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WO2007039458A2 - Conjugues peptidiques du vih et leurs utilisations - Google Patents

Conjugues peptidiques du vih et leurs utilisations Download PDF

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Publication number
WO2007039458A2
WO2007039458A2 PCT/EP2006/066526 EP2006066526W WO2007039458A2 WO 2007039458 A2 WO2007039458 A2 WO 2007039458A2 EP 2006066526 W EP2006066526 W EP 2006066526W WO 2007039458 A2 WO2007039458 A2 WO 2007039458A2
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WIPO (PCT)
Prior art keywords
peptide
vlp
composition
hiv
seq
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PCT/EP2006/066526
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English (en)
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WO2007039458A3 (fr
Inventor
Martin Bachmann
Philippe Saudan
Andrea Jegerlehner
Till Alexander RÖHN
Gary Jennings
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Cytos Biotechnology Ag
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Publication of WO2007039458A2 publication Critical patent/WO2007039458A2/fr
Publication of WO2007039458A3 publication Critical patent/WO2007039458A3/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—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
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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

  • the present invention is in the fields of medicine, public health, immunology, molecular biology and virology.
  • the invention provides composition comprising a virus-like particle (VLP) linked to at least one antigen, wherein said antigen is HIV gpl60 peptide.
  • VLP virus-like particle
  • the invention also provides a process for producing the composition.
  • the compositions of this invention are useful in the production of vaccines, in particular, for the prevention and treatment of diseases.
  • the compositions of the invention induce efficient immune responses, in particular antibody responses.
  • HIV envelope glycoproteins are synthesized as a gpl60 precursor which is subsequently cleaved to the gpl20 outer surface and gp41 transmembrane proteins which form a complex.
  • gpl20 determines viral tropism by binding to target-cell receptors, while gp41 mediates fusion between viral and cellular membranes.
  • Dong et al. identified conserved domains within gp41 which can be used to develop novel effective vaccines (Dong et al., 2001, Immunology Letters, 75, 215). Moreover several epitopes within the HIV envelope proteins have been shown to induce antibodies that could inhibit or neutralize HIV infection in vitro (Ho et al, 1987, J. Virol. 2024, Zwick et al., 2000, J.Virol., Kennedy et al., 1987, JBC 5769, Hovanessian et al, 2004, Immunity, 21, 617). Most of these epitopes are relatively well conserved between different HIV clades and hence represent good targets for broadly neutralizing vaccines. However, despite the identification of such epitopes to date no vaccine which induces neutralizing antibodies has been developed for humans.
  • inventive compositions and vaccines respectively, comprising at least one HIV gpl60 peptide
  • inventive compositions and vaccines are capable of inducing immune responses, in particular antibody responses, leading to high antibody titer against HIV.
  • inventive compositions and vaccines, respectively, comprising at least one HIV gpl60 peptide are capable of inducing immune responses, in particular antibody responses, with protective and/or therapeutic effect against the HIV infection.
  • the immune responses in particular the antibodies generated by the inventive compositions and vaccines, respectively, are, thus, capable of specifically recognizing HIV and/or HIV infected cells in vivo, and neutralizing and inhibiting the infection of the virus.
  • the present invention provides a composition which comprises (a) a virus-like particle (VLP) with at least one first attachment site; and (b) at least one antigen with at least one second attachment site, wherein said at least one antigen is a HIV gpl60 peptide, and wherein (a) and (b) are linked through said at least one first and said at least one second attachment site, preferably to form an ordered and repetitive antigen array.
  • the virus-like particle suitable for use in the present invention comprises recombinant protein, preferably recombinant coat protein, mutants or fragments thereof, of a virus, preferably of a RNA bacteriophage.
  • the composition of the invention further comprises at least one immunostimulatory substance, an immunostimulatory nucleic acid, even more preferably an immunostimulatory nucleic acid comprising at least one unmethylated CpG motif.
  • immunostimulatory substance bound to, preferably packaged inside, the VLP enhances the immune response against HIV.
  • the present invention provides a method of preventing and/or treating HIV infection, wherein the method comprises administering the inventive composition or the inventive vaccine composition, respectively, to a human.
  • Antigen refers to a molecule capable of being bound by an antibody or a T cell receptor (TCR) if presented by MHC molecules.
  • TCR T cell receptor
  • An antigen is additionally capable of being recognized by the immune system and/or being capable of inducing a humoral immune response and/or cellular immune response leading to the activation of B- and/or T-lymphocytes. This may, however, require that, at least in certain cases, the antigen contains or is linked to a Th cell epitope and is given in adjuvant.
  • An antigen can have one or more epitopes (B- and T- epitopes).
  • the specific reaction referred to above is meant to indicate that the antigen will preferably react, typically in a highly selective manner, with its corresponding antibody or TCR and not with the multitude of other antibodies or TCRs which may be evoked by other antigens.
  • Antigens as used herein may also be mixtures of several individual antigens.
  • Antigenic site refers to continuous or discontinuous portions of a polypeptide, which can be bound immunospecifically by an antibody or by a T-cell receptor within the context of an MHC molecule. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity. Antigenic site typically comprise 5- 10 amino acids in a spatial conformation which is unique to the antigenic site.
  • HIV gpl60 the term “HIV gpl60” as used herein, refers to the polypeptide encoded by env gene comprised by the genome of all HIV strains.
  • HIV gpl60 should further encompass post-translational modifications including but not limited to glycosylations, acetylations, phosphorylations of the HIV gpl60 as defined above.
  • HIV gpl60 peptide The term "HIV gpl60 peptide”, as used herein, should encompass any polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of, at least 4, 5, preferably at least 6, 7, 8, 9, 10, 15, contiguous amino acids of a HIV gpl60 and the same polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of at most 60, 55, more preferably 50, 45, still more preferably at most 40 amino acids contiguous amino acids of a HIV gpl60 as defined herein - A - as well as any polypeptide having more than 70%, 80%, more preferably more than 90% and even more preferably more than 95% amino acid sequence identity thereto.
  • HIV gpl60 peptide should encompass any polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of, at least 6 contiguous amino acids of a HIV gpl60 and the same polypeptide comprising, consisting essentially of, or alternatively or preferably consisting of at most 45, still more preferably at most 40 amino acids contiguous amino acids of a HIV gpl60 as defined herein as well as any polypeptide having more than 70%, 80%, preferably more than 90% and even more preferably more than 95% amino acid sequence identity thereto.
  • HIV gpl60 peptide is capable of inducing the production of antibodies in vivo, which specifically bind to HIV and/or to HIV infected cells.
  • HIV gpl60 peptide should furthermore encompass post-translational modifications including but not limited to glycosylations, acetylations, phosphorylations of the HIV gpl60 peptide as defined above.
  • associated or its noun association as used herein refers to all possible ways, preferably chemical interactions, by which two molecules are joined together. Chemical interactions include covalent and non-covalent interactions.
  • non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds
  • covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon- sulfur bonds such as thioether, or imide bonds.
  • first attachment site refers to an element which is naturally occurring with the VLP or which is artificially added to the VLP, and to which the second attachment site may be linked.
