An hmpv virus fusion protein f variant, an mrna vaccine and application thereof

Abstract

The application discloses an hMPV virus fusion protein F variant, an mRNA vaccine and application thereof. The hMPV virus fusion protein F variant has one or more differences in amino acid residues at one or more sites relative to an hMPV virus fusion protein F with a GeneBank database accession number of AGJ74096.1, wherein the amino acid sequence of the hMPV virus fusion protein F variant is shown as SEQ ID NO: 1 or 2. The mRNA vaccine designed based on the hMPV virus fusion protein F variant can induce a stronger specific antibody response, the antibody has strong persistence in the body, the induced antibody can better neutralize hMPV virus strains, and the hMPV virus amplification in the lung is controlled, so that the specific antibody induced by the mRNA vaccine provides an effective basis for preventing hMPV virus infection related diseases and developing a multiple vaccine.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an hMPV virus fusion protein F variant, an mRNA vaccine and application thereof. BACKGROUND

[0002] Human metapneumovirus (hMPV) is a negative-strand RNA single-stranded virus with an envelope, belonging to the family Pneumoviridae and the genus Metapneumovirus. It was first detected by Bernadette G. van den Hoogen and her colleagues from the Netherlands in 2001 from a nasopharyngeal aspirate sample of a child with respiratory tract infection caused by an unknown pathogen by using RAP-PCR (RNA arbitrarily primed PCR) technology. Serological studies have shown that hMPV has been spreading in the human population for at least 70 years and is distributed worldwide, and is one of the common respiratory pathogens. It is genetically closely related to human respiratory syncytial virus (hRSV) and is the second most common cause of acute respiratory disease after RSV. Its clinical features and severity are similar to those of RSV, and it is often mixedly infected with other respiratory viruses. From mild cough, runny nose and fever to more severe bronchiolitis and pneumonia. In susceptible populations with underlying diseases, hMPV infection can lead to death. Serological studies have shown that almost all children in the world have been exposed to the virus by the age of five; reinfection occurs throughout life. hMPV infection is detected throughout the year, with the highest detection rate in winter and spring, and the epidemic season is usually after RSV and influenza virus.

[0003] hMPV is closely related to avian metapneumovirus subtype C and exhibits a paramyxovirus-like morphology. Its size ranges from 150 to 600 nm. Its genome is approximately 13,300 nucleotides long and consists of eight genes encoding nine proteins: 3'-NPMF-M2-SH-GL-5'. These proteins are nucleoprotein N, phosphoprotein P, matrix protein M, fusion protein F, matrix 2 proteins M2-1 and M2-2, small hydrophobin SH, glycoprotein G, and large polymerase L. The genome is similar to RSV but lacks the nonstructural genes NS1 and NS2, and the gene order differs from that of RSV. The interior of the virus particle is composed of nucleoprotein N, tetrameric phosphoprotein P, and large polymerase L, bound to viral RNA. This is surrounded by matrix protein M and encapsulated in a lipid bilayer containing fusion protein F, glycoprotein G, and small hydrophobin SH. Based on the responses of the F and G proteins to monoclonal antibodies, hMPV is divided into two antigenic subtypes: A and B, which include A1, A2, A3, and B1, B2, respectively. These two subtypes circulate simultaneously or alternately during local epidemics. Subtype B tends to cause increased coughing compared to A. However, to date, no single strain appears to be more epidemiologically dominant than the others, nor does any single strain cause distinct pathological changes.

[0004] The hMPV-F protein sequence is highly conserved between strains and exists on the surface of the virus in a homotrimeric structure. The F protein is first translated into an immature 539 amino acid precursor F0 in the host cytoplasm, and the extracellular domain of the monomer is composed of 489 amino acids at the N-terminus. The monomer is cleaved by serine proteases to form two subunits, F1 and F2, which are connected by disulfide bonds. Then, three F2 / F1 heterodimers combine to form a metastable pre-fusion trimer, which constitutes the active form of the protein. This is a typical feature of class I fusion glycoproteins. The pre-fusion conformation is a metastable pre-F with a membrane-proximal coiled coil stem and a globular head structure. It contains 6 major antigenic sites, namely I, II, III, IV and V. After binding to the target on the host cell surface, Pre-F undergoes conformational changes to close the distance between the viral membrane and the cell membrane. The F protein plays an important role in the entry of hMPV into host cells, so it is the target of neutralizing antibodies and the main subject of vaccine development.

[0005] Since the discovery of hMPV, vaccine research and development has been ongoing, with attempts at inactivated vaccines, cryogenically cultured vaccines, attenuated vaccines, viral vector vaccines, subunit vaccines, and virus-like particle vaccines. However, efforts to develop vaccines based on monomeric wild-type hMPV-F have proven unsuccessful, and currently no vaccine or effective drug is available. SUMMARY

[0006] The present application provides an hMPV virus fusion protein F variant, an mRNA vaccine and application thereof to solve the technical problems in the prior art.

[0007] The present application solves the above technical problems through the following technical solutions.

[0008] The first aspect of the present application provides an hMPV virus fusion protein F variant, wherein the hMPV virus fusion protein F variant has one or more amino acid residue differences at one or more sites relative to the hMPV virus fusion protein F with the accession number AGJ74096.1 in the GeneBank database, and wherein the amino acid sequence of the hMPV virus fusion protein F variant is as shown in SEQ ID NO: 1 or 2, preferably as shown in SEQ ID NO: 1.

[0009] The second aspect of the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the hMPV virus fusion protein F variant according to the first aspect of the present application.

[0010] In some embodiments of the present application, the nucleic acid molecule is double-stranded DNA.

[0011] In some embodiments of the present application, the nucleotide sequence encoding the hMPV virus fusion protein F variant comprises a sequence as shown in SEQ ID NO: 13 or 14.

[0012] In some embodiments of the present application, the nucleic acid molecule further comprises one or more selected from the following:

[0013] (1) a T7 promoter; the nucleotide sequence of the T7 promoter is preferably as shown in SEQ ID NO: 3;

[0014] (2) a coding sequence of 5'UTR; the 5'UTR is preferably the 5'UTR of human cytochrome B-245a polypeptide, and the coding sequence thereof is for example as shown in SEQ ID NO: 5;

[0015] (3) a coding sequence of 3'UTR; the 3'UTR is preferably the 3'UTR derived from human AES / TLE5, and the coding sequence thereof is for example as shown in SEQ ID NO: 6;

[0016] (4) a coding sequence of poly A; the nucleotide sequence of the poly A preferably has a spacer, and the coding sequence of the spacer is TGCAT or GCATATGACT (SEQ ID NO: 20); more preferably, the nucleotide sequence of the poly A is as shown in SEQ ID NO: 7 or 8;

[0017] (5) a coding sequence of Kozak; the coding sequence of Kozak is preferably as shown in SEQ ID NO: 4;

[0018] In some embodiments of the present application, the nucleic acid molecule comprises, in order from 5' end to 3' end, a coding sequence of 5' UTR, a coding sequence of Kozak, a nucleotide sequence encoding the hMPV virus fusion protein F variant, and a coding sequence of 3' UTR.

[0019] In some embodiments of the present application, the 5' end of the nucleic acid molecule is linked to the T7 promoter, and the 3' end is linked to the coding sequence of poly A; preferably, the T7 promoter is linked to the coding sequence of 5' UTR through AGG or GGG.

[0020] The third aspect of the present application provides a nucleic acid construct containing the nucleic acid molecule according to the second aspect of the present application.

[0021] In some embodiments of the present application, the nucleic acid molecule is constructed into the nucleic acid construct by, for example, a restriction endonuclease selected from Hind III and Sap I.

[0022] In some embodiments of the present application, the nucleic acid construct is an expression cassette, which, in addition to containing the nucleic acid molecule, can optionally include regulatory elements such as terminators, enhancers, and the like.

[0023] The fourth aspect of the present application provides a recombinant expression vector containing the nucleic acid molecule according to the second aspect of the present application or the nucleic acid construct according to the third aspect of the present application.

[0024] The fifth aspect of the present application provides a transformant containing the nucleic acid molecule according to the second aspect of the present application, the nucleic acid construct according to the third aspect of the present application, or the recombinant expression vector according to the fourth aspect of the present application, or expressing the hMPV virus fusion protein F variant according to the first aspect of the present application.

[0025] In some embodiments of the present application, the host cell used in the construction of the transformant is selected from E. coli, insect cells, yeast cells, and mammalian cells, for example, E. coli Stable cells.

[0026] The sixth aspect of the present application provides an mRNA comprising mRNA encoding the hMPV virus fusion protein F variant according to the first aspect of the present application.

[0027] In some embodiments of the present application, the mRNA is an mRNA obtained by transcription of the nucleic acid molecule according to the second aspect of the present application.

[0028] In some embodiments of the present application, the sequence of the mRNA encoding the hMPV virus fusion protein F variant comprises the sequence as shown in SEQ ID NO: 15 or 16.

[0029] In some embodiments of the present application, the sequence of the mRNA is as shown in SEQ ID NO: 11 or 12.

[0030] In some embodiments of the present application, the 5' end of the mRNA has a cap structure; the cap structure is preferably a Cap1 structure.

[0031] A seventh aspect of the present application provides a method for preparing a nucleic acid molecule, the method comprising culturing the transformant as described in the fifth aspect of the present application, and obtaining the nucleic acid molecule.

[0032] In some embodiments of the present application, the nucleic acid molecule is DNA or mRNA.

