HMPV virus fusion protein F variant, mRNA vaccine and application thereof

Abstract

The invention discloses an hMPV virus fusion protein F variant, an mRNA vaccine and application of the hMPV virus fusion protein F variant and the mRNA vaccine. The hMPV fusion protein F variant has amino acid residue difference on one or more sites relative to an hMPV fusion protein F with the login number of AGJ74096.1 in a GeneBank database, wherein the amino acid sequence of the hMPV fusion protein F variant is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The mRNA vaccine designed based on the hMPV fusion protein F variant can induce a strong specific antibody reaction, the durability of the antibody in vivo is strong, the induced antibody can well neutralize an hMPV strain, the amplification of the hMPV in the lung is controlled, and the immune response of the hMPV in the lung is inhibited. Therefore, the specific antibody induced by the mRNA vaccine provides an effective basis for preventing hMPV virus infection related diseases and developing multi-combined vaccines.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an hMPV virus fusion protein F variant, an mRNA vaccine and applications thereof. Background Art

[0002] Human metapneumovirus (hMPV) is an enveloped, negative-stranded RNA single-stranded virus belonging to the Pneumoviridae family and the genus Metapneumovirus. In 2001, Dutch scientist Bernadette G. van den Hoogen and her colleagues first detected hMPV using RAP-PCR (RNA arbitrarily primed PCR) in nasopharyngeal aspirates from children with respiratory infections caused by unknown pathogens. Serological studies have shown that hMPV has been circulating in the human population for at least 70 years and is distributed throughout the world. It is one of the common respiratory pathogens. It is genetically closely related to human respiratory syncytial virus (hRSV) and is a common cause of acute respiratory disease after RSV. Its clinical features and severity are also similar to RSV, and mixed infections with other respiratory viruses are common. 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 by the age of five, almost all children worldwide have been exposed to the virus; reinfection occurs throughout the lifespan. hMPV infection is detected throughout the year, with the highest rates in winter and spring, and the epidemic season is usually after RSV and influenza viruses.

[0003] hMPV is closely related to avian metapneumovirus subtype C, exhibits paramyxovirus-like morphology, has a size range of 150 to 600 nm, and a genome length of approximately 13,300 nucleotides, consisting of eight genes encoding nine proteins: 3'-NPMF-M2-SH-GL-5', including nucleoprotein N, phosphoprotein P, matrix protein M, fusion protein F, matrix 2 proteins M2-1 and M2-2, small hydrophobic protein 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 is also different from that of RSV. The interior of the virus particle is composed of nucleoprotein N, tetrameric phosphoprotein P, and large polymerase protein L combined with viral RNA, which is surrounded by matrix protein M and is wrapped in a lipid bilayer, in which fusion protein F, glycoprotein G, and small hydrophobic protein SH are distributed. Based on the response of F and G proteins to monoclonal antibodies, hMPV viruses are divided into two antigenic subtypes: A and B, which include A1, A2, A3 and B1, B2, respectively. The two subtypes circulate simultaneously or alternately during local epidemics. Compared with A, subtype B tends to cause increased cough. But so far, no strain seems to be more epidemiologically dominant than other strains, nor does any strain cause different pathological changes.

[0004] The hMPV-F protein sequence is highly conserved between strains and exists on the virus surface 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 to constitute 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 bring the viral membrane and cell membrane closer together. 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 body of vaccine development.

[0005] Since the discovery of hMPV, the research and development of vaccines has never stopped, and attempts have been made to develop inactivated vaccines, low-temperature passaging vaccines, attenuated vaccines, viral vector vaccines, subunit vaccines, virus-like particle vaccines, etc. However, vaccine development efforts based on monomeric wild-type hMPV-F have proven to be unsuccessful, and there is currently no vaccine or specific drug. Summary of the invention

[0006] In order to solve the technical problems existing in the prior art, the present invention provides an hMPV virus fusion protein F variant, an mRNA vaccine and applications thereof.

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

[0008] The first aspect of the present invention provides a hMPV virus fusion protein F variant, which has differences in amino acid residues 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 as shown in SEQ ID NO:1 or 2, preferably as shown in SEQ ID NO:1.

[0009] The second aspect of the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the hMPV viral fusion protein F variant as described in the first aspect of the present invention.

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

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

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

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

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

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

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

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

[0018] In some embodiments of the present invention, the nucleic acid molecule 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.

[0019] In some embodiments of the present invention, 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; preferably, the T7 promoter and the 5'UTR coding sequence are connected via AGG or GGG.

[0020] The third aspect of the present invention provides a nucleic acid construct, wherein the nucleic acid construct contains the nucleic acid molecule as described in the second aspect of the present invention.

[0021] In some embodiments of the invention, 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 invention, the nucleic acid construct is an expression cassette, which, in addition to the nucleic acid molecule, may optionally further include regulatory elements such as a terminator and an enhancer.

[0023] The fourth aspect of the present invention provides a recombinant expression vector, which contains the nucleic acid molecule as described in the second aspect of the present invention or the nucleic acid construct as described in the third aspect of the present invention.

[0024] The fifth aspect of the present invention provides a transformant, which contains the nucleic acid molecule as described in the second aspect of the present invention, the nucleic acid construct as described in the third aspect of the present invention, or the recombinant expression vector as described in the fourth aspect of the present invention, or expresses the hMPV virus fusion protein F variant as described in the first aspect of the present invention.

[0025] In some embodiments of the present invention, the host cell used in constructing the transformant is selected from Escherichia coli, insect cells, yeast cells and mammalian cells, such as Escherichia coli Stable cells.

[0026] The sixth aspect of the present invention provides an mRNA, wherein the mRNA comprises an mRNA encoding the hMPV virus fusion protein F variant as described in the first aspect of the present invention.

[0027] In some embodiments of the present invention, the mRNA is mRNA obtained by transcribing the nucleic acid molecule as described in the second aspect of the present invention.

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

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

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

[0031] The seventh aspect of the present invention provides a method for preparing a nucleic acid molecule, the method comprising culturing the transformant as described in the fifth aspect of the present invention to obtain the nucleic acid molecule.

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

[0033] The eighth aspect of the present invention provides a drug-loaded lipid nanoparticle, wherein the lipid nanoparticle comprises the mRNA as described in the sixth aspect of the present invention.

[0034] In some embodiments of the present invention, the lipid nanoparticles further comprise a cationic lipid and a helper lipid.

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

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

[0037] In some embodiments of the present invention, the lipid nanoparticles include the mRNA, SM-102, DSPC, cholesterol and DMG-PEG-2000.

[0038] In some embodiments of the present invention, 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 invention, in the lipid nanoparticles, the N / P ratio is 4 to 8, for example 6. In the present invention, "N / P" refers to the molar ratio between the nitrogen atom (N) in the cationic lipid and the phosphate group (P) in the nucleic acid.

[0040] In the present invention, the lipid nanoparticles can be administered as a vaccine.

[0041] The ninth aspect of the present invention provides a pharmaceutical composition, which comprises the mRNA as described in the sixth aspect of the present invention and / or the lipid nanoparticles as described in the eighth aspect of the present invention, and an optional pharmaceutical carrier.

[0042] The tenth aspect of the present invention provides the use of the hMPV viral fusion protein F variant as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the nucleic acid construct as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the mRNA as described in the sixth aspect of the present invention, the lipid nanoparticles as described in the eighth aspect of the present invention and / or the pharmaceutical composition as described in the ninth aspect of the present invention in the preparation of drugs for preventing and / or treating diseases caused by hMPV virus infection and / or inhibiting hMPV virus.

[0043] In some embodiments of the present invention, the disease caused by hMPV virus infection is a respiratory disease or ear infection, and the respiratory disease is, for example, bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease or asthma.

