplong) or Q ⁇ served as negative controls.
  • female Prnpo/o mice H. R.
  • Bueler et al, Cell 1993 were injected with mPrPs-Q ⁇ or mPrPl-Q ⁇ .
  • 50 ⁇ g of total protein of mPrPs-Q ⁇ , mPrPl-Q ⁇ or Q ⁇ and 100 ⁇ g of cprpshort and cprplong were diluted in PBS to a final volume of 200 ⁇ l and administered subcutaneously on day 0, day 14, and day 28.
  • mPrPs-Q ⁇ or mPrPl-Q ⁇ were applied intraperitoneally in 500 ⁇ l (1 mg) alum or subcutaneously as CFA-emulsion (100 ⁇ l) on day 0 or intraperitoneally as IFA-emulsion (100 ⁇ l) on day 14 and day 28.
  • Mice were bled retroorbitally four and thirteen days after immunization. Serum was analyzed using a prion protein-specific ELISA.
  • the plates were blocked for two hours with 5% bovine serum albumin (BSA), PBS, 0.1% Tween at room temperature. After washing, the plates were incubated with 30 ⁇ l of two- or threefold serially diluted mouse serum in 1% BSA, PBS, 0.1% Tween in duplicates for two hours at room temperature.
  • BSA bovine serum albumin
  • HRP horse-radish peroxidase
  • ABTS 2.2'-azino-di-etnyl-benzthiazolinsulfonat
  • Optical density (OD) was measured at 405 nm.
  • Endpoint ELISA titers were determined by counting the number of titrations possible above two times the average of the background. Omission of serum and recombinant PrP served as background and corresponded to preimmune serum values.
  • FIG. 3 shows total immunoglobulins (IgG+A+M) detected on day 13 and day 4 after the first boost.
  • mPrPs-Q ⁇ raised a higher anti-PrP titer than mPrPl-Q ⁇ .
  • FIG. 4 ELISA of total immunoglobulins.
  • an anti-PrP titer was detected in wild-type and Prnpo/o mice.
  • three immunizations were sufficient to induce an anti-PrP antibody response lasting two and a half months.
  • immunoglobulin isotypes were analysed on day 13 after the second boost resulting mainly in anti-PrP antibodies of the IgG2a class.
  • mice were inoculated intraperitoneally with scrapie prions (RML strain, passage 5) (Bueler, H.R., et al, Cell 73:1339-1347 (1993)). Spleens were harvested at 62 days after inoculation (dpi) and accumulation of PrP Sc was analysed. 10% (w/v) homogenates of spleen were prepared as described (Bueler, H.R., et al, Cell 73:1339-1347 (1993)).
  • NaPTA Sodium phosphorungstic acid precipitation was performed according to Safar J., et al, Nat Med 4:1157-1165 (1998).
  • PrP Sc accumulation was detected in scrapie-infected mice that had received no treatment, mPrPs-Q ⁇ or PrPs-peptide. However, the level of deposition of PrP Sc was markedly reduced in mice immunized with mPrPs-Q ⁇ (FIG. 5). A two- to fourfold reduction was observed upon quantification, possibly indicating delayed progression of pathogenesis. [0330] Further, since PrP is expressed in most tissues of the body, putative autoimmune phenomena are investigated in immunized mice, e.g. by analyzing deposition of autoimmune complexes in kidney and skin as well as in organs with high PrP levels, such as CNS.
  • E.coli JM109 was transformed with plasmid pAP283-58. 5 ml of LB liquid medium with 20 ⁇ g/ml ampicillin were inoculated with a single colony, and incubated at 37 °C for 16-24 h without shaking.
  • the prepared inoculum was diluted 1:100 in 100-300 ml of LB medium, containing 20 ⁇ g/ml ampicillin and incubated at 37 °C ovemight without shaking.
  • the resulting second inoculum was diluted 1:50 in 2TY medium, containing 0.2 % glucose and phosphate for buffering, and incubated at 37 °C overnight on a shaker. Cells were harvested by centrifugation and frozen at -80°C.
  • Frozen cells were resuspended in lysis buffer at 2 ml/g cells. The mixture was sonicated with 22 kH five times for 15 seconds, with intervals of lmin to cool the solution on ice. The lysate was then centrifuged for 20 minutes at 12 000 rpm, using a F34-6-38 rotor (Ependorf). The centrifugation steps described below were all performed using the same rotor, except otherwise stated. The supernatant was stored at 4° C, while cell debris were washed twice with lysis buffer. After centrifugation, the supernatants of the lysate and wash fractions were pooled.
  • VLP VLP.
  • a concentration of ammonium-sulphate at which AP205 VLP does not precipitate is chosen.
  • the resulting pellet is discarded.
