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WO2025119162A1 - Vaccin contre le métapneumovirus (mpv) - Google Patents

Vaccin contre le métapneumovirus (mpv) Download PDF

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Publication number
WO2025119162A1
WO2025119162A1 PCT/CN2024/136380 CN2024136380W WO2025119162A1 WO 2025119162 A1 WO2025119162 A1 WO 2025119162A1 CN 2024136380 W CN2024136380 W CN 2024136380W WO 2025119162 A1 WO2025119162 A1 WO 2025119162A1
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Prior art keywords
mutant
amino acid
seq
acid sequence
nucleic acid
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Chinese (zh)
Inventor
李林鲜
李惠
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Shenzhen Shenxin Biotechnology Co Ltd
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Shenzhen Shenxin Biotechnology Co Ltd
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Publication of WO2025119162A1 publication Critical patent/WO2025119162A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/155—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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/115—Paramyxoviridae, e.g. parainfluenza virus

Definitions

  • the invention belongs to the field of biotechnology and relates to a metapneumovirus (MPV) vaccine.
  • MPV metapneumovirus
  • hMPV Human metapneumovirus
  • hMPV-related acute lower respiratory tract infections 643,000 hMPV-related hospitalizations, 7,700 hMPV-related hospitalization deaths, and 16,100 hMPV-related acute lower respiratory tract infection deaths (hospitals and communities) in children under 5 years old worldwide.
  • hMPV-induced immune protection is weak, so it can be repeatedly infected and can cause severe diseases (such as bronchitis or pneumonia) in adults (especially the elderly and immunocompromised patients).
  • the hospitalization rate of hMPV is comparable to that of RSV and influenza virus.
  • the present disclosure provides a nucleic acid, the protein or polypeptide encoded by the nucleic acid can induce a specific immune response to prevent or at least reduce metapneumovirus (MPV) infection.
  • MPV metapneumovirus
  • the present disclosure also provides a delivery vector, a pharmaceutical composition and an MPV vaccine comprising the above-mentioned nucleic acid and their use in preparing drugs.
  • a nucleic acid comprising a polynucleotide encoding a mutant of an MPV F protein, wherein the mutant comprises an F1 polypeptide and an F2 polypeptide, and relative to a wild-type MPV F protein, the mutant comprises a disulfide bond mutation, wherein the disulfide bond mutation comprises one or more of the following:
  • the disulfide bond mutations include one or more of the following: A147C and A159C, L165C and F196C, N145C and A161C, S149C and V157C, T59C and N180C.
  • the disulfide bond mutation is selected from one of the following: A147C and A159C, L165C and F196C, N145C and A161C.
  • the mutant is selected from one of the following (1) to (38):
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 1 or 2;
  • mutants E146C and T160C comprising mutations E146C and T160C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 3 or 4;
  • a mutant comprising mutations F168C and F196C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 5 or 6;
  • a mutant comprising mutations L165C and F196C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:7 or 8;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 11 or 12;
  • a mutant comprising mutations T59C and N180C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 13 or 14;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 15 or 16;
  • a mutant comprising mutations T59C and N180C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 15 or 16;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 17 or 18;
  • a mutant comprising mutations T59C and N180C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 19 or 20;
  • (22) a mutant comprising mutations T150C and R156C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 21 or 22;
  • a mutant comprising mutations V52C and L165C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 23 or 24;
  • a mutant comprising mutations V148C and L158C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 29 or 30;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 81 or 82;
  • (32) a mutant comprising mutations V148C and L158C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO:81 or 82;
  • a mutant comprising mutations A147C and A159C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:83;
  • a mutant comprising mutations N145C and A161C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO:85.
  • the wild-type MPV is subtype A or subtype B.
  • the wild-type MPV is wild-type hMPV.
  • the MPV F protein is hMPV F protein.
  • the nucleic acid is RNA; preferably, the RNA is mRNA; more preferably, the mRNA comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR and a poly(A) tail.
  • the nucleic acid is DNA.
  • the DNA is capable of being transcribed into RNA.
  • the DNA sequence corresponding to the 5'-UTR is shown in SEQ ID NO: 31;
  • DNA sequence corresponding to the 3’-UTR is shown in SEQ ID NO: 32;
  • the nucleotides constituting the poly(A) tail contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides; preferably, the nucleotides constituting the poly(A) tail contain at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides in succession; preferably, the nucleotides constituting the poly(A) tail contain one or more nucleotides other than A nucleotides; more preferably, the DNA sequence corresponding to the poly(A) tail is as shown in SEQ ID NO: 33.
  • the nucleic acid contains modified nucleotides
  • the nucleic acid contains modified nucleosides; preferably, the modified nucleosides include at least one of modified uridine, modified cytidine, modified adenosine and modified guanosine.
  • the nucleic acid comprises a polynucleotide encoding one or more mutants of the MPV F protein.
  • the nucleic acid also includes a polynucleotide encoding other proteins or polypeptides other than mutants of MPV F protein.
  • the nucleic acid further comprises one or more of the following: a polynucleotide encoding the transmembrane domain of the MPV F protein, a polynucleotide encoding a ferritin polypeptide, a polynucleotide encoding a signal peptide, and a polynucleotide encoding a trimerization domain.
  • a genetic engineering vector comprising any of the above nucleic acids, or comprising a polynucleotide capable of being transcribed into any of the above nucleic acids.
  • a host cell comprising any one of the above-mentioned genetic engineering vectors.
  • the mutant is a trimer.
  • a delivery vector comprising a nucleic acid composition, wherein the nucleic acid composition comprises a first nucleic acid or a first genetic engineering vector, wherein the first nucleic acid is any of the above nucleic acids, and the first genetic engineering vector is any of the above genetic engineering vectors;
  • the nucleic acid composition includes multiple first nucleic acids or multiple first genetic engineering vectors, the first nucleic acid is any of the above nucleic acids, the first genetic engineering vector is any of the above genetic engineering vectors, the multiple first nucleic acids are independent of each other or the multiple first genetic engineering vectors are independent of each other.
  • the nucleic acid composition further comprises a second nucleic acid or a second vector, wherein the second nucleic acid comprises a polynucleotide encoding other proteins or polypeptides other than the mutant of the MPV F protein, and the second vector comprises a polynucleotide encoding other proteins or polypeptides other than the mutant of the MPV F protein;
  • the first nucleic acid and the second nucleic acid are RNA; more preferably, the first nucleic acid and the second nucleic acid are mRNA.
  • the delivery vehicle is a lipid nanoparticle (LNPs), a cationic liposome, a cationic protein, or a lipid polymer (LPP);
  • LNPs lipid nanoparticle
  • LPP lipid polymer
  • the delivery vehicle is a lipid nanoparticle, wherein the lipid nanoparticle comprises a cationic lipid, wherein the cationic lipid comprises the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:
  • L 3 and L 4 are the same or different, and are each independently C1-C12 alkylene, C2-C12 alkenylene or C2-C12 alkynylene; preferably L 3 and L 4 are the same or different, and are each independently C3-C10 alkylene, C3-C10 alkenylene or C3-C10 alkynylene; in some embodiments, L 3 and L 4 are the same or different, and are each independently C3-C10 alkylene; in some embodiments, L 3 and L 4 are the same or different, and are each independently C5-C8 alkylene;
  • R 18 and R 19 are the same or different and are each independently C5-C27 alkyl or C5-C27 alkenyl containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently C8-C20 alkyl or C8-C20 alkenyl containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently C9-C17 alkyl or C9-C18 alkenyl containing one or two double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently C9-C17 alkyl or C9-C18 alkenyl containing one or two double bonds.
  • R 20 is halogen, hydroxyl, cyano, C1-C6 alkyl, nitro, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxyl, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; in
  • z is 1, 2 or 3.
  • the cationic lipid is the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:
  • the cationic lipid is the following compound, or a pharmaceutically acceptable salt thereof:
  • a pharmaceutical composition comprising any of the above nucleic acids, any of the above genetic engineering vectors, any of the above host cells, any of the above mutants or any of the above delivery vectors, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes a plurality of said delivery vehicles
  • the pharmaceutical composition comprises a plurality of said nucleic acids.
  • the pharmaceutical composition comprises two nucleic acids, the two nucleic acids are co-formulated or separately formulated in lipid nanoparticles, the two nucleic acids encode a mutant of subtype A MPV F protein and a mutant of subtype B MPV F protein, respectively, and the mutant of subtype A MPV F protein and the mutant of subtype B MPV F protein encoded by the two nucleic acids are selected from the following group:
  • a mutant comprising mutations A147C and A159C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:1
  • a mutant comprising mutations A147C and A159C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:2;
  • a mutant comprising mutations A147C and A159C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:81, and a mutant comprising mutations A147C and A159C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:82.
  • the medicament is used to prevent or treat MPV infection or a disease caused by MPV infection.
  • the medicament is used to prevent or treat hMPV infection or a disease caused by hMPV infection.
  • the medicament is a vaccine.
  • a vaccine comprising any of the above nucleic acids, any of the above genetic engineering vectors, any of the above mutants or any of the above delivery vectors.
  • Figures 1A to 1F are the results of IgG antibody titer detection after two immunizations of mice with different mRNA vaccines ( Figures 1C to 1F, GMT), wherein the day of the first immunization was counted as day 0 (D0), and the second immunization was on day 22 (D22);
  • "hMPV/AF protein” represents the F protein of hMPV subtype A (the same applies to "hMPV/AF protein” in other figures)
  • “hMPV/BF protein” represents the F protein of hMPV subtype B (the same applies to "hMPV/B F protein” in other figures)
  • “1155+1171” represents a bivalent hMPV mRNA vaccine, which means that the LNP preparation encapsulating mRNA numbered 1155 and the LNP preparation encapsulating mRNA numbered 1171 were mixed and injected into mice.
  • Figures 2A and 2B are the test results (GMT) of IgG antibody titers induced by mice after two immunizations with different mRNA vaccines, where the day of the first immunization was counted as day 0 (D0) and the day of the second immunization was on day 21 (D21).
  • Figure 2A is the IgG antibody titer specifically binding to hMPV/AF protein
  • Figure 2B is the IgG antibody titer specifically binding to hMPV/B F protein.
  • Figures 3A to 3D are the results of neutralizing antibody detection (GMT) against hMPV/A pseudovirus induced by two immunizations of mice with different mRNA vaccines.
  • the antibody titer is NT50, where the day of the first immunization is counted as D0, and the day of the second immunization is D22.
  • hMPV/A means hMPV subtype A.
  • FIGS 4A to 4G are the results of neutralizing antibody detection (GMT) against hMPV/A pseudovirus induced by two immunizations of mice with different mRNA vaccines.
  • the antibody titer is NT50, where the day of the first immunization is counted as D0, and the day of the second immunization is D21.
  • hMPV/A means hMPV subtype A.
  • Figures 5A to 5C are the ELISpot test results of spleen lymphocytes after mice were immunized twice with different mRNA vaccines, where the day of the first immunization was counted as D0, and the second immunization was on D22.
  • Figure 5A is the ELISpot test result of IFN- ⁇ on D74
  • Figure 5B is the ELISpot test result of IFN- ⁇ on D116
  • Figure 5C is a comparison of the ELISpot test results of IFN- ⁇ and IL-4 on D116.
  • Figures 6A to 6H are the ELISpot detection results of spleen lymphocytes of mice after two immunizations with different mRNA vaccines, wherein the day of the first immunization was counted as D0, and the second immunization was on D21
  • Figure 6A is the ELISpot detection result of IFN- ⁇ on D7
  • Figure 6B is the ELISpot detection result of IL-2 on D7
  • Figure 6C is the ELISpot detection result of IL-4 on D7
  • Figure 6D is a comparison of the ELISpot detection results of IFN- ⁇ and IL-4 on D7
  • Figure 6E is the ELISpot detection result of IFN- ⁇ on D77
  • Figure 6F is the ELISpot detection result of IL-2 on D77
  • Figure 6G is the ELISpot detection result of IL-4 on D77
  • Figure 6H is a comparison of the ELISpot detection results of IFN- ⁇ and IL-4 on D77.
  • Figures 7A to 7C are the results of neutralizing antibody detection (GMT) against hMPV/A pseudovirus induced by two immunizations of mice with different mRNA vaccines.
  • the antibody titer is NT50, where the day of the first immunization is counted as D0 and the day of the second immunization is D28.
  • hMPV/A represents subtype A hMPV.
  • Figures 8A to 8D are the ELISpot test results of spleen lymphocytes after two immunizations of mice with different mRNA vaccines, where the day of the first immunization was counted as D0, and the second immunization was on D28.
  • Figures 8A and 8B are the ELISpot test results of IFN- ⁇ on D49
  • Figure 8C is the ELISpot test result of IFN- ⁇ on D57
  • Figure 8D is a comparison of the ELISpot test results of IFN- ⁇ and IL-4 on D57.
  • the expressions “comprises,” “comprising,” “containing,” and “having” are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components.
  • the expression “consisting of” excludes any element, step, or component not specified.
  • the expression “consisting essentially of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions “consisting essentially of” and “consisting of” are encompassed within the meaning of the expression “comprising.”
  • the numerical ranges described herein should be understood to include any and all subranges contained therein.
  • the range “1 to 10” should be understood to include not only the explicitly stated values of 1 and 10, but also any single value (e.g., 2, 3, 4, 5, 6, 7, 8, and 9) and subranges (e.g., 1 to 2, 1.5 to 2.5, 1 to 3, 1.5 to 3.5, 2.5 to 4, 3 to 4.5, etc.) within the range of 1 to 10.
  • This principle also applies to ranges with only one value as the minimum or maximum value.
  • naturally occurring refers to the fact that a substance can be found in nature.
  • peptides, amino acids, proteins, or nucleic acids that are present in organisms (including viruses) and can be isolated from sources in nature and have not been modified experimentally by man are naturally occurring.
  • non-naturally occurring is used herein to describe nucleic acids with the intent to mean that the nucleic acid is not found in nature.
  • a non-naturally occurring nucleic acid encoding a viral peptide or protein has at least one genetic alteration or chemical modification that is not normally found in a wild-type strain of the virus in question.
  • the genetic alteration includes, for example, the introduction of an expressible nucleic acid sequence encoding a peptide or polypeptide that is heterologous to the virus in question, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and/or other functional disruptions to viral genetic material.
  • Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.
  • wild type means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.
  • mutation refers to the deletion, addition or substitution of an amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide.
  • amino acid substitution at a specific position in a protein sequence is referred to using the code "(amino acid residue in wild-type protein) (amino acid position) (amino acid residue in engineered protein)".
