WO2024103728A1 - Vaccin à base d'acide ribonucléique messager contre un poxvirus - Google Patents

Vaccin à base d'acide ribonucléique messager contre un poxvirus Download PDF

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WO2024103728A1
WO2024103728A1 PCT/CN2023/102077 CN2023102077W WO2024103728A1 WO 2024103728 A1 WO2024103728 A1 WO 2024103728A1 CN 2023102077 W CN2023102077 W CN 2023102077W WO 2024103728 A1 WO2024103728 A1 WO 2024103728A1
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protein
seq
sequence shown
fusion
proteins
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PCT/CN2023/102077
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Chinese (zh)
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刘小虎
穆拉德亚纳尔
侯富军
贾为国
余志斌
丁隽
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上海复诺健生物科技有限公司
复诺健生物科技加拿大有限公司
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Publication of WO2024103728A1 publication Critical patent/WO2024103728A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24122—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an anti-pox virus vaccine and a kit for preparing the anti-pox virus vaccine.
  • the present invention also relates to the use of the vaccine and the kit for preparing the vaccine.
  • Poxviruses are a class of large, pathogenic double-stranded DNA viruses. Among them, smallpox virus has caused great disasters to human society. Since 2022, the number of cases of monkeypox virus infection in humans has increased rapidly, with a cumulative total of more than 50,000 people. In order to curb the large-scale outbreak of the epidemic, corresponding vaccines are urgently needed.
  • An infectious disease vaccine is a substance that can induce an immune response in the body and protect the body from infection or serious infection. It can be the pathogen itself, such as a virus or bacteria, or a part of the pathogen, such as a protein, or it can be genetic information encoding the pathogen protein, such as its ribonucleic acid sequence (RNA) or deoxyribonucleic acid sequence (DNA).
  • RNA ribonucleic acid sequence
  • DNA deoxyribonucleic acid sequence
  • ACAM2000 is a replication-competent vaccinia virus, which may cause significant side effects after vaccination, such as systemic infection, eczema, myocarditis and even death. Therefore, people are more concerned about the safety of this vaccine and are not very willing to get vaccinated.
  • JYNNEOS is a replication-deficient vaccinia virus, which cannot replicate in the human body.
  • An anti-pox virus vaccine comprising mRNA molecules encoding the following proteins and/or fusion proteins:
  • monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or
  • fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.
  • the A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,
  • the M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,
  • the B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and
  • the A29L protein comprises the amino acid sequence shown in SEQ ID NO:25.
  • the A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.
  • the M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15.
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,
  • the B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and
  • the A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.
  • the mRNA molecule further comprises: a 5' cap, a 5' UTR, a 3' UTR and a poly A tail.
  • the 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or
  • the 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.
  • the mRNA molecule encoding the A35R protein comprises the RNA sequence shown in SEQ ID NO: 4,
  • the mRNA molecule encoding the M1R protein comprises the RNA sequence shown in SEQ ID NO: 8,
  • the mRNA molecule encoding the B6R protein comprises the RNA sequence shown in SEQ ID NO: 24, and
  • the mRNA molecule encoding A29L protein contains the RNA sequence shown in SEQ ID NO: 28.
  • the two or more mRNA molecules are encapsulated separately and then mixed with each other, or
  • the two or more mRNA molecules are first mixed with each other and then encapsulated as a whole.
  • LNPs lipid nanoparticles
  • monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or
  • the fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.
  • the A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,
  • the M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,
  • the B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and
  • the A29L protein comprises the amino acid sequence shown in SEQ ID NO:25.
  • the A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.
  • the M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15.
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,
  • the B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and
  • the A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.
  • the mRNA molecule further comprises: a 5' cap, a 5' UTR, a 3' UTR and a poly A tail.
  • the 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or
  • the 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.
  • mRNA molecule is selected from:
  • the mRNA molecule encoding the A35R protein comprises the RNA sequence shown in SEQ ID NO: 4,
  • the mRNA molecule encoding the M1R protein comprises the RNA sequence shown in SEQ ID NO: 8,
  • the mRNA molecule encoding the B6R protein comprises the RNA sequence shown in SEQ ID NO: 24, and
  • the mRNA molecule encoding A29L protein contains the RNA sequence shown in SEQ ID NO: 28.
  • the two or more mRNA molecules are encapsulated separately and then mixed with each other, or
  • the two or more mRNA molecules are first mixed with each other and then encapsulated as a whole.
