Metapneumovirus (MPV) vaccine

Abstract

Provided is a metapneumovirus (MPV) vaccine. Specifically, provided is a nucleic acid comprising a polynucleotide for encoding a mutant of an MPV F protein, compared with a wild-type MPV F protein, the mutant comprises a disulfide bond mutation.

Description

Metapneumovirus (MPV) vaccine Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a metapneumovirus (MPV) vaccine. Background Art

[0002] Since its initial discovery in the Netherlands in 2001, human metapneumovirus (hMPV) has been reported worldwide. hMPV is a single-stranded RNA virus and a major cause of acute lower respiratory tract infections. hMPV comprises two genotypes, A and B, and is further divided into four subtypes: A1, A2, B1, and B2.

[0003] Initial hMPV infection typically occurs in children under the age of five. Epidemiological studies show that in 2018, there were 14.2 million cases of hMPV-related acute lower respiratory tract infections (ALRIs) in children under the age of five worldwide, 643,000 hMPV-related hospitalizations, 7,700 hMPV-related hospitalization deaths, and 16,100 hMPV-related ALRI deaths (both in hospitals and the community). hMPV-induced immune protection is weak, allowing repeated infection. It can cause severe illness (such as bronchitis or pneumonia) in adults (particularly the elderly and immunocompromised patients), with hMPV hospitalization rates comparable to those of RSV and influenza viruses.

[0004] However, there is currently no approved vaccine or effective antiviral drug for hMPV, and current treatment for hMPV infection is limited to symptomatic supportive care. Summary of the Invention

[0005] 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.

[0006] In addition, 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.

[0007] 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, wherein the mutant comprises a disulfide bond mutation relative to a wild-type MPV F protein, wherein the disulfide bond mutation comprises one or more of the following:

[0008] 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.

[0009] In some embodiments, 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.

[0010] In some embodiments, the disulfide bond mutation is selected from one of the following: A147C and A159C, L165C and F196C, N145C and A161C.

[0011] In some embodiments, the mutant is selected from one of the following (1) to (38):

[0012] (1) a mutant comprising the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0013] (2) 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 set forth in SEQ ID NO: 1 or 2;

[0014] (3) a mutant comprising the amino acid sequence shown in SEQ ID NO: 3 or 4;

[0015] (4) a mutant 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 set forth in SEQ ID NO: 3 or 4;

[0016] (5) a mutant comprising the amino acid sequence shown in SEQ ID NO: 5 or 6;

[0017] (6) 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 set forth in SEQ ID NO: 5 or 6;

[0018] (7) a mutant comprising the amino acid sequence shown in SEQ ID NO: 7 or 8;

[0019] (8) 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 set forth in SEQ ID NO: 7 or 8;

[0020] (9) a mutant comprising the amino acid sequence shown in SEQ ID NO: 9 or 10;

[0021] (10) 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 set forth in SEQ ID NO: 9 or 10;

[0022] (11) a mutant comprising the amino acid sequence shown in SEQ ID NO: 11 or 12;

[0023] (12) a mutant comprising mutations S149C and V157C 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: 11 or 12;

[0024] (13) a mutant comprising the amino acid sequence shown in SEQ ID NO: 13 or 14;

[0025] (14) 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 set forth in SEQ ID NO: 13 or 14;

[0026] (15) a mutant comprising the amino acid sequence shown in SEQ ID NO: 15 or 16;

[0027] (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 set forth in SEQ ID NO: 15 or 16;

[0028] (17) a mutant comprising the amino acid sequence shown in SEQ ID NO: 17 or 18;

[0029] (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 set forth in SEQ ID NO: 17 or 18;

[0030] (19) a mutant comprising the amino acid sequence shown in SEQ ID NO: 19 or 20;

[0031] (20) 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 set forth in SEQ ID NO: 19 or 20;

[0032] (21) a mutant comprising the amino acid sequence shown in SEQ ID NO: 21 or 22;

[0033] (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 set forth in SEQ ID NO: 21 or 22;

[0034] (23) a mutant comprising the amino acid sequence shown in SEQ ID NO: 23 or 24;

[0035] (24) 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;

[0036] (25) a mutant comprising the amino acid sequence shown in SEQ ID NO: 25 or 26;

[0037] (26) a mutant comprising mutations V55C and V169C 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: 25 or 26;

[0038] (27) a mutant comprising the amino acid sequence shown in SEQ ID NO: 27 or 28;

[0039] (28) a mutant comprising mutations L58C and T174C 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: 27 or 28;

[0040] (29) a mutant comprising the amino acid sequence shown in SEQ ID NO: 29 or 30;

[0041] (30) 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 set forth in SEQ ID NO: 29 or 30;

[0042] (31) a mutant comprising the amino acid sequence shown in SEQ ID NO: 81 or 82;

[0043] (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 set forth in SEQ ID NO: 81 or 82;

[0044] (33) a mutant comprising the amino acid sequence shown in SEQ ID NO: 83;

[0045] (34) 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 set forth in SEQ ID NO: 83;

[0046] (35) a mutant comprising the amino acid sequence shown in SEQ ID NO: 84;

[0047] (36) 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 set forth in SEQ ID NO: 84;

[0048] (37) a mutant comprising the amino acid sequence shown in SEQ ID NO: 85; and

[0049] (38) 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.

[0050] In some embodiments, the wild-type MPV is subtype A or subtype B.

[0051] In some embodiments, the wild-type MPV is wild-type hMPV.

[0052] In some embodiments, the MPV F protein is hMPV F protein.

[0053] In some embodiments, 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.

[0054] In some embodiments, the nucleic acid is DNA.

[0055] In some embodiments, the DNA is capable of being transcribed into RNA.

[0056] In some embodiments, the DNA sequence corresponding to the 5'-UTR is shown in SEQ ID NO: 31;

[0057] and / or, the DNA sequence corresponding to the 3'-UTR is shown in SEQ ID NO: 32;

[0058] And / or, 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 consecutively; 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.

[0059] In some embodiments, the nucleic acid contains modified nucleotides;

[0060] Preferably, the nucleic acid contains modified nucleosides; preferably, the modified nucleosides include at least one of modified uridine, modified cytidine, modified adenosine and modified guanosine.

[0061] In some embodiments, the nucleic acid comprises a polynucleotide encoding one or more mutants of the MPV F protein.

[0062] In some embodiments, the nucleic acid further comprises a polynucleotide encoding other proteins or polypeptides other than a mutant of the MPV F protein.

[0063] In some embodiments, 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.

[0064] A genetic engineering vector, comprising any one of the above nucleic acids, or comprising a polynucleotide capable of being transcribed into any one of the above nucleic acids.

[0065] A host cell comprising any one of the above genetic engineering vectors.

[0066] A mutant of the MPV F protein encoded by any of the above nucleic acids;

[0067] In some embodiments, the mutant is a trimer.

[0068] A delivery vector comprising a nucleic acid composition, wherein the nucleic acid composition comprises a first nucleic acid or a first genetically engineered vector, wherein the first nucleic acid is any of the nucleic acids described above, and the first genetically engineered vector is any of the genetically engineered vectors described above;

[0069] Alternatively, 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, and the multiple first nucleic acids are independent of each other or the multiple first genetic engineering vectors are independent of each other.

[0070] In some embodiments, the nucleic acid composition further comprises a second nucleic acid or a second vector, wherein the second nucleic acid comprises a polynucleotide encoding a protein or polypeptide other than a mutant of the MPV F protein, and the second vector comprises a polynucleotide encoding a protein or polypeptide other than a mutant of the MPV F protein;

[0071] And / or, the first nucleic acid and the second nucleic acid are RNA; more preferably, the first nucleic acid and the second nucleic acid are mRNA.

[0072] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNPs), a cationic liposome, a cationic protein, or a lipid polymer (LPP);

[0073] In some embodiments, the delivery vehicle is a lipid nanoparticle comprising a cationic lipid comprising the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0074] in:

[0075] L 3 and L 4 The same or different, 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 The same or different, each independently a C5-C8 alkylene group;

[0076] G 4 and G 5 are the same or different and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S- or -SC(=O)-; in some embodiments, G 4 and G 5 are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, or -O-; in some embodiments, G 4 and G 5 the same or different, each independently selected from -O-(C=O)- or -(C=O)-O-;

[0077] R 18 and R 19 are the same or different, and are each independently a C5-C27 alkyl group, or a C5-C27 alkenyl group containing one or more double bonds; in some embodiments, R 18 and R 19 are the same or different and are each independently a C8-C20 alkyl group or a C8-C20 alkenyl group 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 the same or different, each independently

[0078] R 20 is halogen, hydroxy, 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, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; in some embodiments, R 20 is fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl or acetamido;

[0079] z is 1, 2 or 3.

[0080] In some embodiments, the cationic lipid is the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0081] Alternatively, the cationic lipid is the following compound, or a pharmaceutically acceptable salt thereof:

[0082] A pharmaceutical composition comprising any of the aforementioned nucleic acids, any of the aforementioned genetic engineering vectors, any of the aforementioned host cells, any of the aforementioned mutants or any of the aforementioned delivery vectors, and a pharmaceutically acceptable carrier.

[0083] In some embodiments, the pharmaceutical composition includes a plurality of said delivery vehicles;

[0084] Alternatively, the pharmaceutical composition comprises a plurality of said nucleic acids.

[0085] In some embodiments, the pharmaceutical composition comprises two nucleic acids, the two nucleic acids are formulated together or separately in lipid nanoparticles, the two nucleic acids respectively encode a mutant of the subtype A MPV F protein and a mutant of the subtype B MPV F protein, and the mutant of the subtype A MPV F protein and the mutant of the subtype B MPV F protein encoded by the two nucleic acids are selected from the group consisting of:

[0086] (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: 1, 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: 2; and

[0087] (2) a mutant comprising mutations A147C and A159C and 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 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.

[0088] Use of any of the aforementioned nucleic acids, any of the aforementioned genetic engineering vectors, any of the aforementioned host cells, any of the aforementioned mutants, any of the aforementioned delivery vectors, or any of the aforementioned pharmaceutical compositions in the preparation of a drug;

[0089] In some embodiments, the medicament is used to prevent or treat MPV infection or a disease caused by MPV infection.

[0090] In some embodiments, the medicament is used to prevent or treat hMPV infection or a disease caused by hMPV infection.

[0091] In some embodiments, the drug is a vaccine.

[0092] A vaccine comprising any of the aforementioned nucleic acids, any of the aforementioned genetic engineering vectors, any of the aforementioned mutants, or any of the aforementioned delivery vectors. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figures 1A to 1F are the results of IgG antibody titer detection induced by mice after two immunizations with different mRNA vaccines (Figures 1C to 1F, GMT), where the day of the first immunization is counted as day 0 (D0) and the second immunization is on day 22 (D22); in Figures 1A to 1F, "hMPV / AF protein" represents the F protein of hMPV subtype A (the same applies to "hMPV / AF protein" appearing in other figures), "hMPV / BF protein" represents the F protein of hMPV subtype B (the same applies to "hMPV / BF protein" appearing in other figures), and "1155+1171" represents a bivalent hMPV mRNA vaccine, which means that the LNP preparation encapsulating the mRNA numbered 1155 and the LNP preparation encapsulating the mRNA numbered 1171 are mixed and injected into mice. The same applies to the bivalent hMPV mRNA vaccines represented by other groups, and the same applies to other figures without special instructions.

[0094] Figures 2A and 2B are the IgG antibody titer test results (GMT) 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 second immunization was on day 21 (D21). Figure 2A is the IgG antibody titer specifically binding to hMPV / AF protein, and Figure 2B is the IgG antibody titer specifically binding to hMPV / BF protein.

[0095] Figures 3A to 3D are the neutralizing antibody test results (GMT) against hMPV / A pseudovirus induced by mice after two immunizations with different mRNA vaccines. The antibody titer is NT50. The day of the first immunization is counted as D0, and the second immunization is on D22. "hMPV / A" indicates hMPV subtype A.

[0096] Figures 4A to 4G are the neutralizing antibody test results (GMT) against hMPV / A pseudovirus induced by mice after two immunizations with different mRNA vaccines. The antibody titer is NT50. The day of the first immunization is counted as D0, and the second immunization is on D21. "hMPV / A" represents hMPV subtype A.

[0097] 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-γ on D74, Figure 5B is the ELISpot test result of IFN-γ on D116, and Figure 5C is a comparison of the ELISpot test results of IFN-γ and IL-4 on D116.

[0098] Figures 6A to 6H are the ELISpot detection 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 D21. Figure 6A is the ELISpot detection result of IFN-γ 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-γ and IL-4 on D7, Figure 6E is the ELISpot detection result of IFN-γ 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, and Figure 6H is a comparison of the ELISpot detection results of IFN-γ and IL-4 on D77. "*" in Figures 6D and 6H indicates P<0.05, "***" indicates P<0.001, and "ns" indicates P>0.05.

[0099] Figures 7A to 7C are the neutralizing antibody test results (GMT) against hMPV / A pseudovirus induced by mice after two immunizations with different mRNA vaccines. The antibody titer is NT50. The day of the first immunization is counted as D0, and the second immunization is on D28. hMPV / A represents hMPV subtype A.

[0100] 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-γ on D49, Figure 8C is the ELISpot test result of IFN-γ on D57, and Figure 8D is a comparison of the ELISpot test results of IFN-γ and IL-4 on D57.

[0101] Detailed Description of the Invention

[0102] 1. Definition

[0103] All patents, patent applications, scientific publications, manufacturer's instructions and guidelines, etc., cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such publication.

[0104] Unless otherwise indicated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, and microbiology used herein are widely used terms in the corresponding fields (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). At the same time, in order to better understand this application, definitions and explanations of related terms are provided below.

[0105] As used herein, the expressions "comprise," "include," "contain," and "have" are open ended and mean 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."

[0106] As used herein, unless the context indicates otherwise, the singular expressions "a," "an," "the," and similar references used in the context of describing the present application (particularly in the context of the claims) should be interpreted to cover both the singular and the plural. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. The terms "at least one" or "one or more" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9 or more.

[0107] The numerical ranges described herein should be understood to encompass any and all subranges contained therein. For example, the range "1 to 10" should be understood to include not only the explicitly stated values ​​of 1 and 10, but also any individual 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 that use only one value as a minimum or maximum value.

[0108] As used herein, the terms "and / or," "any combination thereof," and their grammatical equivalents are used interchangeably. These terms may expressly refer to any combination. For example, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may refer to "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C."

[0109] As used herein, the term "naturally occurring" or "naturally occurring" refers to the fact that a substance can be found in nature. For example, a peptide, amino acid, protein, or nucleic acid that is present in an organism (including viruses) and can be isolated from a source in nature and has not been experimentally modified by man is naturally occurring.

[0110] As used herein, the term "non-naturally occurring" when used to describe a nucleic acid herein is intended to mean that the nucleic acid is not found in nature. For example, 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 wild-type strains of the virus in question. Such genetic alterations include, 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 the viral genetic material. Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.

[0111] Unless otherwise stated, all methods described herein can be performed in any suitable order.

[0112] As used herein, the term "wild type" means that the sequence is naturally occurring and has not been artificially modified, including naturally occurring mutants.

[0113] The term "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. Throughout the specification and claims, an amino acid substitution at a specific position in a protein sequence is referred to using the code "(amino acid residue in the wild-type protein) (amino acid position) (amino acid residue in the engineered protein)". For example, the code A147C refers to the substitution of an alanine (A) residue at position 147 of the amino acid sequence of a reference protein by a cysteine ​​(C) residue (in a mutant of the reference protein). In the case where there are differences in the amino acid residues at the same position between different wild-type sequences, the amino acid code before the position number, such as "147C", can be omitted in the code.

[0114] As used herein, the term "% identity" or "% identity" refers to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences to be compared. The differences between the two sequences can be distributed over local regions (segments) or over the entire length of the sequences to be compared. The identity between the two sequences is usually determined after optimal alignment of a segment or "comparison window." 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. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or the similarity search method described by computer programs such as GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the publicly available BLASTN or BLASTP algorithms available on the National Center for Biotechnology Information (NCBI) website.

[0115] "% 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 % identity. In some embodiments, the degree of identity 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 identity is given for the entire length of the reference sequence. Alignment for determining sequence identity can be performed using tools known in the art, preferably utilizing optimal sequence alignment, e.g., utilizing Align, utilizing standard settings, preferably EMBOSS::needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0116] As used herein, "nucleotides" include deoxyribonucleotides, deoxyribonucleotides, deoxyribonucleotide derivatives, and ribonucleotide derivatives. As used herein, "ribonucleotides" are constituents of ribonucleic acid (RNA), consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Deoxyribonucleotides" are constituents of deoxyribonucleic acid (DNA), also consisting of one base molecule, one pentose molecule, and one phosphate molecule. They refer to nucleotides in which the hydroxyl group at the 2' position of the β-D-ribofuranosyl group is replaced by hydrogen. They are the main chemical components of chromosomes.