  • the code A147C refers to the replacement of the 147th alanine (A) residue of the amino acid sequence of the reference protein by a cysteine (C) residue (in a mutant of the reference protein).
  • the amino acid code before the position number such as "147C” can be omitted in the code.
  • % identity refers to the percentage of identical nucleotides or amino acids in the optimal alignment between the sequences to be compared, and the differences between the two sequences can be distributed over a local region (segment) or over the entire length of the sequences to be compared.
  • identity between the two sequences is determined after the optimal alignment of the segment or "comparison window".
  • the optimal alignment can be performed manually or with the aid of algorithms known in the art. Algorithms known in the art include, but are not limited to, the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol.
  • % identity or % homology can be obtained by determining the number of identical positions corresponding to the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100 to obtain % homology.
  • the degree of homology is given for a region of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%. In some embodiments, the degree of homology is given for the entire length of the reference sequence.
  • Alignment to determine sequence identity can be performed using tools known in the art, preferably using optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.
  • nucleotide includes deoxyribonucleotide, deoxyribonucleotide, deoxyribonucleotide derivative, and ribonucleotide derivative.
  • ribonucleotide is a constituent substance of ribonucleic acid (RNA), composed of one molecule of base, one molecule of pentose and one molecule of phosphoric acid, which refers to a nucleotide having a hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group
  • deoxyribonucleotide is a constituent substance of deoxyribonucleic acid (DNA), also composed of one molecule of base, one molecule of pentose and one molecule of phosphoric acid, which refers to a nucleotide in which the hydroxyl group at the 2' position of the ⁇ -D-ribofuranosyl group is replaced by hydrogen, and is the main chemical component of
  • Nucleotide is usually referred to by a single letter representing the base, “A” or “A nucleotide” refers to adenine deoxyribonucleotide or adenine ribonucleotide containing adenine, “C” or “C nucleotide” refers to cytosine deoxyribonucleotide or cytosine ribonucleotide containing cytosine, “G” or “G nucleotide” refers to guanine deoxyribonucleotide or guanine ribonucleotide containing guanine, “U” or “U nucleotide” refers to uracil ribonucleotide containing uracil, and “T” or “T nucleotide” refers to thymine deoxyribonucleotide containing thymine.
  • nucleic acid generally refers to a polymer containing deoxyribonucleotides (deoxyribonucleic acid, referred to as DNA) or a polymer containing ribonucleotides (ribonucleic acid, referred to as RNA) or any compound of a combination thereof.
  • nucleic acids herein also include derivatives of nucleic acids.
  • derivatives of nucleic acids includes chemical derivatization of nucleic acids on the bases, sugars or phosphates of nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs.
  • nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.
  • Polynucleotide sequence can be used interchangeably to refer to the order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that the DNA coding strand (sense strand) and the RNA it encodes can be regarded as having the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.
  • the RNA corresponding to the DNA refers to the polynucleotide after all T in the DNA is replaced by U.
  • the mRNA corresponding to the DNA refers to the polynucleotide after all T in the DNA is replaced by U.
  • the polynucleotide may comprise one segment or multiple segments (nucleic acid fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments).
  • the polynucleotide may comprise a segment encoding a polypeptide of interest.
  • the polynucleotide may comprise a segment encoding a polypeptide of interest and a regulatory segment (including but not limited to a segment for transcriptional regulation and translational regulation).
  • the regulatory segment comprises a polynucleotide corresponding to one or more of the following regulatory elements: a promoter, a 5' untranslated region (5'-UTR), a 3' untranslated region (3'-UTR), and a poly (A) tail.
  • promoter refers to a polynucleotide located upstream of the 5' end of the coding region of a gene, which contains a conserved sequence required for specific binding of RNA polymerase and transcription initiation, can activate RNA polymerase, enable RNA polymerase to accurately bind to template DNA and have the specificity of transcription initiation. Promoters can be derived from viruses, bacteria, fungi, plants, insects and animals.
  • promoters include bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or SV 40 late promoter and CMV IE promoter.
  • the term "5' untranslated region" or "5'-UTR” can be an RNA sequence in an mRNA that is located upstream of a coding sequence and is not translated into a protein.
  • the 5'-UTR in a gene usually starts at the transcription start site and ends at the nucleotide upstream of the translation start codon of the coding sequence.
  • the 5'-UTR can contain elements that control gene expression, such as a ribosome binding site, a 5'-terminal oligopyrimidine tract, and a translation initiation signal such as a Kozak sequence.
  • mRNA can be post-transcriptionally modified by adding a 5' cap. Therefore, the 5'-UTR in a mature mRNA can also refer to the RNA sequence between the 5' cap and the start codon.
  • 3' untranslated region can be an RNA sequence in mRNA that is located downstream of a coding sequence and is not translated into a protein.
  • the 3'-UTR in mRNA is located between the stop codon and the poly(A) sequence of the coding sequence, for example, starting from the nucleotide downstream of the stop codon and ending at the nucleotide upstream of the poly(A) sequence.
  • poly (A), poly (A) sequence” and “poly (A) tail” are used interchangeably, and the naturally occurring poly (A) sequence is usually composed of adenine ribonucleotides.
  • modified poly (A) sequence refers to a poly (A) sequence comprising nucleotides or nucleotide segments other than adenine ribonucleotides.
  • the poly (A) sequence is usually located at the 3' end of the mRNA, for example, the 3' end (downstream) of the 3'-UTR.
  • the term "5'-cap structure” the 5'-cap structure is generally located at the 5' end of the mature mRNA. In some embodiments, the 5'-cap structure is connected to the 5'-end of the mRNA via a 5'-5'-triphosphate bond.
  • the 5'-cap structure is generally formed by modified (e.g., methylated) ribonucleotides (especially by guanine nucleotide derivatives).
  • m7GpppN (cap 0 or "cap0", which is a cap structure formed by the 5' phosphate group of hnRNA reacting with the 5'-phosphate group of m7GTP under the action of guanylyl transferase to form a 5', 5'-phosphodiester bond), wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure.
  • the 5'-cap structure includes but is not limited to cap 0, cap 1 (a cap structure formed by further methylating the 2'-OH of the first nucleotide sugar group of hnRNA on the basis of cap 0, or "cap1”), cap 2 (a cap structure formed by further methylating the 2'-OH of the second nucleotide sugar group of hnRNA on the basis of cap 1, or "cap2”), cap 4, cap 0 analogs, cap 1 analogs, cap 2 analogs or cap 4 analogs.
  • cap 1 a cap structure formed by further methylating the 2'-OH of the first nucleotide sugar group of hnRNA on the basis of cap 0, or "cap1”
  • cap 2 a cap structure formed by further methylating the 2'-OH of the second nucleotide sugar group of hnRNA on the basis of cap 1, or "cap2”
  • cap 4 cap 0 analogs, cap 1 analogs, cap 2 analogs or cap 4 analogs.
  • the term "expression” includes transcription and/or translation of a nucleotide sequence. Thus, expression may involve the production of transcripts and/or polypeptides.
  • transcription refers to the process of transcribing the genetic code in a DNA sequence into RNA (transcript).
  • in vitro transcription refers to the in vitro synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation.
  • a vector that can be used to produce a transcript is also referred to as a "transcription vector,” which contains regulatory sequences required for transcription.
  • transcription encompasses "in vitro transcription.”
  • the term "host cell” refers to a cell for receiving, maintaining, replicating, expressing a polynucleotide or a vector.
  • the term "host cell” includes prokaryotic cells (e.g., Escherichia coli) or eukaryotic cells (e.g., yeast cells and insect cells). For example, cells from humans, mice, hamsters, pigs, goats, primates.
  • the cell can be derived from a variety of tissue types and includes primary cells and cell lines. Some specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells, and embryonic stem cells.
  • the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte, or a macrophage.
  • Nucleic acid can be present in a host cell with a single copy or with several copies.
  • the host cell can be a cell expressing the polypeptide of the application therein.
  • the term "plasmid” generally refers to a circular DNA molecule, but the term can also encompass linearized DNA molecules. Specifically, the term “plasmid” also encompasses molecules obtained by, for example, digesting a circular plasmid with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule and linearizing the circular plasmid. Plasmids can replicate, i.e., amplify the genetic information stored as chromosomal DNA in cells independently, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In an alternative specific example, the DNA plasmid is a medium copy or high copy plasmid.
  • the DNA plasmid is a high copy plasmid.
  • high copy plasmids include, for example, pUC and pTZ plasmids or any other plasmids (e.g., pMB1, pCoIE1) comprising a replication origin that supports high copies of the plasmid.
  • vaccine is typically understood as a prophylactic or therapeutic material that provides at least one antigen or has antigenic function and can stimulate the body's adaptive immune system to provide an adaptive immune response.
  • treatment and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. Therefore, the treatment of the present application may relate to the treatment of a certain disease state, but may also relate to a preventive treatment for preventing a disease or its symptoms completely or in part. In some embodiments, the term “treatment” is understood to be therapeutic in terms of partially or completely curing a disease and/or attributing to the adverse effects and/or symptoms of the disease. Treatment may also be prophylactic or preventive treatment, i.e., measures taken to prevent a disease, such as to prevent infection and/or the onset of a disease.
  • the terms "subject” and “patient” can be used interchangeably.
  • the subject is a mammal, such as a human, a non-human primate (e.g., ape, chimpanzee, monkey and orangutan), a domestic animal (including dogs and cats and livestock (e.g., horses, cattle, pigs, sheep and goats)) or other mammals.
  • Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.
  • the subject is a human.
  • the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease.
  • the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.
  • administer refers to providing or administering a medicament to a subject by any effective route.
  • routes of administration include, but are not limited to, one or more of the following: injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous), oral, intracavitary, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation.
  • injection e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, intrathecal, intracerebroventricular, or intravenous
  • oral intracavitary, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation.
  • administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom thereof.
  • prevent a disease, disorder, condition, or symptom administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom.
  • the nucleic acid comprises a polynucleotide encoding a mutant of the MPV F protein, and the mutations of the mutant include: A147C and A159C relative to the wild-type MPV F protein, and in another embodiment, the nucleic acid is mRNA, then the following scheme is also an embodiment of the present application: a nucleic acid comprising a polynucleotide encoding a mutant of the MPV F protein, and the mutations of the mutant include: A147C and A159C relative to the wild-type MPV F protein, and the nucleic acid is mRNA.
  • the genome of hMPV is about 13kb in length and contains 8 genes, encoding the following 9 proteins: nucleoprotein (N protein for short), phosphoprotein (P protein for short), matrix protein (M protein for short), fusion protein (F protein for short), small hydrophobic protein (SH protein for short), adsorption protein (G protein for short), RNA polymerase (L protein for short), transcription elongation factor encoded by M2 gene (M2-1 for short) and RNA synthesis regulatory factor (M2-2 for short).
  • N protein for short nucleoprotein
  • P protein for short phosphoprotein
  • M protein for short matrix protein
  • F protein for short small hydrophobic protein
  • G protein for short adsorption protein
  • L protein for short small hydrophobic protein
  • M2-1 transcription elongation factor encoded by M2 gene
  • M2-2 RNA synthesis regulatory factor
  • F protein, G protein and SH protein are viral envelope glycoproteins, and F protein is essential for infection.
  • the F protein monomer encoded by the F gene is initially an inactive precursor F0, which is hydrolyzed and cut into two subunits F1 and F2 by the action of proteases. F1 and F2 are connected by a disulfide bond.
  • the amino acid sequence of the hMPV F protein Given the substantial conservation of the amino acid sequence of the hMPV F protein, one of ordinary skill in the art can readily compare the amino acid positions between different native hMPV F protein sequences to determine the amino acid positions of the corresponding hMPV F protein between different hMPV strains and subtypes. Thus, the conservation of the amino acid sequence of the native hMPV F protein between strains and subtypes allows the use of the amino acid sequence of the reference hMPV F protein to compare the amino acids at specific positions in the hMPV F protein.
  • the amino acid positions of the hMPV F protein herein are given with reference to the sequence of hMPV F0 shown in SEQ ID NO: 64 (i.e., the amino acid sequence of the full-length native F precursor polypeptide of the A2 subtype strain CAN97-83 (GenBank: AY297749.1)).
  • SEQ ID NO: 64 i.e., the amino acid sequence of the full-length native F precursor polypeptide of the A2 subtype strain CAN97-83 (GenBank: AY297749.1)
  • different hMPV F0 may have different numbering systems (e.g., additional amino acid residues may be added or deleted compared to SEQ ID NO: 64).
  • a particular amino acid residue is referred to by a number, it is not limited to the amino acid residue at exactly that numbered position when counting from the beginning of a given amino acid sequence, but also includes the equivalent/corresponding amino acid residues in the amino acid sequence of any and all hMPV F proteins, even if the amino acid residue is not at the same exact numbered position (for example if the hMPV F protein sequence is shorter or longer than SEQ ID NO:64, or has insertions or deletions compared to SEQ ID NO:64).
  • One embodiment of the present disclosure provides a nucleic acid comprising a polynucleotide for encoding a mutant of the MPV F protein.
  • the mutant of the MPV F protein (hereinafter referred to as "mutant") comprises or is a disulfide bond mutation.
  • the MPV F protein is hMPV F protein.
  • the disulfide bond mutations include or are one or more of the following: A147C and A159C, E146C and T160C, F168C and F196C, L165C and F196C, N145C and A161C, S149C and V157C, T59C and N180C, T150C and R156C, V52C and L165C, V55C and V169C, L58C and T174C, V148C and L158C. It should be noted that, in this article, “multiple” means at least two, for example, two, three, four, five or more.
  • the disulfide bond mutations include or are one or more of the following: A147C and A159C, L165C and F196C, N145C and A161C, S149C and V157C, T59C and N180C.
  • the disulfide bond mutation is selected from one of the following: A147C and A159C, L165C and F196C, N145C and A161C.
  • the mutant is selected from one of the following (1) to (38):
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 1 or 2;
  • mutants E146C and T160C comprising mutations E146C and T160C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 3 or 4;
  • a mutant comprising mutations F168C and F196C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 5 or 6;
  • a mutant comprising mutations L165C and F196C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:7 or 8;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 11 or 12;
  • a mutant comprising mutations T59C and N180C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 13 or 14;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 15 or 16;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 17 or 18;
  • a mutant comprising mutations T59C and N180C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 19 or 20;
  • (22) a mutant comprising mutations T150C and R156C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 21 or 22;
  • a mutant comprising mutations V52C and L165C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 23 or 24;
  • a mutant comprising mutations V148C and L158C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO: 29 or 30;
  • a mutant comprising the amino acid sequence shown in SEQ ID NO: 81 or 82;
  • (32) a mutant comprising mutations V147C and L159C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO:81 or 82;
  • a mutant comprising mutations A147C and A159C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as set forth in SEQ ID NO:83;
  • a mutant comprising mutations N145C and A161C and comprising an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to the amino acid sequence as shown in SEQ ID NO:85.