[0033] An eighth aspect of the present application provides a drug-loaded lipid nanoparticle, the lipid nanoparticle comprising the mRNA as described in the sixth aspect of the present application.

[0034] In some embodiments of the present application, the lipid nanoparticle further comprises a cationic lipid and a helper lipid.

[0035] In some embodiments of the present application, the cationic lipid is SM-102.

[0036] In some embodiments of the present application, the helper lipid is DSPC, cholesterol and / or DMG-PEG-2000.

[0037] In some embodiments of the present application, the lipid nanoparticle comprises the mRNA, SM-102, DSPC, cholesterol and DMG-PEG-2000.

[0038] In some embodiments of the present application, the molar ratio of the cationic lipid, DSPC, cholesterol and DMG-PEG-2000 is (40-60):(2-15):(30-40):(0.8-1.6), for example 50:10:38.5:1.5.

[0039] In some embodiments of the present application, the N / P ratio in the lipid nanoparticle is 4-8, for example 6. In the present application, “N / P” represents the molar ratio between nitrogen atoms (N) in the cationic lipid and phosphate groups (P) in the nucleic acid.

[0040] In the present application, the lipid nanoparticle can be administered as a vaccine.

[0041] The ninth aspect of the present application provides a pharmaceutical composition comprising the mRNA according to the sixth aspect of the present application and / or the lipid nanoparticle according to the eighth aspect of the present application, and optionally a pharmaceutically acceptable carrier.

[0042] The tenth aspect of the present application provides use of the hMPV virus fusion protein F variant according to the first aspect of the present application, the nucleic acid molecule according to the second aspect of the present application, the nucleic acid construct according to the third aspect of the present application, the recombinant expression vector according to the fourth aspect of the present application, the transformant according to the fifth aspect of the present application, the mRNA according to the sixth aspect of the present application, the lipid nanoparticle according to the eighth aspect of the present application, and / or the pharmaceutical composition according to the ninth aspect of the present application in the preparation of a medicament for preventing and / or treating a disease caused by hMPV virus infection and / or inhibiting hMPV virus.

[0043] In some embodiments of the present application, the disease caused by hMPV virus infection is a respiratory disease or an ear infection, such as bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease, or asthma.

[0044] The eleventh aspect of the present application provides a multivalent vaccine comprising the mRNA according to the sixth aspect of the present application, the lipid nanoparticle according to the eighth aspect of the present application, and / or the pharmaceutical composition according to the ninth aspect of the present application, and an mRNA, a lipid nanoparticle, an mRNA vaccine, and / or a pharmaceutical composition for inhibiting other viruses, such as a syncytial virus.

[0045] In some embodiments of the present application, the syncytial virus is a human respiratory syncytial virus (e.g., a human respiratory syncytial virus of type B).

[0046] In some embodiments of the present application, the nucleotide sequence of the mRNA for inhibiting other viruses is shown in SEQ ID NO: 17.

[0047] In some embodiments of the present application, the multivalent vaccine comprises the mRNA according to the sixth aspect of the present application or a human respiratory syncytial virus of type B with a nucleotide sequence shown in SEQ ID NO: 17, which can be respectively encapsulated in a lipid nanoparticle to prepare the multivalent vaccine, or can be jointly encapsulated in one lipid nanoparticle to prepare the multivalent vaccine.

[0048] The twelfth aspect of the present application provides a kit-of-parts comprising a kit A and a kit B, wherein:

[0049] The kit A contains the mRNA according to the sixth aspect of the present application, the lipid nanoparticle according to the eighth aspect of the present application, and / or the pharmaceutical composition according to the ninth aspect of the present application.

[0050] The kit B contains an mRNA inhibiting other viruses, a lipid nanoparticle, an mRNA vaccine and / or a pharmaceutical composition; the other viruses are, for example, syncytial viruses.

[0051] In some embodiments of the present application, the syncytial virus is human respiratory syncytial virus (for example, human respiratory syncytial virus type B).

[0052] In some embodiments of the present application, the nucleotide sequence of the mRNA inhibiting other viruses is shown in SEQ ID NO: 17.

[0053] In the present application, the kit can be used as a premixed state of the above-mentioned multivalent vaccine, that is, the reagents in the kit A and the kit B are mixed to obtain the multivalent vaccine before administration; or the reagents in the kit A and the kit B can be administered separately in sequence; the actual situation can be adjusted.

[0054] The thirteenth aspect of the present application provides a method for inhibiting hMPV virus, which comprises contacting the hMPV virus with the mRNA according to the sixth aspect of the present application, the lipid nanoparticle according to the eighth aspect of the present application, the pharmaceutical composition according to the ninth aspect of the present application and / or the multivalent vaccine according to the eleventh aspect of the present application, inducing an immune response to inhibit the hMPV virus, or using the kit according to the twelfth aspect of the present application, the induced immune response inhibits the hMPV virus. In the present application, "inhibiting hMPV virus" means reducing or preventing the infection or replication of hMPV virus.

[0055] In some embodiments of the present application, the method is for non-therapeutic purposes. In the present application, "non-therapeutic purposes" means that the above-mentioned products are used as a positive control group when studying hMPV virus in the laboratory, for example, when studying the effect of certain reagents on hMPV virus.

[0056] The fourteenth aspect of the present application provides a method for preventing and / or treating diseases caused by hMPV virus infection, which comprises administering an effective dose of the mRNA according to the sixth aspect of the present application, the lipid nanoparticle according to the eighth aspect of the present application, the pharmaceutical composition according to the ninth aspect of the present application and / or the multivalent vaccine according to the eleventh aspect of the present application to a patient in need thereof, or using the kit according to the twelfth aspect of the present application for a patient in need thereof.

[0057] In the present application, "effective dose" means the amount of mRNA that is effective to produce the expected pharmacological, prophylactic or therapeutic results.

[0058] In the present application, "prevention" means that the patient / subject in need has never been infected or is not currently infected with the hMPV virus, and after administration of the above product, the patient / subject will not be infected or, if infected, the symptoms will be less severe or will resolve faster than in a subject who has not been administered the above product when exposed to the hMPV virus.

[0059] In some embodiments of the present application, the disease caused by the hMPV virus infection is a respiratory disease or an ear infection, such as bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease, or asthma.

[0060] In some embodiments of the present application, the patient is infected or not infected with the hMPV virus.

[0061] In some embodiments of the present application, the single administration dose of the lipid nanoparticle can be 1-20 μg, 20-60 μg, 60-110 μg, 110-160 μg, or 160-200 μg, such as 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 μg, or a dose between these values; it can also be adjusted according to the weight of the subject.

[0062] In some embodiments of the present application, the number of administrations is at least 1, such as 1, 2, 3, or 4, which can be adjusted according to the actual situation.

[0063] In some embodiments of the present application, the frequency of administration is at least two weeks, preferably three to eight weeks, such as three, four, five, six, or seven weeks, which can be adjusted according to the actual situation.

[0064] The fifteenth aspect of the present application provides the hMPV virus fusion protein F variant of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, the nucleic acid construct of the third aspect of the present application, the recombinant expression vector of the fourth aspect of the present application, the transformant of the fifth aspect of the present application, the mRNA of the sixth aspect of the present application, the liposome nanoparticle of the eighth aspect of the present application, the pharmaceutical composition of the ninth aspect of the present application, the multiple vaccine of the eleventh aspect of the present application, and / or the kit of the twelfth aspect of the present application, for use in preventing and / or treating a disease caused by hMPV virus infection and / or inhibiting hMPV virus.

[0065] In some embodiments of the present application, the disease caused by the hMPV virus infection is a respiratory disease or an ear infection, such as bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease, or asthma.

[0066] On the basis of common general knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0067] The reagents and raw materials used in the present application are commercially available or can be prepared according to the regulations and can be repeatedly prepared.

[0068] The positive progress effect of the present application is that:

[0069] The mRNA vaccine designed based on the hMPV virus fusion protein F variant of the present application can induce a strong specific antibody response, and the antibody has strong persistence in vivo, and the induced antibody can better neutralize the hMPV virus strain and control the amplification of the hMPV virus in the lung, so the specific antibody induced by the vaccine provides an effective basis for preventing hMPV virus infection related diseases and the development of multivalent vaccines. BRIEF DESCRIPTION OF DRAWINGS

[0070] Figure 1 The mRNA MFE secondary structure of JY-hMPV-081 and JY-hMPV-060 is shown.

[0071] Figure 2A and Figure 2B The nucleic acid sequence of JY-hMPV-081 is shown, wherein Figure 2A is a DNA sequence, Figure 2B is an mRNA sequence.

[0072] Figure 3A and Figure 3B The nucleic acid sequence of JY-hMPV-060 is shown, wherein Figure 3A is a DNA sequence, Figure 3B is an mRNA sequence.

[0073] Figure 4 The agarose gel electrophoresis map of restriction enzyme digestion of JY-hMPV-F series plasmid is shown.

[0074] Figure 5 The in vitro synthesis of JY-hMPV-F series T7 RNA polymerase induced mRNA is shown by agarose gel electrophoresis.

[0075] Figure 6 The expression of hMPV F protein in cells after transfection of 293T cells by JY-hMPV-F series mRNA / LNP nanoparticles is shown by Western Blot detection.

[0076] Figure 7 The JY-hMPV-F series mRNA / LNP vaccine induces strong specific binding antibodies in mice in vivo is shown.