[0117] "Nucleotide" is generally 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.

[0118] As used herein, the term "nucleic acid" generally refers to a polymer comprising deoxyribonucleotides (deoxyribonucleic acid, referred to as DNA) or a polymer comprising ribonucleotides (ribonucleic acid, referred to as RNA) or any compound of a combination thereof. In addition, nucleic acids herein also include derivatives of nucleic acids. The term "derivatives of nucleic acids" includes chemical derivatization of nucleic acids on the bases, sugars or phosphates of the nucleotides, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. In addition, herein, nucleic acids can be in the form of single-stranded or double-stranded linear or covalently closed circular molecules.

[0119] "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to refer to the order of nucleotides in a polynucleotide. Those skilled in the art will understand that a DNA coding strand (sense strand) and the RNA it encodes can be considered to have the same nucleotide sequence, with deoxythymidylate in the DNA coding strand sequence corresponding to uridine in the RNA sequence it encodes. The RNA corresponding to a DNA is a polynucleotide in which all Ts in the DNA are replaced by Us. The mRNA corresponding to a DNA is a polynucleotide in which all Ts in the DNA are replaced by Us.

[0120] The polynucleotide may comprise one or more segments (nucleic acid fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8 segments). For example, the polynucleotide may comprise a segment encoding a polypeptide of interest. In a specific embodiment, the polynucleotide may comprise a segment encoding a polypeptide of interest and a regulatory segment (including but not limited to segments for transcriptional regulation and translational regulation). In one embodiment, 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.

[0121] As used herein, the term "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. Representative examples of 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.

[0122] As used herein, the term "5' untranslated region" or "5'-UTR" can be an RNA sequence in an mRNA that is upstream of the coding sequence and is not translated into protein. The 5'-UTR in a gene generally begins at the transcription start site and ends at the nucleotides 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 the addition of 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.

[0123] As used herein, the term "3' untranslated region" or "3'-UTR" can be an RNA sequence in an mRNA that is located downstream of a coding sequence and is not translated into protein. The 3'-UTR in an mRNA is located between the stop codon and the poly(A) sequence of the coding sequence, for example, starting from the nucleotides downstream of the stop codon and ending at the nucleotides upstream of the poly(A) sequence.

[0124] As used herein, the terms "poly(A)", "poly(A) sequence", and "poly(A) tail" are used interchangeably. Naturally occurring poly(A) sequences are typically composed of adenine ribonucleotides. In the present application, the term "modified poly(A) sequence" refers to a poly(A) sequence that contains nucleotides or nucleotide segments other than adenine ribonucleotides. The poly(A) sequence is typically located at the 3' end of the mRNA, for example, at the 3' end (downstream) of the 3'-UTR.

[0125] As used herein, the term "5'-cap structure": The 5'-cap structure is typically located at the 5' end of the mature mRNA. In some embodiments, the 5'-cap structure is linked to the 5'-end of the mRNA via a 5'-5'-triphosphate bond. The 5'-cap structure is typically formed by modified (e.g., methylated) ribonucleotides (particularly guanine nucleotide derivatives). For example, m7GpppN (cap 0 or "cap0" is a cap structure formed by the 5' phosphate group of the hnRNA reacting with the 5'-phosphate group of the m7GTP under the action of guanylyltransferase to form a 5',5'-phosphodiester bond), wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap structure. In some embodiments, 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 referred to as "cap 1"), 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 referred to as "cap 2"), cap 4, cap 0 analogs, cap 1 analogs, cap 2 analogs, or cap 4 analogs.

[0126] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve the production of transcripts and / or polypeptides. The term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA (transcript). The term "in vitro transcription" refers to the synthesis of RNA, particularly mRNA, in a cell-free system (e.g., in an appropriate cell extract) in vitro (see, e.g., Pardi N., Muramatsu H., Weissman D., Karikó K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.). 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. The term "transcription" encompasses "in vitro transcription."

[0127] As used herein, 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. In other embodiments, the host cell is an antigen presenting cell, particularly a dendritic cell, a monocyte, or a macrophage. The nucleic acid can be present in the host cell with a single copy or with several copies. In some embodiments, the host cell can be a cell expressing the polypeptide of the present application therein.

[0128] In the context of the present application, 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, for example, a circular plasmid digested with a restriction enzyme, thereby converting the circular plasmid molecule into a linear molecule and the resulting molecule of the circular plasmid linearization. Plasmids can replicate, i.e., amplify the genetic information stored as chromosomal DNA in the cell, and can be used for cloning, i.e., for amplifying genetic information in bacterial cells. In an alternative embodiment, the DNA plasmid is a medium copy or high copy plasmid. In another alternative embodiment, the DNA plasmid is a high copy plasmid. Examples of such high copy plasmids include, for example, pUC and pTZ plasmids or any other plasmid (e.g., pMB1, pCoIE1) comprising a replication origin that supports high copies of the plasmid.

[0129] The term "vaccine" is typically understood as a prophylactic or therapeutic material that provides at least one antigen or antigenic function that can stimulate the body's adaptive immune system to provide an adaptive immune response.

[0130] The term "treatment" or the like is used herein to generally refer to 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 prophylactic treatment for preventing a disease or its symptoms in whole 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 owing 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.

[0131] As used herein, the terms "subject" and "patient" can be used interchangeably. In certain embodiments, the subject is a mammal, such as a human, non-human primate (e.g., ape, chimpanzee, monkey, and orangutan), domesticated animal (including dog and cat and livestock (e.g., horse, cattle, pig, sheep, and goat)), or other mammal. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or a neoplastic disease.

[0132] As used herein, the term "administer" refers to providing or administering a medicament to a subject by any effective route. Exemplary 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. When used to treat a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom. When used to prevent a disease, disorder, condition, or symptom, administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom.

[0133] Herein, some elements of the present application are described. These elements are listed together with specific embodiments, but it should be understood that they can be combined in any manner and in any number to produce additional embodiments. The various described examples and preferred embodiments should not be construed to limit the present application to only the explicitly described embodiments. This specification should be understood to support and include embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. In addition, unless the context indicates otherwise, any permutation and combination of all described elements in this application should be considered to be disclosed by the specification of this application. For example, in one embodiment, the nucleic acid comprises a polynucleotide encoding a mutant of the MPV F protein, wherein the mutations of the mutant relative to the wild-type MPV F protein include: A147C and A159C, and in another embodiment, the nucleic acid is mRNA. Then the following scheme is also an embodiment claimed in this application: a nucleic acid comprising a polynucleotide encoding a mutant of the MPV F protein, wherein the mutations of the mutant relative to the wild-type MPV F protein include: A147C and A159C, wherein the nucleic acid is mRNA.

[0134] 2. Nucleic Acids

[0135] The hMPV genome is approximately 13kb in length and contains 8 genes, encoding a total of 9 proteins: nucleoprotein (N protein), phosphoprotein (P protein), matrix protein (M protein), fusion protein (F protein), small hydrophobic protein (SH protein), adsorption protein (G protein), RNA polymerase (L protein), transcription elongation factor encoded by the M2 gene (M2-1) and RNA synthesis regulatory factor (M2-2). Among them, F protein, G protein and SH protein are viral envelope glycoproteins, and F protein is essential for infection.

[0136] 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 under the action of proteases. F1 and F2 are connected by a disulfide bond.

[0137] Given the substantial conservation of the hMPV F protein amino acid sequence, one of ordinary skill in the art can readily compare amino acid positions between different native hMPV F protein sequences to determine the corresponding amino acid positions of the 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 a reference hMPV F protein amino acid sequence to compare amino acids at specific positions within the hMPV F protein. For the purposes of this disclosure (unless the context indicates otherwise), amino acid positions of the hMPV F protein herein are given with reference to the sequence of hMPV F0 set forth in SEQ ID NO: 64 (i.e., the amino acid sequence of the full-length native F precursor polypeptide of subtype A2 strain CAN97-83 (GenBank: AY297749.1)). However, it will be appreciated by those skilled in the art that 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). Thus, it should be understood that when a particular amino acid residue is referred to by a number, this is not limited to the amino acid residue at exactly that numbered position when counting from the start of a given amino acid sequence, but also includes the equivalent / corresponding amino acid residues in any and all amino acid sequences of the hMPV F protein, even if the amino acid residue is not at the same exact numbered position (e.g., 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).

[0138] One embodiment of the present disclosure provides a nucleic acid comprising a polynucleotide encoding a mutant of MPV F protein. Compared to the wild-type MPV F protein, the mutant of MPV F protein (hereinafter referred to as "mutant") comprises or is a disulfide bond mutation.

[0139] In some embodiments, the MPV F protein is hMPV F protein.

[0140] In some embodiments, 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, herein, "multiple" means at least two, for example, two, three, four, five or more.

[0141] In some embodiments, 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.

[0142] In some embodiments, the disulfide bond mutation is selected from one of the following: A147C and A159C, L165C and F196C, N145C and A161C.

[0143] In some embodiments, the mutant is selected from one of the following (1) to (38):

[0144] (1) a mutant comprising the amino acid sequence shown in SEQ ID NO: 1 or 2;

[0145] (2) 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 set forth in SEQ ID NO: 1 or 2;

[0146] (3) a mutant comprising the amino acid sequence shown in SEQ ID NO: 3 or 4;

[0147] (4) a mutant 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 set forth in SEQ ID NO: 3 or 4;

[0148] (5) a mutant comprising the amino acid sequence shown in SEQ ID NO: 5 or 6;

[0149] (6) 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 set forth in SEQ ID NO: 5 or 6;

[0150] (7) a mutant comprising the amino acid sequence shown in SEQ ID NO: 7 or 8;

[0151] (8) 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 set forth in SEQ ID NO: 7 or 8;

[0152] (9) a mutant comprising the amino acid sequence shown in SEQ ID NO: 9 or 10;

[0153] (10) 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 set forth in SEQ ID NO: 9 or 10;

[0154] (11) a mutant comprising the amino acid sequence shown in SEQ ID NO: 11 or 12;

[0155] (12) a mutant comprising mutations S149C and V157C 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: 11 or 12;

[0156] (13) a mutant comprising the amino acid sequence shown in SEQ ID NO: 13 or 14;

[0157] (14) 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 set forth in SEQ ID NO: 13 or 14;

[0158] (15) a mutant comprising the amino acid sequence shown in SEQ ID NO: 15 or 16;

[0159] (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 set forth in SEQ ID NO: 15 or 16;

[0160] (17) a mutant comprising the amino acid sequence shown in SEQ ID NO: 17 or 18;

[0161] (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 set forth in SEQ ID NO: 17 or 18;

[0162] (19) a mutant comprising the amino acid sequence shown in SEQ ID NO: 19 or 20;

[0163] (20) 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 set forth in SEQ ID NO: 19 or 20;

[0164] (21) a mutant comprising the amino acid sequence shown in SEQ ID NO: 21 or 22;

[0165] (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 set forth in SEQ ID NO: 21 or 22;

[0166] (23) a mutant comprising the amino acid sequence shown in SEQ ID NO: 23 or 24;

[0167] (24) 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;

[0168] (25) a mutant comprising the amino acid sequence shown in SEQ ID NO: 25 or 26;

[0169] (26) a mutant comprising mutations V55C and V169C 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: 25 or 26;

[0170] (27) a mutant comprising the amino acid sequence shown in SEQ ID NO: 27 or 28;

[0171] (28) a mutant comprising mutations L58C and T174C 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: 27 or 28;

[0172] (29) a mutant comprising the amino acid sequence shown in SEQ ID NO: 29 or 30;

[0173] (30) 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 set forth in SEQ ID NO: 29 or 30;

[0174] (31) a mutant comprising the amino acid sequence shown in SEQ ID NO: 81 or 82;

[0175] (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 set forth in SEQ ID NO: 81 or 82;

[0176] (33) a mutant comprising the amino acid sequence shown in SEQ ID NO: 83;

[0177] (34) 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 set forth in SEQ ID NO: 83;

[0178] (35) a mutant comprising the amino acid sequence shown in SEQ ID NO: 84;

[0179] (36) 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 set forth in SEQ ID NO: 84;

[0180] (37) a mutant comprising the amino acid sequence shown in SEQ ID NO: 85; and

[0181] (38) 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.

[0182] In some embodiments, the wild-type MPV is subtype A or subtype B.

[0183] In some embodiments, the wild-type MPV is wild-type hMPV.

[0184] In some embodiments, the wild-type hMPV is subtype A1, subtype A2, subtype B1, or subtype B2.

[0185] In some embodiments, 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), or CN / gz01 / 08 (GenBank: GQ153651.1). In an alternative specific example, the amino acid sequence of the wild-type MPV F protein is shown in SEQ ID NO: 64 or 65. It will be understood that in other embodiments, the wild-type MPV is not limited to the above-mentioned ones, but may be other.

[0186] In some embodiments, the mutant comprises an F1 polypeptide and an F2 polypeptide. The F1 polypeptide and F2 polypeptide of the mutant 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 any other subtype F protein. In addition, the F1 polypeptide and F2 polypeptide can be derived from the F protein of the same genotype MPV or from F proteins of different genotypes. For example, the F1 polypeptide is derived from the F protein of MPV subtype A, and the F2 polypeptide is derived from the F protein of MPV subtype B; or the F1 polypeptide is derived from the F protein of MPV subtype B, and the F2 polypeptide is derived from the F protein of MPV subtype A.

[0187] In some embodiments, the mutant further comprises one or more of the following: a signal peptide, a ferritin polypeptide, a transmembrane domain (TM) of the MPV F protein (e.g., the transmembrane domain of the wild-type MPV F protein), and a trimerization domain (e.g., a foldon). Herein, unless otherwise specified, a ferritin polypeptide refers to a polypeptide derived from ferritin, and can be full-length ferritin, full-length ferritin with mutations, truncated ferritin, or truncated ferritin with mutations. 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 can, for example, be a GCN4 leucine zipper, a trimerization motif from lung surfactant protein, or a phage T4 fibritin foldon. In some embodiments, the foldon is located at the C-terminus of the F1 polypeptide. In an alternative specific example, the amino acid sequence of the foldon is: GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 66). In some embodiments, the ferritin polypeptide is located at the C-terminus of the F1 polypeptide. In an alternative specific example, the amino acid sequence of the ferritin polypeptide is shown in SEQ ID NO: 67.

[0188] In some embodiments, the mutant further contains a transmembrane domain (TM) of an MPV F protein (eg, a transmembrane domain of a wild-type MPV F protein).

[0189] In some embodiments, 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). In some embodiments, the mutant comprises a signal peptide and a trimerization domain. In some embodiments, the mutant comprises a transmembrane domain and a trimerization domain of the MPV F protein. In other embodiments, the mutant comprises a signal peptide, a transmembrane domain, and a trimerization domain of the MPV F protein.

[0190] In some embodiments, 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.

[0191] In some embodiments, the F1 polypeptide of the mutant 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).

[0192] In other embodiments, the mutant Fi polypeptide comprises a deletion relative to the full-length Fi polypeptide of the corresponding wild-type MPV F protein (also referred to as the native Fi polypeptide). In some embodiments, the mutant Fi polypeptide comprises a deletion relative to the full-length Fi polypeptide of the corresponding wild-type MPV F protein: one or more of the following: a portion of the extracellular region, a portion of the transmembrane domain, the entire transmembrane domain, a portion of the cytoplasmic domain, or the entire cytoplasmic domain. For example, the sequence from amino acid residue 474 onward in the native Fi polypeptide may be absent from the mutant Fi polypeptide. For example, the sequence from amino acid residue 478 or 491 onward in the native Fi polypeptide may be absent from the mutant Fi polypeptide, i.e., the mutant Fi polypeptide comprises a deletion of 1 to 62 or 1 to 49 amino acid residues from the C-terminus of the native Fi polypeptide. For example, the mutant Fi polypeptide may lack the entire cytoplasmic domain. In another example, the mutant Fi polypeptide may lack the cytoplasmic domain and a portion or all of the transmembrane domain. As a further example, the mutant Fi polypeptide lacks amino acid residues from positions 489, 490, 491, 510, 512, 513, 514, 515, 520, 525, or 530 to 539 compared to the native Fi polypeptide. Typically, for mutants with attached trimerization domains (such as foldon) or ferritin polypeptides, amino acid residues from positions 489, 490, or 491 to 539 are absent. Thus, in some embodiments, the mutant has attached a trimerization domain or ferritin polypeptide, and the mutant Fi polypeptide lacks amino acid residues from positions 489, 490, or 491 to 539 compared to the native Fi polypeptide. In other embodiments, the mutant Fi polypeptide comprises or consists of amino acid residues 103 to 490 of the native FO polypeptide.

