WO2011054995A2 - Vaccins prophylactiques contre la grippe obtenus à partir de capsides virales de birnavirus contenant l'antigène m2e du virus de la grippe - Google Patents

Vaccins prophylactiques contre la grippe obtenus à partir de capsides virales de birnavirus contenant l'antigène m2e du virus de la grippe Download PDF

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WO2011054995A2
WO2011054995A2 PCT/ES2010/070716 ES2010070716W WO2011054995A2 WO 2011054995 A2 WO2011054995 A2 WO 2011054995A2 ES 2010070716 W ES2010070716 W ES 2010070716W WO 2011054995 A2 WO2011054995 A2 WO 2011054995A2
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subunit
chimeric
viral particle
pseudo
particle according
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PCT/ES2010/070716
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Spanish (es)
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WO2011054995A3 (fr
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Thomas Zurcher
Cayetano Von Kobbe
Juan José BERNAL
Ignacio JIMÉNEZ TORRES
María VELA CUENCA
Ana Diaz Blazquez
Miguel Angel Llamas Matias
Diana Martin Lorenzo
Arcadio GARCÍA DE CASTRO
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Chimera Pharma, S. L. U.
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Publication of WO2011054995A2 publication Critical patent/WO2011054995A2/fr
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    • 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
    • A61K39/145—Orthomyxoviridae, e.g. influenza 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
    • A61K39/12—Viral antigens
    • 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
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10022—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
    • C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
    • C12N2720/00011—Details
    • C12N2720/10011—Birnaviridae
    • C12N2720/10023—Virus like particles [VLP]
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the invention relates to prophylactic vaccines for the prevention of influenza in humans.
  • the vaccines of the present invention are formed by birnavirus chimeric pseudo-viral capsids containing the influenza virus M2e antigen. STATE OF THE PREVIOUS TECHNIQUE
  • the flu represents a major global health problem. It is estimated that each year the flu affects 1 billion people, of which between 3 and 5 million develop a severe form of the disease that causes between 300,000 and 500,000 deaths.
  • M2e antigen is bound or fused to other biological entities, such as the TLR5 flagelin ligand [Huleatt W et al. Vaccine (2008) 26, 201— 214], the hemocyanin of Megathura Crenulata (KLH) or the outer membrane protein complex of Neisseria meningitidis (OMPC, Outer Membrane Protein Complex) [Fan J et al. Vaccine (2004) 22: 2993-3003].
  • TLR5 flagelin ligand Huleatt W et al. Vaccine (2008) 26, 201— 214
  • KLH hemocyanin of Megathura Crenulata
  • OMPC Outer Membrane Protein Complex
  • VLP chimeric pseudo-viral particles
  • M2e antigen in Hepatitis B VLP [WO9907839; Neirynck S et al Nat. Med (1999) 5 (10): 1 157-1163; From Fil ⁇ te M et al. (2006) Vaccine 24 (44-46): 6597-6601; From Fil ⁇ te M et al. (2008) Vaccine 26 (51): 6503-6507].
  • VLPs proposed for the presentation of different antigens are those derived from the Infectious Bursitis Virus (IBDV).
  • IBDV belongs to the Birnaviridae family and is the causative agent of Gumboro disease in birds.
  • the protein components of the viral capsid result from the proteolysis of the precursor polypeptide pVP2-VP4-VP3 (109 kDa) to release the VP2 precursor of 512 amino acids (pVP2 512 ), VP4 and VP3.
  • VP2 of different strains of IBDV have a protein sequence homology of more than 80%.
  • VLP T 1 from fusions of sequences encoding said antigen at the carboxyl end of the gene of the VP2 protein [WO2007009673].
  • sequences coding for VP2 of IBDV and M2e of influenza virus are combined in order to obtain an effective vaccine in the prevention of influenza. It is not obvious which sequences of M2e or VP2 may be suitable in the generation of the vaccine, nor the optimal fusion or insertion site of M2e with VP2.
  • the particles selected in the present invention incorporate two or three copies of M2e, preferably two.