[0044] The eleventh aspect of the present invention provides a multi-vaccine, which comprises the mRNA as described in the sixth aspect of the present invention, the lipid nanoparticles as described in the eighth aspect of the present invention and / or the pharmaceutical composition as described in the ninth aspect of the present invention, and mRNA, lipid nanoparticles, mRNA vaccines and / or pharmaceutical compositions that inhibit other viruses; the other viruses are, for example, syncytial viruses.

[0045] In some embodiments of the invention, the syncytial virus is human respiratory syncytial virus (eg, human respiratory syncytial virus type B).

[0046] In some embodiments of the present invention, the nucleotide sequence that inhibits the mRNA of other viruses is shown as SEQ ID NO:17.

[0047] In some embodiments of the present invention, the multi-vaccine includes the mRNA as described in the sixth aspect of the present invention or the human respiratory syncytial virus type B with a nucleotide sequence as shown in SEQ ID NO: 17. The two can be separately encapsulated in lipid nanoparticles and then mixed to prepare the multi-vaccine, or the two can be co-encapsulated in one lipid nanoparticle to prepare the multi-vaccine.

[0048] The twelfth aspect of the present invention provides a set of medicine kits, the set of medicine kits comprising medicine kit A and medicine kit B, wherein:

[0049] The medicine kit A contains the mRNA as described in the sixth aspect of the present invention, the lipid nanoparticles as described in the eighth aspect of the present invention, and / or the pharmaceutical composition as described in the ninth aspect of the present invention;

[0050] The medicine kit B contains mRNA, lipid nanoparticles, mRNA vaccines and / or pharmaceutical compositions that inhibit other viruses; the other viruses are, for example, syncytial viruses.

[0051] In some embodiments of the invention, the syncytial virus is human respiratory syncytial virus (eg, human respiratory syncytial virus type B).

[0052] In some embodiments of the present invention, the nucleotide sequence that inhibits the mRNA of other viruses is shown as SEQ ID NO:17.

[0053] In the present invention, the set of medicine kits can be used as a premixed state of the above-mentioned multi-vaccine, that is, the reagents in medicine kit A and medicine kit B are mixed before administration to obtain the multi-vaccine; the reagents in medicine kit A and medicine kit B can also be administered separately in sequence; it can be adjusted according to actual conditions.

[0054] The thirteenth aspect of the present invention provides a method for inhibiting hMPV virus, the method comprising contacting hMPV virus with mRNA as described in the sixth aspect of the present invention, lipid nanoparticles as described in the eighth aspect of the present invention, pharmaceutical compositions as described in the ninth aspect of the present invention, and / or multi-combination vaccines as described in the eleventh aspect of the present invention, to induce an immune response to inhibit the hMPV virus, or using the set of medicines as described in the twelfth aspect of the present invention, the induced immune response inhibits the hMPV virus. In the present invention, "inhibiting hMPV virus" refers to reducing or preventing infection or replication of hMPV virus.

[0055] In some embodiments of the present invention, the method is for non-therapeutic purposes. In the present invention, the "non-therapeutic purpose" refers to studying hMPV virus in the laboratory, for example, when studying the effect of certain reagents on hMPV virus, the above product is used as a positive control group.

[0056] The fourteenth aspect of the present invention provides a method for preventing and / or treating diseases caused by hMPV virus infection, the method comprising administering an effective dose of the mRNA as described in the sixth aspect of the present invention, the lipid nanoparticles as described in the eighth aspect of the present invention, the pharmaceutical composition as described in the ninth aspect of the present invention and / or the multi-vaccine as described in the eleventh aspect of the present invention to a patient in need, or using the set of medicines as described in the twelfth aspect of the present invention for a patient in need.

[0057] In the present invention, "effective dose" refers to the amount of mRNA effective to produce the desired pharmacological, preventive or therapeutic results.

[0058] In the present invention, "prevention" means that the patient / subject in need has never been infected or is not currently infected with the hMPV virus, and after administering the above-mentioned product, will not be infected when exposed to the hMPV virus, or even if infected, the symptoms will be milder or the symptoms will subside faster than those of the subject who has not administered the above-mentioned product.

[0059] In some embodiments of the present invention, the disease caused by hMPV virus infection is a respiratory disease or ear infection, and the respiratory disease is, for example, bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease or asthma.

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

[0061] In some embodiments of the present invention, the single administration dose of the lipid nanoparticles may be 1-20 μg, 20-60 μg, 60-110 μg, 110-160 μg or 160-200 μg, for example, 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 invention, the number of administrations is at least 1 time, for example 1 time, 2 times, 3 times or 4 times, and can be adjusted according to actual conditions.

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

[0064] The fifteenth aspect of the present invention provides the hMPV virus fusion protein F variant as described in the first aspect of the present invention, the nucleic acid molecule as described in the second aspect of the present invention, the nucleic acid construct as described in the third aspect of the present invention, the recombinant expression vector as described in the fourth aspect of the present invention, the transformant as described in the fifth aspect of the present invention, the mRNA as described in the sixth aspect of the present invention, the liposome nanoparticles as described in the eighth aspect of the present invention, the pharmaceutical composition as described in the ninth aspect of the present invention, the multi-vaccine as described in the eleventh aspect of the present invention and / or the set of medicines as described in the twelfth aspect of the present invention, for preventing and / or treating diseases caused by hMPV virus infection and / or inhibiting hMPV virus.

[0065] In some embodiments of the present invention, the disease caused by hMPV virus infection is a respiratory disease or ear infection, and the respiratory disease is, for example, bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease or asthma.

[0066] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0067] The reagents and raw materials used in the present invention are all commercially available or homemade in compliance with regulations and can be prepared repeatedly.

[0068] The positive and progressive effects of the present invention are:

[0069] The mRNA vaccine designed based on the hMPV virus fusion protein F variant of the present invention can induce a strong specific antibody response, and the antibodies are highly persistent in the body. The induced antibodies can better neutralize the hMPV virus strain and control the proliferation of the hMPV virus in the lungs. Therefore, the specific antibodies induced by the vaccine provide an effective basis for preventing hMPV virus infection-related diseases and the development of multivalent vaccines. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0073] Figure 4 Agarose gel electrophoresis of restriction enzyme digestion of JY-hMPV-F series plasmids is shown.

[0074] Figure 5 Agarose gel electrophoresis showed that the JY-hMPV-F series T7 RNA polymerase-driven mRNA synthesis in vitro.

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

[0076] Figure 7 It was shown that the JY-hMPV-F series mRNA / LNP vaccines induced strong specific binding antibodies in mice.

[0077] Figure 8A-8C The immunization program and immune dose effect of the JY-hMPV-F series mRNA / LNP vaccines in mice were shown. Fig. 8A The immune program effect corresponding to JY-hMPV-081 is shown. Figure 8B The corresponding immune program effects of JY-hMPV-060 are shown; Figure 8C The immune dose effect of the mRNA / LNP vaccine corresponding to JY-hMPV-81 in mice is shown.

[0078] Fig. 9A It was shown that the specific binding antibodies induced by JY-hMPV-F mRNA / LNP vaccine in mice were persistent. Fig. 9B Shown separately Fig. 9A The effect of JY-hMPV-081mRNA / LNP vaccine; Fig. 9C Shown separately Fig. 9A The effect of JY-hMPV-060mRNA / LNP vaccine.

[0079] Figures 10A-10C The results showed that the antibodies induced by JY-hMPV-F mRNA / LNP vaccine in mice showed antigen-independence characteristics. Fig. 10A Results for binding to hMPV-F antigen are shown; Fig. 10B Results for binding to RSV-F antigen are shown; Fig. 10C for Fig. 10A and Fig. 10B The merged graph of .

[0080] Fig.11 This is the result of the neutralization test, which shows that the antibodies induced by the JY-hMPV-F mRNA / LNP vaccine in mice have the ability to neutralize the hMPV virus.

