A hmpv virus-like particle vaccine based on mrna-lnp delivery and uses thereof

Abstract

The present application relates to the field of biological medicine, and in particular, the present application relates to a recombinant hMPV F protein, and a fusion protein, a vaccine, an immunogenic composition, a kit and a pharmaceutical composition comprising the same. The present application also relates to the use of the recombinant hMPV F protein, the fusion protein, the vaccine, the immunogenic composition, the kit and the pharmaceutical composition for preventing and / or treating hMPV infection or diseases and / or symptoms caused by hMPV infection.

Description

Technical Field

[0001] This application relates to the field of biomedicine. Specifically, this application relates to a recombinant hMPV F protein, as well as fusion proteins comprising the recombinant hMPV F protein, vaccines, immunogenic compositions, kits, and pharmaceutical compositions. This application also relates to the use of the recombinant hMPV F protein, fusion protein, vaccine, immunogenic composition, kit, and pharmaceutical composition for the prevention and / or treatment of hMPV infection or diseases and / or symptoms caused by hMPV infection. Background Technology

[0002] Human metapneumovirus (hMPV) is a major pathogen in the Paramyxoviridae family that causes acute lower respiratory tract infections in infants, the elderly, and immunocompromised individuals. Globally, this virus infects more than 14 million children under the age of five annually, leading to approximately 640,000 hospitalizations and significant mortality, posing a serious public health burden. To date, no specific antiviral drugs or preventative vaccines against hMPV have been approved for marketing; therefore, developing highly effective and safe vaccines has significant clinical and social value.

[0003] The hMPV fusion protein (F protein) plays a crucial role in viral invasion and is also a major antigenic target for inducing protective immunity. This protein can exist in two conformations: pre-F and post-F. Literature indicates that the pre-F conformation contains superior neutralizing epitopes, making it a more ideal antigen design target. However, the pre-F conformation itself is less stable and struggles to maintain its native conformation in traditional vaccine systems, resulting in insufficient immunogenicity and difficulty in evoking sustained and high-level protective immunity.

[0004] In recent years, mRNA vaccine technology has provided a new strategy for the prevention and control of infectious diseases due to its flexible design, rapid production, and ability to effectively induce T-cell immunity. However, conventional mRNA vaccines based on single antigen encoding still face challenges in inducing long-term immune memory. Enveloped virus-like particles (eVLPs), as an antigen presentation system with a virus-like structure, can significantly enhance immunogenicity and promote B cell activation and memory formation through multivalent antigen display, but their traditional construction methods usually rely on complex co-expression systems, which limits their application efficiency.

[0005] The intracellular endosome sorting and transport complex (ESCRT) plays a central role in membrane remodeling and viral budding. Recent research indicates that by embedding short peptides containing motifs such as EABR into the intracellular terminus of target antigens, the ESCRT mechanism can be guided to mediate the self-assembly and budding of antigens on the cell membrane, forming structurally regular eVLP particles. This self-assembly strategy provides a new technological pathway for developing next-generation nanovaccines that combine high immunogenicity with ease of preparation.

[0006] In summary, a vaccine that provides an enveloped virus-like particle (eVLP) form and can stably maintain the pre-F protein conformation has good application prospects. Summary of the Invention

[0007] In order to provide a better hMPV vaccine, this application provides a recombinant hMPV F protein to improve the stability and / or immunogenicity of the pre-fusion conformation of the F protein.

[0008] Recombinant hMPV F protein

[0009] In a first aspect, this application provides a recombinant human metapneumovirus (hMPV) fusion (F) protein, which, compared with wild-type hMPV F protein, contains one or more amino acid mutations that stabilize the pre-fusion (pre-F) conformation of the hMPV F protein.

[0010] The amino acid mutations in the recombinant hMPV F protein are selected from any one or more of the following positions: positions 110, 147, 185, 219, 231, 365, 368 and 463 corresponding to the wild-type hMPV F protein.

[0011] In some embodiments, the recombinant hMPV F protein contains any one, two, three, four, five, six, seven, or all eight amino acid mutations mentioned above.

[0012] In some embodiments, the recombinant hMPV F protein has a more stable pre-F protein conformation compared to the wild-type hMPV F protein. In some embodiments, the recombinant hMPV F protein has a higher protein expression level compared to the wild-type hMPV F protein. In some embodiments, the recombinant hMPV F protein has a higher pre-F protein percentage compared to the wild-type hMPV F protein.