  • the chimeric VLPs of the present invention are obtained from a selection process in which the optimal arrangement of the M2e sequences for VLP formation and the VP2 insertion sites that give rise to chimeric VLPs with a greater efficacy in prophylaxis against influenza virus.
  • the present invention provides pseudo-viral particles originating from insertions of M2e sequences encoding the influenza virus into the gene encoding Birnavirus VP2 and effective in prevention. of the flu caused by different variants of the virus that causes the disease.
  • a first aspect of the invention relates to a chimeric pseudo-viral (VLP) particle (hereinafter, chimeric VLP of the invention) formed by a fusion protein (hereinafter, fusion protein of the invention) which understands:
  • subunit (a) consisting of the Birnavirus pVP2 protein or a fragment thereof
  • subunit (b) comprising an influenza virus M2e antigen
  • pseudo-viral capsid refers to a three-dimensional nanometric structure formed by the assembly of structural viral proteins.
  • the structural viral proteins that form the pseudo-viral particle of the invention are fusion proteins comprising the pVP2 protein of a Birnavirus or a fragment thereof and at least one M2e antigen of the Influenza virus (Influenzavirus ).
  • Birnavirus refers to any virus of the family Birnaviridae, belonging to Group I I I according to the Baltimore Classification.
  • the Birnaviridae family consists of the genera Avibirnavirus, Aquabirnavirus, Blosnavirus and Entomobirnavirus.
  • the Birnavirus is from the Avibirnavirus family, and more preferably, the infectious Bursitis Virus (IBDV).
  • infectious bursitis virus or "IBDV” (IBDV) refers to viruses of the family Birnaviridae and genus Avibirnavirus causing Gumboro disease in chickens and belonging to Group II I of the Baltimore Classification.
  • the IBDV is the IBDV strain Soroa.
  • the Birnavirus genome consists of two linear double-stranded RNA molecules called A and B, which encode 5 proteins.
  • pVP2 protein refers to the VP2 precursor protein encoded by the VP2 gene of a Birnavirus. Preferably, this term refers to the 512 amino acid VP2 precursor protein (VP2 5 and 2 ) of IBDV.
  • VP2 5 12 protein of the IBDV strain Soroa (SEQ ID NO: 1) is deposited with the accession number AAD30136 in NCBI (from the National Center for Biotechnology Information).
  • VP2 512 protein from other strains of IBDV has at least 80% identity with SEQ ID NO: 1. Therefore, in a preferred embodiment, the term pVP2 refers to a protein with at least 80%, 85%, 90%, 95%, 98% or 99% identity, with SEQ ID NO: one . In a more preferred embodiment the term pVP2 refers to SEQ ID NO: 1.
  • identity refers to the proportion of identical amino acids between two amino acid sequences that are compared.
  • the percentage of identity existing between two amino acid sequences can be easily identified by one skilled in the art, for example, with the help of an appropriate computer program to compare sequences.
  • fragment refers to a portion of the pVP2 protein, of at least 400 amino acids, capable of forming VLP. This term includes, therefore, the mature VP1 protein of 441 amino acids of the IBDV (VP2 44 i).
  • the subunit (a) of the fusion protein that forms the chimeric VLP of the invention consists of a protein with at least 80% identity with SEQ ID NO: 1 or A fragment of it.
  • the subunit (a) of the fusion protein that forms the chimeric VLP of the invention consists of a protein with SEQ ID NO: 1 or a fragment thereof.
  • the subunit (a) of the fusion protein that forms the chimeric VLP of the invention consists of SEQ ID NO: 2.
  • the subunit (b) is inserted into the P region of the subunit (a).
  • P region refers to the four external domains (BC, DE, FG and H 1) of the pVP2 protein or the amino acid sequences corresponding to said domains in a pVP2 protein fragment.
  • the four external domains of the IBVV strain Soroa strain pVP2 protein correspond to the sequences between amino acids Q219-G224 (BC domain), R249-L255 (DE domain), T283-D287 (FG domain) and S315-Q324 (domain Hl).
