WO2023202711A1 - Vaccin à arnm basé sur un nouveau coronavirus - Google Patents

Vaccin à arnm basé sur un nouveau coronavirus Download PDF

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WO2023202711A1
WO2023202711A1 PCT/CN2023/089871 CN2023089871W WO2023202711A1 WO 2023202711 A1 WO2023202711 A1 WO 2023202711A1 CN 2023089871 W CN2023089871 W CN 2023089871W WO 2023202711 A1 WO2023202711 A1 WO 2023202711A1
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rna
cov
sars
variant
composition
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PCT/CN2023/089871
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Chinese (zh)
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岑山
王静
衣岽戎
董翊洁
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仁景(苏州)生物科技有限公司
仁景国际香港有限公司
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Publication of WO2023202711A1 publication Critical patent/WO2023202711A1/fr

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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
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    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/28—Steroids, e.g. cholesterol, bile acids or glycyrrhetinic acid
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    • A61K9/0012—Galenical forms characterised by the site of application
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    • 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
    • A—HUMAN NECESSITIES
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    • 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
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    • 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
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
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    • 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
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • 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
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present disclosure relates to the field of preventing or treating coronavirus infection, and specifically relates to the novel coronavirus SARS-CoV-2 mRNA vaccine and its preparation method and application.
  • the present disclosure relates to methods and agents for vaccination against coronavirus infection and inducing effective coronavirus antigen-specific immune responses, such as antibody and/or T cell responses. These methods and agents are particularly useful in preventing or treating coronavirus infections.
  • This disclosure also describes methods for preparing the vaccines and immunological evaluation of the vaccines.
  • the 2019 novel coronavirus (SARS-CoV-2) is a new strain of coronavirus that has never been found in humans before. This virus is the seventh coronavirus (CoV) that can infect humans.
  • the incubation period for human infection with SARS-CoV-2 is generally 1 to 14 days. Common signs after infection include respiratory symptoms, fever, cough, shortness of breath, and difficulty breathing. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death.
  • the latest data from the WHO website shows that as of November 26, 2021, a total of 259,502,031 confirmed cases and 5,183,003 deaths have been reported globally.
  • the new coronavirus is a positive-sense single-stranded RNA ((+)ssRNA) enveloped virus encoding four structural proteins: spike protein (S), envelope protein (E), membrane protein (M) and nucleocapsid protein (N).
  • the S protein is divided into two sub-domains, S1 and S2.
  • S1 domain is responsible for recognizing virus-specific receptors and binding to host cells.
  • S2 has a transmembrane domain and is responsible for membrane fusion.
  • the SARS-CoV-2 Delta mutant virus strain was first discovered in India in October 2020. It has since spread to at least 185 countries and regions, becoming the main popular virus mutant strain in the world.
  • the new coronavirus vaccine developed based on early epidemic strains targets SARS- The neutralizing ability of CoV-2 Delta variant strains was significantly reduced.
  • New coronavirus vaccines currently on the market and under development mainly include mRNA vaccines, inactivated vaccines, adenovirus vector vaccines, DNA vaccines, recombinant protein vaccines, etc.
  • mRNA epidemic Vaccines deliver in vitro-transcribed mRNA to cells, where they are translated to produce proteins, thereby stimulating the body's specific immune response.
  • mRNA vaccines do not need to enter the nucleus and are only expressed in the cytoplasm, so there is no risk of insertional mutations in the host genome.
  • the rapid, simple preparation method and low cost of mRNA are also one of the advantages of vaccines, which greatly shortens the response time to respond to sudden infectious diseases and reduces the cost of prevention and control.
  • the purpose of the present invention is to prepare a new coronavirus mRNA vaccine.
  • the S protein of SARS-CoV-2 for vaccine development. More specifically, the mRNA vaccine provided by the invention includes at least one RNA polynucleotide having an open reading frame encoding a SARS-CoV-2 Delta mutant strain antigen.
  • SARS-CoV-2 antigens include spike protein (S protein) and its variants. The pre-fusion conformation of the S protein is crucial to establishing an effective immune system. Therefore, in order to develop a more targeted mRNA vaccine, we selected the S protein gene coding sequence of the Delta virus strain and the specific proline on this basis.
  • substitution variants of the sites resulted in the antigen sequences of Delta S, Delta S-2P (K984P, V985P) and Delta S-6P (F815P, A890P, A897P, A940P, K984P, V985P) respectively.
  • the invention provides an RNA comprising an open reading frame encoding an antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof, wherein the antigenic polypeptide is selected from the group consisting of SARS-CoV-2 2's receptor binding domain, S protein, variants thereof or immunogenic fragments thereof, preferably the SARS-CoV-2 is a SARS-CoV-2 Delta variant strain.