[0081] Figures 12A-12B It showed that JY-hMPV mRNA / LNP vaccine as a booster vaccine could elicit higher anti-hMPV-F antibodies. Fig. 12A the procedures for immunizing and sampling animals; Fig. 12B This is the result of Elisa experiment.

[0082] Figures 13A-13B It showed that the JY-hMPV-F mRNA / LNP vaccine can significantly reduce the infection of hMPV in mouse lungs. Fig.13A Experimental flow chart for animal pre-infection, immunization and sampling; Fig. 13B The results of qPCR detection of mouse lung viral load. DETAILED DESCRIPTION

[0083] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

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

[0085] 1.1 Initial virus strain screening

[0086] According to traditional serological classification, hMPV is divided into subtype A and subtype B, and their amino acid homology is 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 the severity of the disease. Therefore, the design of the present invention takes both into account, but mainly focuses on hMPV A. After analyzing hundreds of complete hMPV F gene sequences, a strain discovered in Australia in 2003 was selected: HMPV / AUS / 150229278 / 2003 / A, GenBank: KC562226.1 (2003), from which JY-hMPV-081 (A) and JY-hMPV-060 (A) were transformed, and the specific information is shown in Table 1 below.

[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] JY-hMPV-060:

[0092]

[0093] 1.2 Sequence Optimization:

[0094] mRNA sequence design is one of the most core and critical issues in the entire mRNA pharmaceutical process. It is closely related to the drugability of mRNA. It affects the yield of mRNA in vitro transcription, the immunogenicity of mRNA molecules, the translation efficiency and the stability of mRNA molecules. Currently, the common mRNA is composed of five elements: 5'Cap (cap structure), 5'UTR (5' non-coding region), ORF (open reading frame encoding protein), 3'UTR (3' non-coding region) and 3'poly A tail (polyadenylic acid tail).

[0095] The 5′Cap structure can protect mRNA from being degraded by the cytoplasmic exonuclease Xrn1 and the nuclear Xrn2 enzyme. Capped mRNA has a longer half-life and higher stability than uncapped mRNA, which significantly increases the yield of intracellular protein synthesis. The chemical nature of the cap structure is a special structure located at the 5' end of mRNA formed by modification during mRNA transcription, namely the m7GPPPN structure, also known as the methylguanosine cap. It is formed under the catalytic action 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 will be recognized by the natural immune receptor RIG-1, and Cap 1 and Cap 2 can protect themselves from being recognized by the innate immune sensor, the present invention selects the Cap1 structure.

[0096] The T7 promoter sequence can be divided into two domains: the binding domain and the 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 coding sequence corresponding to Kozak is GCCGCCACC (SEQ ID NO: 4).

[0097] The regulation of eukaryotic mRNA translation initiation is mainly determined by the characteristics of the 5'UTR, including secondary structure, sequence elements and 5'UTR length, which will affect the stability of mRNA. The human cytochrome B-245a polypeptide (CYBA) UTR with relatively high protein expression was used, and its coding sequence is AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC (SEQ ID NO: 5). Of course, the performance of the UTR also depends on the cell type.

[0098] For the DNA sequence of hMPV-F, choosing the right codons can optimize the overall translation efficiency of mRNA. The first is to get closer to the host cell as a whole, 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 retaining a small number of viral codons slows down the speed of ribosome advancement and provides enough time for the correct folding of proteins, thereby more accurately restoring the antigenic protein structure of the virus. Another point to note is the GC content. A slightly higher GC content is believed to increase the stability of mRNA and increase protein expression in the body. 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 and help improve the stability of mRNA.

[0099] Similar to the 5′UTR, the 3′UTR also contains many regulatory elements, which play an important role in the stability, subcellular localization and translation efficiency of mRNA. The 3′UTR used is from human AES / TLE5, whose coding sequence is CTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACC (SEQ ID NO: 6).

[0100] The poly A tail plays an indispensable role in the protein translation process. It binds to the poly A tail binding protein (PAPB), which then interacts with the translation initiation factor eIF4G to form a "closed loop" structure, recruiting the 40S translation initiation complex to the mRNA, and synergizing with the 5' end cap structure to stimulate translation initiation. During bacterial amplification, plasmids carrying long poly A nucleotide sequences will produce unpredictable recombination events. The poly A tail on the plasmid shortens as the bacteria continue to amplify, causing trouble for plasmid cloning and amplification. Studies have shown that spacing out poly A sequences can significantly reduce recombination events during plasmid DNA amplification, maintain tail length, and not affect the translation efficiency and half-life of mRNA generated by in vitro transcription. Following this principle, the poly A structure adopted is 60-spacer-60 bases, or 40-spacer-80 bases, and the corresponding coding sequence is AGATCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATGCATAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAATTCGAAGTGACT (SEQ ID NO: 7) or TAGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCATATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 8).

[0101] The optimized JY-hMPV-F series coding sequence is as follows Figure 2A-2B , Figure 3A-3B As shown, Figure 2A Shown is the DNA coding sequence of JY-hMPV-081 (SEQ ID NO: 9). Figure 2B The mRNA sequence after transcription is shown (SEQ ID NO: 11); Figure 3A Shown is the DNA coding sequence of JY-hMPV-060 (SEQ ID NO: 10), Figure 3B Shown is the mRNA sequence after transcription (SEQ ID NO: 12).

[0102] The optimized sequences of the JY-hMPV-F series are as follows:

[0103] 1. JY-hMPV-081

[0104] ORF DNA sequence:

[0105]

[0106] ORF mRNA sequence:

[0107]

[0108]

[0109] Full-length mRNA sequence:

[0110]

[0111]

[0112] 2. JY-hMPV-060

[0113] ORF DNA sequence:

[0114]

[0115]

[0116] ORF mRNA sequence:

[0117] Full-length mRNA sequence:

[0118]

[0119] Example 2 Plasmid Synthesis and DNA Extraction of JY-hMPV-F Series mRNA Vaccines

[0120] All designed sequences were cloned into the vector via HindIII and SapI. The specific experimental steps are as follows:

[0121] 2.1 Plasmid synthesis

[0122] After the plasmid sequence design is completed, it will be synthesized by CRO company.

[0123] 2.2 Plasmid DNA extraction

[0124] 2.2.1 Reagent preparation

[0125] The plate and liquid LB culture medium contain peptone, sodium chloride, yeast extract and corresponding antibiotics. The specific formulas are shown in Tables 2 and 3 below.

[0126] Table 2 LB liquid culture medium (500 mL)

[0127] Sodium chloride 5g Peptone 5g Yeast Extract 2.5g

[0128] Table 3 LB plate (500 mL)

[0129] Sodium chloride 5g Peptone 5g Yeast Extract 2.5g Agar powder 7.5g

[0130] 2.2.2 Plasmid transformation

[0131] Plasmid DNA was added to Stable competent cells and placed in an ice box for 30 minutes; heat-shocked in a 42°C water bath for 45 seconds, quickly placed in an ice box for 2 minutes, 500 μL of LB culture medium without antibiotics was added, and cultured in a shaking incubator at 37°C for 1 hour, then plated and cultured at 37°C overnight. The next day, clones were picked from the plate.

[0132] 2.2.3 Plasmid DNA extraction

[0133] The overnight cultured bacterial solution is extracted using a small extraction or large extraction kit.

[0134] 2.3 Plasmid DNA restriction enzyme digestion verification

[0135] Restriction enzymes Hind III and EcoR I were used for digestion verification ( Figure 4 ). Qualified plasmids enter the next step of in vitro transcription experiment.

[0136] Example 3 In vitro transcription and capping of JY-hMPV-F series

[0137] 3.1 Plasmid DNA linearization

[0138] This design uses a reverse Sap I at the 3' end of poly A to linearize the plasmid (e.g. Figure 2A and Figure 3A After 3 hours of reaction at 37°C, SDS-PAGE gel electrophoresis was used to determine whether the enzyme cleavage was complete. The DNA was purified by 3M sodium acetate precipitation method, and then the DNA concentration was determined by Nanodrop method.