[0193] In some embodiments, the mutant F2 polypeptide can have the same length as the full-length F2 polypeptide of the corresponding wild-type MPV F protein, or can have a deletion, for example, 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.

[0194] In addition, the nucleic acid may further comprise one or more of the following: a polynucleotide encoding a protease cleavage site (e.g., a thrombin cleavage site (LVPRGS, SEQ ID NO: 68)), a polynucleotide encoding a protein tag (e.g., a 6×His tag (HHHHHH, SEQ ID NO: 69) and a streptomycin tag II (WSHPGFEK, SEQ ID NO: 70), and a polynucleotide encoding a linker sequence (e.g., GG, SAIG, and GS). The polypeptides encoded by these sequences are not essential for the function of the MPV F protein (e.g., induction of an immune response). Those skilled in the art are familiar with such sequences and will appreciate that these sequences are not limited to the above.

[0195] In some embodiments, the nucleic acid is isolated.

[0196] In some embodiments, the nucleic acid is a non-naturally occurring nucleic acid.

[0197] In some embodiments, the nucleic acid is a codon-optimized polynucleotide. Codon optimization methods are known in the art and can be used as provided herein. In some embodiments, codon optimization can be used to: match the codon frequency of the target and host organism 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 structure 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; regulate translation rate so that the various domains of the protein can fold properly; or reduce or eliminate problematic secondary structures within polynucleotides. 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. In some embodiments, an optimization algorithm is used to optimize the open reading frame (ORF) sequence. In some embodiments, 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.

[0198] In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is RNA, and the RNA contains an open reading frame (ORF).

[0199] In some embodiments, the nucleic acid is RNA, and the RNA is mRNA.

[0200] In some embodiments, the nucleic acid further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail. In an alternative specific example, the nucleic acid further comprises a 5'-cap structure, a 5'-UTR, a 3'-UTR, and a poly(A) tail.

[0201] In some embodiments, the 5'-cap structure is selected from m 7 GpppG、m2 7,3′-O GpppG、m 7 Gppp(5')N1 and m 7 Gppp(m 2′-O) At least one of 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 guanosine nucleoside, "7" represents the methyl group at the 7-position of guanine, and "m 2′-O " represents a methyl group at the 2'-O position of the nucleotide. In some embodiments, the 5'-cap structure is m 7 Gppp(5')N1 or m 7 Gppp(m 2′-O )N1. It will be appreciated that, in other embodiments, the 5'-cap structure is not limited to the above.

[0202] In some embodiments, 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. It should be noted that 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.

[0203] It is understood that, in other embodiments, 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.

[0204] In some embodiments, the nucleotides comprising 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 comprising the poly(A) tail comprise at least 20, at least 40, at least 80, at least 100, or at least 120 A nucleotides consecutively.

[0205] In some embodiments, 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.

[0206] In some embodiments, the nucleic acids described above do not contain modified nucleotides.

[0207] In some embodiments, the nucleic acids described above contain modified nucleotides.

[0208] In some embodiments, the nucleic acid comprises modified nucleosides. In some embodiments, the modified nucleosides comprised by the nucleic acid comprise at least one of modified uridine, modified cytidine, modified adenosine, and modified guanosine.

[0209] In some embodiments, the modified nucleoside in the nucleic acid is a modified uridine. In some embodiments, 0.1% to 100% of the uridine in the 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 (ψ), N1-methyl pseudouridine, pyridin-4-one 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-oxyethyl acid (cmo5U), uridine-5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio -uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine Uridine (τm5s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3 -carboxypropyl) pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl -uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-methoxyformylvinyl)uridine (5-(2-carbomethoxyvinyl)uridine) and 5-[3-(1-E-propenylamino)uridine.

[0210] In some embodiments, the modified nucleosides in the nucleic acids are modified cytidines. In some embodiments, 0.1% to 100% of the cytidines in the 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-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-cytidine, 5-methyl-cytidine, 6-aza-cytidine, 5-methyl-cytidine, 6-aza-cytidine, 6-methyl-cytidine, 4-acetyl-cytidine, 5-acetyl-cytidine, 6 ... iso-zebulin, 5-methyl-zebulin, 5-aza-2-thio-zebulin, 2-thio-zebulin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl Cm), 2'-F-arabino-cytidine, 2'-F-cytidine and 2'-OH-arabino-cytidine.

[0211] In some embodiments, 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-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio -adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabino-adenosine, 2'-F-adenosine, 2'-OH-arabino-adenosine and N6-(19-amino-pentaoxahonadecyl)-adenosine.

[0212] In some embodiments, the modified nucleoside in the above nucleic acid is a modified guanosine. In some embodiments, 0.1% to 100% of the guanosine in the above 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), yW, peroxyyW (o2yW), hydroxyyW (OHyW), undermodified hydroxyyW (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxy queuosine (o Q), galactosyl-braided guanosine (galQ), mannosyl-braided guanosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2,7G), N2,N2,7-dimethyl-guanosine (m2,2,7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine (m1Gm), N2,7-dimethyl-2'-O-methyl-guanosine (M2,7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thioguanosine, O6-methyl-guanosine, 2'-F-arabinoguanosine and 2'-F-guanosine.

[0213] In some embodiments, the modified nucleotides in the nucleic acids described above comprise nucleotides containing isotopes.

[0214] In some embodiments, the nucleic acid comprises nucleotides containing isotopes of hydrogen. Hydrogen isotopes are not limited to deuterium and tritium. In addition, in some embodiments, the nucleic acid further comprises or contains nucleotides containing isotopes of elements other than hydrogen, including but not limited to carbon, oxygen, nitrogen, and phosphorus.

[0215] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide having a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide as set forth in one of SEQ ID NOs: 34-63 and 86-93 and encoding the corresponding MPV F protein as set forth in SEQ ID NOs: 1-30 and 81-85. For example, the nucleic acid comprises an RNA corresponding to a polynucleotide 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 the polynucleotide sequence of SEQ ID NO: 34 and encodes the MPV F protein of SEQ ID NO: 1.

[0216] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as shown in SEQ ID NO: 1 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: 34 or 86.

[0217] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence 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.

[0218] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:3 and having a nucleotide sequence 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:36.

[0219] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:4 and having a nucleotide sequence 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.

[0220] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:5 and having a nucleotide sequence 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:38.

[0221] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:6 and having a nucleotide sequence 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.

[0222] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as set forth in SEQ ID NO:7 and having a nucleotide sequence 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.

[0223] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence set forth in SEQ ID NO:8 and having a nucleotide sequence 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:41.

[0224] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:9 and having a nucleotide sequence 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:42.

[0225] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO: 10 and having a nucleotide sequence 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: 43.

[0226] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as set forth in SEQ ID NO: 11 and having a nucleotide sequence 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: 44.

[0227] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:12 and having a nucleotide sequence 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:45.

[0228] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:13 and a nucleotide sequence 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:46.

[0229] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:14 and having a nucleotide sequence 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:47.

[0230] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:15 and having a nucleotide sequence 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:48.

[0231] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as set forth in SEQ ID NO:16 and having a nucleotide sequence 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:49.

[0232] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as set forth in SEQ ID NO:17 and having a nucleotide sequence 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:50.

[0233] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence set forth in SEQ ID NO: 18 and having a nucleotide sequence 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: 51.

[0234] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO: 19 and having a nucleotide sequence 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: 52.

[0235] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a variant of the amino acid sequence as set forth in SEQ ID NO:20 and having a nucleotide sequence 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:53.

[0236] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as set forth in SEQ ID NO:21 and having a nucleotide sequence 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:54.

[0237] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as set forth in SEQ ID NO:22 and having a nucleotide sequence 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:55.

[0238] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as shown in SEQ ID NO:81 and having a nucleotide sequence 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:88.

[0239] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as shown in SEQ ID NO:82 and having a nucleotide sequence 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:89.

[0240] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as shown in SEQ ID NO:83 and a nucleotide sequence 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.

[0241] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as shown in SEQ ID NO:84 and having a nucleotide sequence 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:91.

[0242] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide encoding a mutant of the amino acid sequence as shown in SEQ ID NO:85 and a nucleotide sequence 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:93.

[0243] In some embodiments, the nucleic acid comprises RNA corresponding to a polynucleotide having a nucleotide sequence as shown in any one of SEQ ID NOs: 34-63 and 86-93.

[0244] In some embodiments, the nucleic acid of any of the above embodiments is mRNA, and all uridine in the nucleic acid is replaced by N1-methylpseudouridine.

[0245] In some embodiments, the nucleic acid comprises an RNA corresponding to a polynucleotide having a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a polynucleotide as set forth in one of SEQ ID NOs: 34-63 and 86-93 and encoding the corresponding MPV F protein as set forth in SEQ ID NOs: 1-30 and 81-85, wherein all uridines in the nucleic acid are replaced with N1-methylpseudouridine.

[0246] In some embodiments, the nucleic acid comprises RNA corresponding to a polynucleotide having a nucleotide sequence as shown in any one of SEQ ID NOs: 34 to 63 and 86 to 93, and all uridine in the nucleic acid is replaced by N1-methyl pseudouridine.

[0247] In some embodiments, the nucleic acid is mRNA, which comprises RNA corresponding to a polynucleotide having a nucleotide sequence as shown in any one of SEQ ID NOs: 34 to 63 and 86 to 93, and all uridine in the nucleic acid is replaced by N1-methyl pseudouridine.

[0248] In other embodiments, the nucleic acid is DNA.

[0249] In some embodiments, the DNA is capable of being transcribed into RNA in vitro.

[0250] In some embodiments, the nucleic acid comprises a polynucleotide 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 a polynucleotide sequence as set forth in one of SEQ ID NOs: 34-63 and 86-93 and encodes the corresponding MPV F protein as set forth in SEQ ID NOs: 1-30 and 81-85.

[0251] In some embodiments, the nucleic acid comprises a polynucleotide having a nucleotide sequence as shown in one of SEQ ID NOs: 34-63 and 86-93.

[0252] In some embodiments, the nucleic acid comprises a polynucleotide encoding a mutant of MPV F protein. It should be noted that "a mutant of MPV F protein" herein and below refers to any one of the multiple mutants of MPV F protein mentioned above.

[0253] In other embodiments, the nucleic acid comprises polynucleotides encoding multiple mutants of the MPV F protein. In this case, the coding strands for the multiple mutants of the MPV F protein are located on the same nucleic acid strand, and the polynucleotides encoding the multiple mutants of the MPV F protein are directly linked or indirectly linked via a linker. It should be noted that the "multiple mutants of the MPV F protein" herein and below refers to any multiple (e.g., two, three, four, or more) of the multiple MPV F protein mutants mentioned above.

[0254] For example, the multiple mutants of the MPV F protein are different MPV F proteins from the same subtype strain but with different mutations. For a further example, the multiple mutants of the MPV F protein are two, the first mutant being an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, and the second mutant being an MPV F protein derived from strain CAN97-83 with L165C and F196C mutations. Accordingly, the 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.

[0255] For another example, the multiple mutants of the MPV F protein are different MPV F proteins from different subtypes but with the same mutations. For a further example, the multiple mutants of the MPV F protein are two, the first mutant being an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, and the second mutant being an MPV F protein derived from strain NL / 1 / 99 with A147C and A159C mutations. Accordingly, the 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.

[0256] For another example, the multiple mutants of the MPV F protein are different MPV F proteins from different subtypes and have different mutations. For a further example, the multiple mutants of the MPV F protein are two, the first mutant being an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, and the second mutant being an MPV F protein derived from strain NL / 1 / 99 with L165C and F196C mutations. Accordingly, the nucleic acid comprises a nucleic acid encoding a polynucleotide for 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 L165C and F196C mutations.

[0257] For another example, there are three multiple mutants of the MPV F protein, the first mutant being a polynucleotide encoding an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, the second mutant being a polynucleotide encoding an MPV F protein derived from strain NL / 1 / 99 with L165C and F196C mutations, and the third mutant being a polynucleotide encoding an MPV F protein derived from strain CAN97-83 with N145C and A161C mutations. Accordingly, the nucleic acid comprises a polynucleotide encoding an MPV F protein derived from strain CAN97-83 with A147C and A159C mutations, a polynucleotide encoding an MPV F protein derived from strain NL / 1 / 99 with L165C and F196C mutations, and a polynucleotide encoding an MPV F protein derived from strain CAN97-83 with N145C and A161C mutations.

[0258] In some embodiments, the nucleic acid further comprises a polynucleotide encoding other proteins or polypeptides other than mutants of the MPV F protein. It is understood that other proteins or polypeptides other than mutants of the MPV F protein are proteins or polypeptides with biological significance (e.g., immunogenicity), such as other proteins or polypeptides of MPV, or mutants thereof (e.g., MPV G protein or mutants thereof), or proteins or polypeptides of other viruses, or mutants thereof.

[0259] Furthermore, it is understood that in the aforementioned nucleic acids, whether encoding a single mutant of the MPV F protein or multiple mutants of the MPV F protein, multiple (e.g., two or three) repeating units may be present on the same chain. For example, if a polynucleotide encoding a single mutant of the MPV F protein is referred to as "fragment A," the same nucleic acid may contain multiple A fragments. For another example, if a polynucleotide encoding multiple mutants of the MPV F protein is referred to as "fragment A," the same nucleic acid may contain multiple A fragments and multiple B fragments, one A fragment and multiple B fragments, or multiple A fragments and one B fragment.

[0260] In some embodiments, the nucleic acid is any one of the mRNAs listed in Table 1. The mRNA in Table 1 is an mRNA encoding a mutant of the hMPV F protein, comprising a Cap1-type cap structure, a 5'-UTR corresponding to the sequence set forth in SEQ ID NO:31, a 3'-UTR corresponding to the sequence set forth in SEQ ID NO:32, a poly(A) tail corresponding to the sequence set forth in SEQ ID NO:33, and an F protein coding region corresponding to one of SEQ ID NOs:34-63 and 86-93, wherein the F protein coding region is an RNA, wherein all uridine residues in all mRNAs listed in Table 1 are replaced with N1-methylpseudouridine. For example, the mRNA designated as No. 1154 comprises a Cap1-type cap structure, a 5'-UTR corresponding to the sequence set forth in SEQ ID NO:31, a 3'-UTR corresponding to the sequence set forth in SEQ ID NO:32, a poly(A) tail corresponding to the sequence set forth in SEQ ID NO:33, and an F protein coding region corresponding to the sequence set forth in SEQ ID NO:34, wherein all uridine residues in the mRNA designated as No. 1154 are replaced with N1-methylpseudouridine. In addition, in Table 1, all mRNAs in the G7 group also contain a polynucleotide encoding Ferritin with an amino acid sequence as shown in SEQ ID NO: 67 at its C-terminus; all mRNAs in the G8 group also contain a polynucleotide encoding a foldon derived from T4 phage fiber protein with a sequence as shown in SEQ ID NO: 66 at its C-terminus.

[0261] Table 1

[0262] In some embodiments, 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. When using the above-mentioned nucleic acid that does not contain a stop codon, those of ordinary skill in the art know that a stop codon (such as UGA or TGA) should be added at the appropriate position. It is understood that the above-mentioned nucleic acid can comprise one or more stop codons.

[0263] The nucleic acid comprises a polynucleotide encoding a mutant of MPV F protein having a disulfide bond mutation, which can induce a specific immune response and prevent or at least reduce human metapneumovirus infection.

[0264] In some embodiments, the nucleic acid or the mutant of the MPV F protein encoded by the nucleic acid can induce a significant humoral immune response in the body, causing the body to produce high levels of IgG antibodies and neutralizing antibodies against MPV.

[0265] In some embodiments, the nucleic acid or the MPV F protein mutant encoded thereby can induce a significant IFN-γ positive cellular immune response. In some embodiments, the nucleic acid or the MPV F protein mutant encoded thereby can induce a significant IFN-γ positive cellular immune response that is Th1 biased.

[0266] In some embodiments, the nucleic acid or the mutant of the MPV F protein encoded thereby can induce the production of neutralizing antibodies against subtype A hMPV.

[0267] In some embodiments, the nucleic acid or the mutant of the MPV F protein encoded thereby can induce the production of neutralizing antibodies against subtype B hMPV.

[0268] In some embodiments, the nucleic acid or the mutant of the MPV F protein encoded thereby can induce the production of neutralizing antibodies against subtype A hMPV and subtype B hMPV.