  • the antigenic polypeptide or immunogenic fragment or variant thereof comprises one or more immunogenic epitopes of a SARS-CoV-2 polypeptide or variant thereof; e.g. , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10 or more immunogenic epitopes; preferably, the The antigenic polypeptide or the immunogenic fragment or variant thereof is selected from the (full-length) S protein of the SARS-CoV-2 Delta variant virus strain, preferably Is a (full-length) S protein variant of the SARS-CoV-2 Delta variant strain, more preferably the S protein variant is selected from Delta S-2P, which contains mutations K984P and V985P, and Delta S-6P, which Contains mutations F815P, A890P, A897P, A940P, K984P and V985P,
  • the antigenic polypeptide or immunogenic fragment or variant thereof comprises the amino acid sequence of amino acids 17-1271 of SEQ ID NO: 1, 2 or 3, and the amino acid sequence 17-1271 of SEQ ID NO: 1, 2 or 3.
  • the amino acid sequence of 1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical; and/or
  • RNA encoding the antigenic polypeptide or an immunogenic fragment or variant thereof includes the nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4, 5 or 6, which is the same as SEQ ID NO: 4, 5 or the nucleotide sequence of nucleotides 49-3813 of 6 has a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
  • the open reading frame encoding an antigenic polypeptide of SARS-CoV-2 or an immunogenic fragment or variant thereof further comprises a secretion signal peptide, preferably N-terminally fused to The antigenic polypeptide or the immunogenic fragment or variant thereof, the secretion signal peptide is preferably the secretion signal peptide of S protein.
  • the secretion signal peptide comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3, which is at least 99% identical to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3.
  • amino acid sequence that is 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or a functional fragment of the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3 or is identical to SEQ ID NO: 1, 2 or 3.
  • the RNA encoding the secretion signal peptide comprises the nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6, and the nucleic acid sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6.
  • the RNA is mRNA, circular RNA (cRNA) and self-replicating RNA (saRNA), preferably the RNA is suitable for intracellular expression of the polypeptide.
  • the RNA is a modified RNA modified by replacing some or all of the uridine residues with a modified uridine residue, preferably the modified uridine is N1-methyl-pseudouridine glycosides.
  • the RNA also includes one or more structural elements optimized for maximum effectiveness of the RNA in terms of stability and translation efficiency.
  • the structural elements include: 5' cap, 5' UTR, 3'UTR and polyA tail sequence.
  • the 5' cap is or contains a cap1 structure; more preferably, the 5' cap is m7G(5')ppp(5')(2'-OMeA)pG.
  • the 5'-UTR is the 5'-UTR sequence of human ⁇ -globin mRNA, optionally with an optimized Kozak sequence; more preferably, the 5'UTR comprises the nucleoside of SEQ ID NO: 7 acid sequence, or a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the nucleotide sequence of SEQ ID NO: 7,
  • the 3'-UTR is a two-repeat 3'-UTR of human ⁇ -globin mRNA; more preferably, the 3'UTR comprises the nucleotide sequence of SEQ ID NO: 8, or is identical to The nucleotide sequence of SEQ ID NO: 8 is a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
  • the polyA tail sequence comprises at least 50, at least 60 or at least 100 A nucleotides; more preferably, the polyA tail sequence comprises the nucleotide sequence of SEQ ID NO: 9, or consists of SEQ ID NO: It consists of 9 nucleotide sequences.
  • the invention provides a composition comprising an RNA as described herein.
  • the composition is formulated or to be formulated as a liquid, solid, or a combination thereof, preferably the composition is formulated or to be formulated for injection or other mode of administration, preferably the composition Formulated or to be formulated for intramuscular injection.
  • the RNA is combined with proteins and/or lipids complex to produce RNA-particles for administration.
  • the RNA is formulated in lipid nanoparticles comprising cationic/ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG) - Lipid; preferably the lipid nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)((( 4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 2-[(polyethylene glycol)-2000]-N, N-ditetradecyl acetamide (2 -[(polyethyleneglyco1)-2000]-N,N-ditetradecylacetamide), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distaroyl-sn-glycero-3-phosphocholine
  • the molar ratio of cationic/ionizable lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids is (40-55):(10-15):(35-45) ⁇ (0.5-2.5),
  • a preferred molar ratio of cationic/ionizable lipid, phospholipid, cholesterol and polyethylene glycol (PEG)-lipid is 50:10:38.5:1.5.
  • the RNA is formulated or to be formulated as a colloid; preferably, the RNA is formulated as particles and is present in 50% or more, 75% of the colloidal dispersed phase formed or more or 85% or more RNA; more preferably said particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase, wherein preferably said organic phase comprises ethanol; further Preferably, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase, wherein preferably the lipids dispersed in the aqueous phase form liposomes.