  • the first attachment site may be a protein, a polypeptide, an amino acid, a peptide, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, histidinyl group, or a combination thereof.
  • a preferred embodiment of a chemically reactive group being the first attachment site is the amino group of an amino acid such as lysine.
  • the first attachment site is located, typically on the surface, and preferably on the outer surface of the VLP. Multiple first attachment sites are present on the surface, preferably on the outer surface of virus-like particle, typically in a repetitive configuration.
  • the first attachment site is associated with the VLP, through at least one covalent bond, preferably through at least one peptide bond.
  • the first attachment site is naturally occurring with the VLP.
  • the first attachment site is artificially added to the VLP.
  • Attachment Site refers to an element which is naturally occurring with or which is artificially added to the HIV gpl60 peptide and to which the first attachment site may be linked.
  • the second attachment site of the HIV gpl60 peptide may be a protein, a polypeptide, a peptide, an amino acid, a sugar, a polynucleotide, a natural or synthetic polymer, a secondary metabolite or compound (biotin, fluorescein, retinol, digoxigenin, metal ions, phenylmethylsulfonylfluoride), or a chemically reactive group such as an amino group, a carboxyl group, a sulfhydryl group, a hydroxyl group, a guanidinyl group, histidinyl group, or a combination thereof.
  • a preferred embodiment of a chemically reactive group being the second attachment site is the sulfhydryl group, preferably of an amino acid cysteine.
  • the terms "antigen with at least one second attachment site” and the interchangeably used term "HIV gpl60 peptide with at least one second attachment site”, as used herein, refers, to a construct comprising the HIV gpl60 peptide and at least one second attachment site.
  • the second attachment site is naturally occurring within the HIV gpl60 peptide.
  • the second attachment site is artificially added to the HIV gpl60 peptide.
  • the second attachment site is associated with the HIV gpl60 peptide through at least one covalent bond, preferably through at least one peptide bond.
  • the HIV gpl60 peptide with at least one second attachment site further comprises a linker, preferably said linker comprises at least one second attachment site, preferably said linker is fused to the HIV gpl60 peptide by a peptide bond.
  • capsid protein within this application, refers to a viral protein, preferably a subunit of a natural capsid of a virus, preferably of a RNA-pahge, which is capable of being incorporated into a virus capsid or a VLP.
  • Linked refers to all possible ways, preferably chemical interactions, by which the at least one first attachment site and the at least one second attachment site are joined together. Chemical interactions include covalent and non-covalent interactions. Typical examples for non-covalent interactions are ionic interactions, hydrophobic interactions or hydrogen bonds, whereas covalent interactions are based, by way of example, on covalent bonds such as ester, ether, phosphoester, amide, peptide, carbon-phosphorus bonds, carbon-sulfur bonds such as thioether, or imide bonds.
  • first attachment site and the second attachment site are linked through at least one covalent bond, preferably through at least one non-peptide bond, and even more preferably through exclusively non-peptide bond(s).
  • Linker A "linker”, as used herein, either associates the second attachment site with a HIV gpl60 peptide or already comprises, essentially consists of, or consists of the second attachment site.
  • a “linker”, as used herein already comprises the second attachment site, typically and preferably - but not necessarily - as one amino acid residue, preferably as a cysteine residue.
  • a “linker” as used herein is also termed “amino acid linker", in particular when a linker according to the invention contains at least one amino acid residue.
  • linker and “amino acid linker” are interchangeably used herein.
  • linker consists exclusively of amino acid residues, even if a linker consisting of amino acid residues is a preferred embodiment of the present invention.
  • the amino acid residues of the linker are, preferably, composed of naturally occurring amino acids or unnatural amino acids known in the art, all-L or all-D or mixtures thereof.
  • Further preferred embodiments of a linker in accordance with this invention are molecules comprising a sulfhydryl group or a cysteine residue and such molecules are, therefore, also encompassed within this invention.
  • linkers useful for the present invention are molecules comprising a C1-C6 alkyl-, a cycloalkyl such as a cyclopentyl or cyclohexyl, a cycloalkenyl, aryl or heteroaryl moiety.
  • linkers comprising preferably a C1-C6 alkyl-, cycloalkyl- (C5, C6), aryl- or heteroaryl- moiety and additional amino acid(s) can also be used as linkers for the present invention and shall be encompassed within the scope of the invention.
  • Association of the linker with the HIV gpl60 peptide is preferably by way of at least one covalent bond, more preferably by way of at least one peptide bond.
  • the linker is associated to the at least one second attachment site, for example, a cysteine, preferably, by way of at least one covalent bond, more preferably by way of at least one peptide bond.
  • ordered and repetitive antigen array generally refers to a repeating pattern of antigen or, characterized by a typically and preferably high order of uniformity in spacial arrangement of the antigens with respect to virus-like particle, respectively. In one embodiment of the invention, the repeating pattern may be a geometric pattern.
  • Certain embodiments of the invention are typical and preferred examples of suitable ordered and repetitive antigen arrays which, moreoever, possess strictly repetitive paracrystalline orders of antigens, preferably with spacings of 1 to 30 nanometers, preferably 2 to 15 nanometers, even more preferably 2 to 10 nanometers, even again more preferably 2 to 8 nanometers, and further more preferably 1.6 to 7 nanometers.
  • the term "packaged” as used herein refers to the state of an immunostimulatory substance, preferably of an immunostimulatory nucleic acid, in relation to the modified VLP.
  • the term “packaged” as used herein refers to the enclosement, or partial enclosement, of the immunostimulatory substance, preferably of the immunostimulatory nucleic acid substance.
  • the term “packaged” as used herein includes binding that may be covalent, e.g., by chemically coupling, or non-covalent, e.g., ionic interactions, hydrophobic interactions, hydrogen bonds, etc.
  • the immunostimulatory substance such as the unmethylated CpG-containing oligonucleotide can be enclosed by the VLP even without the existence of an actual covalent binding.
  • the immunostimulatory nucleic acid is packaged inside the VLP and thus typically and preferably not accessible to DNase or RNase hydrolysis.
  • Polypeptide refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). It indicates a molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides and proteins are included within the definition of polypeptide. Post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, and the like are also encompassed.
  • 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.
  • Virus particle The term “virus particle” as used herein refers to the morphological form of a virus.
  • Virus-like particle refers to a non-replicative or noninfectious, 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 due to physical, chemical inactivation or due to genetic manipulation.
  • 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-phage.
  • 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.
  • 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.
  • virus particle and virus-like particle are highly ordered and repetitive arrangement of its subunits.
  • virus-like particle of a RNA phage refers to a virus-like particle comprising, or preferably consisting essentially of or consisting of coat proteins, mutants or fragments thereof, of a RNA phage.
  • virus-like particle of a RNA phage resembling the structure of a RNA phage, being non replicative or non-infectious, and typically and preferably being non replicative and noninfectious.
  • virus-like particle of a RNA phage should furthermore refer to a virus-like particle of a RNA phage which lacks at least one of the genes, preferably all of the genes, encoding for the replication machinery of the RNA phage, and typically and further preferably even at least one of the genes, preferably all of the genes, encoding the protein or proteins responsible for viral attachment to or entry into the host.