[0139] 3.2 In vitro transcription capping

[0140] The design of the JY-hMPV-F series plasmids all used agg, which is suitable for the co-transcriptional capping method.

[0141] Add the components in order according to Table 4 below and react at 37°C for 3 hours.

[0142] Table 4 Components of co-transcription and capping systems

[0143]

[0144]

[0145] After the reaction is complete, add 5 μL of DNase I to digest the template for every 100 μL, and react at 37°C for 30 min. Agarose gel was used to determine whether the DNA template was completely digested.

[0146] 3.3 Purification

[0147] Add 150 μL of lithium chloride solution (containing 7.5 M lithium chloride and 50 mM EDTA) and 150 μL of DEPC water to every 100 μL of the above reaction volume. The final concentration of lithium chloride should be maintained at 2.5-2.8 M. After mixing, place it at -20°C for at least 30 minutes, or overnight.

[0148] Take out the sample from -20℃, centrifuge it at 12000rpm for 15min, remove the supernatant and collect the precipitate. Then add 1mL of 70% aqueous ethanol to wash, centrifuge it at 12000rpm for 5min, and discard the supernatant; repeat once. Open the lid in the clean bench and place it at room temperature for 5min to allow the ethanol to evaporate.

[0149] Add an appropriate amount of DEPC water to re-dissolve the mRNA, and use agarose gel electrophoresis to determine the quality of the mRNA and the concentration.

[0150] The results of gel detection of in vitro mRNA synthesis promoted by T7 RNA polymerase in JY-hMPV-F series are shown in Figure 5 .

[0151] Example 4 Preparation of JY-hMPV-F Series mRNA / LNP Nanoparticles

[0152] This study uses the commonly used method for preparing nanoparticles LNP in the industry. The specific steps are as follows:

[0153] LNP was first prepared from a mother solution dissolved in ethanol in a proportion of ionizable lipid (SM-102): DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine): cholesterol (cholesterol): DMG-PEG-2000 in 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 increase to form lipid nanoparticles (mRNA / LNP) in which mRNA was encapsulated. 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.

[0154] 4.1. Buffer preparation

[0155] All the following reagents were sterilized by autoclave at 121°C for 20 min and prepared with DEPC water:

[0156] 1) 50 mM acetate buffer, pH 4.0;

[0157] 2) 25 mM Tris-HCl buffer, pH 7.5;

[0158] 3) 0.6 g / mL sucrose solution.

[0159] 4.2. Refer to Table 5 and Table 6 below to prepare lipid solution and mRNA solution

[0160] Table 5 Preparation of lipid solution

[0161]

[0162] 1) Before weighing lipids, they need to be restored to room temperature;

[0163] 2) All ingredients are dissolved in anhydrous ethanol, and an ultrasonic device is used to ensure complete dissolution;

[0164] 3) The prepared lipid solutions were mixed in a ratio of 1:1:1:1 to obtain a 12 mg / mL lipid mixed solution that met the formula.

[0165] Table 6 Preparation of mRNA solution

[0166]

[0167] According to the calculation method and buffer type shown in the above table, the in vitro transcribed and capped mRNA stock solution prepared in Example 3 was diluted to the required concentration of 0.2 mg / mL.

[0168] 4.3 Preparation of mRNA / LNP (taking a total volume of 4 mL as an example)

[0169] 4.3.1 INano LTM, the rapid nanomedicine preparation system of Maianna. Set the parameters as needed.

[0170] 4.3.2 Rinse the chip before use

[0171] Prepare three 5mL syringes, one for absolute ethanol, one for acetate buffer, and one for air. On the left, 5mL of acetate buffer → air 3 times; on the right, 5mL of ethanol → 5mL of ethanol → air 3 times, and clean the chip by hand. After cleaning, install the chip into the card slot.

[0172] 4.3.3 Microfluidic device operation to prepare LNPs

[0173] Use a 3mL syringe to draw 3mL of the above-configured mRNA solution, remove the bubbles, and load it into the syringe slot on the left side of the machine. Use a 1mL syringe to draw 1mL of the above-configured lipid mixture solution, remove the bubbles, and load it into the syringe slot on the right side of the machine. Insert a 15mL centrifuge tube into the waste liquid collection area and sample collection area of ​​the machine for waste liquid and sample collection. After confirming that the parameters are correct, click "Start", and the Myanna Rapid Nanodrug Preparation System INano LTM will start immediately, that is, prepare LNP. After the preparation is completed, take out the centrifuge tube at the sample collection area.

[0174] 4.3.4 Processing of collected mRNA / LNP samples

[0175] The collected mRNA / LNP suspension was immediately diluted with 25mM Tris-HCl pH7.5 buffer to about 25 times the volume of mRNA / LNP, and then ultrafiltration centrifuged at 3000rpm, 4°C, 1h. This process was repeated until the solution volume was less than or equal to the volume before dilution (3.6mL, i.e., the total volume of 4mL minus 0.4mL of waste liquid). Then, it was sterilized by a 0.22μm filter membrane, and 0.6g / mL sucrose solution of 0.2 times the volume of the solution was added to finally obtain the mRNA / LNP preparation product.

[0176] 4.3.5 Detection of mRNA / LNP physical and chemical properties

[0177] The prepared mRNA / LNP should be tested for physical properties as soon as possible to determine the quality of the product and facilitate the next step of biological activity research. The test uses a Malvern Zetasizer to obtain particle size, PDI, and ZP data (Table 7 below).

[0178] Encapsulation efficiency analysis:

[0179] Use 1×TE buffer and 2% Triton TE buffer to dilute the mRNA control (100μg / ml) from 2000ng / mL to 6 standard curve points (2000, 1600, 1200, 800, 400, 200ng / mL). When measuring the total RNA content, use 2% Triton TE buffer to dilute the test LNP 50 times, and when measuring the free RNA content, use 1×TE buffer to dilute the test sample 20 times. Transfer 100μL of the above standard curve solution and the test solution to a black ELISA plate, then add 100μL 0.005×Ribogreen (200-fold dilution) respectively, react at room temperature in the dark for 2-5min, read the fluorescence response value with the ELISA plate (excitation wavelength 480nm, emission wavelength 520nm), and calculate the encapsulation efficiency according to the following formula:

[0180] Encapsulation efficiency EE (%) = [(C total RNA - C free RNA) / C total RNA] * 100%

[0181] The effective concentration and encapsulation efficiency of the sample mRNA obtained according to the above method are shown in Table 7.

[0182] Table 7 Physical properties of JY-hMPV series mRNA / LNP lipid particles

[0183] Sample name Particle size PDI Zeta potential Effective concentration μg / mL Encapsulation rate% JY-hMPV-060 84.18 0.051 -5.709 207 96.6 JY-hMPV-081 85.46 0.058 -3.432 201 96.4

[0184] Example 5 JY-hMPV-F series mRNA / LNP nanoparticles transfected into 293T cells for expression

[0185] To ensure that the mRNA in the lipid nanoparticles can express the correct protein, the first step is to transfect the specific mRNA / LNP into appropriate cells. Then, Elisa, Western Blot, flow cytometry and other methods can be used to detect the correctness of protein expression.

[0186] 5.1 Cell transfection

[0187] One day before transfection, 293T (CRL-3216, ATCC) cells were plated at 2×10^5 / well in a 24-well plate. The complete culture medium was DMEM plus 10% fetal bovine serum and double antibody. The culture conditions were 37°C, 5% CO 2On the second day, the cell culture medium in each well was aspirated and gently rinsed with 1× PBS buffer; 500 μL of complete culture medium was added to each well, and then a gradient of hMPV F mRNA / LNP nanoparticles containing 0.5 to 3 μg RNA / well was added to the cells. After gently shaking to disperse the nanoparticles, the cells were placed at 37°C and 5% CO 2 Incubate for 48-72 hours.

