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WO2024260359A1 - Vaccin à arnm contre les virus respiratoires syncytiaux et son procédé de préparation - Google Patents

Vaccin à arnm contre les virus respiratoires syncytiaux et son procédé de préparation Download PDF

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Publication number
WO2024260359A1
WO2024260359A1 PCT/CN2024/100015 CN2024100015W WO2024260359A1 WO 2024260359 A1 WO2024260359 A1 WO 2024260359A1 CN 2024100015 W CN2024100015 W CN 2024100015W WO 2024260359 A1 WO2024260359 A1 WO 2024260359A1
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Prior art keywords
ome
mrna
seq
mol
vaccine
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Pending
Application number
PCT/CN2024/100015
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English (en)
Chinese (zh)
Inventor
范超
苏晓晔
淡墨
赵璐
魏立帆
王凌宇
杨华
杨思聪
吴磊彬
刘苗苗
王玲玲
钟强
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Cspc Megalith Biopharmaceutical Co Ltd
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Cspc Megalith Biopharmaceutical Co Ltd
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Priority to CN202480001176.0A priority Critical patent/CN119497628A/zh
Publication of WO2024260359A1 publication Critical patent/WO2024260359A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7115—Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—Paramyxoviridae, e.g. parainfluenza virus
    • 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
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the present application belongs to the field of biomedicine technology, and specifically, relates to an mRNA vaccine against respiratory syncytial virus and a preparation method thereof.
  • Respiratory syncytial virus is a genus of Pneumovirus belonging to the family Paramyxoviridae.
  • RSV can be divided into two subtypes, A and B, based on antigenic and genetic characteristics.
  • Type A (such as RSV/A2) is more common in the population.
  • Robert Chanock first isolated the RSV Long strain from a child with bronchopneumonia, which is the first prototype strain of RSV and the prototype strain of RSV-A type RSV/A2. Although there are a few antigenic differences between types A and B, most studies have found that there is no significant difference between the two subtypes, and RSV has only one serotype, so RSV typing does not affect RSV serological detection.
  • RSV mainly causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months old, as well as upper respiratory tract infections such as rhinitis and colds in older children and adults.
  • the virus is the most common pathogen causing viral pneumonia in children.
  • the transmission route is air droplets and close contact. Since maternal antibodies cannot prevent infection, newborn babies can get sick.
  • the reinfection rate of the virus is as high as 65% within 10 years.
  • Studies have shown that RSV susceptible groups are children, the elderly and adults with impaired immune function. More than 90% of children will be infected with RSV before the age of two.
  • RSV infection is the leading cause of hospitalization and death due to viral infection in children under the age of five worldwide.
  • the virus of respiratory syncytial virus is spherical in shape, with a diameter of 120 to 300 nm. It has an envelope and a typical single-stranded negative-strand RNA genome, which mainly encodes 10 proteins, namely three transmembrane proteins: fusion protein (F), adhesion protein (G) and small hydrophobic protein (SH), two matrix proteins M1 and M2, three proteins that combine with viral RNA to form the nucleocapsid (N, P and L), two non-structural proteins (NS1 and NS2), and spikes composed of glycoproteins on the viral envelope.
  • fusion protein F
  • adhesion protein G
  • SH small hydrophobic protein
  • M1 and M2 three proteins that combine with viral RNA to form the nucleocapsid
  • N, P and L three proteins that combine with viral RNA to form the nucleocapsid
  • NS1 and NS2 two non-structural proteins
  • the G protein mediates the binding of the virus to the host cell, and the F protein mediates the fusion of the virus and the host cell membrane, allowing the virus to enter the cell. Both are essential for viral replication and contain B cell and T cell epitopes. They are the most important viral antigen proteins that stimulate the body to produce humoral and cellular immunity.
  • the G protein coding region varies greatly and can be divided into subtype A and subtype B based on its variation.
  • the neutralizing antibodies induced by G protein are subtype specific; the F protein coding region is highly conserved, and the amino acid sequences of the F proteins of subtypes A and B are up to The F protein is at least 90% identical, so the neutralizing antibodies induced by the F protein can inhibit both A and B subtype RSV infection.
  • the structure of the F protein changes dynamically. It is first transcribed and translated into a single inactive polypeptide (F0) in the host cell; then it is cut for the first time by the host cell furin protease to generate a partially cut prefusogenic protein; then the second furin protease cleavage occurs to generate F2 and F1 subunits.
  • F0 inactive polypeptide
  • the two subunits are connected to a monomer by two disulfide covalent bonds, and then the three monomers form a metastable functional prefusion F protein trimer; thereafter, no further processing is required to rearrange the conformation to form a thermodynamically stable postfusion F protein, which can cause the viral envelope to bind to the host cell membrane, facilitate the virus to enter the host cell, and play an important role in RSV infection.
  • the antigenic epitopes related to neutralization activity are mainly I, II, III, IV, V and Species: Epitopes I, II, III, and IV are present in the F protein before and after fusion; V and It is a specific antigenic site of the F protein before fusion.
  • RSV neutralizing antibodies can prevent severe RSV-ALRI.
  • the neutralizing activity of monoclonal antibodies is 10 to 100 times that of Table II monoclonal antibodies. Therefore, most of the RSV neutralizing activity in serum is only directed against the prefusion F protein antigenic site. Inducing high titer neutralizing antibodies is the main goal of developing RSV vaccines, and the prefusion F protein with specific antigenic epitopes has become the most popular RSV vaccine target.
  • an immune composition e.g., mRNA vaccine
  • mRNA vaccine which contains RNA encoding a highly immunogenic antigen capable of inducing an effective neutralizing antibody response against respiratory syncytial virus.
  • the inventors of the present application designed antigens using the surface F protein of RSV virus as a target, and provided mRNA capable of efficiently expressing antigenic proteins, and used lipid nanoparticle (LNP) technology to encapsulate mRNA and deliver it to the body to release mRNA and translate it into F protein, thereby inducing humoral immunity and cellular immunity.
  • LNP lipid nanoparticle
  • the present application provides the following optional implementation scheme:
  • the present application provides a polynucleotide (e.g., mRNA) encoding at least one antigenic peptide or protein derived from respiratory syncytial virus (RSV) or an immunogenic fragment or immunogenic variant thereof, wherein the nucleotide comprises at least one open reading frame connected to a heterologous untranslated region (UTR), and the nucleotide has a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence of any one of SEQ ID NO.1-3.
  • the polynucleotide is suitable for producing a vaccine.
  • the at least one antigenic peptide or protein comprises or consists of at least one protein derived from a structural protein, an accessory protein, or a replication protein, or a fragment or immunogenic variant of any of the foregoing.
  • the structural protein is or is derived from glycoprotein (G), fusion protein (F), phosphoprotein (P), nucleocapsid protein (N, L), non-structural protein (NS1, NS2), matrix protein (M1, M2) and small hydrophobic protein.
  • G glycoprotein
  • F fusion protein
  • P phosphoprotein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocapsid protein
  • N nucleocap
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding a fusion (F) protein and/or an RSV RNA polynucleotide having an open reading frame encoding an attachment protein (G).
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding the F protein.
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding the N protein.
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding the M protein.
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding the L protein.
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding the P protein.
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding an SH protein.
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding NS1 protein.
  • the RSV RNA vaccine comprises an RSV RNA polynucleotide having an open reading frame encoding an NS2 protein.
  • the fusion protein (F) is selected from F1 protein or its immune fragment and variants thereof, F2 protein or its immune fragment and variants thereof.
  • the polynucleotide of the present application may contain or consist of a nucleotide sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO.1, 2 and 3.
  • the respiratory syncytial virus antigen encoded by the polynucleotide of the present application includes at least one or at least two mutations selected from the following relative to SEQ ID NO: 13: S155C, S190F, V207L, S290C, A149C, L373R, I379V, M447V and Y458C, and the coding region includes a nucleotide sequence optimized for G/C content.
  • S155C means that the 155th position relative to the reference sequence SEQ ID NO: 13 is mutated from S to C.
  • the heterologous untranslated region includes at least one heterologous 3'UTR and/or 5'UTR.
  • the heterologous 3'UTR can be selected from the following 3'UTRs used in the translation process of one or more proteins: PSMB3, ALB7, ⁇ -globin, CASP1, COX6B1, GNAS, NDUFA1, DH143, gp130, hHBB, hHBA1, CYBA (cytochrome b-245 alpha chain), rabbit ⁇ -globin, hepatitis B virus (HBV), VEEV (Venezuelan equine encephalitis virus) virus, rps9 (Ribosomal Protein S9), FIG4 (FIG4 Phosphoinositide 5-Phosphatase), human albumin hHBB (human hemoglobin subunit beta) and HBA1 (human Hemoglobin Subunit Alpha 1), or homologs, fragments or variants derived from these 3'UTRs.
  • the heterologous 3'UTR when the heterologous 3'UTR is selected from 3'UTRs used by multiple proteins in the translation process, the heterologous 3'UTR comprises a polynucleotide sequence formed by connecting 0, 1 or more nucleotides to the 3'UTRs used by the multiple proteins in the translation process.
  • the heterologous 3'UTR is as shown in SEQ ID NO: 9.
  • the heterologous 5'UTR is derived from the 5'UTR of Xenopus laevis or human ⁇ -globin or ⁇ -globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, tobacco etch virus, alpha-1-globin, HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and/or UBQLN2, or a homolog, fragment or variant of any of these 5'UTRs.
  • the heterologous 5'UTR is as shown in SEQ ID NO: 6.