[0269] III. Genetic Engineering Vectors, Nucleic Acid Preparation Methods, Host Cells, and Protein Mutants

[0270] The disclosure also provides a kind of genetic engineering vector, this genetic engineering vector comprises the nucleic acid of any one of above-mentioned embodiment, or this genetic engineering vector comprises the polynucleotide that can be transcribed into the nucleic acid of any one of above-mentioned embodiment.In some embodiments, genetic engineering vector is plasmid, cosmid, virus, phage or another carrier conventionally used in genetic engineering.In an alternative specific example, genetic engineering vector is plasmid.In some embodiments, 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 the restriction site that allows to insert DNA element.Preferably the restriction site of multiple cloning site (MCS) form.

[0271] In some embodiments, the genetic engineering vector is an expression vector. In some embodiments, the genetic engineering vector comprises a promoter, a 5'-UTR, a coding region of a mutant of the MPV F protein according to 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.

[0272] In other embodiments, 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.

[0273] 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.

[0274] In addition, 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 the solid phase phosphoramidite method.

[0275] It is understood that the method for preparing the nucleic acid in any of the above embodiments is not limited to the above, and other methods may also be used.

[0276] In addition, 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.

[0277] In some embodiments, the host cell is an isolated cell.

[0278] In some embodiments, the host cells are used to store and / or amplify the nucleic acids described above.

[0279] In some embodiments, the host cell is a bacterial cell. Bacterial host cells include Escherichia coli (E. coli) cells well known to those skilled in the art.

[0280] Host cells of the present disclosure can be prepared by transforming competent host cells using the genetically engineered vectors of any of the above-mentioned embodiments. Competent host cells are cells with the ability of free extracellular genetic material (such as DNA plasmids) that does not rely on sequence uptake. Various bacterial cells well known to those skilled in the art are naturally able to take in exogenous DNA from the environment, and therefore can serve as bacterial host cells according to the present disclosure. In addition, it is known to those skilled in the art that competent bacterial host cells can be obtained from natural non-competent bacterial cells using, for example, electroporation or chemicals (such as, for example, calcium ion treatment and accompanying high temperature exposure). After uptake, exogenous DNA is preferably neither degraded nor integrated in the genome of the bacterial host cell.

[0281] In addition, the present disclosure also provides a mutant of MPV F protein, which is encoded by the nucleic acid of any of the above embodiments.

[0282] In some embodiments, the mutant of the MPV F protein is a unimolecular or multimolecular complex (eg, a trimer).

[0283] In addition, the present disclosure also provides an MPV immunogen, which comprises a mutant of the MPV F protein encoded by the nucleic acid of any one of the above embodiments.

[0284] In some embodiments, the MPV immunogen is a trimer. In some embodiments, the mutations of the trimer monomers are identical. In other embodiments, the mutations of the three trimer monomers are partially identical or completely different.

[0285] In addition, the present disclosure also provides a method for preparing a mutant of MPV F protein, the method comprising: transcribing a nucleic acid comprising a polynucleotide encoding the mutant of MPV F protein into mRNA; and translating the transcribed mRNA into a polypeptide or protein.

[0286] In some embodiments, the method for preparing the mutant of MPV F protein is performed entirely or partially in vitro.

[0287] 4. RNA and RNA Preparation Methods

[0288] The present disclosure also provides a method for preparing RNA, which comprises the step of performing transcription using the genetic engineering vector according to any of the above embodiments of the present disclosure.

[0289] In some embodiments, the method for preparing RNA is an in vitro method. In some embodiments, the method for preparing RNA comprises contacting the genetic engineering vector (e.g., plasmid) of any of the above embodiments with an RNA polymerase. In some embodiments, the method for preparing RNA further comprises the step of linearizing the genetic engineering vector (e.g., plasmid). In some embodiments, before linearization, the supercoil rate of the genetic engineering vector (e.g., plasmid) is at least about 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, etc.). In some embodiments, the method for preparing RNA further comprises the step of purifying the linearized genetic engineering vector. In some embodiments, the method for preparing RNA further comprises the step of purifying RNA.

[0290] The present disclosure also provides another method for preparing RNA, comprising the step of preparing the RNA by chemical synthesis based on 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 the solid-phase phosphoramidite method.

[0291] In some embodiments, the RNA is mRNA.

[0292] In some embodiments, any of the above methods for preparing RNA further comprises the steps of capping and optionally purifying the capped product. In some embodiments, the cap is a Cap1 cap. The Cap1 cap structure is as follows:

[0293] cap G 1 G 2 =m 7 G-5'-ppp-5'-Gm2'-3'-p-[m7=7-CH3; m2'=2'-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-=-PO2H-].

[0294] The capping reaction is as follows:

[0295] pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi

[0296] ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi

[0297] G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc

[0298] m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3')+AdoHyc.

[0299] In other embodiments, the method for preparing RNA is a partially in vitro method. In this case, the RNA preparation method includes the following steps: preparing a genetically engineered vector according to any of the above embodiments in vitro; and introducing the genetically engineered vector into the body (e.g., in the form of a plasmid). In some embodiments, the genetically engineered vector is encapsulated in a delivery vehicle. In this case, the genetically engineered vector is delivered into the body via the delivery vehicle.

[0300] In some embodiments, the RNA prepared in the method for preparing RNA according to any of the above embodiments comprises modified nucleosides or modified nucleotides. Correspondingly, the raw materials for preparing the RNA include 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.

[0301] In addition, the present disclosure also provides an RNA, which is prepared by the method for preparing RNA according to any of the above embodiments. In some embodiments, the RNA is mRNA.

[0302] 5. Nucleic Acid Composition

[0303] 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.

[0304] In some embodiments, the first nucleic acid is RNA. In some embodiments, the first nucleic acid is mRNA.

[0305] In some embodiments, the nucleic acid composition comprises a first nucleic acid or a first genetic engineering vector, wherein 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, and 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.

[0306] In some embodiments, the nucleic acid composition includes a first nucleic acid or a first genetic engineering vector, wherein the first nucleic acid is a nucleic acid comprising a polynucleotide encoding one of the multiple mutants of the above-mentioned MPV F protein, and the first genetic engineering vector is a genetic engineering vector comprising a polynucleotide encoding one of the multiple mutants of the above-mentioned MPV F protein.

[0307] In other embodiments, the nucleic acid composition includes a first nucleic acid or a first genetic engineering vector, wherein the first nucleic acid is a nucleic acid comprising a polynucleotide encoding multiple mutants among the multiple mutants of the above-mentioned MPV F protein, and the first genetic engineering vector is a genetic engineering vector comprising 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.

[0308] In some embodiments, the nucleic acid composition includes multiple first nucleic acids or multiple first genetic engineering vectors, wherein 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 the MPV F protein, and the multiple first genetic engineering vectors are used to clone or express multiple polynucleotides encoding the MPV F protein. It should be noted that "independent of each other" herein means that there is no linker connecting the multiple first nucleic acids, or that there is no linker connecting the multiple first genetic engineering vectors. In this case, the polynucleotides encoding the multiple mutants of the MPV F protein are not on the same nucleic acid chain.

[0309] In some embodiments, 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 mutation, 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.

[0310] For example, 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). In other words, 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.

[0311] For example, 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). In other words, 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.

[0312] For example, 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). In other words, 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.

[0313] For another example, the plurality of first nucleic acids include a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 having A147C and A159C mutations, a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain NL / 1 / 99 having A147C and A159C mutations, and a nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 having L165C and F196C mutations. In other words, the nucleic acid composition includes three first nucleic acids, a first first nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 having A147C and A159C mutations, a second first nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain NL / 1 / 99 having A147C and A159C mutations, and a third first nucleic acid comprising a polynucleotide encoding an MPV F protein derived from strain CAN97-83 having L165C and F196C mutations.

[0314] It is understandable that the above-mentioned 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.).

[0315] In some embodiments, the nucleic acid composition further comprises a second nucleic acid or a second vector, wherein the second nucleic acid comprises a polynucleotide encoding a protein or polypeptide other than a mutant MPV F protein, and the second vector comprises a polynucleotide encoding a protein or polypeptide other than a mutant MPV F protein. Proteins or polypeptides other than mutant MPV F protein are described above. In some embodiments, the second nucleic acid is RNA. In some embodiments, the second nucleic acid is mRNA. It is understood that the nucleic acid composition may include not only one second nucleic acid but also multiple second nucleic acids. For example, the nucleic acid composition may further comprise one second nucleic acid comprising a polynucleotide encoding a protein or polypeptide other than a mutant MPV F protein; or the nucleic acid composition may further comprise multiple second nucleic acids, each of which is independent of the other nucleic acids and comprises multiple polynucleotides encoding proteins or polypeptides other than a mutant MPV F protein. Similarly, the second vector is not limited to one but may also comprise multiple second vectors.

[0316] VI. Delivery vectors, pharmaceutical compositions, and their applications

[0317] The present disclosure also provides a delivery vector comprising the nucleic acid according to any of the above embodiments, the RNA according to any of the above embodiments, the genetic engineering vector according to any of the above embodiments, or the nucleic acid composition according to any of the above embodiments.

[0318] In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is mRNA.

[0319] In some embodiments, 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.

[0320] In some embodiments, the delivery vehicle is a lipid nanoparticle (LNP) comprising the nucleic acid of any of the above embodiments, the RNA of any of the above embodiments, the genetically engineered vector of any of the above embodiments, or the nucleic acid composition of any of the above embodiments.

[0321] In some embodiments, lipid nanoparticles refer to particles having a nanometer scale (eg, 1 nm to 1000 nm) that include one or more lipids.

[0322] In some embodiments, the average diameter of the lipid nanoparticles is 20 nm to 800 nm, 20 nm to 500 nm, 20 nm to 400 nm, 20 nm to 300 nm, 20 nm to 200 nm, 20 nm to 100 nm, 30 nm to 700 nm, 30 nm to 500 nm, 30 nm to 300 nm, 30 nm to 200 nm, 30 nm to 100 nm, 40 nm to 800 nm, 40 nm to 600 nm, 40 nm to 500 nm, 40 nm to 300 nm, 00nm, 40nm~200nm, 40nm~100nm, 50nm~800nm, 50nm~600nm, 50nm~500nm, 50nm~400nm, 50nm~300nm, 50nm~200nm, 50nm~100nm, 60nm~800nm, 60nm~600nm, 60nm~500nm, 60nm~400nm, 60nm~300nm, 60nm~200nm or 60nm~100nm. In some optional specific examples, the average diameter of the lipid nanoparticles is 26 nm, 31 nm, 36 nm, 41 nm, 46 nm, 51 nm, 56 nm, 61 nm, 66 nm, 71 nm, 76 nm, 81 nm, 86 nm, 91 nm, 96 nm, 101 nm, 106 nm, 111 nm, 116 nm, 121 nm, 126 nm, 131 nm, 136 nm, 141 nm, 146 nm, 151 nm, 156 nm, 161 nm, 166 nm, 171 nm, 176 nm, 181 nm, 186 nm, 191 nm, 196 nm, 201 nm, 206 nm, 211 nm, 216 nm, 221 nm, 226 nm, 231 nm, 236 nm, 241 nm, 246 nm, or 249 nm. Herein, the average diameter of lipid nanoparticles can be expressed as the z-average value determined by dynamic light scattering.

[0323] In some embodiments, the lipid nanoparticles include one of the following: cationic lipid nanoparticles, solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC), nonlamellar lipid nanoparticles. In an optional specific example, the lipid nanoparticles are cationic lipid nanoparticles.

[0324] In some embodiments, the lipid nanoparticles contain one or more of the following: cationic lipids, helper lipids, structural lipids, and polymer-lipids.

[0325] The term "cationic lipid" refers to a lipid that becomes positively charged when the pH drops below the pKa of the ionizable group of the lipid, but gradually becomes neutral at higher pH values. At pH values ​​below the pKa, the positively charged lipid is able to bind to negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid.

[0326] In some embodiments, the cationic lipid comprises the following compound (I), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0327] in:

[0328] R1 is selected from the group consisting of: C5-C 30 Alkyl, C5-C 20 alkenyl, -R*YR", -YR", and -R"'M'R';

[0329] R2 and R3 are independently selected from the group consisting of: H, C1-C 14 Alkyl, C2-C 14 alkenyl, -R*YR", -YR" and -R*OR", or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring;

[0330] R4 is selected from the group consisting of hydrogen, C3-C6 carbocycle, -(CH2) n Q, -(CH2) n CHQR, -(CH2) o C(R 10 )2(CH2) n-o Q, -CHQR, -CQ(R)2 and unsubstituted C1-C6 alkyl, wherein Q is selected from carbocyclic, heterocyclic, -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(S)N(R)2, N(R)R8, -N(R)S(O)2R8, -O(CH2) n OR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, - N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N( R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9) R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, each o is independently selected from 1, 2, 3 and 4, and each n is independently selected from 1, 2, 3, 4 and 5;

[0331] each R5 is independently selected from the group consisting of OH, C1-C3 alkyl, C2-C3 alkenyl, and H;

[0332] each R6 is independently selected from the group consisting of OH, C1-C3 alkyl, C2-C3 alkenyl, and H;

[0333] 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-, aryl groups and heteroaryl groups, wherein M" is a bond, C1-C 13 Alkylene or C 2- -C 13 alkenylene;

[0334] R7 is selected from C 1-3 a group consisting of an alkyl group, a C2-C3 alkenyl group, and H;

[0335] R8 chooses from C 3-6 a group consisting of carbocyclic and heterocyclic rings;

[0336] R9 is selected from the group consisting of H, CN, NO2, C1-C6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-C6 alkenyl, C3-C6 carbocycle and heterocycle;

[0337] R 10 selected from the group consisting of H, C1-C3 alkyl and C2-C3 alkenyl;

[0338] Each R is independently selected from the group consisting of: C1-C3 alkyl, C2-C3 alkenyl, (CH2) q OR** and H,

[0339] and each q is independently selected from 1, 2, and 3;

[0340] Each R' is independently selected from the group consisting of: C1-C 18 Alkyl, C2-C 18 alkenyl, -R*YR", -YR", H and And R 11 Choose from C1-C 12 Alkylene and C2-C 12 A group consisting of alkenylene, R 12 and R 13 Each independently selected from C1-C 12 Alkyl and C2-C 12 a group consisting of alkenyl groups;

[0341] Each R" is independently selected from C3-C 15 Alkyl and C3-C 15 a group consisting of alkenyl groups;

[0342] Each R'' is independently selected from C3-C 15 Alkylene and C3-C 15 a group consisting of alkenylene;

[0343] Each R* is independently selected from the group consisting of absent, C1-C 12 Alkylene and C2-C 12 a group consisting of alkenylene;

[0344] Each R** is independently selected from the group consisting of absent, C1-C 12 Alkyl and C2-C 12 a group consisting of alkenyl groups;

[0345] Each Y is independently a C3-C6 carbocycle;

[0346] each X is independently selected from the group consisting of: F, Cl, Br, and I; and

[0347] m is selected from 5, 6, 7, 8, 9, 10, 11, 12 and 13; and wherein when R4 is -(CH2) n Q, -(CH2) nWhen 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 a 5-, 6- or 7-membered heterocycloalkyl.

[0348] In an optional specific example, the cationic lipid is the following compound (I), its N-oxide, its salt or its isomer:

[0349] wherein R1-R7, M and m are as defined above.

[0350] In some embodiments, the cationic lipid comprises the following compound (II), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0351] in:

[0352] R1, R2, R3, R5, R6, M and R7 are as described above,

[0353] R N is H or C1-C3 alkyl;

[0354] X a and X b each independently O or S;

[0355] R 14 Selected from H, halogen, -OH, R b 、-N(R b )2, -CN, -N3, -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)2OR b 、-NO2、-S(O)2N(R b )2、-N(R b )S(O)2R b 、-NH(CH2) t1 N(R b )2、-NH(CH2) p1 O(CH2) q1 N(R b )2、-NH(CH2) s1 OR b 、-N((CH2) S OR b )2、-N(R b )-carbocyclic ring, -N(R b)-heterocyclic, -N(R b )-aryl, -N(R b )-heteroaryl, -N(R b )(CH2) t1 -Carbocyclic ring, -N(R b )(CH2) t1 -heterocycle, -N(R b )(CH2) t1- Aryl, -N(R b )(CH2) t1 - the group consisting of heteroaryl, carbocycle, heterocycle, aryl and heteroaryl;

[0356] Each R b independently selected from the group consisting of C1-C3 alkyl, C2-C3 alkenyl, and H;

[0357] u is 5, 6, 7, 8, 9, 10, 11, 12, or 13;

[0358] w is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0359] r is 0 or 1;

[0360] t 1 is 1, 2, 3, 4, or 5;

[0361] p 1 is 1, 2, 5, 4, or 5;

[0362] q 1 is 1, 2, 5, 4, or 5; and

[0363] s 1 1, 2, 3, 4, or 5.