  • the wild-type MPV is subtype A or subtype B.
  • the wild-type MPV is wild-type hMPV.
  • the wild-type hMPV is subtype A1, subtype A2, subtype B1, or subtype B2.
  • the wild-type hMPV strain is CAN97-83 (GenBank: AY297749.1), NL/1/99 (GenBank: AY525843.1), NL/1/00 (GenBank: AF371337.2), CA/83/97 (GenBank: AY297749), JP/240/03 (GenBank: AY530095.1), CN/gz01/08 (GenBank: GQ153651.1).
  • the amino acid sequence of the wild-type MPV F protein is shown in SEQ ID NO: 64 or 65. It is understood that in other embodiments, the wild-type MPV is not limited to the above list, but can also be others.
  • the mutant comprises an F1 polypeptide and an F2 polypeptide.
  • the F1 polypeptide and the F2 polypeptide of the mutant of the MPV protein into which one or more mutations are introduced can be derived from any wild-type MPV F protein known in the art or discovered in the future, including but not limited to MPV subtype A and subtype B (e.g., subtype A1, subtype A2, subtype B1, or subtype B2) or F proteins of any other subtype.
  • the F1 polypeptide and the F2 polypeptide can be derived from the F protein of the same genotype MPV or from F proteins of different genotypes of MPV.
  • the F1 polypeptide is derived from the F protein of the A subtype MPV, and the F2 polypeptide is derived from the F protein of the B subtype MPV; or the F1 polypeptide is derived from the F protein of the B subtype MPV, and the F2 polypeptide is derived from the F protein of the A subtype MPV.
  • the mutant further comprises one or more of the following: a signal peptide, a ferritin polypeptide, a transmembrane domain (TM) of an MPV F protein (e.g., a transmembrane domain of a wild-type MPV F protein), and a trimerization domain (e.g., a foldon).
  • a ferritin polypeptide refers to a polypeptide derived from ferritin, which may be a full-length ferritin, a full-length ferritin with a mutation, a truncated ferritin, or a truncated ferritin with a mutation.
  • the trimerization domain is used to promote the formation of a trimer of three F1/F2 heterodimers.
  • the trimerization domain is not particularly limited, and may be, for example, a GCN4 leucine zipper, a trimerization motif from a lung surfactant protein, a phage T4 fibritin foldon, and the like.
  • the foldon is located at the C-terminus of the F1 polypeptide.
  • the amino acid sequence of the foldon is: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 66).
  • the ferritin polypeptide is located at the C-terminus of the F1 polypeptide.
  • the amino acid sequence of the ferritin polypeptide is shown as SEQ ID NO: 67.
  • the mutant also contains a transmembrane domain (TM) of the MPV F protein (e.g., the transmembrane domain of the wild-type MPV F protein).
  • TM transmembrane domain
  • the mutant further comprises one or more of the following: a signal peptide, a transmembrane domain of the MPV F protein, and a trimerization domain (e.g., a foldon).
  • the mutant comprises a signal peptide and a trimerization domain.
  • the mutant comprises a transmembrane domain of the MPV F protein and a trimerization domain.
  • the mutant comprises a signal peptide, a transmembrane domain of the MPV F protein, and a trimerization domain.
  • the mutant comprises one or more of the following: a signal peptide, a transmembrane domain of an MPV F protein, and a ferritin polypeptide. In some embodiments, the mutant comprises a signal peptide and a ferritin polypeptide. In some embodiments, the mutant comprises a ferritin polypeptide and a transmembrane domain of an MPV F protein. In other embodiments, the mutant comprises a signal peptide, a transmembrane domain of an MPV F protein, and a ferritin polypeptide.
  • the mutant F1 polypeptide has the same length as the full-length F1 polypeptide of the corresponding wild-type MPV F protein.
  • the full-length F1 polypeptide of the mutant of the MPV F protein corresponds to amino acid positions 103 to 539 of the native MPV F0 precursor, including (from N-terminus to C-terminus) the extracellular region (residues 103 to 488), the transmembrane domain (residues 489 to 513), and the cytoplasmic domain (residues 514 to 539).
  • the mutant F1 polypeptide has a deletion relative to the full-length F1 polypeptide (or native F1 polypeptide) of the corresponding wild-type MPV F protein. In some embodiments, the mutant F1 polypeptide has a deletion of one or more of the following relative to the full-length F1 polypeptide of the corresponding wild-type MPV F protein: a portion of the extracellular region, a portion of the transmembrane domain, the entire transmembrane domain, a portion of the cytoplasmic domain, and the entire cytoplasmic domain. For example, the optional sequence from amino acid residue 474 onwards of the native F1 polypeptide may not exist in the mutant F1 polypeptide.
  • the optional sequence from amino acid residue 478 or 491 onwards of the native F1 polypeptide may not exist in the mutant F1 polypeptide, that is, the mutant F1 polypeptide is missing 1 to 62 or 1 to 49 amino acid residues from the C-terminus of the native F1 polypeptide.
  • the mutant F1 polypeptide lacks the entire cytoplasmic domain.
  • the mutant F1 polypeptide lacks the cytoplasmic domain and a portion or all of the transmembrane domain.
  • the F1 polypeptide of the mutant lacks amino acid residues from positions 489, 490, 491, 510, 512, 513, 514, 515, 520, 525, or 530 to 539 compared to the native F1 polypeptide.
  • amino acid residues from positions 489, 490, or 491 to 539 are absent.
  • the mutant has attached trimerization domains or ferritin polypeptides, and the F1 polypeptide of the mutant lacks amino acid residues from positions 489, 490, or 491 to 539 compared to the native F1 polypeptide.
  • the F1 polypeptide of the mutant comprises or consists of amino acid residues 103 to 490 of the native F0 polypeptide.
  • the mutant F2 polypeptide may have the same length as the full-length F2 polypeptide of the corresponding wild-type MPV F protein, or may have a deletion, such as a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues from the N-terminus or C-terminus of the F2 polypeptide.
  • the above-mentioned nucleic acid also contains one or more of the following: polynucleotides encoding protease cleavage sites (such as thrombin cleavage sites (LVPRGS, SEQ ID NO: 68)), polynucleotides encoding protein tags (such as 6 ⁇ His-tag (HHHHHH, SEQ ID NO: 69) and streptomycin tag II (WSHPGFEK, SEQ ID NO: 70), polynucleotides encoding linker sequences (such as GG, SAIG and GS)).
  • protease cleavage sites such as thrombin cleavage sites (LVPRGS, SEQ ID NO: 68
  • polynucleotides encoding protein tags such as 6 ⁇ His-tag (HHHHHH, SEQ ID NO: 69) and streptomycin tag II (WSHPGFEK, SEQ ID NO: 70
  • polynucleotides encoding linker sequences such as
  • the above-mentioned nucleic acid is an isolated nucleic acid.
  • the above-mentioned nucleic acid is a non-naturally occurring nucleic acid.
  • the nucleic acid is a codon-optimized polynucleotide.
  • Codon optimization methods are known in the art and can be used as provided herein.
  • codon optimization can be used to: match the codon frequencies of the target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base stretches that can impair gene construction or expression; customize transcription and translation control regions; insert or remove protein trafficking sequences; remove/add post-translational modification sites in the encoded protein (e.g., glycosylation sites); add, remove or reorganize protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust the translation rate so that various domains of the protein can fold properly; or reduce or eliminate problematic secondary structures within a polynucleotide.
  • Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), and/or patented methods.
  • an open reading frame (ORF) sequence is optimized using an optimization algorithm.
  • the codon-optimized nucleic acid is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the nucleic acid before codon optimization.
  • the nucleic acid is RNA. In some embodiments, the nucleic acid is RNA, and the RNA contains an open reading frame (ORF).
  • ORF open reading frame
  • the nucleic acid is RNA
  • the RNA is mRNA
  • the nucleic acid further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.
  • the nucleic acid further comprises a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.
  • the 5'-cap structure is selected from at least one of m 7 GpppG, m 2 7,3'-O GpppG, m 7 Gppp(5')N1 and m 7 Gppp(m 2'-O )N1; wherein “m 7 G” represents 7-methylguanosine cap nucleoside, “ppp” represents the triphosphate bond between the 5' carbon of the cap nucleoside and the first nucleotide of the primary RNA transcript, N1 is the 5'-most nucleotide, "G” represents guanine nucleoside, "7” represents the methyl group at the 7-position of guanine, and "m 2'-O " represents the methyl group at the 2'-O position of the nucleotide.
  • the 5'-cap structure is m 7 Gppp(5')N1 or m 7 Gppp(m 2'-O )N1. It is understood that in other embodiments, the 5'-
  • the DNA sequence corresponding to the 5'-UTR is shown in SEQ ID NO: 31; and/or, the DNA sequence corresponding to the 3'-UTR is shown in SEQ ID NO: 32.
  • the "DNA sequence corresponding to the 5'-UTR” refers to the nucleotide sequence of the 5'-UTR in DNA form, and the same applies to the "DNA sequence corresponding to the 3'-UTR” and the "DNA sequence corresponding to the poly(A) tail" below.
  • the 5'-UTR and 3'-UTR are not limited to the above, and may be others, such as the 5'-UTR and 3'-UTR described in patents such as CN108291230A, CN104321432A, and CN107849574A.
  • the nucleotides constituting the poly (A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides. In some embodiments, the nucleotides constituting the poly (A) tail comprise at least 20, at least 40, at least 80, at least 100 or at least 120 A nucleotides consecutively.
  • the nucleotides constituting the poly(A) tail include one or more nucleotides other than A nucleotides. In some embodiments, the poly(A) tail includes two or more consecutive nucleotides other than A nucleotides. In an optional specific example, the nucleotide DNA sequence corresponding to the poly(A) tail is shown in SEQ ID NO: 33. It is understood that the poly(A) tail contained in the nucleic acid of the present disclosure is not limited to the above, and may also be other poly(A) tails, such as the poly(A) tails described in patents such as US20170166905A1 and WO2020074642A1.
  • the nucleic acids described above do not contain modified nucleotides.
  • the above-mentioned nucleic acids contain modified nucleotides.
  • the nucleic acid contains modified nucleosides.
  • the modified nucleosides contained in the nucleic acid include at least one of modified uridine, modified cytidine, modified adenosine and modified guanosine.
  • the modified nucleoside in the above-mentioned nucleic acid is a modified uridine. In some embodiments, 0.1% to 100% of the uridine in the above-mentioned nucleic acid is modified. In some embodiments, 80% to 100% of the uridine is modified. In some embodiments, 100% of the uridine is modified.
  • Exemplary modified uridines include pseudouridine ( ⁇ ), N1-methyl pseudouridine, pyridine-4-ketone ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine-5-oxyacetic acid (cmo5U), uridine-5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (ch
  • the modified nucleosides in the above nucleic acids are modified cytidines. In some embodiments, 0.1% to 100% of the cytidines in the above nucleic acids are modified. In some embodiments, 80% to 100% of the cytidines are modified. In some embodiments, 100% of the cytidines are modified.
  • Exemplary modified cytidines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-d
  • 5-Methoxy-zebularine 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), ⁇ -thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl Cm), N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arabino-cytidine, 2'-F-cytidine and 2'-OH-arabino-cytidine.
  • the modified nucleoside in the above nucleic acid is a modified adenosine. In some embodiments, 0.1% to 100% of the adenosine in the above nucleic acid is modified. In some embodiments, 80% to 100% of the adenosine is modified. In some embodiments, 100% of the adenosine is modified.
  • Exemplary modified adenosines include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl -adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-
  • the modified nucleoside in the above-mentioned nucleic acid is a modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the above-mentioned nucleic acid is modified. In some embodiments, 80% to 100% of the guanosine is modified. In some embodiments, 100% of the guanosine is modified.
  • Exemplary modified guanosines include inosine (I), 1-methyl-inosine (m1I), wyosine (imG), methyl wyosine (mimG), 4-demethyl-wyosine (imG-14), iso-wyosine (imG2), wyosine (yW), peroxy wyosine (o2yW), hydroxy wyosine (OHyW), undermodified hydroxy wyosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (o Q), galactosyl-braided glycoside (galQ), mannosyl-braided glycoside (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-
  • the modified nucleotides in the above-mentioned nucleic acids include nucleotides containing isotopes.
  • the nucleic acid comprises nucleotides containing isotopes of hydrogen.
  • the isotopes of hydrogen are not limited to deuterium and tritium.
  • the nucleic acid further comprises or contains nucleotides of isotopes of other elements besides hydrogen, wherein the other elements include but are not limited to carbon, oxygen, nitrogen and phosphorus.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 34-63 and 86-93 and encodes a corresponding MPV F protein as shown in SEQ ID NOs: 1-30 and 81-85.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the nucleotide sequence of the polynucleotide shown in SEQ ID NO: 34 and encoding the MPV F protein shown in SEQ ID NO: 1.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:1 and a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:34 or 86.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:2 and having a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35 or 87.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:3 and a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:36.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:4 and having a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:5 and a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:38.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:6 and having a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:39.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:7 and having a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:40.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:8 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:41.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:9 and a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:42.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:10 and a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:43.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:11 and a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:44.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:12 and a nucleotide sequence that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:45.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:13 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:46.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:14 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:47.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:15 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:48.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:16 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:49.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:17 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:50.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:18 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:51.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:19 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:52.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:20 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:53.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:21 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:54.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:22 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:55.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:81 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:88.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:82 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:89.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:83 and a nucleotide sequence that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:90 or 92.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:84 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:91.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide encoding an amino acid sequence as shown in SEQ ID NO:85 and a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO:93.
  • the above-mentioned nucleic acid contains RNA corresponding to a polynucleotide whose nucleotide sequence is shown in any one of SEQ ID NOs: 34 to 63 and 86 to 93.
  • the nucleic acid of any of the above embodiments is mRNA, and all uridine in the nucleic acid is replaced by N1-methylpseudouridine.
  • the nucleic acid comprises an RNA corresponding to a polynucleotide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 34 to 63 and 86 to 93 and encoding a corresponding MPV F protein as shown in SEQ ID NOs: 1 to 30 and 81 to 85, and all the uridine in the nucleic acid is replaced by N1-methyl pseudouridine.
  • the above-mentioned nucleic acid contains RNA corresponding to a polynucleotide with a nucleotide sequence as shown in any one of SEQ ID NOs: 34 to 63 and 86 to 93, and all the uridine in the nucleic acid is replaced by N1-methylpseudouridine.