  • the RNA is present in the composition in an amount ranging from 1 ⁇ g to 100 ⁇ g per dose.
  • the present invention provides the use of the RNA or composition described herein in the preparation of a medicament, the medicament being a vaccine, the medicament further comprising one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the medicaments of the present invention are used to induce response to coronavirus in a subject.
  • An immune response to a coronavirus preferably a specific immune response to a coronavirus antigen.
  • the medicaments of the invention are used for the treatment or prophylactic treatment of coronavirus infections.
  • the coronavirus is a betacoronavirus, preferably the coronavirus is a sarbecovirus, more preferably the coronavirus is SARS-CoV-2, and further preferably the coronavirus Viruses include: original strain of new coronavirus (GD108), SARS-CoV-2 Alpha variant strain, SARS-CoV-2 Beta variant strain, SARS-CoV-2 Delta variant strain and SARS-CoV-2 Omicron variant virus strain.
  • GD108 original strain of new coronavirus
  • SARS-CoV-2 Alpha variant strain SARS-CoV-2 Beta variant strain
  • SARS-CoV-2 Delta variant strain SARS-CoV-2 Omicron variant virus strain.
  • RNA, composition or medicament when said RNA, composition or medicament is administered to cells of human origin, detectable expression of said antigenic polypeptide or immunogenic fragment or variant thereof is achieved, and preferably such expression Lasts for at least 24 hours or longer.
  • RNA, composition or medicament produces an immune effect in a subject, said immune effect comprising the production of SARS-CoV-2 neutralizing antibodies and/or T cell responses, in particular Robust TH1 type T cell response, preferably CD4+ and/or CD8+ T cell response.
  • RNA, composition or medicament produces an immune response in a subject
  • said immune response comprises the production of an immune response directed against the S1 subunit of the SARS-CoV-2 spike protein.
  • the immune response includes the production of neutralizing antibody titers against the SARS-CoV-2 virus.
  • the serum of the subject e.g., a mouse
  • the serum of the subject exhibits antibodies directed against a polypeptide encoded by said open reading frame of production.
  • the serum of the subject eg, mouse
  • the subject is a mammal, preferably the subject is a mouse, further preferably the subject is a human.
  • the invention also provides a method for preparing a vaccine, comprising formulating the RNA described herein in lipid nanoparticles, the lipid nanoparticles comprising cationic ionizable lipids.
  • lipids, phospholipids, cholesterol and polyethylene glycol (PEG)-lipids preferably the lipid nanoparticles comprise ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl ) bis(2-hexyl decanoate), 2-[(polyethylene glycol)-2000]-N,N-distetradecyl acetamide, 1,2-distearoyl-sn-glycerol-3 -Phosphocholine and cholesterol; more preferably the lipid nanoparticles comprise SM-102, distearoylphosphatidylcholine (DSPC), cholesterol and DMG-PEG2000.
  • DSPC distearoylphosphatidylcholine
  • the present invention also provides methods of inducing an immune response against a coronavirus, preferably a specific immune response against a coronavirus antigen, in a subject.
  • methods for the treatment or prophylactic treatment of coronavirus infections are also provided.
  • the method includes administering to a subject an RNA, composition or medicament described herein.
  • the subject is a mammal, preferably the subject is a mouse, and further preferably the subject is a human.
  • the coronavirus is a beta coronavirus, preferably the coronavirus is Sabei virus, more preferably the coronavirus is SARS-CoV-2, and further preferably the coronavirus is SARS-CoV-2.
  • the above-mentioned coronaviruses include: the original strain of the new coronavirus (GD108), the SARS-CoV-2 Alpha variant strain, the SARS-CoV-2 Beta variant strain, the SARS-CoV-2 Delta variant strain and the SARS-CoV-2 Omicron Mutated virus strains.
  • FIG. 1 Expression of LNP-mRNA in 293T cells.
  • Figure 2 Immunological evaluation of novel coronavirus mRNA vaccine in mouse model.
  • Figure 3 Immunological evaluation of the new coronavirus mRNA vaccine in the rhesus monkey model.
  • Figure 4 Physiological evaluation of rhesus monkeys after immunization with the new coronavirus mRNA vaccine.
  • Figure 5 Score of pathological changes in the lungs of rhesus monkeys.
  • the term "about” or “approximately” means a change of up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of quantity, level, value, quantity, frequency, percentage, scale, size, quantity, weight or length.