  • This definition should, however, also encompass virus-like particles of RNA phages, in which the aforementioned gene or genes are still present but inactive, and, therefore, also leading to non- replicative and/or noninfectious virus-like particles of a RNA phage.
  • virus-like particle of a RNA phage should therefore also encompass in its broadest definition a virus particle of a RNA phage, the genome of which has been inactivated by physical or chemical or genetic methods so that the virus particle is not capable of infecting and/or replicating.
  • Preferred VLPs derived from RNA-phages exhibit icosahedral symmetry and consist of 180 subunits.
  • subunit and “monomer” are interexchangeably and equivalently used within this context.
  • RNA -phage and the term “RNA-bacteriophage” are interchangeably used.
  • antibodies are defined to be specifically binding if they bind to the antigen with a binding affinity (Ka) of 10 6 M “1 or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, and most preferably 10 9 M "1 or greater.
  • Ka binding affinity
  • the affinity of an antibody can be readily determined by one of ordinary skill in the art (for example, by Scatchard analysis.)
  • the amino acid sequence identity of polypeptides can be determined conventionally using known computer programs such as the Bestfit program.
  • 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.
  • Conservative amino acid substitutions include isosteric substitutions, substitutions where the charged, polar, aromatic, aliphatic or hydrophobic nature of the amino acid is maintained.
  • Typical conservative amino acid substitutions are substitutions between amino acids within one of the following groups: GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr, Cys; Lys, Arg; and Phe and Tyr.
  • compositions and methods for enhancing immune responses against HIV in an animal or in human comprises: (a) a virus-like particle (VLP) with at least one first attachment site; and (b) at least one antigen with at least one second attachment site, wherein the at least one antigen is a HIV gpl60 peptide and wherein (a) and (b) are linked through the at least one first and the at least one second attachment site.
  • VLP virus-like particle
  • the HIV gpl60 peptide is linked to the VLP, so as to form an ordered and repetitive antigen-VLP array.
  • At least 20, preferably at least 30, more preferably at least 60, again more preferably at least 120 and further more preferably at least 180 HIV gpl60 peptides are linked to the VLP.
  • Any virus known in the art having an ordered and repetitive structure may be selected as a VLP of the invention.
  • 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.
  • Virus or virus-like particle can be produced and purified from virus-infected cell culture.
  • the resulting virus or virus-like particle for vaccine purpose needs to be devoid of virulence.
  • physical or chemical methods can be employed to inactivate the viral genome function, such as UV irradiation, formaldehyde treatment.
  • the VLP is a recombinant VLP. Almost all commonly known viruses have been sequenced and are readily available to the public. The gene encoding the coat protein can be easily identified by a skilled artisan. The preparation of VLPs by recombinantly expressing the coat protein in a host is within the common knowledge of a skilled artisan.
  • the virus-like particle comprises, or alternatively consists of, recombinant proteins, mutants or fragments thereof, of a virus selected form the group consisting of: a) RNA phages; b) bacteriophages; c) Hepatitis B virus, preferably its capsid protein (Ulrich, et al, Virus Res.
  • the VLP comprises, or consists of, more than one amino acid sequence, 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 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 recombinant protein or the term “mutant of a recombinant protein” as interchangeably used in this invention, or the term “mutant coat protein” or the term “mutant of a coat protein”, as interchangeably used in this invention, 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 virus-like particle of the invention is of
  • Hepatitis B virus The preparation of Hepatitis B virus-like particles have been disclosed, inter alia, in WO 00/32227, WO 01/85208 and in WO 01/056905. All three 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 WO 01/056905. [0042] In one further preferred embodiments of the invention, a lysine residue is introduced into the HBcAg polypeptide, to mediate the linking of HIV gpl60 peptide to the VLP of HBcAg.
  • 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:20, 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.
  • This modification changes the SEQ ID NO:20 to SEQ ID NO:21.
  • the cysteine residues at positions 48 and 110 of SEQ ID NO:21, 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.
  • compositions and vaccines comprising HBcAg polypeptides which comprise, or alternatively consist of, amino acid sequences which are at least 80%, 85%, 90%, 95%, 97% or 99% identical to SEQ ID NO:21.
  • the virus-like particle of the invention comprises, consists essentially of, or alternatively consists of, recombinant coat proteins, mutants or fragments thereof, of a RNA-phage.
  • the RNA-phage is selected from the group consisting of a) bacteriophage Q ⁇ ; b) bacteriophage Rl 7; c) bacteriophage fr; d) bacteriophage GA; e) bacteriophage SP; f) bacteriophage MS2; g) bacteriophage Mi l; h) bacteriophage MXl; i) bacteriophage NL95; k) bacteriophage f2; 1) bacteriophage PP7 and m) bacteriophage AP205.
  • the composition comprises coat protein, mutants or fragments thereof, of RNA phages, wherein the coat protein has amino acid sequence selected from the group consisting of: (a) SEQ ID NO: 1; referring to Q ⁇ CP; (b) a mixture of SEQ ID NO: 1 and SEQ ID NO:2 .(referring to Q ⁇ Al protein); (c) SEQ ID NO:3; (d) SEQ ID NO:4; (e) SEQ ID NO:5; (f) SEQ ID NO:6, (g) a mixture of SEQ ID NO:6 and SEQ ID NO:7; (h) SEQ ID NO:8; (i) SEQ ID NO:9; (j) SEQ ID NO: 10; (k) SEQ ID NO: 11; (1) SEQ ID NO: 12; (m) SEQ ID NO: 13; and (n) SEQ ID NO: 14.
  • 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 phage.
  • the VLP comprises or alternatively consists of two different coat proteins of a RNA phage, said two coat proteins have an amino acid sequence of SEQ ID NO: 1 and SEQ ID NO:2, or of SEQ ID NO:6 and SEQ ID NO:7.
  • 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- phage 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.
  • the capsid or VLP of Q ⁇ shows unusual resistance to organic solvents and denaturing agents. Surprisingly, we have observed that DMSO and acetonitrile concentrations as high as 30%, and guanidinium concentrations as high as 1 M do not affect the stability of the capsid.
  • the high stability of the capsid or VLP of Q ⁇ is an advantageous feature, in particular, for its use in immunization and vaccination of mammals and humans in accordance of the present invention.
  • RNA-phages in particular of Q ⁇ and fr in accordance of this invention are disclosed in WO 02/056905, the disclosure of which is herewith incorporated by reference in its entirety.
  • Particular 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 phage AP205.
  • Assembly-competent mutant forms of AP205 VLPs including AP205 coat protein with the substitution of proline at amino acid 5 to threonine or AP205 coat protein with the substitution of asparigine at amino acid 14 to aspartic acid, 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.
  • the VLP of the invention comprises or consists of a mutant coat protein of a virus, preferably a RNA phage, wherein the mutant coat protein has been modified by removal of at least one lysine residue by way of substitution and/or by way of deletion.