[0188] 5.2 Western Blot Detection of Antigen Protein Expression

[0189] By WB method, it is possible to qualitatively detect whether mRNA / LNP is translated into the designed antigen target protein after entering the cell. Since the designed antigen protein has a signal peptide, the extracellular protein is measured here.

[0190] 5.2.1 Electrophoresis sample preparation

[0191] Collect the cell supernatant 48-72 hours after transfection, centrifuge at 2500rpm, 5min, 4℃, and take the supernatant into a new 1.5mL tube. If the expression level is too low, choose a Millipore ultrafiltration tube of appropriate specifications to do moderate concentration first. Then determine the protein content, adjust each sample to the same loading volume, and add 6×SDS loading buffer to a final concentration of 1×. Heat the sample at 92℃ for 5min to denature the protein before loading.

[0192] 5.2.2 SDS-PAGE electrophoresis

[0193] The electrophoresis used 10% separation gel and 4% concentration gel, and the sample was immediately loaded after heating. During electrophoresis, the voltage of the concentration gel section was 110V, and the voltage of the separation gel section was 150V.

[0194] 5.2.3 Transfer

[0195] Depending on the molecular weight, choose nitrocellulose membrane or PVDF membrane. Generally, use 80V for transfer for 1 hour, or 60V for transfer for 2 hours, and use ice packs to control the heat. After transfer, use 1× Ponceau red dye solution for 5 minutes to check the transfer effect.

[0196] 5.2.4 Blockade and immune response

[0197] First, the membrane was blocked with 5% skim milk powder overnight at 4°C. The next day, after washing 3 times with PBST solution, an appropriately diluted primary antibody (mouse anti-hMPV-F) was added for hybridization and shaken at room temperature for 90 minutes. Then, the membrane was washed 3 times with PBST solution on a shaker for 5 to 10 minutes each time. The secondary antibody was anti-mouse-HRP and shaken at room temperature for 60 minutes. Then, the membrane was washed 3 times with PBST solution on a shaker for 5 to 10 minutes each time.

[0198] 5.2.5 Development and Exposure

[0199] According to the instructions of the ECL kit, test solution A and test solution B were mixed in a volume ratio of 1:1 and placed in the membrane. The reaction was properly shaken in a dark room, and then exposed to the chemiluminescence image analyzer for detection. The correctness of the target protein size was determined based on the marker band.

[0200] The results are as follows Figure 6 The results showed that after JY-hMPV-F series transfected 293T cells, mRNA / LNP was internalized into cells, released into the cytoplasm under the acidic environment of lysosomes, completed protein translation and modification in the endoplasmic reticulum and Golgi apparatus, and released into the extracellular space under the guidance of signal peptides. The encoded hMPV-F antigen protein can bind to F antibody, and the protein specificity and protein molecular weight also meet the design requirements.

[0201] Example 6 Immunization of mice with JY-hMPV-F series mRNA / LNP vaccines and expression and identification of virus-specific antibodies

[0202] In order to detect whether the vaccine has the expression and manifestation of specific antigens in the body, mice were used as an animal model for testing.

[0203] 6.1 Animal immunization

[0204] Balb / c mice, female, 6 weeks old, were acclimated for one week after arriving at the animal house. The vaccine was the JY-hMPV series mRNA / LNP vaccine prepared in Example 4. The intramuscular injection was 20 μg / mouse, divided into three injections with an interval of 14 days. Blood was collected 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.

[0205] 6.2 Elisa detection of anti-hMPV antibodies

[0206] One of the key indicators of vaccines is to induce specific antiviral antibodies in animals. Elisa method was used to detect the expression of anti-hMPV-F antibodies.

[0207] 6.2.1 Antigen Coating

[0208] Due to the lack of commercial anti-hMPV-F antigen, the supernatant of 293T cells transfected with hMPV-F plasmid was diluted and coated in a 96-well ELISA plate, 100 μL per well, at 4°C for 16 h.

[0209] 6.2.2 Washing and blocking

[0210] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% milk powder) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0211] 6.2.3 Sample dilution and loading

[0212] After the serum samples of immunized mice were appropriately diluted in a 4-fold decreasing gradient, 100 μL was added to the ELISA plate in triplicate, incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0213] 6.2.4 Add detection antibodies

[0214] According to the number of wells to be loaded with samples, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, peroxidase-coupled, H+L), dilute to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 h, and wash the plate 5 times with 300 μL / well PBST.

[0215] 6.2.5 Color development and termination

[0216] Mix the colorimetric solution A (containing H 2 O 2 ) and color development solution B (containing TMB), add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL of 1M HCl stop solution to each well, and then read with a microplate reader at 450 nm.

[0217] from Figure 7 It can be seen from the results that the JY-hMPV-F series mRNA / LNP vaccines can induce strong antibody responses in mice.

[0218] Example 7 Immunization schedule and dose effect of JY-hMPV-F mRNA / LNP vaccine in mice

[0219] As a vaccine, the immunization program and immunization dose are two key considerations. The specific experimental steps are as follows:

[0220] 7.1 Animal immunization

[0221] 7.1.1 Immunization Procedure

[0222] Balb / c mice, female, 6 weeks old, were first adapted for one week after arriving at the animal room. 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. Y-hMPV-F mRNA / LNP vaccine was administered intramuscularly at 20 μg / mouse / time, 14 days apart, twice. Blood was collected from the eye sockets before immunization, on the 14th day after the first immunization, and on the 14th day after the second immunization, and the serum was tested.

[0223] 7.1.2 Dose effect

[0224] Balb / c mice, female, 6 weeks old, were acclimated for one week upon arrival in the animal room. The experiment was divided into four groups: the first three groups were immunized with JY-hMPV-081 at doses of 7.5, 15, and 30 μg / mouse, 14 days apart, and the fourth group was the control group. Blood was collected before immunization and on the 7th day after the second immunization, and the serum was tested.

[0225] 7.2 Elisa detection of anti-hMPV antibodies

[0226] 7.2.1 Antigen Coating

[0227] Due to the lack of commercial anti-hMPV-F antigen, the supernatant of 293T cells transfected with hMPV-F plasmid was diluted and coated in a 96-well ELISA plate, 100 μL per well, at 4°C for 16 h.

[0228] 7.2.2 Washing and sealing

[0229] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% milk powder) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0230] 7.2.3 Sample dilution and loading

[0231] After the serum samples of immunized mice were appropriately diluted in a 4-fold decreasing gradient, 100 μL was added to the ELISA plate in triplicate, incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0232] 7.2.4 Add detection antibodies

[0233] According to the number of wells to be loaded with samples, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, peroxidase-coupled, H+L), dilute to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0234] 7.2.5 Color development and termination

[0235] Mix the colorimetric solution A (containing H 2 O 2 ) and color development solution B (containing TMB), add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL of 1M HCl stop solution to each well, and then read with a microplate reader at 450 nm.

[0236] The results of the immune program exploration are as follows Fig. 8A and Figure 8BIt was 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 second dose of "immunization-boosting" will achieve better results than the first dose. Figure 8C ), there was no difference between 15μg / mouse and 30μg / mouse, both were better than 7.5μg / mouse, which should be that the antigen produced by 15μg / mouse was around the saturation range.

[0237] Example 8 Exploration of the persistence of antibodies induced by JY-hMPV-F mRNA / LNP vaccine

[0238] As a vaccine, how long the antibodies it induces can last in the body is an important factor related to the durability of the vaccine's effectiveness, because it is related to the question of how many immunization injections are needed in a viral infection season.

[0239] 8.1 Animal immunization

[0240] Balb / c mice, female, 6 weeks old, were first adapted for one week after arriving at the animal room. The experiment was divided into three groups: the first group, JY-hMPV-081 immunization; the second group, JY-hMPV-060 immunization; the third group was the control group (PBS buffer treatment group). Y-hMPV-F mRNA / LNP vaccine was administered intramuscularly at 20μg / mouse / time, 14 days apart, and administered twice. Before immunization, blood was collected from the eye sockets on the 14th day after the first immunization, the 7th day after the second immunization, 30 days, 3 months, 6 months, and 9 months, and the serum was tested.