  • Consisting essentially of is meant to include any of the elements listed after the phrase “consisting essentially of” and is limited to not interfering with or contributing to the disclosure of the listed elements Other elements of the activity or action specified in the content. Thus, the phrase “consisting essentially of” is an indication that the listed elements are required or mandatory, but that no other elements are optional and depend on whether they affect the listed elements An activity or action that may or may not exist.
  • mutants and variants refer to molecules that retain the same or substantially the same biological activity as that of the original sequence.
  • the mutant or variant may be from the same or different species, or may be based on a natural molecule or a synthetic sequence of an existing molecule.
  • the terms “mutant” and “variant” refer to a polypeptide having an amino acid sequence that differs from the corresponding wild-type polypeptide by at least one amino acid.
  • mutants and variants may contain conservative amino acid substitutions: substitution of amino acids with similar properties for corresponding amino acids.
  • Conservative substitutions can be polar to polar amino acids (glycine (G, Gly), serine (S, Ser), threonine (T, Thr), tyrosine (Y, Tyr), cysteine (C, Cys), asparagine (N, Asn) and glutamine (Q, Gln)); non-polar versus non-polar amino acids (alanine (A, Ala), valine (V, Val), chrom Acid (W, Trp), leucine (L, Leu), proline (P, Pro), methionine (M, Met), phenylalanine (F, Phe)); acidic versus acidic Amino acids (aspartic acid (D, Asp), glutamic acid (E, Glu)); basic versus basic amino acids (arginine (R, Arg), histidine (H, His), lysine (K, Lys)); charged amino acids versus charged amino acids (aspartic acid (D, Asp), glutamic acid (E, Glu), his
  • a mutant or variant polypeptide can have about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80 , 90, 100 or more, or substitution, addition, insertion or deletion of amino acids in a range consisting of any two of the aforementioned values.
  • a mutant or variant may have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or any two of the aforementioned values.
  • the invention generally encompasses the immunotherapy of a subject comprising the administration of RNA, i.e., vaccine RNA, encoding amino acids, i.e., vaccine antigens, comprising the SARS-CoV-2 S protein or immunogenic fragments or variants thereof. Therefore, the vaccine antigen contains an epitope of the SARS-CoV-2 S protein and is used to induce an immune response in a subject against the coronavirus S protein, particularly the SARS-CoV-2 S protein.
  • RNA encoding a vaccine antigen is administered to provide (after expression of the polynucleotide by an appropriate target cell) an antigen for inducing, i.e., stimulating, priming and/or amplifying an immune response, e.g., antibodies and/or immune effector cells, which target To the target antigen (coronavirus S protein, especially SARS-CoV-2 S protein) or its processed products.
  • an immune response e.g., antibodies and/or immune effector cells, which target To the target antigen (coronavirus S protein, especially SARS-CoV-2 S protein) or its processed products.
  • an immune response induced by disclosure is a B cell-mediated immune response, that is, an antibody-mediated immune response, especially an anti-SARS-CoV-2 immune response.
  • the vaccine described herein contains as an active ingredient single-stranded RNA, which can be translated into the corresponding protein after entering the recipient cells.
  • the RNA may also contain one or more structural elements that are optimized for maximum potency of the RNA in terms of stability and translation efficiency (5' cap, 5′UTR, 3′UTR, polyA tail).
  • the m7G(5’)ppp(5’)(2’-OMeA)pG formed by CleanCap can be used as a specific capping structure at the 5’-end of RNA drug substances.
  • the 5'-UTR sequence of human ⁇ -globin mRNA can be used, optionally with an optimized "Kozak sequence" to improve translation efficiency.
  • the 3'-UTR may be the two repeated 3'-UTR of human ⁇ -globin mRNA.
  • polyA tail sequences of 50-120 nucleotides in length can be used.
  • a secretion signal peptide can be fused to the antigen coding region, preferably in such a way that sec is translated into an N-terminal tag.
  • sec corresponds to the secretion signal peptide of the S protein.
  • the vaccine RNA described herein can be complexed with proteins and/or lipids (preferably lipids) to generate RNA-particles for administration. If a combination of different RNAs is used, the RNAs can be complexed together or separately with proteins and/or lipids to generate RNA-particles for administration.
  • the invention relates to a composition or medical preparation comprising an RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof.
  • the amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof is capable of forming a multimeric complex, in particular a trimer complex.
  • the amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof may comprise a domain that allows the formation of a multimeric complex, in particular comprising the SARS-CoV-2 S protein or an immunogen thereof.
  • the domain that allows the formation of multimeric complexes comprises a trimerization domain, eg, a trimerization domain described herein.
  • the amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof is encoded by a coding sequence that is codon-optimized and/or compared to a wild-type coding sequence. or an increase in G/C content, wherein said codon optimization and/or increase in G/C content preferably does not alter the sequence of the encoded amino acid sequence.