  • the VLP of the invention comprises or consists of a mutant coat protein of a virus, preferably a RNA phage, wherein the mutant coat protein has been modified by addition of at least one lysine residue by way of substitution and/or by way of insertion.
  • the deletion, substitution or addition of at least one lysine residue allows varying the degree of coupling, i.e. the amount of antigen per subunits of the VLP of a virus, preferably of a RNA-phages, in particular, to match and tailor the requirements of the vaccine.
  • the compositions and vaccines of the invention have an antigen density being from 0.5 to 4.0.
  • antigen density refers to the average number of antigen which is linked per subunit, preferably per coat protein, of the VLP, and hereby preferably of the VLP of a RNA phage. Thus, this value is calculated as an average over all the subunits or monomers of the VLP, preferably of the VLP of the RNA -phage, in the composition or vaccines of the invention.
  • VLPs or capsids of Q ⁇ coat protein display a defined number of lysine residues on their surface, with a defined topology with three lysine residues pointing towards the interior of the capsid and interacting with the RNA, and four other lysine residues exposed to the exterior of the capsid.
  • the at least one first attachment site is a lysine residue, pointing to or being on the exterior of the VLP.
  • 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: 15, Lysl3-Arg of SEQ ID NO: 1) b) Q ⁇ -243 (SEQ ID NO: 16, AsnlO-Lys of SEQ ID NO: 1); c) Q ⁇ -250 (SEQ ID NO: 17, Lys2-Arg of SEQ ID NO: 1) d) Q ⁇ -251 (SEQ ID NO: 18, Lysl6-Arg of SEQ ID NO: 1); and e) Q ⁇ -259" (SEQ ID NO: 19, Lys2-Arg, Lysl6-Arg of SEQ ID NO: 1).
  • 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 Al protein.
  • the virus-like particle comprises, or alternatively consists essentially of, or alternatively consists of mutant coat protein with amino acid sequence SEQ ID N0: 15, 16, 17, 18, or 19 and the corresponding Al protein.
  • RNA phage coat proteins have also been shown to self-assemble upon expression in a bacterial host (Kastelein, RA. et al., Gene 23:245-254 (1983), Kozlovskaya, TM. et al., Dokl. Akad. Nauk SSSR 287:452-455 (1986), Adhin, MR. et al., Virology 170:238-242 (1989), Priano, C. et al., J. MoI. Biol. 249:283-297 (1995)).
  • GA Biochemical and biochemical properties of GA (Ni, CZ., et al., Protein Sci.
  • the at least one antigen comprises, consists essentially of, or consists of a HIV gpl60 peptide, wherein preferably the HIV gpl60 peptide is highly conserved among all HIV strains (more than 70% identity) or wherein preferably the HIV gpl60 peptide induces neutralizing antibodies or wherein preferably the HIV gpl60 peptide is a blocking peptide.
  • the HIV gpl60 peptide is selected from the group consisting of:
  • HIV env 1 SLEQIWNNMTWMQWDK (SEQ ID NO:22);
  • HIV env 2 SLEQIWNNMTWMQWDR (SEQ ID NO:23);
  • HIV env 3 IWNNMTWMQWDR (SEQ ID NO:24);
  • HIV env 4 WASLWNW (SEQ ID NO:25);
  • HIV env 5 NWFDISNWLW (SEQ ID NO:26);
  • HIV env 6 LLELDKWASLWNWFNL (SEQ ID NO:27);
  • HIV env 7 ELDKWA, (SEQ ID NO:28);
  • HIV env 8 WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL
  • HIV env 9 CSKLIC (SEQ ID NO:30);
  • HIV env 10 GFLGAAGSTMGAASITLVQ (SEQ ID NO:31);
  • HIV env 12 GIVQQQ (SEQ ID NO:33);
  • HIV env 13 QLLGIWGCSGKLICTTA VP WNSSWS (SEQ ID NO:
  • HIV env 14 NAKTIIVQLNQSVE (SEQ ID NO:35);
  • HIV env 15 GGNSNNESEIFRPGGGD (SEQ ID NO:36); AND
  • VAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSGC SEQ ID NO:37.
  • the at least one antigen comprises, consists essentially of, or consists of a mutein of any one of SEQ ID NOs:22-37 and SEQ ID NO:51.
  • a mutein of any one of SEQ ID NOs:22-37 and SEQ ID NO:51 encompasses any polypeptide having at least 70%, preferably at least 80%, more preferably 90%, still more preferably at least 95%, still more preferably at least 97% amino acid sequence identity with any one of the SEQ ID NOs:22-37 and SEQ ID NO:51.
  • sequence difference between any of the SEQ ID NOs:22-37 and SEQ ID NO:51 and its corresponding mutein protein is introduced by at least one genetic engineering, wherein said genetic engineering is selected from the group consisting of addition, insertion, deletion, truncation (refers to deletion from the end of the protein), substitution and a combination thereof.
  • the mutein has the amino acid sequence of any one of SEQ ID NOs:22-37 and SEQ ID NO:51, wherein at most six amino acid residues, preferably at most five, four or three amino acid residues, more preferably at most two amino acid residues, and even more preferably one amino acid residue of any one of SEQ ID NOs:22-37 and SEQ ID NO: 51 is, deleted, inserted, or substituted, wherein preferably at least one, more preferably at least two, three or four, and even more preferably all of said substitutions are conservative substitutions.
  • the VLP with at least one first attachment site is linked to the HIV gpl60 peptide with at least one second attachment site via at least one peptide bond.
  • Gene encoding HIV gpl60 peptide is in-frame ligated, either internally or preferably to the N- or the C-terminus to the gene encoding the coat protein of the VLP. Fusion may also be effected by inserting sequences of the HIV gpl60 peptide into a mutant of a coat protein where part of the coat protein sequence has been deleted, that are further referred to as truncation mutants. Truncation mutants may have N- or C-terminal, or internal deletions of part of the sequence of the coat protein. For example for the specific VLP HBcAg, amino acids 79-80 are replaced with a foreign epitope.
  • the fusion protein shall preferably retain the ability of assembly into a VLP upon expression which can be examined by electromicroscopy.
  • Flanking amino acid residues may be added to increase the distance between the coat protein and foreign epitope. Glycine and serine residues are particularly favored amino acids to be used in the flanking sequences. Such a flanking sequence confers additional flexibility, which may diminish the potential destabilizing effect of fusing a foreign sequence into the sequence of a VLP subunit and diminish the interference with the assembly by the presence of the foreign epitope.
  • the HIV gpl60 peptide can be fused to a number of other viral coat protein, as way of examples, to the C-terminus of a truncated form of the Al protein of Q ⁇ (Kozlovska, T. M., et al, Intervirology 39:9-15 (1996)), or being inserted between position 72 and 73 of the CP extension.
  • Kozlovska et al., (Intervirology, 39: 9-15 (1996)) describe Q ⁇ Al protein fusions where the epitope is fused at the C-terminus of the Q ⁇ CP extension truncated at position 19.
