US20070184068A1 - Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity - Google Patents

Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity Download PDF

Info

Publication number
US20070184068A1
US20070184068A1 US11/638,664 US63866406A US2007184068A1 US 20070184068 A1 US20070184068 A1 US 20070184068A1 US 63866406 A US63866406 A US 63866406A US 2007184068 A1 US2007184068 A1 US 2007184068A1
Authority
US
United States
Prior art keywords
composition
allergy
bacteriophage
particle
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/638,664
Other languages
English (en)
Inventor
Wolfgang Renner
Martin Bachmann
Indulis Cielens
Conrad Coester
Klaus Dietmeier
Sebastian Fuchs
Vania Manolova
Patrik Maurer
Paul Pumpens
Regina Renhofa
Alain Tissot
Yu Zou
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytos Biotechnology AG
Original Assignee
Cytos Biotechnology AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Cytos Biotechnology AG filed Critical Cytos Biotechnology AG
Priority to US11/638,664 priority Critical patent/US20070184068A1/en
Publication of US20070184068A1 publication Critical patent/US20070184068A1/en
Priority to US12/576,086 priority patent/US8574564B2/en
Assigned to CYTOS BIOTECHNOLOGY AG reassignment CYTOS BIOTECHNOLOGY AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: TISSOT, ALAIN, ZOU, YU, DIETMEIER, KLAUS, CIELENS, INDULIS, PUMPENS, PAUL, RENHOFA, REGINA, BACHMANN, MARTIN F., MANOLOVA, VANIA, MAURER, PATRIK, RENNER, WOLFGANG A.
Assigned to CYTOS BIOTECHNOLOGY AG reassignment CYTOS BIOTECHNOLOGY AG ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: LUDWIG-MAXIMILIANS-UNIVERSITAT-MUNCHEN
Assigned to LUDWIG-MAXIMILIANS-UNIVERSITAT-MUNCHEN reassignment LUDWIG-MAXIMILIANS-UNIVERSITAT-MUNCHEN ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: COESTER, CONRAD JOHANNES, FUCHS, SEBASTIAN
Assigned to VENBIO GLOBAL STRATEGIC FUND L.P. reassignment VENBIO GLOBAL STRATEGIC FUND L.P. SECURITY AGREEMENT Assignors: CYTOS BIOTECHNOLOGY AG
Priority to US13/947,577 priority patent/US20140056933A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0097—Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/04—Antipruritics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/14—Decongestants or antiallergics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/18011—Details ssRNA Bacteriophages positive-sense
    • C12N2795/18111—Leviviridae
    • C12N2795/18123—Virus like particles [VLP]