  • the heterologous 5'UTR is as shown in SEQ ID NO: 7.
  • the nucleotide of any of the foregoing, wherein the nucleotide comprises at least one 3'-poly(A) sequence preferably comprises 30 to 200 adenosine nucleotides and/or at least one 3'-poly(A) sequence.
  • the 3'-poly(A) sequence is as shown in SEQ ID NO: 8.
  • the mRNA further comprises a 5' guanosine cap selected from the group consisting of: m7Gppp (2'OMeA) pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7GpppUpU, m7Gpppm6ApG, m7G 3'Ome pppApA, m7G 3'Ome pppApC, m7G 3'Ome
  • the polynucleotide comprises at least the following structure:
  • a 5'-cap structure wherein the 5'-cap structure is preferably any one of the following: m7G, cap0, cap1, cap2, a modified cap0 or a modified cap1 structure, preferably a cap1 structure, and most preferably m7G(5')ppp(5')(2'OMeA)pG;
  • (c) 5'UTR with a length of 10-200 nucleotides, preferably 15-150 nucleotides, preferably comprising alpha-1-globin 5'UTR, KOZAK sequence, pVAX and/or TEV (tobacco etch virus) sequence, further preferably as shown in SEQ ID NO:6;
  • (d) 3’UTR preferably including human gp130, DH143, hHBB and/or hHBA1 3’UTR sequences, further preferably as shown in SEQ ID NO: 9.
  • the present application relates to an mRNA encoding a respiratory syncytial virus antigen comprising amino acids that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence of SEQ ID NO. 4.
  • the 5'UTR is a TEV sequence or a KOZAK sequence.
  • its KOZAK sequence is shown as SEQ ID NO.5.
  • its pVAX.1+TEV is as shown in SEQ ID NO.6.
  • its pVAX.1+alpha-1-globin is shown as SEQ ID NO.7.
  • the 3’ poly A sequence is preferably a nucleotide sequence as shown in SEQ ID NO.8.
  • the 3’UTR preferably has a hemoglobin alpha-1 (h HBA1) sequence, as shown in SEQ ID NO.9.
  • the 3’UTR is preferably composed of the sequences of gp130 and DH143 connected in series from 5’ to 3’, the gp130 is shown as SEQ ID NO.10, and the DH143 is shown as SEQ ID NO.11.
  • the aforementioned polynucleotide is DNA or RNA.
  • the aforementioned polynucleotides encode RNA.
  • the coding RNA is mRNA, self-replicating RNA, circular RNA or replicon RNA, preferably mRNA.
  • the mRNA contains chemically modified bases or analogs, including 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), and pseudouridine; preferably, 1-methyl pseudouridine partially replaces uridine; and most preferably, 1-methyl pseudouridine completely replaces uracil, so that each uracil in the sequence is replaced by 1-methyl pseudouridine.
  • chemically modified bases or analogs including 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), and pseudouridine; preferably, 1-methyl pseudouridine partially replaces uridine; and most preferably, 1-methyl pseudouridine completely replaces uracil
  • the present invention relates to an mRNA, the fusion protein (F) encoded by which may comprise or consist of an amino acid sequence having at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity with SEQ ID NO.4.
  • the present application provides a composition, preferably an immunogenic composition comprising at least one polynucleotide of the first aspect.
  • the composition may comprise at least one polynucleotide, such as at least one encoding RNA, the polynucleotide being complexed with one or more lipids and encapsulated in a or a plurality of lipids or associated with one or more lipids to form lipid nanoparticles.
  • at least one polynucleotide such as at least one encoding RNA
  • the polynucleotide being complexed with one or more lipids and encapsulated in a or a plurality of lipids or associated with one or more lipids to form lipid nanoparticles.
  • the composition relates to a nucleic acid vaccine against respiratory syncytial virus RSV, wherein the vaccine carrier is a lipid nanoparticle (LNP), comprising ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants, and the molar content of ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants totals 100% in terms of molar percentage (mol%).
  • LNP lipid nanoparticle
  • the lipid nanoparticles comprise 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% helper lipids, and 0.5-15 mol% surfactant.
  • the cationic lipid is selected from SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, C12-200, DlinDMA, preferably SM-102; the structure of SM-102 is as follows:
  • the structured lipid is selected from cholesterol, and cholesterol derivatives, preferably cholesterol;
  • the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC;
  • the surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.
  • the lipid nanoparticles comprise 20-50 mol% ionizable cationic lipids.
  • the lipid nanoparticles may comprise 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mol% ionizable cationic lipids.
  • the lipid nanoparticles comprise 50-60 mol% ionizable cationic lipids.
  • the lipid nanoparticles may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mol% ionizable cationic lipids.
  • the lipid nanoparticle comprises 5-25 mol% DSPC, preferably 2-15 mol% DSPC; for example, the lipid nanoparticle may comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 mol% DSPC.
  • the lipid nanoparticles comprise 25-55 mol% cholesterol, preferably 30-40 mol% cholesterol.
  • the lipid nanoparticles may comprise 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 mol% cholesterol.
  • the lipid nanoparticles comprise 0.5-15 mol% DMG-PEG, preferably 1-2 mol% DMG-PEG.
  • the lipid nanoparticles may comprise 1, 1.5 or 2 mol% DMG-PEG.
  • the lipid nanoparticles comprise 50 mol% ionizable cationic lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.
  • the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.
  • the lipid nanoparticles of the present application comprise an N:P ratio of about 2:1 to about 30:1.
  • the lipid nanoparticles of the present application comprise an N:P ratio of about 6:1.
  • the lipid nanoparticles of the present application comprise an N:P ratio of about 3:1.
  • the lipid nanoparticles of the present application comprise a wt/wt ratio of the ionizable cationic lipid component to the RNA of about 10:1 to about 100:1.
  • the lipid nanoparticles of the present application comprise a wt/wt ratio of ionizable cationic lipid component to RNA of about 20:1.
  • the lipid nanoparticles of the present application comprise a wt/wt ratio of an ionizable cationic lipid component to RNA of about 10: 1. In some embodiments, the lipid nanoparticles of the present application have an average diameter of from about 50 nm to about 150 nm.
  • the lipid nanoparticles of the present application have an average diameter of about 70 nm to about 120 nm, preferably 100-120 nm, and most preferably 100 nm.
  • the mRNA solution is diluted with water for injection.
  • the mRNA solution is dissolved in a buffer to obtain an aqueous phase, each lipid component of the liposome nanoparticle is measured and dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed and purified to obtain a nucleic acid immune composition.
  • the mass ratio of the lipid nanoparticle to the mRNA is between 1: 1 and 30: 1.
  • the lipid carrier is preferably a LNP composition, and the mass ratio is preferably 20:1.
  • the respiratory syncytial virus nucleic acid vaccine of the present application further comprises: a buffer component and a cryoprotectant;
  • the buffer can be selected from: Examples of buffers include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, calcium hydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, dibasic potassium phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, magnesium hydroxide, aluminum hydroxide, alginic acid, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride,
  • the cryoprotectant can be selected from substances such as sugars/polyols, polymers, surfactants, amino acids and salts, wherein the sugar can be selected from: lactose, sucrose, trehalose, galactose and the like.
  • the amount of the cryoprotectant is 1 to 50% w/w, such as from 2 to 50% w/w, or from 4 to 45% w/w, or from 6 to 12% w/w, or preferably from 6 to 10% w/w, or most preferably from 7 to 9% w/w.
  • the pharmaceutical composition of the present application includes the aforementioned lipid nanoparticle composition and an aqueous phase (also referred to as “aqueous phase” or “external aqueous phase” in the present application) buffer.
  • aqueous phase also referred to as “aqueous phase” or “external aqueous phase” in the present application
  • the content of tromethamine is selected from 10-30mmol/L, preferably 15-25mmol/L, preferably 15mmol/L, 15.5mmol/L, 16mmol/L, 16.5mmol/L, 17mmol/L, 17.5mmol/L, 18mmol/L, 18.5mmol/L, 19mmol/L, 19.5mmol/L, 20mmol/L, 20.5mmol/L, 21mmol/L, 21.5mmol/L, 22mmol/L, 22.5mmol/L, 23mmol/L, 23.5mmol/L, 24mmol/L, 24.5mmol/L, 25mmol/L, and most preferably 20mmol/L.
  • the content of sodium acetate is selected from 0-20mmol/L, preferably 5-11mmol/L, preferably 5mmol/L, 5.5mmol/L, 6mmol/L, 6.5mmol/L, 7mmol/L, 7.5mmol/L, 8mmol/L, 8.5mmol/L, 9mmol/L, 9.5mmol/L, 10mmol/L, 10.5mmol/L, 10.6mmol/L, 10.7mmol/L, 10.8mmol/L, 10.9mmol/L, 11mmol/L, 11.5mmol/L, 12mmol/L, 12.5mmol/L, 13mmol/L, and most preferably 10.7mmol/L.
  • the sucrose content (w/v, concentration) is selected from: 5-15%, preferably 7.5-10%, more preferably 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 9%, 9.5%, 10%, and most preferably 8.7%.
  • the present application provides a respiratory syncytial virus vaccine, preferably comprising an antigenic polypeptide of RSV.
  • it comprises or consists of an amino acid sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.4.
  • the present application provides a respiratory syncytial virus vaccine, wherein the vaccine comprises at least one polynucleotide of the first aspect, or the composition of the second aspect, or at least one polypeptide of the third aspect.