  • the above-mentioned nucleic acid is mRNA, which contains RNA corresponding to a polynucleotide with a nucleotide sequence as shown in any one of SEQ ID NOs: 34 to 63 and 86 to 93, and all the uridine in the nucleic acid is replaced by N1-methylpseudouridine.
  • the nucleic acid is DNA.
  • the nucleic acid comprises a polynucleotide that has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a nucleotide sequence as shown in one of SEQ ID NOs: 34-63 and 86-93 and encodes a polynucleotide of the corresponding MPV F protein as shown in SEQ ID NOs: 1-30 and 81-85.
  • the nucleic acid comprises a polynucleotide having a nucleotide sequence as shown in one of SEQ ID NOs: 34-63 and 86-93.
  • the nucleic acid comprises a polynucleotide encoding a mutant of MPV F protein.
  • a mutant of MPV F protein herein and below refers to any one of the multiple mutants of MPV F protein mentioned above.
  • the nucleic acid comprises polynucleotides encoding multiple mutants of MPV F protein.
  • the coding chains of the multiple mutants of MPV F protein are located on the same nucleic acid chain, and the polynucleotides encoding the multiple mutants of MPV F protein are directly connected or indirectly connected through a connecting element.
  • the "multiple mutants of MPV F protein" here and below refers to any multiple (e.g., two, three, four or more) of the multiple mutants of MPV F protein mentioned above.
  • the multiple mutants of the MPV F protein are different MPV F proteins from the same subtype strain but with different mutations.
  • the multiple mutants of the MPV F protein are two, the first mutant is an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, and the second mutant is an MPV F protein derived from strain CAN97-83 with L165C and F196C mutations.
  • the above-mentioned nucleic acid comprises a polynucleotide encoding an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations and a polynucleotide encoding an MPV F protein derived from strain CAN97-83 with L165C and F196C mutations.
  • the multiple mutants of the MPV F protein are different MPV F proteins from different subtype strains but with the same mutations.
  • there are two multiple mutants of the MPV F protein the first mutant is an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, and the second mutant is an MPV F protein derived from strain NL/1/99 with A147C and A159C mutations.
  • the above nucleic acid comprises a polynucleotide encoding an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations and a polynucleotide encoding an MPV F protein derived from strain NL/1/99 with A147C and A159C mutations.
  • the multiple mutants of the MPV F protein are different MPV F proteins from different subtype strains and with different mutations.
  • there are two multiple mutants of the MPV F protein the first mutant is an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, and the second mutant is an MPV F protein derived from strain NL/1/99 with L165C and F196C mutations.
  • the above-mentioned nucleic acid comprises a nucleic acid encoding a polynucleotide of an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations and a polynucleotide of an MPV F protein derived from strain NL/1/99 with L165C and F196C mutations.
  • the first mutant is a polynucleotide of MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations
  • the second mutant is a polynucleotide of MPV F protein derived from strain NL/1/99 and having L165C and F196C mutations
  • the third mutant is a polynucleotide of MPV F protein derived from strain CAN97-83 and having N145C and A161C mutations.
  • the above nucleic acid includes a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations, a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having L165C and F196C mutations, and a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having N145C and A161C mutations.
  • the nucleic acid further comprises a polynucleotide encoding other proteins or polypeptides other than mutants of MPV F protein.
  • proteins or polypeptides other than mutants of MPV F protein refer to proteins or polypeptides with biological significance (e.g., immunogenicity), such as other proteins or polypeptides of MPV, or mutants thereof (e.g., G protein of MPV or mutants thereof), or proteins or polypeptides of other viruses, or mutants thereof.
  • nucleic acid whether it is a polynucleotide encoding a mutant of MPV F protein or a polynucleotide encoding multiple mutants of MPV F protein, there can be multiple (for example, two or three) repeating units on the same chain.
  • a polynucleotide encoding a mutant of MPV F protein a polynucleotide encoding one of the mutants of MPV F protein is referred to as "A fragment", and multiple A fragments can be contained in the same nucleic acid.
  • a polynucleotide encoding one of the mutants of MPV F protein is referred to as "A fragment”
  • a polynucleotide encoding another mutant of MPV F protein is referred to as "B fragment”
  • the same nucleic acid can contain multiple A fragments and multiple B fragments, 1 A fragment and multiple B fragments, or multiple A fragments and 1 B fragment.
  • the nucleic acid is any one of the mRNAs in Table 1.
  • the mRNA in Table 1 is an mRNA encoding a mutant of hMPV F protein, which comprises a Cap1-type cap structure, a 5'-UTR corresponding to the sequence shown in SEQ ID NO: 31, a 3'-UTR corresponding to the sequence shown in SEQ ID NO: 32, a poly(A) tail corresponding to the sequence shown in SEQ ID NO: 33, and an F protein coding region corresponding to one of SEQ ID NO: 34-63 and 86-93, wherein the F protein coding region is RNA, and all uridines in all mRNAs in Table 1 are replaced by N1-methyl pseudouridine.
  • the mRNA shown in No. 1154 contains a Cap1-type cap structure, a 5'-UTR corresponding to the sequence shown in SEQ ID NO: 31, a 3'-UTR corresponding to the sequence shown in SEQ ID NO: 32, a poly(A) tail corresponding to the sequence shown in SEQ ID NO: 33, and an F protein coding region corresponding to the sequence shown in SEQ ID NO: 34, and all the uridines in the mRNA shown in No. 1154 are replaced by N1-methyl pseudouridine.
  • all mRNAs in the G7 group also contain a polynucleotide encoding an amino acid sequence such as Ferritin shown in SEQ ID NO: 67 at their C-terminus; all mRNAs in the G8 group also contain a polynucleotide encoding a foldon derived from T4 bacteriophage fiber protein shown in SEQ ID NO: 66 at their C-terminus.
  • the above-mentioned nucleic acid comprises a stop codon. It is understood that in other embodiments, the above-mentioned nucleic acid does not contain a stop codon.
  • a stop codon e.g., UGA or TGA
  • the above-mentioned nucleic acid may comprise one or more stop codons.
  • the above-mentioned nucleic acid contains a polynucleotide encoding a mutant of MPV F protein with a disulfide bond mutation, which can induce a specific immune response to prevent or at least reduce human metapneumovirus infection.
  • the nucleic acid or the mutant of the MPV F protein encoded by it can induce a significant IFN- ⁇ positive cellular immune response. In some embodiments, the nucleic acid or the mutant of the MPV F protein encoded by it can induce a significant IFN- ⁇ positive cellular immune response, which is Th1 biased.
  • the above-mentioned nucleic acid or a mutant of the MPV F protein encoded therein can induce the production of neutralizing antibodies against subtype A hMPV.
  • the above-mentioned nucleic acid or a mutant of the MPV F protein encoded therein can induce the production of neutralizing antibodies against subtype B hMPV.
  • the above-mentioned nucleic acid or a mutant of the MPV F protein encoded therein can induce the production of neutralizing antibodies against subtype A hMPV and subtype B hMPV.
  • this genetic engineering vector comprises the nucleic acid of any one of the above-mentioned embodiments, or this genetic engineering vector comprises the polynucleotide that can be transcribed into the nucleic acid of any one of the above-mentioned embodiments.
  • genetic engineering vector is plasmid, cosmid, virus, phage or another carrier conventionally used in genetic engineering.
  • genetic engineering vector is plasmid.
  • genetic engineering vector also at least comprises following one or more replication origins (ORI), marker gene or its fragment, reporter gene or its fragment and restriction site that allows insertion of DNA element.Preferably restriction site of multiple cloning site (MCS) form.
  • ORI replication origins
  • MCS multiple cloning site
  • the genetic engineering vector is an expression vector.
  • the genetic engineering vector comprises a promoter, a 5'-UTR, a coding region of a mutant of the MPV F protein of any of the above embodiments (referred to as "mutant coding region"), a 3'-UTR and a poly(A) tail, wherein the poly(A) tail, the promoter, the 5'-UTR, the mutant coding region and the 3'-UTR are operably linked to each other.
  • the genetic engineering vector is a cloning vector. It is understood that the nucleic acid contained in the genetic engineering vector does not contain a 5'-cap structure.
  • the present disclosure also provides a method for preparing the nucleic acid of any of the above embodiments, which comprises the steps of introducing (for example, in the form of a plasmid) the genetic engineering vector of any of the above embodiments into a host cell (for example, Escherichia coli) and then culturing the host cell containing the nucleic acid.
  • a host cell for example, Escherichia coli
  • the present disclosure also provides another method for preparing the above-mentioned nucleic acid, which comprises the step of preparing the nucleic acid by chemical synthesis according to the nucleotide sequence of the nucleic acid of any of the above-mentioned embodiments. It is understood that the specific method of the chemical synthesis method can be a method known in the art, such as a solid phase phosphoramidite method.
  • the present disclosure also provides a host cell, which comprises the nucleic acid of any of the above embodiments or the genetic engineering vector of any of the above embodiments.
  • the host cell described above is an isolated cell.
  • the host cells are used to store and/or amplify the above-mentioned nucleic acids.
  • the host cell is a bacterial cell.
  • Bacterial host cells include Escherichia coli (E. coli) cells well known to those skilled in the art.
  • Host cells of the present disclosure can be prepared by transforming competent host cells by the genetic engineering vector of any of the above-mentioned embodiments.
  • Competent host cells are cells with the ability of free extracellular genetic material (e.g., DNA plasmid) that is independent of sequence uptake.
  • DNA plasmid DNA plasmid
  • Various bacterial cells known to those skilled in the art are naturally capable of taking in exogenous DNA from the environment, and therefore can serve as bacterial host cells according to the present disclosure.
  • competent bacterial host cells can be obtained from natural non-competent bacterial cells using, for example, electroporation or chemicals (e.g., treated with calcium ions and accompanied by high temperature exposure). After uptake, exogenous DNA is preferably neither degraded nor integrated in the genome of the bacterial host cell.
  • the present disclosure also provides a mutant of MPV F protein, which is encoded by the nucleic acid of any of the above embodiments.
  • the mutant of MPV F protein is a unimolecular or multimolecular complex (e.g., a trimer).
  • an MPV immunogen which comprises a mutant of the MPV F protein encoded by the nucleic acid of any of the above embodiments.
  • the MPV immunogen is a trimer.
  • the mutations of the monomers of the trimer are completely identical. In other embodiments, the mutations of the three monomers of the trimer are partially identical or completely different.
  • the present disclosure also provides a method for preparing a mutant of MPV F protein, the preparation method comprising: transcribing a nucleic acid containing a polynucleotide encoding a mutant of MPV F protein into mRNA; and translating the transcribed mRNA into a polypeptide or protein.
  • the method for preparing the mutant of the MPV F protein is carried out entirely in vitro or partially in vitro.
  • the present disclosure also provides a method for preparing RNA, which comprises the step of using the genetic engineering vector of any of the above embodiments of the present disclosure to perform transcription.
  • the method for preparing RNA is an in vitro method.
  • the method for preparing RNA includes contacting the genetic engineering vector (e.g., plasmid) of any of the above embodiments with RNA polymerase.
  • the method for preparing RNA also includes the step of linearizing the genetic engineering vector (e.g., plasmid).
  • the supercoil rate of the genetic engineering vector e.g., plasmid
  • the method for preparing RNA also includes the step of purifying the linearized genetic engineering vector.
  • the method for preparing RNA also includes the step of purifying RNA.
  • the present disclosure also provides another method for preparing RNA, which comprises the step of preparing the RNA by chemical synthesis according to the nucleotide sequence of the RNA or DNA corresponding to any of the above embodiments. It is understood that the specific method of the chemical synthesis method can be a method known in the art, such as a solid phase phosphoramidite method.
  • the RNA is mRNA.
  • any of the above methods for preparing RNA further comprises the steps of capping and optionally purifying the capped product.
  • the cap is a Cap1 type cap.
  • the Cap1 type cap structure is as follows:
  • the capping reaction is as follows:
  • the method for preparing RNA is a partially in vitro method.
  • the RNA method for preparing RNA at this time includes the following steps: preparing the genetic engineering vector of any of the above embodiments in vitro; and introducing the genetic engineering vector into the body (for example, in the form of a plasmid).
  • the genetic engineering vector is wrapped in a delivery vector. At this time, the genetic engineering vector is delivered to the body via a delivery vector.
  • the RNA prepared in the method for preparing RNA of any of the above embodiments comprises modified nucleosides or modified nucleotides.
  • the raw material for preparing the RNA comprises one or more modified nucleosides or nucleotides. It is understood that the amount and type of the modified nucleosides or nucleotides correspond to the RNA to be prepared.
  • the present disclosure also provides an RNA, which is prepared by the method for preparing RNA according to any of the above embodiments.
  • the RNA is mRNA.
  • the present disclosure also provides a nucleic acid composition, which comprises a first nucleic acid or a first genetic engineering vector, wherein the first nucleic acid is the nucleic acid of any of the above embodiments, and the first genetic engineering vector is the genetic engineering vector of any of the above embodiments.
  • the first nucleic acid is RNA. In some embodiments, the first nucleic acid is mRNA.
  • the nucleic acid composition comprises a first nucleic acid or a first genetic engineering vector
  • a first nucleic acid is a nucleic acid comprising a polynucleotide encoding any one or more of the multiple mutants of the above-mentioned MPV F protein
  • a first genetic engineering vector is a genetic engineering vector comprising a polynucleotide encoding any one or more of the multiple mutants of the above-mentioned MPV F protein.
  • the nucleic acid composition includes a first nucleic acid or a first genetic engineering vector
  • a first nucleic acid is a nucleic acid containing a polynucleotide encoding one of the multiple mutants of the above-mentioned MPV F protein
  • a first genetic engineering vector is a genetic engineering vector containing a polynucleotide encoding one of the multiple mutants of the above-mentioned MPV F protein.
  • the nucleic acid composition includes a first nucleic acid or a first genetic engineering vector
  • a first nucleic acid is a nucleic acid containing a polynucleotide encoding multiple mutants among the multiple mutants of the above-mentioned MPV F protein
  • a first genetic engineering vector is a genetic engineering vector containing a polynucleotide encoding multiple mutants among the multiple mutants of the above-mentioned MPV F protein, wherein the polynucleotides encoding multiple mutants among the multiple mutants of the above-mentioned MPV F protein are located on the same nucleic acid chain.