  • the RNA encoding the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises the nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4, 5 or 6 , having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity to the nucleotide sequence of nucleotides 49-3813 of SEQ ID NO: 4, 5 or 6 the nucleotide sequence; and/or
  • SARS-CoV-2 S protein or an immunogenic fragment or variant thereof contains the amino acid sequence of amino acids 17-1271 of SEQ ID NO: 1, 2 or 3, and the amino acid sequence of SEQ ID NO: 1, 2 or 3
  • the amino acid sequence of 17-1271 has an amino acid sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
  • the amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein, or an immunogenic fragment or variant thereof comprises secreted signal peptide.
  • the secretion signal peptide is fused, preferably by N-terminal fusion, to the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof.
  • the RNA encoding the secretion signal peptide comprises a nucleotide sequence of nucleotides 1-48 of SEQ ID NO: 4, 5 or 6, identical to SEQ ID NO: 4, 5 or 6 A nucleotide sequence of nucleotides 1-48 that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to a nucleotide sequence, or SEQ ID NO: A fragment of the nucleotide sequence of nucleotides 1-48 of 4, 5 or 6 or has at least 99%, 98%, A fragment of a nucleotide sequence that is 97%, 96%, 95%, 90%, 85% or 80% identical; and/or
  • the secretion signal peptide comprises the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3, and has at least 99%, An amino acid sequence that is 98%, 97%, 96%, 95%, 90%, 85% or 80% identical, or amino acids 1-16 of SEQ ID NO: 1, 2 or 3 A functional fragment of the acid sequence or is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical to the amino acid sequence of amino acids 1-16 of SEQ ID NO: 1, 2 or 3 A functional fragment of a sexual amino acid sequence.
  • the RNA is modified RNA, in particular stabilized mRNA.
  • the RNA contains a modified nucleoside in place of uridine.
  • the modified nucleoside is N1-methyl-pseudouridine (ml ⁇ ).
  • the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises a 5'UTR comprising the nucleotide sequence of SEQ ID NO: 7, or the same sequence as SEQ ID NO: 7
  • the nucleotide sequence of ID NO: 7 is a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
  • the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises a 3'UTR that comprises the nucleotide sequence of SEQ ID NO: 8, or is identical to SEQ ID NO: 8
  • the nucleotide sequence of ID NO: 8 is a nucleotide sequence that is at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identical.
  • the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein or an immunogenic fragment or variant thereof comprises a polyA tail sequence.
  • the polyA tail sequence contains at least 100 nucleotides.
  • the polyA tail sequence comprises or consists of the nucleotide sequence of SEQ ID NO: 9.
  • the RNA or composition is formulated or to be formulated as a liquid, solid, or a combination thereof. In one embodiment, the RNA or composition is or is to be formulated for injection or other administration. In one embodiment, the RNA or composition is or is to be formulated for intramuscular injection.
  • the RNA is formulated or to be formulated as particles.
  • the particles are lipid nanoparticles (LNPs).
  • the LNP particles comprise (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)((4-hydroxybutyl )azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)), 2-[(polyethylene glycol)-2000]-N, N-ditetradecyl acetamide (2-[( polyethylene glyc01)-2000]-N,N-ditetradecylacetamide), 1,2-distearoyl-sn-glycero-3-phosphocholine (1,2-Distearoyl-sn-glycero-3-phosphocholine) and cholesterol.
  • the RNA is formulated or to be formulated as a colloid. In one embodiment, the RNA is formulated or to be formulated as particles forming a colloidal dispersed phase. In one embodiment, 50% or more, 75% or more, or 85% or more RNA is present in the dispersed phase. In one embodiment, the RNA is formulated or to be formulated as particles comprising RNA and lipids. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dissolved in an organic phase. In one embodiment, the organic phase contains ethanol. In one embodiment, the particles are formed by exposing RNA dissolved in an aqueous phase to lipids dispersed in the aqueous phase. In one embodiment, the lipids dispersed in the aqueous phase form liposomes.
  • the RNA is mRNA, circular RNA (cRNA), and self-replicating RNA (saRNA).
  • the composition or pharmaceutical preparation is a pharmaceutical composition. In one embodiment, the composition or pharmaceutical preparation is a vaccine. In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the invention relates to a composition or pharmaceutical preparation described herein for pharmaceutical use.
  • the pharmaceutical use includes inducing an immune response against coronavirus in a subject.
  • the pharmaceutical use includes treatment or prophylactic treatment of coronavirus infection.
  • the coronavirus is a betacoronavirus.
  • the coronavirus is a sarbecovirus.