  • the vaccine is used against poxviruses of one or more genera selected from the group consisting of Orthopoxvirus, Capripoxvirus, Cervidpoxvirus, Suipoxvirus, Leporipoxvirus, Molluscipoxvirus, Yatapoxvirus, Avipoxvirus, Crocodylidpoxvirus and Parapoxvirus.
  • the vaccine is used against one or more poxviruses selected from the group consisting of: Variola virus/Smallpox virus, Vaccinia virus, Cowpox virus, Camelpox virus, Ectromelia virus, Monkeypox virus, Uasin Gishu disease virus, Tatera poxvirus, Raccoonpox virus, Volepox virus, Skunkpox virus, Sheeppox virus, Goatpox virus, Lumpy skin disease virus, Deerpox virus, Swinepox virus, Myxoma virus, Rabbit fibroma virus fibroma virus), Hare fibroma virus, Squirrel fibroma virus, Molluscum contagiosum virus, Yabapox virus, Tanapox virus, Fowlpox virus, Canarypox virus, Crowpox virus, Juncopox virus, Mynahpox virus, Pigeonpox virus, Psitt
  • poxviruses selected from the group consist
  • kits for preparing an anti-pox virus vaccine comprising:
  • monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or
  • a fusion protein formed by fusing two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and
  • fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.
  • the A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,
  • the M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,
  • the B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and
  • the A29L protein contains the amino acid sequence shown in SEQ ID NO: 25.
  • the A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.
  • the M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15.
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,
  • the B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and
  • the A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.
  • kits according to the preceding embodiment wherein the reagent for transcribing the DNA molecule of (i) into an mRNA molecule is a reagent for transcribing the DNA molecule of (i) into an mRNA molecule in vitro.
  • kits according to the preceding embodiment, wherein the reagent for in vitro transcription of the DNA molecule of (i) into an mRNA molecule comprises:
  • a nucleic acid vector for in vitro transcription comprising a promoter linked from 5' to 3', a coding DNA of a 5'UTR and a coding DNA of a 3'UTR,
  • Adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate Adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate
  • the 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or
  • the 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.
  • kits according to the preceding embodiment wherein the uridine triphosphate is N1-methylpseudouridine triphosphate.
  • RNA polymerase is T7 RNA polymerase.
  • monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or
  • a fusion protein formed by fusing two or more monkeypox virus proteins or parts of proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and
  • fusion protein is a fusion protein formed by fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.
  • the A35R protein comprises the amino acid sequence shown in SEQ ID NO: 1,
  • the M1R protein comprises the amino acid sequence shown in SEQ ID NO: 5,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 14,
  • the fusion protein of A35R and M1R comprises the amino acid sequence shown in SEQ ID NO: 18,
  • the B6R protein comprises the amino acid sequence shown in SEQ ID NO: 21, and
  • the A29L protein contains the amino acid sequence shown in SEQ ID NO: 25.
  • the A35R protein is encoded by a DNA sequence shown in SEQ ID NO: 3.
  • the M1R protein is encoded by a DNA sequence shown in SEQ ID NO: 7.
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 15,
  • the fusion protein of A35R and M1R is encoded by a DNA sequence shown in SEQ ID NO: 19,
  • the B6R protein is encoded by a DNA sequence shown in SEQ ID NO: 23, and
  • the A29L protein is encoded by a DNA sequence shown in SEQ ID NO: 27.
  • reagent for transcribing the DNA molecule of (i) into an mRNA molecule is a reagent for transcribing the DNA molecule of (i) into an mRNA molecule in vitro.
  • a nucleic acid vector for in vitro transcription comprising a promoter linked from 5' to 3', a coding DNA of a 5'UTR and a coding DNA of a 3'UTR,
  • Adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate Adenosine triphosphate, cytidine triphosphate, guanosine triphosphate and uridine triphosphate
  • the 5'UTR is encoded by the DNA sequence shown in SEQ ID NO: 29, and/or
  • the 3'UTR is encoded by the DNA sequence shown in SEQ ID NO: 30.
  • RNA polymerase is T7 RNA polymerase.
  • the vaccine is used against poxviruses of one or more genera selected from the group consisting of Orthopoxvirus, Capripoxvirus, Cervidpoxvirus, Suipoxvirus, Leporipoxvirus, Molluscipoxvirus, Yatapoxvirus, Avipoxvirus, Crocodylidpoxvirus and Parapoxvirus.