Definitions

  • compositions of the invention comprise particles, preferably virus-like particles, nanoparticles, microparticles or liposomes which are packaged with an immunostimulatory nucleic acid.
  • the compositions of the invention are useful in the treatment of hypersensitivity, preferably allergy, including diseases such as atopic eczema, asthma and IgE-mediated allergy (type-I allergy), especially pollen allergy (hay fever).
  • the invention therefore also provides methods for the treatment of these diseases.
  • the invention provides a composition for use as a medicament, the composition comprising, essentially consisting of, or consisting of a particle and an immunostimulatory nucleic acid (ISS-NA), wherein said particle is packaged with said ISS-NA.
  • ISS-NA immunostimulatory nucleic acid
  • the inventive compositions are useful in the prophylactic and therapeutic treatment of hypersensitivity, preferably allergy.
  • the invention thus provides a composition for use in a method of treating hypersensitivity, preferably allergy in an animal, the composition comprising a particle and immunostimulatory nucleic acid (ISS-NA), wherein said particle is packaged with said ISS-NA.
  • the invention provides a composition for use in a method of treating hypersensitivity, preferably allergy in an animal, the composition comprising a virus-like particle and an unmethylated CpG-containing oligonucleotide, wherein said virus-like particle is packaged with said unmethylated CpG-containing oligonucleotide.
  • said hypersensitivity is an allergy, preferably IgE-mediated asthma, atopic eczema or IgE-mediated allergy (type I allergy).
  • said particle is selected from a nanoparticle, a microparticle and a liposome.
  • said particle is a VLP, preferably a VLP of an RNA-bacteriophage, again preferably a VLP of bacteriophage Q ⁇ .
  • said unmethylated CpG-containing oligonucleotide exclusively consists of phosphodiester bound nucleotides, most preferably of G10 (SEQ ID NO:27).
  • the invention provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising (a) a virus-like particle; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b), said process comprising the steps of (i) incubating said VLP (a) with said unmethylated CpG-containing oligonucleotide (b); (ii) adding RNase; and (iii) purifying said composition.
  • the invention provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising (a) a virus-like particle; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b), said process comprises the steps of (i) incubating said VLP with RNase; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying the composition.
  • the invention provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising (a) a virus-like particle; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b) said process comprising the steps of (i) disassembling said VLP; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) reassembling said VLP.
  • the invention provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising (a) a virus-like particle; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b) said process comprises the steps of (i) incubating said VLP with solutions comprising metal ions capable of hydrolyzing the nucleic acids of said VLP; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying said composition, wherein preferably said metal ions of step (i) are selected from the group consisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe) ions; (d) any mixtures of at least one ion of (a), (b) and/or (c).
  • a virus-like particle comprising
  • the invention provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising (a) a virus-like particle; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b) said process comprises the steps of (i) incubating said VLP under alkaline conditions, preferably in the presence of NaOH, most preferably in the presence of about 25 mM NaOH; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying said composition.
  • the invention provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising (a) a virus-like particle; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b), said process comprising the steps of (i) incubating said VLP with a solution capable of destabilizing said VLP, wherein preferably said VLP is a VLP of bacteriophage Q ⁇ ; (ii) purifying the coat protein from said solution; and (iii) reassembling said coat protein to a VLP in the presence of unmethylated CpG-containing oligonucleotide and an oxidizing agent.
  • compositions for use as a medicament wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with unmethylated CpG-containing oligonucleotides; (ii) adding RNase; and (iii) purifying said composition.
  • compositions for use as a medicament wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with RNase; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) purifying the composition.
  • compositions for use as a medicament wherein said compositions are obtainable by a process comprising the steps of (i) disassembling a VLP; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) reassembling said VLP.
  • compositions for use as a medicament wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with solutions comprising metal ions capable of hydrolyzing the nucleic acids of said VLP; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) purifying said composition.
  • compositions for use in a method of treating allergy in an animal wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with unmethylated CpG-containing oligonucleotides; (ii) adding RNase; and (iii) purifying said composition.
  • compositions for use in a method of treating allergy in an animal wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with RNase; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) purifying the composition.
  • compositions for use in a method of treating allergy in an animal wherein said compositions are obtainable by a process comprising the steps of (i) disassembling a VLP; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) reassembling said VLP.
  • compositions for use in a method of treating allergy in an animal wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with solutions comprising metal ions capable of hydrolyzing the nucleic acids of said VLP; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) purifying said composition.
  • compositions comprising a composition of the invention.
  • the invention provides a method of treating hypersensitivity, preferably allergy in an animal, said method comprising introducing into said animal a composition of the invention.
  • a further aspect of the invention is the use of a composition of the invention or of a pharmaceutical composition of the invention for the manufacture of a pharmaceutical for the treatment of hypersensitivity in an animal, wherein preferably said hypersensitivity is an allergy, wherein further preferably said allergy is selected from the group consisting of: (a) IgE-mediated asthma, (b) atopic eczema; and (c) IgE-mediated allergy (type I allergy), preferably pollen allergy (hay fever) or house dust allergy.
  • FIG. 1 Characterization of purified Q ⁇ coat protein by analytical size exclusion chromatography.
  • B sample of the supernatant of the disassembly reaction. Released coat protein is indicated by the presence of the protein-like peak at approx. 12 min. Furthermore several species of non-precipitated RNA molecules are present in the range 6.8 to 11 min.
  • C sample of purified Q ⁇ coat protein. Analysis was performed in PBS on column TSK G5000PWx1 (Tosoh Bioscience).
  • FIG. 2 Analytical size exclusion chromatography of (A) native Q ⁇ VLP, (D) Q ⁇ G10 VLP and the packaging components (B) oligo nucleotide G10 and (C) Q ⁇ coat protein.
  • FIG. 3 Non-reducing SDS-PAGE analysis of native Q ⁇ VLP and in vitro assembled Q ⁇ G10. The position of the coat protein pentamers and hexamers is indicated ((a) molecular weight marker, (b) Q ⁇ VLP, (c) Q ⁇ G10).
  • FIG. 4 Ratio of the number of VLP+ cells at 2 h over the number of VLP+ cells at 24 h in the myeloid-DC, lymphoid-DC, Macrophage, pDC and B-cell populations after subcutaneous injection in the footpad, as a measure of DC-mediated or free draining of VLP to the Lymphnode.
  • Anti-CD11c, -CD11b, -B220 and -CD19 antibodies were used to identify myeloid- and lymphoid-DC, Macrophages, pDCs and B cells by FACS analysis.
  • FIG. 5 Ratio between percentages of particle-positive DC and macrophages. Mice were injected with nanoparticles of different size. 48 h later cells were analyzed by FACS.
  • FIG. 6 Activation of BMDCs by Q ⁇ G10.
  • BMDCs were activated by Q ⁇ G10 and hence secreted IL-12 in a dose dependent manner (dose is given as equivalent of G10 oligonucleotide packaged in the Q ⁇ VLPs) while untreated control cells did not secrete any IL-12.
  • hypersensitivity In the context of the invention the term “hypersensitivity” is to be understood as suggested in Johansson et al. 2001, Allergy 56:813-824 as any objectively reproducible symptom or signs, initiated by exposure to a defined stimulus at a dose tolerated by normal subjects. Hypersensitivity reactions are reproducible in the sense that there is reasonable evidence from history, examination, or investigation of a link between the symptoms and the environmental factors to which the patients attribute their symptoms.
  • hypersensitivity encompasses non-allergic hypersensitivity and allergic hypersensitivity (allergy).
  • Non-allergic hypersensitivity is any hypersensitivity which does not comprise an involvement of the immune system or at least where no involvement of the immune system can be detected, wherein allergic hypersensitivity always comprises an involvement of the cellular or humoral immune system.
  • Hypersensitivity preferably refers to allergic and non-allergic forms of a disease selected from the group consisting of: (a) asthma, (b) rhinitis, (c) conjunctivitis, (d) rhinoconjuctivitis, (e) dermatitis, (e) urticaria, (e) food hypersensitivity, (e) drug hypersensitivity, (e) insect sting or bite hypersensitivity, and (e) anaphylaxis.
  • hypersensitivity also includes any type of allergy.
  • hypersensitivity refers to allergic forms of a disease selected from the group consisting of: (a) asthma, (b) rhinitis, (c) conjunctivitis, (d) rhinoconjuctivitis, (e) dermatitis, (e) urticaria, (e) food hypersensitivity, (e) drug hypersensitivity, (e) insect sting or bite hypersensitivity, and (e) anaphylaxis.
  • hypersensitivity also includes any type of allergy.
  • allergy In the context of the application the term “allergy” stands for “allergic hypersensitivity” and is to be understood as suggested by Johansson et al. 2001, Allergy 56:813-824 and Johannson et al. 2004, J. Allergy Clin. Immunol. 113(5) 832-835. Unless otherwise indicated, the application follows the nomenclature for allergy as set forth therein. Allergy or allergic hypersensitivity is a hypersensitivity reaction initiated by immunologic mechanisms in response to a substance (allergen), often in a genetically predisposed individual (atopy). Allergy can be antibody- or cell-mediated.
  • the antibody typically responsible for an allergic reaction belongs to the IgE isotype (see “antibodies”) and these patients may be said to suffer from IgE-mediated allergy (type-I allergy). It must be noted that not all IgE-mediated allergic reactions occur in atopic subjects. In non IgE-mediated allergy, the antibody may belong to the IgG isotype. Thus, within the meaning of the application, “allergy” refers to both, IgE-mediated allergy (type-I allergy) and non IgE-mediated allergy. IgE-mediated allergy is preferably addressed by the invention. Therefore, in the context of the invention allergy preferably refers to IgE-mediated allergy. Allergies are classified according to the source of the antigen evoking the hypersensitive reaction.
  • allergy is selected from (a) food allergy, (b) drug allergy, (c) house dust allergy, (d) insect venom or bite allergy, and (e) pollen allergy.
  • allergies are classified based on the major symptoms of the hypersensitive reaction.
  • allergy refers to any allergic form of a disease selected from the group of (a) asthma, (b) rhinitis, (c) conjunctivitis, (d) rhinoconjuctivitis, (e) dermatitis, (f) urticaria and (g) anaphylaxis.
  • type-I allergy the terms “type-I allergy” and “IgE-mediated allergy” are used interchangeably and relate to IgE-mediated hypersensitivities to allergens.
  • Preferred embodiments of the invention relate to IgE-mediated allergy selected from the group consisting of (a) pollen allergy (hay fever); (b) house dust allergy; (c) food allergy; (d) drug allergy; (e) insect venom or bite allergy, preferably bee venom allergy; and (f) animal allergy, preferably cat allergy.
  • hay fever typical form of an IgE-mediated allergy (type-I allergy) against pollen which may comprise rhinitis, conjunctivitis and/or asthma, wherein asthma preferably occurs in chronic forms of hay fever.
  • Atopy is a personal or familial tendency to produce IgE antibodies in response to low doses of allergens, usually proteins, and to develop typical symptoms such as asthma, rhinoconjunctivitis, or eczema/dermatitis.
  • allergens usually proteins
  • typical symptoms such as asthma, rhinoconjunctivitis, or eczema/dermatitis.
  • the first manifestations of atopy in a child are often allergic symptoms, such as diarrhea, wheezing, and skin rashes, and only later can the responsible IgE antibody be detected.
  • Allergic symptoms in a typical atopic individual may be referred to as atopic.
  • hypersensitivity is an atopic disease, preferably an atopic disease selected from the group consisting of (a) atopic asthma, (b) atopic eczema, (c) atopic IgE-mediated allergy, preferably pollen allergy (hay fever), house dust allergy or house dust mite allergy.
  • atopic allergy preferably to IgE-mediated atopic allergy.
  • rhinitis The term “rhinitis” relates to hypersensitivity symptoms from the nose, for example, itching, sneezing, increased secretion, and blockage. Rhinitis relates to non-allergic as well as allergic, i.e. immunologically mediated, rhinitis. Preferred embodiments of the invention relate to allergic rhinitis, preferably to IgE-mediated and non IgE-mediated forms of allergic rhinitis. Specifically preferred embodiments relate to IgE-mediated allergic rhinitis.
  • conjunctivitis The term conjunctivitis relates to irritations of the eye which can be of allergic as well as non-allergic origin, wherein allergic conjunctivitis encompasses IgE-mediated and non IgE-mediated allergic conjunctivitis. Allergic conjunctivitis, especially IgE mediated allergic conjunctivitis is commonly accompanied by allergic rhinitis, so this disorder is appropriately termed allergic rhinoconjuctivitis. Besides IgE-mediated conjunctivitis, contact allergic conjunctivitis involving TH1 mechanisms occurs. Non-allergic conjunctivitis also often accompanies non-allergic rhinitis.
  • Preferred embodiments of the invention relate to allergic conjunctivitis, including IgE-mediated and non IgE-mediated forms of allergic conjunctivitis. Specifically preferred embodiments relate to IgE-mediated allergic conjunctivitis. Further preferred embodiments relate to IgE-mediated allergic rhinoconjunctivitis.
  • asthma bronchiale is a chronic respiratory disease due to inflammation of the air passages in the lungs and affects the sensitivity of the nerve endings in the airways so they become easily irritated. In an attack, the lining of the passages swell causing the airways to narrow and reducing the flow of air in and out of the lungs. Asthma can occur in a intermittent form (2 attacks per week or less during daytime, 2 attacks per month or less at night), in persistent form (permanent attacks during daytime, frequent attacks at night) and in any intermediate form.
  • asthma relates to non-allergic as well as to allergic asthma.
  • Preferred embodiments of the invention relate to allergic asthma, including IgE-mediated and non IgE-mediated forms of asthma. Specifically preferred embodiments relate to IgE-mediated allergic asthma, most preferably to atopic asthma.
  • atopic asthma IgE-mediated form of asthma in patients with a genetic predisposition which often occurs in conjunction with atopic eczema and IgE-mediated allergies, for example pollen allergy (hay fever), house dust or dust mite.
  • dermatitis The term “dermatitis” relates to local inflammation of the skin and encompasses, besides other forms, “eczema” and “contact dermatitis” (see definitions below). Preferred embodiments of the invention relate to dermatitis, preferably to eczema and contact dermatitis.
  • eczema The term “eczema” relates to the atopic eczema/dermatitis syndrome (AEDS), describing an aggregation of several skin diseases with certain clinical characteristics in common involving a genetically determined skin barrier defect. This genetically determined target organ sensitivity constitutes the basis for eczema. In children and young adults of the atopic constitution, the underlying inflammation is dominated by an IgE-antibody associated reaction (atopic eczema). In chronic cases, the inflammation seems to be less influenced by IgE antibody, and the dominating cells in biopsies are lymphocytes. Eczema relates to non-allergic eczema and allergic eczema.
  • AEDS atopic eczema/dermatitis syndrome
  • Preferred embodiments of the invention relate to eczema, preferably allergic eczema including atopic (IgE-mediated) eczema and non atopic forms of eczema. Most preferably, the invention relates to atopic (IgE-mediated) eczema.
  • contact dermatitis The term “contact dermatitis” relates to local inflammatory reaction in the skin caused by close contact with low molecular weight chemicals or irritants. Contact dermatitis can be of allergic as well as non-allergic nature. Allergic contact dermatitis is mediated by immunological mechanisms, predominantly TH1 lymphocytes. Typical allergens acting as haptens and causing allergic contact dermatitis are nickel, chromium ions, fragrances, preservatives, and urushiol, from the poison ivy plant. Exposure can occur through oral uptake, so-called systemic allergic contact dermatitis.
  • a subgroup of contact dermatitis, protein contact dermatitis, is an IgE-associated reaction caused by absorption of proteins through damaged skin.
  • Preferred embodiments of the invention relate to contact dermatitis, preferably allergic contact dermatitis. Further preferred embodiments relate to protein contact dermatitis.
  • urticaria The term “urticaria” relates to a non inflammatory reaction in the skin caused by an irritant or allergen and includes non-allergic urticaria as well as allergic urticaria. Allergic urticaria is mediated by immunological mechanisms, which commonly is IgE-mediated but can also be immune complex-associated. Urticaria can also develop locally after topical contact with the allergen, as occurs on the hands of a person with latex allergy wearing latex gloves or in a person with dog allergy licked by a dog. Preferred embodiments of the invention relate to urticaria, preferably allergic urticaria, most preferably IgE-mediated allergic urticaria.
  • “food hypersensitivity” The term “food hypersensitivity” relates to adverse reaction to food, which can be of non-allergic as well as allergic nature. Allergic food hypersensitivities can be IgE-madiated and are referred to as food allergies. Severe, generalized allergic reactions to food can be classified as anaphylaxis (see below). Preferred embodiments of the invention relate to food allergy, preferably to IgE-mediated food allergy.
  • drug hypersensitivity The term “drug hypersensitivity” relates to hypersensitive reactions of the body towards drugs which can be of non-allergic as well as of allergic nature. When immunologic mechanisms have been shown, either antibody or cell mediated, the reactions are referred to as drug allergy. Drug allergies can be mediated by IgE. Preferred embodiments of the invention relate to drug hypersensitivity, preferably to drug allergy, most preferably to IgE-mediated drug allergy.
  • insect sting hypersensitivity or “insect bite hypersensitivity”: these terms relate to hypersensitive reactions towards insect venom and saliva which can be of non-allergic as well as allergic nature. Insect sting hypersensitivity or insect bite hypersensitivity mediated by an immunologic mechanism is referred to as venom or saliva allergy, as in bee venom allergy.
  • venom or saliva allergy The large quantity of venom allergen in a sting is comparable with years of inhaled pollen allergen. This high-dose sensitization probably explains why there is no need for a genetic predisposition for developing such an allergy.
  • Preferred embodiments of the invention relate to venom allergy, preferably to IgE-mediated venom allergy, most preferably to IgE mediated bee venom allergy.
  • anaphylaxis refers to a severe, life-threatening, generalized or systemic hypersensitive reaction. The reaction usually develops gradually, most often starting with itching of the gums/throat, the palms, or the soles, and local urticaria; developing to a multiple organ reaction often dominated by severe asthma; and culminating in hypotension and shock. Hypotension and severe bronchospasm do not have to be present for a reaction to be classified as anaphylaxis. Anaphylaxis can be of non-allergic as well as of allergic nature. Allergic anaphylaxis involves an immunologic mechanism like an IgG immune complex, complement related, or immune cell-mediated mechanism.
  • Anaphylaxis preferably relates to an anaphylactic reaction mediated by IgE antibodies (IgE-mediated anaphylaxis), most preferably to peanut-induced food anaphylaxis or bee venom-induced anaphylaxis.
  • allergen refers to a substance causing allergy. Preferred allergens are allergens disclosed in Shough, H. et al., REMINGTON'S PHARMACEUTICAL SCIENCES, 19th edition, (Chap. 82), Mack Publishing Company, Mack Publishing Group, Easton, Pa. (1995), the entire contents of which is hereby incorporated by reference. Allergens serve as antigens in vertebrate animals.
  • allergen as used herein, also refers to “allergen extracts” and “allergenic epitopes.” Very preferred allergens are selected from the group consisting of: pollens (e.g.
  • grass, ragweed, birch and mountain cedar house dust and dust mites; mammalian epidermal allergens and animal danders; mold and fungus; insect bodies and insect venom; feathers; food; and drugs (e.g. penicillin).
  • allergen extracts are components of provocation test solutions to be used for conjunctival, nasal and bronchial challenges. Such allergen extracts are commercially available and methods for producing such extracts are well-known.
  • Preferred are single allergen provocation solutions comprising a single allergen extract which is prepared from a source selected from the group consisting of (i) tree species or a grass species, most preferably selected from the group consisting of alder, ash, birch, hazel, orchard grass, velvet grass, rye grass, timothy grass, Kentucky blue grass, Meadow fescue, Bermuda grass, ragweed, rye and wheat; (ii) epithelia of different animal species, preferably epithelia from an animal species selected from the group consisting of cat, dog and horse; (iii) moulds, preferably moulds selected from the group consisting of aspergillus, candida,retemaria , and saccharomyces ; and (iv) mite species, preferably mite species selected from the group consisting of Dermatophagoides farinae, Dermatophagoides pteronyssinus and Acarus siro .
  • allergen extracts comprising allergen mixtures can also be used in provocation test solutions.
  • Provocation solutions are usually prepared in physiological saline and can be preserved by addition of 0.4% phenol.
  • antibody refers to molecules belonging to the class of immunoglobulins which are capable of binding an epitope or antigenic determinant.
  • 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.
  • Antigens as used herein may also be mixtures of several individual antigens. However “antigen” does not encompass any of the components of the compositions of the invention. In particular, the term “antigen” does not refer to the particle nor to the ISS-NA of the invention. The term “antigen” also does not refer to any component forming the particle of the invention, such as, for example capsid protein.
  • 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.
  • 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 antigen presenting cells and/or other cells of the innate immune system, such as pDC. Alternatively, the immune response may also result in an altered function of effector cells, such as mast cells.
  • a substance preferably an ISS-NA is capable of inducing an immune response when upon exposure of a cell or an organism to said substance, preferably to an effective amount of said substance, an immune response is detectable in said cell or animal which does not occur in the untreated control.
  • IFN-alpha production by a cell can be determined by various methods generally known in the art, preferably by a method selected from (a) ELISA, most preferably by ELISA essentially as described in Example 14; (b) flow cytometry analysis using fluorochrom-conjugated antibodies, preferably as described in Example 14; and (c) cytopathicity inhibition bioassays.
  • a typical cytopathicity inhibition bioassay is based on bovine MDBK cells infected with vesicular stomatitis virus, as previously described in Pestka, S. (1986) “Interferon Standards and General Abbreviations”, in Methods in Enzymology, Academic Press, New York 119, 14-23.
  • a substance preferably an immunostimulatory nucleic acid
  • a substance is regarded as being “capable of stimulating IFN-alpha production”, when the production of IFN-alpha by a cell as detected by any one of the above described methods, preferably by ELISA, most preferably as described in Example 14, is significantly increased upon exposure of said cell to said substance as compared to a control cell, wherein typically and preferably, said IFN-alpha production is increased by a factor of at least about 2, more preferably by a factor of about 3 or more.
  • enhancing an immune response refers to a substance, preferably to an ISS-NA, which is capable of intensifying or modulating the immune response of a cell or an animal upon exposure of said cell or said animal to said substance, as compared to a suitable control.
  • This observation can relate to any parameter known in the art to be indicative for an immune response, preferably to the formation of cytokines and to cytotoxicity.
  • the lytic activity of cytotoxic T cells can be measured, e.g. using a 51 Cr release assay, with and without the substance, preferably the ISS-NA.
  • 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.
  • the immune response is 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.
  • immunostimulatory nucleic acid refers to a nucleic acid capable of inducing and/or enhancing an immune response.
  • ISS-NA as used herein, comprise ribonucleic acids and in particular desoxyribonucleic acids, wherein both, ribonucleic acids and desoxyribonucleic acids may be either double stranded or single stranded.
  • Preferred ISS-NA are desoxyribonucleic acids, wherein further preferably said desoxyribonucleic acids are single stranded.
  • ISS-NA contain at least one CpG motif comprising an unmethylated C.
  • Very preferred ISS-NA comprise at least one CpG motif, wherein said at least one CpG motif comprises or preferably consist of at least one, preferably one, CG dinucleotide, wherein the C is unmethylated.
  • said CG dinucleotide is part of a palindromic sequence.
  • ISS-NA not containing CpG motifs as described above encompass, by way of example, nucleic acids lacking CG dinucleotides, as well as nucleic acids containing CG dinucleotides with a methylated C.
  • the term “immunostimulatory nucleic acid” as used herein also refers to nucleic acids that contain modified bases, preferably 4-bromo-cytosine.
  • ISS-NA which are capable of stimulating IFN-alpha production in dendritic cells.
  • oligonucleotide refers to a nucleic acid sequence comprising 2 or more nucleotides, preferably at least about 6 nucleotides to about 100,000 nucleotides, more preferably about 6 to about 2000 nucleotides, and still more preferably about 6 to about 300 nucleotides, even more preferably about 20 to about 300 nucleotides, and even more preferably about 20 to about 100 nucleotides, and most preferably 20 to 40 nucleotides.
  • oligonucleotides comprise about 30 nucleotides, more preferably oligonucleotides comprise exactly 30 nucleotides, and most preferably oligonucleotides consist of exactly 30 nucleotides.
  • the term oligonucleotide also refers to a nucleic acid comprising more than 100 to about 2000 nucleotides, preferably more than 100 to about 1000 nucleotides, and more preferably more than 100 to about 500 nucleotides.
  • Oligonucleotides are polyribonucleotides or polydeoxribonucleotides and are preferably selected from (a) unmodified RNA or DNA, and (b) modified RNA or DNA.
  • the modification may comprise the backbone or nucleotide analogues.
  • Oligonucleotides are preferably selected from the group consisting of (a) single- and double-stranded DNA, (b) DNA that is a mixture of single- and double-stranded regions, (c) single- and double-stranded RNA, (d) RNA that is mixture of single- and double-stranded regions, and (e) hybrid molecules comprising DNA and RNA that are single-stranded or, more preferably, double-stranded or a mixture of single- and double-stranded regions.
  • oligonucleotides are triple-stranded regions and higher-ordered structures comprising RNA or DNA or both RNA and DNA.
  • oligonucleotide are synthetic, genomic or recombinant.
  • oligonucleotides are selected from the group consisting of ⁇ -DNA, cosmid DNA, artificial bacterial chromosome, yeast artificial chromosome and filamentous bacteriophage, preferably M13.
  • oligonucleotide refers to (a) DNA or RNA containing at least one modified nucleotide or at least one nucleotide analogue, or (b) to DNA or RNA with backbones modified for stability or for other reasons.
  • Preferred nucleotide modifications/analogs are selected from the group consisting of (a) peptide nucleic acid, (b) inosin, (c) tritylated bases, (d) phosphorothioates, (e) alkylphosphorothioates, (f) 5-nitroindole desoxyribofuranosyl, (g) 5-methyldesoxycytosine, and (h) 5,6-dihydro-5,6-dihydroxydesoxythymidine.
  • Phosphothioated nucleotides are protected against degradation in a cell or an organism and are therefore preferred nucleotide modifications.
  • oligonucleotides capable of stimulating IFN-alpha production in cells, preferably in dendritic cells.
  • Very preferred oligonucleotides capable of stimulating IFN-alpha production in cells are selected from A-type CpGs and C-type CpGs.
  • CpG motif refers to a pattern of nucleotides that includes an unmethylated central CpG, i.e. the unmethylated CpG dinucleotide, in which the C is unmethylated, surrounded by at least one base, preferably one or two nucleotides, flanking (on the 3′ and the 5′ side of) the central CpG.
  • the CpG motif as used herein comprises or alternatively consists of the unmethylated CpG dinucleotide and two nucleotides on its 5′ and 3′ ends.
  • the bases flanking the CpG confer a significant part of the activity to the CpG oligonucleotide.
  • CpG /“unmethylated CpG-containing oligonucleotide”: As used herein, the term “unmethylated CpG-containing oligonucleotide” or “CpG” refers to an oligonucleotide, preferably to an oligodesoxynucleotide, containing at least one CpG motif. Thus, a CpG contains at least one unmethylated cytosine, guanine dinucleotide. Preferred CpGs stimulate/activate, e.g. have a mitogenic effect on, or induce or increase cytokine expression by, a vertebrate bone marrow derived cell.
  • CpGs can be useful in activating B cells, NK cells and antigen-presenting cells, such as dendritic cells, monocytes and macrophages.
  • CpG relates to an oligodesoxynucleotide, preferably to a single stranded oligodesoxynucleotide, containing an unmethylated cytosine followed 3′ by a guanosine, wherein said unmethylated cytosine and said guanosine are linked by a phosphate bond, wherein preferably said phosphate bound is a phosphodiester bound or a phosphothioate bound, and wherein further preferably said phosphate bond is a phosphodiester bound.
  • CpGs can include nucleotide analogs such as analogs containing phosphorothioester bonds and can be double-stranded or single-stranded. Generally, double-stranded molecules are more stable in vivo, while single-stranded molecules have increased immune activity.
  • a CpG is an oligonucleotide that is at least about ten nucleotides in length and comprises at least one CpG motif, wherein further preferably said CpG is 10 to 60, more preferably 15 to 50, still more preferably 20 to 40, still more preferably about 30, and most preferably exactly 30 nucleotides in length.
  • a CpG may consist of methylated and/or unmethylated nucleotides, wherein said at least one CpG motif comprises at least one CG dinucleotide wherein the C is unmethylated.
  • the CpG may also comprise methylated and unmethylated sequence stretches, wherein said at least one CpG motif comprises at least one CG dinucleotide wherein the C is unmethylated.
  • Very preferred CpGs consist exclusively of unmethylated nucleotides.
  • CpG relates to a single stranded oligodesoxynucleotide containing an unmethylated cytosine followed 3′ by a guanosine, wherein said unmethylated cytosine and said guanosine are linked by a phosphodiester bound. Still more preferably, CpG relates to a single stranded oligodesoxynucleotide containing an unmethylated cytosine followed 3′ by a guanosine, wherein said unmethylated cytosine and said guanosine are linked by a phosphodiester bound, and wherein said CpG consist exclusively of unmethylated nucleotides.
  • CpG relates to a single stranded oligodesoxynucleotide of about 30 nucleotides in length, containing an unmethylated cytosine followed 3′ by a guanosine, wherein said unmethylated cytosine and said guanosine are linked by a phosphodiester bound, and wherein said CpG consist exclusively of unmethylated nucleotides.
  • the CpGs can include nucleotide analogs such as analogs containing phosphorothioester bonds and can be double-stranded or single-stranded.
  • phosphodiester CpGs are A-type CpGs as indicated below, while phosphothioester stabilized CpGs are B-type CpGs or C-type CpGs.
  • Preferred CpG oligonucleotides in the context of the invention are A-type CpGs and C-type CpG, most preferred are A-type CpGs.
  • A-type CpG As used herein, the term “A-type CpG” or “D-type CpG” refers to an oligodesoxynucleotide (ODN) comprising at least one CpG motif.
  • ODN oligodesoxynucleotide
  • A-type CpGs preferentially stimulate activation of T cells and the maturation of dendritic cells and are capable of stimulating IFN-alpha production.
  • the nucleotides of the at least one CpG motif are linked by at least one phosphodiester bond.
  • A-type CpGs comprise at least one phosphodiester bond CpG motif which may be flanked at its 5′ end and/or, preferably and, at its 3′ end by phosphorothioate bound nucleotides.
  • the CpG motif and hereby preferably the CG dinucleotide and its immediate flanking regions comprising at least one, preferably two nucleotides, are composed of phosphodiester nucleotides.
  • Preferred A-type CpGs exclusively consist of phosphodiester (PO) bond nucleotides. Further preferred A-type CpGs do not comprise phosphothioate bounds.
  • the poly G motif comprises or alternatively consists of at least one, preferably at least three, at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gs (guanosines), most preferably by at least 10 Gs.
  • the 5′ and/or 3′ ends typically and preferably at least one G of the poly G motifs at the 5′ and/or 3′ ends, preferably at least two, three or four, even more preferably all Gs of the poly G motif, are phoshorothioate modified.
  • all Gs of the poly G motif are linked by phosphodiester bonds.
  • the A-type CpG of the invention comprises or alternatively consists of a palindromic sequence.
  • the CpG motif is part of said palindromic sequence.
  • all nucleotides, but at least the CpG motif of the palindromic sequence are composed of phosphodiester nucleotides.
  • the palindromic sequence is SEQ ID NO:28.
  • Very preferred A-type CpGs are 16 to 30 nucleotides in length, consist exclusively of phosphodiester bound nucleotides, comprise a palindromic sequence, preferably the palindromic sequence of SEQ ID NO:28, and are flanked at their 5′ and at their 3′ end by a poly G motif consisting of 3 to 10 Gs.
  • B-type CpG As used herein, the term “B-type CpG” (K-type) relates to a CpG oligonucleotide which predominantly or preferably exclusively consists of modified nucleotides, preferably phosphorothioate modified nucleotides. B-type CpGs stimulate preferentially B-cell and to some extent NK-cell activation and cytokine production.
  • C-type CpG As used herein, the term “C-type CpG” relates to a CpG oligonucleotide which like a B-type oligonucleotide predominantly or preferably exclusively consists of modified nucleotides, preferably phosphorothioate modified nucleotides. Examples of C-type CpGs have been described in WO2005/042018A2 and in Vollmer et al. 2004, Eur. J. Immunol. 43:351-262 which are incorporated herein by reference. Specific reference is made to SEQ IDs NO: 1 to 69 of WO2005/042018A2.
  • C-type CpGs combine effects of A-type and B-type CpGs and stimulate B-cell or NK-cell activation and IFN-alpha production, preferably IFN-alpha production in dendritic cells.
  • C-type CpCs which are capable of stimulating IFN-alpha production, preferably in dendritic cells, are generally preferred in the context of the invention.
  • C-type CpGs do not typically comprise poly-G stretches.
  • C-type CpGs preferably comprise or alternatively consist of palindromic sequences comprising CpG motifs, preferably palindromic sequences as depicted in SEQ ID NOs:53 to 60.
  • C-type CpGs comprise a sequence selected from the group consisting of (a) TCGTCGTTTTA (SEQ ID NO:61), (b) CGGCGCCGTGCCG (SEQ ID NO:62) and (c) CGGCGTCGTGCCG (SEQ ID NO:63), wherein the 5′ end of said C-type CpG preferably consists of SEQ ID NO:61 and/or wherein the 3′ end of said C-type CpG preferably consists of a nucleotide sequence selected from SEQ ID NO:62 and SEQ ID NO:63, most preferably the 3′ end of said C-type CpG consists of SEQ ID NO:63.
  • C-type CpGs are selected from the group consisting of (a) TCGTCGTTTTACGGCGCCGTGCCG (SEQ ID NO:64) and (b) TCGTCGTTTTACGGCGTCGTGCCG (SEQ ID NO:65), wherein preferably all nucleic acids of said C-type CpGs are phosphorothioate bound.
  • C-type CpGs are selected from the group consisting of (a) TCpGTCGTTTTACGGCGCCGTGCCG (SEQ ID NO:64); (b) TCGTCGTTTTACpGGCpGCCpGTGCCG (SEQ ID NO:64); (c) TCGTCGTTT TACpGGCGCCpGTGCCG (SEQ ID NO:64); (d) TCGTCpGTTTTACpGGCGCCpGTGCCG (SEQ ID NO:64); wherein p indicates phosphodiester bounds while all other nucleotides are phosphorothioate bound.
  • C-type CpGs selected from the group consisting of (a) TCGTCGTTTTCGGCGCGCGCCG (SEQ ID NO:66); (b) TCGTCGTTTTCGACGGCCGTCG (SEQ ID NO:67); (c) TCGTCGTTTTCCGGCGCGCCGG (SEQ ID NO:68); (d) TCGTCGTTTTCGGCGCGCGTCG (SEQ ID NO:69); (e) TCGGCGCGCGCCGTCGTCGTTT (SEQ ID NO:70); (f) TTGGCGCGCGCCGTCGTCGTTT (SEQ ID NO:71); (g) TCGTCGTTTTCGTCGGCCGCCG (SEQ ID NO:72); (h) TCGTCGTTTTCGGCTTTTGCCG (SEQ ID NO:73); (i) TCGTCGTTTTCGGCGTTTTTTT (SEQ ID NO:74); and (j) TCGTCGTTTTCGGCGGCCGCCG (SEQ ID NO:75) are potent inducers of IFN
  • palindromic sequence is a nucleotide sequence which, when existing in the form of a double stranded nucleic acid with regular base pairing (A/T; C/G), would consist of two single strands with identical sequence in 5′-3′ direction.
  • An immunostimulatory nucleic acids of the invention preferably comprises a palindromic sequence, preferably a palindromic sequence consisting of at least 6, preferably of at least 7, 8, 9 or 10, most preferably of exactly 10 nucleotides, wherein most preferably said palindromic sequence preferably comprises a CpG motif.
  • Palindromic sequences of immunostimulatory nucleic acids useful in the context of the invention are, for example, described in Yamamoto et al.
  • Preferred palindromic sequences comprise a CpG motif and are selected from the group consisting of (a) GACGTC (SEQ ID NO:35), (b) AGCGCT (SEQ ID NO:36), (c) AACGTT (SEQ ID NO:37), (d) ATCGAT (SEQ ID NO:38); (e) CGATCG (SEQ ID NO:39); (f) CGTACG (SEQ ID NO:40); (g) CGCGCG (SEQ ID NO:41); (h) GCGCGC (SEQ ID NO:42); (i) TCGCGA (SEQ ID NO:43); (j) ACGATCGT (SEQ ID NO:44); (k) CGACGATCGTCG (SEQ ID NO:45); (l) CGACGACGATCGTCGTCG (SEQ ID NO:46
  • A-type CpGs preferably comprise a palindromic sequence selected from palindromic sequences (a) to (s), while C-type CpGs preferably comprise a palindromic sequence selected from palindromic sequences (t) to (aa).
  • Packaged refers to the state of an ISS-NA, in particular an unmethylated CpG-containing oligonucleotide, in relation to the particle, in particular the VLP.
  • the term “packaged” as used herein refers to covalent binding, preferably by chemically coupling. More preferably, the term “packaged” refers to non-covalent binding, preferably to ionic interactions, hydrophobic interactions, or hydrogen bonds. Covalent bonds are preferably selected from the group consisting of ester, ether, phosphoester, amide, peptide, imide, carbon-sulfur bonds, and carbon-phosphorus bonds.
  • the term “packaged” as used herein refers to the enclosement, or partial enclosement, of said ISS-NA within the particle.
  • the unmethylated CpG-containing oligonucleotide can be enclosed by the VLP without the existence of an actual binding, neither covalently nor non-covalently, or with a non-covalent binding.
  • a particle packaged with ISS-NA protects said ISS-NA from degradation, preferably from DNAse or RNAse hydrolysis. Therefore, in the preferred meaning, the term “packaged” indicates that the ISS-NA, preferably the unmethylated CpG-containing oligonucleotide, in a packaged state is not accessible to DNAse or RNAse hydrolysis. More preferably, the term “packaged” indicates that the ISS-NA, preferably the unmethylated CpG-containing oligonucleotide, is not accessible to DNAse hydrolysis, wherein further preferably the DNAse is DNAseI or Benzonase. Still more preferably, the term “packaged” indicates that the unmethylated CpG-containing oligonucleotide is not accessible to Benzonase hydrolysis.
  • the accessibility of the ISS-NA, in particular the of the unmethyated CpG-containing oligonucleotide for DNAse is preferably assayed as described in Examples 11-17 of WO2003/024481A2 (see p. 111 therein).
  • a VLP is regarded as being packaged with an unmethylated CpG-containing oligonucleotide, when after treatment with Benzonase (190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h) at least 90%, preferably at least 95%, most preferably at least 98% of said unmethylated CpG-containing oligonucleotide can be recovered from said VLP (e.g. in an ethidiumbromide stained gel).
  • Benzonase 190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h
  • Benzonase 190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h
  • a VLP of an RNA bacteriophage is regarded as being packaged with an unmethylated CpG-containing oligonucleotide, when after treatment with Benzonase (190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h) at least 90%, preferably at least 95%, most preferably at least 98% of said unmethylated CpG-containing oligonucleotide can be recovered from said VLP of an RNA bacteriophage.
  • Benzonase 190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h
  • a VLP of a RNA bacteriophage is regarded as being packaged with G10 (SEQ ID NO:27) oligonucleotide, when after treatment with Benzonase (190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h) at least 90%, preferably at least 95%, most preferably at least 98% of said unmethylated CpG-containing oligonucleotide can be recovered from said VLP of an RNA bacteriophage.
  • Benzonase 190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h
  • a VLP of a RNA bacteriophage Q ⁇ , AP205, GA or fr is regarded as being packaged with G10 (SEQ ID NO:27) oligonucleotide, when after treatment with Benzonase (190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h) at least 90%, preferably at least 95%, most preferably at least 98% of said unmethylated CpG-containing oligonucleotide can be recovered from said VLP of an RNA bacteriophage.
  • Benzonase 190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h
  • Benzonase 190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2
  • a VLP of a RNA bacteriophage Q ⁇ is regarded as being packaged with G10 (SEQ ID NO:27) oligonucleotide, when after treatment with Benzonase (190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h) at least 90%, preferably at least 95%, most preferably at least 98% of said unmethylated CpG-containing oligonucleotide can be recovered from said VLP of RNA bacteriophage Q ⁇ .
  • Benzonase 190 U Benzonase/mg capsid protein in a buffer comprising 2 mM MgCl 2 , pH 7.2, 20-25° C., 18 h
  • the packaging state of an ISS-NA in a particle in particular of ISS-NA which do not constitute a substrate for DNAse or RNAse hydrolysis, can be assessed by size exclusion chromatography or SDS-PAGE and subsequent spectroscopic analysis as described in Example 4.
  • a further possibility to verify the packaging state of a particle packaged with an ISS-NA is dialysis or tangential flow filtration of the particle, for example under conditions as described in Example 1, wherein non-packaged nucleic acids are removed while packaged nucleic aids remain associated with said particle.
  • the particle is a virus particle or a virus-like particle of a bacteriophage, preferably of an RNA-bacteriophage, further preferably of an RNA bacteriophage Q ⁇ , and most preferably of a virus-like particle of a RNA-bacteriophage Q ⁇
  • said ISS-NA is a unmethylated CpG-containing oligonucleotide, preferably a A-type CpG, further preferably the SEQ ID NO:27
  • the term “packaged” indicates that the particle packaged with said ISS-NA elutes at the same retention time as the virus-like particle of said bacteriophage, preferably of said RNA-bacteriophage, further preferably of said RNA bacteriophage Q ⁇ obtained by recombinant expression of the coat protein in E. coli , preferably wherein said retention time is determined by size exclusion chromatography, preferably as described in Example 4 of the present application, and comprises said ISS-NA as
  • the ISS-NA preferably the unmethylated CpG-containing oligonucleotide
  • the particle preferably VLP capsids, most preferably in a non-covalent manner.
  • Protocols for the preparation of VLPs packaged with unmethylated CpG-containing oligonucleotide are provided in the prior art, e.g. in WO2003/024481A2 (see Examples 2, 3, 7, 8, 10, 11, 12, 13, 14, 15, 16, and 17 therein, in particular Examples 14-17 therein) and WO2004/000351A1. The disclosure of both publications is incorporated to this application by reference. Further Protocols for the preparation of VLPs packaged with unmethylated CpG-containing oligonucleotide are provided Examples 1, 3 5 and 6 of the present application.
  • some synthetic particles which are packaged with said ISS-NA may release a certain limited amount of ISS-NA, wherein said release typically follows a bi- or more phasic kinetic, wherein said kinetic may comprise a fast initial burst release phase and at least one slow release phase.
  • a certain percentage of the ISS-NA packaged in a synthetic particle may be released in a burst release phase, when incubated at 37° C. or 30° C. in physiological buffers, in vitro. In this case the burst release phase is followed by at least one slow release phase.
  • the second release phase will be flat, meaning that no or very limited amounts of ISS-NA are released from the synthetic particle in that phase.
  • the two or more phases are identified by examination of the release kinetic of ISS-NA from the synthetic particle.
  • an initial burst release phase will be complete in a few hours but may last up to 24 hours, while the slow release phase may last from 2-3 days up to 6 days or longer.
  • the slow release phase may be nearly flat, with no or very little ISS-NA released after the burst release phase.
  • burst release phase may be concomitant to nuclease or serum exposure in an assay to assess packaging, protection from degradation by nucleases or serum assessed in this assay may not be complete.
  • protection is assessed during the time span of the assay corresponding to the slow release phase.
  • the term “packaged” as related to particles being synthetic particles but not being virus particles or virus-like particles also encompasses compositions comprising, essentially consisting of, or consisting of synthetic particles and ISS-NA's, in which such release of ISS-NA by said synthetic particle takes place, provided that at least 30%, preferably at least 40%, more preferably at least 50%, still more preferably at least 60%, even more preferably at least 70%, and most preferably at least 80% of the ISS-NA packaged in said synthetic particle is remaining associated with said synthetic particle at the end of the assay.
  • Particles of the invention have a diameter of 10 to 10000 nm, preferably of 20 to 1000 nm, more preferably 20 to 500 nm, still more preferably 20 to 300 nm, still more preferably 20 to 200 nm, and most preferably 20 to 100 nm, wherein these particles can preferably be packaged with an ISS-NA.
  • Particles of the invention are preferably selected from the group consisting of synthetic particles and VLPs.
  • Very preferred particles of the invention are VLPs, most preferably VLPs of RNA phages.
  • size of a particle The size of a spherical or nearly spherical particle is determined as the medium diameter of a population of particles; the size of an elliptic, longitudinal or irregular formed particle refers to the arithmetic medium of the longest axis in a population of particles.