  • BC domain BC domain
  • R249-L255 DE domain
  • T283-D287 FG domain
  • S315-Q324 domain Hl
  • BC domain of the P region therefore refers to the amino acid sequence between the amino acid of position 219 and the amino acid of position 224 of the pVP2 protein.
  • this term refers to the amino acid sequence between Q219 and G224 of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between the Q219 and G 2 24 SEQ ID NO: 2.
  • DE domain of the P region thus refers to the amino acid sequence between the amino acid of position 249 and the amino acid of position 255 of the pVP2 protein.
  • this term refers to the amino acid sequence between R 24 g and l 25 of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between R 24 g and L 25 5 of SEQ ID NO: 2.
  • FG domain of the P region thus refers to the amino acid sequence between the amino acid of position 283 and the amino acid of position 287 of the pVP2 protein.
  • this term refers to the amino acid sequence between T 28 3 and D 28 7 of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between T 28 3 and D 28 7 of SEQ ID NO: 2.
  • Hl domain of the P region thus refers to the amino acid sequence between the amino acid of position 315 and the amino acid of position 324 of the pVP2 protein.
  • this term refers to the amino acid sequence between S315 and Q3 24 of SEQ ID NO: 1. More preferably, this term refers to the amino acid sequence between S315 and Q324 of SEQ ID NO: 2.
  • the subunit (b) is inserted into the P region of SEQ ID NO: 2.
  • the subunit (b) of the fusion protein that forms the VLP of the invention is inserted into the Hl domain of the P region pVP2 or a fragment of pVP2.
  • the subunit (b) of the fusion protein that forms the VLP of the invention is inserted between amino acids D 32 3 and Q324 of pVP2 or a fragment of pVP2.
  • the subunit (b) of the fusion protein that forms the VLP of the invention is inserted in the Hl domain of the P region of SEQ ID NO: 2.
  • the subunit (b) of the fusion protein that forms the VLP of the invention is inserted between amino acids D 323 and Q 324 of SEQ ID NO: 2.
  • M2e antigen refers to the flu virus M2 matrix protein antigen that contains the extracellular region of that protein.
  • M2e antigen includes antigens of the influenza virus M2 matrix protein, such as, but not limited to, the human, swine or avian influenza virus.
  • M2e antigen refers to the antigen of the human influenza virus M2 matrix protein that contains the extracellular region of said protein, whose sequence is SEQ ID NO: 3. Therefore, the term M2e, preferably, it refers to a polypeptide with at least 60%, 70%, 80%, 90%, 95% or 99% identity, with SEQ ID NO: 3. More preferably, the term is refers to a polypeptide with the amino acid sequence SEQ ID NO: 3.
  • the term M2e antigen refers to the influenza virus M2 matrix protein antigen containing the extracellular region of said protein in which some or all tanks have been replaced by serines.
  • this term refers to a polypeptide with the amino acid sequence SEQ ID NO: 4.
  • the subunit (b) of the fusion protein comprises two M2e antigens linked by a hydrophilic polypeptide. Subunit (b) is inserted into subunit (a) to give rise to the fusion protein that forms the chimeric VLP of the invention.
  • inserted means that the amino acid sequence of the subunit (a) is divided into two parts (a1) and (a2), among which is the amino acid sequence of the subunit (b).
  • the union between the amino acid sequence of the subunit (b) and the amino acid sequence of each of the subunits (a1) or (a2) can be direct or by one or two spacer polypeptides.
  • spacer polypeptide refers to a short amino acid sequence, preferably, up to 15 amino acids in length, more preferably, up to 10 amino acids in length, even more preferably, up to 5 amino acids in length, located either between the amino acid sequence of the subunit (b) and the amino acid sequence of the subunit (a), or between the M2e antigens that are part of the subunit (b ).
  • the spacer polypeptide (p) is a hydrophilic polypeptide.
  • the term "hydrophilic spacer polypeptide” or “hydrophilic linker” are sequences comprising between 2 and 8 hydrophilic amino acids (h), such as histidine (H), glutamine (Q), asparagine (N), lysine (K), aspartic acid (D), glutamic acid (E), arginine (R), serine (S) and glycine (G).