[0241] 8.2 Elisa detection of anti-hMPV-F antibodies

[0242] 8.2.1 Antigen Coating

[0243] The cell supernatant containing hMPV-F protein antigen was appropriately diluted with 1×PBS buffer and 100 μL / well was used to coat the 96-well ELISA plate with the antigen at 4°C for 16 h.

[0244] 8.2.2 Washing and blocking

[0245] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (5% skim milk) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0246] 8.2.3 Sample dilution and loading

[0247] After the serum samples of immunized mice were appropriately diluted in a 4-fold decreasing gradient, 100 μL was added to the ELISA plate in triplicate, incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0248] 8.2.4 Add detection antibodies

[0249] According to the number of wells to be loaded with samples, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, peroxidase-coupled, H+L), dilute to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0250] 8.2.5 Color development and termination

[0251] Mix the colorimetric solution A (containing H 2 O 2 ) and color development solution B (containing TMB), add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL of 1M HCl stop solution to each well, and then read with a microplate reader at 450 nm.

[0252] The results of the exploration of viral antibody persistence are as follows Figure 9A-9C It shows that the mRNA / LNP vaccines corresponding to JY-hMPV-081 and JY-hMPV-060 can induce high-titer virus-specific antibodies in animals, and the antibody titer is still high 6 months after immunization. At 9 months, JY-hMPV-081 still maintains the antibody titer, while JY-hMPV-060 begins to decline.

[0253] Example 9 Study on the Independence of Immunogenicity of Different mRNA / LNP Vaccines in Mice

[0254] In order to expand the protection scope of vaccines and improve the popularity of vaccines, the development of multivalent vaccines has become a trend. To this end, two different vaccines were used to immunize mice at the same time to observe the potential of this vaccine series in the development of multivalent vaccines.

[0255] 9.1 Animal Immunization

[0256] Balb / c mice, female, 6 weeks old, were acclimated for one week after arriving at the animal room. The vaccine was injected intramuscularly, and the dosage was as shown in Table 8:

[0257] Table 8 Mouse immunization program, dosage and time

[0258]

[0259] The first group, RSV B type mRNA / LNP vaccine, JY-RSV-001 (the preparation method thereof is described in Example 4 of patent application CN202410420671.7), 10 μg / mouse; wherein the sequence of RSV B type mRNA is as follows:

[0260]

[0261]

[0262] Group 2, JY-hMPV-081 mRNA / LNP, 20 μg / mouse;

[0263] Group 3, JY-RSV-001 (10 μg / mouse) and JY-hMPV-081 (20 μg / mouse) were mixed and encapsulated before administration;

[0264] Group 4, 1× PBS buffer control; used for later RSV infection;

[0265] The fifth group, 1× PBS buffer control, was used for later hMPV infection.

[0266] The interval between the two vaccinations was 14 days, and blood was collected from the eye sockets on the 14th day after the second vaccination.

[0267] 9.2 Elisa for detection of anti-RSV antibodies and anti-hMPV antibodies

[0268] 9.2.1 Antigen Coating

[0269] The antigen (RSV-F (Sino-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, 100 μL per well, at 4°C for 16 h.

[0270] 9.2.2 Washing and sealing

[0271] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (including 5% milk powder) at room temperature for 30 minutes, and then wash the plate 3 times with 300 μL / well PBST.

[0272] 9.2.3 Sample dilution and loading

[0273] After the serum samples of immunized mice were appropriately diluted in a 4-fold decreasing gradient, 100 μL was added to the ELISA plate in triplicate, incubated at room temperature for 1.5 h, and then washed three times with 300 μL / well PBST.

[0274] 9.2.4 Add detection antibodies

[0275] According to the number of wells to be loaded with samples, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, peroxidase-coupled, H+L), dilute to 0.8 ng / mL, add 80 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0276] 9.2.5 Color development and termination

[0277] Mix the colorimetric solution A (containing H 2 O 2 ) and color development solution B (containing TMB), add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL of 1M HCl stop solution to each well, and then read with a microplate reader at 450 nm.

[0278] The results are as follows Figures 10A-10C As shown, it can be seen that the performance of JY-hMPV-081 did not change whether it was immunized alone or with RSV vaccine ( Fig. 10A ); JY-RSV-001 did not show any changes in performance whether immunized alone or with hMPV vaccine ( Fig. 10B ). The mRNA / LNP vaccine showed good antigenic independence, although in general the virus-specific antibodies induced by RSV were higher than those induced by hMPV ( Fig. 10C ). This result provides a basis for the subsequent development of multi-combination vaccines.

[0279] Example 10 Detection of Antibody Neutralizing Ability to Viruses

[0280] One of the golden indicators of vaccine testing is to detect whether the specific antiviral antibodies induced in animals have the ability to neutralize the virus and prevent viral replication and infection. The neutralization virus test is designed for this purpose. Since hMPV is a biosafety level 2 virus, all the following steps must be completed in a biosafety level 2 laboratory in accordance with the corresponding requirements and specifications.

[0281] 10.1 Preparation of cells

[0282] 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, and then inoculated into 96-well plates, 1×10^4 / well, in DMEM complete medium (containing 5% FBS), at 37°C and 5% CO 2 Incubate overnight.

[0283] 10.2 Antibodies and Virus Neutralization

[0284] 10.2.1 Serum Serial Dilution

[0285] The serum dilution gradient and dilution method commonly used in this laboratory are shown in Table 9 below:

[0286] Table 9 Serum dilution and dilution method

[0287]

[0288] 10.2.2 Serum Antibodies and Virus Neutralization

[0289] The dosage of hMPV virus is preferably 30-40 PFU / well, and the dilution medium is DMEM without fetal bovine serum. The volume after serum dilution is 60μL, and then 60μL of virus dilution solution is added and gently mixed. A virus control group (i.e., virus solution without diluted serum) needs to be set up. Incubate in a 37℃ incubator for 1h.

[0290] 10.2.3 Cell inoculation

[0291] Take 35 μL / well of the mixture of the above virus and serum and add it to a 96-well plate with a monolayer of MK2 cells. Inoculate 3 wells of cells for each dilution. At the same time, set up 8 wells of normal cell control and 8 wells of virus infection control. Place the culture plate at 35°C and 5% CO 2 Incubate in the incubator for 1 hour, shaking gently every 15 minutes. After the incubation, discard the mixture in the wells and add 120 μL / well of the pre-prepared warm culture medium, which is DMEM, 3% FBS, and TPCK-treated trypsin, 5 μg / ml. 35°C, 5% CO 2 Cultivate for 6-8 days.

[0292] 10.3 Result color display

[0293] 10.3.1 Virus and cell fixation

[0294] On the 6th to 8th day of culture, discard the culture medium in the wells, wash once with 1×PBS buffer, then add 100 μL / well of freshly prepared 1% paraformaldehyde fixative and fix for 30 minutes at room temperature. Then discard the solution in the wells and wash once with 1×PBS buffer.

[0295] 10.3.2 Blocking and washing

[0296] Add 120 μL / well 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 PBST.

[0297] 10.3.3 Adding detection antibodies

[0298] The detection antibody was homemade mouse anti-hMPV serum. After appropriate dilution, 100 μL was added to each well. After incubation at room temperature for 90 minutes, the plate was washed three times with 150 μL / well PBST.

[0299] 10.3.4 Adding secondary antibody

[0300] Take the matching anti-mouse secondary antibody (HRP-conjugated), dilute it at 1:5000, add 100 μL to each well, incubate at room temperature for 60 minutes, and then wash the plate 5 times with 150 μL / well PBST.