  • the vaccine is administered by intravenous injection, intramuscular injection or subcutaneous injection, preferably intramuscular injection;
  • the vaccine may be in a dosage form selected from a freeze-dried powder injection, a liquid injection formulation, and an inhalation formulation;
  • the present application provides a kit or a kit of parts comprising at least one nucleotide of the first aspect, and/or at least one composition of the second aspect, and/or at least one polypeptide of the third aspect, and/or at least one vaccine of the fourth aspect.
  • the present application provides a pharmaceutical composition comprising at least two separated components, wherein the at least two separated components are selected from the two nucleotides of the first aspect, and/or at least two compositions of the second aspect, and/or at least two polypeptides of the third aspect, and/or at least two vaccines of the fourth aspect.
  • the present invention provides a method for treating or preventing respiratory syncytial virus infection in a subject, comprising administering to the subject At least one polynucleotide of the first aspect, and/or at least one composition of the second aspect, and/or at least one antigenic polypeptide of the third aspect, and/or at least one vaccine of the fourth aspect can effectively induce a neutralizing antibody response against respiratory syncytial virus (RSV) in the subject.
  • RSV respiratory syncytial virus
  • the present application relates to a method for preparing a respiratory syncytial virus vaccine, comprising mixing a vaccine vector with the mRNA described in the first aspect to obtain a respiratory syncytial virus vaccine.
  • the vaccine carrier is a lipid nanoparticle
  • the preparation method specifically comprises the following steps:
  • step (3) mixing the organic phase of step (1) and the aqueous phase of step (2) to generate a mixed solution to obtain a respiratory syncytial virus vaccine;
  • the organic solution comprises anhydrous ethanol
  • the total concentration of ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants in the organic phase is 10-15 mg/ml;
  • the concentration of the mRNA is 0.01-1 mg/ml, preferably 0.1-0.2 mg/ml;
  • the volume ratio of the organic phase to the aqueous phase is 1:2-4;
  • the mixing is performed using a microfluidic device, and the flow rate is controlled to be ⁇ 12 ml/min.
  • the present application also relates to the use of the polynucleotide of the first aspect, and/or the composition of the second aspect, and/or the antigenic polypeptide of the third aspect, and/or the vaccine of the fourth aspect in preparing a vaccine.
  • the vaccines include multi-combination vaccines and multivalent vaccines.
  • the present application also designs a combination vaccine, comprising a first vaccine and a second vaccine used simultaneously or sequentially, wherein the first vaccine is selected from the polynucleotide of the first aspect, and/or the composition of the second aspect, and/or the pharmaceutical composition of the sixth aspect.
  • the second vaccine is selected from: attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, and recombinant protein vaccines.
  • the second vaccine is selected from: Moderna (mRNA-1345), Advaccine (ADV110), MVA-BN-RSV (Bavarian Nordic), Arexvy (GSK), ABRYSVO (Pfizer), Ad26.RSV.preF, MEDI7510.
  • the mRNA of the first vaccine and the second vaccine is selected from the group consisting of the nucleotide sequences shown in SEQ ID NO:1-3.
  • the vaccine described in the present application is suitable for sequential vaccination with one or more vaccines selected from the following groups, and the vaccine can be based on any technical route, including but not limited to attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccine, DNA vaccine, recombinant protein vaccine, etc.
  • the one or more vaccines are selected from: Moderna (mRNA-1345), Advaccine (ADV110), MVA-BN-RSV (Bavarian Nordic), Arexvy (GSK), ABRYSVO (Pfizer), Ad26.RSV.preF, MEDI7510.
  • the mRNA of one or more vaccines can be mixed into one LNP for delivery, or the above mRNA can be mixed into different LNPs, and then the LNPs are mixed in a certain ratio and administered.
  • the number of vaccinations of the one or more vaccines required to complete immunization can be 1 time, 2 times, 3 times or 4 times, and the interval between each vaccination can be 0 days, 7 days, 21 days, 28 days, 35 days, 2 months, 3 months, 4 months, 5 months, or 6 months.
  • the present application also relates to a method for inducing an antigen-specific immune response in a subject, comprising administering to the subject an amount of the respiratory syncytial virus vaccine effective to produce an antigen-specific immune response.
  • the antigen-specific immune response comprises a T cell response.
  • the antigen-specific immune response comprises a B cell response.
  • the subject is about 5 years old or younger, wherein the subject is between about 1 year old and about 5 years old, wherein the subject is between about 6 months old and about 1 year old, wherein the subject is about 6 months old or younger, or wherein the subject is about 12 months old or younger.
  • the subject is an elderly subject of about 60 years old, about 70 years old or older.
  • the subject is an adult between about 20 and about 50 years old.
  • the subject is a full-term or premature infant.
  • the subject is a pregnant woman.
  • An mRNA comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the nucleotide sequence shown in any one of SEQ ID NO.1-3.
  • the mRNA comprises a nucleotide sequence that is 100% identical to the nucleotide sequence shown in SEQ ID NO.1. In some embodiments, the mRNA has 100% identity to the nucleotide sequence shown in SEQ ID NO.1. In some embodiments, the mRNA comprises a nucleotide sequence that is 100% identical to the nucleotide sequence shown in SEQ ID NO.1, and wherein all uridines are 1-methylpseudouridine. In some embodiments, the mRNA has 100% identity to the nucleotide sequence shown in SEQ ID NO.1, and wherein all uridines are 1-methylpseudouridine.
  • An mRNA encoding a respiratory syncytial virus antigen comprising amino acids that are at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to the amino acid sequence shown in SEQ ID NO. 4.
  • the sequence encoding the respiratory syncytial virus antigen in the mRNA is codon-optimized according to human codon preference.
  • mRNA according to any one of items 1 to 2, wherein the mRNA comprises a 5' untranslated region (UTR), a 3'UTR and a 3'-poly(A).
  • UTR 5' untranslated region
  • 3'UTR 3'UTR
  • 3'-poly(A) 3'-poly(A)
  • the 5'UTR includes the nucleotide sequence shown in SEQ ID NO.6 or SEQ ID NO.7 and/or the 3'UTR is selected from the nucleotide sequence shown in SEQ ID NO.9-11.
  • m7G3’OmepppG2’OmepA m7G3’OmepppG2’OmepU
  • m7G3’OmepppG2’OmepG m7G3’OmepppG2’OmepC
  • m7G3’OmeppU2 'OmepA preferably m7G(5')ppp(5')(2'OMeA)pG.
  • the respiratory syncytial virus antigen encoded by the mRNA comprises amino acids that are 100% identical to the amino acid sequence shown in SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA further comprises a 5'UTR, a 3'UTR and a 3'-polyadenylic acid, the 5'UTR comprising the 5'UTR of TEV, and the 3'UTR comprising the 3'UTR of hemoglobin alpha-1 (h HBA1).
  • the respiratory syncytial virus antigen encoded by the mRNA comprises amino acids that are 100% identical to the amino acid sequence of SEQ ID NO.4, and the sequence encoding the respiratory syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA utilizes the 5'UTR of TEV and the 3'UTR of hHBA1 to regulate the transcription of the viral antigen.
  • the respiratory syncytial virus antigen encoded by the mRNA has 100% identity with the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA further comprises a 5'UTR, a 3'UTR and a 3'-polyadenylic acid, wherein the 5'UTR comprises the 5'UTR of TEV, the 3'UTR comprises the 3'UTR of hemoglobin alpha-1 (h HBA1), and the 3'-polyadenylic acid
  • the number of adenosines in the adenylate is 100.
  • the respiratory syncytial virus antigen encoded by the mRNA has 100% identity with the amino acid sequence of SEQ ID NO.4, and the sequence encoding the respiratory syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA utilizes the 5'UTR of TEV, the 3'UTR of hHBA1, and a 3'-polyadenylate with 100 adenosines to regulate the transcription of the respiratory syncytial virus antigen.
  • the respiratory syncytial virus antigen encoded by the mRNA contains amino acids that are 100% identical to the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized based on human codon preference.
  • the mRNA further contains a 5'UTR, a 3'UTR and a 3'-poly(A), the 5'UTR contains the 5'UTR of TEV, the 3'UTR contains the 3'UTR of hemoglobin alpha-1 (h HBA1), and the number of adenosines in the 3'-poly(A) is 100.
  • the respiratory syncytial virus antigen encoded by the mRNA contains amino acids that are 100% identical to the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized based on human codon preference.
  • the mRNA utilizes the 5'UTR of TEV, the 3'UTR of hHBA1, and a 3'-polyadenylation with 100 adenosines to regulate the transcription of the syncytial virus antigen.
  • the respiratory syncytial virus antigen encoded by the mRNA has 100% identity with the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA further comprises a 5'UTR, a 3'UTR and a 3'-polyadenylic acid, wherein the 5'UTR comprises the 5'UTR of TEV, the 3'UTR comprises the 3'UTR of hemoglobin alpha-1 (h HBA1), and the number of adenosines in the 3'-polyadenylic acid is 100.
  • the 5'UTR comprises the 5'UTR of TEV
  • the 3'UTR comprises the 3'UTR of hemoglobin alpha-1 (h HBA1)
  • the number of adenosines in the 3'-polyadenylic acid is 100.
  • the respiratory syncytial virus antigen encoded by the mRNA has 100% identity with the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA utilizes the 5'UTR of TEV, the 3'UTR of hHBA1, and the 3'-polyadenylic acid with 100 adenosines to regulate the transcription of the viral antigen.