  • the vaccine is used against one or more poxviruses selected from the group consisting of smallpox virus, vaccinia virus, cowpox virus, camelpox virus, ectromelia virus, monkeypox virus, Uasin Gishu disease virus, Tatera poxvirus, Raccoonpox virus, Volepox virus, Skunkpox virus virus), Sheeppox virus, Goatpox virus, Lumpy skin disease virus, Deerpox virus, Swinepox virus, Myxoma virus, Rabbit fibroma virus, Hare fibroma virus, Squirrel fibroma virus, Molluscum contagiosum virus, Yabapox virus Yabapox virus, Tanapox virus, Fowlpox virus, Canarypox virus, Crowpox virus, Juncopox virus, Mynahpox virus, Pigeonpox virus, Psittacinepox
  • poxviruses selected from the group consist
  • a fusion protein formed by the fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein, M1R protein, B6R protein and A29L protein.
  • a fusion protein formed by the fusion of the following monkeypox virus proteins or parts of the proteins: A35R protein and M1R protein.
  • fusion protein described in the above embodiment, wherein the fusion protein comprises the amino acid sequence shown in SEQ ID NO: 14 or 18.
  • a DNA molecule encoding A35R protein which comprises the DNA sequence shown in SEQ ID NO: 3.
  • a DNA molecule encoding M1R protein which comprises the DNA sequence shown in SEQ ID NO: 7.
  • a DNA molecule encoding a fusion protein of A35R and M1R which comprises the DNA sequence shown in SEQ ID NO: 15.
  • a DNA molecule encoding a fusion protein of A35R and M1R which comprises the DNA sequence shown in SEQ ID NO: 19.
  • a DNA molecule encoding B6R protein which comprises the DNA sequence shown in SEQ ID NO: 23.
  • a DNA molecule encoding A29L protein comprising the DNA sequence shown in SEQ ID NO: 27.
  • the mRNA molecule encoding A35R protein contains the RNA sequence shown in SEQ ID NO: 4.
  • the mRNA molecule encoding M1R protein contains the RNA sequence shown in SEQ ID NO: 8.
  • the mRNA molecule encoding the fusion protein of A35R and M1R contains the RNA sequence shown in SEQ ID NO: 16.
  • the mRNA molecule encoding the fusion protein of A35R and M1R comprises the RNA sequence shown in SEQ ID NO: 20.
  • the mRNA molecule encoding B6R protein contains the RNA sequence shown in SEQ ID NO: 24.
  • the mRNA molecule encoding A29L protein contains the RNA sequence shown in SEQ ID NO: 28.
  • the mRNA vaccine of the present invention does not require the aid of cell expression and has no risk of integration into the human genome.
  • the preparation of mRNA is also simple and easy. Vaccines can be produced without contacting poxviruses that are capable of infection and reproduction, thus avoiding biosafety risks.
  • the mRNA vaccine of the present invention has no obvious side effects and is highly safe.
  • FIG. 1 Schematic diagram of the mRNA molecular structure, from 5' to 3': 5' cap-5' UTR-coding sequence-3' UTR-poly A tail.
  • the coding sequence is the RNA sequence of the following protein (or fusion protein) antigens: A35R, M1R, SP-A35R_IECD-M1R, SP-A35R_sECD-M1R, B6R and A29L, where "SP" represents signal peptide.
  • Figures 2A to 2D show the levels of total anti-A35R antibodies, total anti-M1R antibodies, total anti-B6R antibodies, and total anti-A29L antibodies in the blood collected on the 29th day after each group of mRNA vaccines were administered to mice.
  • Figure 2E shows the level of neutralizing antibodies to vaccinia virus in the serum collected on the 29th day after each group of mRNA vaccines were administered to mice (PFU/well).
  • Figure 2F shows the continuous weight changes of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus.
  • Figure 2G shows the viral load in the lungs of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus.
  • Figures 3A and 3B show the levels of total anti-A35R antibodies and total anti-M1R antibodies in the blood collected at 1 month, 2 months, 3 months, 4 months and 5 months after each group of mRNA vaccines were administered to mice.
  • Figure 3C shows the level (%) of neutralizing antibodies to vaccinia virus in the serum collected at 1 month, 2 months, 3 months, 4 months and 5 months after each group of mRNA vaccines were administered to mice.
  • Figure 3D shows the continuous weight changes of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus at 5.5 months after vaccination.
  • Figures 4A to 4E show the weight change (%) relative to the initial body weight of mice intravenously injected with mixed sera obtained from mice administered with DPBS, A-B Group 1, A-B Group 2, and A+B Group 1 vaccines and VACV-WR when challenged with vaccinia virus.
  • Figure 4F compares the weight change (%) relative to the initial body weight of mice intravenously injected with mixed sera obtained from mice administered with different vaccines when challenged with vaccinia virus.