[0301] 10.3.5 Color development and termination

[0302] Add 100 μL of TrueBlue to each well. TM Peroxidase Substrate, shake gently, virus-positive plaques will appear quickly. Then wash away the reaction solution with water. Dry in the dark. Count the virus-positive wells the next day.

[0303] from Fig.11 It can be seen that the antibodies induced by JY-hMPV-081 and JY-hMPV-060 vaccines can neutralize hMPV virus strains well, IC 50 The range was between serum dilution factors of 1300 (JY-hMPV-060) and 3500 (JY-hMPV-081), and the results were better than those of similar vaccines currently being developed during the same period.

[0304] Example 11 Simulating the real world, boosting immunity of animals that have been infected with hMPV virus

[0305] hMPV respiratory infection is a very common respiratory infection. Except for newborn babies who are infected for the first time within one year of age, almost everyone faces repeated infections from viruses brought by the environment. For most people with a complete immune system, reinfection is not a very serious problem, but for young children whose immune systems are still developing, the elderly with declining immune systems, and patients with defective immune systems, hMPV infection is often a serious threat. This experimental design is to explore whether the vaccine has a protective effect on animals that have been infected with hMPV, and the extent of the protective effect.

[0306] 11.1 Animal immunization and sampling

[0307] The animals used were Balb / c mice, female, 6 weeks old. After arriving at the CRO biosafety level 2 animal room, the animals were first acclimated for one week. The animal infection and vaccination schedule is shown in Fig. 12A. The experiment was divided into four groups: the first and second groups were given two immunizations at intervals of two weeks according to the general process, each intramuscular injection, 20μg / mouse, JY-hMPV-081 or JY-hMPV-060; the third group, mice were first infected with hMPV (hMPV 9, A1 type) by nasal inhalation, with a virus dose of 7×10^4 / mouse. Blood was collected for testing on the 30th day, and a booster injection of 20μg / mouse JY-hMPV-081 was given intramuscularly on the second day of blood collection. Blood was collected for testing again 10 days later; the last group was PBS control. The attack virus was the same as the pre-infection virus.

[0308] 11.2 Elisa for detection of anti-hMPV antibody titer

[0309] 11.2.1 Antigen Coating

[0310] The cell supernatant containing hMPV-F protein antigen was appropriately diluted with 1×PBS buffer and 100 μL / well was used to coat the 96-well ELISA plate with the antigen at 4°C for 16 h.

[0311] 11.2.2 Washing and sealing

[0312] Take out the ELISA plate coated with antigen, wash the plate once with 300 μL / well PBST, then add 100 μL blocking solution (containing 5% milk powder) at room temperature for 30 min, and then wash the plate 3 times with 300 μL / well PBST.

[0313] 11.2.3 Sample dilution and loading

[0314] The mouse serum samples were diluted in a 4-fold decreasing gradient, and 100 μL was added to the ELISA plate. After incubation at room temperature for 1.5 h, the plate was washed three times with 300 μL / well PBST.

[0315] 11.2.4 Adding detection antibodies

[0316] According to the number of sample wells to be added, take an appropriate amount of matching detection antibody (goat anti-mouse IgG, peroxidase-coupled, H+L), dilute to 0.8 ng / mL, add 60 μL to each well, incubate at room temperature for 1 hour, and wash the plate 5 times with 300 μL / well PBST.

[0317] 11.2.5 Color development and termination

[0318] Mix the colorimetric solution A (containing H 2 O 2 ) and color development solution B (containing TMB), add 100 μL to each well, leave at room temperature for 15-30 minutes, add 30 μL of 1M HCl stop solution to each well, and then read with a microplate reader at 450 nm.

[0319] The results are as follows Fig. 12B The results showed that one month after hMPV infection, the antibody levels were significantly higher than those of mice that received only one dose of immune induction; however, when the mice completed the "prime-boost regimen", the antibody titer increased significantly to the level of natural infection; at the same time, the booster shot brought the antibodies of the pre-infected mice to a higher level. Antibody neutralization test results ( Fig.11 ) also reflects that booster shots after virus pre-infection can effectively increase the level of neutralizing antibodies. These results fully confirm the role of this vaccine as a booster immunization, that is, if individuals who have been infected with hMPV are given a booster immunization, it can greatly stimulate hMPV-specific memory B cells to quickly start dividing and produce antibodies, thereby playing a protective role.

[0320] Example 12 JY-hMPV-F series mRNA / LNP vaccines inhibit hMPV infection in mouse lungs

[0321] The ultimate testing indicator of a vaccine is to test whether the specific immune response induced by the vaccine in the body has the ability to protect the host and reduce viral infection.

[0322] 12.1 Animal pre-infection, immunization, challenge and sampling

[0323] Balb / c mice, female, 6 weeks old, were acclimated for one week after arrival in the animal room. The vaccines were JY-hMPV-081 and JY-hMPV-060; the pre-infection was hMPV 9 (type A1). Each group had 5 mice, the pre-infection was by nasal inhalation, and the vaccine was injected intramuscularly. The time and dose were as shown in Table 10 and Fig.13A :

[0324] Table 10 Mouse infection or immunization procedures, doses and time

[0325]

[0326] The interval between pre-infection and booster injection was 31 days; the interval between the two doses of vaccine was 14 days; the virus was challenged in the nasal cavity after isoflurane anesthesia; the experiment was terminated on the fourth day after the challenge. After the animals were euthanized by inhalation anesthesia, the complete lungs of the mice were removed and weighed according to the SOP autopsy process.

[0327] 12.2 Lung Viral Load Detection

[0328] Lung viral load can be detected by plaque method or qPCR. This study adopted the latter method. RNA was extracted from mouse lung homogenate, reverse transcribed into cDNA, and the hMPV-F gene was amplified by qPCR to quantify the virus.

[0329] 12.2.1 Lung tissue sample processing

[0330] Prepare cryotubes and add 2 mL of virus protection solution precooled at 4°C; place fresh lung tissue in the cryotube containing virus protection solution and homogenize on the same day to avoid freeze-thaw affecting the experimental results. Use a high-throughput tissue grinder to homogenize the lung tissue at low temperature until there are no obvious particles, draw tissue fluid into 1.5 mL EP tubes, and freeze at -80°C in separate tubes.

[0331] 12.2.2 Extraction of total RNA from samples

[0332] 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.

[0333] Add an equal amount of anhydrous ethanol to the supernatant at a ratio of 1:1. Vortex to mix thoroughly, then centrifuge through the RB column. Repeat this step until the sample is fully bound. Afterwards, wash the RB column once with wash buffer.

[0334] Next, the DNA on the RB column was digested with DNase I solution at 25°C for 15 minutes.

[0335] The RB column was then washed by adding 700 μL of pre-wash buffer, followed by centrifugation and discarding the flow-through. This was repeated three times. Finally, the RB column was centrifuged at 14,000 g for 3 minutes to dry the column matrix.

[0336] 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 14,000 g for 1 minute to obtain RNA.

[0337] 12.2.3 Reverse transcription

[0338] Prepare the cDNA synthesis reaction mixture according to the reverse transcription kit requirements. Each reaction contains 100-500 ng total RNA, 5 μL of 4xRT Mix, and make up the total volume to 20 μL with water. After mixing, incubate at 45°C for 30 minutes. Then inactivate at 85°C for 5 minutes to obtain cDNA.