  • the respiratory syncytial virus antigen encoded by the mRNA comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized based on human codon preference, and the mRNA further comprises a 5'UTR, a 3'UTR and a 3'-poly(A), the 5'UTR comprises a polynucleotide sequence as shown in SEQ ID NO: 6, the 3'UTR comprises a polynucleotide sequence as shown in SEQ ID NO: 9, and the 3'-poly(A) comprises a polynucleotide sequence as shown in SEQ ID NO: 8.
  • the respiratory syncytial virus antigen encoded by the mRNA comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized based on human codon preference.
  • the mRNA utilizes a 5’UTR as shown in SEQ ID NO: 6, a 3’UTR as shown in SEQ ID NO: 9, and a 3’-polyadenylation as shown in SEQ ID NO: 8 to regulate the transcription of the syncytial virus antigen.
  • the respiratory syncytial virus antigen encoded by the mRNA has 100% identity with the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA further comprises a 5'UTR, a 3'UTR and a 3'-polyadenylate, wherein the 5'UTR
  • the mRNA comprises a polynucleotide sequence as shown in SEQ ID NO: 6, the 3'UTR comprises a polynucleotide sequence as shown in SEQ ID NO: 9, and the 3'-poly A comprises a polynucleotide sequence as shown in SEQ ID NO: 8.
  • the respiratory syncytial virus antigen encoded by the mRNA has 100% identity with the amino acid sequence of SEQ ID NO. 4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized according to human codon preference, and the mRNA utilizes the 5'UTR as shown in SEQ ID NO: 6, the 3'UTR as shown in SEQ ID NO: 9, and the 3'-poly A as shown in SEQ ID NO: 8 to regulate the transcription of the respiratory syncytial virus antigen.
  • the respiratory syncytial virus antigen encoded by the mRNA has 100% identity with the amino acid sequence of SEQ ID NO.4, and the sequence encoding the syncytial virus antigen in the mRNA is codon-optimized according to human codon preference.
  • the mRNA further comprises 5'UTR, 3'UTR and 3'-poly(A), the polynucleotide sequence of the 5'UTR is as shown in SEQ ID NO: 6, the polynucleotide sequence of the 3'UTR is as shown in SEQ ID NO: 9, and the polynucleotide sequence of the 3'-poly(A) is as shown in SEQ ID NO: 8.
  • composition comprising lipid nanoparticles and messenger RNA (mRNA), wherein the mRNA is the mRNA described in any one of items 1-7.
  • mRNA messenger RNA
  • composition described in item 8 comprises ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants.
  • auxiliary lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC.
  • composition as described in any one of items 8-14, wherein the lipid nanoparticles contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.
  • the composition comprises lipid nanoparticles and messenger RNA (mRNA), the mRNA is the mRNA described in any one of items 1-7, and the lipid nanoparticles are composed of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.
  • mRNA messenger RNA
  • the composition comprises lipid nanoparticles and messenger RNA (mRNA), the mRNA is the mRNA described in any one of items 1-7, wherein all the uridine in the mRNA is 1-methylpseudouridine, and the lipid nanoparticles are composed of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.
  • mRNA messenger RNA
  • the composition comprises lipid nanoparticles and messenger RNA (mRNA), the mRNA comprises the polynucleotide sequence shown in SEQ ID NO.1, and the lipid nanoparticles are composed of 50mol% SM-102, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% DMG-PEG.
  • mRNA messenger RNA
  • the composition comprises lipid nanoparticles and messenger RNA (mRNA), the mRNA comprising a polynucleotide sequence as shown in SEQ ID NO.1, wherein the uridine in the mRNA is 1-methylpseudouridine, and the lipid nanoparticles are composed of 50mol% SM-102, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% DMG-PEG.
  • mRNA messenger RNA
  • the composition comprises lipid nanoparticles and messenger RNA (mRNA), the polynucleotide sequence of the mRNA is as shown in SEQ ID NO.1, and the lipid nanoparticles are composed of 50mol% SM-102, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% DMG-PEG.
  • mRNA messenger RNA
  • the composition comprises lipid nanoparticles and messenger RNA (mRNA), the polynucleotide sequence of the mRNA is shown in SEQ ID NO.1, wherein the uridine in the mRNA is 1-methylpseudouridine, and the lipid nanoparticles are composed of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG.
  • mRNA messenger RNA
  • a method comprising administering to a subject the composition described in items 8-16, which can effectively induce a neutralizing antibody response against respiratory syncytial virus in the subject.
  • a pharmaceutical composition comprising the composition described in items 8-16, Tris buffer, sodium acetate and 8.7% sucrose, pH 7.4.
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase
  • mRNA messenger RNA
  • the mRNA comprises a polynucleotide sequence as shown in SEQ ID NO.1
  • the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG
  • the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose
  • the pH is 7.4.
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase
  • mRNA comprises a polynucleotide sequence as shown in SEQ ID NO.1
  • the uridine in SEQ ID NO: 1 is 1-methylpseudouridine
  • the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG
  • the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose
  • the pH is 7.4.
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase, wherein the polynucleotide sequence of the mRNA is as shown in SEQ ID NO.1, and the lipid nanoparticles comprise 50 mol% SM-102, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% DMG-PEG, the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose, and the pH is 7.4.
  • mRNA messenger RNA
  • aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase
  • the polynucleotide sequence of the mRNA is shown in SEQ ID NO.1, wherein the uridine in the mRNA is 1-methyl pseudouridine, the lipid nanoparticles comprise 50mol% SM-102, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% DMG-PEG, the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose, and the pH is 7.4.
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase
  • the polynucleotide sequence of the mRNA is as shown in SEQ ID NO.1
  • the lipid nanoparticles are composed of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG
  • the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose
  • the pH is 7.4.
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase
  • the polynucleotide sequence of the mRNA is as shown in SEQ ID NO.1
  • the lipid nanoparticles are composed of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% DMG-PEG
  • the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose
  • the pH is 7.4.
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase
  • the polynucleotide sequence of the mRNA is shown in SEQ ID NO.1, wherein the uridine in the mRNA is 1-methylpseudouridine, the lipid nanoparticles are composed of 50mol% SM-102, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% DMG-PEG, the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose, and the pH is 7.4.
  • the composition comprises lipid nanoparticles, messenger RNA (mRNA) and an aqueous phase
  • the polynucleotide sequence of the mRNA is shown in SEQ ID NO.1, wherein the uridine in the mRNA is 1-methylpseudouridine, the lipid nanoparticles are composed of 50mol% SM-102, 10mol% DSPC, 38.5mol% cholesterol and 1.5mol% DMG-PEG, the aqueous phase comprises Tris buffer, sodium acetate and 8.7% sucrose, and the pH is 7.4.
  • a combination vaccine comprising a first vaccine and a second vaccine used sequentially, wherein the first vaccine is selected from the mRNA described in items 1-7, the composition described in items 8-16 and the pharmaceutical composition of item 21.
  • the second vaccine is selected from: attenuated or inactivated vaccines, adenovirus vaccines, mRNA vaccines, DNA vaccines, and recombinant protein vaccines.
  • the present application also provides a method for preventing or treating respiratory syncytial virus infection, the method comprising administering any of the foregoing compositions or mRNA to a subject.
  • the present application also provides a method for preventing or treating lung infection caused by respiratory syncytial virus, the method comprising administering any of the foregoing compositions or mRNA to a subject.
  • the present application also provides a method for preventing lower respiratory symptoms caused by respiratory syncytial virus infection, the lower respiratory symptoms can be selected from any one or more of the following: perivascular infiltration, peribronchial infiltration, alveolar infiltration and interstitial infiltration.
  • the present application also provides an mRNA vaccine comprising any of the foregoing mRNA.
  • the present application also provides an mRNA vaccine comprising or consisting of any of the foregoing compositions.
  • F protein can form a homotrimeric spike structure on the RSV shell. This protein plays a decisive role in mediating the entry of the virus into cells and is the best antigen choice.
  • this application has modified the preferred RSV/A2 strain F protein, and optimized its coding sequence and expression regulatory elements, so that the antigen expression efficiency in vivo is higher and can induce better immune effects.
  • Figure 1 Relative mRNA expression results after codon optimization.
  • Figure 2 Bioanalyzer analysis of the purity and size of F protein mRNA.
  • Figure 4 Transmission electron microscopy shows the morphology of RSV F protein mRNA and lipid nanoparticle (LNP) complex.
  • FIG. 5 Immunoblotting results of the expression of F protein in host cells.
  • Figure 6 FACS flow cytometry detected the expression of target antigen protein in cells.
  • Figure 7 Results of the test on binding antibody and neutralizing antibody in mouse serum after secondary immunization with RSV-1 mRNA.
  • Figure 8 RSV challenge test results. One-way ANOVA was used for analysis. ***p ⁇ 0.001.
  • FIG. 10 RSV pre-F protein-specific IgG binding antibody titer results in mouse serum.
  • Figure 11 Virus titer in mouse lung tissue.
  • One-way ANOVA was used for analysis. Compared with the model group, ****p ⁇ 0.0001.
  • Figure 12 Pathological scoring results of mouse lung tissue; One-way ANOVA was used for analysis. Compared with the model group, *p ⁇ 0.05, **p ⁇ 0.01; compared with the FI-RSV group, ##p ⁇ 0.01, ###p ⁇ 0.001, ####p ⁇ 0.0001.
  • FIG. 13 RSV pre-F protein-specific IgG binding antibody titer results in cotton rat serum.