[0077] Figures 8A-8C The immunization schedule and immunization dose effect of JY-hMPV-F series mRNA / LNP vaccine in mice are shown. Among them Figure 8A The immunization schedule effect of JY-hMPV-081 is shown, Figure 8B The immunization schedule effect of JY-hMPV-060 is shown; Figure 8C The immunization dose effect of JY-hMPV-81 in mice is shown.

[0078] Figure 9A The specificity of the antibodies induced by JY-hMPV-F mRNA / LNP vaccine in mice is shown to be persistent. Figure 9B The effect of JY-hMPV-081 mRNA / LNP vaccine is shown alone; Figure 9A The effect of JY-hMPV-081 mRNA / LNP vaccine is shown alone; Figure 9C The effect of JY-hMPV-060 mRNA / LNP vaccine is shown alone. Figure 9A The effect of JY-hMPV-060 mRNA / LNP vaccine is shown alone.

[0079] Figures 10A-10C The antibodies induced by JY-hMPV-F mRNA / LNP vaccine in mice are shown to exhibit the characteristics of antigen independence. Among them Figure 10A The results of binding to hMPV-F antigen are shown; Figure 10B The results of binding to RSV-F antigen are shown; Figure 10C The results of binding to hMPV-F antigen are shown; Figure 10A The results of binding to hMPV-F antigen are shown; Figure 10B The combined figures of and are shown.

[0080] Figure 11 The results of neutralization test detection show that the antibodies induced by JY-hMPV-F mRNA / LNP vaccine in mice have the ability to neutralize hMPV virus.

[0081] Figures 12A-12B The JY-hMPV mRNA / LNP vaccine as a booster vaccine can elicit higher anti-hMPV-F antibodies. Figure 12A The flow of animal immunization and sampling is shown; Figure 12B The results of Elisa experiment are shown.

[0082] Figures 13A-13B The JY-hMPV-F mRNA / LNP vaccine can significantly reduce the infection of hMPV in the lungs of mice. Among them Figure 13A The flow chart of animal pre-infection, immunization and sampling experiment is shown; Figure 13B The results of qPCR detection of viral load in mouse lungs are shown. DETAILED DESCRIPTION

[0083] The application will be further described in the following by way of examples without limiting the application to the examples described. The experimental methods in the following examples, where no specific conditions are indicated, were carried out in accordance with standard methods and conditions, or as selected by the manufacturer's instructions.

[0084] Example 1 Screening, modification and mRNA sequence optimization of virus strains

[0085] 1.1 Screening of starting virus strains

[0086] According to traditional serological classification, hMPV is divided into A and B subtypes, with amino acid homology of 93%-96%. Epidemiological studies have found that nearly 70% of hMPV strains belong to group A, among which A2 strains are more common. The proportion of B strains in adults may be higher than that in children. However, there seems to be no difference between the two subtypes in terms of severity of disease. Therefore, the present application is designed to take both into account, but mainly for hMPV A. After analyzing several hundred complete F gene sequences of hMPV, a strain discovered in Australia in 2003: HMPV / AUS / 150229278 / 2003 / A, GenBank: KC562226.1 (2003) was selected, from which JY-hMPV-081 (A) and JY-hMPV-060 (A) were constructed, with specific information as shown in Table 1.

[0087] Table 1 Basic information of JY-hMPV-F series

[0088]

[0089] The amino acid sequences of the proteins corresponding to JY-hMPV-081 and JY-hMPV-060 are as follows:

[0090] JY-hMPV-081:

[0091]

[0092]

[0093] JY-hMPV-060:

[0094]

[0095]

[0096]

[0097] 1.2 Sequence optimization:

[0098] mRNA sequence design is one of the most core and key problems in the whole process of mRNA manufacturing, which is inseparable from the mRNA manufacturing. It affects the yield of mRNA in vitro transcription, the immunogenicity of mRNA molecules, the translation efficiency and the stability of mRNA molecules. The commonly used mRNA is composed of 5'Cap (cap structure), 5'UTR (5' non-coding region), ORF (protein coding open reading frame), 3'UTR (3' non-coding region) and 3'poly A tail (poly A tail).

[0099] 5'Cap structure can protect mRNA from degradation by cytoplasmic exonuclease Xrn1 and nuclear Xrn2 enzyme. Capped mRNA has a longer half-life and higher stability than uncapped mRNA, which significantly improves the yield of protein synthesis in cells. The chemical nature of the cap structure is a special structure at the 5' end of mRNA formed during mRNA transcription, which is m7GPPPN structure, also known as methyl guanosine cap. It is formed under the catalysis of RNA triphosphatase, guanylyltransferase, mRNA (guanine-N7) methyltransferase and mRNA (nucleoside-2') methyltransferase. According to the degree of methylation modification, the cap can be divided into three structures: Cap 0, Cap 1 and Cap 2. Since uncapped mRNA or Cap 0 structure can be recognized by natural immune receptor RIG-1, Cap 1 and Cap 2 can protect themselves from being recognized by innate immune sensors, and Cap 1 structure is selected in the present application.

[0100] The T7 promoter sequence can be divided into two domains: binding domain and transcription initiation domain. For the transcription initiation domain, any base substitution will seriously affect the strength of the promoter. Our design is TAATACGACTCACTATA (SEQ ID NO: 3), followed by agg for co-transcriptional capping. The corresponding coding sequence of Kozak is GCCGCCACC (SEQ ID NO: 4).

[0101] The translation initiation regulation of eukaryotic mRNA is mainly determined by the characteristics of 5'UTR, including secondary structure, sequence elements and 5'UTR length, which will affect the stability of mRNA. Human cytochrome B-245a polypeptide (CYBA) UTR with relatively high protein expression is used, and its coding sequence is AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC (SEQ ID NO: 5). Of course, the performance of UTR also depends on the cell type.

[0102] For the DNA sequence of hMPV-F, the overall translation efficiency of mRNA can be optimized by choosing appropriate codons. One is to bring the overall codon usage closer to that of the host cell, that is, to replace the codons in the viral sequence with synonymous codons that are frequently used in human cells, to ensure that the codon usage bias is more consistent, and to avoid too many rare codons. However, it is also noted that a small number of viral codons are retained to slow down the speed of ribosome advancement, providing sufficient time for the correct folding of the protein, thereby more accurately restoring the antigenic protein structure of the virus. Another point to note is the GC content, which is slightly higher, which is believed to increase the stability of mRNA and improve the expression of protein in vivo. Finally, the secondary structure of the mRNA sequence is also considered in the design Figure 1 ). It is reported that the hairpin structure of the secondary structure can affect the degradation of mRNA, which helps to improve the stability of mRNA.

[0103] Similar to the 5'UTR, the 3'UTR also contains many regulatory elements that have important effects on the stability, subcellular localization and translation efficiency of mRNA. The 3'UTR used is from human AES / TLE5, and its coding sequence is CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 6).

[0104] Poly A tail plays an indispensable role in protein translation process. It binds to poly A binding protein (PAPB), which in turn interacts with translation initiation factor eIF4G, forming a "closed loop" structure, recruiting 40S translation initiation complex to mRNA, and cooperating with 5' cap structure to stimulate translation initiation. During bacterial amplification, plasmids carrying long poly A nucleotide sequences can produce unpredictable recombination events. The poly A tail on the plasmid is shortened with the continuous amplification of bacteria, causing trouble for plasmid cloning and amplification. Studies have shown that spacing the poly A sequence can significantly reduce recombination events during plasmid DNA amplification, maintain the length of the tail, and at the same time, not affect the translation efficiency and half-life of the mRNA generated by in vitro transcription. Following this principle, the poly A structure used is 60-spaced-60 bases, or 40-spaced-80 bases, which corresponds to the coding sequence AGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTCGAAGTGACT (SEQ ID NO: 7) or TAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 8).

[0105] The optimized JY-hMPV-F series coding sequences are shown in Figures 2A-2B , Figures 3A-3B Figure 2A The DNA coding strand sequence of JY-hMPV-081 (SEQ ID NO: 9) is shown, Figure 2B The mRNA sequence after transcription (SEQ ID NO: 11) is shown; Figure 3A The DNA coding strand sequence of JY-hMPV-060 (SEQ ID NO: 10) is shown, Figure 3B The mRNA sequence after transcription (SEQ ID NO: 12) is shown.

[0106] The sequences of the optimized JY-hMPV-F series are as follows:

[0107] 1. JY-hMPV-081 ​

[0108] ORF DNA sequence:

[0109]

[0110]

[0111] ORF mRNA sequence:

[0112]

[0113]

[0114]

[0115] Full-length mRNA sequence:

[0116]

[0117]

[0118]

[0119] 2. JY-hMPV-060

[0120] ORF DNA sequence:

[0121]

[0122]

[0123]

[0124] ORF mRNA sequence:

[0125]

[0126]

[0127] Full-length mRNA sequence:

[0128]

[0129]

[0130]

[0131] Example 2 Plasmid synthesis and DNA extraction of JY-hMPV-F series mRNA vaccine

[0132] All designed sequences were cloned into the vector by HindIII and SapI. The specific experimental steps are as follows:

[0133] 2.1 Plasmid synthesis

[0134] After the plasmid sequence design is completed, it is synthesized by a CRO company.

[0135] 2.2 Plasmid DNA extraction

[0136] 2.2.1 Reagent preparation

[0137] LB plates and liquid media containing peptone, sodium chloride, yeast extract and corresponding antibiotics, the specific formula is as follows Table 2 and Table 3.