  • the hydrophilic polypeptide is a polypeptide consisting of 4 hydrophilic amino acids (hhhh).
  • the amino acid of the carboxy-terminal end of the part (a1) of the subunit (a) forms a bond peptide with the amino acid of the amino-terminal end of the subunit (b) and the amino acid of the carboxyl-terminal end of the subunit (b) forms a peptide bond with the amino acid of the amino-terminal end of part (a2) of the subunit (a) , as represented in the following scheme:
  • (a1) represents a part of the subunit (a)
  • (a2) represents the other part of the subunit (a)
  • (b) represents the subunit (b)
  • Nt represents the amino-terminal end of the corresponding subunit
  • Ct represents the carboxyl-terminal end of the corresponding subunit
  • ⁇ represents a peptide bond between the different units of the protein of fusion of the invention.
  • the amino acid of the carboxyl-terminal end of the part ( a1) of the subunit (a) forms a peptide bond with the amino acid of the amino-terminal end of a first spacer polypeptide (p1)
  • the amino acid of the carboxyl-terminal end of this first spacer polypeptide (p1) forms a bond with the amino acid of the amino-terminal end of the subunit (b)
  • the amino acid of the carboxyl-terminal end of the subunit (b) forms a peptide bond with the amino acid of the amino-terminal end of a second spacer polypeptide (p2) and the amino acid of the end
  • the carboxyl-terminal of this second spacer polypeptide (p2) forms a bond with the amino acid of the amino-terminal end of part (a2) of the subunit (a), as depict
  • (a1) represents a part of the subunit (a)
  • (a2) represents the other part of the subunit (a)
  • (b) represents the subunit (b)
  • (p1) represents a first spacer polypeptide
  • (p2 ) represents a second spacer polypeptide
  • Nt represents the amino-terminal end of the subunit or the corresponding spacer polypeptide
  • Ct represents the carboxyl-terminal end of the subunit or the corresponding spacer polypeptide
  • ⁇ represents a peptide bond between the different units of the fusion protein of the invention.
  • the subunit (b) at one end is joined with one of the parts of the subunit (a) directly by peptide bonding and at the other end is linked to the other part of the subunit (a) by a spacer polypeptide, as represented in the following schemes:
  • (a1) represents a part of the subunit (a)
  • (a2) represents the other part of the subunit (a)
  • (b) represents the subunit (b)
  • (p) represents a spacer polypeptide
  • Nt represents the amino-terminal end of the subunit or the corresponding spacer polypeptide
  • Ct represents the carboxyl-terminal end of the subunit or the corresponding spacer polypeptide
  • ⁇ represents a peptide bond between the different fusion protein units of the invention.
  • the fusion protein that forms the chimeric VLP of the invention comprises, in addition to subunit (a) and subunit (b), one or two hydrophilic spacer polypeptides between the amino acid sequence of subunit (b) and the amino acid sequences of the subunit (a).
  • the fusion protein that forms the chimeric VLP of the invention comprises, in addition to the subunit (a) and the subunit (b), two hydrophilic spacer polypeptides between the amino acid sequence of the subunit (b) and the amino acid sequences of the subunit (a).
  • the subunit (b) of the fusion protein that forms the chimeric VLP of the invention comprises two M2e antigens (M2e-1 and M2e-2), which may be the same or different from each other.
  • M2e-1 and M2e-2 M2e antigens
  • the union between the amino acid sequence between these two antigens M2e-1 and M2e-2 of the subunit (b) can be direct or by a spacer polypeptide.