  • the size of a particle preferably of a nanoparticle or a microparticle, most preferably of a nanoparticle, is determined dynamic light scattering (DLS) technology (Example 13).
  • synthetic particles refers to particles which are formed by chemical or physical processes, preferably by polymerization of monomers, precipitation of polymers, assembly of macromolecules brought together, for example by aggregation or heat denaturation, chemical cross-linking of said assembled macromolecules.
  • Preferred synthetic particles are selected from liposomes, microparticles, and nanoparticles. Further preferred synthetic particles are selected from liposomes, microparticles, nanoparticles, and virosomes. Very preferred synthetic particles are nanoparticles.
  • the term “synthetic particles” does not refer to particles formed by assembly of viral proteins. Particles formed from viral coat proteins are specifically referred to as virus particles or virus-like particles. Viroids, retrotransposon particles, and all particles formed from genetically encoded viral proteins are also understood as virus particles or virus-like particles.
  • liposomes refers to phospholipid vesicles comprising one or more, preferably one, two, or three phospholipid bilayer membranes. Liposomes vary in charge and in size depending on the method of preparation and the lipids used.
  • the liposome of the present invention may be neutral, cationic, anionic, stealth, or cationic stealth. Preferably, the liposome of the invention is a cationic liposome.
  • the liposome may have a diameter between 100 and 800 nm, preferably between 100 and 400 nm, more preferably between 100 and 300 nm, even more preferably between 100 and 200 nm, most preferably less than 200 nm.
  • liposome shall also encompass modified liposomes, preferably modified liposomes, wherein the surface of the liposomes may be specifically modified to optimize binding to DC, for example, via specific sugar moieties (Fukasawa et al., (1998), FEBS, 441, 353-356) or antibodies (Serre et al. (1998), J. Immunol., 161, 6059-6067).
  • modified liposomes preferably modified liposomes, wherein the surface of the liposomes may be specifically modified to optimize binding to DC, for example, via specific sugar moieties (Fukasawa et al., (1998), FEBS, 441, 353-356) or antibodies (Serre et al. (1998), J. Immunol., 161, 6059-6067).
  • lipopolyplex particles are liposomes comprising or, preferably, essentially consisting of, most preferably consisting of a cationic lipid, a polycation and ISS-NA or DNA, whereby it is thought that the cationic lipid forms an additional protective layer surrounding the complex of ISS-NA or DNA with the polycation (Pelisek J. et al. J Gene Med 2006; 8:186-197).
  • microparticle refers to synthetic particles of controlled dimension in the order of micrometers (i.e. >1 ⁇ m, and ⁇ 1000 ⁇ m). Preferred microparticles have a size of 1 to 10 ⁇ m, preferably 1 to 5 ⁇ m, more preferably 1 to 2 ⁇ m.
  • nanoparticle refers to synthetic particles of controlled dimension in the order of nanometers, wherein preferably ISS-NA can be entrapped, encapsulated, non-covalently bound, covalently attached or dissolved in said nanoparticles.
  • the size of a nanoparticle is preferably less than 1000, 900, 800, 700, 600, 500, 400, 300, 200, 100 or 50 nm, wherein further preferably said nanoparticle is not smaller than about 10, 15, 20, 30, 40, or 50 nm.
  • the nanoparticle is not smaller than 50 nm.
  • nanoparticles of the invention are preferably 10 to 500 nm, more preferably 20 to 400 nm, still more preferably 40 to 300 nm, still more preferably 50 to 200 nm and most preferably 50 to 100 nm in size.
  • Preferred in the context of the invention are nanoparticles of 100 to 300 nm in size, more preferred are nanoparticles of 100 to 200 nm in size.
  • Very preferred are nanoparticles of 50 to 200 nm in size.
  • nanoparticle encompasses particles of spherical, elliptic, longitudinal or irregular structure, preferably comprising or consisting of at least one a polymer. Nanoparticle also encompasses nanocapsules, and poliplex particles.
  • Nanocapsules are nanoparticles comprising a reservoir, e.g. oily reservoir, wherein said reservoir is surrounded by a polymer wall (Maria J. Alonso, in Microparticulate systems for the delivery of proteins and vaccines, Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p 206).
  • a nanoparticle of a certain material e.g. a polyester nanoparticle, refers to a nanoparticle comprising or preferably essentially consisting, most preferably consisting of said material. In the context of the invention this formulation does not exclude the presence of the ISS-NA in the nanoparticle.
  • Polymers suitable for the production of nanoparticles, including nanocapsules refer to: biodegradable polyesters (e.g. poly-lactic acid, poly-(lactic glycolic acid) copolymer (referred to as PLA and PLGA), poly-e-caprolactone (referred to as PECL)), poly-(alkylcyanoacrylates) (referred to as PACA), chitosan, alginate, cross-linked human serum albumin, human serum albumin, gelatin, Schizofyllan, dextran. Nanoparticles may also be prepared from non-biodegradable materials such as polystyrene, colloidal gold, silica, or metal clusters. In addition, hydrophilic components such as e.g.
  • PEG polysaccharides
  • PEG polysaccharides
  • dextran chitosan
  • polylysine lecithine
  • Complexation agent such as polylysine (PLL), poly(ethylene imine) (PEI), protamine, spermine or positively charged structured oligopeptides may be incorporated into nanoparticles together with nucleic acids, bound to the surface of nanoparticles for incorporation of nucleic acids, covalently attached to reactive groups on the nanoparticle, or incorporated during the polymerization process into the nanoparticle.
  • Alternative complexation agents include metal salts such as Zinc acetate.
  • Polyplex particles are nanoparticles formed by the direct interaction of a cationic polymer, also called polycation, such polylysine (PLL), poly(ethylene imine) (PEI) or the like with a nucleic acid, preferably an ISS-NA.
  • a Polyplex may also be formed by the direct interaction of PEG-PLL, or, for example, of a branched PEI with said nucleic acid, preferably said ISS-NA.
  • Complexation agent a complexation agent is an agent which non-covalently binds to an ISS-Na and neutralizes or at least partially reduces the effective charge of the resulting complex.
  • complexation agents are polylysine (PLL), spermine, protamine, poyethyleneimine (PEI), branched PEI, lysine-rich structured oligopeptides, cationic lipids such as DOTAP and the like, and metal cations as provided by their salts such as zinc acetate, magnesium, calcium chloride, or the like.
  • More complex polymers, such as poly(ethylene)glycol-polylysine (PEG-PLL) copolymers may also be used as complexation agent.
  • biodegradable A material, preferably a particle, most preferably a microparticle or nanoparticle, is referred to as biodegradable when it is degradable or erodable under normal mammalian physiological conditions. Degradation of the particle may occur, for example, by dissolving of the particle, by enzymatic degradation, preferably by hydrolysis or oxidation, or by destabilisation of the particle by any other chemical or physical process. Normal mammalian physiological conditions can be recreated in the test-tube by incubating samples in serum at 37° C. Microparticles or nanoparticles are considered biodegradable, if they are degraded upon incubation for 72 hours at 37° C. in human serum from healthy volunteers.
  • “degraded” means that the microparticle or nanoparticle loses at least 5%, preferably at least 10%, more preferably at least 20%, still more preferably at least 50% of its mass and/or average polymer length. Most preferably, the particle is completely degraded under these conditions.
  • a microparticle or nanoparticle is referred to as non-biodegradable, if it does not loses at least 5% of its mass and/or average polymer length upon incubation in human serum for 72 hours at 37° C.
  • Very preferred are particles which are biodegradable at low pH, preferably at a pH which is found in the endosomes of immune cells, more preferably at ph of 4.5 to 6, most preferably at pH of about 5.
  • coat protein refers to the protein(s) of a bacteriophage or a RNA bacteriophage capable of being incorporated within the capsid assembly of the bacteriophage or the RNA bacteriophage.
  • the term “CP” is used.
  • the specific gene product of the coat protein gene of RNA bacteriophages Q ⁇ is referred to as “Q ⁇ CP”
  • the “coat proteins” of bacteriophage Q ⁇ comprise the “Q ⁇ CP” as well as the A1 protein.
  • the capsid of Bacteriophage Q ⁇ is composed mainly of the Q ⁇ CP, with a minor content of the A1 protein.
  • the VLP Q ⁇ coat protein contains mainly Q ⁇ CP, with a minor content of A1 protein.
  • recombinant VLP refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology.
  • VLP recombinantly produced refers to a VLP that is obtained by a process which comprises at least one step of recombinant DNA technology.
  • the terms “recombinant VLP” and “VLP recombinantly produced” are interchangeably used herein and should have the identical meaning.
  • virus particle The term “virus particle” as used herein 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.).
  • virus-like particle refers to a non-replicative or non-infectious, preferably a non-replicative and non-infectious virus particle, or refers to a non-replicative or non-infectious, preferably a non-replicative and non-infectious structure resembling a virus particle, preferably a capsid of a virus.
  • non-replicative refers to being incapable of replicating the genome comprised by the VLP.
  • non-infectious refers to being incapable of entering the host cell.
  • a virus-like particle in accordance with the invention is non-replicative and/or non-infectious since it lacks all or part of the viral genome or genome function.
  • a virus-like particle is a virus particle, in which the viral genome has been physically or chemically inactivated, removed by disassembly and reassembly, or by assembly of purified proteins into a VLP.
  • a virus-like particle lacks all or part of 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, preferably RNA bacteriophage.
  • viral capsid refers to a macromolecular assembly composed of viral protein subunits. Typically, there are 60, 120, 180, 240, 300, 360 and more than 360 viral protein subunits. Typically and preferably, the interactions of these subunits lead to the formation of viral capsid or viral-capsid like structure with an inherent repetitive organization, wherein said structure is, typically, spherical or tubular.
  • capsids of RNA bacteriophages or HBcAgs have a spherical form of icosahedral symmetry.
  • capsid-like structure refers to a macromolecular assembly composed of viral protein subunits resembling the capsid morphology in the above defined sense but deviating from the typical symmetrical assembly while maintaining a sufficient degree of order and repetitiveness.
  • the invention encompasses VLPs, preferably non-natural VLPs, comprising a icosahedral symmetry.
  • One common feature of virus particle and virus-like particle is its highly ordered and repetitive arrangement of its subunits.
  • virus-like particle of a RNA bacteriophage refers to a virus-like particle comprising, or preferably consisting essentially of or consisting of coat proteins, mutants or fragments thereof, of a RNA bacteriophage.
  • virus-like particle of a RNA bacteriophage resembling the structure of a RNA bacteriophage, being non replicative and/or non-infectious, and lacking at least the gene or genes encoding for the replication machinery of the RNA bacteriophage, and typically also lacking the gene or genes encoding the protein or proteins responsible for viral attachment to or entry into the host.
  • RNA bacteriophages in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non-replicative and/or non-infectious virus-like particles of a RNA bacteriophage.
  • Preferred VLPs derived from RNA bacteriophages exhibit icosahedral symmetry and consist of 180 subunits.
  • subunit and “monomer” are interexchangeably and equivalently used within this context.
  • Preferred methods to render a virus-like particle of a RNA bacteriophage non replicative and/or non-infectious is by physical, chemical inactivation, such as UV irradiation, formaldehyde treatment, typically and preferably by genetic manipulation.
  • individual proteins may be isolated from whole virions and assembled into VLPs in vitro.
  • Synthetic VLP Particles formed from non-naturally occurring proteins or peptides which spontaneously assemble intracellularly or in vitro are referred to as synthetic VLPs. Examples for synthetic VLPs are provided in WO04071493A1 which is incorporated herein by reference. Synthetic VLPs also encompasses particles which require nucleic acids or metal ions for assembly. Preferred synthetic particles have a defined size of 10 to 1000 nm, preferably 10 to 300 nm, more preferably 10 to 150 nm, and most preferably 15 to 100 nm. Other preferred synthetic VLPs may exist in two or more defined conformations, e.g. 15 nm and 25 nm.
  • virosome As used herein, the term virosome relates to a reconstituted virus envelope, preferably to a reconstituted influenza virus envelope, more preferably to a reconstituted envelope of influenza A virus, most preferably to a reconstituted envelope of influenza A/Singapore virus. Virosomes are known in the art as drug carrier systems.
  • a virosome comprises a lipid membrane, wherein said lipid membrane typically and preferably comprises a unilamellar lipid bilayer.
  • virosome relates to a reconstituted influenza virus envelope, preferably to a reconstituted influenza A virus envelope, most preferably to a reconstituted influenza A/Singapore virus envelope, wherein said reconstituted influenza virus envelope, preferably said reconstituted influenza A virus envelope, most preferably said reconstituted influenza A/Singapore virus envelope comprises lipid membrane, wherein said lipid membrane comprises influenza glycoproteins, wherein preferably said influenza glycoproteins are selected from hemagglutinin HA and neuraminidase NA. Typically and preferably virosomes attach to target cells via HA.
  • Very preferred virosomes comprise a lipid membrane, preferably a lipid bilayer, wherein said lipid membrane or said lipid bilayer comprise or preferably predominantly consist of cationic lipids.
  • Virosomes comprising cationic lipids in their lipid membrane are particularly suited for the delivery of ISS-NA, especially of oligonucleotides.
  • Further preferred are specific virosomes, which attach to a target cell by an antibody or a fragment thereof which is comprised in the lipid membrane.
  • the term virosome relates to a specific virosomes comprising a lipid membrane, wherein said lipid membrane comprises specific antibodies or fragments thereof, preferably Fab fragments or Fab′ fragments, wherein preferably the specificity of said antibodies or fragments thereof allows to direct the virosome to a specific target cell.
  • a virosome is taken up by the target cell by receptor-mediated endocytosis.
  • an effective amount of the composition preferably the pharmaceutical 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.
  • treatment refers to prophylaxis and/or therapy.
  • atopic disease for example, the term refers to a therapeutic treatment which reduces the symptom score of the treated patient as assessed by a standard method commonly used to assess the severity of the symptoms of the disease the patient is suffering from.
  • the term can also refer to a prophylactic treatment of an atopic disease which, for example, prevents or ameliorates the symptoms a patient typically develops upon exposure to a challenging agent as compared to an untreated patient.
  • compositions of the invention can be combined, optionally, with a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human or other animal.
  • carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application.
  • composition refers to a formulation which contains the composition of the invention and which is in a form that is capable of being administered to an animal.
  • the pharmaceutical composition 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 pharmaceutical composition Upon introduction into a host, the pharmaceutical composition 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.
  • Preferred pharmaceutical compositions induce a cytokine milieu, typically and preferably the formation of IFN-alpha, which is reducing allergic and/or asthmatic symptoms.
  • the pharmaceutical composition additionally includes an adjuvant which can be present in either a minor or major proportion relative to the composition 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 composition of the invention provides for an even more enhanced and/or prolonged immune response, preferably cytokine production.
  • adjuvants A variety of adjuvants is known in the art and useful in the invention.
  • Preferred adjuvants are selected from the group consisting of incomplete Freund's adjuvant, aluminum containing adjuvants, modified muramyldipeptide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, BCG (bacille Calmette Guerin) Corynebacterium parvum , ligands of toll-like receptors (TLR) which include but are not limited to peptidoglycans, lipopolysaccharides and their derivatives, poly I:C, immunostimulatory oligonucleotides, imidazoquinolines such as resiquimod and imiquimod, flagellins, monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, GPI-0100, CRL1005, MF-59, OM-174, OM-197, OM-294, Viroso
  • a very preferred adjuvant for the purpose of the invention is aluminium containing adjuvant, preferably an aluminium containing mineral gel, most preferably alhydrogel. In a very preferred embodiment said adjuvant is alhydrogel.
  • the term adjuvant also encompasses a mixture of any of the substances listed above.
  • Particles of the invention, preferably VLPs have been generally described as an adjuvant.
  • the term “adjuvant”, as used within the context of this application refers to an adjuvant not being the particle of the invention, in particular not the VLP used for the inventive compositions. In each case, the term adjuvant refers to an adjuvant used in addition to said particle.
  • polypeptide As used herein the term “polypeptide” refers to a polymer composed of amino acid residues, generally natural amino acid residues, linked together through peptide bonds. Although a polypeptide may not necessarily be limited in size, the term polypeptide is often used in conjunction with peptide of a size of about ten to about 50 amino acids.
  • protein refers to a polypeptide of a size of above 20, more preferably of above 50 amino acid residues. Proteins generally have a defined three dimensional structure although they do not necessarily need to, and are often referred to as folded, in contrast to peptides and polypeptides which often do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.
  • sequence identity The amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as the Bestfit program. When using Bestfit or any other sequence alignment program, preferably using Bestfit, 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. This aforementioned method in determining the percentage of identity between polypeptides is applicable to all proteins, polypeptides or a fragment thereof disclosed in this invention.
  • Sequence homology The homology of nucleotide sequences is preferably determined by the program blastn which is an implementation of the BLAST algorithm, most preferably using the default settings of the software.
  • fragment of a protein in particular fragment of a recombinant protein or recombinant coat protein, as used herein, is defined as a polypeptide, which is of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% the length of the wild-type recombinant protein, or coat protein, respectively and which preferably retains the capability of forming VLP.
  • the fragment is obtained by at least one internal deletion, at least one truncation or at least one combination thereof.
  • fragment of a recombinant protein or “fragment of a coat protein” shall further encompass polypeptide, which has at least 80%, preferably 90%, even more preferably 95% amino acid sequence identity with the “fragment of a recombinant protein” or “fragment of a coat protein”, respectively, as defined above and which is preferably capable of assembling into a virus-like particle.
  • mutant coat protein refers to a polypeptide having an amino acid sequence derived from the wild type recombinant protein, or coat protein, respectively, wherein the amino acid sequence is at least 80%, preferably at least 85%, 90%, 95%, 97%, or 99% identical to the wild type sequence and preferably retains the ability to assemble into a VLP.
  • animal refers to any animal, preferably to any animal comprising an immune system, including non-vertebrates, preferably arachnids and insects, and vertebrates. Typically and preferably, animal relates to vertebrates, more preferably to mammals, most preferably to humans. Thus, animal includes, for example, humans, sheep, horses, cattle, pigs, dogs, cats, rats, mice, birds, reptiles, fish, insects and arachnids.
  • VLPs packaged with unmethylated CpG-containing oligonucleotides can enhance B and T cell responses. It was also previously observed, that application of a CpG-containing oligonucleotide together with an allergen ameliorated allergic response. It was now surprisingly found that no covalent linkage, coupling or mixing of allergen with the particle of the invention, preferably the VLP, is required to achieve an effect in the treatment of hypersensitivity, preferably allergy. Even more surprisingly, it was found that no administration or co-administration of a specific antigen or allergen is required in the treatment of hypersensitivity, preferably allergy, using the compositions of the invention. Furthermore, said effect is not limited to VLPs packaged with unmethylated CpG-containing oligonucleotides but can be generalized to particles of the invention packaged with immunostimulatory nucleic acids (ISS-NA).
  • ISS-NA immunostimulatory nucleic acids
  • Applicants have discovered that hypersensitivities may be cured by treatment with particles packaged with ISS-NA. Applicants have also shown that particles of appropriate size diffuse readily to draining lymph nodes after intradermal or subcutaneous injection, where they are taken up mainly by dendritic cells (DC) and macrophages.
  • DCs including plasmacytoid dendritic cells (pDC), monocyte derived and leucophil dendritic cells or macrophages are activated upon uptake of particles, preferably VLPs, packaged with unmethylated CpG-containing oligonucleotide via the Toll-9 receptor.
  • polystyrene nanoparticles with sizes of 50 to 200 nm also readily diffuse to the draining lymph node, while larger particles (>500 nm) remain at the site of injection during the same time span after subcutaneous injection.
  • PLGA nanoparticles have been shown to be phagocytosed by dendritic cells (DCs) (Diwan et al. J Drug Target. 2003; 11:495-507).
  • Mast cells are active in phagocytosis, and it is possible that particles of the invention, preferably nanoparticles or VLPs, wherein said nanoparticles or VLPs, preferably said VLPs, are packaged with ISS-NA, preferably with unmethylated CpG-containing oligonucleotide, mediate their effect in part via inhibiting mast cells, providing for another alternative or complementary possible mechanism of action of the VLP or nanoparticle packaged with unmethylated CpG-containing oligonucleotide.
  • the mode of action of the compositions of the invention is not limited to this mechanism.
  • the invention provides compositions, pharmaceutical compositions and methods for the treatment or prevention of hypersensitivity, preferably allergy in an animal.
  • the invention provides a composition for use in a method of treating hypersensitivity in an animal, the composition comprising a particle and an ISS-NA, wherein said particle is packaged with said ISS-NA.
  • the invention provides methods and compositions which are especially useful for treating and/or preventing hypersensitivity, preferably allergy, more preferably atopic eczema, atopic asthma and type I allergies like, for example, pollen allergy (hay fever).
  • the term particle refers to any structure which, with respect to its chemical an physical characteristics, can be packaged with ISS-NA, wherein preferably said particle is capable of protecting said ISS-NA from degradation in the body of said animal and/or wherein said particle is capable of specifically delivering and releasing said ISS-NA to immune cells.
  • the particle of the invention preferably is able to (i) package an ISS-NA and (ii) to deliver said ISS-NA to immune cells in the body.
  • the particle of the invention is therefore to be understood in a very broad sense.
  • said particle is selected from the group consisting of synthetic particle, virus particle and VLP.
  • the particles of the invention can be biodegradable or non biodegradable.
  • Biodegradable particles are generally preferred to prevent accumulation of said particle in the body of said animal and to avoid toxicity effects which might be associated with such accumulation. Therefore, in a preferred embodiment said particle, preferably said synthetic particle, most preferably said nanoparticle, comprises or, preferably, essentially consists of, most preferably consists of a biodegradable material. In a further preferred embodiment said particle, preferably said synthetic particle, most preferably said nanoparticle, is biodegradable. In a very preferred embodiment said synthetic particle packaged with said ISS-NA is biodegradable.
  • said particle, preferably said synthetic particle releases said ISS-NA, preferably inside the target cell, most preferably in the endosome of the target cell.
  • said particle, preferably said synthetic particle is degraded in the endosomal compartment of the target cell containing proteases and exhibiting a low pH, preferably about pH 5.
  • said particle is a synthetic particle, preferably a synthetic particle selected from the group consisting of liposome, microparticle and nanoparticle, wherein more preferably said synthetic particle is liposome or a nanoparticle, and even more preferably a nanoparticle.
  • said particle is a synthetic particle, preferably a synthetic particle selected from the group consisting of liposome, microparticle, nanoparticle and virosome.
  • Synthetic particles of the invention comprise or preferably essentially consist of, most preferably consist of non-biodegradable materials, of biodegradable materials or of a mixture of both, wherein said biodegradable materials may be organic or inorganic, or a combination of both.
  • polyamide polyamide
  • macromolecules and modified macromolecules of biological origins preferably selected from proteins (e.g. gelatin, human serum albumin) and (l) polysaccharides (e.g.
  • said synthetic particle is a microparticle or a nanoparticle, preferably a nanoparticle, wherein said synthetic particle comprise or preferably essentially consist of, most preferably consist of a non-biodegradable material selected from the group consisting of: (a) colloidal gold, (b) polystyrene, (c) polyethylene, (d) polypropylene, (e) latex, (f) ferromagnetic or paramagnetic materials, (g) dextran and (h) hydroxyapatite.
  • a non-biodegradable material selected from the group consisting of: (a) colloidal gold, (b) polystyrene, (c) polyethylene, (d) polypropylene, (e) latex, (f) ferromagnetic or paramagnetic materials, (g) dextran and (h) hydroxyapatite.
  • said synthetic particle comprises or preferably essentially consists of, most preferably consists of a biodegradable material selected from the group consisting of: (a) polyesters, preferably selected from PLA, poly(glycolic acid) and PECL, (b) copolymers of polyesters, preferably PLGA, (c) block copolymers of polyester and PEG, (d) polyorthoesters, (e) poly(anhydrides), (f) poly(sebacic acid), (g) polyanhydrides based on sebacic acid monomers incorporating amino acids, (h) polyanhydride esters, (i) polyphosphazene, preferably polyphosphazene containing hydrolysis-sensitive ester groups (Andrianov A.
  • a biodegradable material selected from the group consisting of: (a) polyesters, preferably selected from PLA, poly(glycolic acid) and PECL, (b) copolymers of polyesters, preferably PLGA, (c) block copolymers of polyester and P
  • said biodegradable material is selected from (a) polyesters, preferably selected from PLA, poly(glycolic acid) and PECL, (b) copolymers of polyesters, preferably PLGA, (c) block copolymers of polyester and PEG, (d) polyorthoesters, (e) poly(anhydrides), (f) poly(sebacic acid), (g) polyanhydrides based on sebacic acid monomers incorporating amino acids, (h) polyanhydride esters, (i) polyphosphazene, preferably polyphosphazene containing hydrolysis-sensitive ester groups (Andrianov A K and Payne L G, in Microparticulate systems for the delivery of proteins and vaccines, Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p. 127-147), and (j) polyamide.
  • polyesters preferably selected from PLA, poly(glycolic acid) and PECL
  • copolymers of polyesters preferably PLGA
  • said biodegradable material is selected from (a) polyesters, preferably polyesters selected from PLA, poly(glycolic acid) and PECL, and (b) copolymers of polyesters, preferably PLGA.
  • said biodegradable material is PLA or PLGA, most preferably PLA.
  • said particles, preferably said synthetic particles, most preferably said nanoparticles comprise or preferably essentially consist of at least one, preferably exactly three, more preferably exactly two, most preferably exactly one biodegradable polymer(s), wherein preferably said polymer is degraded into biocompatible non-toxic monomers, wherein still more preferably said biodegradable polymer is a polyester (see Maria J. Alonso et al., in Microparticulate systems for the delivery of proteins and vaccines, Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p 203-242).
  • Nanoparticles may be formed by chemical or physical processes such as polymerization or condensation of monomers, for example in nanodroplets of an emulsion, precipitation of polymers, coacervation of macromolecules, assembly of macromolecules brought together, for example, by the process of emulsification, by ionic interactions, aggregation, heat denaturation or chemical cross-linking of said assembled macromolecules.
  • Nanoparticles may also have a composite structure involving two or more layers with different properties, such as core-shell particles with a hydrophobic core layer of a polymer and a hydrophilic outer shell layer of another polymer, such as poly(ethylene)glycol (PEG).
  • PEG poly(ethylene)glycol
  • core-shell nanoparticles include nanoparticles where a charged polymer, such as polylysine, is adsorbed to the surface of the nanoparticle.
  • a charged polymer such as polylysine
  • more complex nanoparticles may be produced, where co-monomers carrying a cationic moiety and co-monomers carrying a PEG moiety are included in the polymerization process and preferentially locate to the nanoparticle surface during the polymerization process.
  • said nanoparticle comprises an outer shell layer, wherein preferably said outer shell layer comprises or alternatively essentially consists of PEG.
  • said nanoparticle comprises a core and a shell layer, wherein said core comprises or alternatively essentially consists of polyester and wherein said shell layer comprises or alternatively essentially consists of PEG.
  • said nanoparticle comprises a core and a shell layer, wherein said core comprises or alternatively essentially consists of polyalkylcyanoacrylat and wherein preferably said shell layer comprises or alternatively essentially consists of PEG.
  • said nanoparticle is a polyplex.
  • a polyplex nanoparticle is formed by the direct association of a complexation agent such as PEI, PLL or PEG-PLL with an ISS-NA, forming a so called polyplex (Boeckle S. et al. J Gene Med 2004; 6:1102-1111; Wagner E. et al. Adv Genet. 2005; 53:333-54; Walker G. et al. Mol. Ther. 2005; 11:418-25).
  • the size of nanoparticles can be determined with known methods such as scanning- and transmission-electron microscopy, or photon correlation spectroscopy or dynamic light scattering, as reviewed in Alonso M. J. (in Microparticulate systems for the delivery of proteins and vaccines, Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p 203-242).
  • Surface properties such as surface charge, and in particular the zeta-potential, can be measured by determining the velocities of nanoparticles in an electrical field using methods such as electrophoretic mobility measurement.
  • Laser Doppler anemometry or Electrophoretic light scattering may also be used.
  • the sonic response to an alternating electric field may be used as well.
  • the measurements can be performed with commercially available devices.
  • the zeta potential is informative to predict the aggregation behaviour of a nanoparticle, or its ability to adsorb charged components on its surface, and thus helps optimizing the properties of nanoparticles.
  • said nanoparticle comprises or alternatively essentially consists of polyesters, preferably of aliphatic polyesters.
  • Aliphatic polyesters are degraded by random hydrolytic cleavage, into physiologically occurring metabolites. These materials have been used for resorbable sutures, and a polyester microsphere formulation for parenteral injection of Leuprolide acetate, a Gonadotropin Releasing Hormone analog, is a marketed product.
  • polymers can be used in the preparation of the nanoparticles of the invention and have been described: poly(lactic acid) (PLA), poly(glycolic acid) (PGA), or Copolymers of PLAA and PGA, poly(-lactic-co-glycolic acid) named PLGA, are particularly suited.
  • the racemic, amorphous form is preferred.
  • These polymers are commercially available, and their synthesis is well known in the art. Their synthesis is described, for example, by Avgoustakis K (Curr Drug Deliv. 2004; 1:321-33).
  • the properties of polyester microsphere, and PLGA in particular, as well as methods to produce them have been reviewed and described for example by Kissel T and Koneberg R (in Microparticulate systems for the delivery of proteins and vaccines, Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p. 51-87). These methods include spray drying methods, water-in-oil-in-water emulsion solvent evaporation methods and phase separation methods.
  • said nanoparticle comprises or alternatively essentially consists of PLGA, wherein preferably the molar ratio of the monomers constituting PLGA is 50% mol LA and 50% mol GA.
  • Increasing the proportion of either of the monomer leads to slower degradation of the polymer.
  • a polymer with a ratio of LA to GA of 85:15 has a rate of degradation about two-and a half time slower than with a ratio of 50:50.
  • the properties of PLGA polymer can be manipulated by changing the proportion of the monomers.
  • the solvent extraction-evaporation technique is the solvent extraction-evaporation technique.
  • an organic solvent with poor water solubility such as methylene chloride, ethyl acetate or chloroform is used.
  • the solvent is nonetheless extracted from the organic phase into the water phase due to the large excess of water, a process which is further facilitated by subsequent evaporation of the solvent.
  • the solvent extraction-evaporation technique can be essentially performed in two general ways to incorporate molecules into nanoparticles, depending on the hydrophobicity of the molecule to be incorporated.
  • a hydrophobic molecule, or a water insoluble complex can be dissolved or suspended in the same organic solvent as the polymer, and then be emulsified in an external water phase containing a stabilizer, typically a surfactant such as poly(vinyl alcohol) (PVA), a poloxamer, or for example dextran, whereby the nanodroplets formed during the emulsion process form nanoparticles upon solvent extraction and evaporation.
  • PVA poly(vinyl alcohol)
  • the emulsion is prepared using high-speed or high-pressure homogenizers, microfluidization or sonication. Intensive stirring or vortexing may also be appropriate. This method is also referred to as the oil-in-water solvent extraction and evaporation technique.
  • More hydrophilic or amphiphilic compounds are incorporated as aqueous solutions into a larger volume of an organic solvent containing the polymer.
  • the aqueous solution is emulsified with the organic solvent, for example using high-speed or high-pressure homogenizers, microfluidization, sonication, vortexing or intensive stirring.
  • the resulting water-in-oil emulsion or suspension is further emulsified in a larger volume of water, yielding a water-in-oil-in water emulsion, from which the nanoparticles form upon solvent extraction and evaporation.
  • This method is also referred to as the water-in-oil-in-water emulsion solvent evaporation technique.
  • the process of solvent extraction and evaporation can be further accelerated for example by increasing the temperature, applying vacuum or adding an alcohol such as isopropanol (e.g. 2%) to the external water phase to increase the organic solvent solubility in that external water phase.
  • an alcohol such as isopropanol (e.g. 2%)
  • a rotavapor may be used to accelerate solvent extraction and evaporation.
  • Nanoparticles are then typically isolated by freeze-drying, whereby a cryoprotectant such as a sugar like trehalose or glucose is added to prevent aggregation of the nanoparticles.
  • a cryoprotectant such as a sugar like trehalose or glucose
  • Another polymer precipitation technique is the solvent displacement or nanoprecipitation method. It involves the use of an organic solvent that is completely soluble in the external water phase. The polymer is dissolved in acetone, ethanol or methanol, and precipitates upon incorporation under stirring into an aqueous solution of a surfactant, such as for example Poloxamer 188.
  • a surfactant such as for example Poloxamer 188.
  • a further polymer precipitation method is the salting-out technique.
  • the polymer is dissolved in, for example acetone, and a saturated aqueous solution of PVA is added under stirring to form an oil-in-water emulsion. Water is further added, and the polymer precipitates to form nanoparticles when acetone diffuses into the external water phase.
  • said nanoparticle is a PEG-polyester nanoparticle, such as for example, PEG-PLA or PEG-PLGA nanoparticles.
  • PLA and PLGA particles absorb plasma proteins such as opsonin, and activate the complement system.
  • core-shell nanoparticles have been produced, where a layer of a hydrophilic PEG, the shell, surrounds the polymer core of the particle, and prevents or reduces attachments of plasma proteins or opsonin to the hydrophobic polymer surface.
  • Coating of nanoparticles and in particular of PLGA nanoparticles with poly(ethylene glycol) has been shown to strongly reduce complement activation and increase blood circulation time (Gref R et al.
  • PEG-PLA and PEG-PLGA nanoparticles are similar to the methods used for the preparation of PLA and PLGA nanoparticles, and have been reviewed by Avgoustakis K (Curr Drug Deliv. 2004; 1:321-33) and Gref R. et al. (in Microparticulate systems for the delivery of proteins and vaccines, Eds. S. Cohen and Howard Bernstein, Marcel Dekker, New York 1996, p 279-305). These methods include the emulsion-solvent evaporation method, the solvent displacement method and the salting-out method. Use of the salting-out method for the preparation of nanocapsules is also described therein. Methods for synthesizing block copolymers are know in the art.
  • diblock copolymers e.g. PEG-PLA
  • PEG-PLA poly(ethylene glycol)
  • monomers lactide, glycolide, caprolactone or mixtures of them
  • stannous octoate monomethoxy-PEG catalyzed by stannous octoate
  • Variation of the PLA/PEG and PLGA/PEG allows to manipulate the structure of the nanoparticles produced. For example, a low ratio generates more dynamic type of particles, while higher ratios favour more solid-like structures.
  • a shorter PLA or PLGA chain length (x ⁇ 30) can lead to nanoparticle formation via micelle formation process, while with longer chain length an agglomeration-precipitation process is taking place Avgoustakis K (Curr Drug Deliv. 2004; 1:321-33).
  • the nanoparticle is a polyalkylcyanoacrylate nanoparticle.
  • Polyalkylcyanoacrylate nanoparticles may be prepared as described by Fattal E. et al. (J. Contr. Release 1998; 53:137-143), using an emulsion polymerization process.
  • the nanoparticles are then coated with a cationic hydrophobic reagent, such as cetyltrimethylammonium bromide (CTAB), and the ISS-NA is adsorbed on the nanoparticle.
  • CTAB cetyltrimethylammonium bromide
  • ISS-NA is adsorbed on the nanoparticle.
  • Zobel et al. Antisense Nucleic Acid Drug Dev.
  • nanoparticles are produced in an aqueous dispersion polymerization process.
  • the surfactant used is DEAE-dextran, which allows subsequent adsorption of ISS-NA on the surface of the nanoparticle through electrostatic interactions. Particles of sizes ranging from about 170-1000 nm were obtained by Zobel et al.
  • the nanoparticle is a PEG-coated polycyanoacrylate nanoparticle, such as described by Li Y. et al. (Int J Pharm. 2003; 259:93-101), whereby the additional covalent attachment of transferrin to the nanoparticles is omitted.
  • the nanoparticles are prepared by a water-in-oil-in-water emulsion solvent evaporation technique, using poly(aminopoly(ethylene glycol)-cyanoacrylate-co-hexadecyl cyanoacrylate) (poly(H 2 NPEGCA-co-HDCA)) as polymer. Briefly, the ISS-NA diluted in buffer (e.g.
  • NaHCO 3 , pH 8 is emulsified in dichloromethane/ethyl acetate (1:1) containing poly(H 2 NPEGCA-co-HDCA) by sonication.
  • the resulting emulsion is poured into a 1% w/v PVA aqueous solution, and further emulsified.
  • the percentage of PVA may be varied, and in particular a higher percentage (3% in 0.1 M NaHCO 3 , pH 8) has been shown to limit damage to dsDNA (Li Y. et al. (Int J Pharm. 2003; 259:93-101).
  • the resulting double emulsion is then diluted in a 0.3% w/v PVA aqueous solution, under magnetic stirring.
  • the organic solvents are then eliminated by evaporation under reduced pressure in a Rotavapor at 37° C., and the nanoparticles collected by centrifugation at 39000 g.
  • Particle sizes obtained by Li et al. ranged from about 130 to 150 nm.
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide.
  • Packaging of ISS-NA, and in particular unmethylated CpG-containing oligonucleotide in a particle produced by an emulsion-solvent evaporation technique or spray-dry technique will be exemplified in the following for polyester nanoparticles. However, it is apparent for the artisan that similar procedures can be applied for PACA and PEG-PACA nanoparticles.
  • Packaging of ISS-NA, such as for example unmethylated CpG-containing oligonucleotide, within polyester nanoparticles such as PLA, PLGA, PECL, PEG-PLA, PEG-PLGA or PEG-PECL nanoparticles can be performed in several ways.
  • ISS-NA is added to the initial water phase of the water-in-oil-in-water emulsion solvent evaporation technique, as has been reported by Aukunuru J. V. et al. (J. Pharm. Pharmacol. 2003; 55:1199-206).
  • the ISS-NA is suspended in the organic solvent, e.g. dichloromethane, by sonication, and then spray dried to yield nanoparticles having packaged the ISS-NA.
  • the capacity of a nanoparticle to package an ISS-NA, and in particular an ODN is increased by adding a complexation agent, such as lysine rich oligopeptide (Emile C. et al.
  • a solution of the polymer, a complexation agent such as a lysine rich peptide, and of a ISS-NA such as an ODN, in acetone is poured dropwise in an aqueous solution as reported by Emile C. et al. (Drug Deliv 1996; 3:187-195).
  • the complex of the ISS-NA and the complexation agent precipitates with the polymer to form nanoparticles packaged with the ISS-NA.
  • the complex of the ISS-NA and the complexation agent is suspended in an organic solvent, e.g. methylene chloride, and incorporated and packaged in a PLGA nanoparticle using a spray-dry technique (Putney S D et al. Antisense Nucleic Acid Drug Dev. 1999; 9:451-8; Pamujula S et al. J Pharm Pharmacol 2004; 56:1119-25).
  • said nanoparticle is a Block copolymer-coated calcium phosphate nanoparticle (CaP-PEG-PAA; Kakizawa et al. (2004) J. Contr. Release 97:345-56).
  • CaP-PEG-PAA Block copolymer-coated calcium phosphate nanoparticle
  • These are core-shell particles suitable for packaging ISS-NA, preferably unmethylated CpG-containing oligonucleotides. They comprise a core of nanocrystals of CaP, surrounded by a hydrophilic tethered layer of PEG. Mixing of a calcium-, and a phosphate-containing solution in the presence of ISS-NA and PEG-block-poly(aspartic acid) (PEG-PAA) leads to the spontaneous formation of nanoparticles incorporating ISS-NA.
  • PEG-PAA PEG-block-poly(aspartic acid)
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide.
  • the PAA-segment of PEG-PAA has high binding affinity for CaP, and the non-ionic and hydrophilic PEG has a steric stabilization function.
  • Particle size can be adjusted by varying the PEG-PAA and phosphate concentration, as seen in FIG. 1 of Kakizawa et al. (2004) J. Contr. Release 97:345-56. Typical sizes obtained are between about 100 to about 300 nm.
  • PEG-PAA A critical minimal amount of PEG-PAA is required to prevent aggregation of the nanoparticles, while it higher levels, it starts to compete with ISS-NA for CaP binding, and optimal concentration and ratios of the components have to be determined empirically as taught by Kakizawa et al. Increasing phosphate concentration from 1.5 mM to 3 mM, for example, led to a higher ODN binding capacity (Kakizawa et al. (2004) J. Contr. Release 97:345-56).
  • ISS-NA of the present invention envisages the use of Ca-P-PEG-PAA nanoparticles for delivery of siRNA to the cytoplasm, where they expect fast dissolution of the CaP core in the cytoplasm due to the lower Ca 2+ concentration and higher phosphate concentration.
  • the ISS-NA of the present invention to be packaged into nanoparticles do not require delivery to the cytoplasm.
  • ODN release can take place in low-pH compartments such as endosomes.
  • said nanoparticle is an alginate-PLL nanoparticle.
  • Sodium alginate is a natural polysaccharide with mannuronic and guluronic acid as its constituents.
  • Aynié et al. (Antisense Nucleic Acid Drug Dev. 1999; 9:301-12) have described alginate nanoparticles cross-linked by PLL to which ODN are bound.
  • the preparation of the particles is a two-step process, where an initial alginate pre-gel is produced by the addition of calcium chloride under magnetic stirring, and second PLL is added to form a polyelectrolyte complex with the remaining free charges of the pre-gel.
  • ISS-NA is added either simultaneously with PLL to the pre-gel, or after nanoparticle formation.
  • said nanoparticle comprises or alternatively essentially consists of Polylysine polymers of MW 3900 and 7900, wherein preferably said ISS-NA is an unmethylated CpG-containing oligonucleotide.
  • Aynié et al. report particle sizes of 200-600 nm, and ODN loading of approximately 10%. The loading capacity of the alginate nanoparticles however did not reach saturation under the conditions described by Aynié et al., while the encapsulation efficiency was 100%.
  • Alginate-PLL nanoparticles have thus a high ODN loading capacity and are used in embodiments of the invention to package ODN or ISS-NA.
  • the nanoparticle is a chitosan nanoparticle.
  • Chitosan is a positively charged polysaccharide polymer prepared from chitin by deacetylation. The preparation of chitosan nanoparticles has been described for example by Roy K. et al. (Nat Med. 1999; 5:387-91) or Mao H Q et al. (J Control Release. 2001; 70:399-421), who obtained nanoparticles of 150-300 nm in size. These authors used a coacervation process, whereby a Chitosan solution in sodium acetate, is mixed to a solution of ISS-NA in sodium sulphate under vortexing at 55° C.
  • chitosan nanoparticles are further reacted with an N-hydroxy-succinimide derivative of PEG (Leong K W. et al. J Control Release. 1998; 53:183-193).
  • the chitosan nanoparticles are further concomitantly reacted with the homobifunctional cross-linking agent DSS (a bis-N-hydroxysuccinimide compound) (Leong K W. et al. J Control Release. 1998; 53:183-193).
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide.
  • said nanoparticle is a cationized-gelatin nanoparticle, wherein preferably said ISS-NA is an unmethylated CpG-containing oligonucleotide, preferably G10 (SEQ ID NO:27).
  • ISS-NA is an unmethylated CpG-containing oligonucleotide, preferably G10 (SEQ ID NO:27).
  • the preparation of cationized-gelatin nanoparticle has been described by Zwiorek K. et al. (J Pharm Pharmaceut Sci 2004; 7:22-28) or Zillies J and Coester C (J Pharm Pharmaceut Sci 2004; 7:17-21), who report nanoparticles with sizes of 180-280 nm.
  • a two-step desolvation technique is used to generate nanoparticles, which after resuspension in a buffer at pH 4.5, are further derivatized with cholamine in the presence of EDC.
  • ISS-NA is subsequently packaged in the nanoparticles by resuspending the nanoparticles in a solution of ISS-NA and further incubating the resulting mixture.
  • Zwiorek et al. obtained particles of sizes ranging from 180-290 nm. Further guidance in preparing gelatin nanoparticles is provided by Azarmi S. et al. (J. Pharm. Pharmaceut. Sci. 2006; 9:124-132), who uses glutaraldehyde instead of EDC for cross-linking the nanoparticles.
  • said nanoparticle is a gelatin nanosphere, wherein preferably said ISS-NA is an unmethylated CpG-containing oligonucleotide.