  • Figure 14 Results of virus titer in cotton rat nasal turbinates. One-way ANOVA was used for analysis. Compared with the model group, ****p ⁇ 0.0001.
  • polynucleotides encoding the protein or immunogenic fragment of the present application include all polynucleotide sequences that are degenerate from one another and encode the same amino acid sequence.
  • the "uridine” of the present application covers natural uridine and its derivatives, including but not limited to: 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), pseudouridine, 1-ethyl-pseudouridine, and 5-methoxy-uridine.
  • all or part of the nucleic acid of the present application can be replaced with a modified base, such as 1-methyl pseudouridine or pseudouridine.
  • N:P ratio used herein, also referred to as “N/P” or “N:P” in this application, represents the molar ratio of the protonable nitrogen element of the ionizable cationic lipid to the phosphate group of the mRNA.
  • the N:P ratio describes the ratio between the cationic charge of the amino group (N+) in the ionizable cationic lipid and the anionic charge of the phosphate group (PO4-) in the nucleic acid backbone, and is the basis for the complexation of the ionizable cationic lipid and the nucleic acid through electrostatic interaction.
  • the N:P ratio is a key formulation factor for LNP, affecting the physicochemical properties of LNP and the in vivo release of the drug.
  • Antigens used herein are proteins that can induce an immune response (e.g., cause the immune system to produce antibodies against an antigen).
  • the use of the term “antigen” includes immunogenic proteins and immunogenic fragments (immunogenic fragments that induce or can induce an immune response to at least one respiratory syncytial virus).
  • protein includes peptides and the term “antigen” includes antigenic fragments.
  • Other molecules may also be antigenic, such as bacterial polysaccharides or a combination of protein and polysaccharide structures, and the viral vaccine antigens described herein include viral proteins, viral protein fragments, and proteins designed and/or mutated from respiratory syncytial virus.
  • an antigen as used herein will be recognized and understood by those of ordinary skill in the art to mean a substance that can be recognized by the immune system, preferably by the adaptive immune system, and is capable of triggering an antigen-specific immune response, for example, by forming antibodies and/or antigen-specific T cells as part of an adaptive immune response.
  • An antigen can be or can include Peptides or proteins that can be presented to T cells by MHC. Also included are fragments, variants and derivatives derived from, for example, peptides or proteins.
  • Respiratory syncytial virus fusion protein (F) comprising at least one epitope.
  • protein fragments, functional protein domains and homologous proteins are also considered to be within the scope of respiratory syncytial virus antigens of interest.
  • T cell antigen epitopes refer to antigen epitopes recognized by T cell receptors (T-cell receptor/TCR).
  • Epitope components are polypeptides after protein degradation, mostly present inside antigen molecules, and need to be processed by antigen presenting cells (antigen presenting cells/APC) and combined with MHC molecules to form a complex before they can be recognized by TCR. It can usually contain fragments preferably having a length of about 6 to about 20 or more amino acids, for example.
  • Fragments processed and presented by MHC class I molecules preferably have a length of about 8 to about 10 amino acids, for example: 8, 9 or 10 (or 11 or 12 amino acids) or fragments processed and presented by MHC class II molecules preferably have a length of about 13 to about 20 or more amino acids.
  • These fragments are usually recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, i.e., these fragments are usually not recognized in their native form.
  • B cell epitopes are usually fragments located on the outer surface of a (native) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably 5 to 12 amino acids, and even more preferably 6 to 9 amino acids, which can be recognized by antibodies, i.e., recognized in their native form.
  • a (native) protein or peptide antigen preferably having 5 to 15 amino acids, more preferably 5 to 12 amino acids, and even more preferably 6 to 9 amino acids, which can be recognized by antibodies, i.e., recognized in their native form.
  • Such epitopes of proteins or peptides can also be selected from any variants of such proteins or peptides mentioned herein.
  • an epitope can be a conformational or discontinuous epitope, which consists of fragments of a protein or peptide defined herein, which are discontinuous in the amino acid sequence of a protein or peptide defined herein, but are aggregated together in a three-dimensional structure or are continuous or linear epitopes consisting of a single polypeptide chain.
  • nucleic acid or “nucleic acid molecule” will be recognized and understood by those of ordinary skill in the art.
  • the term “nucleic acid” or “nucleic acid molecule” preferably refers to DNA (molecule) or RNA (molecule). It is preferably used synonymously with the term polynucleotide.
  • a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other via phosphodiester bonds of a sugar/phosphate backbone.
  • nucleic acid molecule also includes modified nucleic acid molecules, such as base-modified, sugar-modified or backbone-modified DNA or RNA molecules as defined herein.
  • composition of the present application comprises (at least one) RNA having an open reading frame (ORF) encoding a respiratory syncytial virus antigen (e.g., F protein).
  • ORF open reading frame
  • the RNA is a messenger RNA (mRNA).
  • the nucleic acid comprises at least one heterologous untranslated region (UTR).
  • UTR or “UTR element” will be recognized and understood by those of ordinary skill in the art to mean a portion of a nucleic acid molecule, usually located 5' or 3' to a coding sequence. The 5' end is referred to as a 5'UTR, and the 3' end is referred to as a 3'UTR.
  • UTRs are not translated into proteins; UTRs can be part of a nucleic acid, such as DNA or RNA. UTRs can contain elements for controlling gene expression, also referred to as regulatory elements.
  • RNA e.g., mRNA
  • RNA can further contain a 5'UTR, a 3'UTR, a 3'-poly A and/or a 5' cap analog.
  • the 5'UTR is a heterologous UTR, i.e., a UTR found in nature and associated with a different ORF; in another embodiment, the 5'UTR is a synthetic UTR; the 5'UTR is a region of the mRNA that is located upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5'UTR does not encode a protein.
  • the natural 5'UTR has characteristics that play a role in translation initiation, and it has features such as the Kozak sequence, which has a common CCR(A/G)CCAUGG; exemplary 5'UTRs also include African clawed frog or human ⁇ -globin or ⁇ -globin, human cytochrome (human cytochrome) b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, and tobacco etch virus (Tobacco etch virus), alpha-1-globin (alpha-1-globin) 5'UTR, etc.
  • the 3'UTR can be heterologous or synthetic; for example: globin UTR, including African clawed frog ⁇ -globin UTR and human ⁇ -globin UTR; other 3'UTRs can also be CYBA (cytochrome b-245 alpha chain), rabbit ⁇ -globin, hepatitis B virus (HBV), ⁇ -globin 3'UTR and VEEV (Venezuelan equine encephalitis virus) virus 3'UTR sequences.
  • CYBA cytochrome b-245 alpha chain
  • rabbit ⁇ -globin hepatitis B virus
  • VEEV Venezuelan equine encephalitis virus
  • rps9 Ribosomal Protein S9 3'UTR
  • FIG4 FIG4 Phosphoinositide 5-Phosphatase
  • gp130 human hemoglobin subunit beta
  • HBA1 human Hemoglobin Subunit Alpha 1
  • the 3'-polyadenylic acid also known as the poly A tail; the poly (A) tail is located downstream of the 3'UTR, for example, the mRNA region directly downstream (i.e., 3'), which contains multiple consecutive adenosine monophosphates.
  • the poly (A) tail may contain 10 to 300 adenosine monophosphates, and may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates.
  • the poly (A) tail contains 50 to 250 adenosine monophosphates, more preferably 50-100 adenosine monophosphates; most preferably 100 adenosine monophosphates; in relevant biological environments (e.g., in cells, in vivo), the function of the 3'-poly (A) tail is to protect the mRNA from enzymatic degradation, such as in the cytoplasm, and to facilitate transcription termination and/or export of the mRNA from the nucleus and translation.
  • the RNA further comprises a 5' guanosine cap;
  • the 5' guanosine cap is a eukaryotic mRNA transcript, the 5' cap is composed of an inverted 7-methylguanosine, connected to the rest of the eukaryotic mRNA via a 5'-5' triphosphate bridge, the so-called cap 0 (cap0), which mainly serves as a quality control for correct mRNA processing and helps to stabilize the eukaryotic mRNA; on the basis of cap 0, 2'-OH methylation is performed on the first nucleotide, called cap 1 (cap1); in addition to cap 0 and cap 1, further methylation modification can be performed on the second nucleotide, called cap 2; generally speaking, the synthesis method of the 5'-cap can be: different synthetic routes of 5' capped mRNA based on enzymatic, chemical or chemoenzymatic methods;
  • a cap analog is directly added to the in vitro transcription (IVT) system , and the 5' cap analog includes but is not limited to : m7Gppp (2'OMeA)pG, m7GpppApA , m7GpppApC , m7GpppApG , m7GpppApU , m7GpppCpA, m7GpppCpC , m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC , m7GpppGpG , m7GpppGpU , m7GpppUpA , m7GpppUpC , m7GpppUpG , m7GpppUpU , m7GpppUpU , m7GpppUpC , m7GpppUpC , m7GpppUp
  • the capped analogs may also be other structures, such as tetramers, pentamers, hexamers, heptamers, octamers, nonamers or decamers, etc.
  • the specific sequence thereof may be determined according to the situation of the template.
  • the respiratory syncytial virus mRNA vaccine of the present application may include any 5' untranslated region (UTR) and/or any 3' untranslated region (UTR).
  • Nucleic acid comprises a polymer (nucleotide monomer) of nucleotides. Therefore, nucleic acid is also referred to as polynucleotide.