  • the HIV gpl60 peptide can be inserted between amino acid 2 and 3 of the fr CP(Pushko P. et al., Prot. Eng. 6:883-891 (1993)). Furthermore, the HIV gpl60 peptide can be fused to the N-terminal protuberant ⁇ -hairpin of the coat protein of RNA phage MS-2 (WO 92/13081). Alternatively, the HIV gpl60 peptide can be fused to a capsid protein of papillomavirus, preferably to the major capsid protein Ll of bovine papillomavirus type 1 (BPV-I) (Chackerian, B. et al., Proc. Natl.
  • BPV-I bovine papillomavirus type 1
  • HIV gpl60 peptide is fused to either the
  • the fusion protein further comprises a spacer, wherein said spacer is positioned between the coat protein, fragments or mutants thereof, of AP205 and the HIV gpl60 peptide.
  • said spacer composed of less than 20, even more preferably less than 15, more preferably less than 12, 10, still more preferably less than 8, less than 5 amino acids.
  • the composition comprises or alternatively consists essentially of a virus-like particle with at least one first attachment site linked to at least one HIV gpl60 peptide with at least one second attachment site via at least one covalent bond, preferably the covalent bond is a non-peptide bond.
  • the first attachment site does not comprise or is not sulfhydryl group of cysteine.
  • the first attachment site does not comprise or is not sulfhydryl group.
  • the first attachment site comprises, or preferably is, an amino group, preferably the amino group of a lysine residue.
  • the second attachment site comprises, or preferably is, a sulfhydryl group, preferably a sulfhydryl group of a cysteine
  • the at least one first attachment site is an amino group, preferably an amino group of a lysine residue and the at least one second attachment site is a sulfhydryl group, preferably a sulfhydryl group of a cysteine.
  • the HIV gpl60 peptide is linked to the VLP by way of chemical cross-linking, typically and preferably by using a heterobifunctional cross-linker.
  • the hetero-bifunctional cross- linker contains a functional group which can react with the preferred first attachment sites, preferably with the amino group, more preferably with the amino groups of lysine residue(s) of the VLP, and a further functional group which can react with the preferred second attachment site, i.e. a sulfhydryl group, preferably of cysteine(s) residue inherent of, or artificially added to the HIV gpl60 peptide, and optionally also made available for reaction by reduction.
  • a functional group which can react with the preferred first attachment sites preferably with the amino group, more preferably with the amino groups of lysine residue(s) of the VLP
  • a further functional group which can react with the preferred second attachment site, i.e. a sulfhydryl group, preferably of cysteine(s) residue inherent of, or artificially added to the HIV gpl60 peptide, and optionally also made available for reaction by reduction.
  • cross-linkers include the preferred cross-linkers SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-SIAB, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers available for example from the Pierce Chemical Company, and having one functional group reactive towards amino groups and one functional group reactive towards sulfhydryl groups.
  • the above mentioned cross-linkers all lead to formation of an amide bond after reaction with the amino group and a thioether linkage with the sulfhydryl groups.
  • cross-linkers suitable in the practice of the invention is characterized by the introduction of a disulfide linkage between the HIV gpl60 peptide and the VLP upon coupling.
  • Preferred cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce).
  • the composition of the invention further comprises a linker.
  • a linker is associated to the HIV gpl60 peptide by way of at least one covalent bond, preferably, by at least one, typically one peptide bond.
  • the linker comprises, or alternatively consists of, the second attachment site.
  • the linker comprises a sulfhydryl group, preferably of a cysteine residue.
  • the amino acid linker is a cysteine residue.
  • Linking of the HIV gpl60 peptide to the VLP by using a hetero-bifunctional cross-linker allows coupling of the HIV gpl60 peptide to the VLP in an oriented fashion.
  • Other methods of linking the HIV gpl60 peptide to the VLP include methods wherein the HIV gpl60 peptide is cross-linked to the VLP, using the carbodiimide EDC, and NHS.
  • the HIV gpl60 peptide may also be first thiolated through reaction, for example with SATA, SATP or iminothiolane. The HIV gpl60 peptide, after deprotection if required, may then be coupled to the VLP as follows.
  • the HIV gpl60 peptide is reacted with the VLP, previously activated with a hetero-bifunctional cross-linker comprising a cysteine reactive moiety, and therefore displaying at least one or several functional groups reactive towards cysteine residues, to which the thiolated HIV gpl60 peptide can react, such as described above.
  • a hetero-bifunctional cross-linker comprising a cysteine reactive moiety, and therefore displaying at least one or several functional groups reactive towards cysteine residues, to which the thiolated HIV gpl60 peptide can react, such as described above.
  • low amounts of a reducing agent are included in the reaction mixture.
  • the HIV gpl60 peptide is attached to the VLP, using a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO]4, BS3, (Pierce) or other known homo- bifunctional cross-linkers with functional groups reactive towards amine groups or carboxyl groups of the VLP.
  • a homo-bifunctional cross-linker such as glutaraldehyde, DSG, BM[PEO]4, BS3, (Pierce) or other known homo- bifunctional cross-linkers with functional groups reactive towards amine groups or carboxyl groups of the VLP.
  • the composition comprises or alternatively consists essentially of a virus-like particle linked to HIV gpl60 peptide via chemical interactions, wherein at least one of these interactions is not a covalent bond. Such interactions include but not limited to antigen-antibody interaction, receptor-ligand interaction.
  • Linking of the VLP to the HIV gpl60 peptide can be effected by biotinylating the VLP and expressing the HIV gpl60 peptide as a streptavidin- fusion protein.
  • One or several antigen molecules i.e. HIV gpl60 peptide, can be attached to one subunit of the VLP, preferably of RNA phage coat proteins, preferably through the exposed lysine residues of the coat proteins of RNA phage VLP, if sterically allowable.
  • a specific feature of the VLPs of RNA phage and in particular of the Q ⁇ coat protein VLP is thus the possibility to couple several antigens per subunit. This allows for the generation of a dense antigen array.
  • the HIV gpl60 peptide is linked via a cysteine residue, having been added to either the N-terminus or the C-terminus of, or a natural cysteine residue within HIV gpl60 peptide, to lysine residues of coat proteins of the VLPs of RNA phage, and in particular to the coat protein of Q ⁇ .
  • lysine residues are exposed on the surface of the VLP of Q ⁇ coat protein. Typically and preferably these residues are derivatized upon reaction with a cross-linker molecule. In the instance where not all of the exposed lysine residues can be coupled to an antigen, the lysine residues which have reacted with the cross-linker are left with a cross-linker molecule attached to the ⁇ -amino group after the derivatization step. This leads to disappearance of one or several positive charges, which may be detrimental to the solubility and stability of the VLP.
  • the composition of the invention further comprises at least one immunostimulatory substance.
  • the immunostimulatory substance is a Toll-like receptor ligand, preferably selected from the group consisting of: (a) immunostimulatory nucleic acids; (b) peptidoglycans; (c) lipopolysaccharides; (d) lipoteichonic acids; (e) imidazoquinoline compounds; (f) flagellines; (g) lipoproteins; (h) immunostimulatory organic molecules; (i) unmethylated CpG-containing oligonucleotides; and (j) any mixtures of substance of (a), (b), (c), (d), (e), (f), (g), (h) and (i).