  • the coronavirus is SARS-CoV-2.
  • the coronavirus includes: the original strain of the new coronavirus (GD108), the SARS-CoV-2 Alpha variant strain, the SARS-CoV-2 Beta variant strain, and the SARS-CoV-2 Delta variant Virus strains and SARS-CoV-2 Omicron variant strains
  • the invention relates to a method of inducing an immune response against a coronavirus in a subject, the method comprising administering to the subject a protein comprising an encoding amino acid sequence.
  • a composition of RNA, the amino acid sequence comprising SARS-CoV-2 S protein or an immunogenic fragment or variant thereof.
  • the method is a method of vaccination against a coronavirus. In one embodiment, the method is a method for the treatment or prophylactic treatment of coronavirus infection. In one embodiment, the subject is a mouse. In one embodiment, the coronavirus is a betacoronavirus. In one embodiment, the coronavirus is a sarbecovirus. In one embodiment, the coronavirus is SARS-CoV-2. In further preferred embodiments, the coronavirus includes: the original strain of the new coronavirus (GD108), the SARS-CoV-2 Alpha variant strain, the SARS-CoV-2 Beta variant strain, and the SARS-CoV-2 Delta variant Virus strains and SARS-CoV-2 Omicron variant strains
  • the invention relates to a composition or pharmaceutical preparation described herein for use in the methods described herein.
  • the present disclosure demonstrates that a composition comprising lipid nanoparticle-encapsulated mRNA can achieve detectable antibody drops in serum against an epitope within 7 days after administration to mice according to a protocol that includes administration of at least one dose of the vaccine composition.
  • the mRNA encodes at least a portion (e.g., is or includes an epitope) of a polypeptide encoded by SARS-CoV-2 (e.g., the S protein encoded by SARS-CoV-2).
  • compositions e.g., compositions comprising lipid nanoparticle-encapsulated mRNA encoding a polypeptide encoded by SARS-CoV-2 (e.g., SARS- At least a portion of the S protein encoded by CoV-2 (e.g., is or contains an epitope)
  • provided methods involving such compositions are characterized by the absence of intrinsic adjuvant effects, or are associated with unmodified results Reduced intrinsic adjuvant effect compared to other comparable compositions (or methods).
  • such compositions (or methods) are characterized in that they induce an antibody response and/or a CD4+ T cell response.
  • modified nucleotides may be present, for example, in the 3'UTR sequence, the antigen-encoding sequence, and/or the 5'UTR sequence.
  • the modified nucleotide is or includes one or more modified uracil residues.
  • compositions e.g., compositions comprising lipid nanoparticle-encapsulated mRNA encoding a SARS-CoV-2-encoded polypeptide (e.g., SARS-CoV-2-encoded S protein)
  • an encoded polypeptide e.g., a protein encoded by SARS-CoV-2 (e.g., S protein)
  • said portion may be or comprise an epitope thereof.
  • such compositions and/or methods are characterized in that they achieve detectable polypeptide expression when administered to cells of human origin, and in some embodiments, such expression persists for at least 24 hours or longer time period.
  • the spike protein may comprise embodiments in which at least one or more amino acid substitutions are made, e.g., a proline substitution as described herein, and/or in which the mRNA sequence is codon-optimized for the subject (e.g., mammal, e.g., human) implementation).
  • one of ordinary skill reading this disclosure will understand that it describes certain features and/or advantages of certain mRNA constructs comprising nucleic acid sequences encoding the full-length SARS-CoV-2 spike protein. There is no wish to be bound by any particular theory.
  • the provided mRNA constructs encoding the full-length SARS-CoV-2 S protein can be implemented for use in or for use in immunogenic compositions (e.g., vaccines) Immune effects (eg, generation of SARS-CoV-2 neutralizing antibodies, and/or T cell responses (eg, CD4+ and/or CD8+ T cell responses)) as described herein are particularly useful and/or effective.
  • the disclosure provides an RNA (e.g., mRNA), which RNA is suitable for intracellular expression of polypeptides.
  • RNA e.g., mRNA
  • such RNA can be formulated in lipid nanoparticles (eg, lipid nanoparticles described herein).
  • immunogenic compositions provided herein can comprise multiple (e.g., at least 2 or more, including, For example, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, etc.) immunogenic epitopes.
  • multiple immunogenic epitopes may be encoded by a single RNA (eg, mRNA).
  • multi-epitope immunogenic compositions provided may be particularly useful in providing protection against multiple viral variants and/or may provide greater opportunities to develop diverse and/or robust neutralizing antibody and/or T cell responses, In particular, a particularly robust TH1 type T cell (eg, CD4+ and/or CD8+ T cell) response.