[0138] Table 2 LB liquid medium (500 mL)

[0139] Sodium chloride 5g Peptone 5g Yeast extract 2.5g

[0140] Table 3 LB plate (500 mL)

[0141] Sodium chloride 5g Peptone 5g Yeast extract 2.5g Agar powder 7.5g

[0142] 2.2.2 Plasmid transformation

[0143] The plasmid DNA is added to the Stable competent cells, and is placed in an ice box for 30 min; 42°C water bath heat shock for 45 seconds, quickly placed in an ice box for 2 min, 500 μL of antibiotic-free LB culture solution is added, 37°C shaking table culture for 1 h, then plated, and cultured at 37°C overnight. The next day, the clones are picked from the plate.

[0144] 2.2.3 Plasmid DNA extraction

[0145] The overnight culture is extracted by the method of small or large extraction kit.

[0146] 2.3 Plasmid DNA restriction verification

[0147] Restriction enzymes Hind III and EcoR I are used for restriction verification Figure 4 ). The qualified plasmid enters the next step of in vitro transcription experiment.

[0148] Example 3 In vitro transcription and capping of JY-hMPV-F series

[0149] 3.1 Plasmid DNA linearization

[0150] This design uses a reverse Sap I at the 3' end of poly A to linearize the plasmid (such as Figure 2A and Figure 3A ). After 37°C reaction for 3 hours, SDS-PAGE gel electrophoresis is used to determine whether the enzyme cutting is complete. 3M sodium acetate precipitation method is used for purification; then the DNA concentration is determined by Nanodrop method.

[0151] 3.2 In vitro transcription and capping

[0152] The JY-hMPV-F series plasmids were designed, all of which used agg, suitable for co-transcriptional capping.

[0153] The components were added in turn according to Table 4 below, and reacted at 37°C for 3 hours.

[0154] Table 4 Co-transcription and capping system components

[0155]

[0156]

[0157] After the reaction was completed, 5 μL of DNase I was added to 100 μL of the template for digestion, and reacted at 37°C for 30 min. Agarose gel was used to determine whether the DNA template was completely digested.

[0158] 3.3 Purification

[0159] 150 μL of lithium chloride solution (containing 7.5 M lithium chloride and 50 mM EDTA) and 150 μL of DEPC water were added to each 100 μL of the above reaction volume, and the final concentration of lithium chloride was maintained at 2.5-2.8 M. After mixing, it was placed in -20°C for at least 30 min, or overnight.

[0160] The sample was taken out from -20°C, and centrifuged at 12000 rpm for 15 min, and the supernatant was removed and the precipitate was collected. Then 1 mL of 70% aqueous ethanol was added for washing, and centrifuged at 12000 rpm for 5 min, and the supernatant was discarded; this was repeated once. The lid was opened in a clean bench, and it was placed at room temperature for 5 min to allow the ethanol to evaporate.

[0161] The mRNA was resuspended in an appropriate amount of DEPC water, and agarose gel electrophoresis was used to determine the quality of the mRNA, and the concentration was determined.

[0162] The results of gel detection of JY-hMPV-F series T7 RNA polymerase-induced in vitro synthesis of mRNA are shown in Figure 5 .

[0163] Example 4 Preparation of JY-hMPV-F series mRNA / LNP nanoparticles

[0164] The preparation method of nanoparticles LNP commonly used in the industry was used in this study. The specific steps are as follows:

[0165] LNP was first prepared by proportionally mixing the stock solution dissolved in ethanol, ionizable lipid (SM-102): DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine): cholesterol: DMG-PEG-2000 at a molar ratio of 50:10:38.5:1.5; then mixed with mRNA dissolved in acetate buffer (pH 4) in a high-impact nanoparticle preparation device, followed by dilution and pH elevation to form lipid nanoparticles (mRNA / LNP) with mRNA wrapped therein. The sample was concentrated and purified by ultrafiltration centrifugation. The final product was tested for particle size, polydispersity index (PDI), zeta potential (ZP), encapsulation efficiency, and effective mRNA concentration.

[0166] 4.1. Buffer preparation

[0167] All the following reagents were autoclaved at 121 °C for 20 min and prepared with DEPC water:

[0168] 1) 50 mM acetate buffer, pH 4.0;

[0169] 2) 25 mM Tris-HCL buffer, pH 7.5;

[0170] 3) 0.6 g / mL sucrose solution.

[0171] 4.2. Preparation of lipid solution and mRNA solution, see Table 5 and Table 6 below

[0172] Table 5 Preparation of lipid solution

[0173]

[0174] 1) The lipid needs to be restored to room temperature before weighing;

[0175] 2) All ingredients are dissolved in anhydrous ethanol, and an ultrasonic instrument is used to ensure complete dissolution;

[0176] 3) The above prepared lipid solution is mixed according to the ratio of 1:1:1:1, and the 12 mg / mL lipid mixed solution according to the formula is obtained.

[0177] Table 6 Preparation of mRNA solution

[0178]

[0179] Dilute the in vitro transcribed and capped mRNA stock prepared in Example 3 to the desired concentration 0.2 mg / mL according to the calculation method and buffer type shown in the table above.

[0180] 4.3 Preparation of mRNA / LNP (for example with a total volume of 4 mL)

[0181] 4.3.1 INano LTM, the rapid nanomedicine preparation system of Maianna. Set the parameters as needed.

[0182] 4.3.2 Pre-rinse of the chip

[0183] Prepare 3 5 mL syringes, one of which draws anhydrous ethanol, one draws acetate buffer, and one is empty. Left side acetate buffer 5 mL → air 3 times; right side ethanol 5 mL → ethanol 5 mL → air 3 times, and push the chip to clean it. After cleaning, put the chip into the card slot.

[0184] 4.3.3 Run the microfluidic device to prepare LNP

[0185] Use a 3 mL syringe to draw 3 mL of the mRNA solution configured above, and remove the air bubbles, and put it into the left syringe card slot of the machine. Use a 1 mL syringe to draw 1 mL of the lipid mixture solution configured above, and remove the air bubbles, and put it into the right syringe card slot of the machine. Insert a 15 mL centrifuge tube for waste and sample collection at the waste collection and sample collection of the machine respectively. After confirming that the parameters are correct, click "Start", and the INano LTM, the rapid nanomedicine preparation system of Maianna, will immediately run, i.e. prepare LNP. After the preparation is completed, take out the centrifuge tube at the sample collection.

[0186] 4.3.4 Processing of the collected mRNA / LNP sample

[0187] Immediately dilute the collected mRNA / LNP suspension with 25 mM Tris-HCl pH 7.5 buffer to about 25 times the volume of mRNA / LNP, and then centrifuge ultrafiltration at 3000 rpm, 4°C, for 1 h. Repeat this process until the solution volume is less than or equal to the pre-dilution volume (3.6 mL, i.e. total volume 4 mL minus waste 0.4 mL). Then, sterilize through a 0.22 μm filter membrane, and add 0.2 times the solution volume of 0.6 g / mL sucrose solution, to finally obtain the mRNA / LNP preparation product.

[0188] 4.3.5 Detection of the physical and chemical properties of mRNA / LNP

[0189] The prepared mRNA / LNP should be as soon as possible for physical property detection to determine the quality of the product, and facilitate the next step of biological activity research. The Malvern Zetasizer is used for detection, and the particle size, PDI, and ZP data can be obtained (Table 7 below).

[0190] Encapsulation efficiency analysis:

[0191] The mRNA control (100 μg / ml) was diluted to 6 standard curve points (2000, 1600, 1200, 800, 400, 200 ng / mL) using lx TE buffer and 2% Triton TE buffer, respectively. The test sample LNP was diluted 50 times using 2% Triton TE buffer when measuring total RNA content, and the test sample was diluted 20 times using lx TE buffer when measuring free RNA content. 100 μL of the above standard curve solution and test sample solution were transferred into a black microplate, and then 100 μL of 0.005x Ribogreen (200-fold dilution) was added, respectively, and reacted at room temperature for 2-5 min in the dark. The fluorescence response value (excitation wavelength 480 nm, emission wavelength 520 nm) was read by a microplate reader, and the encapsulation efficiency was calculated according to the following formula:

[0192] Encapsulation efficiency EE (%) = [(Ctotal RNA - Cfree RNA) / Ctotal RNA] * 100%

[0193] The effective concentration and encapsulation efficiency of the sample mRNA obtained according to the above method are shown in Table 7.

[0194] Table 7 Physical properties of JY-hMPV series mRNA / LNP lipid particles

[0195] Sample name Particle size PDI Zeta potential Effective concentration pg / mL Encapsulation efficiency % JY-hMPV-060 84.18 0.051 -5.709 207 96.6 JY-hMPV-081 85.46 0.058 -3.432 201 96.4

[0196] Example 5 JY-hMPV-F series mRNA / LNP nanoparticle transfection into 293T cells for expression

[0197] To determine whether the mRNA in the lipid nanoparticle can express the correct protein, the first step is to transfect the specific mRNA / LNP into suitable cells, and then Elisa, Western Blot, flow cytometry, etc. can be used to detect the correctness of protein expression.

[0198] 5.1 Cell transfection

[0199] One day before transfection, 293T (CRL-3216, ATCC) cells were plated at 2 x 10^5 per well in a 24-well plate in complete medium, DMEM plus 10% fetal bovine serum and antibiotics, in a 37°C, 5% CO2 incubator. The next day, the cell culture medium was aspirated from each well and gently washed with 1 x PBS buffer. After adding 500 μL of complete medium to each well, the cells were transfected with a gradient of hMPV F mRNA / LNP nanoparticles containing 0.5 to 3 μg of RNA per well. After gently shaking to disperse the nanoparticles, the cells were incubated at 37°C, 5% CO2 for 48-72 hours.