[0013] In some embodiments, the recombinant hMPV F protein lacks all or part of the extracellular domains, transmembrane domains, and / or cytoplasmic tail region of the wild-type hMPV F protein. In some embodiments, the recombinant hMPV F protein lacks all or part of the extracellular domains of the wild-type hMPV F protein. In some embodiments, the recombinant hMPV F protein is truncated at the C-terminus of the wild-type hMPV F protein by 10-30, 30-50, 50-70, 70-100, or more amino acids. In some embodiments, the recombinant hMPV F protein is truncated at the C-terminus of the wild-type hMPV F protein by 50 amino acids.

[0014] In some embodiments, the protease cleavage site of the recombinant hMPV F protein contains a mutation of one or more amino acids. In some embodiments, the amino acid mutation corresponds to positions 99 to 102 of the wild-type hMPV F protein. In some embodiments, the amino acid mutation in the protease cleavage site of the recombinant hMPV F protein is located at positions 100 and 101 of the wild-type hMPV F protein.

[0015] Natural hMPV F proteins exhibit high sequence conservation across different subtypes. For example, subtypes A and B share 90% sequence identity. Furthermore, within the same subtype, the sequence identity of the F protein is even higher. Given the sequence conservation of hMPV F proteins, those skilled in the art can readily compare the amino acid positions of different natural hMPV F proteins to identify the corresponding amino acid positions of hMPV F proteins from different hMPV F strains or subtypes.

[0016] As used herein, when referring to the amino acid sequence of wild-type hMPV F protein, the sequence shown in SEQ ID NO: 1 is used for description. For example, the statement "position 110 of wild-type hMPV F protein" refers to the 110th amino acid residue of the protein shown in SEQ ID NO: 1. However, those skilled in the art will understand that wild-type hMPV F protein can have multiple versions that have substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), and substantially the same biological function, but may still have minor differences in amino acid sequence from one another. Therefore, in this application, wild-type hMPV F protein is not limited to the protein shown in SEQ ID NO: 1, but is intended to cover all known wild-type hMPV F proteins. Therefore, in this application, the term "wild-type hMPV F protein" should include various naturally occurring, biologically functional hMPV F proteins, including, for example, the hMPV F protein shown in SEQ ID NO: 1 and its naturally occurring variants.

[0017] In some embodiments, the wild-type hMPV is a strain of subtype A, a strain of subtype B, or a strain derived from subtype A or subtype B.

[0018] In some embodiments, the wild-type hMPV F protein comprises, or is composed of, sequences selected from, the following: (i) the sequence shown in SEQ ID NO: 1; or (ii) a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 1.

[0019] In some embodiments, the wild-type hMPV F protein comprises a sequence selected from the following: (i) the sequence shown in SEQ ID NO: 2; or (ii) a sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 2.

[0020] In some embodiments, the amino acid mutations in the recombinant hMPV F protein are located at positions 110, 147, 185, 219, 231, 365, 368, and 463 corresponding to positions in the wild-type hMPV F protein. In some embodiments, the amino acid mutations in the recombinant hMPV F protein are L110C, A147C, D185P, L219K, V231I, T365C, H368N, and V463C. In some embodiments, the sequence of the recombinant hMPV F protein is as shown in SEQ ID NO: 3.

[0021] In some embodiments, the amino acid mutations contained in the protease cleavage site of the recombinant hMPV F protein are Q100R and S101R.

[0022] In some embodiments, the sequence of the recombinant hMPV F protein is shown in SEQ ID NO: 9.

[0023] In this paper, an amino acid mutation at a specific position in a protein sequence is described as follows: (amino acid residue in wild-type protein) (amino acid position) (amino acid residue in engineered protein). For example, "T365C" means that the threonine (T) residue at position 365 of the amino acid sequence of the wild-type hMPV F protein is replaced by a cysteine ​​(C) residue.

[0024] Fusion protein

[0025] In a second aspect, this application provides a fusion protein comprising the recombinant hMPV F protein described in the first aspect, wherein the fusion protein further comprises additional proteins or peptides. It is understood that these additional proteins or peptides will not adversely affect the activity and / or function of the recombinant hMPV F protein.

[0026] In some embodiments, the additional protein or polypeptide is selected from: human-derived EPM motif, linker peptide, EABR motif (ESCRT-Activating Budding Region motif), signal peptide, polymerization domain, tag, transmembrane domain™, or any combination thereof.

[0027] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, the recombinant hMPV F protein described in the first aspect, and a transmembrane domain TM.

[0028] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the recombinant hMPV F protein described in the first aspect, a polymerization domain, and a transmembrane domain TM.

[0029] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the following components in sequence: the recombinant hMPV F protein described in the first aspect, a transmembrane domain TM, a human EPM motif, a linker peptide, and an EABR motif.

[0030] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the following components in sequence: the recombinant hMPV F protein described in the first aspect, a polymerization domain, a transmembrane domain TM, a human EPM motif, a linker peptide, and an EABR motif.