  • the carboxyl-terminal amino acid of a first M2e-1 antigen forms a peptide bond with the amino acid of the amino-terminal end of a second M2e-1 antigen, as depicted in the following scheme:
  • the amino acid of the carboxyl-terminal end of a first M2e antigen -1 forms a peptide bond with the amino-terminus amino acid of a spacer polypeptide (p) and the amino acid of the carboxy-terminal end of the spacer polypeptide (p) forms a peptide bond with the amino acid of the amino-terminal end of a second M2e-2 antigen, as represented in the following scheme: ⁇ Nt- (M2e-1) -Ct ⁇ Nt- (p) -Ct ⁇ Nt- (M2e-2) -Ct ⁇ where (M2e-1) represents one of the M2e antigens, (M2e-2) represents the other M2e antigens, (p) represents a spacer polypeptide, Nt represents the amino-terminal end of
  • the subunit (b) of the fusion protein that forms the chimeric VLP of the invention comprises three M2e antigens (M2e-1, M2e-2 and M2e-3), which may be the same or different from each other. .
  • M2e antigens M2e-1, M2e-2 and M2e-3
  • the union between the amino acid sequence between these different antigens M2e-1, M2e-2 and Me-3 of subunit (b) can be direct or by one or two spacer polypeptides.
  • the amino acid of the carboxyl-terminal end of a first M2e-1 antigen forms a peptide bond forms a peptide bond with the amino acid of the amino-terminal end of a first spacer polypeptide (p1)
  • the amino acid of the carboxyl-terminal end of this first Spacer polypeptide (p1) forms a bond with the amino acid of the amino-terminal end of a second M2e-1 antigen
  • the amino acid of the carboxyl-terminal end of this second M2e-2 antigen forms a peptide bond with the amino acid of the amino-terminal end of a second spacer polypeptide (p2)
  • the carboxyl-terminal amino acid of this second spacer polypeptide (p2) forms a bond with the amino acid of the amino-terminal end of a te rcer M2e-3 antigen, as depicted in the following scheme: ⁇ Nt- (M2e-1) -Ct ⁇ Nt- (p1) -Ct ⁇ N
  • one of the junctions between the M2e antigens is a direct binding by peptide bond, while another of the junctions takes place by means of a spacer polypeptide, as represented in the following schemes:
  • the subunit (b) of the fusion protein that forms the chimeric VLP of the invention comprises two M2e antigens, linked together in addition to the subunit (a) and subunit (b), one or two polypeptides hydrophilic spacers between the amino acid sequence of the subunit (b) and the amino acid sequences of the subunit (a).
  • the fusion protein where the subunit (b) comprises two M2e antigens, linked together by a hydrophilic polypeptide, wherein said subunit (b) is inserted into the subunit (a) by two hydrophilic spacer polypeptides, such and as represented in the following scheme: ⁇ Nt- (a1) -Ct ⁇ Nt- (p1) -Ct ⁇ Nt- (M2e-1) -Ct ⁇ Nt- (p2) - Ct ⁇ Nt- (M2e-2 ) -Ct ⁇ Nt- (p3) -Ct ⁇ Nt- (a2) -Ct ⁇ , where (M2e-1) represents a first M2e antigen, (M2e-2) represents a second M2e antigen, (p1) represents a first spacer polypeptide, (p2) represents a second spacer polypeptide, (p3) represents a third spacer polypeptide, Nt represents the amino-terminal end of the
  • amino acid sequence of ⁇ Nt- (p) -Ct ⁇ Nt- (M2e-1) -Ct ⁇ Nt- (p) -Ct ⁇ Nt- (M2e-2) -Ct ⁇ Nt- ( p) -Ct ⁇ according to the previous scheme is SEQ ID NO: 7.
  • a preferred embodiment of this first aspect of the invention relates to a chimeric VLP formed by a fusion protein comprising SEQ ID NO: 7 inserted between amino acids D323 and Q324 of SEQ ID NO: 2.
  • a more preferred embodiment refers to a chimeric VLP formed by a fusion protein whose amino acid sequence is SEQ ID NO: 8.
  • a second aspect of the invention relates to a process for obtaining the chimeric VLPs of the invention, which comprises culturing a host cell comprising a nucleic acid encoding the fusion protein of the invention, under conditions that allow expression. from said fusion proteins, and the assembly of said fusion proteins to form chimeric VLPs.