  • inventive compositions further comprising at least one immunostimulatory substance may be ideal vaccine compositions for prophylactic or therapeutic treatment against AIDS.
  • the immunostimulatory nucleic acid is preferably selected from the group consisting of: (a) a nucleic acid of bacterial origin; (b) a nucleic acid of viral origin; (c) a nucleic acid comprising unmethylated CpG motif; (d) a double-stranded RNA; (e) a single stranded RNA; and (g) a nucleic acid free of unmethylated CpG motif.
  • Immunostimulatory nucleic acids that do not contain unmethylated CpG motif have been disclosed in the art, for example in WO 01/22972 which is incorporated herein by reference in its entirety.
  • the bond between the nucleotides is typically and preferably phosphodiester bond.
  • the immunostimulatory nucleic acid is preferably selected from the group consisting of: (a) DNA that contains immunostimulatory sequences, in particular unmethylated CpG dinucleotides within flanking bases (referred to as CpG motifs) and (b) double-stranded RNA synthesized by various types of viruses.
  • the immunonucleic acid comprises or consists essentially of, or alternatively consists of double-stranded RNA poly LC.
  • the immunostimulatory oligonucleotides contains at least one unmethylated CpG motif, in particularly C is unmethylated.
  • the unmethylated CpG-containing oligonucleotide comprises the sequence: 5' X1X2CGX3X4 3', wherein Xl, X2, X3 and X4 are any nucleotide.
  • the oligonucleotide can comprise about preferably about 20 to about 300 nucleotides.
  • the CpG-containing oligonucleotide contains one or more phosphorothioate modifications of the phosphate backbone.
  • the CpG-containing oligonucleotide is devoid of phosphorothioate modifications of the phosphate backbone.
  • the unmethylated CpG-containing oligonucleotide comprises or consists of TCCATGACGTTCCTGAATAAT (SEQ ID NO:38).
  • the unmethylated CpG-containing oligonucleotide comprises, or alternatively consists essentially of, or alternatively consists of a palindromic sequence.
  • said palindromic sequence is flanked by guanine nucleotides, preferably by at least 4 or 6, still more preferably by at least 8 or 10 guanine nucleotides.
  • unmethylated CpG-containing oligonucleotide comprises or consists of GGGGTC AACGTTGAAGGGGGG (SEQ ID NO:39).
  • said palindromic sequence comprises, or alternatively consists essentially of, or alternatively consists of GACGATCGTC (SEQ ID NO: 40).
  • the unmethylated CpG-containing oligonucleotide comprises, or alternatively consists essentially of, or alternatively consists of the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO:41).
  • Other useful immunostimulatory nucleic acid sequences have been disclosed in the published WO2004/085635 and the disclosure is incorporated herein by way of reference.
  • Immunostimulatory substance particularly immunostimulatory nucleic acid, more particularly oligonucleotides comprising unmethylated CpG have been disclosed in WO 03/024480, WO 03/024481 and PCT/EP/04/003165.
  • the immunostimulatory substance is mixed with the composition of the invention.
  • the immunostimulatory substance is bound to, preferably packaged inside, the VLP of the invention.
  • Methods of mixing the immunostimulatory substances with the VLP-antigen have been disclosed in WO03/024480.
  • Methods of packaging the immunostimulatory substances inside the VLP have been disclosed in WO 03/024481.
  • the entire applications of WO 03/024480, 03/024481 and PCT/EP/04/003165 are therefore incorporated herein by way of reference.
  • the packaged nucleic acids and CpGs, respectively are protected from degradation, i.e., they are more stable.
  • non-specific activation of cells from the innate immune system is dramatically reduced
  • the invention provides a vaccine composition comprising the composition of the invention.
  • the vaccine composition further comprises at least one adjuvant.
  • the administration of the at least one adjuvant may hereby occur prior to, contemporaneously or after the administration of the inventive composition.
  • adjuvant refers to non-specific stimulators of the immune response or substances that allow generation of a depot in the host which when combined with the vaccine and pharmaceutical composition, respectively, of the present invention may provide for an even more enhanced immune response.
  • the at least one adjuvant include and preferably consist of complete and incomplete Freund's adjuvant, aluminum hydroxide, aluminium salts, and modified muramyldipeptide.
  • Further adjuvants are 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. Such adjuvants are also well known in the art.
  • 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, and Virosomal adjuvant technology.
  • Still further adjuvant include immunostimulatory nucleic acid, preferably the immunostimulatory nucleic acid contains one or more modifications in the backbone, preferably phosphorothioate modifications. The modification is to stabilize the nucleic acid against degradation.
  • the vaccine composition is devoid of adjuvant.
  • An advantageous feature of the present invention is the high immunogenicity of the composition, even in the absence of adjuvants.
  • the administration of the vaccine of the invention to a patient will preferably occur without administering at least one adjuvant to the same patient prior to, contemporaneously or after the administration of the vaccine.
  • VLP has been generally described as an adjuvant.
  • adjuvant refers to an adjuvant not being the VLP used for the inventive compositions, rather in addition to said VLP.
  • the invention further discloses a method of immunization comprising administering the vaccine of the present invention to an animal or a human.
  • the animal is preferably a mammal, such as rabbit, rat, mouse, monkey, chimpanzee and particularly human.
  • the vaccine may be administered to an animal or a human by various methods known in the art, but will normally be administered by injection, infusion, inhalation, oral administration, or other suitable physical methods.
  • the conjugates may alternatively be administered intramuscularly, intravenously, transmucosally, transdermally, intranasally, intraperitoneal ⁇ or subcutaneously.
  • Components of conjugates for administration include sterile aqueous (e.g., physiological saline) or non-aqueous solutions and suspensions.
  • non-aqueous solvents examples include 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.
  • Vaccines of the invention are said to be "pharmacologically acceptable” if their administration can be tolerated by a recipient individual. Further, the vaccines of the invention will be administered in a "therapeutically effective amount” (i.e., an amount that produces a desired physiological effect).
  • the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the composition as taught in the present invention and an acceptable pharmaceutical carrier.
  • vaccine of the invention When administered to an individual, it may be in a form which contains salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the conjugate.
  • materials suitable for use in preparation of pharmaceutical compositions are provided in numerous sources including REMINGTON'S PHARMACEUTICAL SCIENCES (Osol, A, ed., Mack Publishing Co., (1990)).
  • the invention teaches a process for producing the composition of the invention comprising the steps of: (a) providing a VLP with at least one first attachment site; (b) providing a HIV gpl60 peptide with at least one second attachment site, and (c) combining said VLP and said HIV gpl60 peptide to produce a composition, wherein said HIV gpl60 peptide and said VLP are linked through the first and the second attachment sites.
  • the present invention provides a method of preventing and/or treating HIV infection, wherein the method comprises administering the inventive composition or the inventive vaccine composition, respectively, to an animal or to a human.
  • the invention provides for the use of the composition for the manufacture of a medicament for prevention and/or treatment of HIV infection in human.
  • HIV gpl60 peptides 1-16 (SEQ ID NO:22-37) with C fused to the N-terminus or with C fused to the C-terminus are chemically synthesized according to standard procedures.