  • Gelatine nanospheres are prepared as described by Truong-Le V L et al. Hum Gene Ther. 1998; 9:1709-1717), by a coacervation technique with Na 2 SO 4 at low pH, and whereby the particles are cross-linked with EDC but omitting transferrin in the reaction mixture. Particle of sizes 300-600 nm were obtained by Truong-Le et al.
  • said nanoparticle is a gelatine-cholamin nanoparticle.
  • said nanoparticle is an albumin nanoparticle, wherein preferably said ISS-NA is an unmethylated CpG-containing oligonucleotide.
  • Preparation of albumin nanoparticles has been described, for example, by Irache J M et al. (Mini Rev Med Chem. 2005; 5:293-305), who report nanoparticles with a size of 250-300 nm.
  • the albumin nanoparticles are made from human serum albumin.
  • Albumin nanoparticles are prepared by a coacervation or desolvation process, and subsequently stabilized by cross-linking with glutaraldehyde.
  • ISS-NA is incubated with an albumin aqueous solution (2% w/v), and the mixture is desolvated with ethanol, which induces formation of nanoparticles. These are then cross-linked with glutaraldehyde.
  • the albumin nanoparticle is a protamine-albumin-ISS-NA nanoparticle (Vogel V. et al. J control release. 2005; 103: 99-11).
  • the albumin nanoparticle is a protamine-albumin-ISS-NA nanoparticle (Weyermann et al. J control release. 2004; 100: 411-423; Vogel V. et al. J control release. 2005; 103: 99-11; Mayer G. et al. J control release 2005; 106:181-187).
  • Nanoparticles useful in the practice of the invention also include the nanoparticles described by Kumar MNVR (J. Pharm. Pharmaceut. Sci. 2000; 3:234-258).
  • said nanoparticle is a methacrylate-based hydrogel nanoparticle.
  • Preparation of such nanoparticle as well as the adsorption of ISS-NA has been described by Jain S. et al. (Biomacromolecules. 2005; 6:2590-600), who obtained particles of about 500 nm in size.
  • These nanoparticles are prepared by a two-phase miniemulsion polymerization process. The emulsion is formed in a near saturated salt solution of pluronic F-68 as described by Jain S. et al, except that no ovalbumin is included in the process.
  • PEG-methacrylate, methacrylic acid and PEG-dimethacrylate are added to the pluronic salt solution under stirring.
  • the resulting solution is heated to 40° C., causing phase separation and emulsion, and polymerization is initiated by addition of ammonium persulfate and sodium meta bisulfite.
  • the nanoparticles are isolated by centrifugation, and poly(L-arginine) followed by ISS-NA are adsorbed onto the hydrophobic nanoparticles.
  • said nanoparticle is a methyl methacrylate-based cationic nanoparticle.
  • Preparation of such nanoparticles has been described by Tondelli L. et al. (J Biomater Sci Polymer Edn 2003; 14:1209-1227), who obtained nanoparticles of 500-1000 nm.
  • Methylmethacrylate is mixed with a PEG-derivatized methyl methacrylate and a methacryl methacrylate derivative containing a quaternary ammonium ion in an emulsion polymerization reaction initiated with potassium persulfate.
  • ISS-NA is then incubated with the cationic nanoparticle.
  • said nanoparticle is a PLL-modified silica nanoparticle.
  • Preparation of the nanoparticles is described by Zhu S G. et al. (Biotechnol Appl Biochem 2004; 39:179-187), who obtained nanoparticles of 20 nm in size.
  • the nanoparticles are prepared using a water-in-oil microemulsion where hydrolysis and condensation reactions of tetraethoxysilane is initiated with ammoniumhydroxide.
  • Silica nanoparticles are activated in carbonate buffer before addition of PLL, and ISS-NA is further incubated with washed PLL-silica nanoparticles.
  • Nanoparticles suitable in the practice of the invention can be tested for activation of bone marrow derived dendritic cells (BMDCs) as described in Example 11.
  • BMDCs bone marrow derived dendritic cells
  • activity of the nanoparticle in this assay may be identified by detecting interleukin-6 (IL-6) or IFN-alpha in supernatants, or by quantifying CD-86 upregulation on BMDCs upon nanoparticle incubation by fluorescent associated cell-sorting.
  • IL-6 interleukin-6
  • IFN-alpha interleukin-12
  • these assays are reproducible when using an identical batch of BMDCs, which can be frozen. Comparison between active agents should therefore be performed with the same batch of BMDCs.
  • nanoparticles with unmethylated CpG-containing oligonucleotide induce activation of BMDCs in the cellular assay described in Example 11 in a similar way to Q ⁇ packaged with G10 oligonucleotide and reach the lymph node upon subcutaneous injection in mice footpad with a kinetic similar to Q ⁇ -VLP or a polystyrene bead of 20-500 nm size (see Examples 7 and 9).
  • Activation of BMDCs in a way similar to Q ⁇ packaged with G10 oligonucleotide is meant to express that an identical dose of G10 oligonucleotide package in nanoparticles give a half-maximal amount of IL-12 secretion within 80%, preferably 60%, more preferably 50%, 40% and 30% of the amount induced by the same dose of G10 oligonucleotide packaged in Q ⁇ .
  • the nanoparticles are protective in the animal models of allergy described in the Examples.
  • the nanoparticle of the invention is packaged with ISS-NA, preferably with unmethylated CpG-containing oligonucleotide, most preferably with Q ⁇ G10, amounting to 0.5 to 80% (w/w) of said particle, preferably 0.5 to 40% (w/w), more preferably 2 to 40% (w/w), still more preferably 6 to 40% (w/w), even more preferably 10 to 40% (w/w), even more preferably 15 to 40% (w/w), most preferably 18 to 30% (w/w).
  • microparticles described therein include polyester microparticles, pegylated-polyester microparticles, polyphosphazene microparticles, lipospheres and gelatin microparticles. Additional microparticles include alginate microparticles (Aggarwal N. et al. Can J Vet Res. 1999; 63:148-52), chitosan microparticles (Aral C and Akbuga J. J Pharm Pharm Sci. 2003; 6:321-6; Xu W. Et al. Vaccine.
  • Processes used for the preparation of nanoparticles where resulting particle size is determined by the size of the droplet within the emulsion used to prepare the nanoparticle can be readily adapted for the preparation of microparticles, as is well-known in the art, whereby the mixing, vortexing, high-speed homogenization steps are modified such that microparticles are produced.
  • the spray-drying method may also be adapted such that microparticles are generated.
  • Microparticles useful in the practice of the invention also include the microparticles described in Kumar MNVR (J Pharm Pharmaceut Sci 2000; 3:234-258).
  • Processes for packaging of ISS-NA into nanoparticles described above can be adapted for packaging ISS-NA into microparticles, whereby the homogenization or mixing step is modified as would be known to those skilled in the art, such that the emulsion produced result in microparticle generation.
  • the spray-drying method may also be adapted such that microparticles are generated.
  • polyester microsphere, and PLGA microsphere in particular, as well as methods to produce them have been reviewed and described for example by Kissel T and Koneberg R (in Microparticulate systems for the delivery of proteins and vaccines, Eds. S. Cohen and H. Bernstein, Marcel Dekker, New York 1996, p. 51-87). These methods include spray drying methods, water-in-oil-in-water emulsion solvent evaporation methods and phase separation methods.
  • a favored molar ratio of the monomers constituting PLGA is 50% mol LA and 50% mol GA. Increasing the proportion of either of the monomer leads to slower degradation of the polymer.
  • a polymer with a ratio of LA to GA of 85:15 has a rate of degradation about two-and a half time slower than with a ratio of 50:50.
  • the properties of PLGA polymer can be manipulated by changing the proportion of the monomers.
  • said synthetic particle is a liposome, wherein said liposome is a lipid vesicles consisting of a lipid bilayer.
  • Liposomes can be packaged with ISS-NA using methods known in the art.
  • the liposome of the invention may be selected from the group consisting of neutral liposome, anionic liposome, cationic liposome, stealth, or cationic stealth.
  • the liposome is a cationic liposome.
  • the liposome may have a diameter between 100 and 800 nm, preferably between 100 and 400 nm, more preferably between 100 and 300 nm, even more preferably between 100 and 200 nm, most preferably 200 nm.
  • the liposome exhibits positive charges in order to facilitate interaction of liposomes with target cells.
  • the liposome comprises a cationic lipid, a colipid, and a stabilizing additive.
  • the liposome comprises dimethylaminoethane-carbamol-cho Listerol, and/or dioleoylphosphatidylethanolamine, and/or polyethylene glycol derivatized phosphatidylethanolamine.
  • the liposome comprises phosphatidylcholine, and/or cholesterol, and/or DL- ⁇ -tocopherol, preferably phosphatidylcholine, cholesterol, and DL- ⁇ -tocopherol.
  • ISS-NA may be mixed with preformed vesicles comprising or preferably essentially consisting of, most preferably consisting of cationic lipids, may be mixed directly with cationic lipid, resulting in lipoplexes, or in a preferred embodiment, encapsulated within the aqueous space enclosed by a lipid bilayer.
  • said liposome is a lipopolyplex (Pelisek J. et al. J Gene Med 2006; 8:186-197).
  • the liposome is a microencapsulated liposome, in an alginate-PLL coat, as described by Cohen S. et al. Proc. Natl. Acad. Sci. USA 1991; 88:10440-10444).
  • the ISS-NA preferably an unmethylated CpG-containing oligonucleotide, is packaged within a “stabilized antisense-lipid particle” containing preferably PEG-ceramide-C14, as described by Semple S. C. et al. Methods Enzymol. 2000; 313:322-41.
  • the antisense oligonucleotide is replaced by an ISS-NA, and in particular an unmethylated CpG-containing oligonucleotide.
  • ISS-NA ISS-NA
  • unmethylated CpG-containing oligonucleotide bound to the outer surface of liposomes during the liposome preparation at low pH can be subsequently dissociated and eliminated by anion exchange chromatography once the preparation has been brought back to neutral pH.
  • Suitable further liposomes, methods of preparation as well as methods for ISS-NA and in particular oligonucleotides, can be found in Semple S. C.
  • Methods for preparing liposomes include the dry lipid hydration method, the reverse phase hydration method, the detergent dialysis method, the minimal volume entrapment method.
  • packaging of the ISS-NA in particular an unmethylated CpG-containing oligonucleotide is facilitated by using an ISS-NA or unmethylated CpG-containing oligonucleotide substituted by a residue selected from the group consisting of C6-C30 alkyl chain, bile acids, cholic acid, taurocholic acid, desoxycholate, cholesterol, oleyl litocholic acid, oleoyl cholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids, steroids, vitamins, vitamin E, saturated fatty acids, unsaturated fatty acids, fatty acid esters, triglycerides.
  • the ISS-NA in liposomes are used to induce systemically increased levels of IFN-alpha.
  • IFN-alpha are known to be therapeutically active in hypersensitivity, preferably allergy.
  • said synthetic particle is a virosome, wherein preferably said visosome is a reconstituted virus envelope of a influenza virus, wherein further preferably said influenza virus is a influenza A virus, wherein still further preferably said influenza A virus is influenza A/Singapore virus.
  • Virosomes comprising cationic (positively charged) lipids are especially suited to deliver nucleic acids to a target cell.
  • said synthetic particle is a virosome, wherein said virosome comprises a lipid membrane, wherein said lipid membrane comprises or preferably essentially consists of cationic lipids.
  • said synthetic particle is a virosome and said ISS-NA is a unmethylated CpG-containing oligonucleotide, wherein preferably said unmethylated CpG containing oligonucleotide is G10 (SEQ ID NO:27), and wherein further preferably said virosome comprises a lipid membrane, wherein said lipid membrane comprises or preferably essentially consists of cationic lipids.
  • said virosome comprises a lipid membrane, wherein said lipid membrane comprises antibodies or fragments thereof, wherein preferably said antibodies specifically interact with a receptor of a target cell.
  • Synthetic particles of the invention preferably microparticles and nanoparticles, most preferably nanoparticles may be injected subcutaneously, intravenously, intradermally, intraperitoneally, administered intranasally, orally, transdermally or inhaled.
  • said particle is a virus particle or a virus-like particle (VLP), preferably a VLP.
  • VLP virus-like particle
  • Any virus known in the art may be selected as a VLP or a virus particle of the invention. Most commonly known viruses have been sequenced and are readily available to the public. The taxonomy of viruses is well known to the artisan and summarized, for example, in H. V. Van Regenmortel et al.
  • Virus Taxonomy 7 th Report of the International Committee on Taxonomy of Viruses (2000) (Academic Press/elsevier, Burlington Mass., USA), on the Virus Taxonomy web-page of the University of Leicester (UK) (http://www-micro.msb.le.ac.uk/3035/Virusgroups.html) and by the Taxonomy Browser of the National Center for Biotechnology Information (NCBI, Washington D.C., USA) (http://www.ncbi.nlm.nih.gov/ICTVdb/).
  • viral coat proteins can be identified by a skilled artisan and their nucleotide and amino acid sequences may, for example, be obtained from Genbank (http://www.ncbi.nlm.nih.gov/). Viruses which are particularly useful in the context of the invention are generally disclosed in “Artificial DNA—Methods and Applications”, Yury Khudyakov and Howard Fields, eds., CCR Press, 2003.
  • virus particles or VLPs can be produced and purified from virus-infected cell cultures.
  • said virus particles or VLPs are be preferably non-replicative or non-infectious, more preferably non-replicative and non-infectious.
  • UV irradiation, chemical treatment, such as with formaldehyde, ⁇ -propione or chloroform, are the general methods known to skilled person to inactivate a virus.
  • said non-replicative and non-infectious virus particle or said non-replicative and non-infectious VLP can be produced by purification and reassembly of core proteins of said virus.
  • said virus particle or VLP is a virus particle or VLP, preferably VLP, of a virus, wherein said virus may be a DNA virus, including DNA reverse transcribing viruses, or a RNA virus.
  • said virus is a DNA virus, wherein said DNA virus is a single stranded DNA virus, wherein said single stranded DNA virus is preferably selected from the group consisting of: (a) Parvovirus, preferably parvovirus B19, porcine parvovirus (PPV) or canine parvovirus (CPV), (b) Erythrovirus, (c) Dependovirus, (d) recombinant of CPV with feline panleucopenia virus (FPV) (Saliki. T.
  • said DNA virus is a double stranded DNA virus, including double stranded DNA reverse transcribing viruses, wherein said double stranded DNA virus is preferably selected from the group consisting of: (a) nucleopolyhedrovirus, preferably Autographa californica nucleopolyhedrovirus (AcMNPV) or a chimera of AcMNPV polyhedrin and Trichoplusioa ni granulosis virus (TnGV) (Eason J. E. et al.
  • AcMNPV Autographa californica nucleopolyhedrovirus
  • TnGV Trichoplusioa ni granulosis virus
  • papillomavirus preferably selected from (i) human papilloma virus (HPV, most preferably HPV6, HPV11, HPV16, HPV18, or HPV33), (ii) bovine papillomavirus (BPV, preferably BPV1), and (iii) cottontail rabbit papillomavirus (CRPV), (c) polyomavirus, preferably selected from (i) murine polyomavirus (preferably Py or SV40), (ii) budgerigar fledgling virus, (iii) human polyomavirus JC, (iv) hamster polyomavirus (HaPV), (v) monkey B-lympotropic papovirus (LPV), (vi) avian polyomavirus (APV) and (vii) recombinant human and non-human polyomaviruses (Sasnauskas K.
  • HPV human papilloma virus
  • BPV bovine
  • said virus is a RNA virus, wherein said RNA virus may be a single stranded RNA virus or a double stranded RNA virus.
  • said RNA virus is a single stranded RNA virus, wherein preferably said single stranded RNA virus is a single stranded positive sense RNA virus, wherein preferably said single stranded positive sense RNA virus is selected from: (a) bromoviridae, preferably selected from (i) alfamovirus (e.g. alfalfa mosaic virus (AlMV)), and (ii) ilarvirus (e.g. prunus necrotic ringspot ilarvirus (PNRSV, Pallas V.
  • alfamovirus e.g. alfalfa mosaic virus (AlMV)
  • ilarvirus e.g. prunus necrotic ringspot ilarvirus (PNRSV, Pallas V.
  • calicivirus preferably selected from (i) norwalk virus (NV), (ii) norwalk-like calcivirus, (iii) human calcivirus, (iv) Lorsdale calcivirus, (v) rabbit hemorrhagic disease virus (RHDV), (vi) European brown hare syndrom virus (EBHSV), (vii) Toronto virus, (viii) Hawaii virus, (ix) Sapporo-like virus, and (x) Grimsby feline calcivirus, (h) RNA bacteriophage, (i) luteovirus, preferably potato leaf roll virus (PLRV), (j) flock house virus, (k) retroid viruses, preferably selected from (i) oncoretrovirus, (ii) lentivirus, and (iii) yeast retrotransposon Ty1, (l) tick-borne encephalitis virus (TBEV, Leibl H. (1998) Vaccine 16(4):340-345) and (m
  • RNA virus is a single stranded positive sense RNA virus selected from: (a) bromoviridae, preferably selected from (i) alfamovirus (e.g. alfalfa mosaic virus (AIMV)), and (ii) ilarvirus (e.g. prunus necrotic ringspot ilarvirus (PNRSV, Pallas V. (1998) Arch. Virol. 144:797-803); prune dwarf virus (PDV, Abou-Jawdah Y. et al. (2004) J. Virological Methods 121:31-38)), (iii) bromovirus (e.g.
  • alfamovirus e.g. alfalfa mosaic virus (AIMV)
  • ilarvirus e.g. prunus necrotic ringspot ilarvirus (PNRSV, Pallas V. (1998) Arch. Virol. 144:797-803
  • prune dwarf virus PDV, Abou-Jawdah Y. e
  • cowpea chlorotic mottle virus CCMV
  • brome mosaic virus BMV
  • cucumovirus e.g. cucumber mosaic virus, Natilla A. et al. Arch Virol 2004 149(1):137-154
  • tombusviridae preferably (i) tombusvirus, preferably tomato bushy stunt virus (TBSV, Joelson T. et al. (1997) J. Gen. Virol. 78:1213-1217),
  • carmovirus, turnic crinkle virus TCV, Qu F. and Morris T. J. (1997) J. Virol.
  • potyvirus preferably Johnsongrass mosaic virus (JGMV) and plum pox potyvirus (PPV, Fernandez-Fernandes M. R. et al. (2002) J. Virol. 76(24):12646-12653)
  • tobacco mosaic virus TMV
  • comovirus preferably cowpea mosaic virus (CPMV)
  • CPMV cowpea mosaic virus
  • PVX potato virus X
  • calicivirus preferably selected from (i) norwalk virus (NV), (ii) norwalk-like calcivirus, (iii) human calcivirus, (iv) Lorsdale calcivirus, (v) rabbit hemorrhagic disease virus (RHDV), (vi) European brown hare syndrom virus (EBHSV), (vii) Toronto virus, (viii) Hawaii virus, (ix) Sapporo-like virus, and (x) Grimsby feline calcivirus, (h) RNA bacteriophage, (i) luteovirus, preferably potato leaf roll virus (PLRV), (j) flock house virus, (k) retroid viruses, preferably selected from (i) oncoretrovirus, (ii) lentivirus, and (iii) yeast retrotransposon Ty1, (l) tick-borne encephalitis virus (TBEV, Leibl H.
  • NV norwalk virus
  • norwalk-like calcivirus preferably human calcivirus
  • togaviridae preferably alphavirus, most preferably Sindbis virus
  • Nodaviridae preferably Alphanodavirus, most preferably Pariacoto virus
  • RNA virus is a double stranded RNA virus, wherein preferably said double stranded RNA virus is selected from: (a) birnavirus, (b) cypovirus, preferably Bombyx mori cytoplasmic polyhedrovirus (BmCPV), (c) orbivirus, preferably bluetoung virus (BTV) or African horse sickness virus (AHSV), (d) rotavirus and, very preferably, (e) double stranded RNA bacteriophages, preferably selected from (i) bacteriophage 8, (ii) bacteriophage phi6, (iii) bacteriophage phi12, and (iv) bacteriophage phi12.
  • BmCPV Bombyx mori cytoplasmic polyhedrovirus
  • BTV bluetoung virus
  • AHSV African horse sickness virus
  • said virus particle or VLP is a virus particle or VLP of a virus, wherein said virus is a bacteriophage, wherein said bacteriophage may be a DNA bacteriophage or an RNA bacteriophage.
  • said bacteriophage is a DNA bacteriophage, wherein said DNA bacteriophage may be a single stranded DNA bacteriophage or a double stranded bacteriophage.
  • said DNA bacteriophage is a single stranded DNA bacteriophage, wherein said single stranded DNA bacteriophage is preferably selected from (a) Microviridae, preferably Phi X 174 and (b) Inoviridae, preferably fd and M13.
  • said DNA bacteriophage is a double stranded DNA bacteriophage, wherein said double stranded DNA bacteriophage is preferably selected from the group consisting of: (a) Myoviridae, preferably T2, T4 or T6, (b) Siphoviridae, preferably bacteriophage Lambda, T1, T5 or HK97, (c) Podoviridae, preferably T2, T7 or P22, (d) Tectiviridae, preferably PRD1, (e) Corticoviridae, preferably PM2, (f) Plasmaviridae, preferably mycoplasma phages, (g) Lipothrixviridae, preferably Thermoproteus bacteriophage TTV1 and (h) Fuselloviridae, preferably sulfolobus bacteriophage 1.
  • Myoviridae preferably T2, T4 or T6
  • Siphoviridae preferably bacteriophage Lambda
  • said bacteriophage is an RNA bacteriophage, wherein said RNA bacteriophage may be a single stranded or a double stranded RNA bacteriophage.
  • said RNA bacteriophage is a single stranded RNA bacteriophage, wherein preferably said single stranded RNA bacteriophage is an enterobacteriophage, wherein preferably said enterobacteriophage is a representative of the Leviviridae, wherein preferably said representative of the Leviviridae is selected from the group consisting of: (a) taxonomically not assigned family member Acinetobacter phage 205 (AP205), (b) levivirus, and, preferably (c) allolevivirus.
  • said representative of the Leviviridae is a levivirus, wherein preferably said levivirus is selected from the group consisting of: (a) bacteriophage BZ13, (b) bacteriophage GA, (c) bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophage TH1, (e) bacteriophage MS2, (f) bacteriophage f2, (g) bacteriophage fr, (h) bacteriophage JP501, (i) bacteriophage M12, (j) bacteriophage R17, and (k) bacteriophage PP7.
  • levivirus is selected from the group consisting of: (a) bacteriophage BZ13, (b) bacteriophage GA, (c) bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophage TH1, (e) bacteriophage MS2, (f) bacterioph
  • said representative of the Leviviridae is an allolevivirus, wherein preferably said allolevivirus is selected from the group consisting of: (a) bacteriophage FI, (b) bacteriophage ID2, (c) bacteriophage NL95, (d) bacteriophage SP, (d) bacteriophage TW28, (e) bacteriophage Q ⁇ , (f) bacteriophage M11, (g) bacteriophage MX1, (h) bacteriophage ST, (i) bacteriophage TW18, and (j) bacteriophage VK.
  • said allolevivirus is selected from the group consisting of: (a) bacteriophage FI, (b) bacteriophage ID2, (c) bacteriophage NL95, (d) bacteriophage SP, (d) bacteriophage TW28, (e) bacteriophage Q ⁇ , (f) bacteriophage M11, (g
  • RNA bacteriophage is selected from the group consisting of: (a) bacteriophage BZ13, (b) bacteriophage GA, (c) bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophage TH1, (e) bacteriophage MS2, (f) bacteriophage f2, (g) bacteriophage fr, (h) bacteriophage JP501, (i) bacteriophage M12, (j) bacteriophage R17, (k) bacteriophage PP7, (l) bacteriophage FI, (m) bacteriophage ID2, (n) bacteriophage NL95, (o) bacteriophage SP, (p) bacteriophage TW28, (q) bacteriophage Q ⁇ , (r) bacteriophage M11, (s) bacteriophage MX1, (t) bacteriophage ST, (
  • RNA bacteriophage 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 M11, (h) bacteriophage MX1, (i) bacteriophage NL95, (k) bacteriophage f2, (l) bacteriophage PP7, and (m) bacteriophage AP205.
  • RNA bacteriophage is a double stranded RNA bacteriophage, wherein preferably said double stranded RNA bacteriophage is a representative of the Cystoviridae, more preferably said representative of the Cystoviridae is a Cystovirus, most preferably said Cystovirus is pseudomonas bacteriophage Phi 6.
  • said particle is a virus particle of a bacteriophage, and wherein preferably said bacteriophage is a RNA bacteriophage, wherein further preferably said RNA bacteriophage is a single stranded positive sense RNA bacteriophage, and wherein still further preferably said single stranded positive sense RNA bacteriophage is a single stranded positive sense RNA bacteriophage selected from the group consisting of: (a) bacteriophage Q ⁇ , (b) bacteriophage fr, (c) bacteriophage GA, and (d) bacteriophage AP205, most preferably said single stranded positive sense RNA bacteriophage is Q ⁇ .
  • said particle is a VLP, preferably a VLP of an RNA virus, more preferably a VLP of a single stranded positive sense RNA virus, most preferably a VLP of an RNA bacteriophage.
  • said particle is a VLP of a bacteriophage, more preferably a VLP of a enterobacteriophage, still more preferably a VLP of a representative of the Leviviridae, most preferably a VLP of a levivirus or an allolevivirus.
  • said VLP is a VLP of an allolevivirus.
  • said particle is a virus particle or a VLP, preferably a VLP, of a icosahedral virus, wherein said icosahedral virus is preferably a plant-infectious icosahedral virus.
  • VLPs of plant-infectious icosahedral viruses are for example disclosed in WO2005/067478A2 which is incorporated herein by reference.
  • said icosahedral virus is selected from a representative of any one taxon selected from the group consisting of (a) Papillomaviridae, (b) Totiviridae, (c) Dcistroviridae, (d) Hepadnaviridae, (e) Togaviridae, (f) Polyomaviridae, (g) Nodaviridae, (h) Tectiviridae, (i) Leviviridae, (j) Microviridae, (k) Sipoviridae, (l) Picomoviridae, (m) Parvoviridae, (n) Calciviridae, (o) Tetraviridae, and (p) Satellite viruses.
  • said icosahedral virus is a plant-infectious icosahedral virus, wherein said plant-infectious icosahedral virus is a representative of any one taxon selected from the group consisting of (a) Bunyaviridae, (b) Reoviridae, (c) Rhabdoviridae, (d) Luteoviridae, (e) Nanoviridae, (f) Partitiviridae, (g) Sequiviridae, (h) Tymoviridae, (i) Ourmiavirus, (j) Tobacco Necrosis Virus Satellite, (k) Caulimoviridae, (l) Geminiviridae, (m) Comoviridae, (n) Sobemovirus, (o) Tombusviridae, and (p) Bromoviridae.
  • said plant-infectious icosahedral virus is a representative of any one taxon selected from the group consisting of (a) Luteoviridae, (b) Nanoviridae, (c) Partitiviridae, (d) Sequiviridae, (e) Tymoviridae, (f) Ourmiavirus, (g) Tobacco Necrosis Virus Satellite, (h) Caulimoviridae, (i) Geminiviridae, (j) Comoviridae, (k) Sobemovirus, (l) Tombusviridae, and (m) Bromoviridae.
  • said plant-infectious icosahedral virus is a representative of any one taxon selected from the group consisting of (a) Caulimoviridae, (b) Geminiviridae, (c) Comoviridae, (d) Sobemovirus, (e) Tombusviridae, and (e) Bromoviridae.
  • said plant-infectious icosahedral virus is a representative of any one taxon selected from the group consisting of the (a) Comoviridae, (b) Sobemovirus, (c) Tombusviridae, and (d) Bromoviridae.
  • said plant-infectious icosahedral virus is a representative of any one taxon selected from Comoviridae and Bromoviridae.
  • said plant-infectious icosahedral virus is a Cowpea Mosaic Virus or a Cowpea Chlorotic Mottle Virus.
  • said plant-infectious icosahedral virus is a representative of the Bromoviridae, preferably Bromovirus, Cucumovirus, Ilarvirus or Alfamovirus.
  • said plant-infectious icosahedral virus is selected from: brome mosaic virus, cowpea chlorotic mottle virus, cucumber mosaic virus, Tobacco streak virus and alfalfa mosaic virus (AMV, including AMV1 and AMV2).
  • said VLP is a synthetic VLP.
  • the VLP is a recombinant VLP.
  • the preparation of VLPs by recombinantly expressing the coat protein in a host is within the common knowledge of a skilled artisan.
  • Illustrative DNA or RNA viruses, the coat or capsid protein of which can be used for the preparation of VLPs have been disclosed in WO 2004/009124 on page 25, line 10-21, on page 26, line 11-28, and on page 28, line 4 to page 31, line 4. These disclosures are incorporated herein by way of reference.
  • said VLP comprises, or alternatively consists of, recombinant proteins, mutants or fragments thereof, of a virus, wherein preferably said virus is selected from any virus listed above.
  • said VLP comprises, or alternatively consists of, recombinant proteins, mutants or fragments thereof, of a virus, wherein said virus is selected form the group consisting of: (a) RNA bacteriophages, (b) bacteriophages, (c) Hepatitis B virus, preferably its capsid protein (Ulrich, et al., Virus Res.
  • VLP comprises, or alternatively consists of, recombinant proteins, mutants or fragments thereof, of a virus, wherein said virus is selected form the group consisting of: (a) Hepatitis B virus, and (b) Polyoma virus.
  • said VLP comprises, or alternatively consists of, recombinant proteins, mutants or fragments thereof, of a virus, wherein said virus is a plant-infectious icosahedral virus, wherein preferably said plant-infectious icosahedral virus is selected from (a) Comoviridae, (b) Sobemovirus, (c) Tombusviridae, and (d) Bromoviridae.
  • the VLP comprises or consists of more than one amino acid sequences, preferably two amino acid sequences, of the recombinant proteins, mutants or fragments thereof.
  • VLP comprises or consists of more than one amino acid sequence is referred, in this application, as mosaic VLP.
  • fragment of a recombinant protein or the term “fragment of a coat protein”, as used herein, is defined as a polypeptide, which is of at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95% the length of the wild-type recombinant protein, or coat protein, respectively and which preferably retains the capability of forming VLP.
  • the fragment is obtained by (i) at least one, preferably exactly one, internal deletion, (ii) at least one, preferably exactly one, truncation, or (iii) at least one, preferably exactly one, combination thereof.
  • the fragment is obtained by at most 5, 4, 3 or 2 internal deletions, at most 2 truncations or exactly one combination thereof. Further preferably, the fragment is obtained by at most 5, 4, 3 or 2 internal deletions, wherein still further preferably each of said deletions comprises 1 to 5, preferably 1 to 4, more preferably 1 to 3, still more preferably 1 to 2, and most preferably exactly 1 amino acid.
  • fragment of a recombinant protein or “fragment of a coat protein” shall further encompass polypeptide, which has at least 80%, preferably 90%, even more preferably 95% amino acid sequence identity with the “fragment of a recombinant protein” or “fragment of a coat protein”, respectively, as defined above and which is preferably capable of assembling into a virus-like particle.
  • mutant coat protein refers to a polypeptide having an amino acid sequence derived from the wild type recombinant protein, or coat protein, respectively, wherein the amino acid sequence is at least 80%, preferably at least 85%, 90%, 95%, 97%, or 99% identical to the wild type sequence and preferably retains the ability to assemble into a VLP.
  • the VLP of the invention is VLP of Hepatitis B virus.
  • the preparation of Hepatitis B virus-like particles has been disclosed, inter alia, in WO00/32227, WO01/85208, WO01/056905 and WO2004/000351. All four documents are explicitly incorporated herein by way of reference.
  • Other variants of HBcAg suitable for use in the practice of the present invention have been disclosed in page 34-39 of WO 01/056905.
  • Specifically preferred Hepatitis B virus VLPs are described on page 43, line 12 to page 49, line 8 of WO2004/000351 and in SEQ IDs NO:19-68, 71 and 97 of WO2004/000351.
  • a lysine residue is introduced into the HBcAg polypeptide.
  • VLPs and 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:1, which is modified so that the amino acids at positions 79 and 80 are replaced with a peptide having the amino acid sequence of Gly-Gly-Lys-Gly-Gly (SEQ ID NO:24). This modification changes the SEQ ID NO:1 to SEQ ID NO:2.
  • cysteine residues at positions 48 and 110 of SEQ ID NO:2, or its corresponding fragments, preferably 1-144 or 1-149 are mutated to serine.
  • the invention further includes compositions comprising Hepatitis B core protein mutants having above noted corresponding amino acid alterations.
  • the virus-like particle comprises, consists essentially of, or alternatively consists of, recombinant coat proteins, mutants or fragments thereof, of a RNA bacteriophage.
  • the RNA bacteriophage is selected from the group consisting of: (a) bacteriophage BZ13, (b) bacteriophage GA, (c) bacteriophage JP34, (d) bacteriophage KU1, (d) bacteriophage TH1, (e) bacteriophage MS2, (f) bacteriophage f2, (g) bacteriophage fr, (h) bacteriophage JP501, (i) bacteriophage M12, (j) bacteriophage R17, (k) bacteriophage PP7, (l) bacteriophage FI, (m) bacteriophage ID2, (n) bacteriophage NL95, (o) bacteriophage SP, (p)
  • the RNA bacteriophage 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 M11, (h) bacteriophage MX1, (i) bacteriophage NL95, (k) bacteriophage f2, (l) bacteriophage PP7, and (m) bacteriophage AP205.
  • the virus-like particle comprises at least one coat protein, mutant or fragment thereof, of an RNA bacteriophage, wherein the coat protein has an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:3 referring to Q ⁇ CP; (b) a mixture of SEQ ID NO:3 and SEQ ID NO:4 (Q ⁇ A1 protein); (c) SEQ ID NO:5 (R17 capsid protein); (d) SEQ ID NO:6 (fr capsid protein); (e) SEQ ID NO:7 (GA capsid protein); (f) SEQ ID NO:8 (SP capsid protein); (g) a mixture of SEQ ID NO:8 and SEQ ID NO:9; (h) SEQ ID NO:10 (MS2 capsid protein); (i) SEQ ID NO:11 (M11 capsid protein); (j) SEQ ID NO:12 (MX1 capsid protein); (k) SEQ ID NO:13 (NL95 capsid protein); (l
  • the virus-like particle comprises coat proteins having an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:3; (b) a mixture of SEQ ID NO:3 and SEQ ID NO:4; (c) SEQ ID NO:6; (d) SEQ ID NO:7; (e) SEQ ID NO:21.
  • the virus-like particle comprises coat proteins having an amino acid sequence selected from the group consisting of: (a) SEQ ID NO:3; and (b) a mixture of SEQ ID NO:3 and SEQ ID NO:4.
  • the virus-like particle essentially consists of coat proteins having an amino acid sequence of SEQ ID NO:3, or essentially consists of a mixture of coat proteins having amino acid sequences of SEQ ID NO: 4, or mutants thereof, and of SEQ ID NO:3.
  • the VLP is a mosaic VLP comprising or alternatively consisting of more than one amino acid sequence, preferably two amino acid sequences, of coat proteins, mutants or fragments thereof, of a RNA bacteriophage.
  • the virus particle or VLP is a VLP of bacteriophage fr or GA.
  • Fr coat protein in the form of recombinant VLP may be obtained as described by Pushko P et al. ((1993) Prot Engin 6:883-891), while GA VLP may be obtained by cloning GA coat protein cDNA isolated by reverse transcription from GA phage into pQb185, which is described for example in WO2004/007538.
  • Disassembly of Fr and GA VLPs can be readily done by incubating the VLPs in 7 M urea, optionally supplemented with acetic acid at a concentration of 0.1 M.
  • the nucleic acid is further purified from the coat protein by ion exchange chromatography, either at a pH where a significant amount of the coat protein flows through while the nucleic acid is retained, or at a pH where the coat protein is also adsorbed on the column and subsequently eluted with a salt gradient.
  • Reassembly of fr and GA coat protein with ISS-NA is effected essentially as described in WO2003/024481 by slow dialysis, wherein said ISS-NA preferably is an unmethylated CpG-containing oligonucleotide, more preferably G10 (SEQ ID NO:27), and even more preferably aggregated G10 (SEQ ID NO:27) having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • the reassembly mixture is concentrated for example by dialysis against a 50% (v/v) glycerol solution in NET buffer (WO2003/024481) and purified further by gel filtration, for example on a Sepharose CL-4B column. Additional purification methods include ultracentrifugation on a CsCl gradient or sucrose cushion. Further protocols for the disassembly and reassembly of Fr and GA VLPs are disclosed in Examples 5 and 6 of the present application.
  • the VLP comprises or alternatively consists of two different coat proteins of a RNA bacteriophage, said two coat proteins have an amino acid sequence of CP Q ⁇ (SEQ ID NO: 3) and CP Q ⁇ A1 (SEQ ID NO:4), or of CP SP (SEQ ID NO:8) and CP SP A1 (SEQ ID NO:9).
  • the virus-like particle of the invention comprises, or alternatively consists essentially of, or alternatively consists of recombinant coat proteins, mutants or fragments thereof, of the RNA-bacteriophage Q ⁇ , fr, AP205 or GA.
  • the VLP of the invention is a VLP of RNA bacteriophage Q ⁇ .
  • the capsid contains 180 copies of the coat protein, which are linked in covalent pentamers and hexamers by disulfide bridges (Golmohammadi, R. et al., Structure 4:543-5554 (1996)), leading to a remarkable stability of the Q ⁇ capsid.
  • Capsids or VLPs made from recombinant Q ⁇ coat protein may contain, however, subunits not linked via disulfide bonds to other subunits within the capsid, or incompletely linked.
  • VLPs of RNA bacteriophages in accordance with this invention in particular of Q ⁇ and fr, are disclosed in WO 02/056905, the disclosure of which is herewith incorporated by reference in its entirety.
  • Example 18 of WO 02/056905 gave detailed description of preparation of VLP particles from Q ⁇ .
  • the VLP of the invention is a VLP of RNA bacteriophage AP205.
  • Assembly-competent mutant forms of AP205 VLPs, including AP205 coat protein with the substitution of proline at amino acid 5 to threonine, may also be used in the practice of the invention and leads to other preferred embodiments of the invention.
  • WO 2004/007538 describes, in particular in Example 1 and Example 2, how to obtain VLP comprising AP205 coat proteins, and hereby in particular the expression and the purification thereto.
  • WO 2004/007538 is incorporated herein by way of reference.
  • said virus particle or VLP is a virus particle or VLP of RNA bacteriophage AP205, wherein said ISS-NA preferably is an unmethylated CpG-containing oligonucleotide, more preferably G10 (SEQ ID NO:27), and even more preferably aggregated G10 (SEQ ID NO:27) having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • the disassembly and reassembly of AP205 is demonstrated in Example 5.
  • the virus-like particle comprises, consists essentially of or alternatively consists of mutant Q ⁇ coat proteins.
  • these mutant coat proteins comprise or alternatively consist of an amino acid sequence selected from the group of a) Q ⁇ -240 (SEQ ID NO:16, Lys13-Arg of SEQ ID NO: 3) b) Q ⁇ -243 (SEQ ID NO:17, Asn10-Lys of SEQ ID NO:3); c) Q ⁇ -250 (SEQ ID NO:18, Lys2-Arg of SEQ ID NO:3) d) Q ⁇ -251 (SEQ ID NO:19, Lys16-Arg of SEQ ID NO:3); and e) Q ⁇ -259 (SEQ ID NO:20, Lys2-Arg, Lys16-Arg of SEQ ID NO:3).
  • the construction, expression and purification of the above indicated Q ⁇ mutant coat proteins, mutant Q ⁇ coat protein VLPs and capsids, respectively, are described in WO 02/056905. In particular is hereby referred to Example 18 of above mentioned application.
  • said virus particle or VLP is a virus particle or VLP of RNA bacteriophage Q ⁇ , wherein said ISS-NA preferably is an unmethylated CpG-containing oligonucleotide, more preferably G10 (SEQ ID NO:27), and even more preferably aggregated G10 (SEQ ID NO:27) having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • the disassembly and reassembly of Q ⁇ VLPs is demonstrated in Examples 1 and 3.
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of mutant coat protein of Q ⁇ , or mutants or fragments thereof, and the corresponding A1 protein.
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of mutant coat protein with amino acid sequence SEQ ID NO:16, 17, 18, 19, or 20 and the corresponding A1 protein.
  • RNA bacteriophage coat proteins have also been shown to self-assemble upon expression in a bacterial host (Kastelein, R A. et al., Gene 23:245-254 (1983), Kozlovskaya, T M. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, M R. et al., Virology 170:238-242 (1989), Priano, C. et al., J. Mol. Biol. 249:283-297 (1995)).
  • GA Neurogenet al.
  • the virus particle or VLP is a VLP or virus particle of Cowpea cholortic mottle virus (CCMV). Assembly of CCMV virus from coat proteins expressed in E. Coli and nucleic acids has been described (Zhao X. et al. (1995) Virology 207:486-494). In particular, the reassembly of CCMV with RNA was shown to be independent of RNA sequence (Johnson J M. et al. (2004) J Mol Biol 335:455-464).
  • CCMV Cowpea cholortic mottle virus
  • the virus may exist in a swollen form, susceptible to nuclease digestion, which can be disassembled by adding a high NaCl concentration (1 M; Johnson J E and Speir J A (1997) J Mol Biol 269:665-675). Methods for reassembly of CCMV in the presence of nucleic acids are also described therein.
  • the CCMV particle is thus reassembled with ISS-NA, preferably with an unmethylated CpG-containing oligonucleotide.
  • the unmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27), more preferably aggregated G10, still more preferably aggregated G10 having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • native CCMV virus particle is swollen, treated with nucleases, and an ISS-NA, preferably an unmethylated CpG-containing oligonucleotide, and even more preferably G10 (SEQ ID NO:27) is added to the nuclease treated particle after nuclease removal.
  • CCMV capsids are reassembled without nucleic acids, as has been described for example by Zlotnick et al. (2000) Virology 277:450-456, optionally swollen by bringing the solution to the appropriate pH and ionic strength as described by Zlotnick et al. ((2000) Virology 277:450-456) or Johnson J E and Speir J A ((1997) J Mol Biol 269:665-675) and ISS-NA, preferably an unmethylated CpG-containing oligonucleotide, and even more preferably G10 (SEQ ID NO:27) is added to the swollen empty particle.
  • Zlotnick et al. (2000) Virology 277:450-456
  • ISS-NA preferably an unmethylated CpG-containing oligonucleotide, and even more preferably G10 (SEQ ID NO:27) is added to the swollen empty particle.
  • the virus particle or VLP is a VLP or virus particle of Brome mosaic virus (BMV). Reassembly of BMV has been described previously (Choi Y G and Rao L N (2000) Virology 275: 249-257, and references therein). A tRNA-like structure (tls) at the 3′ of each viral RNA has been shown to be required for packaging, and can be added in trans (Choi Y G et al. (2002) Proc. Natl. Acad. Sci. USA 99:655-660) as a nucleotide sequence of about 200 nucleotide in length.
  • BMV Brome mosaic virus
  • Tls from other viruses such as tobacco mosaic virus (TMV) or CMV, or even tRNAs such as wheat germ tRNAs may also be added in trans and facilitate reassembly, although Choi et al. did not detect packaging of tRNAs in the BMV capsid (Choi Y G et al. (2002) Proc. Natl. Acad. Sci. USA 99:655-660).
  • BMV is reassembled with an ISS-NA, preferably an unmethylated CpG-containing oligonucleotide, more preferably G10 and even more preferably aggregated G10 having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • an ISS-NA preferably an unmethylated CpG-containing oligonucleotide, more preferably G10 and even more preferably aggregated G10 having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • compositions of the invention comprise an immunostimulatory nucleic acid (ISS-NA), wherein preferably said ISS-NA is capable of inducing the production of a cytokin, preferably of IFN-alpha, in a cell, preferably in a dendritic cell.
  • said ISS-NA is selected from the group consisting of: (a) ribonucleic acids; (b) desoxyribonucleic acids, (c) chimeric nucleic acids; and (d) any mixtures of at least one nucleic acid of (a), (b) and/or (c).
  • said ISS-NA is a ribonucleic acid, wherein preferably said ribonucleic acid is a double stranded ribonucleic acid, preferably a double stranded ribonucleic acid selected from the group consisting of: (a) double stranded viral RNA, and (b) synthetic double stranded RNA, preferably poly-(A:U) or poly(I:C), most preferably poly(I:C).
  • the innate immune system has the capacity to recognize invariant molecular pattern shared by microbial pathogens. Recent studies have revealed that this recognition is a crucial step in inducing effective immune responses.
  • the main mechanism by which microbial products augment immune responses is to stimulate APC, especially dendritic cells to produce proinflammatory cytokines and to express high levels co-stimulatory molecules for T cells. These activated dendritic cells subsequently initiate primary T cell responses and dictate the type of T cell-mediated effector function.
  • nucleic acids Three classes of nucleic acids, namely (i) bacterial DNA that contains immunostimulatory sequences, in particular unmethylated CpG dinucleotides within specific flanking bases (referred to as CpG motifs), (ii) double-stranded RNA synthesized by various types of viruses represent important members of the microbial components that enhance immune responses, and (iii) single stranded RNA.
  • Synthetic double stranded (ds) RNA such as polyinosinic-polycytidylic acid (poly I:C) are capable of inducing dendritic cells to produce proinflammatory cytokines and to express high levels of costimulatory molecules.
  • ribonucleic acid encompass polyinosinic-polycytidylic acid double-stranded RNA (poly I:C). Ribonucleic acids and modifications thereof as well as methods for their production have been described by Levy, H. B (Methods Enzymol.
  • ribonucleic acids comprise polynucleotides of inosinic acid and cytidiylic acid such poly (I:C) of which two strands form double stranded RNA. Ribonucleic acids can be isolated from organisms.
  • Ribonucleic acids also encompass further synthetic ribonucleic acids, in particular synthetic poly (I:C) oligonucleotides that have been rendered nuclease resistant by modification of the phosphodiester backbone, in particular by phosphorothioate modifications.
  • the ribose backbone of poly (I:C) is replaced by a desoxyribose.
  • said ISS-NA is a single stranded ribonucleic acid, preferably polyuridylic acid (poly-U, Westwood A. (2006), Vaccine 24:1736-1743).
  • said ISS-NA is desoxyribonucleic acid, wherein preferably said desoxyribonucleic acid is a double stranded desoxyribonucleic acid.
  • said ISS-NA is desoxyribonucleic acid, wherein preferably said desoxyribonucleic acid is a single stranded desoxyribonucleic acid, most preferably a oligodesoxynucleotide (ODN).
  • ODN oligodesoxynucleotide
  • said ISS-NA is an oligonucleotide, wherein said oligonucleotide is preferably selected from the group consisting of (a) unmethylated CpG-containing oligonucleotide; and (b) oligonucleotide free of unmethylated CpG motifs.
  • said ISS-NA is an unmethylated CpG-containing oligonucleotide.
  • Unmethylated CpG-dinucleotides within specific flanking bases (referred to as CpG motifs) represent important members of the microbial components that enhance immune responses.
  • Toll-like receptor 9 (TLR9) is activated by bacterial DNA, in particular by unmethylated CpG-containing oligonucleotides.
  • the unmethylated CpG-containing oligonucleotide comprises the sequence: 5′ X 1 X 2 CGX 3 X 4 3′, wherein X 1 , X 2 , X 3 and X 4 are any nucleotide.
  • Preferred unmethylated CpG-containing oligonucleotides further comprise about 6 to about 100,000 nucleotides, more preferably about 6 to about 2000 nucleotides, still more preferably about 20 to about 2000 nucleotides, and even more preferably comprises about 20 to about 300 nucleotides.
  • unmethylated CpG-containing oligonucleotides comprise 100 to about 2000 nucleotides, preferably 100 to about 1000 nucleotides, and more preferably 100 to about 500 nucleotides.
  • oligonucleotides, unmethylated CpG-containing oligonucleotide in the context of the invention comprise 20 to 40, preferably 26, 27, 28, 29, 30, 31 or 32 nucleotides, most preferably 30 nucleotides.
  • the CpG-containing oligonucleotide can contain one or more phosphothioester modifications of the phosphate backbone to enhance the stability of the oligonucleotide.
  • a CpG-containing oligonucleotide having one or more phosphate backbone modifications or having all of the phosphate backbone modified and a CpG-containing oligonucleotide wherein one, some or all of the nucleotide phosphate backbone modifications are phosphorothioate modifications are included within the scope of the present invention.
  • said ISS-NA is a CpG-containing oligonucleotide, wherein preferably said CpG-containing oligonucleotide consisting exclusively of phosphodiester bound, preferably unmethylated nucleotides are preferred in the context of the invention.
  • the CpG-containing oligonucleotide can also be recombinant, genomic, synthetic, cDNA, plasmid-derived and single or double stranded.
  • the nucleic acids can be synthesized de novo using any of a number of procedures well known in the art. For example, the b-cyanoethyl phosphoramidite method (Beaucage, S. L., and Caruthers, M. H., Tet. Let. 22:1859 (1981); nucleoside H-phosphonate method (Garegg et al., Tet. Let. 27:4051-4054 (1986); Froehler et al., Nucl. Acid. Res.
  • oligonucleotide synthesizers available in the market.
  • CpGs can be produced on a large scale in plasmids, (see Sambrook, T., et al., “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor laboratory Press, New York, 1989) which after being administered to a subject are degraded into oligonucleotides.
  • Oligonucleotides can be prepared from existing nucleic acid sequences (e.g., genomic or cDNA) using known techniques, such as those employing restriction enzymes, exonucleases or endonucleases.
  • the ISS-NA preferably the unmethylated CpG-containing oligonucleotide
  • the particle preferably the VLP
  • the particle encloses, fully or partially, the ISS-NA, preferably the unmethylated CpG-containing oligonucleotide.