  • Figures 5A and 5B show the levels of total anti-A35R antibodies and total anti-M1R antibodies in the blood collected on the 7th day after each group of mRNA vaccines were administered to mice.
  • Figure 5C shows the level (%) of neutralizing antibodies to vaccinia virus in the serum collected on the 7th day after each group of mRNA vaccines were administered to mice.
  • Figure 5D shows the continuous weight changes of mice administered with each group of mRNA vaccines after intranasal challenge with vaccinia virus on the 8th day after vaccination.
  • the gene and protein sequences of each virus in the Poxviridae family are highly similar, and a vaccine for one poxvirus can often prevent another poxvirus. Therefore, the mRNA vaccine of the present invention can be used for each of the following listed Immunity against Poxviridae viruses.
  • the 5' end of eukaryotic mRNA usually has a bridged 7-methylguanosine (m7G) cap structure (Cap0).
  • m7G 7-methylguanosine
  • Cap1 Cap1 structure
  • the 5' cap structure can also protect mRNA from degradation by nuclease exonucleases, work with translation initiation factor proteins, recruit ribosomes, and assist ribosomes in binding to mRNA, so that translation starts from AUG.
  • the Cap structure can recognize each other with the eukaryotic initiation factor 4E (eIF4E) at the initiation stage of translation and start the subsequent translation process.
  • eIF4E eukaryotic initiation factor 4E
  • the Cap1 structure can greatly reduce the immunogenicity of mRNA in vivo.
  • the 5' cap that can be used in the present invention is not particularly limited.
  • the 5' cap is Cap1-GAG (3'OMe), that is, m7 (3'OMeG) (5') ppp (5') (2'OMeA) pG, whose molecular formula is C 33 H 45 N 15 O 24 P 4 and whose structural formula is as follows;
  • capping methods for preparing mRNA by in vitro transcription, including enzymatic capping, co-transcriptional capping, etc.
  • Enzymatic capping is a more traditional capping method. After the IVT reaction involving T7 polymerase is completed, the uncapped mRNA is purified first, and then Cap0 is produced by vaccinia virus capping enzyme (which has RNA triphosphatase activity, guanylyltransferase activity and guanine methyltransferase activity), which is then converted into Cap1 by 2'-O-methyltransferase and S-adenosylmethionine, and purified again to obtain the final mRNA.
  • vaccinia virus capping enzyme which has RNA triphosphatase activity, guanylyltransferase activity and guanine methyltransferase activity
  • One-step co-transcriptional capping is to directly add a cap analog to the IVT reaction system involving T7 polymerase, so as to obtain mRNA containing Cap1 structure in one step, and only one purification is required in the whole process.
  • This reaction method reduces the preparation steps, thereby effectively shortening the overall processing time, simplifying the purification steps, and reducing the number of enzymes required. Therefore, the chemical co-transcriptional capping is relatively simple in process, introduces few impurities, and can quickly increase the production capacity of mRNA vaccines and drugs.
  • one-step co-transcriptional capping is gradually becoming the mainstream technical route for mRNA preparation technology.
  • the uridine triphosphate that can be used in the present invention is not particularly limited, and can be natural uridine triphosphate or any modified uridine triphosphate commonly used in the art.
  • UTP is N1- methylpseudouridine triphosphate (N1-Me-pUTP, usually represented as " ⁇ "), whose molecular formula is C10H14N2Na3O15P3 , and whose structural formula is as follows :
  • the 5'UTR and 3'UTR that can be used in the present invention are not particularly limited.
  • the 5'UTR and 3'UTR that can be used in the present invention are not particularly limited.
  • the coding DNA sequence of 5'UTR is as follows (SEQ ID NO: 29):
  • the coding DNA sequence of 3'UTR is as follows (SEQ ID NO: 30):
  • the present invention also relates to 5'UTR and 3'UTR having 80% or more identity, 85% or more identity, 90% or more identity, 91% or more identity, 92% or more identity, 93% or more identity, 94% or more identity, 95% or more identity, 96% or more identity, 97% or more identity, 98% or more identity, or 99% or more identity with the above-mentioned 5'UTR and 3'UTR, respectively.
  • the poxvirus immunogen/antigen used in the present invention is preferably:
  • monkeypox virus proteins selected from the group consisting of A35R protein, M1R protein, B6R protein and A29L protein, and/or
  • the fusion protein is a fusion protein formed by the fusion of the following monkeypox virus proteins or parts of the proteins: A35R and M1R.