[0364] In an optional specific example, the cationic lipid is the following compound (II), its N-oxide, its salt or its isomer:

[0365] Among them, R1-R3, R5-R7, R 14 、X a 、X b 、R N , M, u, w and r are as defined above.

[0366] In some embodiments, the cationic lipid comprises the following compound (III), an N-oxide thereof, a salt thereof, or an isomer thereof:

[0367] in:

[0368] L 1 or L 2One of them is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O-, and L 1 or L 2 The other one is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -SS-, -C(=O)S-, SC(=O)-, -NR a C(=O)-, -C(=O)NR a -、NR a C(=O)NR a -、-OC(=O)NR a -or-NR a C(=O)O- or bond;

[0369] G 1 and G 2 Each independently is an unsubstituted C1-C 12 Alkylene or C1-C 12 alkenylene;

[0370] G 3 C1-C 24 Alkylene, C1-C 24 Alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;

[0371] R a H or C1-C 12 hydrocarbon group;

[0372] R 15 and R 16 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0373] R 17 H, OR 18 、CN、-C(=O)OR 19 、-OC(=O)R 19 or –NR 18 C(=O)R 19 ;

[0374] R19 C1-C 12 hydrocarbon group;

[0375] R 18 is H or a C1-C6 hydrocarbon group; and

[0376] x is 0, 1, or 2.

[0377] In an optional specific example, the cationic lipid is the following compound (III), its N-oxide, its salt or its isomer:

[0378] Where: R 15 -R 17 , G 1 -G 3 , L 1 -L 2 As defined above.

[0379] In some embodiments, the cationic lipid comprises the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0380] in:

[0381] L 3 and L 4 The same or different, each independently C1-C 12 Alkylene, C2-C 12 Alkenylene or C2-C 12 Alkyne; in some embodiments L 3 and L 4 The same or different, each independently C3-C 10 Alkylene, C3-C 10 Alkenylene or C3-C 10 Alkyne; in some embodiments, L 3 and L 4 The same or different, each independently C3-C 10 Alkylene; in some embodiments, L 3 and L 4 The same or different, each independently a C5-C8 alkylene group;

[0382] G 4 and G 5 are the same or different and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S- or -SC(=O)-; in some embodiments, G 4 and G 5are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, or -O-; in some embodiments, G 4 and G 5 the same or different, each independently selected from -O-(C=O)- or -(C=O)-O-;

[0383] R 18 and R 19 The same or different, each independently C5-C 27 Alkyl, or C5-C 27 alkenyl; in some embodiments, R 18 and R 19 The same or different, each independently C8-C 20 Alkyl or C8-C 20 alkenyl; in some embodiments, R 18 and R 19 The same or different, each independently C9-C 17 Alkyl or C9-C 18 alkenyl; in some embodiments, R 18 and R 19 the same or different, each independently

[0384] R 20 is halogen, hydroxy, 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, hydroxy, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; in some embodiments, R 20 is halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; in some embodiments, R 20 is fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl or acetamido;

[0385] z is 1, 2 or 3.

[0386] In some embodiments, the cationic lipid is the following compound (IV), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0387] Where: R18 -R 20 , G 4 -G 5 , L 3 -L 4 and z are as defined above.

[0388] In some embodiments, the cationic lipid comprises the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof:

[0389] In some embodiments, the cationic lipid comprises the following compound, or a pharmaceutically acceptable salt thereof:

[0390] In some embodiments, 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

[0391] In some embodiments, the helper lipids of the lipid nanoparticles include phospholipids. Phospholipids are typically semi-synthetic, but can also be naturally derived or chemically modified. In an alternative specific example, the helper lipids of the lipid nanoparticles are phospholipids. In some embodiments, the phospholipids of the lipid nanoparticles include one or more of the following: DSPC (distearoylphosphatidylcholine), 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. In some embodiments, the helper lipids of the lipid nanoparticles are one or more selected from the following: DSPC, DOPE, DOPC, and DOPS. In some embodiments, the helper lipid of the lipid nanoparticle is DSPC and / or DOPE.

[0392] In some embodiments, the structural lipids of the lipid nanoparticles include sterols. In an alternative specific example, the structural lipids of the lipid nanoparticles are sterols. In some embodiments, the sterols of the lipid nanoparticles include one or more of the following: 20α-hydroxycholesterol, cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, ergosterol, β-sitosterol, and oxidized cholesterol derivatives. In some embodiments, the structural lipids of the lipid nanoparticles include at least one of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, and bile acids. In some embodiments, the structural lipid of the lipid nanoparticles is cholesterol. In an alternative specific example, the structural lipid of the lipid nanoparticles is high-purity cholesterol, particularly injection-grade high-purity cholesterol, such as CHO-HP (produced by AVT). In other embodiments, the structural lipid is 20α-hydroxycholesterol.

[0393] Polymer-lipid refers to a conjugate comprising a polymer and a lipid coupled to the polymer. Polymer-lipid (e.g., polyethylene glycol-lipid) in lipid nanoparticles can improve the stability of lipid nanoparticles in vivo.

[0394] In some embodiments, 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).

[0395] In some embodiments, 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.

[0396] In some embodiments, 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.

[0397] In some embodiments, the hydrophilic polymer comprises one or more of the following: polyethylene glycol (PEG), polyoxazolines (POX), polyglycerols (PGs), polyhydroxypropyl methacrylate (PHPMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), polyvinylpyrrolidone (PVP), poly(N,N-dimethyl acrylamide) (PDMA), poly(N-acryloyl morpholine) (PAcM), polyaminoacids, glycosaminoglycans (GAGs), heparin, hyaluronic acid (HA), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond (PEG-60 peptide bond), PEG-60 peptide bond acid (HA), polysialic acid (PSA), elastin-like polypeptides (ELPs), serum albumin and CD47.

[0398] Correspondingly, polymer-lipid includes one or more of the following: polyethylene glycol-lipid (PEG-lipid), polyoxazoline-lipid, polyglycerol-lipid, polyhydroxypropyl methacrylate-lipid, polymethacrylate-2-hydroxyethyl ester-lipid, poly N-(2-hydroxypropyl) methacrylamide-lipid, polyvinyl pyrrolidone-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. It should be noted that "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, and the same applies to other polymer-lipids. In an optional specific example, the hydrophilic polymer includes polyethylene glycol.

[0399] In some embodiments, the polymer-lipid includes a PEG-lipid. In an alternative specific example, the polymer-lipid is a PEG-lipid. In some embodiments, 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-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). The PEG-lipid is preferably PEG-DMG, PEG-DSG, or PEG-DPG. PEG-DMG is a polyethylene glycol derivative of 1,2-dimyristoylglycerol. In some embodiments, the average molecular weight of the PEG in the PEG-lipid is about 2000 to 5000. In an alternative specific example, the average molecular weight of PEG in the PEG-lipid is about 2000. In some embodiments, the amphoteric polymer includes one or more of the following: polycarboxybetaine (pCB), polysulfobetaine (pSB), phosphobetaine-based polymers, and phosphorylcholine polymers.In some embodiments, 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 vinylimidazole, poly(sulfobetaine) based on vinylimidazole, poly(sulfobetaine) based on vinylpyridine.

[0400] Correspondingly, the polymer-lipid comprises one or more of the following: polyhydroxybetaine-lipid, polysulfobetaine-lipid, phosphobetaine-based polymer-lipid and phosphorylcholine polymer-lipid. In some embodiments, the polymer-lipid comprises one or more of the following: poly(carboxybetaine acrylamide)-lipid, poly(carboxybetaine methacrylate)-lipid, poly(sulfobetaine methacrylate)-lipid, poly(methacryloyloxyethylphosphorylcholine)-lipid, poly(vinylpyridylpropanesulfonate)-lipid, polyvinylimidazolylbetaine-lipid, polyvinylimidazolylsulfobetaine-lipid, polyvinylpyridylsulfobetaine-lipid.

[0401] In addition, in some embodiments, the polymers used in nanoparticles in "The Importance of Poly(ethylene glycol) Alternatives for Overcoming PEG Immunogenicity in Drug Delivery and Bioconjugation." Polymers vol. 12, 2 298 by Hoang Thi, Thai Thanh et al. are also incorporated herein.

[0402] In some embodiments, the lipid nanoparticles contain cationic lipids, helper lipids, structural lipids and polymer-lipids. In some embodiments, the lipid nanoparticles include the following amount (molar percentage) of cationic lipids based on the total amount of cationic lipids, helper lipids, structural lipids and polymer-lipids: about 25.0% to 75.0%, such as 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%.

[0403] In some embodiments, the lipid nanoparticles comprise cationic lipids, helper 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 helper 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.

[0404] In some embodiments, the cationic lipid in the lipid nanoparticles comprises 25 mol% to 75 mol% of the total lipid present in the lipid nanoparticles, for example, 25 mol%, 28 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, 49.5 mol%, 50 mol%, 50.5 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, 60 mol%, 61 mol%, 62 mol%, 63 mol%, 64 mol%, 65 mol%, 66 mol%, 67 mol%, 68 mol%, 69 mol%, 70 mol%, 71 mol%, 72 mol%, 73 mol%, 74 mol%, 75 mol%, 76 mol%, 77 mol%, 78 mol%, 79 mol%, 80 mol%, 81 mol%, 82 .5mol%, 53mol%, 53.5mol%, 54mol%, 54.5mol%, 55mol%, 55.5mol%, 56mol%, 56.5mol%, 57mol%, 57.5mol%, 58mol%, 58.5mol%, 59mol%, 59. 5 mol%, 60 mol%, 60.5 mol%, 61 mol%, 61.5 mol%, 62 mol%, 62.5 mol%, 63 mol%, 63.5 mol%, 64 mol%, 64.5 mol%, 65 mol%, 68 mol%, 70 mol% or 75 mol%. In some embodiments, the cationic lipid in the above-mentioned lipid nanoparticles accounts 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.

[0405] In some embodiments, 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. For example, 0.5 mol%, 1 mol%, 3 mol%, 5 mol%, 7.5 mol%, 8 mol%, 8.5 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 11.5 mol%, 12 mol%, 12.5 mol%, 13 mol%, 13.5 mol%, 14 mol%, 14.5 mol%, 15 mol%, 15.5 mol%, 16 mol%, 16.5 mol%, 17 mol%, 17.5 mol%, 18 mol%, 18.5 mol%, 19 mol%, 19.5 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%. In some embodiments, the helper 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.

[0406] In some embodiments, 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. For example, 0 mol%, 1 mol%, 5 mol%, 8 mol%, 10 mol%, 12 mol%, 14 mol%, 15 mol%, 16 mol%, 18 mol%, 20 mol%, 22 mol%, 24 mol%, 25 mol%, 27 mol%, 27.5 mol%, 28 mol%, 28.5 mol%, 29 mol%, 29.5 mol%, 30 mol%, 30.5 mol%, 31 mol%, 31.5 mol%, 32 mol%, 32.5 mol%, 33 mol%, 33.5 mol%, 34 mol%, 34.5 mol%, 35 mol%, 35.5 mol%. , 36mol%, 36.5mol%, 37mol%, 37.5mol%, 38mol%, 38.5mol%, 39mol%, 39.5mol%, 40mol%, 40.5mol%, 41mol%, 41.5mol%, 42mol%, 42.5mol%, 43mol% , 44mol%, 45mol%, 46mol%, 47mol%, 48mol%, 49mol%, 50mol%, 51mol%, 52mol%, 53mol%, 54mol%, 55mol%, 56mol%, 57mol%, 58mol%, 59mol% or 60mol%. In some embodiments, 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%, or 25 mol% to 40 mol% of the total lipids present in the lipid nanoparticles.

[0407] In some embodiments, the polymer-lipid (e.g., PEG-lipid) in the 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%. In some embodiments, the polymer-lipid (e.g., PEG-lipid) in the 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. In some embodiments, the non-lamellar lipid nanoparticles are selected from one of the following: alcohol-containing liposomes and echogenic liposomes.

[0408] In some embodiments, the delivery vector is a liposome containing 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. The liposome encapsulates the nucleic acid, RNA, vector, or nucleic acid composition according to any of the above embodiments using a vesicle formed by a phospholipid bilayer membrane. In some embodiments, the liposome comprises phospholipids and cholesterol.

[0409] In some embodiments, the delivery vector is a cationic protein loaded with the nucleic acid of any of the above embodiments, the RNA of any of the embodiments, the genetic engineering vector of any of the embodiments, or the nucleic acid composition of any of the embodiments. In some embodiments, the cationic protein includes but is not limited to protamine.

[0410] In some embodiments, the above-mentioned delivery vector is a polymer comprising the nucleic acid of any of the above-mentioned embodiments, the RNA of any of the above-mentioned embodiments, the genetic engineering vector of any of the above-mentioned embodiments, or the nucleic acid composition of any of the above-mentioned embodiments. In some embodiments, the polymer is a lipid polymer (lipopolyplex, LPP) and / or a hyaluronic acid polymer (such as hyaluronic acid gel) comprising the nucleic acid, RNA, genetic engineering vector or nucleic acid composition of any of the above-mentioned embodiments. In an alternative specific example, the polymer is a lipid polymer or a hyaluronic acid gel. Lipid polymer is a double-layer structure with a polymer-encapsulated nucleic acid (such as mRNA) as a core and a lipid (such as phospholipid) wrapped as an outer shell.

[0411] It is understood that 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 genetically engineered 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).

[0412] In addition, the present disclosure also provides a method for preparing the above-mentioned compound (IV), and the reaction of the preparation method is as follows:

[0413] Among them, R 18 ~R 20 , G 4 -G 5 , L 3 -L 4 and z are as defined above, and X is halogen, preferably bromine.

[0414] The above preparation method includes step S11 and step S12.

[0415] Step S11: Intermediate compound (V) and intermediate compound (VI) are reacted at room temperature (16°C to 30°C, the same below) in an organic solvent in the presence of an acid-binding agent to obtain intermediate compound (VII). The organic solvent in 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. For example, the organic solvent in step S1 is selected from one or more of acetonitrile, methanol, ethanol, dichloromethane, and dichloroethane (DCE). The acid-binding agent in step S1 is selected from one or more of organic bases and inorganic bases. For example, the acid-binding agent in step S1 is selected from one or more of sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA.

[0416] Step S12: by making intermediate compound (VII) and intermediate compound (VIII) carry out 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. For example, 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 organic bases and inorganic bases. For example, 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.

[0417] In addition, 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:

[0418] Wherein, X is a halogen, preferably bromine.

[0419] Specifically, 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.

[0420] Step S21: Compound (1-X) and compound (1-1) are subjected to an N-alkylation reaction at 30°C to 50°C in a solvent (e.g., a nitrile, alcohol, halogenated hydrocarbon, amide, or aromatic hydrocarbon solvent, specifically acetonitrile, methanol, ethanol, dichloromethane, or dichloroethane (DCE)) to obtain compound (1-2);

[0421] Step S22: In a solvent (e.g., nitriles, alcohols, halogenated hydrocarbons, amides, aromatic hydrocarbons, or ether solvents, specifically acetonitrile, methanol, ethanol, dichloromethane, dichloroethane (DCE), cyclopentane methyl ether, or methyl tert-butyl ether), in the presence or absence of an acid-binding agent and a catalyst, compound (1-2) is subjected to an N-alkylation reaction with 8-halogenated octanoic acid nonyl ester 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. For example, one or more of the following: sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, triethylamine, and DIPEA; the catalyst is an iodide, preferably KI.

[0422] In some embodiments, according to the above method, when the compound (1-X) used is (1S, 3R)-3-aminocyclohexanol and the compound (1-2) is represented by the structural formula (1-2-1):

[0423] Obtain the optical isomer compound (IV-1-1) of compound (IV-1):

[0424] When the compound (1-X) used is (1S,3S)-3-aminocyclohexanol and the compound (1-2) is as shown in the structural formula (1-2-2):

[0425] Obtain the optical isomer compound (IV-1-2) of compound (IV-1):

[0426] When the compound (1-X) used is (1R, 3R)-3-aminocyclohexanol and the compound (1-2) is represented by the structural formula (1-2-3):

[0427] Obtain the optical isomer compound (IV-1-3) of compound (IV-1):

[0428] When the compound (1-X) used is (1R, 3S)-3-aminocyclohexanol and the compound (1-2) is as shown in the structural formula (1-2-4):

[0429] Obtain the optical isomer compound (IV-1-4) of compound (IV-1):

[0430] In addition, 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 genetically engineered 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.