  • said particle, preferably said VLP is packaged with said ISS-NA, wherein further preferably said ISS-NA is a unmethylated CpG-containing oligonucleotide, most preferably G10 (SEQ ID NO:27).
  • the ISS-NA preferably the unmethylated CpG-containing oligonucleotide
  • the VLP binding site comprises an arginine-rich repeat or a lysine-rich repeat.
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein preferably said palindromic sequence is selected from any one of SEQ ID NO:28 and SEQ ID NOs: 35 to 60.
  • said palindromic sequence is GACGATCGTC (SEQ ID NO:28).
  • said ISS-NA is an A-type CpG or an C-type CpG.
  • said unmethylated CpG containing oligonucleotide is an A-type CpG, wherein preferably the nucleotides are exclusively linked by phosphodiester bonds.
  • said ISS-NA is a A-type CpG comprising a palindromic sequence, wherein preferably said palindromic sequence is selected from the group consisting of: (a) GACGTC (SEQ ID NO:35), (b) AGCGCT (SEQ ID NO:36), (c) AACGTT (SEQ ID NO:37), (d) ATCGAT (SEQ ID NO:38); (e) CGATCG (SEQ ID NO:39); (f) CGTACG (SEQ ID NO:40); (g) CGCGCG (SEQ ID NO:41); (h) GCGCGC (SEQ ID NO:42); (i) TCGCGA (SEQ ID NO:43); (j) ACGATCGT (SEQ ID NO:44); (k) CGACGATCGTCG (SEQ ID NO:45); (l) CGACGACGATCGTCGTCG (SEQ ID NO:46); (m) GACGATCGTC (SEQ ID NO:28), (n)
  • said palindromic sequence is flanked at its 3′-terminus and at its 5′-terminus by guanosine entities. In a further preferred embodiment said palindromic sequence is flanked at its 5′-terminus by at least 3 and at most 25 guanosine entities, wherein said palindromic sequence is flanked at its 3′-terminus by at least 3 and at most 25 guanosine entities.
  • said palindromic sequence is flanked at its 5′-terminus by at least 3 and at most 15, preferably at most 10, guanosine entities, wherein said palindromic sequence is flanked at its 3′-terminus by at least 3 and at most 15, preferably at most 10 guanosine entities.
  • the palindromic sequence is flanked at its 5′-terminus and at its 3′-terminus by at least 3 and at most 15, preferably at most 10, guanosine entities.
  • the palindromic sequence is flanked at its 5′-terminus by at least 3 and at most 15, preferably at most 10, guanosine entities, and wherein said palindromic sequence is flanked at its 3′-terminus by at least 6 and at most 15, preferably at most 10, guanosine entities.
  • the palindromic sequence is flanked at its 5′-terminus by at least 5 and at most 10 guanosine entities, and wherein said palindromic sequence is flanked at its 3′-terminus by at least 5 and at most 10 guanosine entities.
  • the palindromic sequence preferably SEQ ID NO:28, is flanked at its 3′-terminus by at least 10, preferably exactly 10, guanosine entities and at its 5′-terminus by at least 10, preferably exactly 10, guanosine entities.
  • said ISS-NA is a A-type CpG comprising a palindromic sequence, wherein said palindromic sequence is flanked at its 5′-terminus by 3 to 10 guanosine entities, and wherein said palindromic sequence is flanked at its 3′-terminus by 3 to 10 guanosine entities.
  • said ISS-NA is a A-type CpG comprising a palindromic sequence, wherein said palindromic is SEQ ID NO:28, and wherein said palindromic sequence is flanked at its 5′-terminus by 3 to 10 guanosine entities, and wherein said palindromic sequence is flanked at its 3′-terminus by 3 to 10 guanosine entities.
  • These immunostimulatory substances can be efficiently packaged into VLPs, wherein the packaging efficiency is typically decreasing with increasing number of flanking guanosine entities at the 5′ and/or 3′ terminus.
  • the unmethylated CpG-containing oligonucleotide comprises, or alternatively consists essentially of, or alternatively consists of the a sequence selected from the group consisting of (a) “G8-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25); (b) “G9-9” GGGGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:26); or (c) “G10” GGGGGGGGGG GACGATCGTC GGGGGGGGGG (SEQ ID NO:27). The latter was previously found to be able to stimulate blood cells in vitro (Kuramoto E. et al., Japanese Journal Cancer Research 83, 1128-1131 (1992)).
  • the unmethylated CpG-containing oligonucleotide comprises, or alternatively consists essentially of, or alternatively consists of “G10” (SEQ ID NO:27), wherein preferably said G10 consists exclusively of phosphodiester bound nucleotides, wherein further preferably said G10 is aggregated G10 having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • the unmethylated CpG-containing oligonucleotide comprises, or alternatively consists essentially of, or alternatively consists of “G9-9” (SEQ ID NO:26). In a further specifically preferred embodiment the unmethylated CpG-containing oligonucleotide comprises, or alternatively consists essentially of, or alternatively consists of “G8-8” (SEQ ID NO:25).
  • said ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein said unmethylated CpG-containing oligonucleotide has a nucleic acid sequence selected from (a) “G3-6” GGG GACGATCGTC GGGGGG (SEQ ID NO:29); (b) “G4-6” GGGG GACGATCGTC GGGGGG (SEQ ID NO:30); (c) “G5-6” GGGGG GACGATCGTC GGGGGG (SEQ ID NO:31); (d) “G6-6” GGGGGG GACGATCGTC GGGGGG (SEQ ID NO:32); and (e) “G7-7” GGGGGGG GACGATCGTC GGGGGGG (SEQ ID NO:33); (f) “G8-8” GGGGGGGG GACGATCGTC GG
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein said palindromic sequence is GACGATCGTC (SEQ ID NO:28), and wherein said palindromic sequence is flanked at its 5′-terminus of at least 4 and at most 9 guanosine entities and wherein said palindromic sequence is flanked at its 3′-terminus of at least 6 and at most 9 guanosine entities.
  • the immunostimulatory substance is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein said unmethylated CpG-containing oligonucleotide has a nucleic acid sequence selected from (a) “G4-6” GGGG GACGATCGTC GGGGGG (SEQ ID NO:30); (b) “G5-6” GGGGG GACGATCGTC GGGGGG (SEQ ID NO:31); (c) “G6-6” GGGGGG GACGATCGTC GGGGGG (SEQ ID NO:32); (d) “G7-7” GGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:33); (e) “G8-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25); and (f) “G9-9” GGGGGGGGG
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein said unmethylated CpG-containing oligonucleotide has a nucleic acid sequence selected from (a) “G4-6” GGGG GACGATCGTC GGGGGG (SEQ ID NO:30); (b) “G5-6” GGGGG GACGATCGTC GGGGGG (SEQ ID NO:31); (c) “G6-6” GGGGGG GACGATCGTC GGGGGG (SEQ ID NO:32); (d) “G7-7” GGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:33); (e) “G8-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25); and (f) “G9-9” GGGGGGGGGGG
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein said palindromic sequence is GACGATCGTC (SEQ ID NO:28), and wherein said palindromic sequence is flanked at its 5′-terminus of at least 5 and at most 8 guanosine entities and wherein said palindromic sequence is flanked at its 3′-terminus of at least 6 and at most 8 guanosine entities.
  • the experimental data show that the ease of packaging said ISS-NA, preferably the guanosine flanked, palindromic and unmethylated CpG-containing oligonucleotides, wherein the palindromic sequence is GACGATCGTC (SEQ ID NO:28), and wherein the palindromic sequence is flanked at its 3′-terminus and at its 5′-terminus by less than 10 guanosine entities, into particles, preferably VLPs, increases if the palindromic sequences are flanked by fewer guanosine entities.
  • decreasing the number of guanosine entities flanking the palindromic sequences leads to a decrease of stimulating blood cells in vitro.
  • packagability is paid by decreased biological activity of the indicated inventive immunostimulatory substances.
  • the preferred embodiments represent, thus, a compromise between packagability and biological activity.
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein said unmethylated CpG-containing oligonucleotide has a nucleic acid sequence selected from (a) “G5-6” GGGGG GACGATCGTC GGGGGG (SEQ ID NO:31); (b) “G6-6” GGGGGG GACGATCGTC GGGGGG (SEQ ID NO:32); (c) “G7-7” GGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:33); and (d) “G8-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25).
  • the ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein the CpG motif of said unmethylated CpG-containing oligonucleotide is part of a palindromic sequence, wherein said unmethylated CpG containing oligonucleotide has the nucleic acid sequence of SEQ ID NO:25 (G8-8).
  • the optimal sequence used to package into VLPs is a compromise between packagability and biological activity.
  • the G8-8 immunostimulatory substance is a further very preferred embodiment of the present invention since it is biologically highly active while it is still reasonably well packaged.
  • said ISS-NA is a unmethylated CpC containing oligonucleotide, wherein said unmethylated CpC containing oligonucleotide is capable of inducing the production of IFN-alpha in a cell, preferably in PBMCs, spleenocytes or human pDCs, and wherein further preferably said unmethylated CpC containing oligonucleotide is selected from: (a) T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*T*T*T*T*G*T*C*G*T*C*G*T*T (2006-PS, SEQ ID NO: 76); (b) GGGGGACGAT CGTCGGGGGG (2216-PO, SEQ ID NO:77); (c) G*G*GGGACGATCGTC*G*G*G*G*G*G*G (2216-PO core, SEQ ID NO:77); (
  • said unmethylated CpG containing oligonucleotide is capable of inducing the production of IFN-alpha in human pDCs, wherein preferably said unmethylated CpG containing oligonucleotide is T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*G*T*C*G*T*C*G*T*C*G*T*C*G*T*C*G*T*C*G*T*C*G*T*T*T (2006-PS, SEQ ID NO: 76), wherein * indicates a phosphorothioate modification, while all other nucleotides are phosphodiester bound.
  • said unmethylated CpG containing oligonucleotide is capable of inducing the production of IFN-alpha in PBMCs or spleen cells, wherein preferably said unmethylated CpG containing oligonucleotide is selected from the group consisting of: (a) GGGGGACGAT CGTCGGGGGG (2216-PO, SEQ ID NO:77); (b) G*G*GGGACGATCGTC*G*G*G*G*G*G*G*G (2216-PO core, SEQ ID NO:77); (c) GGTGCATCGATGCAGGGGGG (D 19-PO, SEQ ID NO:78); (d) G*G*TGCATCGATGCAG*G*G*G*G*G*G (D19-PO core, SEQ ID NO:78); (e) GGGGACGATCGTCGGGGGG (G3-6, SEQ ID NO:29); (f) GGGGGACGATCGTCGGGGGG (G4-6, SEQ ID NO
  • said unmethylated CpG containing oligonucleotide is GGGGGGGGG GACGATCGTC GGGGGGGGGGGGGGGGGG GACGATCGTC GGGGGGGGGGGG (G102x, SEQ ID NO:79), wherein preferably all nucleotides are phosphodiester bound.
  • said ISS-NA is a C-type CpG, wherein preferably said C-type CpG comprises a palindromic sequence, wherein further preferably said palindromic sequence is selected from any one of the sequences depicted in SEQ ID NOs:53 to 60.
  • said C-type CpG is SEQ ID NO:64 or SEQ ID NO:65, wherein preferably all nucleic acids of said C-type CpG are phosphorothioate bound.
  • C-type CpGs are selected from the group consisting of (a) TCpGTCGTTTT ACGGCGCCGTG CCG (SEQ ID NO:64); (b) TCGTCGTTTT ACpGGCpGCCpGTGCCG (SEQ ID NO:64); (c) TCGTCGTTT TACpGGCGCCpGTGCCG (SEQ ID NO:64); and (d) TCGTCpGTTTT ACpGGCGCCpGTGCCG (SEQ ID NO:64); wherein p indicates phosphodiester bounds while all other nucleotides are phosphorothioate bound.
  • C-type CpGs selected from the group consisting of (a) TCGTCGTTTTCGGCGCGCGCCG (SEQ ID NO:66); (b) TCGTCGTTTTCGACGGCCGTCG (SEQ ID NO:67); (c) TCGTCGTTTTCCGGCGCGCCGG (SEQ ID NO:68); (d) TCGTCGTTTTCGGCGCGCGTCG (SEQ ID NO:69); (e) TCGGCGCGCGCCGTCGTCGTTT (SEQ ID NO:70); (f) TTGGCGCGCGCCGTCGTCGTTT (SEQ ID NO:71); (g) TCGTCGTTTTCGTCGGCCGCCG (SEQ ID NO:72); (h) TCGTCGTTTTCGGCTTTTGCCG (SEQ ID NO:73); (i) TCGTCGTTTTCGGCGTTTTTTT (SEQ ID NO:74); and (j) TCGTCGTTTTCGGCGGCCGCCG (SEQ ID NO:75) are potent inducers of IFN
  • One embodiment of the invention is a composition for use in a method of treating or preventing hypersensitivity in an animal, preferably a mammal, most preferably a human, the composition comprising a particle and an immunostimulatory nucleic acid, wherein said particle is packaged with said immunostimulatory nucleic acid, and wherein preferably said hypersensitivity is an allergy or a non-allergic hypersensitivity.
  • the compositions and pharmaceutical compositions of the invention can be used in a therapeutic as well as in a prophylactic treatment.
  • said hypersensitivity is selected from the group consisting of: (a) asthma, (b) rhinitis, (c) conjunctivitis, (d) rhinoconjuctivitis, (e) dermatitis, (f) urticaria, and (g) anaphylaxis.
  • said hypersensitivity is an allergy, wherein said allergy is preferably selected from IgE-mediated allergy and non IgE-mediated allergy.
  • said hypersensitivity is asthma, preferably IgE-mediated asthma, wherein said asthma can be intermittent or persistent asthma.
  • said hypersensitivity is atopic asthma.
  • said hypersensitivity is dermatitis, preferably eczema, most preferably atopic eczema.
  • said hypersensitivity is an IgE mediated allergy (type I allergy), wherein preferably said IgE-mediated allergy is an IgE-mediated allergy against a naturally occurring allergen, i.e. an allergen occurring in a natural source such as pollen, animal hair, house dust, dust mite etc.
  • a naturally occurring allergen i.e. an allergen occurring in a natural source such as pollen, animal hair, house dust, dust mite etc.
  • said allergy preferably said IgE-mediated allergy
  • said allergy is selected from the group consisting of: (a) pollen allergy (hay fever), (b) house dust allergy, (c) food allergy, (d) drug allergy, (e) insect venom allergy, preferably bee venom allergy, and (f) animal allergy, preferably cat allergy.
  • said allergy is an allergy against an allergen occurring in a source selected from the group consisting of (a) pollen; (b) dust, preferably house dust; (c) dust mite; (d) fungi, preferably aspergillus ; (e) mammalian epidermis, (f) bird feather; (g) insects, preferably bee venom; (h) food, preferably strawberry, kiwi, peanut, or wheat protein; (i) mammalian dander, preferably cat dander; (j) saliva; (k) serum; and (l) urine.
  • a source selected from the group consisting of (a) pollen; (b) dust, preferably house dust; (c) dust mite; (d) fungi, preferably aspergillus ; (e) mammalian epidermis, (f) bird feather; (g) insects, preferably bee venom; (h) food, preferably strawberry, kiwi, peanut, or wheat protein; (i) mammalian
  • said allergy preferably said IgE-mediated allergy, is an allergy against an allergen occurring in a source selected from the group consisting of: (a) trees, (b) grasses, (c) animal products, and (d) plant products.
  • said allergy is an allergy against an antigen selected from the group consisting of (a) bee venom phospholipase A2; (b) ragweed pollen Amb a 1; (c) birch pollen Bet v I; (d) white faced hornet venom 5 Dol m V; (e) house dust mite Der p 1; (f) house dust mite Der f 2; (g) house dust mite DerP 2; (h) dust mite Lep d; (i) fungus allergen Alt a 1; (j) fungus allergen Asp f 1; (k) fungus allergen Asp f 16; and (l) peanut allergens.
  • an antigen selected from the group consisting of (a) bee venom phospholipase A2; (b) ragweed pollen Amb a 1; (c) birch pollen Bet v I; (d) white faced hornet venom 5 Dol m V; (e) house
  • said allergy preferably said IgE-mediated allergy, is an allergy against cat allergen, preferably an allergy against FelD1 antigen.
  • said allergy is an allergy against dust mite, wherein preferably said dust mite is selected from: (a) Dermatophagoides pteronyssinus , (b) D. farinae , (c) D. microceras , (d) Euroglyphus maynei , (e) Glycyphagus sp., (f) Gohieria fusca , (g) Blomia tropicalis.
  • dust mite is selected from: (a) Dermatophagoides pteronyssinus , (b) D. farinae , (c) D. microceras , (d) Euroglyphus maynei , (e) Glycyphagus sp., (f) Gohieria fusca , (g) Blomia tropicalis.
  • said allergy preferably said IgE-mediated allergy
  • said allergy is pollen allergy (hay fever).
  • said allergy, preferably said IgE-mediated allergy is house dust allergy, preferably IgE-mediated allergy against house dust mite allergens contained in house dust, wherein said house dust mite allergens are preferably selected from the group consisting of (a) Der p 1; (b) Der f 2; and (c) DerP 2.
  • the present invention relates to the finding that particles, preferably VLPs, can be packaged with ISS-NA, preferably with single stranded DNA oligonucleotides rich in non-methylated C and G (CpGs).
  • a preferred embodiment of the invention is therefore a composition for use in a method of treating or preventing hypersensitivity in an animal, preferably a mammal, most preferably a human, the composition comprising a VLP and an unmethylated CpG containing oligonucleotide, wherein said VLP is packaged with said unmethylated CpG containing oligonucleotide.
  • a further preferred embodiment of the invention is a composition for use in a method of treating or preventing allergy in a human, the composition comprising a VLP of an RNA bacteriophage and an unmethylated CpG containing oligonucleotide, wherein said VLP of an RNA bacteriophage is packaged with said unmethylated CpG containing oligonucleotide, and wherein preferably said unmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27).
  • a very preferred embodiment of the invention is a composition for use in a method of treating or preventing allergy in a human, the composition comprising a VLP of RNA bacteriophage Q ⁇ and unmethylated CpG containing oligonucleotide G10 (SEQ ID NO:27), wherein said VLP of RNA bacteriophage Q ⁇ is packaged with said unmethylated CpG containing oligonucleotide G10.
  • compositions of the invention are processes for the production of the compositions of the invention and methods for treating hypersensitivity using said compositions, wherein preferably said hypersensitivity is atopic asthma, type I allergy or atopic eczema.
  • the invention provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising a VLP and an unmethylated CpG containing oligonucleotide, wherein said VLP is packaged with said unmethylated CpG-containing oligonucleotide, said process comprising the steps of (i) incubating said VLP (a) with said unmethylated CpG-containing oligonucleotide (b); (ii) adding RNase; and (iii) purifying said composition.
  • said VLP is produced in a bacterial expression system.
  • said RNase is RNase A.
  • said process comprises the steps of (i) incubating said VLP with RNase; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying the composition.
  • said VLP is produced in a bacterial expression system.
  • said RNase is RNase A.
  • said process comprising the steps of (i) disassembling said VLP; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) reassembling said VLP.
  • said process further comprises the step of removing nucleic acids from the disassembled VLP.
  • said process further comprises the step of removing nucleic acids of the at least partially disassembled VLP and/or purifying the composition after reassembly.
  • said VLP is produced in a bacterial expression system.
  • said VLP referred to in step (i) of said process is a VLP of an RNA bacteriophage, more preferably a VLP of an RNA bacteriophage selected from the group consisting of (a) bacteriophage Q ⁇ , (b) bacteriophage AP205, (c) bacteriophage GA, and (d) bacteriophage fr.
  • said VLP referred to in step (i) of said process is a VLP of a RNA bacteriophage, more preferably a VLP of RNA bacteriophages AP205 or Q ⁇ , most preferably of Q ⁇ .
  • said unmethylated CpG-containing oligonucleotide referred to in step (ii) of said process consists of 5 to 60 nucleotides, preferably of 20 to 40 nucleotides most preferably of about 30 nucleotides.
  • said unmethylated CpG-containing oligonucleotide is an A-type CpG, preferably an A-type CpG comprising poly G motifs at the 5′ and/or 3′ ends.
  • said unmethylated CpG-containing oligonucleotide is selected from the group consisting of: (a) “G8-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25); (b) “G9-9” GGGGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:26); or (c) “G10” GGGGGGGGGG GACGATCGTC GGGGGGGGGG (SEQ ID NO:27), most preferably SEQ ID NO:27.
  • said process comprises the steps of (i) incubating said VLP in a solution comprising metal ions capable of hydrolyzing the nucleic acids of said VLP; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying said composition, wherein preferably said metal ions of step (i) are selected from the group consisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe) ions; (d) any mixtures of at least one ion of (a), (b) and/or (c).
  • said VLP is produced in a bacterial expression system.
  • said process comprises the steps of (i) incubating said VLP with solutions comprising metal ions capable of hydrolyzing the nucleic acids of said VLP; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying said composition, wherein preferably said metal ions of step (i) are selected from the group consisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe) ions; (d) magnesium (Mg) ions, and any mixtures of at least one ion of (a), (b), (c) and/or (d).
  • said VLP is produced in a bacterial expression system.
  • said process comprises the steps of (i) incubating said VLP with a solution capable of destabilizing said VLP; (ii) purifying the coat protein from said solution; and (iii) reassembling said VLP in the presence of unmethylated CpG-containing oligonucleotide, wherein preferably said solution capable of destabilizing said VLP comprises magnesium chloride, wherein further preferably the concentration of said magnesium chloride is 0.2 to 1.5 M, more preferably 0.4 to 1 M, most preferably about 0.7 M; and wherein still further preferably said VLP is a VLP of bacteriophage Q ⁇ .
  • said process comprises the steps of (i) incubating said VLP with a solution capable of destabilizing said VLP; (ii) purifying the coat protein from said solution; and (iii) reassembling said VLP in the presence of unmethylated CpG-containing oligonucleotide, wherein preferably said solution capable of destabilizing said VLP comprises magnesium chloride, wherein further preferably the concentration of said magnesium chloride is 0.2 to 1.5 M, more preferably 0.4 to 1 M, most preferably about 0.7 M; and wherein still further preferably said VLP is a VLP of bacteriophage Q ⁇ ; and wherein still further preferably said unmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27), most preferably said unmethylated CpG-containing oligonucleotide is aggregated G10 having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 9
  • said process comprises the steps of (i) incubating said VLP under alkaline conditions, preferably in the presence of NaOH, most preferably in the presence of about 25 mM NaOH; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying said composition.
  • the optimal aggregation conditions may vary between different unmethylated CpG-containing oligonucleotides and even between different batches of the same unmethylated CpG-containing oligonucleotide.
  • the actual aggregation state of an unmethylated CpG-containing oligonucleotide can be assessed by HPLC, preferably under conditions as set forth in Example 2.
  • the invention provides a process comprising the steps of (i) disassembling said VLP; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) reassembling said VLP, wherein prior to said adding of said unmethylated CpG-containing oligonucleotide said process comprises the additional step of incubating said unmethylated CpG-containing oligonucleotide under conditions supporting the formation of oligonucleotide aggregates. In a preferred embodiment said incubating is performed at a temperature of 70 to 100° C., preferably at about 85° C., preferably in the presence of sodium ions.
  • said incubating is performed at a concentration of said unmethylated CpG-containing oligonucleotide of 100 to 250 ⁇ m, preferably about 175 ⁇ m, in the presence of 200 to 500 mM sodium ions, preferably in the presence of about 250 mM sodium ions, preferably at 85° C. for about 10 min, wherein further preferably said unmethylated CpG-containing oliginucleotide comprises poly G motifs at their 5′ and/or 3′ ends.
  • said unmethylated CpG-containing oliginucleotide is selected from the group consisting of: (a) “G8-8” GGGGGGGG GACGATCGTC GGGGGGGG (SEQ ID NO:25); (b) “G9-9” GGGGGGGGG GACGATCGTC GGGGGGGGG (SEQ ID NO:26); or (c) “G10” GGGGGGGGGG GACGATCGTC GGGGGGGGGGGG (SEQ ID NO:27), most preferably said unmethylated CpG-containing oliginucleotide is G10 (SEQ ID NO:27).
  • said conditions supporting the formation of oligonucleotide aggregates are chosen in such a way that aggregated unmethylated CpG containing oligonucleotide, preferably aggregated G10 (SEQ ID NO:27) is obtained, wherein said aggregated unmethylated CpG containing oligonucleotide, preferably said aggregated G10 (SEQ ID NO:27) shows a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • the invention further provides a process of producing a composition for use in a method of treating hypersensitivity in an animal, said composition comprising (a) a virus-like particle, a VLP of bacteriophage Q ⁇ ; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b), said process comprising the steps of (i) incubating said VLP with a solution capable of destabilizing said VLP; (ii) purifying the coat protein from said solution; and (iii) reassembling said coat protein to a VLP in the presence of unmethylated CpG-containing oligonucleotide and an oxidizing agent.
  • said solution capable of destabilizing said VLP comprises magnesium chloride and a reducing agent, wherein preferably the concentration of said magnesium chloride is 0.2 to 1.5 M, more preferably 0.4 to 1 M, most preferably about 0.7 M; and wherein further preferably said reducing agent is DTT, and wherein still further preferably the concentration of said DTT is 1 to 100 mM, preferably 2 to 15 mM, more preferably about 10 mM and most preferably about 10 mM.
  • said oxidizing agent is H 2 O 2 , wherein further preferably the concentration of said H 2 O 2 is 1 to 50 mM, preferably 1 to 10 mM, most preferably about 7 mM.
  • said reassembling of said coat protein to a VLP is performed in the presence of salt, wherein preferably said salt is NaCl, and wherein further preferably the concentration of said salt, preferably of said NaCl is 100 mM to 1 M, most preferably 100 mM to 500 mM, most preferably about 250 mM.
  • said purified coat protein is incubated in a solution comprising salt, reducing agent and unmethylated CpG-containing oligonucleotide, wherein preferably (i) said salt is NaCl, and wherein further preferably the concentration of said salt, preferably of said NaCl is 100 mM to 1 M, most preferably 100 mM to 500 mM, most preferably about 250 mM; (ii) the concentration of said urea is 100 mM to 7 M, more preferably 500 mM to 2 M, most more preferably about 1 M; and (iii) said reducing agent ist DTT, wherein preferably the concentration of said DTT is 1 to 10 mM, preferably 1 to 5 mM, most preferably 2.5 mM.
  • said unmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27), wherein most preferably said unmethylated CpG-containing oligonucleotide is aggregated G10 having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • compositions for use in a method of treating hypersensitivity in an animal wherein said compositions are obtainable by any one of the processes disclosed herein.
  • a virus particle instead of said VLP, wherein said virus particle preferably is a virus particle of a bacteriophage, preferably of a RNA bacteriophage.
  • the invention provides compositions for use in a method of treating hypersensitivity in an animal, wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with unmethylated CpG-containing oligonucleotides; (ii) adding RNase; and (iii) purifying said composition.
  • said VLP is produced in a bacterial expression system.
  • said RNase is RNase A.
  • compositions for use in a method of treating hypersensitivity in an animal wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with RNase; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) purifying the composition.
  • said VLP is produced in a bacterial expression system.
  • said RNase is RNase A.
  • the invention further provides compositions for use in a method of treating hypersensitivity in an animal, wherein said compositions are obtainable by a process comprising the steps of (i) disassembling a VLP; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) reassembling said VLP.
  • said process further comprises the step of removing nucleic acids from the disassembled VLP.
  • said process further comprises the step of removing nucleic acids of the at least partially disassembled VLP and/or purifying the composition after reassembly.
  • said VLP is produced in a bacterial expression system.
  • the invention further provides compositions for use in a method of treating hypersensitivity in an animal, wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with solutions comprising metal ions capable of hydrolyzing the nucleic acids of said VLP; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) purifying said composition, wherein preferably said metal ions of step (i) are selected from the group consisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe) ions; (d) any mixtures of at least one ion of (a), (b) and/or (c).
  • said VLP is produced in a bacterial expression system.
  • the invention further provides compositions for use in a method of treating hypersensitivity in an animal, wherein said compositions are obtainable by a process comprising the steps of (i) incubating a VLP with solutions comprising metal ions capable of hydrolyzing the nucleic acids of said VLP; (ii) adding unmethylated CpG-containing oligonucleotides; and (iii) purifying said composition, wherein preferably said metal ions of step (i) are selected from the group consisting of (a) zinc (Zn) ions; (b) copper (Cu) ions; (c) iron (Fe) ions; (d) magnesium (Mg) ions; and (e) any mixtures of at least one ion of (a), (b), (c) and/or (d).
  • said VLP is produced in a bacterial expression system.
  • compositions for use in a method of treating hypersensitivity in an animal wherein said compositions are obtainable by a process comprising the steps of (i) incubating said VLP with a solution capable of destabilizing said VLP, wherein preferably said VLP is a VLP of bacteriophage Q ⁇ ; (ii) purifying the coat protein from said solution; and (iii) reassembling said coat protein to a VLP in the presence of unmethylated CpG-containing oligonucleotide and an oxidizing agent.
  • said solution capable of destabilizing said VLP comprises magnesium chloride and a reducing agent, wherein preferably the concentration of said magnesium chloride is 0.2 to 1.5 M, more preferably 0.4 to 1 M, most preferably about 0.7 M; and wherein further preferably said reducing agent is DTT, and wherein still further preferably the concentration of said DTT is 1 to 100 mM, preferably 2 to 15 mM, more preferably about 10 mM and most preferably about 10 mM.
  • said oxidizing agent is H 2 O 2 , wherein further preferably the concentration of said H 2 O 2 is 1 to 50 mM, preferably 1 to 10 mM, most preferably about 7 mM.
  • said reassembling of said coat protein to a VLP is performed in the presence of salt, wherein preferably said salt is NaCl, and wherein further preferably the concentration of said salt, preferably of said NaCl is 100 mM to 1 M, most preferably 100 mM to 500 mM, most preferably about 250 mM.
  • said purified coat protein is incubated in a solution comprising salt, reducing agent and unmethylated CpG-containing oligonucleotide, wherein preferably (i) said salt is NaCl, and wherein further preferably the concentration of said salt, preferably of said NaCl is 100 mM to 1 M, most preferably 100 mM to 500 mM, most preferably about 250 mM; (ii) the concentration of said urea is 100 mM to 7 M, more preferably 500 mM to 2 M, most more preferably about 1 M; and (iii) said reducing agent ist DTT, wherein preferably the concentration of said DTT is 1 to 10 mM, preferably 1 to 5 mM, most preferably 2.5 mM.
  • said unmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27), wherein most preferably said unmethylated CpG-containing oligonucleotide is aggregated G10 having a retention time relative to Q ⁇ capsid standard under HPLC conditions as set forth in Example 2 of 80 to 120%, most preferably of 80 to 95%.
  • compositions for use in a method of treating hypersensitivity in an animal wherein said compositions are obtainable by a process comprising the steps of (i) incubating said VLP under alkaline conditions, preferably in the presence of NaOH, most preferably in the presence of about 25 mM NaOH; (ii) adding said unmethylated CpG-containing oligonucleotide; and (iii) purifying said composition.
  • compositions for use as a medicament wherein said compositions are obtainable by any one of the processes of the invention, said composition comprising a particle and an ISS-NA, wherein said particle is packaged with said unmethylated CpG-containing oligonucleotide, wherein said particle preferably is a VLP of a RNA bacteriophage, most preferably of RNA bacteriophage Q ⁇ , and wherein preferably said ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein said unmethylated CpG-containing oligonucleotide preferably exclusively consists of phosphodiester bound nucleotides, wherein further preferably said unmethylated CpG-containing oligonucleotide comprises the palindromic sequence of SEQ ID NO:28, wherein most preferably said unmethylated CpG-containing oligonucleotide is G10 (SEQ ID NO:27).
  • the invention further provides compositions for use in a method of treating hypersensitivity, preferably allergy, most preferably atopic asthma, atopic eczema, pollen allergy, house dust or dust mite allergy, in an animal, wherein said compositions are obtainable by any one of the processes of the invention, said composition comprising (a) a VLP; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b), wherein said VLP preferably is a VLP of an RNA bacteriophage, most preferably of RNA bacteriophage Q ⁇ , and wherein said unmethylated CpG-containing oligonucleotide preferably exclusively consists of phosphodiester bound nucleotides, wherein further preferably said unmethylated CpG-containing oligonucleotide comprises the palindromic sequence of SEQ ID NO:28, wherein most
  • the invention further provides compositions for use in a method of treating hypersensitivity, preferably allergy, most preferably atopic asthma, atopic eczema, pollen allergy, house dust or dust mite allergy, in an animal, wherein said compositions are obtainable by any one of the processes of the invention, said composition comprising a VLP and an unmethylated CpG-containing oligonucleotide, wherein said virus-like particle is packaged with said unmethylated CpG-containing oligonucleotide, wherein said VLP preferably is a VLP of a RNA bacteriophage, most preferably of RNA bacteriophage Q ⁇ , and wherein said unmethylated CpG-containing oligonucleotide preferably exclusively consists of phosphodiester bound nucleotides, wherein further preferably said unmethylated CpG-containing oligonucleotide comprises the palindromic sequence of SEQ ID NO:28, wherein most preferably said unmethylated CpG-containing
  • compositions of the invention comprise, or alternatively consist of, an immunologically effective amount of the inventive compositions together with a pharmaceutically acceptable diluent, carrier or excipient.
  • the pharmaceutical composition may also optionally comprise an adjuvant.
  • said pharmaceutical composition comprises a slow release formulation of the composition of the invention.
  • Slow release formulations are well known in the art.
  • Typical and preferred slow release formulations are compositions of the invention formulated in microparticles, emulsions, and gels.
  • the invention provides pharmaceutical compositions for treating or preventing atopic eczema. In another embodiment, the invention provides pharmaceutical compositions for treating or preventing asthma. In another embodiment, the invention provides pharmaceutical compositions for treating or preventing IgE-mediates allergy (type I allergy), preferably pollen allergy, house dust or dust mite allergy.
  • type I allergy preferably pollen allergy, house dust or dust mite allergy.
  • compositions of the invention when administered to an animal, they can be in a composition which contains salts, buffers, adjuvants or other substances which are desirable for improving the efficacy of the composition.
  • materials suitable for use in preparing pharmaceutical compositions are provided in numerous sources including R EMINGTON'S P HARMACEUTICAL S CIENCES (Osol, A, ed., Mack Publishing Co., (1990)).
  • adjuvants can be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum .
  • BCG Bacille Calmette-Guerin
  • compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, AdjuVax 100a, QS-21, QS-18, CRL1005, Aluminum salts (Alum), MF-59, OM-174, OM-197, OM-294, isomatrix, and virosomal adjuvant technology.
  • the adjuvants can also comprise a mixture of these substances.
  • Immunologically active saponin fractions having adjuvant activity derived from the bark of the South American tree Quillaja Saponaria Molina are known in the art.
  • QS21 also known as QA21
  • QA21 is an Hplc purified fraction from the Quillaja Saponaria Molina tree and it's method of its production is disclosed (as QA21) in U.S. Pat. No. 5,057,540.
  • Quillaja saponin has also been disclosed as an adjuvant by Scott et al, Int. Archs. Allergy Appl. Immun., 1985, 77, 409.
  • Monosphoryl lipid A and derivatives thereof are known in the art.
  • a preferred derivative is 3 de-o-acylated monophosphoryl lipid A, and is known from British Patent No. 2220211. Further preferred adjuvants are described in WO00/00462, the disclosure of which is herein incorporated by reference.
  • 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 can be administered by various methods known in the art.
  • the particular mode selected will depend of course, upon the particular composition selected, the severity of the condition being treated and the dosage required for therapeutic efficacy.
  • the methods of the invention generally speaking, can be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects.
  • modes of administration include oral, rectal, parenteral, intracisternal, intravaginal, intraperitoneal, topical (as by powders, ointments, drops or transdermal patch), bucal, or as an oral or nasal spray.
  • compositions of the invention can also be injected directly in a lymph node.
  • compositions comprising microparticles are preferably injected subcutaneously, intravenously, intradermally, intraperitoneally, administered intranasally, orally, transdermally or inhaled.
  • 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.
  • Dosage levels depend on the mode of administration, the nature of the subject, and the quality of the carrier/adjuvant formulation. Typical amounts are in the range of about 0.001 ⁇ g to about 20 mg per subject. Preferred amounts are 50 ⁇ g to 1000 ⁇ g, more preferably 100 ⁇ g to 600 ⁇ g, and most preferably about 300 ⁇ g of a composition of the invention per single administration. Further preferred amounts are at least about 50 ⁇ g to about 500 ⁇ g per subject, most preferably 300 ⁇ g per subject. Multiple administration to treat the subject is preferred, and protocols are those standard in the art adapted to the subject in question.
  • the administration of said composition or said pharmaceutical composition is repeated several times, preferably at least three to 10 times, most preferably three to five times, in weekly, monthly or yearly intervals, preferably in intervals of about 1 week to about 1 month, more preferably in biweekly intervals, most preferably in weekly intervals.
  • the administration of said composition or said pharmaceutical composition is repeated 6 times in weekly intervals, wherein preferably each time 50 ⁇ g to about 500 ⁇ g, most preferably about 300 ⁇ g are administered.
  • compositions of the invention can conveniently be presented in unit dosage form and can be prepared by any of the methods well-known in the art of pharmacy. Methods include the step of bringing the compositions of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compositions of the invention into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • compositions suitable for oral administration can be presented as discrete units, such as capsules, tablets or lozenges, each containing a predetermined amount of the compositions of the invention.
  • Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, an elixir or an emulsion.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compositions of the invention described above, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art.
  • a further aspect of the invention is a method of treating hypersensitivity, preferably atopic eczema, atopic asthma or IgE-mediated allergy (type I allergy), in an animal, preferably a mammal, most preferably a human, said method comprising introducing into said animal (i) a composition comprising a particle and an ISS-NA, wherein said particle is packaged with said ISS-NA or (ii) a pharmaceutical composition comprising an immunologically effective amount of the composition (i) together with a pharmaceutically acceptable diluent, wherein preferably said pharmaceutical composition (ii) further comprises an adjuvant.
  • said composition (i) or said pharmaceutical composition (ii) is introduced into said animal subcutaneously, intramuscularly, intravenously, intranasally or directly into the lymph node.
  • said introducing into said animal of the composition (i) or the pharmaceutical composition (ii) is repeated at least twice, preferably at least three times, most preferably at least four times in intervals of 1 week to 3 months, preferably 1 week.
  • said introducing into said animal of the composition (i) or the pharmaceutical composition (ii) is repeated 6 times in intervals of about 1 week, wherein preferably each time about 300 ⁇ g of said composition (i) are introduced.
  • a preferred embodiment of the invention is a method of treating allergy in an animal, said method comprising introducing into said animal a composition comprising (a) a VLP; and (b) an unmethylated CpG-containing oligonucleotide; wherein said virus-like particle (a) is packaged with said unmethylated CpG-containing oligonucleotide (b), and wherein said allergy preferably is atopic eczema, asthma or type I allergy, preferably pollen allergy (hay fever), wherein further said VLP is a VLP of a RNA bacteriophage, preferably of RNA bacteriophage Q ⁇ , and wherein preferably said unmethylated CpG-containing oligonucleotide (b) consists exclusively of phosphodiester bound nucleotides, most preferably of SEQ ID NO:27.
  • the effectiveness of a treatment of the invention with respect to a particular disease can be assessed by assessing the severity of the symptoms associated with said disease using standard methods known in the art. Generally symptoms are scored directly before the beginning of the treatment, i.e. before the first vaccination, in intervals during the treatment and 1 to 3 months after the last treatment. Symptoms of atopic dermatitis can, for example be scored as described in N. Engl. J. Med 1997, 337:816-21. Symptoms of asthma can be scored by various methods including questionnaires described in Juniper et al., Health Qual. Life Outcomes, 2005 Sep. 16, 3:58, and combinations of questionnaires and spirometric measurements of pulmonary functions as described in N. Engl. J. Med 2000, 343:1054-63.
  • Pollen allergy can, inter alia, be assessed using a nasal provocation test, other allergies, e.g. house dust or dust mite allergy, can be assessed using a conjunctival provocation procedure or a skin prick test. These testing methods are described in detail in the Example section.
  • a further aspect of the invention is the use of a composition of the invention or of a pharmaceutical composition of the invention for the manufacture of a pharmaceutical for the treatment of hypersensitivity in an animal, wherein said hypersensitivity preferably is an allergy, wherein further preferably said allergy is selected from the group consisting of: (a) atopic eczema; (b) atopic asthma; and (c) IgE-mediated allergy (type I allergy), preferably pollen allergy (hay fever) or house dust allergy.
  • a preferred embodiment of the invention is the use of a composition comprising a particle and an ISS-NA, wherein said particle is packaged with said ISS-NA, for the manufacture of a pharmaceutical for the treatment of hypersensitivity in an animal, wherein hypersensitivity is preferably selected from the group consisting of: (a) atopic eczema; (b) atopic asthma; and (c) IgE-mediated allergy (type I allergy), preferably pollen allergy (hay fever), and wherein further preferably said particle is a VLP of a RNA bacteriophage, preferably of RNA bacteriophage Q ⁇ , and wherein preferably said ISS-NA is an unmethylated CpG-containing oligonucleotide, wherein preferably said unmethylated CpG-containing oligonucleotide consists exclusively of phosphodiester bound nucleotides, and wherein more preferably said unmethylated CpG-containing oligonucleotide comprises the palindromic sequence of SEQ ID NO:28
  • said particle is a VLP of bacteriophage Q ⁇
  • said VLP preferrably essentially consists of coat proteins having the amino acid sequence of SEQ ID NO:3.
  • said ISS-NA is the unmethylated CpG-containing oligonucleotide G10 (SEQ ID NO:27)
  • said unmethylated CpG-containing oligonucleotide preferrably consists exclusively of phosphodiester bound nucleotides.
  • said particle is a VLP of bacteriophage Q ⁇ and wherein said ISS-NA is the unmethylated CpG-containing oligonucleotide G10 (SEQ ID NO:27), said VLP of bacteriophage Q ⁇ preferrably essentially consists of coat proteins having the amino acid sequence of SEQ ID NO:3, and, further preferably, said unmethylated CpG-containing oligonucleotide consists exclusively of phosphodiester bound nucleotides.
  • VLPs of bacteriophage Q ⁇ used in the Examples are/were VLPs essentially consisting of coat proteins having the amino acid sequence of SEQ ID NO:3.
  • the unmethylated CpG containing oligonucleotide G10 (SEQ ID NO:27) used in the Examples is/was unmethylated CpG containing oligonucleotide G10 (SEQ ID NO:27) consisting exclusively of phosphodiester bound nucleotides.
  • VLPs Bacterial produced VLPs contain high levels of single stranded RNA, randomly packaged into the VLP during self assembly, which can be visualized on a 1% agarose gel stained with ethidium bromide or Coomassie blue for the detection of RNA/DNA or protein.
  • the RNA has to be removed from the VLP before packaging with CpG DNA by digestion with RNase A. Therefore 1 mg/ml VLP in 20 mM HEPES, pH 7.4 was incubated with 300 ⁇ g RNase A for 3 h at 37° C.
  • the VLP were dialysed for 2-3 days against 20 mM HEPES using a 100'000 or 300'000 MW cut off membrane.
  • CpG-oligonucleotide 1668-CpG TCC ATG ACG TTC CTG AAT AAT, SEQ ID NO:80
  • a phopshorothioate backbone 1 ml VLP (1 mg/ml in 20 mM HEPES, pH 7.4, 2 mM MgCl 2 ) with 100 nmol CpG) allowing free diffusion into the particle during incubation for 3 hrs at 37° C.
  • the CpG-oligonucleotide containing VLPs were purified from unbound CpG-oligonucleotide via tangential flow filtration using a 300'000 MW cut off membrane.
  • a 1% agarose gel stained with ethidium bromide or Coomassie blue visualized the removal of RNA followed by packaging of CpG into VLP and proves the removal of unbound CpG.
  • Aggregation (10.0 ml scale, 175 ⁇ M G10, 250 mM NaOH, 85° C., 9-24 min): 7.1 ml disaggregated G10 solution, 2.13 ml purified water and 770 ⁇ l 3 M NaCl were mixed in a 15 ml tube (175 ⁇ M oligo, 250 mM Na + ). The mixture was incubated for 9 minutes at 85° C. in a water bath. The solution was cooled down in an ice/water bath and stored on ice until use. Aggregated oligonucleotide solutions should be used within 3 hours after preparation.
  • Q ⁇ VLPs 45 mg Q ⁇ VLP (2.5 mg/ml, as determined by Bradford analysis) in PBS (20 mM Phosphate, 150 mM NaCl, pH 7.5), was reduced with 10 mM DTT for 15 min at RT under stirring conditions. Then, magnesium chloride was added to 0.7 M final concentration and the incubation was continued for 15 min at RT under stirring conditions, leading to precipitation of the encapsulated host cell RNA and concomitant disintegration of the VLPs. The solution was centrifuged 10 min at 4000 rpm at 4° C.
  • the isolated Q ⁇ coat protein (the eluted fraction from the cation exchange column) was loaded onto a Sephacryl S-100 HR column (xk26/60, 320 ml, Amersham Bioscience) equilibrated with 20 mM sodium phosphate/250 mM sodium chloride; pH 7.2.
  • the chromatography was carried out at RT with a flow rate of 2.5 ml/min and the absorbance was monitored at 260 nm and 280 nm. Fractions of 5 ml were collected.
  • the solution was then diafiltrated at room temperature against 300 ml of 20 mM sodium phosphate 250 mM NaCl pH 7.2, using a 30 kDa cut off cartridge (Pellicon XL, Millipore) and a cross flow rate of 10 ml/min and a permeate flow rate of 2.5 ml/min.
  • H 2 O 2 was added to 7 mM final concentration and the solution incubated for 1 h at RT in order to induce the formation of disulfide bonds.
  • the solution was then diafiltrated against 500 ml of 20 mM sodium phosphate 150 mM NaCl pH 7.2, using a 300 kDa cut off cartridge (Pellicon XL, Millipore) and a cross flow rate of 10 ml/min and a permeate flow rate of 2.5 ml/min, in order to remove excess of H 2 O 2 and non-packaged G10 oligonucleotides from the assembled Q ⁇ G10 product.
  • a 300 kDa cut off cartridge Pellicon XL, Millipore
  • Characterization of packaged Q ⁇ G10 VLP by analytical size exclusion chromatography A sample of packaged Q ⁇ G10 VLP was analyzed by analytical size exclusion chromatography ( FIG. 2 ) and compared to intact Q ⁇ VLP, which had been purified from E. coli lysate. The presence of correctly assembled VLP in the product was confirmed by a peak migrating at identical retention time as the peak representing native Q ⁇ VLP. The observed peak for Q ⁇ G110 VLP ( FIG. 2D ) is dominated by the nucleic acid content of the VLP, because the absorption coefficient nucleic acids at 260 nm is more than 100-fold higher than the absorption coefficient of the coat protein.