  • HIV gpl60 peptide 1 SLEQIWNNMTWMQWDK (SEQ ID NO:22); HIV gpl60 peptide 2: SLEQIWNNMTWMQWDR (SEQ ID NO:23); HIV gpl60 peptide 3: IWNNMTWMQWDR (SEQ ID NO:24); HIV gpl60 peptide 4: WASLWNW (SEQ ID NO:25); HIV gpl60 peptide 5: NWFDISNWLW (SEQ ID NO:26); HIV gpl60 peptide 6: LLELDKWASLWNWFNL (SEQ ID NO:27); HIV gpl60 peptide 7: ELDKWA, (SEQ ID NO:28);
  • HIV gpl60 peptide 8 WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL (SEQ ID NO:29);
  • HIV gpl60 peptide 9 CSKLIC (SEQ ID NO:30);
  • HIV gpl60 peptide 10 GFLGAAGSTMGAASITLVQ (SEQ ID NO:31); HIV gpl60 peptide 11 : QQNNLLRAIEAQQHLLQLTVWGIKQL (SEQ ID NO:32); HIV gpl60 peptide 12: GIVQQQ (SEQ ID NO:33);
  • HIV gpl60 peptide 13 QLLGIWGCSGKLICTTA VPWNSSWS (SEQ ID NO:34); HIV gpl60 peptide 14: NAKTIIVQLNQSVE (SEQ ID NO:35); HIV gpl60 peptide 15: GGNSNNESEIFRPGGGD (SEQ ID NO: 36); AND HIV gpl60 peptidel ⁇ : VAPTKAKRRVVQREKRAVGIGALFLGFLGAAGSGC (SEQ ID NO:37). HIV gpl60 peptide 17: NKSLDRIWNNMTWMEWER (SEQ ID NO:51)
  • EXAMPLE 2 Coupling of HIV peptides to Q ⁇ VLP
  • a solution of 600 ⁇ l (2.0 mg/ml) Q ⁇ VLP in 20 niM Hepes, pH 7.4 was reacted for 30 minutes with 17.3 ⁇ l SMPH (50 niM in DMSO, Pierce) at 25°C.
  • the reaction was then dialyzed twice for 2 hours against 2 L of 20 niM Hepes, pH 7.4 at 4°C.
  • the dialysed, derivatized Q ⁇ VLP was subsequently used to couple HIV gpl60 peptides.
  • Coomassie blue stained gels of the coupling reaction were monitored for the appearance of bands with molecular weights corresponding to those predicted for peptide 1 covalently linked to Q ⁇ VLP.
  • the coupling efficiency [i.e. mol Q ⁇ -peptide / mol Q ⁇ monomer (total)] was estimated, by densitometric analysis of the Coomassie blue stained gels.
  • the resulting vaccine was named according to the peptide used for coupling.
  • N-terminus refers to the first Alanine, not to the initial
  • Construct 378-2 addition of a short GSGG spacer and Ncol and Kpn2I cloning sites within the nucleic acid sequence coding for the spacer at the N-terminus of the AP205 coat protein.
  • This construction was made by PCR using 378-2 as a template and using an upstream primer p2.589 (SEQ ID NO:47)containing Ncol and a downstream primer pi.46 (SEQ ID NO:46) containing HindIII restriction sites.
  • the PCR fragment was digested with Ncol and HindIII and cloned in the same restriction sites into pQbl85, resulting in plasmid pAP382-2.
  • Construct 409-44: Addition of a short GSG spacer and Kpn2I and MphllO3I cloning sites within the nucleic acid sequence coding for said spacer at the C-terminus of AP205 coat protein.
  • This construction was made by PCR with 409-44 as template using an upstream primer pi.45 (SEQ ID NO:48) containing Xbal and a downstream primer p2.588 (SEQ ID NO: 50) containing Mph 11031 restriction sites.
  • the PCR fragment was digested with Xbal and Mph 11031 and cloned in the same restriction sites into a pQblO, resulting in plasmid pAP405 -61.
  • two complementary oligonucleotides encoding the desired sequence of HIV gpl60 peptide (SEQ ID NO:22-37) to be fused in frame with the AP205 coat protein, flanked by appropriate restriction sites for cloning are synthesized.
  • a stop codon is also included at the end of the peptide coding sequence for fusions to the C-terminus of AP205 (cloning into pAP409, pAP405).
  • An initial methionine codon is also introduced into the primer if the peptide is to be fused at the N-terminus.
  • the complementary oligonucleotides are annealed, digested with the appropriate restriction enzymes and cloned into the respective AP205 fusion vector.
  • the HIV env peptides are cloned into pAP378 and pAP382 to generate N-terminal fusions and into pAP409 and pAP405 to generate C-terminal fusions.
  • E.coli JM 109 cells are transformed with the corresponding AP205 fusion protein plasmids obtained from EXAMPLE 3.
  • a seed culture is prepared by inoculated an individual colony grown on agar containing 100 mg/1 Ampicillin into LB medium containing 20 mg/1 Ampicillin and growing the culture overnight at 37 0 C without shaking.
  • the overnight culture is diluted at 1 :50 in M9 medium supplemented with casaminoacids (Difco) and containing 20 mg/1 Ampicillin and growth of the culture carried out at 37 0 C with vigorous aeration for 14-20 hours.
  • Cells are collected at 6000 rpm for 15'-20' at 4-8 0 C.
  • Cells are lysed by ultrasonication in lysis buffer (50 mM Tris, 5 mM EDTA 0.1 %
  • the fractions containing VLPs are pooled, concentrated with a centrifugal filter unit and dialyzed against 10 mM Hepes, pH 7.5, Particle assembly and display of the M2 peptide is demonstrated by analysis of purified VLPs by SDS-PAGE and EM.
  • HIV peptides 1 or 17 or as a control with Q ⁇ VLP were diluted in PBS to a volume of 200 ⁇ l and injected subcutaneously into the right and the left inguinal region of each animal on days 0 and 14. Mice were bled on day 14 and 21, and the antibody response was measured in an ELISA.
  • For each rabbit 200 ⁇ g of vaccine was diluted in PBS to a volume of 200 ⁇ l and rabbits immmunized at day 0, 21 and 42. Rabbits were bled at days 21, 42 and 48 and antibody response measured by ELISA.
  • mice and rabbits were then bled out by heart puncture and serum of each vaccine was purified as follows: sera from one rabbit or 5 pooled mice of the respective group were centrifuged for five minutes at 14O00 rpm. The supernatant was loaded on a column of prewashed protein G sepharose (Amersham Biosciences). The column was then washed with 10 column volumes of PBS and eluted with 100 mM glycine pH 2.6.
  • RNAse coupled with peptide was coated onto HiTrap NHS activated sepharose colums.
  • HIV neutralisation assay was performed essentially as described previously (Trkola et al, J. Virol, 1999, page 8966).
  • the R5 viruses CCR5 co-receptor specific strains
  • JR-FL and SF162 have been described previously (O'Brien et al., Nature 1990, 348, page 69; and Shioda et al., Nature 1991, 349, page 167).