  • the integral extracellular domain (IECD) of A35R is fused with M1R to form a fusion protein (A35R_IECD-M1R).
  • the small extracellular domain (sECD) of A35R is fused with M1R to form a fusion protein (A35R_sECD-M1R).
  • a signal peptide (SP) may be attached to the 5' end of the fusion protein.
  • SP has the amino acid sequence shown in SEQ ID NO: 9 or 10.
  • the fusion protein is connected via a peptide linker.
  • the peptide linker has a structure represented by (G 4 S) n , wherein G represents glycine (Gly), S represents serine (Ser), and n is an integer from 1 to 7.
  • the peptide linker has an amino acid sequence represented by SEQ ID NO: 11 or 12.
  • the DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins that can be used in the present invention are not particularly limited, and can be DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins of any virus from the Poxviridae family.
  • the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the genus Orthopoxvirus.
  • the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from Monkeypox virus.
  • the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus. In a preferred embodiment, the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus that are codon-optimized.
  • the present invention uses DNA sequences and mRNA sequences of the above-mentioned proteins and fusion proteins from the Zaire79 strain of Monkeypox virus that are codon-optimized for expression in humans.
  • the amino acid sequence, DNA sequence and mRNA sequence of the above-mentioned protein and fusion protein are as follows:
  • Codon-optimized coding DNA for expression in humans (SEQ ID NO: 3)
  • Codon-optimized coding DNA for expression in humans (SEQ ID NO: 7)
  • amino acid sequence of A35R_IECD (SEQ ID NO: 13):
  • Codon-optimized coding DNA for expression in humans (SEQ ID NO: 15)
  • Amino acid sequence of A35R_sECD (SEQ ID NO: 17):
  • Codon-optimized coding DNA for expression in humans (SEQ ID NO: 19)
  • Codon-optimized coding DNA for expression in humans (SEQ ID NO: 23)
  • Codon-optimized coding DNA for expression in humans (SEQ ID NO: 27)
  • the present invention also relates to amino acid sequences, DNA sequences and mRNA sequences that are more than 80% identical, more than 85% identical, more than 90% identical, more than 91% identical, more than 92% identical, more than 93% identical, more than 94% identical, more than 95% identical, more than 96% identical, more than 97% identical, more than 98% identical, or more than 99% identical to the above-mentioned amino acid sequences, DNA sequences and mRNA sequences, respectively.
  • the mRNA vaccine of the present invention is preferably encapsulated in a protective carrier.
  • a protective carrier As long as it is sufficient to keep the mRNA vaccine of the present invention from degrading for a sufficiently long time and does not hinder the realization of the technical effects of the present invention, there is no particular limitation on the encapsulation carrier of mRNA that can be used in the present invention.
  • nanoparticle-type carriers are used to encapsulate mRNA in the present invention.
  • nanoparticles containing lipids are used in the present invention to encapsulate mRNA.
  • LNP may include but is not limited to liposomes and micelles.
  • the lipid nanoparticles may include cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphiphilic lipids, pegylated lipids and/or structural lipids.
  • the LNP may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and/or ionizable lipids.
  • “Cationic lipid” generally refers to a lipid that carries any number of net positive charges at a certain pH (e.g., physiological pH).
  • the cationic lipids may include, but are not limited to, SM102, 3-(didodecylamino)-N1,N1,4-triadecyl-1-piperazineethylamine (KL10), N1-[2-(triadecylamino)ethyl]-N1,N4,N4-triadecyl-1,4-piperazinediethylamine (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaporane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, octyl-CLinDMA, octyl-CLinDMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA and DMRIE.
  • KL10 3-(didodecylamino)-N1,
  • the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%, for example, about 40-65%, about 40-60%, about 45-55% or about 48-53%.
  • the LNP may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) non-cationic lipids.
  • the non-cationic lipids may include anionic lipids.
  • Anionic lipids suitable for lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylphosphatidylethanolamine, and other neutral lipids having anionic groups connected thereto.
  • the non-cationic lipid may include a neutral lipid, which may include, for example, a phospholipid, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (PO ...phosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidyl
  • the molar ratio of the phospholipid in the lipid nanoparticles is about 5-20%.
  • the LNP may include lipid conjugates, for example, polyethylene glycol (PEG)-modified lipids and derived lipids.
  • PEG-modified lipids may include, but are not limited to, polyethylene glycol chains covalently linked to lipids with alkyl chains of lengths of C6 to C20, up to a length of 5 kDa. The addition of these components can prevent lipid aggregation, increase circulation duration, facilitate lipid-nucleic acid composition delivery to target cells, or quickly release nucleic acids.