  • the nucleic acid composition includes multiple first nucleic acids or multiple first genetic engineering vectors, the multiple first nucleic acids are independent of each other or the multiple first genetic engineering vectors are independent of each other, the multiple first nucleic acids are used to encode multiple mutants of MPV F protein, and the multiple first genetic engineering vectors are used to clone or express multiple polynucleotides encoding MPV F protein.
  • independent of each other here means that there are no connecting elements connecting the multiple first nucleic acids, or there are no connecting elements connecting the multiple first genetic engineering vectors.
  • the various polynucleotides encoding the multiple mutants of MPV F protein are not on the same nucleic acid chain.
  • the multiple mutants of the MPV F protein corresponding to the multiple first nucleic acids are MPV F proteins derived from different subtype strains and having the same mutations, MPV F proteins derived from different subtype strains and having different mutations, and/or MPV F proteins derived from the same subtype strain but having different mutations.
  • the plurality of first nucleic acids are nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations (or referred to as a first first nucleic acid), and nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having A147C and A159C mutations (or referred to as a second first nucleic acid).
  • the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid being a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations, and the second first nucleic acid being a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having A147C and A159C mutations.
  • the plurality of first nucleic acids are nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations (a first first nucleic acid), and nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having L165C and F196C mutations (a second first nucleic acid).
  • the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid being a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations, and the second first nucleic acid being a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having L165C and F196C mutations.
  • the plurality of first nucleic acids are nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations (a first first nucleic acid), and nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having L165C and F196C mutations (a second first nucleic acid).
  • the nucleic acid composition comprises two first nucleic acids, the first first nucleic acid being a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations, and the second first nucleic acid being a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having L165C and F196C mutations.
  • the multiple first nucleic acids are nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations, nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having A147C and A159C mutations, and nucleic acids comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having L165C and F196C mutations.
  • the nucleic acid composition comprises three first nucleic acids, the first first nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations, the second first nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having A147C and A159C mutations, and the third first nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having L165C and F196C mutations.
  • nucleic acid composition is not limited to including one or more of the above-mentioned first nucleic acids or first genetic engineering vectors, but also includes other nucleic acids (such as nucleic acids encoding other immunogens) and/or other substances (such as buffers or lyophilization protectants, etc.).
  • the nucleic acid composition further comprises a second nucleic acid or a second vector, the second nucleic acid comprising a polynucleotide encoding other proteins or polypeptides other than the mutant of MPV F protein, and the second vector comprising a polynucleotide encoding other proteins or polypeptides other than the mutant of MPV F protein.
  • Other proteins or polypeptides other than the mutant of MPV F protein are as described above.
  • the second nucleic acid is RNA.
  • the second nucleic acid is mRNA. It is understood that the second nucleic acid contained in the nucleic acid composition is not limited to one, but may be multiple.
  • the nucleic acid composition may also include a second nucleic acid, which comprises a polynucleotide encoding a protein or polypeptide other than the mutant of MPV F protein; the nucleic acid composition may also include multiple second nucleic acids, which are independent of each other and are multiple nucleic acids comprising polynucleotides encoding other proteins or polypeptides other than the mutant of MPV F protein.
  • the second vector is also not limited to one, but may be multiple.
  • the present disclosure also provides a delivery vector comprising the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetic engineering vector of any of the above embodiments, or the nucleic acid composition of any of the above embodiments.
  • the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.
  • the delivery vehicle is selected from a plurality of composites or one of the following: lipid nanoparticles (LNPs), liposomes, cationic proteins, vesicles, microparticles, polymers and micelles. In some embodiments, the delivery vehicle is selected from one of the following: lipid nanoparticles, liposomes, cationic proteins, vesicles, microparticles, polymers and micelles.
  • LNPs lipid nanoparticles
  • cationic proteins vesicles, microparticles, polymers and micelles.
  • the delivery vehicle is a lipid nanoparticle (LNPs) comprising a nucleic acid according to any of the above embodiments, an RNA according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, or a nucleic acid composition according to any of the above embodiments.
  • LNPs lipid nanoparticle
  • lipid nanoparticles refer to particles having a nanometer scale (eg, 1 nm to 1000 nm) that include one or more lipids.
  • the average diameter of the lipid nanoparticles is 20nm to 800nm, 20nm to 500nm, 20nm to 400nm, 20nm to 300nm, 20nm to 200nm, 20nm to 100nm, 30nm to 700nm, 30nm to 500nm, 30nm to 300nm, 30nm to 200nm, 30nm to 100nm, 40nm to 800nm, 40nm to 600nm, 40nm to 500nm, 40nm to 300nm, 40nm to 400nm, 40nm to 500nm, 40nm to 300nm, 40nm to 600nm, 40nm to 700nm, 40nm to 800nm, 40nm to 600nm, 40nm to 3 ...
  • the average diameter of the lipid nanoparticles is 26nm, 31nm, 36nm, 41nm, 46nm, 51nm, 56nm, 61nm, 66nm, 71nm, 76nm, 81nm, 86nm, 91nm, 96nm, 101nm, 106nm, 111nm, 116nm, 121nm, 126nm, 131nm, 136nm, 141nm, 146nm, 151nm, 156nm, 161nm, 166nm, 171nm, 176nm, 181nm, 186nm, 191nm, 196nm, 201nm, 206nm, 211nm, 216nm, 221nm, 226nm, 231nm, 236nm, 241nm, 246nm or 249nm.
  • the average diameter of lipid nanoparticles can be
  • the lipid nanoparticles include one of the following: cationic lipid nanoparticles (cationic lipid nanoparticles), solid lipid nanoparticles (solid lipid nanoparticles, SLN), nanostructured lipid carriers (nanostructured lipid carriers, NLC), nonlamellar lipid nanoparticles (nonlamellar lipid nanoparticles).
  • the lipid nanoparticles are cationic lipid nanoparticles.
  • lipid nanoparticles contain one or more of the following substances: cationic lipids, auxiliary lipids, structural lipids, and polymer-lipids.
  • cationic lipid refers to a lipid that becomes positively charged when the pH is reduced below the pKa of the ionizable group of the lipid, but gradually becomes neutral at higher pH values. When the pH value is below the pKa, the positively charged lipid can bind to negatively charged nucleic acids.
  • the cationic lipid comprises a zwitterionic lipid.
  • the cationic lipid comprises the following compound (I), its N-oxide, its salt or its isomer:
  • R1 is selected from the group consisting of C5 - C30 alkyl, C5 - C20 alkenyl, -R*YR", -YR", and -R"'M'R';
  • R 2 and R 3 are independently selected from the group consisting of H, C 1 -C 14 alkyl, C 2 -C 14 alkenyl, -R*YR", -YR", and -R*OR", or R 2 and R 3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring;
  • R4 is selected from the group consisting of hydrogen, C3 - C6 carbocycle, -( CH2 ) nQ , -( CH2 ) nCHQR , -( CH2 ) oC ( R10 ) 2 ( CH2 ) noQ , -CHQR, -CQ(R) 2 , and unsubstituted C1 - C6 alkyl, wherein Q is selected from carbocycle, heterocycle, -OR, -O( CH2 ) nN (R) 2 , -C(O)OR, -OC(O)R, -CX3 , -CX2H , -CXH2, -CN, -N(R) 2 , -C(O)N(R) 2 , -N(R) C (O)R, -N(R)S(O) 2R , -N(R)C(O)N(R) 2 , -N(R)C(
  • each R 5 is independently selected from the group consisting of OH, C 1 -C 3 alkyl, C 2 -C 3 alkenyl, and H;
  • Each R 6 is independently selected from the group consisting of OH, C 1 -C 3 alkyl, C 2 -C 3 alkenyl, and H;
  • M and M' are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M"-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O) 2- , -SS-, an aryl group, and a heteroaryl group, wherein M" is a bond, a C1 - C13 alkylene group, or a C2 -- C13 alkenylene group;
  • R7 is selected from the group consisting of C1-3 alkyl, C2 - C3 alkenyl and H;
  • R 8 is selected from the group consisting of C 3-6 carbocyclic ring and heterocyclic ring;
  • R 9 is selected from the group consisting of H, CN, NO 2 , C 1 -C 6 alkyl, -OR, -S(O) 2 R, -S(O) 2 N(R) 2 , C 2 -C 6 alkenyl, C 3 -C 6 carbocycle, and heterocycle;
  • R 10 is selected from the group consisting of H, C 1 -C 3 alkyl and C 2 -C 3 alkenyl;
  • each q is independently selected from 1, 2 and 3;
  • Each R' is independently selected from the group consisting of C1 - C18 alkyl, C2- C18 alkenyl , -R*YR", -YR", H and and R 11 is selected from the group consisting of C 1 -C 12 alkylene and C 2 -C 12 alkenylene, and R 12 and R 13 are each independently selected from the group consisting of C 1 -C 12 alkyl and C 2 -C 12 alkenyl;
  • each R" is independently selected from the group consisting of C 3 -C 15 alkyl and C 3 -C 15 alkenyl;
  • each R'' is independently selected from the group consisting of C 3 -C 15 alkylene and C 3 -C 15 alkenylene;
  • each R* is independently selected from the group consisting of absent, C 1 -C 12 alkylene, and C 2 -C 12 alkenylene;
  • each R** is independently selected from the group consisting of absent, C 1 -C 12 alkyl, and C 2 -C 12 alkenyl;
  • Each Y is independently a C 3 -C 6 carbocyclic ring
  • each X is independently selected from the group consisting of: F, Cl, Br and I;
  • n is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; and wherein when R 4 is -(CH 2 ) n Q, -(CH 2 ) n CHQR, -CHQR or -CQ(R) 2 , then (i) when n is 1, 2, 3, 4 or 5, Q is not -N(R) 2 ; or (ii) when n is 1 or 2, Q is not 5-, 6- or 7-membered heterocycloalkyl.
  • the cationic lipid is the following compound (I), its N-oxide, its salt or its isomer:
  • R 1 -R 7 , M and m are as defined above.
  • the cationic lipid comprises the following compound (II), an N-oxide thereof, a salt thereof, or an isomer thereof:
  • R 1 , R 2 , R 3 , R 5 , R 6 , M and R 7 are as described above,
  • RN is H or C1 - C3 alkyl
  • Xa and Xb are each independently O or S;
  • R 14 is selected from H, halogen, -OH, R b , -N(R b ) 2 , -CN, -N 3 , -C(O)OH, -C(O)OR b , -OC(O)R b , -OR b , -SR b , -S(O)R b , -S(O)OR b , -S(O) 2 OR b , -NO 2 , -S(O) 2 N(R b ) 2 , -N(R b )S(O) 2 R b , -NH(CH 2 ) t1 N(R b ) 2 , -NH(CH 2 ) p1 O(CH 2 ) q1 N(R b ) 2 , -NH(CH 2 ) s1 OR b , -N((CH 2 ) S OR b ) 2 , -N(R b
  • Each R b is independently selected from the group consisting of C 1 -C 3 alkyl, C 2 -C 3 alkenyl and H;
  • u is 5, 6, 7, 8, 9, 10, 11, 12, or 13;
  • w is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • r is 0 or 1;
  • t1 is 1, 2, 3, 4, or 5;
  • p1 is 1, 2, 5, 4, or 5;
  • q 1 is 1, 2, 5, 4, or 5;
  • s1 is 1, 2, 3, 4 or 5.
  • the cationic lipid is the following compound (II), its N-oxide, its salt or its isomer:
  • R 1 -R 3 , R 5 -R 7 , R 14 , X a , X b , RN , M, u, w and r are as defined above.
  • the cationic lipid comprises the following compound (III), its N-oxide, its salt or its isomer:
  • G1 and G2 are each independently an unsubstituted C1 - C12 alkylene group or a C1 - C12 alkenylene group;
  • G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C 3 -C 8 cycloalkylene, C 3 -C 8 cycloalkenylene;
  • Ra is H or a C1 - C12 hydrocarbon group
  • R 15 and R 16 are each independently C 6 -C 24 alkyl or C 6 -C 24 alkenyl
  • R 19 is a C 1 -C 12 hydrocarbon group
  • R 18 is H or a C 1 -C 6 hydrocarbon group
  • x 0, 1, or 2.
  • the cationic lipid is the following compound (III), its N-oxide, its salt or its isomer:
  • R 15 -R 17 , G 1 -G 3 , L 1 -L 2 are as defined above.
  • the cationic lipid comprises the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:
  • L 3 and L 4 are the same or different, and are each independently C 1 -C 12 alkylene, C 2 -C 12 alkenylene or C 2 -C 12 alkynylene; in some embodiments, L 3 and L 4 are the same or different, and are each independently C 3 -C 10 alkylene, C 3 -C 10 alkenylene or C 3 -C 10 alkynylene; in some embodiments, L 3 and L 4 are the same or different, and are each independently C 3 -C 10 alkylene; in some embodiments, L 3 and L 4 are the same or different, and are each independently C 5 -C 8 alkylene;
  • R 18 and R 19 are the same or different and are each independently C 5 -C 27 alkyl or C 5 -C 27 alkenyl containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently C 8 -C 20 alkyl or C 8 -C 20 alkenyl containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently C 9 -C 17 alkyl or C 9 -C 18 alkenyl containing one or two double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently C 9 -C 17 alkyl or C 9 -C 18 alkenyl containing one or two double bonds.
  • R 20 is halogen, hydroxy, cyano, C 1 -C 6 alkyl, nitro, C 1 -C 6 alkoxy, C 1 -C 6 alkylcarbonyloxy, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 alkylaminocarbonyl or C 1 -C 6 alkylcarbonylamino ; In some embodiments, R 20 is halogen, hydroxy, cyano, C 1 -C 6 alkoxy, C 1 -C 6 alkylcarbonyloxy, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 alkylaminocarbonyl or C 1 -C 6 alkylcarbonylamino; In some embodiments, R 20 is halogen, hydroxy, cyano, C 1 -C 4 alkoxy, C 1 -C 4 alkylcarbonyloxy, C 1 -C 4 alkoxycarbonyl , C 1 -C 4 alkylaminocarbonyl or C
  • z is 1, 2 or 3.
  • the cationic lipid is the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:
  • R 18 -R 20 , G 4 -G 5 , L 3 -L 4 and z are as defined above.
  • the cationic lipid comprises the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:
  • the cationic lipid comprises the following compound, or a pharmaceutically acceptable salt thereof:
  • the cationic lipids include one or more of the following: ALC-0315 (CAS No. 2036272-55-4), SM-102 (CAS No. 2089251-47-6), and
  • the auxiliary lipids of lipid nanoparticles include phospholipids.
  • Phospholipids are usually semi-synthetic, or they can be of natural origin or chemically modified.
  • the auxiliary lipids of lipid nanoparticles are phospholipids.