  • a preferred embodiment of this second aspect of the invention relates to a process for obtaining the chimeric VLP particles of the invention, which comprises culturing a host cell comprising a nucleic acid encoding the fusion protein of the invention, under conditions that allow the expression of said fusion proteins, and the assembly of said fusion proteins to form chimeric VLPs, and which further comprises isolating or purifying said chimeric VLPs.
  • the fusion protein of the invention can be obtained by genetic or recombinant engineering techniques well known in the state of the art.
  • the sequence of a nucleic acid encoding the fusion protein of the invention (hereinafter, nucleic acid of the invention) can be obtained by any biological or synthetic method, including, for example, but not limited to, the restriction of appropriate sequences or amplification of the DNA sequence of the protein of interest by polymerase chain reaction (PCR).
  • the nucleic acid of the invention comprises the sequence SEQ ID NO: 5.
  • the nucleic acid may be comprised in a gene construct (hereinafter, gene construct of the invention).
  • This gene construct of the invention may comprise the nucleic acid of the invention, operably linked to, a sequence regulating the expression of the nucleic acid of the invention, thereby constituting an expression cassette.
  • "Operationally linked” refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way.
  • a control sequence "operatively linked" to the nucleic acid is linked to it in such a way that expression of the nucleic acid coding sequence is achieved.
  • Control sequence refers to nucleic acid sequences that affect the expression of the sequences to which they are linked. Such control sequences include, for example, but not limited to, promoters, initiation signals, termination signals, enhancers or silencers. The term “control sequences” is intended to include those components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.
  • the gene construct of the invention comprises the nucleic acid of the invention operably linked to at least one control sequence of the list comprising: a. a promoter,
  • promoter refers to a region of DNA located at position 5 'with respect to the starting point of transcription and which is necessary or facilitates such transcription in an animal cell. This term includes, for example, but not limited to, constitutive promoters, cell or tissue specific promoters or inducible or repressible promoters.
  • control sequences depend on the origin of the cell in which the nucleic acid of the invention is to be expressed.
  • expression control sequences linked to the nucleic acid of the invention are functional in prokaryotic cells and organisms, for example, but without limit yourself, bacteria; while in another particular embodiment, said expression control sequences are functional in eukaryotic cells and organisms, for example, yeast cells or animal cells.
  • the nucleic acid of the invention or the gene construct of the invention can be introduced into a cell, called a host cell, for example, but not limited, as a naked nucleic acid or by a vector.
  • cloning vector refers to a DNA molecule in which another DNA fragment can be integrated, without losing the capacity for self-replication.
  • expression vectors are, but are not limited to, plasmids, cosmids, DNA phages or artificial yeast chromosomes.
  • expression vector refers to a cloning vector suitable for expressing a nucleic acid that has been cloned therein after being introduced into a cell, called a host cell. Said nucleic acid is generally operatively linked to control sequences.
  • host cell refers to any prokaryotic or eukaryotic organism that is the recipient of an expression vector, cloning or any other DNA molecule.
  • a third aspect of the invention relates to the use of the chimeric VLP of the invention for the preparation of a medicament, preferably a vaccine.
  • a fourth aspect of the invention relates to the use of the chimeric VLP of the invention for the preparation of a medicament for the prevention and / or treatment of an infection caused by the influenza virus.
  • a sixth aspect of the invention relates to a pharmaceutical composition (hereinafter, pharmaceutical composition of the invention) comprising the chimeric VLP of the invention.
  • a preferred embodiment of this sixth aspect of the invention relates to a pharmaceutical composition comprising the chimeric VLP of the invention and further comprising a pharmaceutically acceptable carrier.
  • Another preferred embodiment of this sixth aspect of the invention relates to a pharmaceutical composition comprising the chimeric VLP of the invention and further comprising another active ingredient.
  • a more preferred embodiment of this sixth aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the chimeric VLP of the invention, a pharmaceutically acceptable carrier and also another active ingredient.