  • Characterization of packaged Q ⁇ G10 VLP by SDS-PAGE analysis A sample of packaged Q ⁇ G10 was analyzed by non-reducing SDS-PAGE ( FIG. 3 ) and compared to intact Q ⁇ VLP, which had been purified from E. coli lysate. The presence of correctly assembled VLP in the product was confirmed by the formation of bands of disulfide-linked pentameric and hexameric forms of the coat protein, similar to the intact Q ⁇ VLPs, indicating the correct structural arrangement of the coat protein units in the in vitro assembled Q ⁇ G10 VLP.
  • the amount of packaged oligonucleotide G10 was 0.2 mg per mg of Q ⁇ coat protein.
  • G10 content of Q ⁇ G10 VLP and yield calculation for the packaging reaction Aggregated G10 was packaged into Q ⁇ VLP by assembly/reassembly of the VLP as described in Example 3. 953 mg G10 oligonucleotide were introduced for reassembly with 4000 mg purified Q ⁇ dimer. The reaction yielded Q ⁇ G10 comprising 20 ⁇ g G10 oligonucleotide per 100 ⁇ g protein (protein content determined by Bradford analysis or HPLC). The G10 yield of the packaging reaction was 63% at a protein yield of 75%.
  • AP205 or GA355 VLPs (as determined by Bradford analysis) in buffer A (5 mM NaPO 4 pH 6.8, 100 mM NaCl, 2 mM MgCl 2 ) were incubated at 30° C. for 16 hours with RNAse A (Sigma) and Benzonase (Novagen) at 1 mg/ml and 5 U/ml, respectively.
  • RNAse A Sigma
  • Benzonase Novagen
  • AP205 VLP deoxidation of the internal disulfide bridges was performed preceding the addition of RNAse A and Benzonase by addition of 20 mM DTT followed by a 30 min incubation at 37° C.
  • Coat proteins were eluted as one fraction with buffer D (20 mM NaPO 4 pH 6.5, 1 M urea, 300 mM NaCl) and analyzed by LDS-PAGE followed by Coomassie staining. Eluted protein fractions were stored at 4° C. as “disassembled coat protein”. Protein concentrations were determined by Bradford analysis.
  • Reassembly Purified AP205 or GA355 coat protein with were used in a five fold excess (w/w) to G10 oligonucleotide.
  • the coat proteins were mixed with the G10 oligonucleotide in a reassembly buffer containing 1 M urea and 2.5 mM DTT and incubated for one hour at room temperature. After incubation the reassembly mix was dialyzed for 24 hours against 5 liter PBS. The resulting suspension was centrifuged for 10 min, 27,000 g at 4° C. A negligible sediment was discarded. The supernatant contained the reassembled and packaged VLPs. Protein concentration was determined by Bradford analysis and the reassembled and packaged VLPs were concentrated with centrifugal filter devices (Amicon Ultra 15, 10K MWCO).
  • FR VLPs (as determined by Bradford analysis) in buffer A (5 mM NaPO 4 pH 6.8, 100 mM NaCl, 2 mM MgCl 2 ) are incubated at 30° C. for 16 hours with RNAse A (Sigma) and Benzonase (Novagen) at 1 mg/ml and 5 U/ml, respectively.
  • RNAse A Sigma
  • Benzonase Novagen
  • 1 M NaCl precipitation of the FR coat proteins is induced by a 15 min incubation at 70° C.
  • Precipitated coat proteins are sedimented by centrifugation for 10 min, 27,000 g at 4° C.
  • the supernatant containing RNAse A. Benzonase and degraded nucleic acids are discarded.
  • the pellet is resuspended in buffer B (20 mM NaPO 4 pH 7.2, 6 M urea) and incubated for 10 min at room temperature.
  • FR coat proteins are eluted as one fraction with buffer D (20 mM NaPO 4 pH 6.5, 1 M urea, 300 mM NaCl) and analyzed by LDS-PAGE followed by Coomassie staining. The eluted protein fractions is stored at 4° C. as “disassembled coat protein”. Protein concentration is determined by Bradford analysis.
  • FR coat protein is used in a five fold excess (w/w) to G10 oligonucleotide.
  • the FR coat proteins are mixed with the G10 oligonucleotide in a reassembly buffer containing 1 M urea and 2.5 mM DTT and incubated for one hour at room temperature. After incubation the reassembly mix is dialyzed for 24 hours against 5 liter PBS. The resulting suspension is centrifuged for 10 min, 27,000 g at 4° C. A negligible sediment is discarded. The supernatant contains the reassembled and packaged FR VLPs. Protein concentration is determined by Bradford analysis and the reassembled and packaged FR VLPs are concentrated with centrifugal filter devices (Amicon Ultra 15, 10K MWCO).
  • FR VLPs Purification of reassembled and packaged FR VLPs: Up to 25 mg total protein is loaded onto a SepharoseTM CL-4B (26/60, Amersham Biosciences) equilibrated with PBS. Size exclusion chromatography is performed with equilibration buffer at room temperature with a flow rate of 1.25 ml/min. During the elution absorbance at 254 nm and 260 nm is monitored. Two peaks are isolated. A major high molecular weight peak precedes a small peak of lower apparent molecular weight. The major peak reveals a apparent molecular weight consistent to purified FR VLPs as shown by SE-HPLC. Analysis of FR VLPs packaged with G10 oligonucleotide is performed essentially as shown in Example 16 of WO 03/024481 (p. 131 ff).
  • yellow-green fluorescent polystyrene nanoparticles with sizes ranging from 20 nm to 2000 nm (Molecular probes) were injected in the footpads (25 ⁇ g/footpads) of C57BL/6 mice and tracked.
  • VLPs of bacteriophage Q ⁇ were coupled to Alexa488 using a protein labeling kit (Molecular probes) and included in the experiment as 30 nm fluorescent particles. Forty eight hours later the nanoparticles and VLPs were tracked in the popliteal draining lymph node (LN) by flow cytometry.
  • Popliteal LN were isolated and digested with 1 mg/ml Collagenase D (Boehringer) and 0.04 mg/ml DNA-se I (Roche) for 30 min at 37° C.
  • LN cells were stained with anti-CD11c-PE (BD).
  • BD anti-CD11c-PE
  • Flow cytometry analysis showed that 48 hours after injection, nanoparticles and VLPs have reached the popliteal LN and were associated to different level with LN cells, including dendritic cells (DC, Table 1). LN cells acquired more efficiently smaller particles (20-500), than larger ones (1000-2000 nm).
  • VLP-Alexa488 (30 nm) showed efficiency of LN uptake between the efficiency of 20 and 100 nm polystyrene nanoparticles. These data suggest that nanoparticles and VLPs traffic from the injection site to the LN in a size-dependent manner, with significant trafficking taking place for particles of sizes between 20 and 500 nm. TABLE 1 Flow cytometry analysis of the trafficking of nanoparticles or VLP to the popliteal LN: 48 h after injection. Anti - CD11c antibodies were used to identify DC. Size [nm] % FL1 + DC sd % FL + LN cells sd 20 0.82 0.08 2.46 0.15 30 1.38 0.18 4.14 0.65 100 2.31 0.36 12.09 1.22
  • VLP-Alexa488 prepared as described in Example 7 were injected in C57BL/6 mice (as described in Example 7) and tracked after 2 to 96 h.
  • LN cells were stained with combinations of the following antibodies: anti-CD11c-PE, anti-CD11b-APC, anti-CD11c-biotin, anti-CD19-APC, anti-B220-PE, antiCD8-CyChr, anti-CD40-APC and streptavidin-CyChr (all from BD).
  • VLP-Alexa488 have associated with antigen presenting cells (APC) from the popliteal LN (Table 2). It is quite unlikely that dendritic cells from the skin had taken up and had trafficked VLP-Alexa488 to the draining LN for the short period of 2 h. These results rather suggest that VLP-Alexa488 had drained in a cell-free manner through the lymphatic system into the draining lymph node. Macrophages and DC were the most prominent populations taking up VLP-Alexa488. Most importantly, 2 h after injection, 1.9% of LN-resident plasmacytoid DC also acquired VLP-Alexa488.
  • VLP-Alexa488 the ability of VLP-Alexa488 to drain free to the LN makes them a very appropriate vehicle to target pDC with the possibility to simultaneously deliver ISS (such as CpG), packaged in VLP.
  • VLP-Alexa488 peaked at 24 h after injection (Table 2), remained quite stable by 48 h and declined after 96 h. It is likely, that at that later time point also skin derived DC contribute to the traffic of VLP-Alexa488 to the draining LN. Indeed, the ratio between the uptake of VLP-Alexa488 at 2 h (draining) and 24 h (DC-mediated transport) is lower for myeloid (including skin-derived, CD11c+CD40hiCD8 ⁇ ) compared to lymphoid (CD11c+CD401oCD8+) DC, which acquire VLP while in the LN ( FIG. 4 ).
  • Macrophages are thought to be non-migratory cells, therefore particle bearing macrophages are supposed to be LN-resident cells that had acquired particles while in the LN.
  • DC have the capacity to engulf particles at the injection site and to migrate to the draining LN.
  • the fluorescent nanoparticles ranging in size from 20 to 2000 nm were injected in the footpads of mice as described in Example 7, and the ratio between the nanoparticle-containing DC and macrophages was calculated ( FIG. 5 ). The larger the injected particle (500-2000 nm), the more DCs are involved in its uptake in LN. Smaller particles (20-200 nm) were detected in comparable percentage of DC and macrophages, suggesting cell-free draining and association with LN-resident APC.
  • mice 11 week old mice from the LPS-resistant (C3H/HeJ) and LPS-responding (C3H/HeN) strains were sacrificed and the femurs and tibias isolated and kept in PBS. After removing the flesh, bones were cut from both sides and the bone marrow flushed out using a syringe filled with 10% FCS RPMI. Released cells were collected and passed through a 70 ⁇ m cell strainer. Cells were washed and re-suspended at 20 ⁇ 10 4 cells/ml in 10% FSC RPMI containing 10 ng/ml mouse GM-CSF (R&D). 2 ⁇ 10 6 cells were plated in bacterial grade Petri dishes in 10 ml medium for 6 days.
  • C3H/HeJ LPS-resistant
  • C3H/HeN LPS-responding
  • BMDC The differentiation status of BMDC was ascertained on day 6 by analyzing cells for the expression of CD11c and CD11b by flow cytometry.
  • BMDC were harvested from the Petri dishes, washed once and plated in 96-well U-bottom plate (BD) at 5 ⁇ 10 4 /well. 6 dilutions (4-fold) of Q ⁇ G10 were prepared in a separate plate and added to BMDC for 20 h.
  • ELISA for determination of IL-12 Microtiters plates (Maxisorp, Nunc) were coated overnight with 2 ⁇ g/ml rat anti-mouse IL-12 (p40/p70) monoclonal antibody (mAb, Pharmingen) in coating buffer (0.1 M NaHCO 3 , pH 9.6). After washing (0.05% Tween 20/PBS) and blocking (2% BSA/0.05% Tween 20/PBS), 70 ⁇ l of supernatants from BMDC or two-fold dilutions of recombinant IL-12 (starting from 20 ⁇ g/ml) were added and incubated for 2 h at room temperature (RT).
  • RT room temperature
  • biotin-labeled rat anti-mouse IL-12 mAb (p40/p70, Pharmingen) was added at 1 ⁇ g/ml for 1 h at RT. Plates were washed and incubated with 1 ⁇ g/ml streptavidin-HRPO (Jackson laboratories) for 1 h at RT. After washing, o-phenylene diamine (OPD, Fluka) in citric acid buffer (pH 5) was added for 5 min and the reaction was stopped with 5% H 2 SO 4 . Optical density at 490 nm was measured in an ELISA reader (Molecular Devices) and data were analyzed using the software Soft max Pro.
  • OPD o-phenylene diamine
  • BMDCs were activated by Q ⁇ G10 and hence secreted IL-12 in a dose dependent way (dose is given as equivalent of G10 oligonucleotide packaged in the Q ⁇ VLPs) while untreated control cells did not secrete any IL-12. Therefore G10 oligonucleotide packaged in Q ⁇ VLPs is able to activate BMDCs, demonstrating that the particles are taken up by the cells and oligonucleotide is made subsequently available for stimulation of the BMDCs.
  • BMDC from C3H/HeJ mice were in vitro generated as described in Example 11.
  • Cells were plated in a 96-well plate and stimulated either mock or with the indicated concentrations of AP205 reassembled with G10 (AP205G10). Eighteen hours later the supernatants were collected and IL-12 was measured by a sandwich ELISA, as described in Example 11.
  • AP205G10 induced a dose-dependent secretion of IL-12, while untreated cells released IL-12 only to basal levels (Table 5). These results demonstrate that AP205G10 is taken up by BMDC and activates them to release IL-12.
  • AP205G10 ⁇ g/ml IL-12 [pg/ml] 300 4699.7 150 4831.3 75 4535.9 37.5 3644.7 18.75 2760.3 9.375 1407.0 0 471.1
  • GNP plain Gelatin Nanoparticles
  • the procedure used was the original one described by (Coester et al. 2000) as follows. 1.25 g of gelatin type A (Bloom175) was dissolved in 25 ml highly purified water (HPW) to 5% (w/w) under gentle heating to 50° C. A first desolvation step was initiated by the addition of 25 ml acetone. After sedimentation of the precipitated gelatin fractions for 60 seconds, the supernatant consisting of dispersed gelatin as well as dissolved gelatin was discarded. The sediment was dissolved again by the addition of 25 ml water under heating to 50° C. and the pH was adjusted to 2.5.
  • HPW highly purified water
  • In situ gelatin nanoparticles were formed during a second desolvation step by drop wise addition of 50 ml acetone under constant stirring (500 rpm). After 10 min, 175 ⁇ l of glutaraldehyde (25%) were added to the reaction vessel to crosslink the nanoparticles. Finally, after stirring for 12 hours in an extractor hood, the particles were purified by three-fold centrifugation (20000 g for 20 min) and redispersion in acetone/water (30/70), the last step in HPW alone. The purified nanoparticles were stored as dispersion in HPW (conductivity ⁇ 0.04 ⁇ s/cm) at 4-8° C.
  • Particle sizes were determined employing dynamic light scattering (DLS) technology. DLS experiments were performed with a Zetasizer ZS (Malvern Instruments, Worcestershire, England) using NIBSTM-technology (Non Invasive Back Scattering) at a static detection angle of 173°. The nanoparticles were diluted in sterile filtered, highly purified water and measured in concentrations between 30 and 100 ⁇ g/ml. Due to these low concentrations, the nanoparticles did not influence the viscosity of the dispersion, so that the viscosity of pure water which is of 0.8872 cP at 25° C. was used. The experiments were performed at 25° C.
  • DLS dynamic light scattering
  • EDC 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride
  • the reaction was stopped after 1 h and the nanoparticles were purified by 3-fold centrifugation and redispersion, analogous to the purification of plain nanoparticles.
  • the final particles were characterized by size determination using dynamic light scattering, and measurement of the Zeta potential. Each assigned size and corresponding polydispersity index was the mean of 10 subruns.
  • the Zeta potential was determined using the same instrument and calculated as the mean of 10 individual measurements in PBS. Particles showing a positive charge of 5 mV or higher in PBS were used for loadings.
  • G10-ODN loading on cationized gelatin nanoparticles G10-ODN was employed either in an aggregated or non-aggregated form (omitting the aggregation step in the protocol for ODN G10 preparation, Example 2) in purified water and in PBS.
  • the contents were 1% for GNP and 0.1% for G10-ODN leading to a targeted loading of 10% (w/w) in PBS.
  • a G10-ODN content of 0.25% and GNP of 2.5% were prepared as well providing a 10% (w/w) loading in PBS.
  • the loading experiment is exemplary described for a targeted (aspired) 10% (w/w) G10-ODN loading on GNP in PBS, and the same procedure was adapted for the other targeted loading percentages using aggregated or non-aggregated G10-ODN.
  • 43.3 ⁇ l of an aqueous GNP dispersion containing 1.2 mg GNP were transferred to an EppendorfTM 1.5 ml cap under aseptic conditions.
  • 1086 ⁇ l of PBS were added and mixed at the same time with the pipette.
  • 71.4 ⁇ l of G10 stock solution containing 120 ⁇ g of aggregated G10 were added into the dispersion by quick but gentle up and down pipetting for at least 20 seconds.
  • the prepared sample was subsequently incubated for 1 h 15 min at 22° C. and 750 rpm on a ThermomixerTM device.
  • G10-ODN loading efficiency (Zillies and Coester 2004): After incubation, the G10-ODN loaded GNP samples to be tested for successful loading were centrifuged for 1 h at 15000 g. After centrifugation the supernatant was carefully separated from the remaining pellet and analyzed spectrophotometrically at a wavelength of 260 nm. A reference of unloaded GNP of the same concentration (1 mg/ml) and a reference of G10-ODN of the same concentration (100 g/ml) were prepared and centrifuged along.
  • G10-ODN loading in percent was calculated from the percentage free G10-ODN, which was obtained by subtracting the measured absorption of the particle reference from the sample result and then dividing this result by the measured absorption of the G10-ODN reference, and multiplying by hundred. Finally, the loading efficiency was calculated by subtracting the percentage free G10-ODN from 100%.
  • Results of G10-ODN loading determination Results of G10-ODN loading determination: Results of loading efficiency were obtained by various GNP batches. However, the preparation process and especially cationization was the same in each case. Hence, loadings can be compared as well as among batches with different particle sizes. All results given in percent are mass percentage (w/w) calculated on the employed masses of G10-ODN and GNP. As shown in Tables I-III for non-aggregated G10-ODN and Tables IV-V for aggregated G10-ODN, G10-GNPs with loading percentage from 3-15% (non-aggregated G10-ODN) or 5-10% (aggregated G10-ODN) were successfully obtained. Optimal loading medium was PBS, where no flocculation was detected in the preparations.
  • G10-loaded gelatin nanoparticles induce IL-12 secretion from BMDC: GNP-G10 were prepared as described above. To evaluate the effect of unbound G10, an aliquot of GNP-G10 preparation was centrifuged at 16000 g for 1 h at 4° C. and the supernatant was collected (supGNP-G10).
  • BMDC from C3H/HeJ mice were generated in vitro, as described in Example 11. Cells were plated in a 96-well plate and stimulated either mock or with the indicated concentrations of GNP-G10 or supGNP-G10 (given as their molar concentration of G10).
  • G10-loaded gelatin nanoparticles are protective in an mouse model of asthma: An experimental asthma model of allergic airway (Hessel E M et al., J. Exp. Med. 2005, 202, 1563) is used to assess the effects of GNP-G10 on total IgE level.
  • groups B-E five mice per group
  • mice are injected intraperitonealy (i.p.) with 150 ⁇ g ragweed ( Ambrosia artemisiifolia ) pollen extract (RW, Greer) mixed with 450 ⁇ g of Alum (Alhydrogel 2.0% Brenntag Biosector, Denmark) on day 0 and 3.
  • mice from one group are treated i.p. with PBS only.
  • mice from group C are injected subcutaneously with 300 ⁇ g QbG10 (equivalent to 60 ⁇ g G10), mice from group D with 600 ⁇ g GNP-G10 (equivalent to 60 ⁇ g G10), mice from group E with 600 ⁇ g GNP-G10 aggregated (equivalent to 60 ⁇ g G10) mice from groups A and B with PBS.
  • Blood is sampled from all animals prior to injection.
  • all animals are bled, and total IgE titer is determined. The total IgE level in blood is measured by ELISA.
  • an ELISA plates (96 well MAXIsorp, NUNC immuno plate #442404) is coated with anti-IgE (BD Pharmingen, # 553413) at a concentration of 5 ⁇ g/ml in coating buffer (0.1 M NaHCO3, pH 9.6) over night at 4° C.
  • the plates are then washed with wash buffer (PBS/0.05% Tween) and blocked for 2 h at 37° C. with 2% BSA in wash buffer.
  • the plate is then extensively washed and then incubated with 1 to 20 diluted mouse sera or serially diluted mouse IgE standard (BD Pharmingen, # 557079). The plate is incubated at RT for 2 h and then extensively washed with wash buffer.
  • Bound specific mouse antibodies are then detected by one hour incubation with a HRPO-labelled, epsilon chain specific, rat anti-mouse IgE antibody (Southern Biotech #H021-NBB4C). After extensive washing with wash buffer, plates are developed with OPD solution (1 OPD tablet, 25 ml OPD buffer and 8 ⁇ l H 2 O 2 ) for 6 minutes and the reaction is stopped with 5% H 2 SO 4 solution. Plates are then read at OD 450 nm on an ELISA reader (Biorad Benchmark). The concentrations of IgE in sera is calculated with GraphPad Prism4 using the mouse IgE standard as reference.
  • PBMC Human peripheral blood mononuclear cells
  • FCS fetal calf serum
  • IFN-alpha in the supernatants was measured by sandwich ELISA, using an antibody set provided by PBL Biomedical Laboratories. Briefly, ELISA plates were coated with 5 ⁇ g/ml of the capture anti-IFN-alpha antibody. After washing, plates were blocked with 0.5% BSA for 1 h at room temperature (RT). Supernatants from treated and control PBMC, as well as recombinant IFN-alpha were added to the plate and incubated for 2 h at 37° C.
  • Rabbit polyclonal anti-IFN-alpha serum was used as detection antibody and incubated for 1 h at RT.
  • Peroxidase-conjugated goat anti-rabbit serum was subsequently added to the plates for 1 h at RT. Washing step was performed after each incubation.
  • the color reaction was developed using the enzyme substrate o-phenylendiamine and read at 490 nm in an ELISA reader device. The data are expressed as pg/ml secreted IFN-alpha according to a standard curve of recombinant IFN-alpha.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • mice from group B received 375 ⁇ g of Q ⁇ G10 subcutaneously (s.c.).
  • mice from group C received 375 ⁇ g of Q ⁇ G10 subcutaneously.
  • mice from of group A received PBS as control.
  • all mice were challenged with 200 ⁇ g of RW in PBS intranasally (i.n.). 48 hours after the challenge mice were sacrificed and the lungs were washed with PBS (Table 6).
  • the cells contained in the broncho alveolar lavage (BAL) were analysed by FACS. Eosinophils were identified by high CCR3 (Mouse CCR3 Phycoerythrin MAb, R & D) expression. As shown in Table 7, the total amount of cells in BAL was strongly reduced in Q ⁇ G10 treated compared to PBS control mice.
  • mice were injected intraperitonealy with 150 ⁇ g RW pollen extract in 450 ⁇ g of alum on day 0 and 3. On day 9 and 13, mice from group B were subcutaneously injected with 375 ⁇ g of Q ⁇ G10. Mice from of group A received PBS as control. On day 11 and 15, mice were challenged with 200 ⁇ g of RW in PBS intranasally.
  • the total IgE level in blood was measured by ELISA. Briefly, an ELISA plates (96 well MAXIsorp, NUNC immuno plate #442404) were coated with anti-IgE (BD Pharmingen, # 553413) at a concentration of 5 ⁇ g/ml in coating buffer (0.1 M NaHCO3, pH 9.6) over night at 4° C. The plates were then washed with wash buffer (PBS/0.05% Tween) and blocked for 2 h at 37° C. with 2% BSA in wash buffer. The plate was then extensively washed and then incubated with 1 to 20 diluted mouse sera or serially diluted mouse IgE standard (BD Pharmingen, # 557079).
  • the plate was incubated at RT for 2 h and then extensively washed with wash buffer. Bound specific mouse antibodies were then detected by one hour incubation with a HRPO-labelled, epsilon chain specific, rat anti-mouse IgE antibody (Southern Biotech #H021-NBB4C). After extensive washing with wash buffer, plates were developed with OPD solution (1 OPD tablet, 25 ml OPD buffer and 8 ⁇ l H 2 O 2 ) for 6 minutes and the reaction was stopped with 5% H 2 SO 4 solution. Plates were then read at OD 450 nm on an ELISA reader (Biorad Benchmark). The concentrations of IgE in sera were calculated with GraphPad Prism4 according to the standard.
  • mice were sensitized with RW mixed with alum as in Example 16 on day 0 and 3. On day 10 and day 12, mice received Q ⁇ G10, Q ⁇ G10 mixed with RW (20 ⁇ g) or PBS subcutaneously. 6 and 14 days after the treatment, mice were challenged with RW. 48 hours after the challenge, IgE level in sera was analyzed (Table 13). The data clearly show that IgE level in Q ⁇ G10 or Q ⁇ G10 mixed with RW treated mice is significantly reduced comparing to PBS treated controls (Table 14) and no clear difference has been seen between Q ⁇ G10 and Q ⁇ G10 mixed with RW. TABLE 13 Experimental Protocol.
  • RW Analysis i.p. i.p. i.n. E RW alum RW alum Q ⁇ G10 s.c. Q ⁇ G10 s.c. RW Analysis i.p. i.p. i.n.
  • mice are sensitized with RW and Fel d1 (Cytos, Switzerland) on day 0 and 3.
  • mice are treated with either PBS (group A and B), or Q ⁇ G10 (group C and D), or Q ⁇ G10 mixed with RW (group E and F) or Q ⁇ G10 mixed with Fel d1.
  • mice from group A, C, E and G are challenged with RW mixed with alum.
  • mice from group B, D, F and H are challenged with Fel d1.
  • all mice receive the same treatment as on day 10.
  • On day 16 all the mice are challenged as on day 12.
  • mice were sensitized with ragweed on day 0 and day 3 by i.p. injection of ragweed mixed with alum.
  • mice from group B and C were treated with G10 or Q ⁇ G10 respectively.
  • mice from group A received PBS as control. All mice were challenged with 200 ⁇ g of RW by i.n. administration and two days after the challenge mice were sacrificed. Infiltrating cells and IL-4 concentration in BAL and IgE level in blood were analysed (Table 16).
  • mice are sensitized by intraperitoneal injection with 5 ⁇ g of Fel d1 on day 1 and 14, and then boosted intranasally with 1 ⁇ g of Fel d1 on days 28, 29, 30 and 33.
  • Mice are injected subcutaneously with 375 ⁇ g of Q ⁇ G10 or PBS on days 37, 39 and 41.
  • mice are challenged intranasally with 1 ⁇ g of Fel d1 and the core temperature of the mice is measured rectally using a rectal probe digital thermometer. Mast cell degranulation is monitored in the cat allergy model.
  • Mice are injected subcutaneously with 375 ⁇ g of Q ⁇ G10 on day 0 and day 3.
  • mice are injected intradermally with 50 ⁇ l of 1:5 diluted cat allergic sera or purified IgE from that serum or control serum. 4 or 24 h later, mice are injected intravenously with 10 ⁇ g of Fel d1 plus Evans blue dye. The mice are sacrificed 30 min after the intravenous challenge and the blue staining reaction is analyzed. Q ⁇ G10 treated mice have reduced blue staining as compared to untreated mice.
  • mice Female 6 to 8 week-old CBA/J mice are obtained from Harlan (Zeist, The Netherlands). Animals are sensitized every other week by s.c. injection in the ventral region and at the base of the tail of 0.1 ⁇ g PLA2 adsorbed to 1 mg alum. Two protocols are applied for Q ⁇ G10 therapy. First, presensitized mice receive daily s.c. injections of 375 ⁇ g of Q ⁇ G10 for 6 consecutive days. IgE levels are measured 48 hours after the last Q ⁇ G10 injection. Second, naive mice are injected s.c. with 375 ⁇ g of Q ⁇ G10 on day 0 and day 3. Two weeks after the last Q ⁇ G10 injection, mice are sensitized with PLA2 in alum.
  • IgE levels are measured 7 days after PLA2 sensitization.
  • Control groups receive either PBS or 30 ⁇ g native PLA2.
  • animals are injected i.p. with 30 ⁇ g native PLA2 in PBS, and rectal temperature is monitored with a calibrated digital thermometer. In comparison to untreated mice, the temperature drop in Q ⁇ G10 treated mice is less pronounced.
  • mice Male CBA/CaH mice are sensitized with an intraperitoneal injection of 5 ⁇ g of Der P1+1 mg of alhydrogel. For intranasal installations, 25 ⁇ l aliquots containing 5 ⁇ g of Der P1 or sterile saline are applied to the noses of anesthetized mice. Two protocols are applied Q ⁇ G10 therapy. First, presensitized mice receive daily s.c. injections of 375 ⁇ g of Q ⁇ G10 for 6 consecutive days in. IgE levels are measured 48 hours after the last Q ⁇ G10 injection. Second, naive mice are injected s.c. with 375 ⁇ g of Q ⁇ G10 on day 0 and day 3. Two weeks after the last Q ⁇ G10 injection, mice are sensitized with Der P1 in alum. IgE levels are measured 7 days after Der P1 sensitization. Q ⁇ G10 treated mice exhibit lower IgE concentrations than the untreated control.
  • Total body involvement is estimated with the use of shapes of 100 to 1000 cm 2 or by the rule of nine which assigns standard measurements to body parts on the basis of the size of the body part.
  • the total body score is the sum of the individual scores, on a scale of 0 to 3, for erythema, edema, pruritus, oozing crusting, excoriation, and lichenification of all involved skin, dryness of noninvolved skin, and sleep loss.
  • test persons grade the pruritus of the selected areas on a 10-cm visual analogue scale, with severe at the bottom and absent at the top; this grade is converted to a score of 0 to 3 for analysis. To ensure consistency in the assessment of efficacy, all the investigators receive a manual and participate in a on-day, centralized training course, as well as on-site training.
  • Q ⁇ G10 A group of test persons suffering from Atopic dermatitis is treated at least three times in intervals of 1 week with 300 ⁇ g Q ⁇ G10 (Q ⁇ VLP packaged with G10 (SEQ ID NO:27)), a control group with comparable symptoms is treated with placebo.
  • Q ⁇ VLP packaged with G10 SEQ ID NO:27
  • a control group with comparable symptoms is treated with placebo.
  • Dependent on the average symptom score of the test group the treatment of the test group can be repeated up to a total of 6 to 10 vaccinations with 300 ⁇ g Q ⁇ G10 each; the control group is always treated with placebo in parallel.
  • the Physician's Global Assessment (PGA) of Atopic Dermatitis (AD) The investigator will rate the overall disease severity using the scale described in Table 21. The rating is to be assigned in consideration of the patient's current condition, without reference to previous assessments.
  • Eczema Area and Severity Index The extent and severity of dermatitis over 4 body surface areas will be calculated using the Eczema Area and Severity Index (EASI) (Hanifin J. M. et al. (2001), Exp. Dermatol. 10:11-18).
  • the four body regions are assigned proportionate body surface areas (BSA) such that the head and neck together comprise 10% BSA, the front and back of the trunk each comprise 15% BSA, each arm comprises 10% BSA, and each leg comprises 20% BSA.
  • BSA body surface areas
  • Table 22 The definitions outlined in Table 22 will be used.
  • the score for each body region is obtained by multiplying the sum of the severity scores of the four key symptoms by the percentage of area involved on the body region, then multiplying the result by the constant weighted value assigned to the particular body region (see Table 23).
  • the EASI score is the sum of the four body region scores.
  • EASI Eczema Area and Severity Calculation (patients of 8 years and older).
  • Q ⁇ G10 Treatment with Q ⁇ G10: A double-blind parallel-group clinical trial is performed with 36 patients with atopic dermatitis. Patients are allocated randomly to two groups of 18 patients each. The first group is treated with 300 ⁇ g Q ⁇ G10 (Q ⁇ VLP filled with G10 (SEQ ID NO:27)) six times in intervals of 1 week. The second group is treated with placebo.
  • PGA Physician Global Assessment
  • EASI Scores EASI Scores
  • Patient's assessment of pruritus are performed prior to the first treatment, bi-weekly during the treatment period, and several times after treatment until the end of the study at 24 weeks after the first treatment.
  • the changes in the symptom scores from before the treatment are compared between the groups at the various assessment points after the treatment.
  • Asthma symptoms are assessed using the standardized Asthma Quality of Life Questionnaire (AQLQ(S) or AQLQ12+, Juniper et al., Health Qual. Life Outcomes September 2005 3:58).
  • AQLQ(S) or AQLQ12+ Juniper et al., Health Qual. Life Outcomes September 2005 3:58.
  • Q ⁇ G10 or AP205G10 Two test groups and one control group of persons suffering from asthma and showing comparable symptom score with AQLQ(S) or AQLQ12+are treated with VLPs of bacteriophage Q ⁇ packaged with G10 (SEQ ID NO:27), VLPs of bacteriophage AP205 packaged with G10 (SEQ ID NO:27) or placebo, respectively.
  • Q ⁇ G10 and AP205G10 are produced according to Examples 16 and 17 of WO03/024481 or preferably according to the Examples herein.
  • the test persons are treated at least three times with 300 ⁇ g Q ⁇ G10 or 300 ⁇ g AP205G10 in intervals of 1 week.
  • Dependent on the average symptom score of the test group the treatment of the test group can be repeated up to a total of 6 to 10 vaccinations with 300 ⁇ g VLP-G10 each; the control group is always treated with placebo in parallel.
  • the severity of asthma symptoms can be assessed by spirometric measurements before and after administration of methacholine according to the following protocol.
  • Asthma symptoms are assessed by Spirometry with measurements obtained before and after the administration of methacholine according to Example 28. Determined are the parameters FEV1 and the ratio of FEV1 to the forced vital capacity FVC expressed as a percentage of the predicted value. Additionally, the test persons (or their parents or guardians) complete a diary card each day throughout the study recording night awakenings due to asthma, morning and evening peak flows as measured by peak-flow meters, use of medication, use of rescue medicine (albuterol) for symptoms and to prevent exercise-induced bronchospasm, use of prednisone, absence from school/work due to asthma, visits to a physician's office or hospital because of asthma and severity of symptoms. Test persons use their usual medication throughout the study.
  • a skin prick test is performed to verify that a subject shows hypersensitization to a certain allergen.
  • the test is useful to test the reaction of a patient to a wide range of allergens, including pollen allergen and allergen mix that is routinely used to screen for atopy.
  • the test is useful to test the reaction towards dust mite allergens (Der p and Der f). At Screening this test must be performed prior to CPT (Example 31).
  • Skin prick tests and intradermal skin tests allow a visualisation of sensitisation.
  • the basic principle of skin testing is the introduction of a small amount of allergen into the dermis.
  • the released mediators cause vasodilatation and increase vascular permeability, which in turn leads to tissue edema and the development of a weal. Histamine triggers the release of the neuromediator substance P by an axon reflex, which causes the surrounding skin to flush.
  • a small drop of about 30 ⁇ l of serial dilutions of the allergen is placed on the volar surface of the forearm.
  • the test sites must be at least 2 cm apart to avoid false-positive reactions.
  • a so called prick needle i.e. a needle that can be perpendicularly inserted into the skin
  • the allergen solution is “pricked” into the dermis, giving highly reproducible results.
  • the diluent of the allergen solution is used for a negative control.
  • Histamine is used as a positive control to detect suppression of cutaneous reactivity by medications (mainly antihistaminic drugs).
  • a histamine solution at 1 mg/ml induces a weal ranging from 2-7 mm in diameter.
  • reaction will then be scored as a percentage of the positive control.
  • the evaluation of the end-point titrations will be based on parallel line bioassay and on median slope.
  • the allergic reaction is always assessed with respect to the control reaction (diluent only).
  • subjects are considered allergic if they exhibit at least one edema or erythema of at least 6 mm diameter or at least one edema or erythema with an area of at least 7 mm2.
  • the response of a subject to an allergen can be assessed using the so called conjunctival provocation test which is performed according to the following procedure.
  • the test is useful to test the reaction of a patient to a wide range of allergens, including pollen allergen.
  • the test is useful to test the reaction towards dust mite allergens (Der p and Der f).
  • CPT Conjunctival Provocation Test
  • the stages are determined for the following solutions (dilution factor of the standard allergen solution): Negative Solution; Concentration I: 1:1000, Concentration II: 1:100; Concentration III: 1:10; and Concentration IV: 1;
  • the CPT is positive if the response is stage II or higher.
  • Symptoms are scored using the Score Sheet for CPT (Table 26) which provides for scores from 0 to 3 for 5 different parameters. Thus, the maximum score is 15 per challenge. The CPT is positive if the total response is >10. TABLE 26 CPT Score Sheet - Challenge Symptom Questionnaire. Symptom None Mild (1) Moderate (2) Severe (3) 10′ Score Conjunctival none Slight Definite severe hyperemia redness redness redness Tearing none Slight Definite need to sensation sensation whipe off Itching none Slight Definite need to rub sensation sensation eyes Burning none Slight Definite severe sensation sensation sensation Swelling of none Slight Definite unable to eyelids sensation sensation open eyes
  • Nasal provocation test is useful to test the reaction of a patient to a wide range of allergens, especially pollen allergens.
  • the nose provides an ideal site for allergen provocation.
  • Allergen can be applied to the mucosa with a high degree of accuracy.
  • the challenge procedure should reflect natural exposure. Quantitative measurements with high reproducibility are important (Andersson M, L. Greiff, C. Svensson and C. Persson. Acta Otolaryngol. 115 (1995), pp. 705-713).
  • Bousquet Bosquet J, P. van Cauwenberge and N. Khaltaev. Aria Workshop Group; WHO, J. Allergy Clin. Immunol. 108 (2001), pp.
  • the solutions of allergen will be delivered from a meter-dose pump spray, as the nasal pump spray delivers the solution over a large area of the nasal mucosa.
  • the allergen solution will be an isotonic, buffered aqueous solution with a neutral pH. Serial 1:3 dilutions with saline will be freshly prepared prior to testing. The exact allergen concentrations are to be determined in a previous study.
  • the allergen solution will be applied at a volume of 100 ⁇ l.
  • Contraindications for NPT Episode of rhinitis in last 4 weeks; Exacerbation of allergic disease; Use of allergen known to have caused anaphylactic reaction; Pregnancy; Nasal surgery in last 8 weeks; Coexisting severe general disease, especially cardiopulmonary diseases; Treatment with medications that may interfere with the treatment of systemic allergic reactions (e.g. ⁇ -blockers or ACE inhibitors); Vaccinations within one week prior to testing.
  • systemic allergic reactions e.g. ⁇ -blockers or ACE inhibitors
  • Antihistamines systemic: 48 hours to 1 week depending on their half life; Antihistmines, nasal: 1 week; Ketotifen: 2 weeks; DNCG, Nedocromil: 3 days; Corticosteroids, nasal: 1 week; Corticosteroids, systemic 1 week; Topical ⁇ -adrenergic agonists: 1 day; Nonsteroidal antiinflammatory drugs (NSAIDs): 1 week; Antihypertensives (e.g. reserpine, clonidine): 3 weeks; Antidepressants (e.g., imipramines, tricyclics): 3 days.
  • NSAIDs Nonsteroidal antiinflammatory drugs
  • Antihypertensives e.g. reserpine, clonidine
  • Antidepressants e.g., imipramines, tricyclics
  • Rhinoscopic examination including anterior or posterior rhinoscopy, preferably anterior rhinoscopy, will be performed preceding NPT in order to evaluate the baseline condition. Patients should be well adapted to room temperature for at least 15 minutes before a baseline evaluation by rhinoscopy and a clinical symptom score, as well as rhinomanometry.
  • the wider side of the nose is used for the challenge. Before administration of the actual allergen solution, the nasal mucosa of the wider side of the nose is challenged for unspecific hyperresponsiveness by 100 ⁇ l of the allergens diluent. Evaluation of the scores and rhinomanometry is repeated after 10 minutes to check on clinical symptoms or significant changes in objective measurements of nasal patency. If no significant changes occur, the actual allergen provocation is performed on the wider side of the nose.
  • the applicator of the delivery device is inserted into the nasal vestibulum and pointed upward and laterally towards the medial canthus of the eye to deposit allergen on the inferior and the middle turbinate mucosa when spraying 100 ⁇ L of allergen test solution into the nose.
  • allergen application the patient must hold his or her breath to avoid inhaling the allergen into the lower airways. Patients are told to inhale before and to exhale right after the application.
  • Nasal symptom recording will be performed in parallel according to three common methods (Bachert C: Nasal provocation test: critical evaluation. In Ring J, Behrendt H D, editors: New trends in allergy, IV, Berlin, 1997, Learnuf Springer-Verlag, p 277).
  • Score 1 Severity of each nasal symptom is recorded on a 10-cm linear visual analog scale. The severity is then evaluated based on the score (mild 1-3 cm; moderate 4-7 cm; severe 8-10 cm) obtained with diluent (negative control) and each provocation dose.
  • Score 2 A practical scoring system for a standardized quantification of these clinical parameters, which has been proposed by the ENT section of the German Society for Allergology and Clinical Immunology is summarized in Table 27 (Reichelmann et al. Position statement. Allergo J (2002) 11:29-36.). TABLE 27 Scoring system for evaluation of clinical symptoms after nasal provocation (“Score 2”).
  • Score 3 This score is often used in both clinical and scientific research studies (Linder, 1988). The end point is considered the amount of stimulant that produces a total symptom score of 5 from a maximum score of 13 points (Table 28). TABLE 28 Scoring system for evaluation of clinical symptoms after nasal provocation (“Score 3”).
  • End Point After the allergen challenge the end point is reached when the patient has more than 3 points in score 2, or if the reduction in nasal flow rate is >40% at 150 Pa. The end point shall also be reached, if the reduction in nasal flow rate is >20% at 150 Pa in conjunction with >2 points in score 2.
  • Nasal air-space volume and patency will be assessed by anterior rhinomanometry. It is designed to analyze the transnasal airflow in one nostril at a time depending on transnasal pressure, which is assessed contralaterally during tidal breathing. For complete and state of the art evaluation of nasal patency, this method is combined with acoustic rhinometry, which documents nasal geometry as cross-sectional area within the nasal cavity.
  • An individual suffering from pollen allergy and atopic eczema was treated four times by subcutaneous injection of 300 ⁇ g Q ⁇ G10 in 1 week intervals.
  • a skin prick test according to Example 30 and nasal provocation test according to Example 32 were performed on the day before the first treated to determine a baseline.
  • the nasal provocation test was performed with a commercial challenging agent containing pollen allergen in dilutions of 1:1000, 1:100, 1:10 and 1:1 and repeated 1 week after the second treatment (directly before the third treatment) and 1 week after the fourth treatment.
  • the reaction of the test person was scored using score 2 and score 3 as described in Example 32. Additionally, the number of sneezes was recorded (Table 31).
  • Q ⁇ G10 Q ⁇ G8-8 After determination of a base line with skin prick and nasal provocation test (see Examples 30 and 32) three groups of test persons suffering from pollen allergy are formed, wherein all groups show a similar test score in average. The first group is treated at least three times in intervals of 1 week with 300 ⁇ g Q ⁇ G10 (Q ⁇ VLP packaged with G10 (SEQ ID NO:27)). The second group is treated in parallel with 300 ⁇ g Q ⁇ packaged with G8-8 (SEQ ID NO 25). The third group is treated with placebo.
  • the treatment of the test group can be repeated up to a total of 6 to 10 vaccinations with 300 ⁇ g of Q ⁇ G10 or Q ⁇ G8-8, respectively; the control group is always treated with placebo in parallel.
  • Q ⁇ G10 Q ⁇ G8-8 After determination of a base line with skin prick and conjunctival provocation test (see Examples 30 and 31) three groups of test persons suffering from house dust allergy are formed, wherein all groups show a similar test score in average. The first group is treated at least three times in intervals of 1 week with 300 ⁇ g Q ⁇ G10 (Q ⁇ VLP packaged with G10 (SEQ ID NO:27)). The second group is treated in parallel with 300 ⁇ g Q ⁇ packaged with G8-8 (SEQ ID NO 25). The third group is treated with placebo.
  • the treatment of the test group can be repeated up to a total of 6 to 10 vaccinations with 300 ⁇ g of Q ⁇ G10 or Q ⁇ G8-8, respectively; the control group is always treated with placebo in parallel.
  • Treatment with HBcG10 HBcG8-8 After determination of a base line with skin prick and conjunctival provocation test (see Examples 30 and 31) three groups of test persons suffering from house dust allergy are formed, wherein all groups show a similar test score in average. The first group is treated at least three times in intervals of 1 week with 300 ⁇ g HBcG10 (HBc VLP packaged with G10 (SEQ ID NO:27)). The second group is treated in parallel with 300 ⁇ g HBc packaged with G8-8 (SEQ ID NO 25). The third group is treated with placebo.
  • the treatment of the test group can be repeated up to a total of 6 to 10 vaccinations with 300 ⁇ g of HBcG10 or HBcG8-8, respectively; the control group is always treated with placebo in parallel.
  • Q ⁇ G10 Treatment with Q ⁇ G10: An open-label clinical trial was performed with patients with allergy to grass pollen. Patients were treated with 300 ⁇ g Q ⁇ G10 (Q ⁇ VLP filled with G10 (SEQ ID NO:27)) six times in intervals of 1 week.
  • Nasal provocation test with a standardized grass pollen extract was performed prior to the first treatment and 2 weeks after the last treatment. Nasal provocation testing is also performed at months 6 and 12 following the first treatment. The changes in the symptom scores from before the treatment are assessed at the various assessment points after the treatment. Efficacy is also measured with the skin prick test (see Example 30) using solutions containing various amounts of grass pollen allergens. Efficacy of the treatment in daily life is measured using a validated patient diary to record symptoms and medication use during the first pollen season following the treatment. The validated patient diary is also used to record symptoms and medication use during the second pollen season following the treatment.
  • Results of the nasal provocation test 5 patients with grass pollen allergy treated with 6 weekly injections of 300 ⁇ g Q ⁇ G10 were subjected to nasal provocation (Example 32) before treatment and 2 weeks after the treatment. As shown in Table 32 these patients showed reduced symptom score as compared to their reaction before the treatment. TABLE 32 Symptoms due to nasal provocation were assessed using score system 2 (Example 32).
  • Treatment with Q ⁇ G10 A double-blind parallel-group clinical trial is performed with 30 patients with allergy to grass pollen. Patients are allocated randomly to three groups of 10 patients each. The first group is treated with 300 ⁇ g Q ⁇ G10 (Q ⁇ VLP packaged with G10 (SEQ ID NO:27)) six times in intervals of 1 week. The second group is treated with 300 ⁇ g Q ⁇ G10 in combination with the adjuvant aluminum hydroxide six times in intervals of 1 week. The third group is treated with placebo.
  • Q ⁇ G10 Q ⁇ VLP packaged with G10 (SEQ ID NO:27)
  • Conjunctival provocation test with a standardized grass pollen extract is performed prior to the first treatment, 2 weeks after the last treatment, and 6 and 12 months after the first treatment. The changes in the symptom scores from before the treatment are compared between the groups at the various assessment points after the treatment. Efficacy is also measured with the skin prick test (see Example 30) using solutions containing various amounts of grass pollen allergens. Efficacy of the treatment in daily life is measured using a validated patient diary to record symptoms and medication use during the first pollen season following the treatment. The validated patient diary is also used to record symptoms and medication use during the second pollen season following the treatment.
  • Q ⁇ G10 Treatment with Q ⁇ G10: A double-blind parallel-group clinical trial is performed with 20 patients with allergy to house dust mites. Patients are allocated randomly to two groups of 10 patients each. The first group is treated with 300 ⁇ g Q ⁇ G10 (Q ⁇ VLP packaged with G10 (SEQ ID NO:27)) in combination with the adjuvant aluminum hydroxide six times in intervals of 1 week. The second group is treated with placebo.
  • Conjunctival provocation test with a standardized house dust mite extract is performed prior to the first treatment, 2 weeks after the last treatment, and 6 and 12 months after the first treatment.
  • the changes in the symptom scores from before the treatment are compared between the groups at the various assessment points after the treatment.
  • Efficacy is also measured with the skin prick test (see Example 30) using solutions containing various amounts of house dust mite allergens.
  • Efficacy of the treatment in daily life is measured using a validated patient diary to record symptoms and medication use during 14 consecutive days prior to treatment, two weeks after treatment, and 6 and 12 months after the first treatment.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Immunology (AREA)
  • Nanotechnology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Pathology (AREA)
  • Hematology (AREA)
  • Pulmonology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Radiology & Medical Imaging (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Medical Informatics (AREA)
  • Dermatology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Diabetes (AREA)
  • Endocrinology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Rheumatology (AREA)
  • Toxicology (AREA)
  • Urology & Nephrology (AREA)
US11/638,664 2005-12-14 2006-12-14 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity Abandoned US20070184068A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US11/638,664 US20070184068A1 (en) 2005-12-14 2006-12-14 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US12/576,086 US8574564B2 (en) 2005-12-14 2009-10-08 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US13/947,577 US20140056933A1 (en) 2005-12-14 2013-07-22 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US75004205P 2005-12-14 2005-12-14
US81259206P 2006-06-12 2006-06-12
US11/638,664 US20070184068A1 (en) 2005-12-14 2006-12-14 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US12/576,086 Continuation US8574564B2 (en) 2005-12-14 2009-10-08 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity

Publications (1)

Publication Number Publication Date
US20070184068A1 true US20070184068A1 (en) 2007-08-09

Family

ID=37891967

Family Applications (3)

Application Number Title Priority Date Filing Date
US11/638,664 Abandoned US20070184068A1 (en) 2005-12-14 2006-12-14 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US12/576,086 Expired - Fee Related US8574564B2 (en) 2005-12-14 2009-10-08 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US13/947,577 Abandoned US20140056933A1 (en) 2005-12-14 2013-07-22 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity

Family Applications After (2)

Application Number Title Priority Date Filing Date
US12/576,086 Expired - Fee Related US8574564B2 (en) 2005-12-14 2009-10-08 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US13/947,577 Abandoned US20140056933A1 (en) 2005-12-14 2013-07-22 Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity

Country Status (8)

Country Link
US (3) US20070184068A1 (ja)
EP (1) EP1973608A1 (ja)
JP (2) JP5484732B2 (ja)
AU (1) AU2006325225B2 (ja)
CA (1) CA2636139A1 (ja)
NZ (1) NZ569741A (ja)
RU (1) RU2451523C2 (ja)
WO (1) WO2007068747A1 (ja)

Cited By (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030099668A1 (en) * 2001-09-14 2003-05-29 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US20030109469A1 (en) * 1993-08-26 2003-06-12 Carson Dennis A. Recombinant gene expression vectors and methods for use of same to enhance the immune response of a host to an antigen
US20040058883A1 (en) * 2002-04-22 2004-03-25 Phillips Nigel C. Oligonucleotide compositions and their use for the modulation of immune responses
US20080095738A1 (en) * 2004-10-05 2008-04-24 Cytos Biotechnology Ag Vlp-Antigen Conjugates and Their Uses as Vaccines
US20080139797A1 (en) * 2006-12-12 2008-06-12 Integrated Dna Technologies, Inc. Oligonucleotides containing high concentrations of guanine monomers
US20100098722A1 (en) * 2003-03-26 2010-04-22 Cytos Biotechnology Ag Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use
US20100247669A1 (en) * 2009-03-30 2010-09-30 Cerulean Pharma Inc. Polymer-agent conjugates, particles, compositions, and related methods of use
US20100285144A1 (en) * 2009-03-30 2010-11-11 Scott Eliasof Polymer-epothilone conjugates, particles, compositions, and related methods of use
US20110135605A1 (en) * 2002-10-29 2011-06-09 Coley Pharmaceutical Group, Inc. Methods and products related to treatment and prevention of hepatitis c virus infection
WO2011119881A1 (en) * 2010-03-24 2011-09-29 Northeastern University Multi-compartmental macrophage delivery
WO2012024526A3 (en) * 2010-08-20 2012-07-05 Cerulean Pharma Inc. Conjugates, particles, compositions, and related methods
US20120322103A1 (en) * 2005-05-26 2012-12-20 Cytos Biotechnology Ag Scalable Fermentation Process
US8343498B2 (en) 2008-10-12 2013-01-01 Massachusetts Institute Of Technology Adjuvant incorporation in immunonanotherapeutics
US8343497B2 (en) 2008-10-12 2013-01-01 The Brigham And Women's Hospital, Inc. Targeting of antigen presenting cells with immunonanotherapeutics
US8354094B1 (en) * 2005-01-04 2013-01-15 Gp Medical, Inc. Pharmaceutical composition of nanoparticles
US20130039973A1 (en) * 2011-08-03 2013-02-14 Henry J. Smith Viral Immunogenic Compositions
US20130209566A1 (en) * 2012-02-15 2013-08-15 Monica Jablonski Nanoparticle composition and methods to make and use the same
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US8591905B2 (en) 2008-10-12 2013-11-26 The Brigham And Women's Hospital, Inc. Nicotine immunonanotherapeutics
US8629151B2 (en) 2009-05-27 2014-01-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US8709483B2 (en) 2006-03-31 2014-04-29 Massachusetts Institute Of Technology System for targeted delivery of therapeutic agents
US8906381B2 (en) 2008-10-12 2014-12-09 Massachusetts Institute Of Technology Immunonanotherapeutics that provide IGG humoral response without T-cell antigen
US9066978B2 (en) 2010-05-26 2015-06-30 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9080014B2 (en) 2006-05-15 2015-07-14 Massachusetts Institute Of Technology Polymers for functional particles
US9217129B2 (en) 2007-02-09 2015-12-22 Massachusetts Institute Of Technology Oscillating cell culture bioreactor
US9267937B2 (en) 2005-12-15 2016-02-23 Massachusetts Institute Of Technology System for screening particles
US9333179B2 (en) 2007-04-04 2016-05-10 Massachusetts Institute Of Technology Amphiphilic compound assisted nanoparticles for targeted delivery
US9381477B2 (en) 2006-06-23 2016-07-05 Massachusetts Institute Of Technology Microfluidic synthesis of organic nanoparticles
WO2016109310A1 (en) 2014-12-31 2016-07-07 Checkmate Pharmaceuticals, Llc Combination tumor immunotherapy
US9404126B2 (en) 2006-06-12 2016-08-02 Kuros Biosciences Ag Processes for packaging aggregated oligonucleotides into virus-like particles of RNA bacteriophages
US9474717B2 (en) 2007-10-12 2016-10-25 Massachusetts Institute Of Technology Vaccine nanotechnology
US9492400B2 (en) 2004-11-04 2016-11-15 Massachusetts Institute Of Technology Coated controlled release polymer particles as efficient oral delivery vehicles for biopharmaceuticals
US9539217B2 (en) 2013-04-03 2017-01-10 Allertein Therapeutics, Llc Nanoparticle compositions
US9597385B2 (en) 2012-04-23 2017-03-21 Allertein Therapeutics, Llc Nanoparticles for treatment of allergy
EP3044339A4 (en) * 2013-09-10 2017-04-26 Mockv Solutions Methods and kits for quantifying the removal of mock virus particles from a purified solution
WO2017075483A1 (en) * 2015-10-28 2017-05-04 University Of Maryland, College Park Multifunctional biodegradable carriers for drug delivery
WO2017173334A1 (en) 2016-04-01 2017-10-05 Checkmate Pharmaceuticals, Inc. Fc receptor-mediated drug delivery
KR20180057766A (ko) * 2016-11-21 2018-05-31 충북대학교 산학협력단 돼지파보바이러스 및 돼지열병바이러스에 대한 동시 항원성 복합 항원 단백질의 제조 방법
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
US10293044B2 (en) * 2014-04-18 2019-05-21 Auburn University Particulate formulations for improving feed conversion rate in a subject
CN110042103A (zh) * 2019-03-25 2019-07-23 华南农业大学 一种对猪具有特异性免疫刺激作用的CpG-ODN及其应用
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US10933129B2 (en) 2011-07-29 2021-03-02 Selecta Biosciences, Inc. Methods for administering synthetic nanocarriers that generate humoral and cytotoxic T lymphocyte responses
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11207428B2 (en) 2015-03-16 2021-12-28 Oregon Health & Science University Cross-linked polymer modified nanoparticles
US11213593B2 (en) 2014-11-21 2022-01-04 Northwestern University Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates
US11224573B2 (en) 2019-07-12 2022-01-18 Oregon Health & Science University Therapeutic constructs for co-delivery of mitotic kinase inhibitor and immune checkpoint inhibitor
US11235058B2 (en) 2019-07-12 2022-02-01 Oregon Health & Science University Immunotherapeutic constructs and methods of their use
WO2022081738A3 (en) * 2020-10-13 2022-06-16 The Board Of Regents Of The University Of Texas System Polymer-conjugated microbubbles for high drug/gene loading
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
US11571474B2 (en) * 2015-09-08 2023-02-07 Universität Zürich Compositions against cat allergy
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties

Families Citing this family (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7615227B2 (en) * 2001-12-20 2009-11-10 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of CpG oligodeoxynucleotides to induce angiogenesis
WO2010122164A1 (en) 2009-04-23 2010-10-28 Cytos Biotechnology Ag VIRUS-LIKE PARTICLES OF BACTERIOPHAGE φCB5
WO2010125202A1 (en) * 2009-04-30 2010-11-04 Cytos Biotechnology Ag Influenza hemagglutinin compositions and uses thereof
EP2399608B1 (en) * 2010-03-05 2020-07-08 Sebastian Fuchs Immunomodulating nanoparticulate composition for use in inhalation therapy
EP3679933A1 (en) 2011-04-29 2020-07-15 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers to reduce immune responses to therapeutic proteins
US9665956B2 (en) * 2011-05-27 2017-05-30 Abbott Informatics Corporation Graphically based method for displaying information generated by an instrument
WO2015024992A2 (en) * 2013-08-22 2015-02-26 Cytos Biotechnology Ag Treatment of asthma
WO2015038746A1 (en) * 2013-09-11 2015-03-19 Georgia Tech Research Corporation Compositions and methods for inhibiting gene expression
WO2015073831A1 (en) * 2013-11-15 2015-05-21 Liquidia Technologies, Inc. Virtual conjugate particles
EP3174828A4 (en) * 2014-07-31 2018-01-17 Amorphical Ltd. Non-aqueous liquid and semi-solid formulations of amorphous calcium carbonate
MA40824A (fr) 2014-10-22 2017-08-29 Saiba Gmbh Particules de cmv de type virus modifié
JP6715775B2 (ja) 2014-12-25 2020-07-01 国立研究開発法人医薬基盤・健康・栄養研究所 非凝集性免疫賦活化オリゴヌクレオチド
US11000472B2 (en) 2015-04-06 2021-05-11 Lg Household & Health Care Ltd. Soluble microneedle for delivering poorly-soluble drug
US10758606B2 (en) 2015-09-04 2020-09-01 Inventprise, Llc VLP stabilized vaccine compositions
EP3347042A4 (en) 2015-09-10 2019-02-20 Inventprise, LLC. MULTIVALENT CONJUGATES WITH VIRUSUAL PARTICLES
WO2017179025A1 (en) 2016-04-15 2017-10-19 Alk-Abelló A/S Epitope polypeptides of ragweed pollen allergens
WO2017186808A1 (en) 2016-04-27 2017-11-02 Allergy Therapeutics (Uk) Limited Treatment of peanut allergy
JP7039561B2 (ja) 2016-04-27 2022-03-22 ベンチマーク アニマル ヘルス エルティーディー． イヌアトピー性皮膚炎の治療
CN107389847B (zh) * 2017-06-05 2020-01-07 中国农业科学院蜜蜂研究所 一种快速分析蜂花粉中脂类成分的方法
EP3775218A1 (en) 2018-04-09 2021-02-17 Checkmate Pharmaceuticals Packaging oligonucleotides into virus-like particles
CN112423766A (zh) * 2018-07-19 2021-02-26 一般财团法人阪大微生物病研究会 含有A型CpG寡脱氧核糖核苷酸的脂质颗粒
EP3897711A1 (en) 2018-12-20 2021-10-27 Saiba AG Virus-like particles of cmv modified by fusion
US20230038487A1 (en) * 2019-11-25 2023-02-09 The Regents Of The University Of California Modified viral therapeutics and uses thereof
WO2021260131A1 (en) 2020-06-25 2021-12-30 Saiba AG Virus-like particles of cmv modified by fusion
WO2024047090A2 (en) 2022-08-30 2024-03-07 Saiba Animal Health Ag Modified virus-like particles of cmv