  • TH1 type T cell eg, CD4+ and/or CD8+ T cell
  • compositions and/or methods are provided that are characterized in that they achieve one or more specific therapeutic outcomes (e.g., an effective immune response as described herein and/or encoded SARS) with a single administration. - detectable expression of CoV-2 S protein).
  • the immune response can include generating binding antibody titers directed against the S1 subunit of the SARS-CoV-2 spike protein. In some embodiments, the immune response can include the production of neutralizing antibody titers against the SARS-CoV-2 virus.
  • the neutralizing antibody titer is (e.g., established to be) sufficient to reduce or block virus compared to vaccinated mice compared to an appropriate control (e.g., unvaccinated control mice). Serum binding titers.
  • the target antigen of this new coronavirus mRNA vaccine is the full length of the S protein of the Delta mutant virus strain.
  • the coding sequence of its gene can be found in GenBank: QWU18818.1.
  • combinatorial mutations at different sites are carried out to obtain Delta S- 2P (K984P, V985P) and Delta S-6P (F815P, A890P, A897P, A940P, K984P, V985P).
  • the encoded nucleotides were deduced and synthesized by a gene synthesis company after optimization of human codons and elimination of relevant enzyme cutting sites.
  • the synthesized S sequence was double digested with BamHI and AscI.
  • the digested product was recovered by 1% agarose gel electrophoresis and then ligated.
  • the 5'UTR, target gene, 3'UTR and polyA were sequentially digested.
  • the luc coding sequence in the pUC57-luc vector of the tail sequence is replaced with the S gene sequence to form the pUC57-S recombinant plasmid.
  • the plasmid was mini-prepared and positive clones were verified by BamHI and AscI restriction endonuclease digestion and DNA sequencing.
  • the plasmid contains the T7 promoter, 5'UTR, ORF, 3'UTR and polyA tail sequence. There is a SapI restriction site after the last A of the polyA tail sequence. Use the restriction endonuclease SapI to linearize the plasmid containing the target gene.
  • the reaction system is shown in Table 1 and digest at 37°C for 3 hours.
  • 20mM acetic acid solution pH 6.0
  • the mRNA and lipid mixture are mixed in a T-flow at a volume ratio of 3:1 to obtain LNP-encapsulated mRNA.
  • Liquid replacement and concentration are then performed by ultrafiltration.
  • the concentration of liposome mRNA was measured using the Ribogreen method, free RNA was directly sampled and measured, and total RNA was measured after cleavage with 5% OTG. The encapsulation efficiency was calculated, and all were above 90%.
  • the particle size of liposome mRNA was measured using a Malvern ZetaSizer particle size analyzer, and both were around 60 nm.
  • 293T cells were seeded into a six-well plate, and 2 ⁇ g of LNP-encapsulated mRNA was added to the cell culture medium 24 hours later for intracellular expression. Collect the cells after 24 hours, add 100 ⁇ L of 1 ⁇ protein loading buffer to each well of the cell pellet, and lyse and denature in a 100°C metal bath for 30 minutes. Protein samples were stored at -20°C or -80°C. Detect the expression of S protein by immunoblotting: take 8 ⁇ L of protein lysate and perform 10% SDS-PAGE electrophoresis to separate the protein samples. Use PVDF membrane to wet transfer at 75V constant pressure for 1.2 hours, then block with 5% skim milk at room temperature for 1 hour.
  • SPF grade female BALB/c mice (6-8 weeks old) were injected and immunized using the mRNA vaccine of the present invention, and the immunization scheme is shown in Figure 2A.
  • the immune groups are LNP control group and different mRNA vaccine groups.
  • the serum used in this experiment was the serum 14 days after the second immunization.
  • PMID: 35412852. to detect the neutralizing activity of serum against SARS-CoV-2 Delta and Omicron strain pseudotypes.
  • Pseudotyped virus is a single-round infection pseudotyped virus packaged by S (Spike) protein with lentivirus as the core and firefly luciferase reporter gene.
  • the serum to be tested was serially diluted 2-fold with DMEM culture medium, and a total of 8 gradients were diluted, starting from 1:128 to 1:16384.
  • Pseudovirus was diluted to 1.5 ⁇ 10 4 TCID 50 /mL in DMEM medium in the P2 laboratory.
  • the neutralizing antibody efficacy of immunized mouse sera against five strains of SARS-CoV-2 (epidemic strain (i.e., the original strain of COVID-19 (GD108)), Alpha, Beta, Delta and Omicron strains) was detected on Vero-E6 cells. price.
  • the serum to be tested was inactivated at 56°C for 30 minutes. Perform 2-fold serial dilutions in DMEM culture medium, starting from 1:8.