[0200] 5.2 Western Blot method for detecting expression of antigen protein

[0201] By the WB method, the translation of mRNA / LNP into the designed antigen protein after entering the cells can be qualitatively detected. Since the designed antigen protein has a signal peptide, the extracellular protein is determined here.

[0202] 5.2.1 Preparation of electrophoresis sample

[0203] The supernatant of the cells after transfection for 48-72 hours was collected, centrifuged at 2500 rpm, 5 min, 4°C, and transferred to a new 1.5 mL tube. If the expression is too low, a suitable size Millipore ultrafiltration tube can be selected for moderate concentration. Then the protein content was determined, and each sample was adjusted to the same sample amount, and 6 x SDS loading buffer was added to a final concentration of 1 x. The sample was heated at 92°C for 5 min to denature the protein before loading.

[0204] 5.2.2 SDS-PAGE electrophoresis

[0205] The electrophoresis used 10% separation gel and 4% concentrated gel, and the sample was loaded immediately after heating. During electrophoresis, the concentrated gel segment was at 110 V and the separation gel segment was at 150 V.

[0206] 5.2.3 Transferring membrane

[0207] According to the size of the molecular weight, nitrocellulose membrane or PVDF membrane was selected. Generally, 80 V was used for 1 h, or 60 V was used for 2 h, and ice bag was used for heat control. After transfer, 1 x rose red staining solution was used for 5 min to detect the transfer effect.

[0208] 5.2.4 Blocking and immunoreaction

[0209] First, the membrane was blocked with 5% skim milk powder at 4°C overnight. The next day, after washing with PBST solution for 3 times, the diluted primary antibody (mouse anti-hMPV-F) was hybridized at room temperature for 90 min. Then, the membrane was washed with PBST solution for 3 times, each for 5-10 min. The secondary antibody was anti-mouse-HRP, which was hybridized at room temperature for 60 min. Subsequently, the membrane was also washed with PBST solution for 3 times, each for 5-10 min

[0210] 5.2.5 Development and exposure

[0211] According to the ECL kit instructions, the test solution A and B were mixed at a volume ratio of 1:1 and placed in the membrane, and then shaken appropriately in the dark room. Then, the membrane was exposed to the chemiluminescence image analyzer for detection. The marker band was used to determine the correctness of the target protein size.

[0212] The results are shown in Figure 6 It was shown that after the JY-hMPV-F series transfected 293T cells, the mRNA / LNP was endocytosed into the cells, released to the cytoplasm under the acidic environment of lysosomes, and completed protein translation and modification in the endoplasmic reticulum and Golgi apparatus, and released to the extracellular under the guidance of the signal peptide. The encoded hMPV-F antigen protein can bind to the F antibody, and the protein specificity and protein molecular weight also meet the design requirements.

[0213] Example 6 JY-hMPV-F series mRNA / LNP vaccine immunization of mice and identification of virus-specific antibody expression

[0214] In order to detect whether the vaccine has specific antigen expression and performance in vivo, mice were used as animal models for detection.

[0215] 6.1 Animal immunization

[0216] Balb / c mice, female, 6 weeks old, were first adapted for a week after arriving at the animal house. The vaccine was the JY-hMPV series mRNA / LNP vaccine prepared in Example 4. Intramuscular injection, the dose was 20 μg per mouse, and the injection was divided into three times with an interval of 14 days. Blood was taken one day before the first immunization, 6 days after the second immunization, and 7 days after the third immunization, and the serum was used for subsequent antibody Elisa detection.

[0217] 6.2 Elisa detection of anti-hMPV antibody

[0218] One of the key indicators of the vaccine is to induce specific anti-virus antibodies in animals. Elisa method was used to detect the expression amount of anti-hMPV-F antibody.

[0219] 6.2.1 Antigen coating

[0220] Since there is no commercialized anti-hMPV-F antigen, the supernatant carrying hMPV-F plasmid transfected by 293T cells was diluted and used to coat the antigen in 96-well enzyme-labeled plate, 100 μL per well, 4°C, 16 h.

[0221] 6.2.2 Washing and blocking

[0222] The enzyme-labeled plate coated with the antigen was taken out and washed with 300 μL / well PBST once. Then, 100 μL blocking solution (5% milk powder) was added, and the plate was incubated at room temperature for 30 min. Subsequently, the plate was washed with 300 μL / well PBST for 3 times.

[0223] 6.2.3 Sample dilution and addition

[0224] After the serum sample of the immunized mouse was diluted by a suitable gradient of 4 times, 100 μL was added to the enzyme-labeled plate, and the plate was incubated at room temperature for 1.5 h. Then, the plate was washed with 300 μL / well PBST for 3 times.

[0225] 6.2.4 Addition of detection antibody

[0226] According to the number of added wells, the appropriate amount of detection antibody (goat anti-mouse IgG, peroxidase conjugated, H+L) was diluted to 0.8 ng / mL, and 80 μL was added to each well. After incubation at room temperature for 1 h, the plate was washed with 300 μL / well PBST for 5 times.

[0227] 6.2.5 Color development and termination

[0228] Color developing solution A (containing H2O2) and color developing solution B (containing TMB) were mixed at a ratio of 1:1, and 100 μL was added to each well. After incubation at room temperature for 15-30 min, 30 μL of 1M HCl termination solution was added to each well. Then, the plate was read at 450 nm by using an enzyme-labeled instrument.

[0229] From the results shown in Table 1, it can be seen that the JY-hMPV-F series mRNA / LNP vaccine can induce a strong antibody response in mice. Figure 7 Example 7 Immunization procedure and dose effect of JY-hMPV-F mRNA / LNP vaccine in mice

[0230] As a vaccine, the immunization procedure and the immunization dose are two key factors. The specific experimental steps are as follows:

[0231] 7.1 Animal immunization

[0232] 7.1.1 Immunization procedure

[0233] 7.1.2 Immunization dose

[0234] Balb / c mice, female, 6 weeks old, first adapt for a week after arriving at the animal room. The experiment is divided into three groups: the first group is immunized with JY-hMPV-081; the second group is immunized with JY-hMPV-060; the third group is the control group. Y-hMPV-F mRNA / LNP vaccine is administered by intramuscular injection at 20 μg per mouse per time, with an interval of 14 days, and administered twice. Before immunization, on the 14th day after the first immunization, and on the 14th day after the second immunization, blood is taken from the orbit, and the serum is tested.

[0235] 7.1.2 Dose effect

[0236] Balb / c mice, female, 6 weeks old, first adapt for a week after arriving at the animal room. The experiment is divided into three groups: the first group is immunized with JY-hMPV-081; the second group is immunized with JY-hMPV-060; the third group is the control group. Y-hMPV-F mRNA / LNP vaccine is administered by intramuscular injection at 20 μg per mouse per time, with an interval of 14 days, and administered twice. Before immunization, on the 14th day after the first immunization, and on the 14th day after the second immunization, blood is taken from the orbit, and the serum is tested.

[0237] 7.2 Elisa detection of anti-hMPV antibody

[0238] 7.2.1 Antigen coating

[0239] Due to the lack of commercial anti-hMPV-F antigen, the supernatant carrying the hMPV-F plasmid transfected by 293T cells is diluted and used for antigen coating in a 96-well enzyme-labeled plate, 100 μL per well, 4°C, 16 h.

[0240] 7.2.2 Plate washing and blocking

[0241] Take out the enzyme-labeled plate with coated antigen, wash the plate once with 300 μL per well PBST. Then add 100 μL blocking solution (5% milk powder), room temperature, 30 min. Then wash the plate 3 times with 300 μL per well PBST.

[0242] 7.2.3 Sample dilution and sample addition

[0243] After the serum samples of immunized mice are diluted by a suitable 4-fold gradient, 100 μL is added to the enzyme-labeled plate, three replicates, incubated at room temperature for 1.5 h, and then washed 3 times with 300 μL per well PBST.

[0244] 7.2.4 Addition of detection antibody

[0245] According to the number of wells to be added, the appropriate amount of detection antibody (goat anti-mouse IgG, peroxidase conjugated, H+L) is taken, diluted to 0.8 ng / mL, and then 80 μL per well is added, incubated at room temperature for 1 h, and then washed 5 times with 300 μL per well PBST.

[0246] 7.2.5 Color development and termination

[0247] Mix color developing solution A (containing H2O2) and color developing solution B (containing TMB) at a ratio of 1:1, add 100 μL per well, and let stand at room temperature for 15-30 minutes. Then add 30 μL of 1 M HCl per well to terminate the reaction, and then read the results at 450 nm using an enzyme label meter.

[0248] The results of the immunization program exploration are shown in Table 1. Figure 8A and Figure 8B It is shown that the mRNA / LNP vaccines corresponding to JY-hMPV-081 and JY-hMPV-060 can induce high titers of virus-specific antibodies in animals, and that "immunization-boosting" secondary administration can achieve better results than a single administration. In terms of dosage (see Table 2), there is no difference between 15 μg / animal and 30 μg / animal, and both are superior to 7.5 μg / animal. It is believed that the antigen produced by 15 μg / animal is in the range of saturation. Figure 8C

[0249] Example 8 Exploration of the persistence of antibodies induced by JY-hMPV-F mRNA / LNP vaccines

[0250] As a vaccine, the length of time for which the antibodies it induces can persist in the body is an important factor related to the persistence of the efficacy of the vaccine, because it relates to the problem of needing to perform several immunizations in a viral infection season.