[0431] In some embodiments, the pharmaceutical compositions described above comprise one or more nucleic acids according to any of the above embodiments.

[0432] In some embodiments, the pharmaceutical compositions described above comprise one or more nucleic acids according to any of the above embodiments, formulated alone (eg, encapsulated) or co-formulated in a delivery vehicle.

[0433] In some embodiments, the pharmaceutical composition comprises a nucleic acid according to any of the above embodiments.

[0434] In some embodiments, the pharmaceutical composition comprises a nucleic acid according to any of the above embodiments, formulated (eg, encapsulated) in a delivery vehicle.

[0435] In some embodiments, the pharmaceutical composition comprises a nucleic acid as described above, formulated (e.g., encapsulated) in a delivery vehicle (e.g., a lipid nanoparticle), wherein 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:

[0436] (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: 1, 2, 81, 82 or 83;

[0437] (2) a mutant comprising mutations E146C and T160C 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: 3 or 4;

[0438] (3) mutants 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;

[0439] (4) a mutant comprising mutations L165C and F196C and having an amino acid sequence 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;

[0440] (5) mutants 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;

[0441] (6) mutants 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;

[0442] (7) mutants 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

[0443] (8) 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.

[0444] In some embodiments, the pharmaceutical composition comprises the nucleic acid described above, which is formulated (e.g., encapsulated) in a delivery vector, and the amino acid sequence of the mutant MPV F protein encoded by the nucleic acid is shown in any one of SEQ ID NOs: 1-22 and 81-85.

[0445] In some embodiments, the pharmaceutical composition comprises one of the nucleic acids described above, formulated (e.g., encapsulated) in a delivery vehicle (e.g., a lipid nanoparticle), and the nucleic acid is selected from one of the following:

[0446] (1) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 1 and having a nucleotide sequence 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;

[0447] (2) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 2 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 35 or 87;

[0448] (3) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence set forth in SEQ ID NO: 3 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 36;

[0449] (4) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence set forth in SEQ ID NO:4 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37;

[0450] (5) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence set forth in SEQ ID NO:5 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:38;

[0451] (6) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 6 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 39;

[0452] (7) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO:7 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:40;

[0453] (8) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 8 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41;

[0454] (9) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence set forth in SEQ ID NO:9 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42;

[0455] (10) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 10 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 43;

[0456] (11) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 11 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 44;

[0457] (12) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 12 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45;

[0458] (13) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 13 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 46;

[0459] (14) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 14 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 47;

[0460] (15) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 15 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 48;

[0461] (16) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 16 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 49;

[0462] (17) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 17 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 50;

[0463] (18) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 18 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 51;

[0464] (19) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 19 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 52;

[0465] (20) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 20 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 53;

[0466] (21) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 21 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 54;

[0467] (22) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 22 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 55;

[0468] (23) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO:81 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:88;

[0469] (24) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 82 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 89;

[0470] (25) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO: 83 and having a nucleotide sequence 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;

[0471] (26) comprising an mRNA corresponding to a DNA encoding a mutant of the amino acid sequence set forth in SEQ ID NO: 84 and having a nucleotide sequence 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

[0472] (27) An mRNA corresponding to a DNA encoding a mutant of the amino acid sequence shown in SEQ ID NO:85 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:93.

[0473] In some embodiments, the pharmaceutical composition comprises one of the nucleic acids described above, which is formulated (e.g., encapsulated) in a delivery vehicle (e.g., lipid nanoparticles), and the nucleic acid is an mRNA corresponding to a DNA comprising a nucleotide sequence as shown in any one of SEQ ID NOs: 34 to 55 and 86 to 93.

[0474] In some embodiments, the pharmaceutical composition comprises a plurality of nucleic acids according to any of the above embodiments.

[0475] In some embodiments, the pharmaceutical composition comprises multiple nucleic acids according to any of the above embodiments, wherein the multiple nucleic acids are co-formulated in a delivery vehicle or the multiple nucleic acids are individually formulated in a delivery vehicle. "Co-formulated" means that multiple nucleic acids are contained (e.g., encapsulated) in a single delivery vehicle, while "individually formulated" means that a single delivery vehicle contains (e.g., encapsulated) one nucleic acid, and the same applies hereinafter.

[0476] In some embodiments, the pharmaceutical composition comprises two of the nucleic acids described above, wherein 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).

[0477] In some embodiments, the pharmaceutical composition comprises two nucleic acids, the two nucleic acids being co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids being separately formulated in a delivery vehicle (e.g., lipid nanoparticles), the two nucleic acids encoding a mutant of the subtype A MPV F protein and a mutant of the subtype B MPV F protein, respectively, the mutant of the subtype A MPV F protein and the mutant of the subtype B MPV F protein encoded by the two nucleic acids being selected from the group consisting of:

[0478] (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: 1, 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: 2;

[0479] (2) a mutant comprising mutations E146C and T160C and 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: 3, and a mutant comprising mutations E146C and T160C and 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: 4;

[0480] (3) a mutant comprising mutations F168C and F196C and 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, and a mutant comprising mutations F168C and F196C and 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: 6;

[0481] (4) a mutant comprising mutations L165C and F196C and 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, and a mutant comprising mutations L165C and F196C and 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: 8;

[0482] (5) a mutant comprising mutations N145C and A161C and 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, and a mutant comprising mutations N145C and A161C and 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: 10;

[0483] (6) a mutant comprising mutations S149C and V157C and 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 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;

[0484] (7) 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 SEQ ID NO: 13, and 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 SEQ ID NO: 14;

[0485] (8) 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 SEQ ID NO: 15, and 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 SEQ ID NO: 16;

[0486] (9) 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 SEQ ID NO: 17, and 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 SEQ ID NO: 18;

[0487] (10) 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 SEQ ID NO: 19, and 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 SEQ ID NO: 20;

[0488] (11) a mutant comprising mutations T150C and R156C and 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 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;

[0489] (12) 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;

[0490] In some embodiments, the pharmaceutical composition comprises two nucleic acids, the two nucleic acids being co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids being separately formulated in a delivery vehicle (e.g., lipid nanoparticles), the two nucleic acids encoding a mutant of the subtype A MPV F protein and a mutant of the subtype B MPV F protein, respectively, the mutant of the subtype A MPV F protein and the mutant of the subtype B MPV F protein encoded by the two nucleic acids being selected from the group consisting of:

[0491] (1) a mutant with an amino acid sequence as shown in SEQ ID NO: 1 and a mutant with an amino acid sequence as shown in SEQ ID NO: 2;

[0492] (2) a mutant with an amino acid sequence as shown in SEQ ID NO: 3 and a mutant with an amino acid sequence as shown in SEQ ID NO: 4;

[0493] (3) a mutant with an amino acid sequence as shown in SEQ ID NO: 5 and a mutant with an amino acid sequence as shown in SEQ ID NO: 6;

[0494] (4) a mutant with an amino acid sequence as shown in SEQ ID NO: 7 and a mutant with an amino acid sequence as shown in SEQ ID NO: 8;

[0495] (5) a mutant with an amino acid sequence as shown in SEQ ID NO: 9 and a mutant with an amino acid sequence as shown in SEQ ID NO: 10;

[0496] (6) a mutant with an amino acid sequence as shown in SEQ ID NO: 11 and a mutant with an amino acid sequence as shown in SEQ ID NO: 12;

[0497] (7) a mutant with an amino acid sequence as shown in SEQ ID NO: 13 and a mutant with an amino acid sequence as shown in SEQ ID NO: 14;

[0498] (8) a mutant with an amino acid sequence as shown in SEQ ID NO: 15 and a mutant with an amino acid sequence as shown in SEQ ID NO: 16;

[0499] (9) a mutant with an amino acid sequence as shown in SEQ ID NO: 17 and a mutant with an amino acid sequence as shown in SEQ ID NO: 18;

[0500] (10) a mutant with an amino acid sequence as shown in SEQ ID NO: 19 and a mutant with an amino acid sequence as shown in SEQ ID NO: 20;

[0501] (11) a mutant with an amino acid sequence as shown in SEQ ID NO: 21 and a mutant with an amino acid sequence as shown in SEQ ID NO: 22; and

[0502] (12) A mutant having an amino acid sequence as shown in SEQ ID NO: 81 and a mutant having an amino acid sequence as shown in SEQ ID NO: 82.

[0503] In some embodiments, the pharmaceutical composition comprises two of the above nucleic acids, the two nucleic acids being co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids being separately formulated in a delivery vehicle (e.g., lipid nanoparticles), the two nucleic acids encoding a mutant of the subtype A MPV F protein and a mutant of the subtype B MPV F protein, respectively, the two nucleic acids being selected from the group consisting of:

[0504] (1) an mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO: 1 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 34, and an mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO: 2 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 35;

[0505] (2) an mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO: 1 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 86, and an mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO: 2 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 87;

[0506] (3) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:3 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:4 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:37;

[0507] (4) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:5 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:38, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:6 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:39;

[0508] (5) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:7 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:40, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:8 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:41;

[0509] (6) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:9 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:42, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:10 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:43;

[0510] (7) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:11 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:44, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:12 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:45;

[0511] (8) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:13 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:46, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:14 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:47;

[0512] (9) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:15 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:48, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:16 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:49;

[0513] (10) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO: 17 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 50, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO: 18 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 51;

[0514] (11) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:19 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:52, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:20 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:53;

[0515] (12) mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:21 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:54, and mRNA corresponding to a DNA encoding a variant of the amino acid sequence set forth in SEQ ID NO:22 and having a nucleotide sequence at least 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:55; and

[0516] (13) An mRNA corresponding to a DNA encoding a variant of the amino acid sequence 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, and an mRNA corresponding to a DNA encoding a variant of the amino acid sequence 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.

[0517] In some embodiments, the pharmaceutical composition comprises two of the above nucleic acids, the two nucleic acids being co-formulated in a delivery vehicle (e.g., lipid nanoparticles) or the two nucleic acids being separately formulated in a delivery vehicle (e.g., lipid nanoparticles), the two nucleic acids being selected from the group consisting of:

[0518] (1) mRNA corresponding to a DNA comprising the nucleotide sequence shown in SEQ ID NO: 34, and mRNA corresponding to a DNA comprising the nucleotide sequence shown in SEQ ID NO: 35;

[0519] (2) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 36, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 37;

[0520] (3) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 38, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 39;

[0521] (4) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 40, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 41;

[0522] (5) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 42, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 43;

[0523] (6) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 44, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 45;

[0524] (7) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 46, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 47;

[0525] (8) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 48, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 49;

[0526] (9) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 50, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 51;

[0527] (10) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 52, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 53;

[0528] (11) mRNA corresponding to a DNA comprising the nucleotide sequence shown in SEQ ID NO: 54, and mRNA corresponding to a DNA comprising the nucleotide sequence shown in SEQ ID NO: 55

[0529] (12) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 86, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 87; and

[0530] (13) mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 88, and mRNA corresponding to the DNA comprising the nucleotide sequence shown in SEQ ID NO: 89.

[0531] In some embodiments, the nucleic acid contained in the pharmaceutical composition is mRNA.

[0532] In some embodiments, the nucleic acid contained in the pharmaceutical composition is mRNA, and all uridine in the mRNA is replaced by N1-methylpseudouridine.

[0533] In some embodiments, the pharmaceutical composition comprises a nucleic acid according to any of the above embodiments, wherein the nucleic acid is formulated in a delivery vehicle, and the nucleic acid is mRNA.

[0534] In some embodiments, the pharmaceutical composition comprises a plurality of nucleic acids according to 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.

[0535] In some embodiments, the pharmaceutical composition comprises two of the above nucleic acids, the two nucleic acids being co-formulated in a delivery vehicle (e.g., a lipid nanoparticle) or the two nucleic acids being separately formulated in a delivery vehicle (e.g., a lipid nanoparticle), and the two nucleic acids being mRNA. In some embodiments, the pharmaceutical composition comprises multiple delivery vehicles, and the MPV F protein mutants encoded by the nucleic acids contained in the multiple delivery vehicles are derived from the same subtype strain but with different mutations, different subtype strains but with the same mutations, or different subtype strains with different mutations.

[0536] For example, 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, and 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.

[0537] For another example, 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, and 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.

[0538] For another example, 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, and 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.

[0539] In some embodiments, the delivery vehicle of the pharmaceutical composition of any of the above embodiments is a lipid nanoparticle.

[0540] In some embodiments, the pharmaceutical composition comprises multiple nucleic acids according to any of the above embodiments, multiple RNAs according to any of the above embodiments, multiple genetically engineered vectors according to any of the above embodiments, or multiple host cells according to any of the above embodiments.

[0541] In some embodiments, the pharmaceutical composition of any of the above embodiments is a vaccine.

[0542] The term "pharmaceutically acceptable" as used herein 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). The term "pharmaceutically acceptable carrier" refers to a substance that can be administered together 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.

[0543] In some embodiments, the above pharmaceutical compositions do not include an adjuvant.

[0544] In some embodiments, the above pharmaceutical composition further comprises an adjuvant.

[0545] In some embodiments, the adjuvant comprises a flagellin adjuvant. In some embodiments, the flagellin adjuvant is mRNA encoding flagellin. Furthermore, the present disclosure provides the use of a nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a host cell according to any of the above embodiments, a nucleic acid composition according to any of the above embodiments, a mutant of the MPV F protein according to any of the above embodiments, an RNA according to any of the above embodiments, an immunogen according to any of the above embodiments, a delivery vector according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments in the preparation of a medicament.

[0546] In some embodiments, the medicament is for preventing or treating MPV infection or a disease caused by MPV infection.

[0547] In some embodiments, the medicament is for preventing or treating hMPV infection or a disease caused by hMPV infection.

[0548] In some embodiments, the drug is a vaccine.

[0549] 7. Vaccines

[0550] 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.

[0551] In some embodiments, the above-mentioned vaccine is a nucleic acid vaccine or a protein vaccine.

[0552] In some embodiments, the above-mentioned vaccine is an MPV vaccine.

[0553] In some embodiments, the above-mentioned vaccine is a multivalent vaccine.

[0554] In some embodiments, the vaccine comprises any of the aforementioned nucleic acids or any of the aforementioned mutants of the MPV F protein.

[0555] In some embodiments, the above vaccine is a combination vaccine.

[0556] In some embodiments, the vaccine is a concatenated nucleic acid vaccine. In some embodiments, in addition to comprising any one or more of the aforementioned nucleic acids comprising polynucleotides encoding mutants of the MPV F protein, the vaccine further comprises nucleic acids encoding proteins or polypeptides other than the MPV F protein, or proteins or polypeptides from other viruses that can serve as immunogens. Of course, the other viruses can be a single subtype, multiple viruses, or multiple subtypes.

[0557] Alternatively, the vaccine is a polyvalent protein vaccine. In some embodiments, in addition to comprising any one or more of the aforementioned MPV F protein mutants, the vaccine further comprises proteins or polypeptides from other viruses that can serve as immunogens. Of course, the other viruses can be one or more.

[0558] In some embodiments, the above vaccines do not include an adjuvant.

[0559] In some embodiments, the above-mentioned vaccine further comprises an adjuvant.

[0560] It is understood that the dosage form of the above-mentioned vaccine is not particularly limited. In some embodiments, the above-mentioned vaccine is an mRNA vaccine.

[0561] In some embodiments, 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).

[0562] 8. Prevention or Treatment Methods

[0563] The present disclosure also provides a method for preventing or treating MPV virus infection, which comprises administering to a subject a nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a nucleic acid composition according to any of the above embodiments, a mutant of the MPV F protein according to any of the above embodiments, an RNA according to any of the above embodiments, an immunogen according to any of the above embodiments, a delivery vector according to any of the above embodiments, a pharmaceutical composition according to any of the above embodiments, or a vaccine according to any of the above embodiments.

[0564] In some 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 mammal. Other mammals include, but are not limited to, mice, rats, guinea pigs, rabbits, hamsters, etc. In an alternative specific example, the subject is a human.

[0565] In some embodiments, the number of administrations is one, two, three, four, or more times.

[0566] In addition, the present disclosure also provides a method for inducing an immune response against MPV in a subject. In some embodiments, 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. Relative to the anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against MPV, the anti-antigen antibody titer in the subject after vaccination with the present disclosure is increased. "Anti-antigen antibodies" are serum antibodies that specifically bind to an antigen.