  • the phospholipids of lipid nanoparticles include one or more of the following: DSPC (distearylphosphatidylcholine), DOPE (dioleoylphosphatidylethanolamine), DOPC (dioleoylphosphatidylcholine), DOPS (dioleoylphosphatidylserine), DSPG (1,2-dioctadecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol)), DPPG (dipalmitoylphosphatidylglycerol), DPPC (dipalmitoylphosphatidylcholine), DGTS (1,2-dipalmitoyl-sn-glycero-3-O-4'-(N,N,N-trimethyl) homoserine) and lysophospholipids.
  • DSPC disearylphosphatidylcholine
  • DOPE dioleoylphosphatidylethanolamine
  • the auxiliary lipids of lipid nanoparticles are selected from one or more of the following: DSPC, DOPE, DOPC and DOPS.
  • the helper lipid of the lipid nanoparticle is DSPC and/or DOPE.
  • the structural lipids of the lipid nanoparticles include sterols.
  • the structural lipids of the lipid nanoparticles are sterols.
  • the sterols of the lipid nanoparticles include one or more of the following: 20 ⁇ -hydroxycholesterol, cholesterol, cholesterol esters, steroid hormones, steroid vitamins, bile acids, ergosterol, ⁇ -sitosterol, and oxidized cholesterol derivatives.
  • the structural lipids of the lipid nanoparticles include at least one of cholesterol, cholesterol esters, steroid hormones, steroid vitamins, and bile acids.
  • the structural lipids of the lipid nanoparticles are cholesterol.
  • the structural lipids of the lipid nanoparticles are high-purity cholesterol, particularly injection-grade high-purity cholesterol, such as CHO-HP (produced by AVT).
  • the structural lipids are 20 ⁇ -hydroxycholesterol.
  • Polymer-lipid refers to a conjugate comprising a polymer and a lipid coupled to the polymer.
  • Polymer-lipid eg, polyethylene glycol-lipid
  • Polymer-lipid in lipid nanoparticles can improve the stability of lipid nanoparticles in vivo.
  • the lipids of the polymer-lipid used to form lipid nanoparticles include one or more of the following: 1,2-dimyristoyl-sn-glycerol (DMG), distearoyl-phosphatidyl-ethanolamine (DSPE), diacylglycerol (DAG), dialkyloxypropyl (DAA), phospholipids, ceramide (Cer), 1,2-distearoyl-rac-glycerol (DSG) and 1,2-dipalmitoyl-rac-glycero (DPG).
  • DMG 1,2-dimyristoyl-sn-glycerol
  • DSPE distearoyl-phosphatidyl-ethanolamine
  • DAG diacylglycerol
  • DAA dialkyloxypropyl
  • phospholipids ceramide
  • Cer 1,2-distearoyl-rac-glycerol
  • DPG 1,2-dipalmitoyl-rac-glycero
  • the polymer of the polymer-lipid used to form the lipid nanoparticles includes one or both of the following: a hydrophilic polymer and an amphiphilic polymer.
  • the polymer of the polymer-lipid used to form the lipid nanoparticles is a hydrophilic polymer. In other embodiments, the polymer of the polymer-lipid used to form the lipid nanoparticles is an amphoteric polymer.
  • the hydrophilic polymer includes one or more of the following: polyethylene glycol (PEG), polyoxazolines (POX), polyglycerols (PGs), polyhydroxypropyl methacrylate (PHPMA), poly(2-hydroxyethyl methacrylate), poly(N-(2-hydroxypropyl)methacrylamide), polyvinylpyrrolidone (poly(vinylpyrrolidone)).
  • ylpyrrolidone PVP
  • PDMA poly(N,N-dimethyl acrylamide)
  • PAcM poly(N-acryloyl morpholine)
  • GAGs glycosaminoglycans
  • HA heparin
  • hyaluronic acid HA
  • PSA polysialic acid
  • ELPs elastin-like polypeptide
  • polymer-lipid includes one or more of the following: polyethylene glycol-lipid (PEG-lipid), polyoxazoline-lipid, polyglycerol-lipid, polyhydroxypropyl methacrylate-lipid, polymethacrylate-2-hydroxyethyl-lipid, poly-N-(2-hydroxypropyl) methacrylamide-lipid, polyvinylpyrrolidone-lipid, poly-N,N-dimethylacrylamide-lipid, poly-N-acryloylmorpholine-lipid, glycosaminoglycan-lipid, heparin-lipid, hyaluronic acid-lipid, polysialic acid-lipid, elastin-lipid, serum albumin-lipid and CD47-lipid.
  • PEG-lipid polyethylene glycol-lipid
  • polyoxazoline-lipid polyglycerol-lipid
  • polyhydroxypropyl methacrylate-lipid polymethacrylate-2-hydroxyethyl-lipid
  • poly-N-(2-hydroxypropyl) methacrylamide-lipid
  • PEG-lipid is a conjugate of polyethylene glycol and lipid
  • polyoxazoline-lipid refers to a conjugate formed by coupling polyoxazoline with lipid
  • polyglycerol-lipid refers to a conjugate formed by coupling polyglycerol with lipid
  • the hydrophilic polymer includes polyethylene glycol.
  • the polymer-lipid includes PEG-lipid.
  • the polymer-lipid is PEG-lipid.
  • the PEG-lipid includes one or more of the following: PEG-myristoyl diglycerol (PEG-DMG), PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-diacylglycerol (PEG-DAG), PEG-dialkyloxypropyl (PEG dialkyloxypropyl, PEG-DAA), PEG-phospholipids, PEG-ceramide (PEG-ceramide, PEG-Cer), PEG-1,2-distearoyl-rac-glycerol (PEG-DSG) and PEG-1,2-dipalmitoyl-rac-glycerol (PEG-DPG).
  • PEG-lipid is preferably PEG-DMG, PEG-DSG, PEG-DPG.
  • PEG-DMG is a polyethylene glycol derivative of 1,2-dimyristoylglycerol.
  • the average molecular weight of PEG in PEG-lipid is about 2000 to 5000. In an optional specific example, the average molecular weight of PEG in the PEG-lipid is about 2000.
  • the amphoteric polymer includes one or more of the following: polycarboxybetaine (pCB), polysulfobetaine (pSB), phosphobetaine-base polymers and phosphorylcholine polymers.
  • the amphoteric polymer includes one or more of the following: poly(carboxybetaine acrylamide, pCBAA), poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine)based on vinylidazole, poly(sulfobetaine)based on vinylidazole, and poly(sulfobetaine)based on vinylpyridine.
  • poly(carboxybetaine acrylamide, pCBAA) poly(carboxybetaine methacrylate), poly(sulfobetaine methacrylate), poly(methacryloyloxyethyl phosphorylcholine), poly(vinyl-pyridinio propanesulfonate), poly(carboxybetaine)based on vinylidazole, poly(sulfobetaine)based on vinylid
  • polymer-lipids include one or more of the following: polyhydroxybetaine-lipids, polysulfobetaine-lipids, phosphobetaine-based polymer-lipids and phosphorylcholine polymer-lipids.
  • polymer-lipids include one or more of the following: poly(carboxybetaine acrylamide)-lipids, poly(carboxybetaine methacrylate)-lipids, poly(sulfobetaine methacrylate)-lipids, poly(methacryloyloxyethyl phosphorylcholine)-lipids, poly(vinylpyridinylpropanesulfonate)-lipids, polyvinylimidazolyl betaine-lipids, polyvinylimidazolyl sulfobetaine-lipids, polyvinylpyridinyl sulfobetaine-lipids.
  • lipid nanoparticles contain cationic lipids, auxiliary lipids, structural lipids and polymer-lipids.
  • the total amount of cationic lipids, auxiliary lipids, structural lipids and polymer-lipids, the lipid nanoparticles include the following amount (molar percentage) of cationic lipids: about 25.0% to 75.0%, for example, about 25.0% to 28.0%, 28.0% to 32.0%, 32.0% to 35.0%, 35.0% to 40.0%, 40.0% to 42.0%, 42.0% to 45.0%, 45.0% to 46.3%, 46.3% to 48.0%, 48.0% to 49.5%, 49.5% to 50.0%, 50.0% to 55.0%, 55.0% to 60.0%, 60.0% to 65.0%, or 65.0% to 75.0%.
  • the lipid nanoparticles comprise cationic lipids, auxiliary lipids, structural lipids and polymer-lipids, wherein the cationic lipids account for 25 mol% to 75 mol% of the total lipids present in the lipid nanoparticles, the auxiliary lipids account for 0 mol% to 45 mol% of the total lipids present in the lipid nanoparticles, the structural lipids account for 0 mol% to 60 mol% of the total lipids present in the lipid nanoparticles, and the polymer-lipids account for 0.5 mol% to 5 mol% of the total lipids present in the lipid nanoparticles.
  • the cationic lipid in the lipid nanoparticles accounts for 25 mol% to 75 mol% of the total lipids present in the lipid nanoparticles.
  • the cationic lipids in the above-mentioned lipid nanoparticles account for 30 mol% to 65 mol%, 30 mol% to 60 mol%, 35 mol% to 60 mol%, 40 mol% to 60 mol%, 45 mol% to 55 mol% or 50 mol% to 55 mol% of the total lipids present in the lipid nanoparticles.
  • the auxiliary lipid (e.g., DSPC) in the above-mentioned lipid nanoparticles accounts for 0 mol% to 45 mol% of the total lipid present in the lipid nanoparticles.
  • mol% 20 mol%, 20.5 mol%, 21 mol%, 21.5 mol%, 22 mol%, 22.5 mol%, 23 mol%, 23.5 mol%, 24 mol%, 24.5 mol%, 25 mol%, 25.5 mol%, 26 mol%, 26.5 mol%, 27 mol%, 27.5 mol%, 28 mol%, 28.5 mol%, 29 mol%, 29.5 mol%, 30 mol%, 34 mol%, 35 mol%, 36 mol%, 38 mol%, 40 mol%, 42 mol%, 44 mol% or 45 mol%.
  • the auxiliary lipid (e.g., DSPC) in the above-mentioned lipid nanoparticles accounts for 1 mol% to 40 mol%, 5 mol% to 40 mol%, 5 mol% to 35 mol%, 5 mol% to 30 mol% or 5 mol% to 25 mol% of the total lipid present in the lipid nanoparticles.
  • the structural lipid (e.g., cholesterol) in the lipid nanoparticles described above accounts for 0 mol% to 60 mol% of the total lipid present in the lipid nanoparticles.
  • the structural lipid (e.g., cholesterol) in the above-mentioned lipid nanoparticles accounts for 1 mol% to 60 mol%, 1 mol% to 55 mol%, 5 mol% to 55 mol%, 10 mol% to 50 mol%, 15 mol% to 50 mol%, 15 mol% to 45 mol%, 20 mol% to 45 mol%, 25 mol% to 40 mol% of the total lipids present in the lipid nanoparticles.
  • the polymer-lipid (e.g., PEG-lipid) in the above-mentioned lipid nanoparticles accounts for 0.5 mol% to 5 mol% of the total lipid present in the lipid nanoparticles. For example, 0.5 mol%, 1 mol%, 1.5 mol%, 2 mol%, 2.5 mol%, 3 mol%, 3.5 mol%, 4 mol%, 4.5 mol% or 5 mol%.
  • the polymer-lipid (e.g., PEG-lipid) in the above-mentioned lipid nanoparticles accounts for 0.5 mol% to 4.5 mol%, 1 mol% to 4.5 mol%, 1 mol% to 4 mol%, 1.5 mol% to 4 mol%, 1.5 mol% to 3.5 mol% or 1.5 mol% to 3 mol% of the total lipid present in the lipid nanoparticles.
  • the non-lamellar lipid nanoparticles are selected from one of the following: alcohol-containing liposomes (ethosomes) and echogenic liposomes (echogenic liposomes).
  • the delivery vector is a liposome containing the nucleic acid of any embodiment, the RNA of any embodiment, the genetic engineering vector of any embodiment, or the nucleic acid composition of any embodiment.
  • the liposome uses a vesicle formed by a phospholipid bilayer membrane to encapsulate the nucleic acid, RNA, vector, or nucleic acid composition of any embodiment.
  • the components of the liposome include phospholipids and cholesterol.
  • the delivery vector is a cationic protein loaded with the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetic engineering vector of any of the above embodiments, or the nucleic acid composition of any of the above embodiments.
  • the cationic protein includes but is not limited to protamine.
  • the delivery vector is a polymer comprising a nucleic acid of any of the above embodiments, an RNA of any of the above embodiments, a genetic engineering vector of any of the above embodiments, or a nucleic acid composition of any of the above embodiments.
  • the polymer is a lipid polymer (lipopolyplex, LPP) and/or a hyaluronic acid polymer (e.g., hyaluronic acid gel) comprising a nucleic acid, RNA, a genetic engineering vector, or a nucleic acid composition of any of the above embodiments.
  • the polymer is a lipid polymer or a hyaluronic acid gel.
  • Lipid polymers are a double-layer structure with a polymer-encapsulated nucleic acid (e.g., mRNA) as a core and a lipid (e.g., phospholipid) as a shell.
  • the delivery vectors applicable to the present disclosure are not limited to the above, and may also be other substances capable of delivering the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetic engineering vector of any of the above embodiments, or the nucleic acid composition of any of the above embodiments into the body, such as vesicles (e.g., exosomes), etc.
  • vesicles e.g., exosomes
  • R 18 -R 20 , G 4 -G 5 , L 3 -L 4 and z are as defined above, and X is halogen, preferably bromine.
  • the above preparation method includes step S11 and step S12.
  • Step S11 The intermediate compound (V) and the intermediate compound (VI) are subjected to substitution reaction in an organic solvent in the presence of an acid-binding agent at room temperature (16°C to 30°C, the same below) to obtain the intermediate compound (VII).
  • the organic solvent of step S1 is selected from one or more of nitrile organic solvents, alcohol organic solvents, halogenated hydrocarbon organic solvents, amide organic solvents, and aromatic hydrocarbon organic solvents.
  • the organic solvent of step S1 is selected from one or more of acetonitrile, methanol, ethanol, dichloromethane and dichloroethane (DCE).
  • the acid-binding agent of step S1 is selected from one or more of organic bases and inorganic bases.
  • the acid-binding agent of step S1 is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA.
  • Step S12 The intermediate compound (VII) and the intermediate compound (VIII) are subjected to substitution reaction in an organic solvent at room temperature in the presence or absence of an acid binding agent and an iodide to obtain a compound of formula (IV).
  • the organic solvent of step S2 is selected from one or more of nitrile organic solvents, alcohol organic solvents, halogenated hydrocarbon organic solvents, amide organic solvents and aromatic hydrocarbon organic solvents.