  • Alternativley, the X4 strains NL4-3 and 2044 have been described previously (Trkola et al (1998), J. Virol. 72:396; Trkoly et al (1998), J. Virol 72-1876).
  • cells were incubated with serial dilutions of purified polyclonal total mouse or rabbit IgG, peptide specific affinity purified rabbit IgG or positive control monoclonal antibodies 2D7, 4E10 or anti CD4 (25 ⁇ g/ml - 25 ng/ml; Pharmingen) in 96-well culture plates for Ih at 37°C.
  • HIV-I inoculums were adjusted to contain approximately 1,000 to 4,000
  • TCID 50 /ml in assay medium (TCID 50 : 50% tissue culture infective dose, Trkola et al., J. Virol., 1999, page 8966).
  • Virus inoculum (100 TCID50; 50% tissue culture infective dose;) was added and plates cultured for 4-14 days. The total infection volume was 200 ⁇ l.
  • the supernatant medium was assayed for the HIV-I p24 antigen production by using an immunoassay, as described previously (Moore et al., 1990. Science 250, page 139).
  • purified IgG fractions were tested for their ability to neutralise primary HIV isolates as described previously (Hovanessian et al., Immunity 2004, page 617-627).
  • Activated CD8+ T cell depleted PBMCs were infected with the indicated HIV virus strains (TABLE 1, upper table: JRFL; lower table: NL4.3). TABLE 1 shows the concentrations of indicated IgG fractions or purified antibodies required to achieve 50 %, 70% or 90% inhibition of HIV infection. TABLE 1 virus:
  • Peptide 1 Total rabbit IgG rabbit 2 1.8 11.2 >50 ⁇ g/ml
  • CD4-lgG2 0.02 0.03 0.08 ⁇ g/ml
  • CD4-lgG2 0.02 0.03 0.07 ⁇ g/ml
  • the HIV gpl60 peptide 1(SLEQIWNNMTWMQWDK; SEQ ID NO:22) with a C linker at the N-terminus was chemically synthesized according to standard procedures.
  • a solution of 5 ml (1.0 mg/ml) Q ⁇ VLP in 20 mM Hepes, pH 7.2 was reacted for 30 minutes with 142 ⁇ l SMPH (50 mM in DMSO, Pierce) at 25°C. The reaction was then dialyzed twice for 2 hours against 5L of 20 mM Hepes, pH 7.2 at 4°C. The dialysed, derivatized Q ⁇ VLP was subsequently used to couple peptide HIV gpl60 peptide 1.
  • the covalent chemical coupling of HIV gpl60 peptide 1 to Q ⁇ VLP was assessed by SDS-PAGE using 12 % Nu-PAGE gels (Invitrogen). Coomassie blue stained gels of the coupling reaction were monitored for the appearance of bands with molecular weights corresponding to those predicted for HIV gpl60 peptide 1 covalently linked to Q ⁇ VLP. The appearance of these additional bands as compared to derivatized Q ⁇ VLP alone, demonstrated the covalent coupling of HIV gpl ⁇ peptide 1 to Q ⁇ . The coupling efficiency was estimated on the coomassie blue stained gels to be approxiamately 1.4 peptides per Q ⁇ subunit . The resulting vaccines was named HIV gpl60 peptide 1- Q ⁇ .
  • mice were then bled out by heart puncture on day 28 and serum was purified as follows: sera from the 10 mice of each group (HIV gpl60 peptide - Q ⁇ and sera from the Q ⁇ immunised mice as a control) were pooled and centrifuged for five minutes at 14O00 rpm. The supernatant was loaded on a column of 3 ml prewashed protein G sepharose (Amersham Biosciences). The column was then washed with 10 column volumes of PBS and eluted with 100 mM glycine pH2.8.

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Abstract

La présente invention concerne les domaines de la médecine, de la santé publique, de l'immunologie, de la biologie moléculaire et de la virologie. L'invention porte sur une composition comprenant une pseudo-particule virale (VLP) liée à au moins un antigène, l'antigène étant le peptide gp160 du VIH. Cette invention concerne également un procédé de production de la composition. Les compositions de cette invention sont utiles dans la production de vaccins utiles, en particulier, dans la prévention et le traitement de maladies. Ces compositions induisent, de plus, des réponses immunes efficientes, notamment des réponses anticorps.
PCT/EP2006/066526 2005-09-21 2006-09-20 Conjugues peptidiques du vih et leurs utilisations WO2007039458A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009011912A1 (fr) * 2007-07-18 2009-01-22 Bristol-Myers Squibb Company Composition de traitement du vih comprenant des particules apparentées au virus
WO2013116656A1 (fr) * 2012-02-03 2013-08-08 Emory University Compositions immunostimulatrices, particules et applications associées
WO2017173334A1 (fr) * 2016-04-01 2017-10-05 Checkmate Pharmaceuticals, Inc. Administration de médicament médiée par un récepteur fc
US12084655B2 (en) 2018-04-09 2024-09-10 Checkmate Pharmaceuticals Packaging oligonucleotides into virus-like particles
US12246031B2 (en) 2018-02-13 2025-03-11 Checkmate Pharmaceuticals, Inc. Compositions and methods for tumor immunotherapy

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JPH01501547A (ja) * 1986-06-12 1989-06-01 バイオジェン　ナームローズ　ベンノットシャップ Ｈｉｖ感染の病因に関するペプチド
EP0330359A3 (fr) * 1988-02-25 1991-06-05 Bio-Rad Laboratories, Inc. Composition utile pour le diagnostic et le traitement de l'infection par le HIV-I
SE9101863D0 (sv) * 1991-06-13 1991-06-13 Replico Medical Ab Peptider, diagnostiskt antigen, vaccinkomposition och foerfarande foer selektering av hiv-stammar
AU2004224762B2 (en) * 2003-03-26 2009-12-24 Kuros Us Llc Packaging of immunostimulatory oligonucleotides into virus-like particles: method of preparation and use
EP1466924B9 (fr) * 2003-04-11 2013-06-19 Institut Pasteur Vaccins de peptide synthétiques pour HIV: l'épitope de CBD comme immunogen efficace pour obtenir des anticorps neutralisants contre VIH
FR2874612A1 (fr) * 2004-08-27 2006-03-03 Commissariat Energie Atomique Epitotes t cd4+ du vih restreints a hla-dp4 et leurs applications

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009011912A1 (fr) * 2007-07-18 2009-01-22 Bristol-Myers Squibb Company Composition de traitement du vih comprenant des particules apparentées au virus
WO2013116656A1 (fr) * 2012-02-03 2013-08-08 Emory University Compositions immunostimulatrices, particules et applications associées
WO2017173334A1 (fr) * 2016-04-01 2017-10-05 Checkmate Pharmaceuticals, Inc. Administration de médicament médiée par un récepteur fc
US12246031B2 (en) 2018-02-13 2025-03-11 Checkmate Pharmaceuticals, Inc. Compositions and methods for tumor immunotherapy
US12084655B2 (en) 2018-04-09 2024-09-10 Checkmate Pharmaceuticals Packaging oligonucleotides into virus-like particles

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