  • Nucleic acid can be or can include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNAs), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and/or chimera and/or its combination.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • TAA threose nucleic acid
  • GAA ethylene glycol nucleic acid
  • PNA peptide nucleic acid
  • LNAs locked nucleic acid
  • ENA ethylene nucleic acid
  • CeNA cyclohexenyl nucleic acid
  • Messenger RNA is any RNA that encodes (at least one) protein (a naturally occurring, non-naturally occurring or modified amino acid polymer) and can be translated in vitro, in vivo to produce the encoded protein, in situ or ex vivo.
  • RNA e.g., mRNA
  • the nucleic acid sequences listed in this application may refer to "T” in the representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T” will be replaced with "U”. Therefore, any DNA disclosed and identified herein by a specific sequence identification number also discloses a corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U”.
  • ORF open reading frame
  • AUG methionine
  • stop codon e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA.
  • sequences disclosed herein may also include additional elements, such as 5' and 3' UTRs, but unlike ORFs, these elements are not necessarily present in the RNA polynucleotides of the present application.
  • the composition comprises RNA (e.g., mRNA) comprising a nucleotide sequence of any one of SEQ ID NO.1-3 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity.
  • RNA e.g., mRNA
  • the open reading frame is preferably at least partially codon optimized. It is now known that translation efficiency can be determined by the different frequencies of transfer RNA (tRNA) occurrence in cells. Therefore, if there are an increasing degree of so-called "rare codons" in the coding region of the nucleic acid of the present application as defined herein, the translation of the corresponding modified nucleic acid sequence is less efficient than when there are codons encoding relatively "common" tRNAs. Those skilled in the art can perform codon optimization for sequences to be translated based on the characteristics of their in vitro expression systems.
  • tRNA transfer RNA
  • RNA e.g., mRNA
  • RNA is not chemically modified, but comprises standard ribonucleotides consisting of adenosine, guanosine, cytosine, and uridine.
  • the nucleotides and nucleosides disclosed herein comprise standard nucleoside residues, such as those present in transcribed RNA (e.g., A, G, C, or U).
  • the nucleotides and nucleosides disclosed herein include standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT);
  • the composition of the present application comprises RNA with an open reading frame encoding respiratory syncytial virus antigen, wherein the nucleic acid comprises standard (unmodified) or modified nucleotides and/or nucleosides known in the art.
  • the nucleotides and nucleosides of the present application include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include modifications of sugars, backbones or core base moieties of nucleotides and/or nucleosides known in the art.
  • the modified nucleic acid base in the nucleic acid includes 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine and/or pseudouridine, pseudouridine.
  • In vitro transcription is the process of generating mRNA by using DNA as a template in an in vitro cell-free system containing components such as RNA polymerase and NTP to mimic the in vivo transcription process.
  • the capped RNA synthesized in the in vitro transcription reaction can be used for subsequent experiments such as microinjection, in vitro translation, and transfection.
  • the in vitro transcription system usually includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase.
  • NTPs can be synthesized by themselves or selected from a supplier.
  • NTPs can be natural or non-natural NTPs.
  • Optional polymerases include, but are not limited to, bacteriophage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and/or polymerase mutants thereof, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and/or modified nucleotides, including chemically modified nucleic acids and/or nucleotides. Some embodiments exclude the use of DNA enzymes. In some embodiments, the RNA contains a 5' guanosine cap.
  • chemical synthesis methods can also be used, including solid phase chemical synthesis and liquid phase chemical synthesis; regarding solid phase chemical synthesis, the nucleic acids disclosed in this application can be prepared in whole or in part using solid phase technology; solid phase chemical synthesis of nucleic acids is an automated method in which molecules are fixed on a solid support and synthesized stepwise in a reactant solution. Solid phase synthesis can be used for site-specific introduction of chemical modifications in nucleic acid sequences; regarding liquid phase chemical synthesis, the nucleic acids of this application can be synthesized in liquid phase by sequentially adding monomer constructs. In addition, the above-mentioned synthesis methods can also be combined for use Because the synthetic methods discussed above each have their own advantages and limitations, it is possible to try to combine these methods together to overcome the above limitations. Combinations of these methods are within the scope of this application.
  • compositions of the present application include RNA encoding respiratory syncytial virus antigen variants (e.g., variant trimeric F proteins, such as stable pre-fusion F proteins).
  • Antigenic variants or other polypeptide variants refer to molecules whose amino acid sequences are different from wild-type, natural or reference sequences. Compared to natural or reference sequences, antigen/polypeptide variants may have substitutions, deletions and/or insertions at certain positions within the amino acid sequence. Typically, variants have at least 50% identity with wild-type, natural or reference sequences. In some embodiments, variants have at least 80% or at least 90% identity with wild-type, natural or reference sequences.
  • Variant antigens/polypeptides encoded by the nucleic acid of the present application may include amino acid changes that confer any of a variety of desired properties, for example, enhancing their immunogenicity, enhancing their expression and/or improving their stability or PK/PD properties.
  • Conventional mutagenesis techniques can usually be used to prepare variant antigens/polypeptides, and analysis is performed as appropriate to determine whether they have the desired properties. Determination of expression levels and immunogenicity is well known in the art, and exemplary such determinations are described in the Examples section.
  • the PK/PD properties of protein variants can be measured using techniques recognized in the art, for example, by determining the expression of antigens over time in the inoculated subject and/or by observing the persistence of the induced immune response.
  • the stability of the protein encoded by the variant nucleic acid can be measured by measuring the thermal stability or stability during urea denaturation, or can be measured using computer prediction. Methods for such tests and computer determinations are known in the art.
  • identity refers to the relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids) determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gap alignments (if any) are solved by a specific mathematical model or computer program (e.g., "algorithm”). The identity of the related antigens or nucleic acids can be easily calculated by known methods.
  • Percentage (%) identity for polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in an amino acid sequence or the nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage identity. Methods and computer programs for alignment are well known in the art. It is understood that identity depends on the calculation of percentage identity, but its value may vary due to gaps and penalties introduced in the calculation.
  • variants of a particular polynucleotide or polypeptide have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a particular reference polynucleotide or polypeptide as determined by the sequence alignment programs and parameters described herein and known to those of skill in the art.
  • LNP Lipid Nanoparticles
  • RNA e.g., mRNA
  • LNPs lipid nanoparticles
  • Lipid nanoparticles generally include ionizable cationic lipids, auxiliary lipids, cholesterol and PEG lipid components and nucleic acids of interest. Nanoparticles can be generated using components, compositions, and methods generally known in the art.
  • compositions provided herein may include RNA or multiple RNAs encoding two or more antigens of the same or different types.
  • the compositions include RNA or multiple RNAs encoding two or more respiratory syncytial virus antigens.
  • the RNA may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more respiratory syncytial virus antigens.
  • Two or more different RNAs (e.g., mRNA) encoding antigens can be formulated in the same lipid nanoparticle.
  • two or more different RNAs encoding antigens can be formulated in separate lipid nanoparticles (each RNA is formulated in a single lipid nanoparticle).
  • the lipid nanoparticles can then be combined and administered as a single vaccine composition (e.g., comprising a variety of RNAs encoding a variety of antigens), or can be administered separately.
  • compositions provided herein may include RNA or multiple RNAs encoding two or more antigens of the same or different virus strains.
  • Combination vaccines are also provided herein, which include RNAs encoding one or more respiratory syncytial viruses and one or more antigens of different organisms. Therefore, the vaccine of the present application may be a combined vaccine targeting one or more antigens of the same strain/species, or one or more antigens of different strains/species, such as other microorganisms found in geographic areas with high risk of respiratory syncytial virus infection or other antigens that may be contacted simultaneously when an individual is exposed to respiratory syncytial virus.
  • Sequential vaccination refers to the interval vaccination of vaccines with different technical routes, including basic immunization sequence and booster immunization sequence; if the first shot is an inactivated vaccine and the second shot is an adenovirus vaccine or mRNA vaccine or any other non-inactivated vaccine, this vaccination method is called basic immunization sequence; if two doses of inactivated vaccine have been completed and a booster shot is needed subsequently, and any other non-inactivated vaccine is used instead, this vaccination method is called booster immunization sequence.
  • compositions e.g., pharmaceutical compositions
  • methods, kits, and reagents for preventing or treating, for example, respiratory syncytial virus in humans and other mammals.
  • the compositions provided herein can be used as therapeutic or prophylactic agents. They can be used in drugs for preventing and/or treating respiratory syncytial virus infection.
  • composition refers to the combination of an active agent and an inert or active carrier, making the composition particularly suitable for in vivo or in vitro diagnostic or therapeutic use.
  • a “pharmaceutically acceptable carrier” does not cause undesirable physiological effects after administration to a subject or after administration to a subject.
  • the carrier in the pharmaceutical composition must be “acceptable”, which also means that it is compatible with the active ingredient and capable of stabilizing it.
  • One or more solubilizing agents can be used as pharmaceutical carriers for delivering the active agent.
  • pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, adjuvants, additives and diluents to obtain a composition that can be used as a dosage form.
  • examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose and sodium lauryl sulfate.
  • Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for them, are described in Remington's Pharmaceutical Sciences.
  • nucleic acid sequences in the following sequences may represent DNA sequences or RNA sequences. When they represent RNA sequences, unless otherwise specified, "T" and "U” are used interchangeably to represent uridine.
  • sequences used in the specific examples later in this application correspond to the following sequences with sequence numbers or names, and when they represent mRNA sequences in the specific examples, the uridines therein are all 1-methyl pseudouridine.