  • the organic solvent of step S2 is selected from one or more of acetonitrile, methanol, ethanol, dichloromethane and dichloroethane.
  • the acid binding agent is selected from one or more of an organic base and an inorganic base.
  • the acid binding agent is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine and DIPEA.
  • the iodide is a common iodide, such as potassium iodide.
  • the present disclosure also provides a method for preparing an optical isomer (IV-1-i) of the above compound (IV-1), and the reaction of the preparation method is as follows:
  • X is a halogen, preferably bromine.
  • the preparation step of the optical isomer (IV-1-i) of the compound (IV-1) of the present disclosure includes step S21 and step S22.
  • Step S21 In a solvent (e.g., nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbon solvents, specifically acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), etc.), compound (1-X) and compound (1-1) are subjected to an N-alkylation reaction at 30° C. to 50° C. to prepare compound (1-2);
  • a solvent e.g., nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbon solvents, specifically acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), etc.
  • Step S22 In a solvent (e.g., nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, ether solvents, such as acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), cyclopentane methyl ether, methyl tert-butyl ether, etc.), in the presence or absence of an acid-binding agent and a catalyst, compound (1-2) and 8-halogenated octanoic acid nonyl ester are subjected to N-alkylation reaction at 60°C to 110°C to prepare compound (IV-1-i), wherein the acid-binding agent is selected from one or more of the following: an organic base and an inorganic base.
  • a solvent e.g., nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, ether solvents, such as acetonitrile, methanol,
  • the present disclosure also provides a pharmaceutical composition, which comprises the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetic engineering vector of any of the above embodiments, the host cell of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the immunogen of any of the above embodiments, the mutant of the MPV F protein of any of the above embodiments or the delivery vector of any of the above embodiments, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition described above comprises one or more nucleic acids according to any of the above embodiments.
  • the pharmaceutical composition described above comprises one or more nucleic acids according to any of the above embodiments, which are formulated alone (eg, encapsulated) or co-formulated in a delivery vehicle.
  • the pharmaceutical composition comprises a nucleic acid according to any of the above embodiments.
  • the pharmaceutical composition described above comprises a nucleic acid according to any of the above embodiments, which is formulated (eg, encapsulated) in a delivery vehicle.
  • the pharmaceutical composition comprises a nucleic acid as described above, which is formulated (e.g., encapsulated) in a delivery vehicle (e.g., a lipid nanoparticle), and the nucleic acid encodes a mutant of the MPV F protein, wherein the mutant of the MPV F protein encoded by the nucleic acid is selected from one of the following:
  • a mutant comprising mutations A147C and A159C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO: 1, 2, 81, 82 or 83;
  • a mutant comprising mutations F168C and F196C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO: 5 or 6;
  • a mutant comprising mutations L165C and F196C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO: 7, 8 or 84;
  • a mutant comprising mutations N145C and A161C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO: 9, 10 or 85;
  • (6) a mutant comprising mutations S149C and V157C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO: 11 or 12;
  • a mutant comprising mutations T59C and N180C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to any one of SEQ ID NOs: 13 to 20; and
  • a mutant comprising mutations T150C and R156C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:21 or 22.
  • the pharmaceutical composition comprises a nucleic acid as described above, which is formulated (e.g., encapsulated) in a delivery vector, and the amino acid sequence of a mutant of the MPV F protein encoded by the nucleic acid is shown in any one of SEQ ID NOs: 1 to 22 and 81 to 85.
  • the pharmaceutical composition comprises one of the above nucleic acids, which is formulated (e.g., encapsulated) in a delivery vehicle (e.g., a lipid nanoparticle), and the nucleic acid is selected from one of the following:
  • (1) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:1 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:34 or 86;
  • (9) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:9 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42;
  • (10) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:10 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43;
  • (11) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:11 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44;
  • (13) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:13 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:46;
  • (14) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:14 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:47;
  • (21) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:21 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:54;
  • (22) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:22 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:55;
  • (23) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:81 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:88;
  • (24) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:82 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:89;
  • (25) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:83 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:90 or 92;
  • (26) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence as shown in SEQ ID NO:84 and having a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:91; and
  • the above-mentioned pharmaceutical composition comprises an above-mentioned nucleic acid, which is formulated (e.g., encapsulated) in a delivery carrier (e.g., lipid nanoparticle), and the nucleic acid is an mRNA corresponding to a DNA comprising a nucleotide sequence as shown in any one of SEQ ID NO: 34 to 55 and 86 to 93.
  • a delivery carrier e.g., lipid nanoparticle
  • the pharmaceutical composition described above comprises a plurality of nucleic acids according to any of the above embodiments.
  • the pharmaceutical composition comprises a plurality of nucleic acids of any of the above embodiments, and the plurality of nucleic acids are co-formulated in a delivery vector or the plurality of nucleic acids are separately formulated in a delivery vector.
  • Co-formulation means that a single delivery vector contains (e.g., encapsulates) a plurality of nucleic acids
  • single formulation means that a single delivery vector contains (e.g., encapsulates) a nucleic acid, and the same applies hereinafter.
  • the pharmaceutical composition comprises two of the above nucleic acids, the two nucleic acids are co-formulated in a delivery vehicle (eg, lipid nanoparticles) or the two nucleic acids are separately formulated in a delivery vehicle (eg, lipid nanoparticles).
  • a delivery vehicle eg, lipid nanoparticles
  • the two nucleic acids are separately formulated in a delivery vehicle (eg, lipid nanoparticles).
  • the pharmaceutical composition comprises two nucleic acids, the two nucleic acids are co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids are separately formulated in a delivery vehicle (e.g., lipid nanoparticles), the two nucleic acids encode a mutant of the A subtype MPV F protein and a mutant of the B subtype MPV F protein, respectively, and the mutant of the A subtype MPV F protein and the mutant of the B subtype MPV F protein encoded by the two nucleic acids are selected from the following group:
  • a mutant comprising mutations A147C and A159C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:1
  • a mutant comprising mutations A147C and A159C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:2;
  • (6) a mutant comprising mutations S149C and V157C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:11, and a mutant comprising mutations S149C and V157C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:12;
  • (11) a mutant comprising mutations T150C and R156C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:21, and a mutant comprising mutations T150C and R156C and having an amino acid sequence that is at least 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% identical to SEQ ID NO:22;
  • the pharmaceutical composition comprises two nucleic acids, the two nucleic acids are co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids are separately formulated in a delivery vehicle (e.g., lipid nanoparticles), the two nucleic acids encode a mutant of the A subtype MPV F protein and a mutant of the B subtype MPV F protein, respectively, and the mutant of the A subtype MPV F protein and the mutant of the B subtype MPV F protein encoded by the two nucleic acids are selected from the following group:
  • the pharmaceutical composition comprises two of the above nucleic acids, the two nucleic acids are co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids are separately formulated in a delivery vehicle (e.g., lipid nanoparticles), the two nucleic acids respectively encode a mutant of the A subtype MPV F protein and a mutant of the B subtype MPV F protein, and the two nucleic acids are selected from the following group:
  • the pharmaceutical composition comprises two of the above nucleic acids, the two nucleic acids are co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids are separately formulated in a delivery vehicle (e.g., lipid nanoparticles), and the two nucleic acids are selected from the following group:
  • mRNA corresponding to a DNA comprising a nucleotide sequence as shown in SEQ ID NO:34 and mRNA corresponding to a DNA comprising a nucleotide sequence as shown in SEQ ID NO:35;
  • the nucleic acid contained in the above pharmaceutical composition is mRNA.
  • the nucleic acid contained in the above pharmaceutical composition is mRNA, and all the uridine in the mRNA is replaced by N1-methylpseudouridine.
  • the pharmaceutical composition comprises a nucleic acid according to any of the above embodiments, wherein the nucleic acid is formulated in a delivery vector, and the nucleic acid is mRNA.
  • the pharmaceutical composition comprises a plurality of nucleic acids of any of the above embodiments, wherein the plurality of nucleic acids are co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the plurality of nucleic acids are individually formulated in a delivery vehicle (e.g., lipid nanoparticles), and the plurality of nucleic acids are all mRNAs.
  • a delivery vehicle e.g., lipid nanoparticles
  • the plurality of nucleic acids are individually formulated in a delivery vehicle (e.g., lipid nanoparticles)
  • the plurality of nucleic acids are all mRNAs.
  • the pharmaceutical composition comprises two of the above nucleic acids, the two nucleic acids are co-formulated in a delivery vector (e.g., lipid nanoparticles) or the two nucleic acids are separately formulated in a delivery vector (e.g., lipid nanoparticles), and the two nucleic acids are mRNA.
  • the pharmaceutical composition comprises multiple delivery vectors, and the mutants of MPV F protein encoded by the nucleic acids contained in the multiple delivery vectors are from the same subtype strain but with different mutations, different subtype strains but with the same mutations, or different subtype strains with different mutations.
  • the above-mentioned pharmaceutical composition comprises a first delivery vector and a second delivery vector
  • the first delivery vector comprises a first nucleic acid
  • the second delivery vector comprises a second nucleic acid
  • the first nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations
  • the second nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having A147C and A159C mutations.
  • the above-mentioned pharmaceutical composition comprises a first delivery vector and a second delivery vector
  • the first delivery vector comprises a first nucleic acid
  • the second delivery vector comprises a second nucleic acid
  • the first nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations
  • the second nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having L165C and F196C mutations.
  • the above-mentioned pharmaceutical composition comprises a delivery vector
  • the delivery vector comprises a first nucleic acid and a second nucleic acid
  • the first nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 and having A147C and A159C mutations
  • the second nucleic acid is a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain NL/1/99 and having A147C and A159C mutations.
  • the delivery vehicle of the pharmaceutical composition of any of the above embodiments is a lipid nanoparticle.
  • the pharmaceutical composition comprises a plurality of nucleic acids according to any of the above embodiments, a plurality of RNAs according to any of the above embodiments, a plurality of genetic engineering vectors according to any of the above embodiments, or a plurality of host cells according to any of the above embodiments.
  • the pharmaceutical composition of any of the above embodiments is a vaccine.
  • the term "pharmaceutically acceptable” means approved for use in animals and/or humans by a regulatory agency (e.g., the State Food and Drug Administration (CFDA), the U.S. Food and Drug Administration (FDA)) or a recognized pharmacopoeia (e.g., the Chinese Pharmacopoeia, the European Pharmacopeia).
  • CFDA State Food and Drug Administration
  • FDA U.S. Food and Drug Administration
  • a recognized pharmacopoeia e.g., the Chinese Pharmacopoeia, the European Pharmacopeia
  • pharmaceutically acceptable carrier refers to a substance that can be administered with the nucleic acid, genetic engineering vector, nucleic acid composition, protein mutant or delivery vector of the present disclosure, including but not limited to diluents, sweeteners, flavoring agents, wetting agents, adjuvants, glidants, preservatives, dyes/colorants, surfactants, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers.
  • the above pharmaceutical compositions do not include an adjuvant.
  • the above pharmaceutical composition further comprises an adjuvant.
  • the adjuvant includes a flagellin adjuvant.
  • the flagellin adjuvant is an mRNA encoding flagellin.
  • the present disclosure also provides a nucleic acid of any of the above embodiments, a genetic engineering vector of any of the above embodiments, a host cell of any of the above embodiments, a nucleic acid composition of any of the above embodiments, a mutant of the MPV F protein of any of the above embodiments, an RNA of any of the above embodiments, an immunogen of any of the above embodiments, a delivery vector of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments for use in the preparation of a drug.
  • the medicament is for preventing or treating MPV infection or a disease caused by MPV infection.
  • the medicament is for preventing or treating hMPV infection or a disease caused by hMPV infection.
  • the medicament is a vaccine.
  • the present disclosure also provides a vaccine, which comprises the nucleic acid of any of the above embodiments, the genetically engineered vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the mutant of the MPV F protein of any of the above embodiments, the RNA of any of the above embodiments, the immunogen of any of the above embodiments, or the delivery vector of any of the above embodiments.
  • the above-mentioned vaccine is a nucleic acid vaccine or a protein vaccine.
  • the above-mentioned vaccine is an MPV vaccine.
  • the above-mentioned vaccine is a multivalent vaccine.
  • the vaccine comprises any multiple of the nucleic acids or any multiple of the mutants of the MPV F protein.
  • the above vaccine is a combined vaccine.
  • the vaccine is a multi-linked nucleic acid vaccine.
  • the vaccine comprises, in addition to any one or more of the nucleic acids comprising the polynucleotides encoding the mutants of the MPV F protein, nucleic acids encoding proteins or polypeptides other than the MPV F protein or other viruses that can serve as immunogens.
  • the other viruses can be one virus subtype, or multiple viruses or multiple virus subtypes.
  • the vaccine is a multi-protein vaccine.
  • the vaccine in addition to any one or more of the MPV F protein mutants, also contains proteins or polypeptides of other viruses that can serve as immunogens.
  • the other viruses can be one or more.
  • the above vaccines do not include an adjuvant.
  • the above-mentioned vaccine further comprises an adjuvant.
  • the dosage form of the above-mentioned vaccine is not particularly limited.
  • the above-mentioned vaccine is an mRNA vaccine.
  • the vaccine is an mRNA vaccine, and the vaccine is administered nasally, intratracheally, or injectably (e.g., intravenously, intraocularly, intravitreally, intramuscularly, intradermally, intracardially, intraperitoneally, and subcutaneously).
  • the present disclosure also provides a method for preventing or treating MPV virus infection, which comprises administering to a subject a nucleic acid of any of the above embodiments, a genetically engineered vector of any of the above embodiments, a nucleic acid composition of any of the above embodiments, a mutant of the MPV F protein of any of the above embodiments, an RNA of any of the above embodiments, an immunogen of any of the above embodiments, a delivery vector of any of the above embodiments, a pharmaceutical composition of any of the above embodiments, or a vaccine of any of the above embodiments.
  • the subject is a mammal (e.g., a human, a non-human primate (e.g., apes, chimpanzees, monkeys, and orangutans)), a domestic animal (e.g., a dog, a cat, and livestock (e.g., a horse, a cow, a pig, a sheep, and a goat)), or other mammals.
  • a mammal e.g., a human, a non-human primate (e.g., apes, chimpanzees, monkeys, and orangutans)
  • a domestic animal e.g., a dog, a cat
  • livestock e.g., a horse, a cow, a pig, a sheep, and a goat
  • Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc.
  • the subject is a human.
  • the number of administrations is one, two, three, four or more times.
  • the present disclosure also provides a method for inducing an immune response against MPV in a subject.