  • an ammonium sulphate concentration at which AP205 VLP quantitatively precipitates is selected, and AP205 VLP is isolated from the pellet of this precipitation step by centrifugation (14 000 rpm, for 20 min).
  • the obtained pellet is solubilised in NET buffer.
  • Sepharose 4B column (2.8 X 70 cm), and eluted with NET buffer, at 4 ml/hour/fraction.
  • Fractions 28-40 were collected, and precipitated with ammonium sulphate at 60% saturation.
  • the fractions were analyzed by SDS- PAGE and Western Blot with an antiserum specific for AP205 prior to precipitation (FIG. 6A and FIG. 6B).
  • the pellet isolated by centrifugation was resolubilized in NET buffer, and loaded on a Sepharose 2B column (2.3 X 65 cm), eluted at 3 ml/h/fraction.
  • Fractions were analysed by SDS-PAGE, and fractions 44-50 were collected, pooled and precipitated with ammonium sulphate at 60% saturation.
  • the pellet isolated by centrifugation was resolubilized in NET buffer, and purified on a Sepharose 6B column (2.5 X 47 cm), eluted at 3 ml/hour/fraction.
  • the fractions were analysed by SDS-PAGE. Fractions 23-27 were collected, the salt concentration adjusted to 0.5 M, and precipitated with PEG 6000, added from a 40% stock in water and to a final concentration of 13.3%.
  • the pellet isolated by centrifugation was resolubilized in NET buffer, and loaded on the same Sepharose 2B column as above, eluted in the same manner. Analysis of the fractions by SDS-PAGE is shown in FIG. 6C.
  • Fractions 43-53 were collected, and precipitated with ammonium sulphate at a saturation of 60%.
  • the pellet isolated by centrifugation was resolubilized in water, and the obtained protein solution was extensively dialyzed against water. About 10 mg of purified protein per gram of cells could be isolated.
  • FIG. 6A shows in the top panel, the silver-stained SDS-PAGE run under reducing conditions of the fractions of the first Sepharose 4B chromatography step.
  • Lane 1- 13 were loaded with every second fraction from fraction 20 to 44.
  • Fraction 50 was loaded in lane 14.
  • a second gel was loaded with the same fractions and analysed by Western blotting with an anti-serum specific for AP205, and is shown in the lower panel (FIG. 6B).
  • FIG. 6C shows the silver-stained SDS-PAGE run under reducing conditions of the fractions of the last Sepharose 2B chromatography step. Fractions 38-54 are loaded in Lane 1- 16.
  • FIG. 7A shows an EM picture of AP205 phage particles, while an EM picture of self assembled particles of recombinant AP205 VLP is shown in FIG. 7B.
  • the dimer of mouse prion protein, mPrPt (aa 121-230), is constructed by assembly PCR by amplifying first the cDNA encoding mPrPt of example 13 with two sets of primers in two individual PCR reactions, and then splicing both fragments in a "splice overlap assembly PCR".
  • the forward primer anneals to a sequence complementary to the 5 '-end of mPrPt cDNA, and contains a restriction site for cloning.
  • the reverse primer is composed of three sequence elements.
  • the forward primer contains three sequence elements as well.
  • the more 5' sequence element anneals to a sequence complementary to the 3' end of mPrPt cDNA.
  • the central sequence element anneals to a sequence complementary to a sequence coding for the amino acid linker GGGGS.
  • the more 3' sequence element anneals to a sequence complementary to the 5 'end of mPrPt cDNA.
  • the reverse primer anneals to the 3' end of mPrPt cDNA, and contains a restriction site for cloning.
  • PCR fragments obtained in the first PCR reactions are assembled and amplified with the forward primer of the first set of primer and the reverse primer of the second set of primer.
  • These primers contain restriction sites suitable for cloning into either the pMod vectors described in example 4 of WO/02056905, filed by the present assignee, or the pCEP-SP-EK-Fc* vector (WO/02056905, filed by the present assignee), leading to the introduction of an amino acid linker containing a cysteine residue as second attachment site to the C-terminus of the mPrPt dimer.
  • the protein is expressed, purified and coupled as described in example 13 or as described for proteins cloned in the pMod vectors described in example 4 of WO/02056905, filed by the present assignee.