  • active substance refers to any component that potentially provides a pharmacological activity or other different diagnostic effect , cure, mitigation, treatment or prevention of a disease, or that affects the structure or function of the body of the human being or other animals.
  • composition of the invention can be formulated for administration in a variety of ways known in the state of the art. Such formulations may be administered to an animal and, more preferably, to a mammal, including a human, by a variety of routes, including, but not limited to parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal, intraaricular, intrasynovial. , intrathecal, intralesional, intraarterial, intracapsular, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular or topical.
  • routes including, but not limited to parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal, intraaricular, intrasynovial.
  • intrathecal intralesional, intraarterial, intracapsular, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intra
  • the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, age, weight, sex or tolerance of the animal.
  • the term "therapeutically effective amount” refers to the amount of the pharmaceutically effective composition that produces the desired effect and, in general, will be determined among other causes, by the characteristics of said pharmaceutical composition and of the therapeutic effect to be achieved.
  • the pharmaceutically acceptable “adjuvants” or “vehicles” that can be used in said compositions are the vehicles known in the state of the art.
  • FIG. 1 Construction of insertion vectors: Represents the insertion of Notl-Spel restriction sites from Serine-Threonine (TS) insertions generated by directed mutagenesis.
  • Step 1 an adapter is cloned by using Xbal-Spel compatible restriction enzymes.
  • Step 2 the adapter is replaced by the insert of interest using the Notl-Spel restriction sites.
  • FIG. 2 DNA sequence of the 2xM2e insert and its corresponding amino acid sequence.
  • the sequence of M2e is represented with gray color.
  • VLPs birnavirus chimeric pseudoviral capsids
  • EXAMPLE 1 SEARCH AND SELECTION OF VIRAL CAPSIDES OF BIRNAVIRUS CONTAINING THE M2e ANTIGEN OF THE VIRUS OF THE FLU. a) SEARCH AND SELECTION, THROUGH A PROCESS OF SUCCESSIVE SCREENING, OF CHEMICAL VLP CONTAINING M2e OF THE VIRUS OF THE FLU.
  • a process of insertion of two copies of M2e of the influenza virus is carried out in different positions of the external P domains of the VP2 protein.
  • a collection of 8 different plasmids containing a Spel site at the preferred positions within the domains is obtained by directed mutagenesis using the yeast expression plasmid pESC-URA (Stratagene TM) -VP2 45 2 external P of the VP2 protein.
  • each plasmid contains a multiple cloning site (MCS).
  • MCS multiple cloning site
  • the insert with the MCS contains several stop codons of translation "stop" in phase, which ensures that the religion of vectors without insert generates a truncated VP2, unable to form VLP.
  • the selection of the 8 vectors includes cloning sites in the 4 main external domains of the P region of the VP2 protein (see Table 1).
  • the 2xM2e sequence containing two copies of the sequence encoding the human influenza virus M2e antigen is generated by PCR (SEQ ID NO: 3) in which methionine in position 1 (Mi) is excluded, and the cysteines of positions 17 and 19 (Ci 7 and C19) are replaced by serines (S) (SEQ ID NO: 4).
  • each of the four hydrophilic amino acids is encoded by a codon of three randomly generated nucleotides as follows: in position 1 nucleotide A, G or C (V according to the IUPAC code), in the position 2 nucleotide A or G (R according to the IUPAC code), and in position 3 nucleotide A, G, C or T (N according to the IUPAC code).
  • VRN codons code for the 9 most hydrophilic amino acids, that is, histidine (H), glutamine (Q), asparagine (N), lysine (K), aspartic acid (D), glutamic acid (E), arginine (R ), serine (S) and glycine (G), as shown in Figure 2.
  • H histidine
  • glutamine Q
  • asparagine N
  • lysine K
  • aspartic acid D
  • E glutamic acid
  • E arginine
  • S serine
  • G glycine
  • the different randomly generated 2xM2e inserts contain seven additional amino acids in its ends whose DNA sequence contains the Not ⁇ and Spel restriction sites, used in molecular cloning [SEQ ID NO: 6].