  • the polyethylene glycol (PEG)-modified lipid molecule can be a PEG-ceramide with a shorter acyl chain (e.g., C14 or C18).
  • the polyethylene glycol (PEG)-modified lipid molecule has a molar ratio of about 0.5 to 2% in lipid nanoparticles, for example, about 1 to 2%, about 1.2 to 1.8%, or about 1.4 to 1.6%.
  • the polyethylene glycol (PEG)-modified lipid molecule can be PEG2000-DMG.
  • the LNP may further comprise cholesterol.
  • the molar ratio in the lipid nanoparticles is about 30-50%, for example, about 35-45%, or about 38-42%.
  • the LNP may include cationic lipids, cholesterol, phospholipids and lipid molecules modified with polyethylene glycol.
  • the molar ratio of the cationic lipids, cholesterol, phospholipids and lipid molecules modified with polyethylene glycol may be 45-55:35-45:5-15:0.5-2.
  • the coding DNA sequence listed in Table 1 above was inserted between the 5' untranslated region (UTR) (SEQ ID NO: 29) and 3'UTR (SEQ ID NO: 30) downstream of the T7 promoter in the T7 RNA polymerase-based in vitro transcription (IVT) vector and used as an IVT template.
  • lipid nanoparticles lipid nanopartical, LNP
  • LNP lipid nanopartical, LNP-mRNA
  • Each group of mRNA vaccines (LNP-mRNA) prepared in Example 1 was initially administered (Prime) to 7-week-old Balb/c female mice at a dose of 10 ⁇ g/dose/mouse by intramuscular injection on day 0, and boosted (Boost) was administered to the mice by intramuscular injection on day 14.
  • the control group was administered with Dulbecco's phosphate buffered saline (DPBS, Thermo Fisher, 14190136).
  • DPBS Dulbecco's phosphate buffered saline
  • the blood collected from the mice on day 29 after vaccination as described above was measured for the concentrations of total anti-A35R antibodies, total anti-M1R antibodies, total anti-B6R antibodies and total anti-A29L antibodies by measuring the absorbance at 450 nm (OD450) ( FIGS. 2A to 2D ).
  • the total anti-B6R antibody level in the blood of mice administered with the vaccines of A+B+C+D Group 1 and A+B+C+D Group 2 was significantly increased compared with the blood of negative control mice administered with DPBS.
  • the total anti-A29L antibody level in the blood of mice administered with the vaccines of A+B+C+D Group 1 and A+B+C+D Group 2 was significantly increased compared with the blood of negative control mice administered with DPBS.
  • the serum dilution series and the virus dilution were mixed in equal volumes in a 96-well plate and incubated at 37°C for 1 hour. The PFU of each mixture was then determined. The serum dilution that reduced the PFU by 50% compared to the control group without serum addition was set as the neutralizing antibody titer of the serum.
  • the neutralizing antibody levels (PFU/well) against vaccinia virus in the sera diluted as above are shown in FIG. 2E .
  • mice administered with vaccines from Group A-B 1, Group A-B 2, and Group A+B 2 achieved complete neutralization of vaccinia virus, indicating that the level of neutralizing antibodies against vaccinia virus was the most abundant.
  • mice vaccinated with each vaccine were challenged with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1 ⁇ 10 6 PFU by intranasal administration.
  • the mice challenged with vaccinia virus were weighed for several consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.
  • mice administered with each group of mRNA vaccines did not experience significant changes in body weight due to challenge with vaccinia virus.
  • mice described in (4) above were killed, and lung tissues were collected to measure the viral load (PFU/g) therein.
  • the vaccines of AB Group 1, AB Group 2 and A+B Group 1 prepared in Example 1 (at a dose of 10 ⁇ g/dose/mouse), live vaccinia virus Western Reserve (VACV-WR, Cat. # VR-1354, ATCC) as a positive control (at a dose of 1 ⁇ 10 4 PFU) and DPBS (Thermo Fisher, 14190136) as a negative control were initially administered (Prime) to 7-week-old Balb/c female mice by intramuscular injection on day 0, and boosted (Boost) to the mice by intramuscular injection on day 14 (0.5 month after vaccination).
  • VACV-WR live vaccinia virus Western Reserve
  • DPBS Thermo Fisher, 14190136
  • the concentrations of anti-A35R antibody and anti-M1R antibody in the blood collected from the mice at 1, 2, 3, 4 and 5 months after vaccination were determined by measuring the absorbance at 450 nm (OD450) ( FIGS. 3A and 3B ).