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Abstract

L'invention concerne les domaines de la biologie moléculaire, de la virologie, de l'immunologie et de la médecine. L'invention concerne également une composition contenant un antigène ordonné et répétitif ou un jeu ordonné de déterminants antigènes, et en particulier, un peptide de prion ou un jeu de particules du type virus et des protéines prions. L'invention concerne plus spécifiquement une composition contenant une particule du type virus et au moins une protéine prion (PrP) ou un dimère de celle-ci ou un peptide PrP lié à celle-ci. L'invention concerne, en outre, une méthode de production de conjugués et de jeux ordonnés et répétitifs respectifs. On utilise lesdites compositions pour produire des vaccins destinés à traiter des maladies à prion et en tant que pharmaccins (vaccins thérapeutiques) permettant de prévenir ou de traiter des maladies à prion et d'induire efficacement des réponses immunitaires, en particulier des réponses d'anticorps. Ces compositions sont particulièrement utiles pour induire des réponses immunitaires auto-spécifiques dans le contexte indiqué.
PCT/EP2003/000460 2002-01-18 2003-01-17 Conjuges-vecteurs de proteines prion Ceased WO2003059386A2 (fr)

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US10/050,902 2002-01-18
US10/050,902 US7264810B2 (en) 2001-01-19 2002-01-18 Molecular antigen array
IBPCT/IB02/00166 2002-01-21
PCT/IB2002/000166 WO2002056905A2 (fr) 2001-01-19 2002-01-21 Jeu ordonne d'echantillons d'antigenes moleculaires
US39372502P 2002-07-08 2002-07-08
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US7517520B2 (en) 2003-03-26 2009-04-14 Cytos Biotechnology Ag Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
US7537767B2 (en) 2003-03-26 2009-05-26 Cytis Biotechnology Ag Melan-A- carrier conjugates
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
WO2014041116A1 (fr) * 2012-09-17 2014-03-20 F. Hoffmann-La Roche Ag Procédé de production de polypeptides dans le périplasme de cellule procaryotes
US8691209B2 (en) 2001-09-14 2014-04-08 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US9902972B2 (en) 2006-06-12 2018-02-27 Kuros Biosciences Ag Processes for packaging oligonucleotides into virus-like particles of RNA bacteriophages
US10086056B2 (en) 2015-01-15 2018-10-02 University Of Copenhagen Virus-like particle with efficient epitope display
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CA2354183A1 (fr) * 1998-11-30 2000-06-08 Cytos Biotechnology Ag Presentation moleculaire ordonnee d'antigenes, procede de preparation et utilisation
DE60032486T2 (de) * 1999-06-23 2007-08-16 Idexx Laboratories, Inc. Prion protein peptide und deren verwendung
WO2001053457A2 (fr) * 2000-01-21 2001-07-26 University Of Connecticut Health Center Vaccins contre certains troubles neurodegeneratifs
AU2001252458A1 (en) * 2000-05-05 2001-11-20 Martin Bachmann Molecular antigen arrays and vaccines
ES2253156T5 (es) * 2000-05-23 2013-04-30 Blood Transfusion Centre Of Slovenia Anticuerpos capaces de detectar de manera selectiva isoformas del prión PrPSc

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US9950055B2 (en) 2001-09-14 2018-04-24 Kuros Biosciences Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US7517520B2 (en) 2003-03-26 2009-04-14 Cytos Biotechnology Ag Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
US7537767B2 (en) 2003-03-26 2009-05-26 Cytis Biotechnology Ag Melan-A- carrier conjugates
WO2006083795A1 (fr) * 2005-02-01 2006-08-10 Ramot At Tel-Aviv University Ltd. Methode destinee a traiter une inflammation associee aux depots amyloides et une inflammation cerebrale impliquant une microglie activee
US7867487B2 (en) 2005-02-01 2011-01-11 Ramot At Tel-Aviv University Ltd. Method for treating inflammation associated with amyloid deposits and brain inflammation involving activated microglia
US8361458B2 (en) 2005-02-01 2013-01-29 Ramot At Tel-Aviv University Ltd. Method and filamentous phage for treating inflammation associated with amyloid deposits and brain inflammation involving activated microglia
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US10358656B2 (en) 2006-06-12 2019-07-23 Kuros Biosciences Ag Oligonucleotides packaged into virus-like particles of RNA bacteriophages
US9902972B2 (en) 2006-06-12 2018-02-27 Kuros Biosciences Ag Processes for packaging oligonucleotides into virus-like particles of RNA bacteriophages
AU2013314264B2 (en) * 2012-09-17 2017-08-31 F. Hoffmann-La Roche Ag Method for the production of polypeptides in the periplasm of prokaryotic cells
RU2644652C2 (ru) * 2012-09-17 2018-02-13 Ф.Хоффманн-Ля Рош Аг Способ получения полипептидов в периплазме прокариотических клеток
JP2015528477A (ja) * 2012-09-17 2015-09-28 エフ．ホフマン−ラ ロシュ アーゲーＦ． Ｈｏｆｆｍａｎｎ−Ｌａ Ｒｏｃｈｅ Ａｋｔｉｅｎｇｅｓｅｌｌｓｃｈａｆｔ 原核細胞のペリプラズムにおけるポリペプチドの産生のための方法
US9926582B2 (en) 2012-09-17 2018-03-27 Hoffmann-La Roche Inc. Method for the production of polypeptides in the periplasm of prokaryotic cells
CN104619716A (zh) * 2012-09-17 2015-05-13 弗·哈夫曼-拉罗切有限公司 在原核细胞周质中产生多肽的方法
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WO2014041116A1 (fr) * 2012-09-17 2014-03-20 F. Hoffmann-La Roche Ag Procédé de production de polypeptides dans le périplasme de cellule procaryotes
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US11129882B2 (en) 2015-10-30 2021-09-28 University Of Copenhagen Virus like particle with efficient epitope display

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