  • SEQ ID NO.1 F protein nucleotide sequence 1 (RSV-1)
  • SEQ ID NO.2 F protein nucleotide sequence 2 (RSV-2)
  • SEQ ID NO.3 F protein nucleotide sequence 3) (RSV-3)
  • U in the above sequence SEQ ID NO.1-3 can be replaced in whole or in part by a modified base, for example: 1-methylpseudouridine or pseudouridine.
  • SEQ ID NO.4 F protein antigen variant amino acid sequence
  • SEQ ID NO.8 (3'-polyadenylic acid)
  • SEQ ID NO.12 F protein original coding sequence 0, RSV-0
  • SEQ ID NO: 13 F protein of RSV wild-type strain
  • RSV mRNA sequence According to the natural coding region sequence of respiratory syncytial virus F protein, this patent designs RSV mRNA sequence.
  • RSV mRNA sequence features also include: 5'UTR, such as pVAX.1+TEV 5'UTR or pVAX.1+alpha-1-glob 5'UTR; 3'UTR, such as hHBA1 3'UTR or gp130 3'UR+DH143 3'UTR; and 3'-polyadenylic acid (PolyA), such as 3'-polyadenylic acid containing 100 A (SEQ ID NO.8).
  • 5'UTR such as pVAX.1+TEV 5'UTR or pVAX.1+alpha-1-glob 5'UTR
  • 3'UTR such as hHBA1 3'UTR or gp130 3'UR+DH143 3'UTR
  • PolyA 3'-polyadenylic acid
  • RSV-1, RSV-2 and RSV-3 mRNA sequences have been optimized in the F protein coding region, and the GC content of the optimized sequences has been improved compared with the RSV F protein natural coding sequence (RSV-0).
  • RSV-0, RSV-1, RSV-2 and RSV-3 mRNA sequence design optimization scheme and relative expression test results are shown in Table 1.
  • RSV-1 SEQ ID NO.1 with the highest relative expression level was selected as the preferred mRNA sequence for subsequent research and development of vaccines.
  • IVT in vitro transcription
  • the IVT kit (Novogene kit catalog number: DD4201-P-01), prepare the IVT reaction system, that is, mix 10x Transcription Buffer, ATP, GTP, CTP, PseudoUTP (1-methylpseudouracil, Zhaowei Technology, catalog number R5-064), 5' cap analog (m7G(5')ppp(5')(2'OMeA)pG, Zhaowei Technology, catalog number ON-134), water for injection, plasmid template (linearized plasmid with T7 promoter, the template contains the DNA sequence corresponding to SEQ ID NO.1) and Enzyme Mix.
  • the purity of the obtained mRNA was analyzed using a Bioanalyzer.
  • the test results showed (see Figure 2) that the mRNA of the F protein was highly pure after being isolated and purified after being synthesized in vitro.
  • the formulation was optimized by comprehensively considering the following four aspects: lipid ratio, buffer system, and N:P (nitrogen-phosphorus ratio).
  • the specific formulation selection is shown in Tables 2 and 3.
  • 3 ⁇ g mRNA (encapsulated with LNPs of different formulations 1-7 as shown in Table 2) was used to immunize normal mice once, and then the binding antibodies in the mouse serum were detected 14 days later. Based on the titer performance of the binding antibodies and the stability of the formulation, formulation 1 was finally determined as the preferred formulation (therefore, the mRNA in the subsequent examples was encapsulated with LNPs of formulation 1) (see Figure 3).
  • the F protein antigen mRNA-LNP preparation was prepared according to the formula selected in Example 2 (i.e., Formula 1), and the specific preparation steps were as follows:
  • the lipid working solution and the mRNA working solution were mixed in a volume ratio of 1:3 to prepare an mRNA-loaded LNP solution.
  • test results show (see Figure 4): F protein mRNA and lipid working solution are mixed by microfluidics to form LNP complexes with uniform particle size and consistent morphology, most of which are about 100 nm. The two-phase ions of the LNP are fully fused, meeting the basic requirements for the next step of the experiment.
  • the F protein mRNA stock solution prepared in Example 1 was transfected into COS-7 cells (Note: African green monkey kidney fibroblasts, commercially available). After 24 hours of transfection, the cells were collected and subjected to immunoblotting. Cells not transfected with F protein mRNA were used as negative controls (Blank), and GAPDH was used as an internal reference.
  • the experimental results show ( Figure 5): The optimized mRNA encoding F protein (RSV-1, 2, 3) can be effectively and stably expressed in large quantities in COS-7 cells, and RSV-1 has a relatively higher expression level.
  • the methanol-activated PVDF membrane was assembled into a membrane transfer device according to the "sandwich method", and then the membrane was transferred at 100 V for 1.5 h in an ice bath.
  • the PVDF membrane was washed three times with PBST and incubated with GAPDH antibody (mouse monoclonal antibody) at room temperature for 1 h.
  • GAPDH antibody mouse monoclonal antibody
  • Example 5 Flow cytometry detection of antigen protein expression on cell surface
  • RSV-1F protein mRNA stock solution prepared in Example 1 i.e., RSV pre-F mRNA as shown in FIG5
  • flow cytometry was used to detect the expression of the target antigen protein in the cells.
  • Cells not transfected with F protein mRNA were used as negative controls (Untransfected). The specific method is as follows:
  • COS-7 cells cultured for more than 24 hours were digested and transferred into 6-well plates, and the cell density was controlled at 300,000 per well.
  • the expression level is close to the peak.
  • the cell supernatant is removed, washed once with PBS, digested with 0.05% trypsin for 1 minute, neutralized with complete culture medium, and the cells are collected. The collected cells are centrifuged at 350g for 5 minutes and the supernatant is discarded. The cells are resuspended in 2ml PBS and collected, centrifuged at 350g for 5 minutes, the supernatant is discarded, and finally the cells are resuspended with 100 ⁇ l PBS, and the cell number is controlled between 200,000 and 1,000,000.
  • mice Female BALB/c mice (6-8 weeks old, randomly divided into groups) were immunized by intramuscular injection of vehicle and test RSV-1 mRNA-LNP preparation (5 ⁇ g/mouse, 25 ⁇ g/mouse) on day 0 and day 28, respectively. Serum samples were collected on day 35 after immunization to detect serum binding antibodies and neutralizing antibody titers.
  • mice immunized with the RSV-1 mRNA-LNP preparation prepared in Example 3 were able to produce high titers of neutralizing antibodies to type A and type B RSV (part B in Figure 7 shows the results of serum true virus neutralization test), and to produce high titers of binding antibodies to type A RSV F protein (part A in Figure 7 shows the results of serum antibody binding test), especially the 25 ⁇ g group was able to produce neutralizing antibodies with a titer of more than 10 4 .
  • Mouse serum antibody binding test method is as follows:
  • a high-binding 96-well plate coated with type A RSV preF protein (commercially available) was prepared one day in advance. The next day, serum diluted into different gradients was added to the coated 96-well plate and incubated for 2 hours. The binding antibodies in the serum were then detected using a universal ELISA method.
  • mice serum true virus neutralization test The specific method of mouse serum true virus neutralization test is as follows:
  • the serum samples were heat-inactivated at 56°C for 30 min, and then diluted ten-fold with culture medium, and then three-fold for a total of 8 dilutions.
  • the virus (RSV A2 and RSV B18537) was thawed, the virus was diluted to 4000TCID50/mL, and 50 ⁇ L of serially diluted serum and 50 ⁇ L of diluted virus were taken to a 96-well microplate and incubated at 37°C for 1 hour. At the same time, a 96-well microplate was taken and the diluted virus was diluted twice, 100 ⁇ L/well, for virus back titration verification. The virus inoculation volume was 200TCID50/well.
  • Hep-2 cells were inoculated into the test plate and the back-titration plate at a certain density, 100 ⁇ L/well, and the cell culture medium was set.
  • Cell control group cells, no virus infection
  • virus control group cells infected with virus, no other treatment. Cells were cultured in an incubator for 5 days.
  • the cell culture supernatant was aspirated and 75 ⁇ L of pre-cooled 80% acetone was added to each well to fix the cells at 4°C for 15 min, the solution was discarded, and the plate was air-dried.
  • the plate was blocked with 100 ⁇ L/well 5% BSA at room temperature for 1 hour, and then washed three times with 200 ⁇ L/well 1 ⁇ TBST.
  • 75 ⁇ L of primary antibody solution (1:5000 dilution) was added to each well, incubated at 37°C for 1 hour, after which the solution was discarded, and the plate was washed three times with 200 ⁇ L/well 1 ⁇ TBST.
  • mice Female BALB/c mice (6-8 weeks old, randomly divided) were immunized by intramuscular injection of vehicle and the test RSV-1 mRNA-LNP preparation (5 ⁇ g/mouse and 25 ⁇ g/mouse) prepared in Example 3 on day 0 and day 28, respectively. On day 49, RSV/A2 virus was inoculated intranasally at a dose of 1.0 ⁇ 10 5 PFU/mouse.
  • R-1) RSV-1F protein mRNA LNP preparation prepared by encapsulating RSV-1 mRNA using LNP of formula 1 is hereinafter referred to as R-1):
  • mice Five days after RSV/A2 virus inoculation (day 54), mice were killed and lung viral load was detected.
  • the test results are shown in Figure 8.
  • the tested vaccine R-1 (5 ⁇ g/mouse, 25 ⁇ g/mouse) can significantly reduce the RSV virus titer in the lung tissue of infected animals, and the virus titer is reduced by 2.00Log, showing good in vivo anti-RSV virus efficacy.