  • the concentration of total anti-A35R antibodies in the blood of mice administered with the vaccine of A-B Group 1, A-B Group 2, and A+B Group 1 was significantly higher than that in the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS.
  • the concentration of anti-A35R antibodies decreased over time, but was still significantly higher than that in the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS.
  • the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccines of A-B Group 1 and A-B Group 2 was significantly higher than that of the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS.
  • the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccine of A+B Group 1 was between that of the blood of positive control mice administered with VACV-WR and the blood of negative control mice administered with DPBS. There was no obvious regularity in the changes over time in the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccines of A-B Group 1, A-B Group 2, and A+B Group 1.
  • the serum dilution series and the virus dilution were mixed in equal volumes in a 96-well plate and incubated at 37°C for 1 hour. The PFU of each mixture was then determined. The serum dilution that reduced the PFU by 50% compared to the control group without serum addition was set as the neutralizing antibody titer of the serum.
  • the neutralizing antibody levels (%) against vaccinia virus in the sera diluted as above are shown in FIG. 3C .
  • the neutralizing antibody levels in the sera of mice administered with the vaccines of Group A-B 1 and Group A-B 2 were significantly higher than those of the positive control mice administered with VACV-WR and the negative control mice administered with DPBS.
  • the neutralizing antibody levels in the sera of mice administered with the vaccine of Group A+B 1 were between those of the positive control mice administered with VACV-WR and the negative control mice administered with DPBS.
  • the neutralizing antibody levels in the sera of mice administered with the vaccines of Group A-B 1, Group A-B 2, and Group A+B 1 were relatively constant over time.
  • mice vaccinated with each vaccine were challenged intranasally with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1 ⁇ 10 6 PFU.
  • the mice challenged with vaccinia virus were weighed on consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.
  • mice administered with vaccines of A-B Group 1, A-B Group 2, and A+B Group 1 and the positive control mice administered with VACV-WR did not undergo significant weight changes due to challenge with vaccinia virus.
  • the vaccines of AB Group 1, AB Group 2 and A+B Group 1 prepared in Example 1 (at a dose of 10 ⁇ g/dose/mouse), live vaccinia virus Western Reserve (VACV-WR, Cat. # VR-1354, ATCC) as a positive control (at a dose of 1 ⁇ 10 4 PFU) and DPBS (Thermo Fisher, 14190136) as a negative control were initially administered (Prime) to 7-week-old Balb/c female mice by intramuscular injection on day 0, and boosted (Boost) to the mice by intramuscular injection on day 14 (0.5 month after vaccination).
  • VACV-WR live vaccinia virus Western Reserve
  • DPBS Thermo Fisher, 14190136
  • Serum was separated from the blood collected from the mice at 1 month, 2 months, 3 months and 4 months after vaccination as above, and equal volumes of the blood were mixed. 100 ⁇ l of the mixed serum was intravenously injected into a new batch of 7-8 week old Balb/c female mice.
  • mice injected with mixed serum were challenged with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1 ⁇ 10 5 PFU via intranasal route on the next day.
  • the mice challenged with vaccinia virus were weighed for 17 consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.
  • FIG. 4A shows the weight change (%) relative to the initial weight of mice intravenously injected with pooled sera obtained from negative control mice administered DPBS when challenged with vaccinia virus.
  • FIG. 4B shows the weight change (%) relative to the initial weight of mice injected intravenously with the pooled sera obtained from mice administered with the vaccine of Group A-B 1 upon challenge with vaccinia virus.
  • FIG. 4C shows the weight change (%) relative to the initial weight of mice injected intravenously with pooled sera obtained from mice administered with vaccines of Groups A-B 2 upon vaccinia virus challenge.
  • FIG. 4D shows the weight change (%) relative to the initial weight of mice intravenously injected with the pooled sera obtained from mice administered with the vaccine of Group A+B 1 when challenged with vaccinia virus.
  • FIG. 4E shows the weight change (%) relative to the initial weight of mice injected intravenously with pooled sera obtained from positive control mice administered VACV-WR when challenged with vaccinia virus.
  • mice injected intravenously with pooled sera from mice given the above different vaccines were compared.
  • Body weight change relative to initial body weight during vaccinia virus challenge (%).
  • mice intravenously injected with pooled sera obtained from mice administered with AB group 2 vaccine and mice intravenously injected with pooled sera obtained from positive control mice administered with VACV-WR had significantly less weight change due to vaccinia virus challenge than mice intravenously injected with pooled sera obtained from negative control mice administered with DPBS.