[0567] In addition, the present disclosure also provides a use of a nucleic acid according to any of the above embodiments, a genetically engineered vector according to any of the above embodiments, a nucleic acid composition according to any of the above embodiments, a mutant of the MPV F protein according to any of the above embodiments, an RNA according to any of the above embodiments, an immunogen according to any of the above embodiments, a delivery vector according to any of the above embodiments, or a pharmaceutical composition according to any of the above embodiments in preparing a detection reagent or kit for an MPV F protein-binding antibody.

[0568] In addition, 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 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, the delivery vector of any of the above embodiments, or the drug composition of any of the above embodiments.

[0569] In addition, 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 a nucleic acid according to any of the aforementioned embodiments, a genetically engineered vector according to any of the aforementioned embodiments, a nucleic acid composition according to any of the aforementioned embodiments, a mutant of an MPV F protein according to any of the aforementioned embodiments, an immunogen according to any of the aforementioned embodiments, or a pharmaceutical composition or vaccine containing the mutant of an MPV F protein; contacting a biological sample from the subject with the mutant of an 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. Example

[0570] To make the purpose and technical solutions of the present disclosure more clear, the following detailed description is given in conjunction with specific embodiments. Obviously, the embodiments described are only a part of the embodiments of the present disclosure, and not all of them. Based on the embodiments of the present disclosure, all other implementation methods obtained by ordinary technicians in this field without making any creative work are within the scope of protection of the present disclosure.

[0571] Example 1

[0572] 1. Synthesis of Compound (IV-1)

[0573] Step 1): Synthesis of 8-((3-hydroxycyclohexyl)amino)nonyl octanoate (Compound (IV-1-p1))

[0574] In a 100 mL reaction flask, 8-bromooctanoic acid nonyl ester (3.50 g, 10 mmol), 3-aminocyclohexanol (11.5 g, 100 mmol), and 30 mL of ethanol were added in sequence. After stirring to dissolve, N,N-diisopropylethylamine (2.58 g, 20 mmol) was added and reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times, dried over anhydrous sodium sulfate, concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester.

[0575] 1H NMR (600MHz, CDCl3) δ4.20-4.13(m,0.5H),4.05(t,2H),3.83(m,0.5H),3.09(m,0.5H),2.87(m,0.5H),2.76-2.59(m,2H), 2.29(t,2H),2.00(m,0.5H),1.93-1.78(m,1.5H),1.78-1.65(m,2H),1.65-1.46(m,8H),1.40-1.18(m,20H),0.88(t,3H).

[0576] LCMS: 384.3 [M+H] + .

[0577] Step 2): Synthesis of compound (IV-1)

[0578] In a 100 mL reaction flask, 8-((3-hydroxycyclohexyl)amino)octanoic acid nonyl ester (383 mg, 1 mmol), 8-bromooctanoic acid 1-octyl nonyl ester (554 mg, 1.2 mmol), and 20 mL of acetonitrile were added in sequence. After stirring and dissolving, potassium carbonate (276 mg, 2 mmol) and potassium iodide (166 mg, 1 mmol) were added. The mixture was reacted at room temperature for 24 h. 100 mL of dichloromethane was added, and the mixture was washed with water three times. It was then dried over anhydrous sodium sulfate and concentrated, and purified using a flash column chromatography system (dichloromethane: methanol = 20:1 to 5:1) to obtain compound (IV-1).

[0579] 1 H NMR (600MHz, CDCl3) δ4.95-4.81(m,1H),4.30-4.12(m,0.5H),4.07(t,2H),3.72-3.61(m,0.5H),2.97(m,0.5H),2.64-2.52(m ,0.5H),2.48-2.37(m,4H),2.30(q,4H),1.93-1.81(m,2H),1.72-1.57(m,8H),1.51(t,4H),1.43-1.18(m,56H),0.89(m,9H).

[0580] LCMS: 765.3 [M+H] + .

[0581] 2. Preparation of Compound (IV-1-1)

[0582] To a 250 mL single-necked flask, 8 g (17.3 mmol) of 9-heptadecyl-8-bromooctanoate, 10 g (76.5 mmol) of (1S,3R)-3-aminocyclohexanol, and 100 mL of ethanol were added and reacted at 50°C for 15 h. The 9-heptadecyl-8-bromooctanoate reacted completely. After removing the solvent by rotary evaporation, the crude product was added to 200 mL of ethyl acetate (EA) and washed twice with 100 mL of water to thoroughly remove the (1S,3R)-3-aminocyclohexanol. The EA phase was then dried to afford crude compound (IV-1-1-p) (i.e., 9-heptadecyl-8-(((1R,3S)-3-hydroxycyclohexyl)amino)octanoate). The crude product was purified by column chromatography (dichloromethane:methanol = 20:1 to 5:1) to afford compound (IV-1-1-p).

[0583] NMR data:

[0584] 1 H NMR (600MHz, CDCl3): δ4.91-4.81(m,1H),3.86-3.82(m,1H),2.84-2.81(m,1H),2.69-2.54(qt,J=11.2,7.3Hz,2H),2.29-2.26(t,J=7.5Hz,2H),1. 93-1.86(m,1H),1.77-1.74(d,J=12.0Hz,1H),1.72-1.57(m,6H),1.54-1 .45(m,6H),1.37-1.30(m,8H),1.30-1.22(m,24H),0.88(t,J=7.0Hz,6H).

[0585] LCMS: 496.7 [M+H] + .

[0586] Take 6 g (12.1 mmol) of the above compound (IV-1-1-p) in a 250 mL single-necked bottle, add 90 mL of acetonitrile and 60 mL of cyclopentane methyl ether, then add 6.7 g of potassium carbonate powder (48.4 mmol), 2 g of potassium iodide (12.1 mmol) and 5 g of 8-bromooctanoic acid nonyl ester (17.5 mmol), and place at 90 ° C to react for 24 hours. The raw material compound ((IV-1-1-p) is completely reacted. Remove the oil bath and wait for the reaction to cool to room temperature. After the solid is removed by filtration, the filtrate is spin-dried to obtain a crude product, and finally purified by column chromatography (dichloromethane: methanol = 20: 1 to 5: 1) to obtain compound (IV-1-1).

[0587] NMR data:

[0588] 1H NMR (600MHz, CDCl3): δ4.93-4.84(m,1H),4.09(t,J=6.8Hz,2H),3.68-3.61(m,1H),2.62-2.54(m,1H),2.53-2.41(m,4H),2.31-2.26(q,J= 7.4Hz,4H),1.97(m,1H),1.91-1.78(m,2H),1.71-1.61(m,7H),1.54(d,J=6.0Hz,4H),1.43-1.37(m,4H),1.39-1.23(m,52H),0.91(m,9H).

[0589] Specific rotation: +4.3°

[0590] LCMS: 765.2 [M+H] + .

[0591] Example 2 Design of hMPV F protein mutants and their coding sequences

[0592] hMPV F protein mutants as shown in Table 1 were designed. Based on the amino acid sequences of the mutants, codon-optimized DNA sequences (DNA sequences corresponding to the protein coding regions in Table 1) were further designed and commissioned to Nanjing GenScript Biotechnology Co., Ltd. for synthesis. Testing results showed that all DNA sequences were correct. In addition, Nanjing GenScript Biotechnology Co., Ltd. was also commissioned to synthesize DNA sequences corresponding to mRNAs numbered 1191, 1192, 1274, and 1275 (see Table 2). Testing results showed that all DNA sequences were correct. Among them, mRNAs numbered 1191 and 1192 encode hMPV post F protein, and mRNAs numbered 1274 and 1275 encode hMPV pre F protein. mRNAs numbered 1191, 1192, 1274, and 1275 use the same 5'-UTR, 3'-UTR, and poly(A) tail as the mRNAs in Table 1.

[0593] Table 2

[0594] Example 3 Construction of engineered plasmid

[0595] The construction method is as follows:

[0596] 1. Construction of 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. New restriction sites HindIII, BamHI, KpnI, and ApaI were added after the T7 promoter, and the NeoR / KanR sequence from 2136 bp to 2930 bp was deleted to obtain plasmid B.

[0597] 2. Construction of plasmid C: Plasmid B was linearized with ApaI and homologously recombined with a nucleic acid fragment synthesized by IDT (Integrated DNA Technologies) with the nucleotide sequence shown in SEQ ID NO: 33 to obtain plasmid C.

[0598] 3. Construction of plasmid D: Plasmid C was double-digested with KpnI and ApaI, and a large fragment of approximately 4.7 kb was recovered by agarose gel electrophoresis. The fragment was then ligated with a nucleic acid fragment synthesized by IDT (SEQ ID NO: 32) using T4 enzyme to obtain plasmid D.

[0599] 4. Construction of plasmid F: Plasmid D was double-digested with BamHI and HindIII, and a large fragment of approximately 4.8 kb was recovered by agarose gel electrophoresis. The fragment was then ligated with the nucleic acid fragment synthesized by IDT (SEQ ID NO: 31) using T4 enzyme to obtain plasmid F.

[0600] 5. Construction of engineered plasmids: Homology 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 large molecular fragments of approximately 4.8 kb were recovered by gel run for homologous recombination. Finally, multiple engineered plasmids with completely correct sequencing were obtained.

[0601] Example 4 Preparation of mRNA encoding mutants of hMPV F protein

[0602] 1. Extract the engineering plasmid and ensure that the supercoil rate of the plasmid is above 85%.

[0603] 2. Plasmid linearization

[0604] (1) Enzyme linearization: 20 μg of different engineered plasmids containing the coding sequence of hMPV F protein mutants prepared in Example 3 were taken and the reaction systems were prepared according to Table 3. After mixing, the mixture was placed in a 37°C incubator for 16 h for enzyme digestion reaction.

[0605] Table 3

[0606] (2) Purification of linearized plasmid: After ensuring that the enzyme digestion is complete, use a DNA fragment purification kit (Takara, catalog number: 9761) to recover the linearized plasmid.

[0607] (3) Identification: 100 ng of the purified linear plasmid and the original circular plasmid were subjected to 1% agarose gel electrophoresis to confirm whether the plasmid linearization was complete.

[0608] 3. In vitro transcription of mRNA

[0609] (1) Prepare the IVT reaction system according to Table 4.

[0610] Table 4

[0611] (2) The reaction was carried out at 37°C for 2 h. After the reaction was completed, 5 μL of DNase was added to the reaction system and incubated at 37°C for 15 min (DNase was used to remove the linearized plasmid template).

[0612] (3) Refer to the purification magnetic beads (Hieff The IVT product was purified by magnetic beads according to the instructions of RNACleaner (YEASEN, catalog number: 12602ES56).

[0613] (4) Capping:

[0614] mRNACap1 capping reaction:

[0615] The Cap1 type cap structure and reaction principle are as follows:

[0616] pppN1(p)Nx-OH(3')→ppN1(pN)x-OH(3')+Pi

[0617] ppN1(pN)x-OH(3')+GTP→G(5')ppp(5')N1(pN)x-OH(3')+PPi

[0618] G(5')ppp(5')N1(pN)x-OH(3')+AdoMet→m7G(5')ppp(5')N1(pN)x-OH(3')+AdoHyc

[0619] m7GpppN1(pN)x-OH(3')+AdoMet→m7Gppp[m2'-O]N1(pN)x-OH(3')+AdoHyc

[0620] 5'-Cap1 type cap structure:

[0621] cap G 1 G 2 =m7 G-5'-ppp-5'-Gm 2 '-3'-p-[m 7 =7-CH3;m 2 '=2'-O-CH3; -ppp-=-PO2H-O-PO2H-O-PO2H)-; -p-=-PO2H-].

[0622] A 58 μL mRNA solution (125 pmol uncapped mRNA was made up to 58 μL with water) was preheated at 65°C for 5 min and then mixed with a mixture containing 10 μL 10× Capping Buffer, 10 μL 10 mM GTP, 1.88 μL 32 mM SAM, 0.187 μL RNase Inhibitor, 5 μL 2'-O-Methyltransferase (50 U / μL), and 15 μL ScriptCap Capping Enzyme (10 U / μL) and incubated at 42°C for 1 h. The above reagents used for the capping reaction were purchased from Suzhou Jinan Protein Technology Co., Ltd.

[0623] (5) Purification of capped product:

[0624] Reference purification magnetic beads (Hieff The capped product was purified by magnetic beads according to the instructions of RNACleaner (YEASEN, Cat. No. 12602ES56). Characterization data: Concentration was determined using onedrop or Qubit.

[0625] Experimental Results: mRNAs encoding different mutants of hMPV F protein were obtained, containing a Cap1-type cap structure, a 5'-UTR, a 3'-UTR, a poly(A) tail, and in which all uridines were replaced by N1-methylpseudouridine. The amino acid sequences of the mutant hMPV F proteins after translation of the protein coding regions of the various mRNAs are shown in Tables 1 and 2.

[0626] Example 5 Mouse Immunization Experiment with Encapsulated hMPV mRNA Vaccine

[0627] (1) Encapsulation: Using a microsyringe and a microfluidic chip (SN.000038), mRNA was encapsulated at a rate of 9 mL / min in the aqueous phase and 3 mL / min in the alcohol phase to prepare crude LNP preparations containing mRNA encoding hMPV F protein mutants. The LNP preparations differed only in the encapsulated mRNA. The mRNA used in this example was prepared according to the method described in Example 4. The compositions of the aqueous and alcohol phases of the various LNP preparations are shown in Table 5 below.

[0628] Table 5

[0629] (2) Dialysis fluid exchange:

[0630] a. Dialysate Preparation: Tris + 8% (m / V) Sucrose Solution: Weigh 1.22g Tris and 4.7g Tris-HCl, dissolve and mix thoroughly in 2L of Watson's distilled water. Add 160g sucrose and mix thoroughly to obtain a Tris + 8% (m / V) sucrose solution.

[0631] b. Dialysis: The crude encapsulated product was transferred into a 100 kDa dialysis bag and immersed in a beaker containing 1 L of dialysis solution. The beaker was wrapped with aluminum foil and dialyzed at 100 rpm for 1 hour at room temperature. The dialysate was then replaced and dialysis continued for another hour. The dialyzed formulation samples were sterile filtered through a 0.22 μm disposable filter membrane to prepare LNP formulations encapsulating mRNA encoding hMPV F protein mutants. The LNP formulations had an mRNA concentration of 0.2 μg / μL, an mRNA:Lipid mass ratio of 1:10, a particle size of 80 nm to 130 nm, and an encapsulation efficiency exceeding 85%.

[0632] (3) In vivo immunization: 7- to 9-week-old Balb / c female mice (17 g to 22 g, housed in an SPF environment) were divided into a blank LNP group and a test group. The test sample was injected intramuscularly at 10 μg per mouse, wherein each test sample included an equal amount of an LNP preparation encapsulating mRNA encoding a subtype A hMPV F protein mutant and an LNP preparation encapsulating mRNA encoding a subtype B hMPV F protein mutant with the same mutation. For example, 5 μg of an LNP preparation encapsulating mRNA numbered 1154 and 5 μg of an LNP preparation encapsulating mRNA numbered 1170 were mixed and injected into mice twice. The first injection time was recorded as Day 1. 0 (D0), the second injection time is D21 or D22, and blood is collected from the eye socket 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°C, 3500 rpm for 10 minutes. After stratification, the upper light yellow liquid is carefully aspirated with a pipette to prepare the immune serum.

[0633] 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).

[0634] Example 6 IgG antibody titer experiment

[0635] The immune serum used in this example was provided by the experiment in Example 5.

[0636] Reagents: 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 min, Goat anti-Mouse IgG (H+L) HRP Conjugate, PBS, FBS, one-component TMB colorimetric solution, and stop solution.

[0637] The steps include:

[0638] (1) Dilute hMPV subtype A or hMPV subtype B F protein to 1 μg / mL using coating solution, mix thoroughly, and set aside. Add 100 μL per well to a 96-well ELISA plate, seal with sealing film, and incubate at 2–8°C overnight (16–20 h).

[0639] (2) After incubation, wash the wells three times with 300 μL of washing solution and pat dry on clean paper.

[0640] (3) Add 250 μL / well of blocking solution to the ELISA plate, seal the plate with a sealing film, and incubate at 37°C for 60 min.

[0641] (4) After blocking, wash the wells three times with 300 μL / well washing solution and pat dry on clean paper.

[0642] (5) Take out the serum that has been separated in advance, vortex mix it, and use it for IgG titer detection.

[0643] (6) Take the separated serum and determine its first dilution factor according to the different immunization times. Use this dilution factor as the first dilution factor and make a gradient dilution. Add the diluted serum to the ELISA plate at 100 μL / well and incubate at 37°C for 1 hour.

[0644] (7) After incubation, wash the wells three times with 300 μL / well washing solution and pat dry on clean paper.