[0251] 8.1 Animal immunization

[0252] Balb / c mice, female, 6 weeks old, were first adapted for one week after arrival in the animal house. The experiment was divided into three groups: the first group was immunized with JY-hMPV-081; the second group was immunized with JY-hMPV-060; and the third group was the control group (treated with PBS buffer). The Y-hMPV-F mRNA / LNP vaccine was administered by intramuscular injection at a dose of 20 μg / animal / time, with a 14-day interval, and administered twice. Before immunization, on the 14th day after the first immunization, on the 7th day, 30th day, 3rd month, 6th month, and 9th month after the second immunization, blood was taken from the orbit, and the serum was tested.

[0253] 8.2 Elisa detection of anti-hMPV-F antibodies

[0254] 8.2.1 Antigen coating

[0255] The cell supernatant containing the hMPV-F protein antigen was diluted with 1x PBS buffer, and 100 μL / well was used to coat the 96-well enzyme-labeled plate, which was then incubated at 4°C for 16 h.

[0256] 8.2.2 Washing and blocking

[0257] ​Take out the enzyme labeled plate coated with antigen, wash the plate with 300 μL / well PBST for 1 time. Then add 100 μL blocking solution (5% skim milk) and incubate at room temperature for 30 min. Then wash the plate with 300 μL / well PBST for 3 times.

[0258] 8.2.3 Sample dilution and sample addition

[0259] After the serum sample of the immunized mouse is diluted by a suitable 4-fold gradient, 100 μL is added to the enzyme labeled plate, three replicates, incubated at room temperature for 1.5 h, and then washed with 300 μL / well PBST for 3 times.

[0260] 8.2.4 Addition of detection antibody

[0261] According to the number of wells to be added, the appropriate amount of detection antibody (goat anti-mouse IgG, peroxidase conjugated, H+L) is added to each well, diluted to 0.8 ng / mL, incubated at room temperature for 1 h, and then washed with 300 μL / well PBST for 5 times.

[0262] 8.2.5 Color development and termination

[0263] Mix color developing solution A (containing H2O2) and color developing solution B (containing TMB) at a ratio of 1:1, add 100 μL to each well, and incubate at room temperature for 15-30 min. Then add 30 μL of 1 M HCl termination solution to each well, and then read at 450 nm using an enzyme labeled instrument.

[0264] The results of the exploration of viral antibody persistence are as follows Figures 9A-9C It is shown that the mRNA / LNP vaccines corresponding to JY-hMPV-081 and JY-hMPV-060 can induce high titers of virus-specific antibodies in animals, and the antibody titers remain high 6 months after immunization. At 9 months, JY-hMPV-081 still maintains the antibody titers, while JY-hMPV-060 starts to decline.

[0265] Example 9 Independent study of immunogenicity of different mRNA / LNP vaccines in mice

[0266] In order to expand the protection range of the vaccine and improve the popularity of the vaccine, the development of multi-link multi-valent vaccine has become a trend. Therefore, two different vaccines are simultaneously immunized in mice to observe the potential of the vaccine series in the development of multi-link vaccine.

[0267] 9.1 Animal immunization

[0268] Balb / c mice, female, 6 weeks old, first adapt for one week after arriving at the animal house. Vaccine intramuscular injection, the dose is as follows in Table 8:

[0269] Table 8 Mouse immunization procedure, dose and time

[0270]

[0271] Group 1, RSV B mRNA / LNP vaccine, JY-RSV-001 (for its preparation method, see Example 4 of patent application CN202410420671.7), 10 μg / mouse; wherein, the sequence of RSV B mRNA is as follows:

[0272]

[0273]

[0274] Group 2, JY-hMPV-081 mRNA / LNP, 20 μg / mouse;

[0275] Group 3, JY-RSV-001 (10 μg / mouse) and JY-hMPV-081 (20 μg / mouse) were mixed and encapsulated before administration;

[0276] Group 4, 1× PBS buffer control; used for later RSV infection;

[0277] The fifth group, 1× PBS buffer control, was used for later hMPV infection.

[0278] The interval between the two vaccinations was 14 days, and blood was collected from the eye sockets on the fourteenth day after the second vaccination.

[0279] 9.2 Elisa for detection of anti-RSV antibodies and anti-hMPV antibodies

[0280] 9.2.1 Antigen Coating

[0281] The antigen (RSV-F (Yiqiao Bio, 11049-V08B) or hMPV-F (obtained by step 6.2.1 of Example 6)) was diluted with 1× PBS buffer and coated in a 96-well ELISA plate with 100 μL per well at 4°C for 16 h.

[0282] 9.2.2 Washing and sealing

[0283] Remove the antigen-coated ELISA plate and wash once with 300 μL / well PBST. Then add 100 μL of blocking solution (containing 5% milk powder) and incubate at room temperature for 30 minutes. Then wash three times with 300 μL / well PBST.

[0284] 9.2.3 Sample dilution and loading

[0285] After the serum samples of the immunized mice were gradiently diluted by 4 times, 100 μL was added to the enzyme-labeled plate, three holes were repeated, and after incubation at room temperature for 1.5 h, the plate was washed with 300 μL / hole PBST for 3 times.

[0286] 9.2.4 Addition of detection antibody

[0287] According to the number of added holes, the appropriate amount of detection antibody (goat anti-mouse IgG, peroxidase conjugated, H+L) was matched and diluted to 0.8 ng / mL, and then 80 μL was added to each hole, and after incubation at room temperature for 1 h, the plate was washed with 300 μL / hole PBST for 5 times.

[0288] 9.2.5 Color development and termination

[0289] Color developing solution A (containing H2O2) and color developing solution B (containing TMB) were mixed according to a ratio of 1:1, 100 μL was added to each hole, and after standing at room temperature for 15-30 min, 30 μL of 1M HCl termination solution was added to each hole, and then the reading was taken at 450 nm by using an enzyme-labeled instrument.

[0290] The results are shown in Figures 10A-10C It can be seen that the performance of JY-hMPV-081 does not change whether it is immunized alone or together with RSV vaccine Figure 10A ; the performance of JY-RSV-001 also does not change whether it is immunized alone or together with hMPV vaccine Figure 10B . The mRNA / LNP vaccine shows good antigen independence, although the virus-specific antibodies induced by RSV are higher than those induced by hMPV in general Figure 10C . The results provide a basis for subsequent development of multi-vaccine.

[0291] Example 10 Detection of the neutralization ability of antibodies to viruses

[0292] One of the golden detection indicators of vaccines is whether the specific anti-virus antibodies induced in the animal body have the ability to neutralize viruses and prevent virus replication and infection. The virus neutralization test is designed for this purpose. Since hMPV belongs to a biosafety level 2 virus, all the following steps must be completed in a biosafety level 2 laboratory according to the corresponding requirements and specifications.

[0293] 10.1 Preparation of cells

[0294] LLC-MK2 cells (ATCC, CCL-7), i.e., monkey kidney epithelial cells, are commonly used hMPV growth cells. On the day before the neutralization experiment, the cells were digested and resuspended and washed, inoculated into a 96-well plate at 1×10^4 / hole, and incubated at 37℃, 5% CO2 in DMEM complete medium (containing 5% FBS) overnight.

[0295] 10.2 Antibody neutralization of virus

[0296] 10.2.1 Serum dilution gradient

[0297] The serum dilution gradient and dilution method commonly used in the laboratory are shown in Table 9 below:

[0298] Table 9 Serum dilution and dilution method

[0299]

[0300] 10.2.2 Serum antibody neutralization of virus

[0301] The amount of hMPV virus used is preferably 30-40 PFU / well, and the dilution medium is DMEM without fetal bovine serum. The volume of the diluted serum is 60 μL, and 60 μL of the virus dilution is added, and mixed gently. A virus control group (i.e., virus solution without diluted serum) is set up. Incubate in a 37°C incubator for 1 h.

[0302] 10.2.3 Inoculation of cells

[0303] Take 35 μL / well of the mixture of the above virus and serum, and add to the 96-well plate with a monolayer of MK2 cells. Inoculate 3 wells of each dilution. At the same time, set up 8 wells of normal cell controls and 8 wells of virus infection controls. Incubate the plate in a 35°C, 5% CO2incubator for 1 h, and gently shake every 15 min. After incubation, discard the mixture in the wells, and add 120 μL / well of pre-prepared warm medium, which is DMEM, 3% FBS, TPCK-treated trypsin, 5 μg / ml. Incubate at 35°C, 5% CO2for 6-8 days.

[0304] 10.3 Color development of results

[0305] 10.3.1 Virus and cell fixation

[0306] On the 6th-8th day of incubation, discard the medium in the wells, and wash once with 1x PBS buffer. Then add 100 μL / well of freshly prepared 1% paraformaldehyde fixing solution, and fix at room temperature for 30 min. Then discard the solution in the wells, and wash once with 1x PBS buffer.

[0307] 10.3.2 Blocking and plate washing

[0308] Add 120 μL / well of blocking solution (5% milk powder), and shake gently on a shaker at room temperature for 30 min. Then wash the plate 3 times with 150 μL / well of PBST.