[0031] signal peptide

[0032] To enhance protein production or secretion, the fusion protein of this application may contain a signal peptide.

[0033] In some embodiments, the signal peptide is a natural signal peptide of hMPV or a variant thereof, or a natural signal peptide or a variant thereof derived from other organisms. In some embodiments, the signal peptide is located at one end of the fusion protein (e.g., the N-terminus). In some embodiments, the N-terminus of the recombinant hMPV F protein is also linked to the signal peptide.

[0034] Multi-domain

[0035] The recombinant hMPV F protein provided in this application can be linked to a multimerization domain to promote the formation of multimers (e.g., dimers, trimers, tetramers, pentameres) of the recombinant hMPV F protein.

[0036] Exogenous multimerizing domains capable of promoting the formation of stable polymers from soluble proteins are known in the art. Specifically, examples of multimerizing domains that can be linked to the fusion protein of this application include, but are not limited to:

[0037] (1) GCN4 leucine zipper (for specific sequence and information, please refer to Harbury et al. 1993 Science 262:1401-1407); (2) trimerization motif from lung surfactant protein (for specific sequence and information, please refer to Hoppe et al. 1994 FEB S Lett 344: 191-195); (3) collagen (for specific sequence and information, please refer to McAlinden et al. 2003 Biol Chem 278:42200-42207); and (4) phage T4 fibritin fold (for specific sequence and information, please refer to Miroshnikov et al. 1998 Protein Eng11:329-414).

[0038] In some embodiments, the multimerizing domain is a trimerizing domain. In some embodiments, the trimerizing domain has a sequence as shown in SEQ ID NO: 10.

[0039] Linking peptides

[0040] The linker peptide of this application can be any suitable flexible peptide that links two polypeptides together, and the specific sequences of such linker peptides include, but are not limited to, G, GG, GGGS, GS, SAIG, GGPG, GPGGG, GGGPG, EAAAK, and PAAAK.

[0041] In some embodiments, the linker peptide comprises at least one glycine (e.g., 1, 2, 3, 4, 5, 6, or 7) and at least one (e.g., 1, 2, or 3) proline. In some embodiments, the amino acid sequence of the linker peptide is selected from GGS (Genomic Glycol). n (GGGS) n Or (G4S) n The group consists of n, where n is an integer from 1 to 10. In some embodiments, the linker peptide has a sequence as shown in SEQ ID NO: 14.

[0042] Human EPM Sequence

[0043] The recombinant hMPV F protein provided in this application can be linked to a human EPM motif to drive the targeted localization of the recombinant hMPV F protein (e.g., targeted localization to the cell membrane).

[0044] In some embodiments, the human EPM motif is located at the C-terminus of the recombinant hMPV F protein. In some embodiments, the human EPM motif has the sequence shown in SEQ ID NO: 13.

[0045] EABR sequence

[0046] The recombinant hMPV F protein provided in this application can be linked to the EABR motif to guide the self-assembly and release of eVLP.

[0047] In some embodiments, the EABR motif is located at the C-terminus of the recombinant hMPV F protein. In some embodiments, the EABR motif has the sequence shown in SEQ ID NO: 15.

[0048] Transmembrane domain™

[0049] In some embodiments, the transmembrane domain TM has a sequence as shown in SEQ ID NO: 11.

[0050] In some embodiments, the fusion protein has a sequence as shown in SEQ ID NO: 12, 9, 17 or 16.

[0051] Nucleic acid molecules

[0052] It is readily understood that the nucleic acid molecules can be used to clone or express the recombinant hMPV F protein or fusion protein of the present invention. In some cases, to improve efficiency, the nucleotide sequence of the nucleic acid molecules can be codon-optimized according to cell preferences.

[0053] Therefore, in a third aspect, this application provides a nucleic acid molecule comprising a nucleotide sequence encoding the recombinant hMPV F protein described in the first aspect or the fusion protein described in the second aspect.

[0054] In some embodiments, the nucleotide sequence may or may not be codon-optimized according to the codon preference of the host cell. In some embodiments, the nucleic acid molecule is DNA, or an RNA (mRNA) product transcribed from said DNA, or a mixture of both.

[0055] carrier

[0056] Vectors for expressing the recombinant hMPV F protein or fusion protein of this application in insect or mammalian cells are well known in the art. These vectors can be cloning vectors or expression vectors. In some preferred embodiments, the vectors of the present invention can be, for example, plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and viral vectors, etc. Viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0057] In a fourth aspect, this application provides a carrier comprising the nucleic acid molecules described in the third aspect.

[0058] In some implementations, the vector is a cloning vector, or it may be an expression vector.