  • the method comprises administering to the subject a nucleic acid of any of the above embodiments, a genetically engineered vector of any of the above embodiments, a nucleic acid composition of any of the above embodiments, a mutant of the MPV F protein of any of the above embodiments, an RNA of any of the above embodiments, an immunogen of any of the above embodiments, a delivery vector of any of the above embodiments, a pharmaceutical composition of any of the above embodiments, or a vaccine of any of the above embodiments.
  • Anti-antigen antibodies are serum antibodies that specifically bind to antigens.
  • the present disclosure also provides a nucleic acid of any of the above embodiments, a genetically engineered vector of any of the above embodiments, a nucleic acid composition of any of the above embodiments, a mutant of the MPV F protein of any of the above embodiments, an RNA of any of the above embodiments, an immunogen of any of the above embodiments, a delivery vector of any of the above embodiments, or a pharmaceutical composition of any of the above embodiments for use in preparing a detection reagent or kit for MPV F protein-binding antibodies.
  • the present disclosure also provides a detection reagent or kit for MPV F protein-binding antibodies, which comprises the nucleic acid of any of the above embodiments, the genetic engineering vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the mutant of the MPV F protein of any of the above embodiments, the RNA of any of the above embodiments, the immunogen of any of the above embodiments, the delivery vector of any of the above embodiments, or the composition of the drugs of any of the above embodiments.
  • a detection reagent or kit for MPV F protein-binding antibodies which comprises the nucleic acid of any of the above embodiments, the genetic engineering vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the mutant of the MPV F protein of any of the above embodiments, the RNA of any of the above embodiments, the immunogen of any of the above embodiments, the delivery vector of any of the above embodiments, or the composition of the drugs of any of the above embodiments.
  • the present disclosure also provides a method for detecting or isolating MPV F protein binding antibodies in a subject, the method comprising: providing an effective amount of the nucleic acid of any of the above embodiments, the genetic engineering vector of any of the above embodiments, the nucleic acid composition of any of the above embodiments, the mutant of the MPV F protein of any of the above embodiments, the immunogen of any of the above embodiments, or a pharmaceutical composition or vaccine containing the mutant of the MPV F protein; contacting a biological sample from the subject with the mutant of the MPV F protein under conditions sufficient to form an immune complex between the mutant and the MPV F binding antibody; and detecting the immune complex, thereby detecting or isolating the MPV F binding antibody in the subject.
  • Step 1) Synthesis of 8-((3-hydroxycyclohexyl)amino)nonyl octanoate (compound (IV-1-p1))
  • N,N-diisopropylethylamine (2.58 g, 20 mmol)
  • the hMPV F protein mutants shown in Table 1 were designed, and codon-optimized DNA sequences (DNA sequences corresponding to the protein coding regions in Table 1) were further designed based on the amino acid sequences of the mutants and commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. The test results showed that all DNA sequences were correct. In addition, Nanjing GenScript Biotechnology Co., Ltd. was also commissioned to synthesize the DNA sequences corresponding to the mRNAs numbered 1191, 1192, 1274 and 1275 (see Table 2).
  • test results showed that all DNA sequences were correct, among which the mRNAs numbered 1191 and 1192 encoded hMPV post F proteins, and the mRNAs numbered 1274 and 1275 encoded hMPV pre F proteins; the mRNAs numbered 1191, 1192, 1274 and 1275 used the same 5'-UTR, 3'-UTR and poly(A) tail as the mRNAs in Table 1.
  • the construction method is as follows:
  • Plasmid B Suzhou GENEWIZ Biotechnology Co., Ltd. (GENEWIZ) was commissioned to replace the Amp resistance gene in plasmid pcDNA3.1 with the Kana resistance gene, and further add new restriction sites HindIII, BamHI, KpnI and ApaI after the T7 promoter, and delete the NeoR/KanR sequence from 2136bp to 2930bp to obtain plasmid B.
  • Plasmid B was linearized with ApaI and homologously recombined with the nucleic acid fragment synthesized by IDT (Integrated DNA Technologies) such as SEQ ID NO: 33 to obtain plasmid C.
  • Plasmid C was double-digested with KpnI and ApaI, and a large fragment of about 4.7 kb was recovered by agarose gel electrophoresis. It was then ligated with the nucleic acid fragment synthesized by IDT (SEQ ID NO: 32) by T4 enzyme to obtain plasmid D.
  • Plasmid D was double-digested with BamHI and HindIII, and a large fragment of about 4.8 kb was recovered by agarose gel electrophoresis. It was then ligated with the nucleic acid fragment synthesized by IDT (SEQ ID NO: 31) by T4 enzyme to obtain plasmid F.
  • engineered plasmids Homologous arm sequences were introduced at both ends of the DNA sequences corresponding to the protein coding regions of different hMPV F protein mutants (see Tables 1 and 2 for details) by PCR. The PCR products were then digested with plasmid F by BamHI and KpnI, and the large molecular fragments of about 4.8 kb were recovered by gel running for homologous recombination. Finally, a variety of engineered plasmids with completely correct sequencing were obtained.
  • Enzyme linearization Take 20 ⁇ g of different engineered plasmids containing the coding sequence of hMPV F protein mutants prepared in Example 3, prepare the reaction system according to Table 3, mix well and place in a 37°C incubator for 16 h for enzyme digestion reaction.
  • Cap1 type cap structure and reaction principle are as follows:
  • 58 ⁇ L of mRNA aqueous solution (125 pmol of uncapped mRNA was supplemented with water to 58 ⁇ L) was preheated at 65°C for 5 min, then mixed with a mixture containing 10 ⁇ L 10 ⁇ Capping Buffer, 10 ⁇ L 10 mM GTP, 1.88 ⁇ L 32 mM SAM, 0.187 ⁇ L RNase Inhibitor, 5 ⁇ L 2'-O-Methyltransferase (50 U/ ⁇ L) and 15 ⁇ L ScriptCap Capping Enzyme (10 U/ ⁇ L), and incubated at 42°C for 1 h.
  • the above reagents for capping reaction were purchased from Suzhou Nearshore Protein Technology Co., Ltd.
  • mRNAs encoding different mutants of hMPV F protein were obtained, which contained Cap1 type cap structure, 5’-UTR, 3’-UTR, poly(A) tail and all uridine replaced by N1-methylpseudouridine.
  • the amino acid sequences of the mutants of hMPV F protein after translation of the protein coding regions of each mRNA are shown in Tables 1 and 2.
  • Encapsulation A microsyringe and a microfluidic chip (SN.000038) were used to encapsulate mRNA at a rate of 9 mL/min in the aqueous phase and 3 mL/min in the alcohol phase to prepare a crude LNP preparation containing mRNA encoding the hMPV F protein mutant. The difference between the LNP preparations was only the encapsulated mRNA.
  • the mRNA used in this example was prepared according to the method described in Example 4. The compositions of the aqueous phase and alcohol phase of the various LNP preparations are shown in Table 5 below.
  • Dialysate preparation Tris + 8% (m/V) sucrose solution: Weigh 1.22g Tris and 4.7g Tris-HCL, dissolve and mix with 2L Watson's distilled water, then add 160g sucrose and mix to obtain Tris + 8% (m/V) sucrose solution.
  • Dialysis The crude products obtained by encapsulation were loaded into 100KD dialysis bags, immersed in a beaker containing 1L of dialysis solution, wrapped in aluminum foil, and dialyzed at 100rpm for 1h at room temperature, then the dialysate was replaced and the dialysis was continued for 1h.
  • the preparation samples after dialysis were sterilized and filtered using a 0.22 ⁇ m disposable filter membrane to prepare various LNP preparations encapsulating mRNA encoding hMPV F protein mutants, wherein the mRNA concentration in the LNP preparation was 0.2 ⁇ g/ ⁇ L, the mass ratio of mRNA:Lipid was 1:10, the particle size was 80nm ⁇ 130nm, and the encapsulation efficiency was above 85%.
  • an LNP preparation encapsulating mRNA numbered 1154 and 5 ⁇ g of an LNP preparation encapsulating mRNA numbered 1170 were mixed and injected into mice for a total of two injections, and the first injection time was recorded as Day 0 (D0), the second injection time is D21 or D22, and blood is collected from the eye sockets on the 7th, 14th, 21st, 27th, 35th, 42nd, 49th, 63rd, 74th (or 77th) and 116th day after immunization (after injection).
  • the blood is collected in a non-anticoagulant tube, placed on ice for 30 minutes, and centrifuged at 4°C3500rpm for 10 minutes. After stratification, the upper light yellow liquid is carefully aspirated with a pipette to prepare the immune serum.
  • mice For some experimental batches of mice, spleens were collected on days 74 and 116 or days 7 and 77 for subsequent ELISpot detection (Example 8).
  • the immune serum used in this example was provided by the experiment in Example 5.
  • hMPV subtype A F protein amino acid sequence as shown in SEQ ID NO: 79
  • hMPV subtype B F protein amino acid sequence as shown in SEQ ID NO: 80
  • mouse serum inactivated by incubation at 56°C for 30 minutes
  • Goat anti-Mouse IgG (H+L) HRP Conjugate PBS, FBS, single-component TMB colorimetric solution, and stop solution.
  • the steps include:
  • IgG antibody titer test results of hMPV mRNA vaccines containing different mRNAs are shown in Figures 1A to 2B. All mRNA vaccines induced IgG antibodies against hMPV subtype A F protein and/or hMPV subtype B F protein.
  • the immune serum required for the pseudovirus neutralization test was provided by the experiment in Example 5.
  • MPV-GFP1 pseudovirus 7.0 log 10 TCID50/mL
  • LLC-MK 2 cells LLC-MK 2 cells
  • Opti-MEM Opti-MEM
  • FBS FBS
  • the steps include:
  • LLC- MK2 cells (2.5 ⁇ 10 4 cells/well) were added to a 96-well plate and cultured in Opti-MEM medium containing 5% FBS for 24 h.
  • Reagents RPMI Medium 1640basic (1 ⁇ ), mouse lymphocyte separation medium, positive reference PMA+Ionomycin, ELISpot Plus Mouse IFN-gamma (HRP), hMPV F protein peptide library, ELISpot Plus Mouse IL-4 (HRP), red blood cell lysis solution.
  • mice spleen used in the ELISpot experiment was from the experiment in Example 5.
  • the specific steps of ELISpot experiment include:
  • the enzyme-linked immunosorbent assay (ELISA) analyzer reads the plate and records various parameters of the spots for statistical analysis.
  • the ELISpot test results of hMPV mRNA vaccines containing different mRNAs are shown in Figures 5A to 6H.
  • the hMPV mRNA vaccines containing different mRNAs can all induce the body to produce obvious T cell immune responses, and the frequency of F protein-specific cells secreting IFN- ⁇ is higher than that of F protein-specific cells secreting IL-4, revealing that the immune response is Th1-biased rather than Th2-biased.
  • mice 7- to 9-week-old Balb/c female mice were immunized (muscularly injected) twice with LNP preparations encapsulated with mRNA.
  • the first immunization time was D0, and the second immunization time was D28.
  • Blood was collected from the eye sockets at D14, D21, D28 (collected before the second intramuscular injection), D35 and D42 to obtain immune serum.
  • the spleens of some batches of mice were collected at D49 or D57 for subsequent ELISpot detection.
  • the immune serum obtained in step 2 was subjected to the MPV-GFP1 pseudovirus neutralization experiment with reference to Example 7, and the results are shown in Figures 7A to 7C.
  • the introduction of mutations (A147C and A159C, L165C and F196C, or N145C and A161C) can enhance the humoral immune response of the antigen.
  • the spleen obtained in step 2 was subjected to ELISpot experiment according to Example 8, and the results are shown in Figures 8A to 8D.
  • the introduction of mutations (A147C and A159C, L165C and F196C, or N145C and A161C) can enhance the cellular immune response to the antigen.

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  • Immunology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un vaccin contre le métapneumovirus (MPV). Plus particulièrement, l'invention concerne un acide nucléique comprenant un polynucléotide pour coder un mutant d'une protéine F MPV, par comparaison avec une protéine F MPV de type sauvage, le mutant comprenant une mutation de liaison disulfure.
PCT/CN2024/136380 2023-12-04 2024-12-03 Vaccin contre le métapneumovirus (mpv) Pending WO2025119162A1 (fr)

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CNPCT/CN2023/136163 2023-12-04
CN2023136163 2023-12-04

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WO2025119162A1 true WO2025119162A1 (fr) 2025-06-12

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022214678A2 (fr) * 2021-04-09 2022-10-13 Valneva Se Vaccin contre le métapneumovirus humain
US20230174587A1 (en) * 2020-04-29 2023-06-08 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Recombinant human metapneumovirus f proteins and their use
WO2023110618A1 (fr) * 2021-12-16 2023-06-22 Janssen Vaccines & Prevention B.V. Protéines de fusion hmpv pré-hybrides stabilisées
CN116615235A (zh) * 2020-10-09 2023-08-18 得克萨斯州大学系统董事会 融合前稳定的hmpv f蛋白
US20230310571A1 (en) * 2021-11-30 2023-10-05 Sanofi Pasteur Inc. Human metapneumovirus vaccines
US20240252614A1 (en) * 2023-01-18 2024-08-01 Pfizer Inc. Vaccines against respiratory diseases

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230174587A1 (en) * 2020-04-29 2023-06-08 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Recombinant human metapneumovirus f proteins and their use
CN116615235A (zh) * 2020-10-09 2023-08-18 得克萨斯州大学系统董事会 融合前稳定的hmpv f蛋白
WO2022214678A2 (fr) * 2021-04-09 2022-10-13 Valneva Se Vaccin contre le métapneumovirus humain
US20230310571A1 (en) * 2021-11-30 2023-10-05 Sanofi Pasteur Inc. Human metapneumovirus vaccines
WO2023110618A1 (fr) * 2021-12-16 2023-06-22 Janssen Vaccines & Prevention B.V. Protéines de fusion hmpv pré-hybrides stabilisées
US20240252614A1 (en) * 2023-01-18 2024-08-01 Pfizer Inc. Vaccines against respiratory diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
STEWART-JONES GUILLAUME B., GORMAN JASON, OU LI, ZHANG BAOSHAN, JOYCE M. GORDON, YANG LIJUAN, CHENG CHENG, CHUANG GWO-YU, FOULDS K: "Interprotomer disulfide-stabilized variants of the human metapneumovirus fusion glycoprotein induce high titer-neutralizing responses", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES (PNAS), vol. 118, no. 39, 28 September 2021 (2021-09-28), pages 1 - 8, XP093323626, ISSN: 0027-8424, DOI: 10.1073/pnas.2106196118 *

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