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5057540A (en) * 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US5071651A (en) * 1986-09-03 1991-12-10 University Of Saskatchewan Rotavirus nucleocapsid protein VP6 as a carrier in vaccine compositions
US5962636A (en) * 1998-08-12 1999-10-05 Amgen Canada Inc. Peptides capable of modulating inflammatory heart disease
US20010044416A1 (en) * 2000-01-20 2001-11-22 Mccluskie Michael J. Immunostimulatory nucleic acids for inducing a Th2 immune response
US6326200B1 (en) * 1997-06-23 2001-12-04 Ludwig Institute For Cancer Research Isolated nona-and decapeptides which bind to HLA molecules, and the use thereof
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
US20030060440A1 (en) * 1999-04-12 2003-03-27 Dennis Klinman Oligodeoxynucleotide and its use to induce an immune response
US20030087848A1 (en) * 2000-02-03 2003-05-08 Bratzler Robert L. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20040005338A1 (en) * 2002-06-20 2004-01-08 Cytos Biotechnology Ag Packaged virus-like particles for use as adjuvants: method of preparation and use
US6949520B1 (en) * 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon

Family Cites Families (90)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2034118A1 (en) 1970-07-09 1972-01-13 Seinfeld, Hugo, Brendel, Walter, Prof Dr . 8000 München Xenogenic nucleic acids for enhancing antigenicity - of peptides and proteins
US4912094B1 (en) 1988-06-29 1994-02-15 Ribi Immunochem Research Inc. Modified lipopolysaccharides and process of preparation
EP0468520A3 (en) 1990-07-27 1992-07-01 Mitsui Toatsu Chemicals, Inc. Immunostimulatory remedies containing palindromic dna sequences
JPH06503821A (ja) 1990-12-20 1994-04-28 スミスクライン・ビーチャム・バイオロジカルス（ソシエテ・アノニム） Ｂ型肝炎表面抗原に基づくワクチン
GB9101550D0 (en) 1991-01-24 1991-03-06 Mastico Robert A Antigen-presenting chimaeric protein
GB9114003D0 (en) 1991-06-28 1991-08-14 Mastico Robert A Chimaeric protein
GB9213601D0 (en) 1992-06-26 1992-08-12 Mastico Robert A Protein based delivery system
RU95104940A (ru) 1992-07-27 1997-01-10 Хайбрайдон Способ введения в олигонуклеотид алкилфосфонотиоатной или арилфосфонотиоатной межнуклеотидной связи, способ получения олигонуклеотида, олигонуклеотиды, способ ингибирования генной экспрессии, способ лечения
FR2695563B1 (fr) * 1992-09-11 1994-12-02 Pasteur Institut Microparticules portant des antigènes et leur utilisation pour l'induction de réponses humorales ou cellulaires.
NO176837B1 (no) 1992-12-30 1995-06-07 Yara Int Asa Fremgangsmate for a fjerne hydrogensulfid fra oljeholdig vann
WO1995026204A1 (en) 1994-03-25 1995-10-05 Isis Pharmaceuticals, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US6727230B1 (en) 1994-03-25 2004-04-27 Coley Pharmaceutical Group, Inc. Immune stimulation by phosphorothioate oligonucleotide analogs
US7935675B1 (en) * 1994-07-15 2011-05-03 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
CA2194761C (en) 1994-07-15 2006-12-19 Arthur M. Krieg Immunomodulatory oligonucleotides
US6239116B1 (en) 1994-07-15 2001-05-29 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US6429199B1 (en) 1994-07-15 2002-08-06 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules for activating dendritic cells
US5935821A (en) 1995-01-17 1999-08-10 Board Of Trustees Of The University Of Kentucky Polynucleotides related to monoclonal antibody 1A7 and use for the treatment of melanoma and small cell carcinoma
WO1996030523A2 (en) 1995-03-31 1996-10-03 Hans Wolf Antigen presentation system based on retrovirus-like particles
US5994315A (en) 1995-06-07 1999-11-30 East Carolina University Low adenosine agent, composition, kit and method for treatment of airway disease
BR9707154A (pt) 1996-01-23 1999-05-25 Icn Pharmaceuticals Modulaç o da express o de citocina em th1/th2 por ribavirina e análogos de ribavirina em linfócitos t ativados
CA2244946C (en) 1996-01-30 2010-04-13 The Regents Of The University Of California Gene expression vectors which generate an antigen specific immune response and methods of using the same
GB9621091D0 (en) 1996-10-09 1996-11-27 Fondation Pour Le Perfectionem Attenuated microorganisms strains and their uses
DE69733020T2 (de) 1996-10-11 2006-02-16 The Regents Of The University Of California, Oakland Immunostimulierende oligonucleotidekonjugate
EP0855184A1 (en) 1997-01-23 1998-07-29 Grayson B. Dr. Lipford Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination
GB9702021D0 (en) 1997-01-31 1997-03-19 Imperial College Medicaments
US6214806B1 (en) 1997-02-28 2001-04-10 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CPC dinucleotide in the treatment of LPS-associated disorders
US6406705B1 (en) 1997-03-10 2002-06-18 University Of Iowa Research Foundation Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant
WO1998049195A1 (en) 1997-04-29 1998-11-05 Universiteit Utrecht Corona virus-like particles as tools for vaccination and therapy
AU750702B2 (en) 1997-05-01 2002-07-25 Chiron Corporation Use of virus-like particles as adjuvants
WO1998052581A1 (en) 1997-05-20 1998-11-26 Ottawa Civic Hospital Loeb Research Institute Vectors and methods for immunization or therapeutic protocols
US6225292B1 (en) 1997-06-06 2001-05-01 The Regents Of The University Of California Inhibitors of DNA immunostimulatory sequence activity
US6169175B1 (en) 1997-08-06 2001-01-02 Centers For Disease Control And Prevention Preparation and use of recombinant influenza A virus M2 construct vaccines
EP1009413B1 (en) 1997-09-05 2007-02-14 The Regents Of The University Of California Use of immunostimulatory oligonucleotides for preventing or treating asthma
US6171591B1 (en) 1997-12-08 2001-01-09 Pentamer Pharmaceuticals, Inc. Recombinant nodavirus compositions and methods
JP2002510644A (ja) 1998-04-03 2002-04-09 ユニバーシティ オブ アイオワ リサーチ ファウンデーション 免疫治療用オリゴヌクレオチドおよびサイトカインを用いる免疫系刺激のための方法および産物
US5990085A (en) 1998-05-04 1999-11-23 Michigan State University Inhibin-HBc fusion protein
AU760795B2 (en) 1998-05-14 2003-05-22 Coley Pharmaceutical Gmbh Methods for regulating hematopoiesis using CpG-oligonucleotides
ATE355266T1 (de) 1998-06-30 2006-03-15 Om Pharma Neue acylierte pseudodipeptide, verfahren zu ihrer herstellung, und diese enthaltende pharmazeutische zusammensetzungen
CA2333854A1 (en) 1998-07-27 2000-02-10 University Of Iowa Research Foundation Stereoisomers of cpg oligonucleotides and related methods
CA2341338A1 (en) 1998-09-03 2000-03-16 Coley Pharmaceutical Gmbh G-motif oligonucleotides and uses thereof
ES2258869T3 (es) 1998-10-21 2006-09-01 The United States Government As Represented By The Department Of Health And Human Services Particulas similares a virus para la induccion de autoanticuerpos.
BR9915771A (pt) 1998-11-30 2001-12-26 Cytos Biotechnology Ag Apresentação molecular ordenada de antìgenos,processo de preparação e utilização
WO2000037610A2 (en) 1998-12-23 2000-06-29 Boyce Thompson Institute For Plant Research At Cornell Expression of immunogenic hepatitis b surface antigens in transgenic plants
JP2003523721A (ja) 1998-12-31 2003-08-12 カイロン コーポレイション 抗原性ｈｉｖｃ型ポリペプチドをコードするポリヌクレオチド、ポリペプチド、およびそれらの使用
WO2000046365A1 (en) 1999-02-02 2000-08-10 Biocache Pharmaceuticals, Inc. Advanced antigen presentation platform
JP2002537403A (ja) 1999-02-25 2002-11-05 スミスクライン ビーチャム バイオロジカルズ ソシエテ アノニム ＩｇＥのＣ−イプシロン−３またはＣ−イプシロン−４ドメイン由来のエピトープまたはミモトープ、その拮抗薬、及びそれらの治療的使用
DE60020677T2 (de) 1999-02-26 2006-05-04 Chiron Corp., Emeryville Mikroemulsionen mit adsorbierten makromolekülen und mikropartikeln
WO2000054803A2 (en) 1999-03-16 2000-09-21 Panacea Pharmaceuticals, Llc Immunostimulatory nucleic acids and antigens
DE60014076T2 (de) 1999-04-19 2005-10-13 Glaxosmithkline Biologicals S.A. Adjuvans-zusammensetzung, enthaltend saponin und ein immunstimulatorisches oligonukleotid
ES2250151T3 (es) 1999-06-29 2006-04-16 Glaxosmithkline Biologicals S.A. Uso de cpg como adyuvante de vacuna contra vih.
US7479285B1 (en) 1999-08-19 2009-01-20 Dynavax Technologies Corporation Methods of modulating an immune response using immunostimulatory sequences and compositions for use therein
AU6801800A (en) 1999-08-30 2001-03-26 Ludwig Institute For Cancer Research Isolated nona and decapeptides which bind to hla molecules, and the use thereof
JP2001151698A (ja) 1999-09-10 2001-06-05 Nichiko Pharmaceutical Co Ltd インフルエンザワクチン
MXPA02003108A (es) 1999-09-25 2003-10-14 Univ Iowa Res Found Acidos nucleicos inmunoestimuladores.
ES2265980T5 (es) 1999-09-27 2010-12-28 Coley Pharmaceutical Group, Inc. Metodos relacionados con interferón inducido por ácidos nucleicos inmunoestimuladores.
DE60043708D1 (de) 1999-10-13 2010-03-04 Novartis Vaccines & Diagnostic Verfahren zur erhaltung zellimmuneantworten gegen proteinen
US7223398B1 (en) 1999-11-15 2007-05-29 Dynavax Technologies Corporation Immunomodulatory compositions containing an immunostimulatory sequence linked to antigen and methods of use thereof
ES2430368T3 (es) 1999-11-24 2013-11-20 Novartis Vaccines And Diagnostics, Inc. Partículas de tipo virus VHB/VHC
AT409085B (de) 2000-01-28 2002-05-27 Cistem Biotechnologies Gmbh Pharmazeutische zusammensetzung zur immunmodulation und herstellung von vakzinen
EP1975182A1 (en) 2000-02-01 2008-10-01 PanGenetics B.V. CD40-binding APC-activating molecules
US6756044B1 (en) 2000-02-09 2004-06-29 Genvec, Inc. Antigenic complexes and methods
US6423539B2 (en) 2000-02-24 2002-07-23 The Board Of Trustees Of The Leland Stanford Junior University Adjuvant treatment by in vivo activation of dendritic cells
EP1278542A2 (en) 2000-05-05 2003-01-29 Cytos Biotechnology AG Molecular antigen arrays and vaccines
AUPQ912000A0 (en) 2000-07-31 2000-08-24 Crown In The Right Of The Queensland Department Of Health, The Improved virus like particles
US20030055014A1 (en) 2000-12-14 2003-03-20 Bratzler Robert L. Inhibition of angiogenesis by nucleic acids
US7094409B2 (en) 2001-01-19 2006-08-22 Cytos Biotechnology Ag Antigen arrays for treatment of allergic eosinophilic diseases
US7264810B2 (en) 2001-01-19 2007-09-04 Cytos Biotechnology Ag Molecular antigen array
US7128911B2 (en) 2001-01-19 2006-10-31 Cytos Biotechnology Ag Antigen arrays for treatment of bone disease
WO2003040308A2 (en) 2001-07-27 2003-05-15 The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Use of sterically stabilized cationic liposomes to efficiently deliver cpg oligonucleotides in vivo
CA2492823A1 (en) * 2001-09-14 2003-03-27 Martin F. Bachmann In vivo activation of antigen presenting cells for enhancement of immune responses induced by virus like particles
ES2335979T3 (es) * 2001-09-14 2010-04-07 Cytos Biotechnology Ag Empaquetamiento de cpg inmunoestimuladores en particulas similares a virus: metodo de preparacion y su uso.
US7115266B2 (en) 2001-10-05 2006-10-03 Cytos Biotechnology Ag Angiotensin peptide-carrier conjugates and uses thereof
NZ531534A (en) 2001-10-05 2005-10-28 Cytos Biotechnology Ag Angiotensin peptides conjugated with a carrier comprising a virus-like particle
DK1455593T3 (da) 2001-10-06 2013-08-26 Merial Ltd Fremgangsmåder og sammensætninger til fremme af vækst og medfødt immunitet hos unge dyr
ATE412428T1 (de) 2001-11-07 2008-11-15 Cytos Biotechnology Ag Antigen-raster enthaltend rankl zur behandlung von knochenerkrankungen
DE60230656D1 (de) 2001-11-07 2009-02-12 Cytos Biotechnology Ag Antigenraster welche il-5, il-13 oder eotaxin präshilen erkrankungen
TW200303759A (en) 2001-11-27 2003-09-16 Schering Corp Methods for treating cancer
AU2003205623A1 (en) 2002-01-18 2003-07-30 Cytos Biotechnology Ag Prion protein carrier-conjugates
US20030219459A1 (en) 2002-01-18 2003-11-27 Cytos Biotechnology Ag Prion protein carrier-conjugates
EP1532167B1 (en) 2002-07-17 2012-01-25 Cytos Biotechnology AG Molecular antigen arrays using a virus like particle derived from the ap205 coat protein
KR20050027168A (ko) 2002-07-19 2005-03-17 사이토스 바이오테크놀로지 아게 그렐린-캐리어 컨쥬게이트
JP4509999B2 (ja) 2003-02-17 2010-07-21 ブルクハルト，ペーター 医薬送達及び抗原提示システムとしてのペプチドナノ粒子
AU2004255470B2 (en) * 2003-07-10 2010-08-19 Cytos Biotechnology Ag Packaged virus-like particles
CA2544240A1 (en) * 2003-07-22 2005-02-17 Cytos Biotechnology Ag Cpg-packaged liposomes
UA88457C2 (ru) 2003-10-30 2009-10-26 Коли Фармасьютикал Гмбх Иммуностимулирующая нуклеиновая кислота с улучшенной иммуностимулирующей эффективностью
DE102004041340A1 (de) 2004-08-20 2006-02-23 Deutsche Gelatine-Fabriken Stoess Ag Nanopartikel und Verfahren zu deren Herstellung
CA2580208A1 (en) * 2004-09-21 2006-03-30 Cytos Biotechnology Ag Virus-like particles comprising a fusion protein of the coat protein of ap205 and an antigenic polypeptide
CN101141979B (zh) * 2005-03-18 2013-05-08 赛托斯生物技术公司 猫变应原融合蛋白及其用途
RU2451523C2 (ru) 2005-12-14 2012-05-27 Цитос Биотехнологи Аг Упакованные иммуностимулирующей нуклеиновой кислотой частицы, предназначенные для лечения гиперчувствительности

Patent Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5071651A (en) * 1986-09-03 1991-12-10 University Of Saskatchewan Rotavirus nucleocapsid protein VP6 as a carrier in vaccine compositions
US5374426A (en) * 1986-09-03 1994-12-20 University Of Saskatchewan Rotavirus nucleocapsid protein VP6 in vaccine compositions
US5057540A (en) * 1987-05-29 1991-10-15 Cambridge Biotech Corporation Saponin adjuvant
US6326200B1 (en) * 1997-06-23 2001-12-04 Ludwig Institute For Cancer Research Isolated nona-and decapeptides which bind to HLA molecules, and the use thereof
US5962636A (en) * 1998-08-12 1999-10-05 Amgen Canada Inc. Peptides capable of modulating inflammatory heart disease
US20030060440A1 (en) * 1999-04-12 2003-03-27 Dennis Klinman Oligodeoxynucleotide and its use to induce an immune response
US6949520B1 (en) * 1999-09-27 2005-09-27 Coley Pharmaceutical Group, Inc. Methods related to immunostimulatory nucleic acid-induced interferon
US20010044416A1 (en) * 2000-01-20 2001-11-22 Mccluskie Michael J. Immunostimulatory nucleic acids for inducing a Th2 immune response
US20030087848A1 (en) * 2000-02-03 2003-05-08 Bratzler Robert L. Immunostimulatory nucleic acids for the treatment of asthma and allergy
US20030050268A1 (en) * 2001-03-29 2003-03-13 Krieg Arthur M. Immunostimulatory nucleic acid for treatment of non-allergic inflammatory diseases
US20040005338A1 (en) * 2002-06-20 2004-01-08 Cytos Biotechnology Ag Packaged virus-like particles for use as adjuvants: method of preparation and use

Cited By (93)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030109469A1 (en) * 1993-08-26 2003-06-12 Carson Dennis A. Recombinant gene expression vectors and methods for use of same to enhance the immune response of a host to an antigen
US9950055B2 (en) 2001-09-14 2018-04-24 Kuros Biosciences Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US8691209B2 (en) 2001-09-14 2014-04-08 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US20030099668A1 (en) * 2001-09-14 2003-05-29 Cytos Biotechnology Ag Packaging of immunostimulatory substances into virus-like particles: method of preparation and use
US20040058883A1 (en) * 2002-04-22 2004-03-25 Phillips Nigel C. Oligonucleotide compositions and their use for the modulation of immune responses
US7371734B2 (en) * 2002-04-22 2008-05-13 Bioniche Life Sciences Inc. Oligonucleotide compositions and their use for the modulation of immune responses
US20110135605A1 (en) * 2002-10-29 2011-06-09 Coley Pharmaceutical Group, Inc. Methods and products related to treatment and prevention of hepatitis c virus infection
US20100098722A1 (en) * 2003-03-26 2010-04-22 Cytos Biotechnology Ag Packaging of Immunostimulatory Substances Into Virus-Like Particles: Method of Preparation and Use
US20080095738A1 (en) * 2004-10-05 2008-04-24 Cytos Biotechnology Ag Vlp-Antigen Conjugates and Their Uses as Vaccines
US7959928B2 (en) 2004-10-05 2011-06-14 Cytos Biotechnology Ag VLP-antigen conjugates and their uses as vaccines
US9492400B2 (en) 2004-11-04 2016-11-15 Massachusetts Institute Of Technology Coated controlled release polymer particles as efficient oral delivery vehicles for biopharmaceuticals
US8354094B1 (en) * 2005-01-04 2013-01-15 Gp Medical, Inc. Pharmaceutical composition of nanoparticles
US9518095B2 (en) 2005-05-26 2016-12-13 Kuros Biosciences Ag Scalable fermentation process
US20120322103A1 (en) * 2005-05-26 2012-12-20 Cytos Biotechnology Ag Scalable Fermentation Process
US8574564B2 (en) 2005-12-14 2013-11-05 Cytos Biotechnology Ag Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
US9267937B2 (en) 2005-12-15 2016-02-23 Massachusetts Institute Of Technology System for screening particles
US8802153B2 (en) 2006-03-31 2014-08-12 Massachusetts Institute Of Technology System for targeted delivery of therapeutic agents
US8709483B2 (en) 2006-03-31 2014-04-29 Massachusetts Institute Of Technology System for targeted delivery of therapeutic agents
US9688812B2 (en) 2006-05-15 2017-06-27 Massachusetts Institute Of Technology Polymers for functional particles
US9080014B2 (en) 2006-05-15 2015-07-14 Massachusetts Institute Of Technology Polymers for functional particles
US9902972B2 (en) 2006-06-12 2018-02-27 Kuros Biosciences Ag Processes for packaging oligonucleotides into virus-like particles of RNA bacteriophages
US10358656B2 (en) 2006-06-12 2019-07-23 Kuros Biosciences Ag Oligonucleotides packaged into virus-like particles of RNA bacteriophages
US9404126B2 (en) 2006-06-12 2016-08-02 Kuros Biosciences Ag Processes for packaging aggregated oligonucleotides into virus-like particles of RNA bacteriophages
US9381477B2 (en) 2006-06-23 2016-07-05 Massachusetts Institute Of Technology Microfluidic synthesis of organic nanoparticles
US8586728B2 (en) 2006-12-12 2013-11-19 Cytos Biotechnology Ag Oligonucleotides containing high concentrations of guanine monomers
US20080139797A1 (en) * 2006-12-12 2008-06-12 Integrated Dna Technologies, Inc. Oligonucleotides containing high concentrations of guanine monomers
US9914746B2 (en) 2006-12-12 2018-03-13 Kuros Biosciences Ag Oligonucleotides containing high concentrations of guanine monomers
US9217129B2 (en) 2007-02-09 2015-12-22 Massachusetts Institute Of Technology Oscillating cell culture bioreactor
US9333179B2 (en) 2007-04-04 2016-05-10 Massachusetts Institute Of Technology Amphiphilic compound assisted nanoparticles for targeted delivery
US9474717B2 (en) 2007-10-12 2016-10-25 Massachusetts Institute Of Technology Vaccine nanotechnology
US11547667B2 (en) 2007-10-12 2023-01-10 Massachusetts Institute Of Technology Vaccine nanotechnology
US10736848B2 (en) 2007-10-12 2020-08-11 Massachusetts Institute Of Technology Vaccine nanotechnology
US9526702B2 (en) 2007-10-12 2016-12-27 Massachusetts Institute Of Technology Vaccine nanotechnology
US9539210B2 (en) 2007-10-12 2017-01-10 Massachusetts Institute Of Technology Vaccine nanotechnology
US8591905B2 (en) 2008-10-12 2013-11-26 The Brigham And Women's Hospital, Inc. Nicotine immunonanotherapeutics
US8906381B2 (en) 2008-10-12 2014-12-09 Massachusetts Institute Of Technology Immunonanotherapeutics that provide IGG humoral response without T-cell antigen
US8343497B2 (en) 2008-10-12 2013-01-01 The Brigham And Women's Hospital, Inc. Targeting of antigen presenting cells with immunonanotherapeutics
US9233072B2 (en) 2008-10-12 2016-01-12 Massachusetts Institute Of Technology Adjuvant incorporation in immunonanotherapeutics
US8562998B2 (en) 2008-10-12 2013-10-22 President And Fellows Of Harvard College Targeting of antigen presenting cells with immunonanotherapeutics
US9308280B2 (en) 2008-10-12 2016-04-12 Massachusetts Institute Of Technology Targeting of antigen presenting cells with immunonanotherapeutics
US8637028B2 (en) 2008-10-12 2014-01-28 President And Fellows Of Harvard College Adjuvant incorporation in immunonanotherapeutics
US8932595B2 (en) 2008-10-12 2015-01-13 Massachusetts Institute Of Technology Nicotine immunonanotherapeutics
US8343498B2 (en) 2008-10-12 2013-01-01 Massachusetts Institute Of Technology Adjuvant incorporation in immunonanotherapeutics
US9439859B2 (en) 2008-10-12 2016-09-13 Massachusetts Institute Of Technology Adjuvant incorporation in immunoanotherapeutics
US20100247669A1 (en) * 2009-03-30 2010-09-30 Cerulean Pharma Inc. Polymer-agent conjugates, particles, compositions, and related methods of use
US20100285144A1 (en) * 2009-03-30 2010-11-11 Scott Eliasof Polymer-epothilone conjugates, particles, compositions, and related methods of use
US9006254B2 (en) 2009-05-27 2015-04-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US8629151B2 (en) 2009-05-27 2014-01-14 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US9884112B2 (en) 2009-05-27 2018-02-06 Selecta Biosciences, Inc. Immunomodulatory agent-polymeric compounds
US10500156B2 (en) 2010-03-24 2019-12-10 Northeastern University Multi-compartmental macrophage delivery
WO2011119881A1 (en) * 2010-03-24 2011-09-29 Northeastern University Multi-compartmental macrophage delivery
US9764031B2 (en) 2010-05-26 2017-09-19 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
US9066978B2 (en) 2010-05-26 2015-06-30 Selecta Biosciences, Inc. Dose selection of adjuvanted synthetic nanocarriers
CN103080313A (zh) * 2010-08-20 2013-05-01 天蓝制药公司 缀合物、粒子、组合物以及相关方法
WO2012024526A3 (en) * 2010-08-20 2012-07-05 Cerulean Pharma Inc. Conjugates, particles, compositions, and related methods
US9994443B2 (en) 2010-11-05 2018-06-12 Selecta Biosciences, Inc. Modified nicotinic compounds and related methods
US10933129B2 (en) 2011-07-29 2021-03-02 Selecta Biosciences, Inc. Methods for administering synthetic nanocarriers that generate humoral and cytotoxic T lymphocyte responses
US20130039973A1 (en) * 2011-08-03 2013-02-14 Henry J. Smith Viral Immunogenic Compositions
US11590078B2 (en) * 2011-08-03 2023-02-28 Henry J. Smith Viral immunogenic compositions
US20150328305A1 (en) * 2011-08-03 2015-11-19 Henry J. Smith Viral Immunogenic Compositions
US20130209566A1 (en) * 2012-02-15 2013-08-15 Monica Jablonski Nanoparticle composition and methods to make and use the same
US9149426B2 (en) * 2012-02-15 2015-10-06 University Of Tennessee Research Foundation Nanoparticle composition and methods to make and use the same
US11071776B2 (en) 2012-04-23 2021-07-27 N-Fold Llc Nanoparticles for treatment of allergy
US9597385B2 (en) 2012-04-23 2017-03-21 Allertein Therapeutics, Llc Nanoparticles for treatment of allergy
US9999600B2 (en) 2013-04-03 2018-06-19 N-Fold Llc Nanoparticle compositions
US9539217B2 (en) 2013-04-03 2017-01-10 Allertein Therapeutics, Llc Nanoparticle compositions
US10837018B2 (en) 2013-07-25 2020-11-17 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US10894963B2 (en) 2013-07-25 2021-01-19 Exicure, Inc. Spherical nucleic acid-based constructs as immunostimulatory agents for prophylactic and therapeutic use
US11754565B2 (en) 2013-09-10 2023-09-12 Mockv Solutions Llc Methods and kits for quantifying the removal of mock virus particles from a purified solution
US10309963B2 (en) 2013-09-10 2019-06-04 MockV Solutions, Inc. Methods for evaluating viral clearance from a process solution employing mock viral particles
EP3044339A4 (en) * 2013-09-10 2017-04-26 Mockv Solutions Methods and kits for quantifying the removal of mock virus particles from a purified solution
US11135288B2 (en) 2014-04-18 2021-10-05 Auburn University Particulate formulations for enhancing growth in animals
US10293044B2 (en) * 2014-04-18 2019-05-21 Auburn University Particulate formulations for improving feed conversion rate in a subject
US11957788B2 (en) 2014-06-04 2024-04-16 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11123294B2 (en) 2014-06-04 2021-09-21 Exicure Operating Company Multivalent delivery of immune modulators by liposomal spherical nucleic acids for prophylactic or therapeutic applications
US11213593B2 (en) 2014-11-21 2022-01-04 Northwestern University Sequence-specific cellular uptake of spherical nucleic acid nanoparticle conjugates
EP3835312A1 (en) 2014-12-31 2021-06-16 Checkmate Pharmaceuticals, Inc. Combination tumor immunotherapy
WO2016109310A1 (en) 2014-12-31 2016-07-07 Checkmate Pharmaceuticals, Llc Combination tumor immunotherapy
US10682365B2 (en) 2014-12-31 2020-06-16 Checkmate Pharmaceuticals, Inc. Combination tumor immunotherapy
US11207428B2 (en) 2015-03-16 2021-12-28 Oregon Health & Science University Cross-linked polymer modified nanoparticles
US11305024B2 (en) 2015-03-16 2022-04-19 Oregon Health & Science University Cross-linked polymer modified nanoparticles
US11571474B2 (en) * 2015-09-08 2023-02-07 Universität Zürich Compositions against cat allergy
WO2017075483A1 (en) * 2015-10-28 2017-05-04 University Of Maryland, College Park Multifunctional biodegradable carriers for drug delivery
WO2017173334A1 (en) 2016-04-01 2017-10-05 Checkmate Pharmaceuticals, Inc. Fc receptor-mediated drug delivery
US11364304B2 (en) 2016-08-25 2022-06-21 Northwestern University Crosslinked micellar spherical nucleic acids
KR101886286B1 (ko) 2016-11-21 2018-08-10 주식회사 옵티팜 돼지파보바이러스 및 돼지열병바이러스에 대한 동시 항원성 복합 항원 단백질의 제조 방법
KR20180057766A (ko) * 2016-11-21 2018-05-31 충북대학교 산학협력단 돼지파보바이러스 및 돼지열병바이러스에 대한 동시 항원성 복합 항원 단백질의 제조 방법
US11696954B2 (en) 2017-04-28 2023-07-11 Exicure Operating Company Synthesis of spherical nucleic acids using lipophilic moieties
CN110042103A (zh) * 2019-03-25 2019-07-23 华南农业大学 一种对猪具有特异性免疫刺激作用的CpG-ODN及其应用
US11235058B2 (en) 2019-07-12 2022-02-01 Oregon Health & Science University Immunotherapeutic constructs and methods of their use
US11224573B2 (en) 2019-07-12 2022-01-18 Oregon Health & Science University Therapeutic constructs for co-delivery of mitotic kinase inhibitor and immune checkpoint inhibitor
US11679082B2 (en) 2019-07-12 2023-06-20 Oregon Health & Science University Therapeutic constructs for co-delivery of mitotic kinase inhibitor and immune checkpoint inhibitor
WO2022081738A3 (en) * 2020-10-13 2022-06-16 The Board Of Regents Of The University Of Texas System Polymer-conjugated microbubbles for high drug/gene loading

Also Published As

Publication number Publication date
CA2636139A1 (en) 2007-06-21
EP1973608A1 (en) 2008-10-01
AU2006325225A1 (en) 2007-06-21
US20140056933A1 (en) 2014-02-27
US8574564B2 (en) 2013-11-05
RU2008128136A (ru) 2010-01-20
WO2007068747A1 (en) 2007-06-21
NZ569741A (en) 2012-02-24
US20100303846A1 (en) 2010-12-02
JP2009519299A (ja) 2009-05-14
JP2014012677A (ja) 2014-01-23
RU2451523C2 (ru) 2012-05-27
JP5484732B2 (ja) 2014-05-07
AU2006325225B2 (en) 2013-07-04

Similar Documents

Publication Publication Date Title
US8574564B2 (en) Immunostimulatory nucleic acid packaged particles for the treatment of hypersensitivity
ES2344303T3 (es) Particulas que comprenden un nucleo de nanoparticulas de fosfato calcico, una biomolecula y un acido biliar, los metodos de produccion y el uso terapeutico de las mismas.
EP1513552B1 (en) Packaged virus-like particles in combination with cpg for use as adjuvants with allergens : method of preparation and use
Sokolova et al. The potential of nanoparticles for the immunization against viral infections
Lugade et al. Single low-dose un-adjuvanted HBsAg nanoparticle vaccine elicits robust, durable immunity
JP2009513532A (ja) パッケージ化ウイルス様粒子
Teng et al. Golden-star nanoparticles as adjuvant effectively promotes immune response to foot-and-mouth disease virus-like particles vaccine
Shah et al. Nanomedicine based approaches for combating viral infections
KR101671376B1 (ko) 돼지 살모넬라 감염 및 prrs 바이러스 예방 또는 치료용 서방형 제제 및 그 제조방법
Feng et al. DEC-205 receptor targeted poly (lactic-co-glycolic acid) nanoparticles containing Eucommia ulmoides polysaccharide enhances the immune response of foot-and-mouth disease vaccine in mice
AU2013204626A1 (en) Immunostimulatory nucleic acid packaged particles for the treatment of hypersenstivity
Stephens et al. Nanoparticle vaccines against respiratory syncytial virus
Rasool et al. Advances in vaccine delivery strategies to promote effective immunization
Patel et al. Cationic nanoparticles for delivery of CpG oligodeoxynucleotide and ovalbumin: In vitro and in vivo assessment
Piva-Amaral et al. Bovine serum albumin nanoparticles containing Poly (I: C) can enhance the neutralizing antibody response induced by envelope protein of Orthoflavivirus zikaense
Jones et al. Small Molecule Antivirals to Nanoparticles: The Need of the Hour to Combat Pandemics
Chen Characterization of Degradation Profiles of Acetalated Dextran Microparticles and Their Application for Immune Modulation
Misra et al. Nanovaccines for Mucosal Immunity
Rahaman Nanomedicine: an essential resource in treatment and diagnosis of viral diseases
McHugh The delivery of DNA vaccines using particulate and non-particulate adjuvants
Zhao et al. M13 Phage-Loaded Quaternized Chitosan Nano-Vaccine Against H9N2 Avian Influenza Virus

Legal Events

Date Code Title Description
STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION

AS Assignment

Owner name: CYTOS BIOTECHNOLOGY AG, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:RENNER, WOLFGANG A.;BACHMANN, MARTIN F.;CIELENS, INDULIS;AND OTHERS;SIGNING DATES FROM 20101122 TO 20101125;REEL/FRAME:027748/0763

Owner name: LUDWIG-MAXIMILIANS-UNIVERSITAT-MUNCHEN, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:COESTER, CONRAD JOHANNES;FUCHS, SEBASTIAN;SIGNING DATES FROM 20101216 TO 20110309;REEL/FRAME:027748/0807

Owner name: CYTOS BIOTECHNOLOGY AG, SWITZERLAND

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:LUDWIG-MAXIMILIANS-UNIVERSITAT-MUNCHEN;REEL/FRAME:027748/0801

Effective date: 20110314

AS Assignment

Owner name: VENBIO GLOBAL STRATEGIC FUND L.P., CALIFORNIA

Free format text: SECURITY AGREEMENT;ASSIGNOR:CYTOS BIOTECHNOLOGY AG;REEL/FRAME:028275/0531

Effective date: 20120515