  • mice intravenously injected with pooled sera obtained from mice administered with AB group 1 vaccine and mice intravenously injected with pooled sera obtained from mice administered with A+B group 1 vaccine had comparable weight changes due to vaccinia virus challenge as mice intravenously injected with pooled sera obtained from negative control mice administered with DPBS.
  • the vaccines of AB Group 1, AB Group 2 and A+B Group 1 prepared in Example 1 (at a dose of 10 ⁇ g/dose/mouse), live vaccinia virus Western Reserve (VACV-WR, Cat. # VR-1354, ATCC) as a positive control (at a dose of 2 ⁇ 10 4 PFU (VACV-WR-low) or 2 ⁇ 10 5 PFU (VACV-WR-high)), and DPBS (Thermo Fisher, 14190136) as a negative control were administered to 7-week-old Balb/c female mice by intramuscular injection on day 0.
  • VACV-WR live vaccinia virus Western Reserve
  • VACV-WR live vaccinia virus Western Reserve
  • DPBS Thermo Fisher, 14190136
  • the concentrations of anti-A35R antibody and anti-M1R antibody in the blood collected from the mice on day 7 after vaccination as described above were determined by measuring absorbance at 450 nm (OD450) ( FIGS. 5A and 5B ).
  • the concentrations of total anti-A35R antibodies in the blood of mice administered with vaccines from Group A-B 1, Group A-B 2, and Group A+B 1 were significantly higher than those in the blood of positive control mice administered with VACV-WR-low and VACV-WR-high and the blood of negative control mice administered with DPBS.
  • the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccines of A-B Group 1 and A-B Group 2 was significantly higher than that in the blood of positive control mice administered with VACV-WR-low and VACV-WR-high and the blood of negative control mice administered with DPBS.
  • the concentration of total anti-M1R antibodies in the blood of mice administered with the vaccine of A+B Group 1 was comparable to that in the blood of positive control mice administered with VACV-WR-low and VACV-WR-high and the blood of negative control mice administered with DPBS.
  • the serum dilution series and the virus dilution were mixed in equal volumes in a 96-well plate and incubated at 37°C for 1 hour. The PFU of each mixture was then determined. The serum dilution that reduced the PFU by 50% compared to the control group without serum addition was set as the neutralizing antibody titer of the serum.
  • the neutralizing antibody levels (%) against vaccinia virus in the sera diluted as above are shown in FIG5C .
  • the neutralizing antibody levels in the sera of mice administered with the vaccines of A-B Group 1 and A-B Group 2 were significantly higher than those in the sera of negative control mice administered with DPBS.
  • the neutralizing antibody levels in the sera of mice administered with the vaccine of A+B Group 1 were between those of the sera of positive control mice administered with VACV-WR-low and VACV-WR-high and those of negative control mice administered with DPBS.
  • mice vaccinated with each vaccine were challenged intranasally with live vaccinia virus Western Reserve (VACV-WR, Cat.# VR-1354, ATCC) at a dose of 1 ⁇ 10 6 PFU.
  • the mice challenged with vaccinia virus were weighed for 18 consecutive days thereafter, and the weight change (%) relative to the initial weight was calculated.
  • mice administered with vaccines from AB Group 1, AB Group 2, and A+B Group 1 and the positive control mice administered with VACV-WR-low and VACV-WR-high did not undergo significant weight changes in response to vaccinia virus challenge.

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Abstract

La présente invention concerne un vaccin anti-poxvirus, qui comprend une molécule d'ARNm codant pour les protéines et/ou protéines de fusion suivantes : (a) plus d'une protéine de poxvirus simien choisie parmi les suivantes : une protéine A35R, une protéine M1R, une protéine B6R et une protéine A29L et/ou (b) une protéine de fusion formée par fusion d'au moins deux protéines ou parties de protéines de poxvirus simien choisies parmi les suivantes : une protéine A35R, une protéine M1R, une protéine B6R et une protéine A29L. La présente invention concerne également un kit de préparation du vaccin anti-poxvirus, qui comprend : (i) une molécule d'ADN codant pour la protéine et/ou la protéine de fusion décrites et éventuellement (ii) un réactif pour transcrire la molécule d'ADN de (i) en molécule d'ARNm. La présente invention concerne également une molécule d'ADN ou une molécule d'ARNm codant pour la protéine et la protéine de fusion décrites.
PCT/CN2023/102077 2022-11-18 2023-06-25 Vaccin à base d'acide ribonucléique messager contre un poxvirus WO2024103728A1 (fr)

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