  • the generation of an insertion library "hhhh- (M2e) -hhhh- (M2e) -hhhh" in the eight possible preselected insertion sites in the P region of the VP2 protein is carried out by a ligation reaction of the library of 8 vectors pre-digested with Not ⁇ and Spel and the library of PCR-generated DNA fragments containing the randomly generated 2xM2e fragments and pre-digested with Not ⁇ and Spel.
  • the ligation result is transformed into electrocompetent E.coli cells to generate a library of clones containing the different 2xM2e fragments inserted in the eight possible points of Preset insert in VP2.
  • ⁇ g of DNA from a mixture of plasmid DNA from all clones of the library is used to transform yeast cells, S.cerevisiae strain Y449 that are subsequently seeded in YNB / CSM-URA medium with 2% glucose
  • the yeast clones isolated and obtained in this selective medium will be transferred to YNB / CSM-URA plates with galactose and the resulting colonies are transferred to polyvinylidene fluoride membranes (English PVDF, Polyvinylidene Fluoride) to study the production of VLP by immunoblots of colonies using specific antibodies against the M2e epitope.
  • chimeric VLP VLP-M2e capable of generating a significant immune response against M2e is carried out by immunizing 6 mice with an initial dose intraperitoneally (i. P.), followeded by two recall doses subcutaneously. (sc) - In each dose 50 ⁇ g is administered for each of the VLP-2xM2e generated, or of the VLPs control without the M2e insert, in 200 ⁇ saline. All three doses are administered at intervals of 3 weeks. One week before the first immunization and two weeks after each booster dose, blood samples are obtained from the animals by retro-orbital puncture.
  • Table 3 Places of insertion in VP2 and sequence of the 2xM2e insert of the constructions resulting from the process of search and selection of chimeric VLPs containing 2xM2e and expressing more efficiently.
  • the efficacy as a vaccine for the protection against influenza virus infection of the three candidates of VLP-M2e with greater title "endpoint" is evaluated by the viral challenge in previously immunized animals. For this, groups of 13 mice are immunized with an initial immunogen dose intraperitoneally (i.p.), followed by two subcutaneous (s.c.) recall doses at 3-week intervals. In each dose 50 ⁇ g of VLP-2xM2e, or of the appropriate control, is administered in 200 ⁇ of saline.
  • mice are sacrificed if their body weight falls below 75% of their initial weight. As shown in Table 4, all mice in the control VLP groups without M2e inserts die within 8 days after the viral challenge. In contrast, none of the animals vaccinated with VLP 4 52-D 3 23 ⁇ 2xM2e ⁇ Q324 (Clone ID 4A) die in the course of the experiment, which means a survival rate of 100%.
  • Table 4 Survival data of mice vaccinated with different VLPs incorporating 2xM2e insertions in P regions of the VP2 of IBDV.

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Abstract

L'invention concerne des vaccins prophylactiques pour la prévention de la grippe chez les êtres humains. En particulier, les vaccins de la présente invention sont formés de capsides pseudo-virales chimériques de birnavirus contenant l'antigène M2e du virus de la grippe.
PCT/ES2010/070716 2009-11-06 2010-11-05 Vaccins prophylactiques contre la grippe obtenus à partir de capsides virales de birnavirus contenant l'antigène m2e du virus de la grippe WO2011054995A2 (fr)

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CN103083656A (zh) * 2011-10-27 2013-05-08 苏州科贝生物技术有限公司 甲型流感病毒保守肽M2e与病毒样颗粒的缀合物及应用

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WO2013059168A2 (fr) * 2011-10-21 2013-04-25 Research Development Foundation Virus espirito santo et procédés de détection et de prévention d'une infection par ledit virus
WO2013059168A3 (fr) * 2011-10-21 2013-08-01 Research Development Foundation Virus espirito santo et procédés de détection et de prévention d'une infection par ledit virus
CN103083656A (zh) * 2011-10-27 2013-05-08 苏州科贝生物技术有限公司 甲型流感病毒保守肽M2e与病毒样颗粒的缀合物及应用

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