[0309] 10.3.3 Addition of detection antibody

[0310] The detection antibody is self-made mouse anti-hMPV serum, after appropriate dilution, 100 μL is added to each well, incubated at room temperature for 90 minutes, and then washed with 150 μL / well PBST for 3 times.

[0311] 10.3.4 Addition of secondary antibody

[0312] Take the matching anti-mouse secondary antibody (HRP conjugated), dilute 1:5000, add 100 μL to each well, incubate at room temperature for 60 minutes, and then wash with 150 μL / well PBST for 5 times.

[0313] 10.3.5 Color development and termination

[0314] Add 100 μL of TrueBlue Peroxidase Substrate to each well, shake gently, and virus positive plaques will appear quickly. Then wash the reaction solution with water. Dry in the dark. The next day, count the virus positive wells. TM Peroxidase Substrate, shake gently, and virus positive plaques will appear quickly. Then wash the reaction solution with water. Dry in the dark. The next day, count the virus positive wells.

[0315] From Figure 11 It can be seen that the antibodies induced by JY-hMPV-081 and JY-hMPV-060 vaccines can well neutralize hMPV strains, and the IC 50 range is between 1300 (JY-hMPV-060) and 3500 (JY-hMPV-081) times of serum dilution, which is better than the similar vaccine development carried out at the same period.

[0316] Example 11 Simulation of the real world, immunization of animals that have been infected with hMPV virus

[0317] hMPV virus respiratory tract infection is a very common respiratory tract infection disease. Except for the first infection of newborns within one year, almost everyone is faced with the repeated infection of the virus from the environment. For most people with a perfect immune system, re-infection is not a very serious problem, but for young children whose immune system is still developing, for the elderly whose immune system is declining, and for patients with defective immune systems, hMPV infection is often a serious threat. The design of this experiment is to explore whether the vaccine has a protective effect on animals that have been infected with hMPV, and the degree of protection.

[0318] 11.1 Animal immunization and sampling

[0319] The animals used are Balb / c mice, female, 6 weeks old. After the animals arrive at the CRO biosafety level 2 animal house, they are adapted for one week. The animal infection and vaccination schedule is shown in Table 1. Figure 12AThe experiment was divided into four groups: the first and second groups were given two immunizations with a two-week interval according to the general procedure, each time 20 μg / mouse of JY-hMPV-081 or JY-hMPV-060 was injected intramuscularly; the third group of mice was first infected with hMPV (hMPV 9, type Al) by intranasal infection at a virus dose of 7 x 104 / mouse. Blood was taken for testing at 30 days, and the second day after blood collection, a booster injection of 20 μg / mouse of JY-hMPV-081 was given intramuscularly. Blood was taken for testing again 10 days later; the last group was a PBS control. The challenge virus was the same as the pre-infection virus.

[0320] 11.2 Elisa detection of anti-hMPV antibody titers

[0321] 11.2.1 Antigen coating

[0322] The cell supernatant containing the hMPV-F protein antigen was diluted with 1 x PBS buffer to 100 μL / well of a 96-well enzyme-labeled plate, and the antigen was coated at 4°C for 16 h.

[0323] 11.2.2 Washing and blocking

[0324] The enzyme-labeled plate with coated antigen was removed, and the plate was washed once with 300 μL / well of PBST. Then 100 μL of blocking solution (containing 5% milk powder) was added, and the plate was incubated at room temperature for 30 min. Subsequently, the plate was washed three times with 300 μL / well of PBST.

[0325] 11.2.3 Sample dilution and sample addition

[0326] After the mouse serum sample was diluted by a suitable 4-fold gradient, 100 μL was added to the enzyme-labeled plate, and the plate was incubated at room temperature for 1.5 h. Then the plate was washed three times with 300 μL / well of PBST.

[0327] 11.2.4 Addition of detection antibody

[0328] According to the number of sample wells, the appropriate amount of detection antibody (goat anti-mouse IgG, peroxidase conjugated, H+L) was added to each well at a dilution of 0.8 ng / mL, and the plate was incubated at room temperature for 1 h. Then the plate was washed five times with 300 μL / well of PBST.

[0329] 11.2.5 Color development and termination

[0330] Color developing solution A (containing H2O2) and color developing solution B (containing TMB) were mixed at a ratio of 1:1, 100 μL was added to each well, and the plate was placed at room temperature for 15-30 min. Then 30 μL of 1 M HCl termination solution was added to each well, and the plate was read at 450 nm using an enzyme-labeled instrument.

[0331] The results are shown in Table 1. Figure 12BResults: hMPV infection one month later, antibody levels were significantly higher than those induced by a single dose of immunization in mice; however, when the mice completed the "prime-boost regimen", the antibody titers increased significantly, reaching the level of natural infection; meanwhile, the booster needle let the pre-infection mice's antibodies go up another step. The results of the antibody neutralization test ( Figure 11 ) also reflect that the administration of a booster needle after viral pre-infection can effectively increase the level of neutralizing antibodies. These results fully affirm the role of this vaccine as a booster, that is, in individuals who have been infected with hMPV, if a booster is given, it can greatly stimulate hMPV-specific memory B cells to rapidly initiate division and produce antibodies, thereby playing a protective role.

[0332] Example 12 JY-hMPV-F series mRNA / LNP vaccine inhibits hMPV virus infection in the lungs of mice

[0333] The ultimate test of a vaccine is to test whether the specific immune response induced by the vaccine in vivo has the ability to protect the host from viral infection.

[0334] 12.1 Animal pre-infection, immunization, challenge and sampling

[0335] Balb / c mice, female, 6 weeks old, were first adapted for a week after arriving at the animal house. The vaccines were JY-hMPV-081 and JY-hMPV-060, respectively; the pre-infection was hMPV 9 (type A1). There were 5 mice in each group, the pre-infection was intranasal, the vaccine was intramuscular, and the time and dose were as follows in Table 10 and Figure 13A :

[0336] Table 10 Mouse infection or immunization program, dose and time

[0337]

[0338] The pre-infection and booster needle were separated by 31 days; the two doses of vaccine were separated by 14 days; the challenge was intranasal after isoflurane anesthesia; the fourth day after the challenge, the experiment ended. After the animals were euthanized by inhalation anesthesia, the mice were dissected according to the SOP dissection procedure, and the whole lung was taken and weighed.

[0339] 12.2 Lung virus load detection

[0340] Lung virus load detection can be by plaque method and qPCR method. The latter was used in this study. RNA was extracted from mouse lung homogenate, reverse transcribed into cDNA, and hMPV-F gene was amplified by qPCR to quantify the virus.

[0341] 12.2.1 Lung tissue sample processing

[0342] Prepare the freezing tube, add 2 mL, 4 ℃ pre-cooled virus protection solution; Put the fresh lung tissue into the freezing tube containing the virus protection solution, homogenize on the same day, avoid freeze-thaw effect on the experimental results. Homogenize the lung tissue to no obvious particles using a high-throughput tissue grinder under low temperature, and suck the tissue liquid into a 1.5 mL EP tube, and freeze at -80℃.

[0343] 12.2.2 Extraction of total RNA from samples

[0344] Take 200 μL of lung homogenate for RNA extraction. Add GENEzoI TM reagent, then vortex briefly. Incubate at room temperature for 5 minutes. Centrifuge to remove debris, and transfer the clear supernatant to a new 1.5 mL centrifuge tube.

[0345] Add an equal amount of anhydrous ethanol to the supernatant at a ratio of 1:1. Vortex thoroughly, then centrifuge over an RB column. Repeat this step until the sample is completely bound. Then, wash the RB column once with wash buffer.

[0346] Next, digest the DNA on the RB column with DNase I solution at 25℃ for 15 minutes.

[0347] Subsequently, wash the RB column, add 700 μL of pre-washed buffer, then centrifuge and discard the flow-through. Repeat three times. Finally, centrifuge the RB column at 14000g for 3 minutes to dry the column matrix.

[0348] RNA elution: Place the dry RB column in a clean 1.5 mL centrifuge tube. Add 25-50 μL of RNase-free water to the center of the column matrix. Let stand for at least 3 minutes, then centrifuge at 14000g for 1 minute to obtain the RNA.

[0349] 12.2.3 Reverse transcription

[0350] Prepare the cDNA synthesis reaction mixture according to the requirements of the reverse transcription kit. Each reaction contains 100-500 ng of total RNA, 5 μL of 4xRT Mix, and water to make the total volume 20 μL. After mixing, incubate at 45℃ for 30 minutes. Then, inactivate at 85℃ for 5 minutes to obtain the cDNA.

[0351] 12.2.4 qPCR

[0352] Use qPCR to quantify hMPV virus, and the primer sequence used is from the F gene:

[0353] hMPV-F-Fw: 5'-CATATAAGCATGCTATATTAAAAGAGTCTC-3' (SEQ ID NO: 18)

[0354] hMPV-F-Rv: 5'-CCTATTTCTGCAGCATATTTGTAATCAG-3' (SEQ ID NO: 19)

[0355] Mouse GAPDH internal control primer (Shanghai Biotechnology, item number B662304)

[0356] According to the kit recommendations, the qPCR reaction system is as follows:

[0357] MonAmp TM ChemoHS qPCR Mix]]> 10 pL Forward primer (10 pM) 0.4 pL Reverse primer (10 pM) 0.4 pL Low concentration ROX dye (100x) or high concentration ROX dye (100x) 0.2 pL cDNA template x pL dd H2O Add to 20 pL

[0358] qPCR reaction program:

[0359]

[0360] 12.2.5 Data analysis

[0361] From lung homogenate to qPCR, there are several steps, and the virus content of each lung is calculated as follows:

[0362] - 2 mL of MEM was added to the mouse lung tissue for homogenization, and 200 μL of sample was taken for RNA extraction, which was diluted by 10 times;

[0363] - The total RNA volume was 30 μL, and 2 μL was used for reverse transcription, which was diluted by 15 times;

[0364] - The reverse transcription output was 20 μL of cDNA, and 2 μL was used for qPCR, which was diluted by 10 times;

[0365] Therefore, the total dilution factor is 1500;

[0366] Using the standard curve external standard method, the viral load of each lung was calculated.