  • the virus was diluted to working concentration in DMEM medium according to the original titer of the virus. Mix the diluted virus liquid with the serum of each dilution gradient, and incubate for 1 hour at 37°C in a 5% CO2 incubator.
  • Delta S-6P mRNA vaccine was used to immunize rhesus monkeys by intramuscular injection, and the immunization time points were D0 (first vaccination) and D21 (second vaccination).
  • the immune groups are LNP control group, low-dose group (30 ⁇ g) mRNA vaccine, and high-dose group (100 ⁇ g) mRNA vaccine. Challenge was performed 28 days after the second immunization.
  • the three groups of experimental animals all used the IND strain (Delta) (virus seed batch 21V05P0345/21V05P0346/21V05P0347, using a titer of 1 ⁇ 10 6 TCID 50 /mL), and were challenged by inoculating 500 ⁇ L of each via nasal instillation + tracheal injection. The amount of virus attacked by each monkey is 1 ⁇ 10 6 TCID 50 .
  • the immunization protocol is shown in Figure 3A.
  • the serum used in further experiments was the serum 14 days after the second immunization.
  • the neutralizing activity of the serum against the original strain of SARS-CoV-2, Delta, Omicron BA.1, Omicron BA.2.12.1, and Omicron BA.4/5 pseudotyped viruses was tested according to the same experimental method as in Example 1.
  • the results shown in Figure 3H show that the vaccine immune group has a strong neutralizing effect against the original strain and Delta strain pseudovirus.
  • the NT 50 values for the Omicron strains in the high and low dose groups are as follows: ⁇ 826 and ⁇ 1172 for the BA.1 strain, respectively; ⁇ 570 and ⁇ 905 for the BA.2.12.1 strain, respectively; and ⁇ 364 and ⁇ 364 for the BA4/5 strain, respectively.
  • ⁇ 463 which indicate that the vaccine has the potential to induce broadly neutralizing antibodies in rhesus monkeys.
  • Rhesus monkeys were anesthetized before challenge and on days 1, 3, 5, and 7 after challenge, and nasal swabs, throat swabs, and anal swabs were collected.
  • Swab sample processing Nasal, pharyngeal, and anal swabs were lysed with 800 ⁇ l Trizol, and 200 ⁇ L was used to extract RNA template using an automatic nucleic acid extractor.
  • qRT-PCR (one-step method) determines SARS-CoV-2 genomic RNA levels.
  • the left lung (upper, middle, and lower lobes) and right lung (upper, middle, and lower) tissues were taken from each monkey.
  • Each lung lobe adopts a multi-point (6 points) random sampling method. The sampling points run through the entire lung lobe. The sampling points are mixed and weighed. The total weight is close to 100mg.
  • Tissue homogenization is performed with 800 ⁇ L Trizol. 400 ⁇ L is taken to extract the RNA template and qRT-PCR is used. (One-step method) Detection of viral load.
  • each rhesus monkey's lung tissue (upper left, middle left, lower left, upper right, middle right, lower right, total 6 lobes) were stained with hematoxylin and eosin (H&E) and scanned in full. Pulmonary inflammation, lung structural changes, and bleeding in rhesus monkeys were graded. The scoring standards for each indicator are shown in Table 4 below.
  • the pathological atlas of each lobe of each rhesus monkey's lung tissue was evaluated according to the scoring table. Score, the score is mainly based on the main pathological characteristics of COVID-19: the degree of lung septal thickening or consolidation, the degree of lung septal bleeding, the degree of inflammatory cell infiltration, vascular thrombus, dust cell distribution area and other characteristic indicators are evaluated. Each indicator is scored. The total number is the pathological score. At least 5 fields of view were selected for scoring, and the average of the pathological scores of all lung lobes was the comprehensive pathological score of the monkey's entire lungs. The results in Figure 5 show that the lung pathological damage scores of the low-dose group and the high-dose group were significantly lower than those of the control group (p ⁇ 0.01).

Abstract

La présente invention concerne un vaccin à ARNm basé sur un nouveau coronavirus, en particulier un vaccin à ARNm pour prévenir ou traiter une infection à coronavirus, un procédé de synthèse pour le vaccin à ARNm, et une composition d'ARN. En particulier, la présente invention concerne un vaccin à ARNm pour prévenir une infection à coronavirus par induction d'une réponse immunitaire efficace spécifique d'un antigène de coronavirus. La présente invention concerne en outre un procédé de préparation du vaccin et une évaluation immunologique du vaccin.
PCT/CN2023/089871 2022-04-22 2023-04-21 Vaccin à arnm basé sur un nouveau coronavirus WO2023202711A1 (fr)

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