[0339] 12.2.4qPCR

[0340] hMPV was quantified by qPCR using primer sequences from the F gene:

[0341] hMPV-F-Fw: 5'-CATATAAGCATGCTATATTAAAAGAGTCTC-3' (SEQ ID NO: 18)

[0342] hMPV-F-Rv: 5'-CCTATTTCTGCAGCATATTTGTAATCAG-3' (SEQ ID NO: 19)

[0343] Mouse GAPDH internal reference primer (Sangon, Cat. No. B662304)

[0344] According to the kit recommendations, the qPCR reaction system is as follows:

[0345] <![CDATA[MonAmp TM ChemoHS qPCR Mix]]> 10μL Forward primer (10 μM) 0.4μL Reverse primer (10 μM) 0.4μL Low concentration ROX dye (100×) or high concentration ROX dye (100×) 0.2μL cDNA template xμL <![CDATA[dd H 2 The]]> Add to 20 μL

[0346] qPCR reaction procedure:

[0347]

[0348] 12.2.5 Data Analysis

[0349] There are several steps from lung homogenization to qPCR, and the viral content of each lung is calculated as follows:

[0350] -2 mL of MEM was added to the mouse lung tissue for homogenization, and 200 μL of the sample was taken for RNA extraction, which was diluted 10 times;

[0351] - The total RNA volume was 30 μL, 2 μL was used for reverse transcription, which was diluted 15 times;

[0352] - Reverse transcription produced 20 μL cDNA, 2 μL was used for qPCR, and diluted 10 times;

[0353] So the total dilution factor is 1500;

[0354] The viral load of each lung was calculated using the standard curve external standard method.

[0355] The results are as follows Fig. 13B The results showed that when mice received two doses of 20μg / mRNA / LNP vaccine, the proliferation of hMPV virus in the lungs was effectively controlled, and the amount of lung virus was reduced by about 100 times. The effect of JY-hMPV-081 was better than that of JY-hMPV-060. It was also found that when mice were previously infected with hMPV once, the effect of giving a booster shot of the vaccine one month later was better than or similar to that of receiving two doses of JY-hMPV-081.

[0356] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hMPV virus fusion protein F variant, characterized in that: The hMPV virus fusion protein F variant has differences in amino acid residues 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 as described in claim 1; preferably, the nucleic acid molecule is double-stranded DNA.

3. The nucleic acid molecule according to claim 2, characterized in that The nucleotide sequence encoding the hMPV viral fusion protein F variant comprises the sequence shown in SEQ ID NO: 13 or 14; Preferably, the nucleic acid molecule further comprises one or more selected from the following: (1) T7 promoter; the nucleotide sequence of the T7 promoter is preferably as shown in SEQ ID NO: 3; (2) a coding sequence of a 5'UTR; the 5'UTR is preferably a 5'UTR of a human cytochrome B-245a polypeptide, and its coding sequence is, for example, as shown in SEQ ID NO: 5; (3) a coding sequence of a 3'UTR; the 3'UTR is preferably a 3'UTR derived from human AES / TLE5, and its coding sequence is, for example, as shown in SEQ ID NO: 6; (4) a coding sequence of poly A; the nucleotide sequence of poly A preferably has a gap, and the coding sequence of the gap is TGCAT or as shown in SEQ ID NO: 20; more preferably, the nucleotide sequence of poly A is as shown in SEQ ID NO: 7 or 8; (5) Kozak coding sequence; the Kozak coding sequence is preferably as shown in SEQ ID NO: 4; Further preferably, the nucleic acid molecule comprises, from the 5' end to the 3' end, the coding sequence of the 5'UTR, the coding sequence of Kozak, the nucleotide sequence encoding the hMPV viral fusion protein F variant, and the coding sequence of the 3'UTR; More preferably, 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; preferably, the T7 promoter and the coding sequence of the 5'UTR are connected via AGG or GGG.

4. A nucleic acid construct, characterized in that The nucleic acid construct contains the nucleic acid molecule according to claim 2 or 3; Preferably, the nucleic acid molecule is constructed into the nucleic acid construct by a restriction endonuclease, for example, selected from Hind III and Sap I.

5. A recombinant expression vector, characterized in that: The recombinant expression vector contains the nucleic acid molecule according to claim 2 or 3 or the nucleic acid construct according to claim 4.

6. A transformant, characterized in that: The transformant contains the nucleic acid molecule according to claim 2 or 3, the nucleic acid construct according to claim 4, or the recombinant expression vector according to claim 5, or expresses the hMPV viral fusion protein F variant according to claim 1; Preferably, the host cell used in constructing the transformant is selected from Escherichia coli, insect cells, yeast cells and mammalian cells, such as Escherichia coli Stable cells.

7. An mRNA, characterized in that The mRNA comprises the mRNA encoding the hMPV virus fusion protein F variant as described in claim 1.

8. The mRNA according to claim 7, characterized in that The mRNA is mRNA obtained by transcription of the nucleic acid molecule according to claim 2 or 3; Preferably, the sequence of the mRNA encoding the hMPV viral fusion protein F variant comprises the sequence shown in SEQ ID NO: 15 or 16; More preferably, the sequence of the mRNA is as shown in SEQ ID NO: 11 or 12; Further preferably, the 5' end of the mRNA has a cap structure; the cap structure is preferably a Cap1 structure.

9. A method for preparing a nucleic acid molecule, characterized in that: The method comprises culturing the transformant according to claim 6 to obtain the nucleic acid molecule; Preferably, the nucleic acid molecule is DNA or mRNA.

10. A drug-loaded lipid nanoparticle, characterized in that: It comprises the mRNA according to claim 7 or 8; Preferably, the lipid nanoparticles further comprise a cationic lipid and an auxiliary lipid; Further preferably, the cationic lipid is SM-102; And / or, the auxiliary lipid is DSPC, cholesterol and / or DMG-PEG-2000; More preferably, the lipid nanoparticles include the mRNA, SM-102, DSPC, cholesterol and DMG-PEG-2000; More preferably, 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.

11. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the mRNA according to claim 7 or 8, the lipid nanoparticle according to claim 10, and an optional pharmaceutically acceptable carrier.

12. Use of the hMPV virus fusion protein F variant according to claim 1, the nucleic acid molecule according to claim 2 or 3, the nucleic acid construct according to claim 4, the recombinant expression vector according to claim 5, the transformant according to claim 6, the mRNA according to claim 7 or 8, the lipid nanoparticle according to claim 10 and / or the pharmaceutical composition according to claim 11 in the preparation of a drug for preventing and / or treating diseases caused by hMPV virus infection and / or inhibiting hMPV virus; Preferably, the disease caused by hMPV virus infection is a respiratory disease or ear infection, and the respiratory disease is, for example, bronchitis, bronchiolitis, pneumonia, chronic obstructive pulmonary disease or asthma.

13. A combined vaccine, characterized in that: The multi-combination vaccine comprises the mRNA according to claim 7 or 8, the lipid nanoparticles according to claim 10 and / or the pharmaceutical composition according to claim 11, and mRNA, lipid nanoparticles, mRNA vaccines and / or pharmaceutical compositions that inhibit other viruses; the other viruses are, for example, syncytial viruses; Preferably, the syncytial virus is human respiratory syncytial virus; More preferably, the nucleotide sequence that inhibits the mRNA of other viruses is as shown in SEQ ID NO:

17.

14. A medicine kit, characterized in that: The kit comprises kit A and kit B, wherein: The kit A contains the mRNA according to claim 7 or 8, the lipid nanoparticles according to claim 10 and / or the pharmaceutical composition according to claim 11; The kit B contains mRNA, lipid nanoparticles, mRNA vaccines and / or pharmaceutical compositions that inhibit other viruses; the other viruses are, for example, syncytial viruses; Preferably, the syncytial virus is human respiratory syncytial virus; More preferably, the nucleotide sequence that inhibits the mRNA of other viruses is as shown in SEQ ID NO:

19.

15. A method for inhibiting hMPV virus, characterized in that: The method comprises contacting the hMPV virus with the mRNA according to claim 7 or 8, the lipid nanoparticles according to claim 10, the pharmaceutical composition according to claim 11 and / or the multi-combination vaccine according to claim 13 to induce an immune response to inhibit the hMPV virus, or using the kit according to claim 14 to induce an immune response to inhibit the hMPV virus; Preferably, the method is for non-therapeutic purposes.

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