[0367] The results are shown in Figure 13B It is found that when the mice receive two doses of 20 μg / each mRNA / LNP vaccine, they can effectively control the amplification of hMPV virus in the lungs, and the lung virus load is reduced by about 100 times, and JY-hMPV-081 is better than JY-hMPV-060. At the same time, it is also found that when the mice are pre-infected with hMPV once, and given a booster vaccine one month later, the effect is better than or similar to that of receiving two doses of JY-hMPV-081 immunization.

[0368] In summary, the above embodiments are only preferred embodiments of the present application and do not limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A hMPV viral fusion protein F variant, characterized in that The hMPV virus fusion protein F variant has amino acid residue differences at one or more sites relative to the hMPV virus fusion protein F with accession number AGJ74096.1 in the GeneBank database, wherein the amino acid sequence of the hMPV virus fusion protein F variant is shown in SEQ ID NO: 1 or 2.

2. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the hMPV viral fusion protein F variant according to claim 1.

3. The nucleic acid molecule according to claim 2, wherein The nucleic acid molecule is double-stranded DNA.

4. The nucleic acid molecule according to claim 2, wherein The nucleotide sequence encoding the hMPV viral fusion protein F variant comprises the sequence shown in SEQ ID NO: 13 or 14.

5. The nucleic acid molecule according to any one of claims 2 to 4, wherein The nucleic acid molecule further comprises one or more selected from the following: (1) T7 promoter; (2) 5'UTR coding sequence; (3) 3'UTR coding sequence; (4) Poly A coding sequence; (5) Kozak coding sequence.

6. The nucleic acid molecule according to claim 5, wherein The nucleotide sequence of the T7 promoter is shown in SEQ ID NO:

3.

7. The nucleic acid molecule according to claim 5, wherein The 5'UTR is the 5'UTR of human cytochrome B-245a polypeptide.

8. The nucleic acid molecule according to claim 7, wherein The coding sequence of the 5'UTR is shown in SEQ ID NO:

5.

9. The nucleic acid molecule according to claim 5, wherein The 3'UTR is derived from the 3'UTR of human AES / TLE5.

10. The nucleic acid molecule according to claim 9, wherein The coding sequence of the 3'UTR is shown in SEQ ID NO:

6.

11. The nucleic acid molecule according to claim 5, wherein The nucleotide sequence of the poly A has a gap, and the coding sequence of the gap is TGCAT or as shown in SEQ ID NO:

20.

12. The nucleic acid molecule according to claim 11, wherein The nucleotide sequence of the poly A is shown in SEQ ID NO: 7 or 8.

13. The nucleic acid molecule according to claim 5, wherein The coding sequence of Kozak is shown in SEQ ID NO:

4.

14. The nucleic acid molecule according to any one of claims 6 to 13, wherein The nucleic acid molecule sequentially comprises, from the 5' end to the 3' end, a 5' UTR coding sequence, a Kozak coding sequence, a nucleotide sequence encoding the hMPV viral fusion protein F variant, and a 3' UTR coding sequence.

15. The nucleic acid molecule according to claim 14, wherein The 5' end of the nucleic acid molecule is connected to the T7 promoter, and the 3' end is connected to the poly A coding sequence.

16. The nucleic acid molecule of claim 15, wherein the T7 promoter is linked to the coding sequence of the 5' UTR via AGG or GGG.

17. A nucleic acid construct, characterized in that The nucleic acid construct contains the nucleic acid molecule according to any one of claims 2 to 16.

18. The nucleic acid construct according to claim 17, wherein The nucleic acid molecule is constructed into the nucleic acid construct by restriction endonucleases.

19. The nucleic acid construct according to claim 18, wherein The restriction endonuclease is selected from Hind III and Sap I.

20. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to any one of claims 2 to 16 or the nucleic acid construct according to any one of claims 17 to 19.

21. A transformant, characterized in that: The transformant contains the nucleic acid molecule according to any one of claims 2 to 16, the nucleic acid construct according to any one of claims 17 to 19, or the recombinant expression vector according to claim 20, or expresses the hMPV virus fusion protein F variant according to claim 1.

22. The transformant according to claim 21, wherein The host cells used in constructing the transformant are selected from Escherichia coli, insect cells, yeast cells and mammalian cells.

23. The transformant according to claim 22, wherein The host cell is an Escherichia coli Stable cell.

24. An mRNA, characterized in that The mRNA comprises the mRNA encoding the hMPV viral fusion protein F variant according to claim 1.

25. The mRNA according to claim 24, wherein The mRNA is mRNA obtained by transcribing the nucleic acid molecule according to any one of claims 2 to 16.

26. The mRNA of claim 25, wherein The sequence of the mRNA encoding the hMPV viral fusion protein F variant comprises the sequence shown in SEQ ID NO: 15 or 16.

27. The mRNA according to claim 26, wherein The sequence of the mRNA is shown in SEQ ID NO: 11 or 12.

28. The mRNA according to claim 27, wherein The 5' end of the mRNA has a cap structure.

29. The mRNA according to claim 28, wherein The cap structure is a Cap1 structure.

30. A method for preparing a nucleic acid molecule, characterized in that: The method comprises culturing the transformant according to any one of claims 21 to 23 to obtain the nucleic acid molecule.

31. The method of claim 30, wherein: The nucleic acid molecule is DNA or mRNA.

32. A drug-loaded lipid nanoparticle, characterized in that: It comprises the mRNA according to any one of claims 24 to 29.

33. The lipid nanoparticle of claim 32, wherein The lipid nanoparticles further comprise a cationic lipid and a helper lipid.

34. The lipid nanoparticle of claim 33, wherein The cationic lipid is SM-102; And / or, the helper lipid is DSPC, cholesterol and / or DMG-PEG-2000.

35. The lipid nanoparticle of claim 34, wherein The lipid nanoparticles include the mRNA, SM-102, DSPC, cholesterol and DMG-PEG-2000.

36. The lipid nanoparticle of claim 35, wherein The molar ratio of the cationic lipid, DSPC, cholesterol and DMG-PEG-2000 is (40-60): (2-15): (30-40): (0.8-1.6); And / or, in the lipid nanoparticles, the N / P ratio is 4-8.

37. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the mRNA according to any one of claims 24 to 29, the lipid nanoparticle according to any one of claims 32 to 36, and an optional pharmaceutically acceptable carrier.

38. Use of the hMPV viral fusion protein F variant according to claim 1, the nucleic acid molecule according to any one of claims 2-16, the nucleic acid construct according to any one of claims 17-19, the recombinant expression vector according to claim 20, the transformant according to any one of claims 21-23, the mRNA according to any one of claims 24-29, the lipid nanoparticles according to any one of claims 32-36 and / or the pharmaceutical composition according to claim 37 in the preparation of drugs for preventing and / or treating diseases caused by hMPV virus infection and / or inhibiting hMPV virus.

39. The use according to claim 38, wherein The disease caused by the hMPV virus infection is a respiratory disease or an ear infection.

40. The use according to claim 39, wherein The respiratory disease is bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease or asthma.

41. A combined vaccine, characterized in that The multi-combination vaccine comprises the mRNA according to any one of claims 24 to 29, the lipid nanoparticles according to any one of claims 32 to 36 and / or the pharmaceutical composition according to claim 37, and mRNA, lipid nanoparticles, mRNA vaccines and / or pharmaceutical compositions that inhibit other viruses.

42. The combination vaccine according to claim 41, wherein The other virus is syncytial virus.

43. The combined vaccine according to claim 42, wherein The syncytial virus is human respiratory syncytial virus.

44. The combination vaccine according to claim 43, wherein The nucleotide sequence of the mRNA that inhibits other viruses is shown in SEQ ID NO:

17.

45. A medicine kit, characterized in that: The kit comprises kit A and kit B, wherein: The kit A contains the mRNA according to any one of claims 24 to 29, the lipid nanoparticles according to any one of claims 32 to 36, and / or the pharmaceutical composition according to claim 37; The kit B contains mRNA, lipid nanoparticles, mRNA vaccines and / or pharmaceutical compositions that inhibit other viruses.

46. ​​The kit of claim 45, wherein: The other virus is syncytial virus.

47. The kit of claim 46, wherein: The syncytial virus is human respiratory syncytial virus.

48. The kit of claim 47, wherein: The nucleotide sequence of the mRNA that inhibits other viruses is shown in SEQ ID NO:

17.

49. A method for inhibiting hMPV virus for non-therapeutic purposes, characterized in that: The method comprises contacting the hMPV virus with the mRNA as described in any one of claims 24-29, the lipid nanoparticles as described in any one of claims 32-36, the pharmaceutical composition as described in any one of claims 37 and / or the multi-vaccine as described in any one of claims 41-44 to induce an immune response to inhibit the hMPV virus, or using the set of kits as described in any one of claims 45-48 to induce an immune response to inhibit the hMPV virus.

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