  • the Buffer group in Figure 8 is used to represent the lung virus situation of healthy mice without virus attack, as a reference.
  • the plaque assay was used to detect RSV titers in lung tissue samples. The specific steps are as follows:
  • Cell plating Add 1 mL of Hep2 cells with a cell density of 3.0*1/mL to each well of a 12-well cell plate and place in a CO2 cell culture incubator overnight to obtain a monolayer of cells;
  • Plaque counting Scan the image and read the number of plaques in it to calculate the virus titer in the sample.
  • the virus titer is expressed as Log 10 (the number of plaques per gram of lung tissue homogenate).
  • Arexvy is a vaccine that contains a recombinant prefusion RSV F glycoprotein antigen (RSV Pre F3) and GSK's proprietary adjuvant AS01E. It is FDA-approved for the prevention of lower respiratory tract disease caused by RSV infection in people aged 60 years and older.
  • Arexvy was injected intramuscularly into BALB/c mice (6-8 weeks old, randomly divided into groups) at 1/10 of the human dose (12 ⁇ g per mouse), and the RSV-1 mRNA-LNP preparation prepared in Example 3 was injected intramuscularly into BALB/c mice at a dose of 3 ⁇ g per mouse.
  • the mice were immunized twice at an interval of 28 days, and the weight of the mice was measured on the day of administration and the day after administration.
  • Serum samples were collected 7 and 14 days after the second immunization, and the titer of IgG antibodies specific to the pre-F protein in the serum of BALB/c mice was detected by ELISA, and the neutralizing antibody titer of the RSV/A2 virus strain was detected by fluorescent spot reduction neutralization test (FRNT).
  • FRNT fluorescent spot reduction neutralization test
  • the neutralizing antibody activity of the RSV-1 mRNA-LNP preparation was significantly better than Arexvy (P ⁇ 0.05), and the neutralization titer was nearly 20% higher than Arexvy, indicating that the immunogenicity of the RSV-1 mRNA-LNP preparation is better (Table 3).
  • the ELISA method was used to detect the titer of IgG antibodies specific to pre-F protein in mouse serum samples (i.e., the binding antibody titer detection method in Table 3).
  • the specific steps were as follows: a high-binding 96-well plate coated with RSV preF protein was prepared one day in advance. The next day, serum diluted into different gradients was added to the coated 96-well plate and incubated for 2 hours. The binding antibodies in the serum were then detected using a universal ELISA method.
  • the neutralizing antibody titer in Table 3 was tested using FRNT (plaque reduction neutralization test) to evaluate the neutralizing antibody titer of RSV/A2 virus strain in serum.
  • the specific steps are as follows: the serum to be tested was graded diluted and mixed with a certain amount of RSV-A2-GFP recombinant virus (commercially available) in equal volumes for pre-incubation for 1 hour, and then HEp-2 cells were infected. After continued cultivation for 24 hours, GFP fluorescent spots were counted using a cell spot reader, and the serum dilution corresponding to the 50% neutralizing antibody inhibition rate of the sample was calculated as the ND50 neutralizing antibody titer.
  • the first generation of WHO anti-RSV serum international standard was introduced for reference to convert and calculate the neutralizing antibody titer IU/mL.
  • mice Female BALB/c mice (6-8 weeks old, randomly divided into groups) were immunized by intramuscular injection of vehicle (Model and Health groups), formalin-inactivated vaccine FI-RSV (0.05 ⁇ g dose) and RSV-1 mRNA-LNP preparation prepared in Example 3 (5 ⁇ g and 15 ⁇ g dose) on days 0 and 28, respectively.
  • RSV/A2 virus was inoculated by intranasal drops on day 42 (Health group was not challenged with virus), with an inoculation dose of 5.0 ⁇ 10 5 PFU.
  • Serum samples were collected on day 35 (D35) to detect pre-F protein-specific IgG binding antibodies for immunogenicity evaluation; lung tissue samples were collected on day 47 to detect lung tissue virus titers to evaluate the in vivo efficacy of the tested vaccines, and vaccine enhanced disease (VED) was evaluated by lung tissue pathological analysis.
  • D35 day 35
  • VED vaccine enhanced disease
  • test results showed that the specific binding antibody titers of serum samples in the RSV-1 mRNA-LNP preparation administration group were higher than those in the model group and FI-RSV group, showing a good RSV specific binding antibody induction effect (Figure 10).
  • the test vaccine RSV-1 mRNA-LNP 5 ⁇ g per mouse and 15 ⁇ g per mouse
  • the virus titer is reduced by 3.1Log, showing good in vivo anti-RSV virus efficacy
  • test vaccine RSV-1 (5 ⁇ g/mouse, 15 ⁇ g/mouse) can significantly reduce the scores of perivascular infiltration, peribronchial infiltration, alveolar infiltration and interstitial infiltration, and the scores are lower than the model group, indicating that the test vaccine RSV-1 did not find obvious lung pathology enhancement, so it has significantly better safety than FI-RSV ( Figure 12).
  • the ELISA method was used to detect the titer of IgG antibodies specific to pre-F protein in mouse serum samples.
  • the specific steps were as follows: a high-binding 96-well plate coated with RSV preF protein was prepared one day in advance. The next day, serum diluted into different gradients was added to the coated 96-well plate and incubated for 2 hours. The binding antibodies in the serum were then detected using a universal ELISA method.
  • the plaque assay was used to detect RSV titers in lung tissue samples. The specific steps are as follows:
  • Cell plating Add 1 mL of Hep2 cells with a cell density of 3.0*10 5 /mL to each well of a 12-well cell plate and place in a CO 2 cell culture incubator overnight to obtain a monolayer of cells;
  • Plaque counting Scan the image and read the number of plaques in it to calculate the virus titer in the sample.
  • the virus titer is expressed as Log10 (the number of plaques per gram of lung tissue homogenate).
  • HE staining was used to detect inflammatory indicators of lung tissue. The specific steps were as follows: the lung tissue was perfused and fixed in 4% paraformaldehyde for more than 24 hours, dehydrated, and then sliced. The slice thickness was 4 ⁇ m.
  • the staining rack loaded with the slices was placed in a 60-65°C oven and baked for 1 hour. After the paraffin melted into a transparent liquid, the staining rack was placed at room temperature for 10 minutes to cool. After cooling, HE staining was performed and finally the slices were sealed.
  • mice Female cotton rats (6-8 weeks old, randomly divided into groups) were immunized by intramuscular injection of vehicle, formalin-inactivated vaccine FI-RSV (0.1 ⁇ g) and RSV-1 mRNA-LNP preparation (7.5 ⁇ g or 15 ⁇ g) prepared in Example 3 on days 0 and 28, respectively.
  • RSV B9320 virus was inoculated intranasally on day 42 at a dose of 1.5 ⁇ 10 6 PFU.
  • Serum samples were collected on day 35 to detect pre-F protein-specific IgG binding antibodies for immunogenicity evaluation; nasal turbinates were collected on day 47 to detect nasal turbinates virus titers to evaluate the in vivo efficacy of the tested vaccines.
  • the test results showed that the specific binding antibody titers of serum samples in the RSV-1 mRNA-LNP preparation group were higher than those in the model group and FI-RSV group, showing a good RSV specific binding antibody induction effect (Figure 13).
  • the tested vaccine RSV-1 mRNA-LNP preparation (at 7.5 ⁇ g and 15 ⁇ g doses) can significantly reduce the RSV virus titer in the nasal turbinate tissue of infected animals, and the virus titer is reduced by 2.05Log, which is better than FI-RSV (reduced by 0.19Log), showing a good in vivo anti-RSV virus efficacy (Figure 14).
  • the ELISA method was used to detect the titer of IgG antibodies specific to pre-F protein in cotton rat serum samples.
  • the specific steps were as follows: a high-binding 96-well plate coated with RSV preF protein was prepared one day in advance. The next day, serum diluted into different gradients was added to the coated 96-well plate and incubated for 2 hours. The binding antibodies in the serum were then detected using a universal ELISA method.
  • the RSV titer in the nasal turbinate tissue sample was detected by plaque assay, and the specific steps are as follows:
  • Cell plating Add 1 mL of Hep2 cells with a cell density of 3.0*10 5 /mL to each well of a 12-well cell plate. Incubate in a CO2 cell culture incubator overnight to obtain a monolayer of cells;
  • Plaque counting Scan the image and read the number of plaques in it to calculate the virus titer in the sample.
  • the virus titer is expressed as Log10 (the number of plaques per gram of tissue homogenate).
  • RSV-1 mRNA-LNP preparation vaccine can fully activate the immune system and produce high titer binding antibody/neutralizing antibody, and has good attack and poison protection effect on RSV A and B subtypes, and has no obvious lung pathological damage enhancement effect.
  • the serum neutralizing antibody level and the binding antibody titer are better than the positive reference of the same dose (GSK's RSV vaccine Arexvy), and unexpected technical effects have been achieved. And while having a higher attack and poison protection effect, it has higher safety.

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Abstract

L'invention concerne un vaccin à ARNm pour la prévention des virus respiratoires syncytiaux, des composants principaux dudit vaccin comprenant un ARNm ayant un site de mutation et des nanoparticules lipidiques. Le vaccin à ARNm présente de bons effets immunitaires sur divers sous-types de virus syncytiaux respiratoires.
PCT/CN2024/100015 2023-06-20 2024-06-19 Vaccin à arnm contre les virus respiratoires syncytiaux et son procédé de préparation Pending WO2024260359A1 (fr)

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