[0645] (8) Dilute the enzyme-labeled secondary antibody 10,000-fold with sample diluent, 100 μL / well, and incubate at 37°C for 1 h.

[0646] (9) After incubation, wash the wells three times with 300 μL / well washing solution and pat dry on clean paper.

[0647] (10) Equilibrate the TMB single-component colorimetric solution to room temperature in advance, add 100 μL to each well, and incubate at 37°C in the dark for 5 min.

[0648] (11) After color development, add 50 μL / well of stop solution.

[0649] (12) Select a detection wavelength of 450 nm and a reference wavelength of 630 nm, and read the results in an enzyme-labeled instrument.

[0650] The 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.

[0651] Example 7 MPV-GFP1 pseudovirus neutralization experiment

[0652] The immune serum required for the pseudovirus neutralization test was provided by the experiment in Example 5.

[0653] Reagent: MPV-GFP1 pseudovirus (7.0log 10 TCID50 / mL), mouse serum inactivated by incubation at 56°C for 30 min, LLC-MK2 cells, Opti-MEM, and FBS.

[0654] The steps include:

[0655] (1) LLC-MK2 cells (2.5×10 4 The cells were cultured in Opti-MEM medium containing 5% FBS for 24 h.

[0656] (2) Take the separated serum and determine its first dilution factor according to different immunization times. Use this dilution factor as the first dilution factor and make a 3-fold serial dilution. Add 310 μL of serum-free Opti-MEM medium to H7-H12 of a 96-well deep-well plate as a cell control (CC), and add 155 μL of Opti-MEM medium to H1-H6 as a virus control (VC).

[0657] (3) Add 233 μL of diluted serum sample to each of A1 to A12 as the initial dilution. Add 155 μL of Opti-MEM serum-free medium to each of B, C, D, E, F, and G. Take 78 μL of each of A1 to A12 and transfer it to the next row, mix thoroughly, and perform a 3-fold serial dilution for a total of 6 serial dilutions. Discard the excess 78 μL of liquid from the last row.

[0658] (4) Except for H7 to H12, 155 μL of diluted virus mixture was added to each well. The titer of the mixed virus was diluted to 1250 TCID50 / mL and incubated at 37°C for 1 h.

[0659] (5) Add 93 μL of the mixture to each well of the 96-well plate in which LLC-MK2 cells were plated one day in advance (the cells were washed once with PBS in advance). Make three replicate wells for each dilution and culture for 48 h.

[0660] (6) Remove the 96-well plate, aspirate the culture medium from the plate, add 100 μL PBS for washing, and immediately read the fluorescence intensity using a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0661] The neutralizing antibody titer test results of hMPV mRNA vaccines containing different mRNA combinations are shown in Figures 3A to 4G. Except for the hMPV mRNA vaccine represented by 1191+1192, other hMPV mRNA vaccines can induce high levels of neutralizing antibodies.

[0662] Example 8 Mouse splenocyte ELISpot experiment

[0663] Reagents: RPMI Medium 1640 basic (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 buffer.

[0664] The mouse spleen used in the ELISpot experiment was from the experiment in Example 5.

[0665] The specific steps of the ELISpot experiment include:

[0666] (1) Place spleen in a 6-well cell culture plate and add 7 mL of mouse lymphocyte separation solution to each well (return to room temperature and shake well before use). Grind with a syringe piston, using the upward rebound force of the cell screen to control the grinding force and minimize possible mechanical damage to the cells.

[0667] (2) Immediately transfer the separation solution containing suspended spleen cells into a 15 mL centrifuge tube and slowly add 10 mL of RPMI 1640 culture medium, keeping a clear liquid boundary.

[0668] (3) Centrifuge at 800 g in a swing-out rotor for 30 min at room temperature.

[0669] (4) Aspirate the lymphocyte layer, add 10 mL of RPMI 1640 medium, and wash by inversion. Centrifuge at 250 g for 10 min at room temperature to collect the cells.

[0670] (5) Perform erythrocyte lysis according to the instructions of the erythrocyte lysis buffer. Add 2 mL of erythrocyte lysis buffer to each tube and resuspend. Incubate at 4°C for 2 minutes, then add 10 mL of DPBS to terminate the erythrocyte lysis reaction. Centrifuge at 250 g for 10 minutes at 4°C to collect the cells.

[0671] (6) Add 10 mL of RPMI 1640 medium to the cells collected in the previous step and wash by inversion. Centrifuge at 250 g for 10 min at room temperature to collect the cells.

[0672] (7) Finally, resuspend the cells in culture medium, count, and dilute.

[0673] (8) Add the cell suspension with adjusted concentration to each experimental well, 5×10 5 cells / 100 μL / well.

[0674] (9) Add stimulants (PMA + Ionomycin working solution (PMA 250 ng / mL + Ionomycin 5 μg / mL) and F protein peptide library (2 μg / mL)), 10 μL / well.

[0675] (10) After all samples and stimulants have been added, cover the plate and place in a 37°C, 5% CO2 incubator for 20 hours.

[0676] (11) Discard the liquid in the wells and add PBS buffer at 200 μL / well. Repeat five times, blot dry on absorbent paper each time.

[0677] (12) Dilute the detection antibody to 1 μg / mL with sample diluent, 100 μL / well. Incubate at room temperature for 2 h.

[0678] (13) Repeat the above plate washing operation.

[0679] (14) Dilute the enzyme-labeled avidin (Streptavidin-HRP) with sample diluent 1000-fold, 100 μL / well. Incubate at room temperature for 1 h.

[0680] (15) Repeat the above plate washing operation.

[0681] (16) Equilibrate the TMB substrate colorimetric solution to room temperature in advance, add 100 μL to each well, and incubate at room temperature in the dark for 10 min.

[0682] (17) Pour out the liquid in the wells, remove the base of the plate, and wash the front and back surfaces and the base with deionized water 3 to 5 times to stop the color development. Place the plate in a cool, dark place at room temperature and allow it to dry naturally before closing the base.

[0683] (18) The enzyme-linked immunosorbent assay (ELISA) analyzer reads the plate and records various parameters of the spots for statistical analysis.

[0684] The ELISpot test results of hMPV mRNA vaccines containing different mRNAs are shown in Figures 5A to 6H. All hMPV mRNA vaccines containing different mRNAs can induce the body to produce obvious T cell immune responses, and the frequency of F protein-specific cells secreting IFN-γ is higher than that of F protein-specific cells secreting IL-4, revealing that the immune response is Th1-biased rather than Th2-biased.

[0685] Example 9

[0686] 1. Referring to Examples 1 to 5, an LNP formulation encapsulating mRNA numbered 1154, 1170, 2309, 2310, 2358, 2359, 2311, 1873, 1874, 1875, 1189, 1893 or 1894 was prepared, wherein: the mRNA numbered 1189 encodes the hMPV F protein with an amino acid sequence as shown in SEQ ID NO: 94, and the DNA sequence corresponding to the F protein coding region thereof is shown in SEQ ID NO: 97; the mRNA numbered 1893 encodes the hMPV F protein with an amino acid sequence as shown in SEQ ID NO: 95, and the DNA sequence corresponding to the F protein coding region thereof is shown in SEQ ID NO: 98; the mRNA numbered 1894 encodes the hMPV F protein with an amino acid sequence as shown in SEQ ID NO: 96, and the DNA sequence corresponding to the F protein coding region thereof is shown in SEQ ID NO: NO: 99; mRNAs numbered 1189, 1893, and 1894 use the same 5'-UTR, 3'-UTR, and poly(A) tail as the mRNAs in Table 1.

[0687] 2. Referring to Example 5, 7- to 9-week-old Balb / c female mice were immunized (intramuscularly) twice with the LNP formulation encapsulated with mRNA. The first immunization time was D0, the second immunization time was D28, and orbital blood was collected on 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 on D49 or D57 for subsequent ELISpot detection.

[0688] 3. The immune serum obtained in step 2 was subjected to an MPV-GFP1 pseudovirus neutralization experiment as described in Example 7. The results are shown in Figures 7A to 7C. As 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 to the antigen.

[0689] 4. The spleen obtained in step 2 was subjected to ELISpot analysis as described in Example 8. The results are shown in Figures 8A to 8D. Figures 8A to 8D show that the introduction of mutations (A147C and A159C, L165C and F196C, or N145C and A161C) can enhance the cellular immune response to the antigen.

Claims

1. 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: 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; Preferably, 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; Preferably, the disulfide bond mutation is selected from one of the following: A147C and A159C, L165C and F196C, N145C and A161C.

2. The nucleic acid according to claim 1, wherein the mutant is selected from one of the following (1) to (38): (1) a mutant comprising the amino acid sequence shown in SEQ ID NO: 1 or 2; (2) 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 shown in SEQ ID NO: 1 or 2; (3) a mutant comprising the amino acid sequence shown in SEQ ID NO: 3 or 4; (4) a mutant 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; (5) a mutant comprising the amino acid sequence shown in SEQ ID NO: 5 or 6; (6) 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; (7) a mutant comprising the amino acid sequence shown in SEQ ID NO: 7 or 8; (8) 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 shown in SEQ ID NO: 7 or 8; (9) a mutant comprising the amino acid sequence shown in SEQ ID NO: 9 or 10; (10) 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: 9 or 10; (11) a mutant comprising the amino acid sequence shown in SEQ ID NO: 11 or 12; (12) a mutant comprising mutations S149C and V157C 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: 11 or 12; (13) a mutant comprising the amino acid sequence shown in SEQ ID NO: 13 or 14; (14) 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; (15) a mutant comprising the amino acid sequence shown in SEQ ID NO: 15 or 16; (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; (17) a mutant comprising the amino acid sequence shown in SEQ ID NO: 17 or 18; (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: 17 or 18; (19) a mutant comprising the amino acid sequence shown in SEQ ID NO: 19 or 20; (20) 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; (21) a mutant comprising the amino acid sequence shown in SEQ ID NO: 21 or 22; (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; (23) a mutant comprising the amino acid sequence shown in SEQ ID NO: 23 or 24; (24) 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; (25) a mutant comprising the amino acid sequence shown in SEQ ID NO: 25 or 26; (26) a mutant comprising mutations V55C and V169C 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: 25 or 26; (27) a mutant comprising the amino acid sequence shown in SEQ ID NO: 27 or 28; (28) a mutant comprising mutations L58C and T174C 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: 27 or 28; (29) a mutant comprising the amino acid sequence shown in SEQ ID NO: 29 or 30; (30) 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; (31) 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; (33) a mutant comprising the amino acid sequence shown in SEQ ID NO: 83; (34) 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 shown in SEQ ID NO:83; (35) a mutant comprising the amino acid sequence shown in SEQ ID NO: 84; (36) 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 shown in SEQ ID NO:84; (37) a mutant comprising the amino acid sequence shown in SEQ ID NO: 85; and (38) 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.

3. The nucleic acid according to any one of claims 1 to 2, wherein the wild-type MPV is subtype A or subtype B; Preferably, the wild-type MPV is wild-type hMPV; Preferably, the MPV F protein is hMPV F protein.

4. The nucleic acid according to any one of claims 1 to 3, wherein 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; Alternatively, the nucleic acid is DNA; preferably, the DNA is capable of being transcribed into RNA.

5. The nucleic acid according to claim 4, wherein 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; And / or, 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.

6. The nucleic acid according to any one of claims 1 to 5, wherein the nucleic acid contains modified nucleotides; Preferably, the nucleic acid contains modified nucleosides; preferably, the modified nucleosides include at least one of modified uridine, modified cytidine, modified adenosine and modified guanosine.

7. The nucleic acid according to any one of claims 1 to 6, comprising a polynucleotide encoding one or more mutants of MPV F protein.

8. The nucleic acid according to any one of claims 1 to 7, further comprising a polynucleotide encoding other proteins or polypeptides except the mutant of MPV F protein.

9. The nucleic acid according to any one of claims 1 to 8, further comprising 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.

10. A genetic engineering vector, comprising the nucleic acid according to any one of claims 1 to 9, or comprising a polynucleotide capable of being transcribed into the nucleic acid according to any one of claims 1 to 9. A host cell comprising the genetic engineering vector according to claim 10 .

12. A mutant of the MPV F protein encoded by the nucleic acid according to any one of claims 1 to 19; Preferably, the mutant is a trimer.

13. 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 the nucleic acid according to any one of claims 1 to 9, and the first genetic engineering vector is the genetic engineering vector according to claim 10; Alternatively, the nucleic acid composition comprises a plurality of first nucleic acids or a plurality of first genetic engineering vectors, the first nucleic acid is the nucleic acid according to any one of claims 1 to 9, the first genetic engineering vector is the genetic engineering vector according to claim 10, and the plurality of first nucleic acids are independent of each other or the plurality of first genetic engineering vectors are independent of each other.

14. The delivery vector according to claim 13, wherein 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 except the mutant of the MPV F protein, and the second vector comprises a polynucleotide encoding other proteins or polypeptides except the mutant of the MPV F protein; And / or, the first nucleic acid and the second nucleic acid are RNA; more preferably, the first nucleic acid and the second nucleic acid are mRNA.

15. The delivery vector according to any one of claims 13 to 14, wherein the delivery vector is lipid nanoparticles (LNPs), cationic liposomes, cationic proteins or lipid polymers (LPP); Preferably, 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: in: 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; further preferably, L 3 and L 4 The same or different, each independently is C3-C10 alkylene; most preferably, L 3 and L 4 The same or different, each independently a C5-C8 alkylene group; G 4 and G 5 are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)-, -O-, -C(=O)-S- or -SC(=O)-; preferably, G 4 and G 5 are the same or different, and are each independently -O-(C=O)-, -(C=O)-O-, -C(=O)- or -O-; most preferably, G 4 and G 5 are the same or different, each independently selected from -O-(C=O)- or -(C=O)-O-; R 18 and R 19 are the same or different, and are each independently a C5-C27 alkyl group, or a C5-C27 alkenyl group containing one or more double bonds; preferably, R 18 and R 19 are the same or different, and are each independently a C8-C20 alkyl group or a C8-C20 alkenyl group containing one or more double bonds; further preferably, R 18 and R 19 are the same or different and are each independently a C9-C17 alkyl group or a C9-C18 alkenyl group containing one or two double bonds; most preferably, R 18 and R 19 the same or different, each independently 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; preferably, R 20 is halogen, hydroxyl, cyano, C1-C6 alkoxy, C1-C6 alkylcarbonyloxy, C1-C6 alkoxycarbonyl, C1-C6 alkylaminocarbonyl or C1-C6 alkylcarbonylamino; further preferably, R 20 is halogen, hydroxy, cyano, C1-C4 alkoxy, C1-C4 alkylcarbonyloxy, C1-C4 alkoxycarbonyl, C1-C4 alkylaminocarbonyl or C1-C4 alkylcarbonylamino; most preferably, R 20 is fluorine, hydroxy, cyano, methoxy, acetoxy, methoxycarbonyl, butylaminocarbonyl or acetamido; z is 1, 2 or 3.

16. The delivery vector according to claim 15, wherein the cationic lipid is the following compound (IV-1), or a pharmaceutically acceptable salt or stereoisomer thereof: Alternatively, the cationic lipid is the following compound, or a pharmaceutically acceptable salt thereof:

17. A pharmaceutical composition comprising the nucleic acid of any one of claims 1 to 9, the genetic engineering vector of claim 10, the host cell of claim 11, the mutant of MPV F protein of claim 12 or the delivery vector of any one of claims 13 to 16, and a pharmaceutically acceptable carrier.

18. The pharmaceutical composition according to claim 17, comprising a plurality of said delivery vectors; Alternatively, the pharmaceutical composition comprises a plurality of said nucleic acids.

19. The pharmaceutical composition according to claim 17 or 18, wherein the pharmaceutical composition comprises two nucleic acids, the two nucleic acids are formulated together or separately 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: (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: 1, 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: 2; and (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.

20. Use of the nucleic acid according to any one of claims 1 to 9, the genetic engineering vector according to claim 10, the host cell according to claim 11, the mutant of the MPV F protein according to claim 12, the delivery vector according to any one of claims 13 to 16, or the pharmaceutical composition according to any one of claims 18 to 19 in the preparation of a drug; Preferably, the drug is used to prevent or treat MPV infection or a disease caused by MPV infection; Preferably, the drug is used to prevent or treat hMPV infection or a disease caused by hMPV infection; Preferably, the drug is a vaccine.

21. A vaccine, comprising the nucleic acid of any one of claims 1 to 9, the genetic engineering vector of claim 10, the mutant of MPV F protein of claim 12, or the delivery vector of any one of claims 13 to 16.

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