[0059] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is selected from: influenza virus vectors, reverse transcriptase virus vectors, adenovirus vectors, adeno-associated virus vectors, herpesvirus vectors, poxvirus vectors, baculovirus vectors, papillomavirus vectors, or papillomavirus vectors.

[0060] host cells

[0061] The recombinant hMPV F protein provided in this application can be prepared by conventional methods known in the art, such as by expression in recombinant host cells using a suitable vector.

[0062] Host cells include, for example, insect cells, mammalian cells, avian cells, bacterial cells, and yeast cells. Examples of insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, and Schneider S2 cells. Examples of mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells.

[0063] In a fifth aspect, this application provides a host cell comprising the recombinant hMPV F protein described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect.

[0064] In some embodiments, the host cell is selected from prokaryotic cells (e.g., Escherichia coli cells) or eukaryotic cells. In some embodiments, the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell.

[0065] Preparation method

[0066] The methods used for expressing and purifying the recombinant hMPV F protein or fusion protein of this application are common in the art, and can be found in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory Press, ColdSpring Harbor, NY, 200; and Ausubel et al., Short Protocols in Molecular Biology, 4th Edition, John Wiley & Sons, Inc., 999.

[0067] Therefore, in another aspect, this application provides a method for expressing or generating the recombinant hMPV F protein or fusion protein of this application, the method comprising culturing the host cell described in the fifth aspect under conditions that allow protein expression, and optionally, recovering or purifying the recombinant hMPV F protein or fusion protein of this application expressed therein.

[0068] vaccine

[0069] The vaccine provided in this application is not limited to protein form, nucleic acid form, or a mixture of both. Furthermore, the nucleic acid can be selected from DNA, cDNA, RNA, or any combination thereof.

[0070] On the other hand, this application provides a vaccine comprising one or more of the following (1) to (4):

[0071] (1) The recombinant hMPV F protein described in the first aspect;

[0072] (2) The fusion protein described in the second aspect;

[0073] (3) The nucleic acid molecules described in the third aspect;

[0074] (4) The carrier described in the fourth aspect.

[0075] In some embodiments, the nucleic acid may be selected from DNA, cDNA, RNA (e.g., mRNA), or any combination thereof.

[0076] In some embodiments, the vaccine further comprises an adjuvant and / or a buffer solution. In some embodiments, the adjuvant is selected from metal salts, 3-D-monophosphoryllipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles (e.g., lipid nanoparticles), or any combination thereof.

[0077] In some embodiments, the recombinant hMPV F protein or fusion protein in the vaccine is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixture thereof.

[0078] The vaccine provided in this application can be administered using standard routes of administration. Non-limiting administration methods include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration. A single dose can be given to the subject, or one or more booster doses. If a booster vaccination is performed, it is typically administered to the same individual at a time between 1 week and 10 years after the first dose (referred to in such cases as the "primitive vaccination"), for example, between 2 weeks and 6 months.

[0079] Reagent test kit

[0080] On the other hand, this application provides a kit comprising an immunogen component selected from one or more of the following (1) to (4): (1) the recombinant hMPV F protein of the first aspect; (2) the fusion protein of the second aspect; (3) the nucleic acid molecule of the third aspect; and (4) the vector of the fourth aspect.

[0081] In some embodiments, the kit further includes a carrier component capable of displaying the immunogen component.

[0082] In some embodiments, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized pedestals, virus-like particles (VLPs), or any combination thereof.

[0083] In some embodiments, the immunogen and carrier components of the kit are provided individually or as a complex. In some embodiments, the immunogen components are in the form of polymers (e.g., dimers, trimers, tetramers), monomers, or a mixture. In some embodiments, the immunogen components of the kit are provided in the form of proteins or nucleic acids. In some embodiments, the carrier components of the kit are provided in the form of proteins or nucleic acids.

[0084] In some embodiments, the VLP is assembled from proteins derived from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0085] Virus-like particles

[0086] In another aspect, this application provides a virus-like particle (VLP) comprising: the recombinant hMPV F protein described in the first aspect or the fusion protein described in the second aspect; and a carrier component displaying the immunogenic component.

[0087] In some embodiments, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized substrates, or any combination thereof.

[0088] In some implementations, the VLP is an enveloped virus-like particle (eVLP).

[0089] Pharmaceutical Composition

[0090] In another aspect, this application provides a pharmaceutical composition comprising:

[0091] (i) Selected from any one or more of the following (1) to (8): (1) the recombinant hMPV F protein as described in the first aspect; (2) the fusion protein as described in the second aspect; (3) the nucleic acid molecule as described in the third aspect; (4) the vector as described in the fourth aspect; (5) the host cell as described in the fifth aspect; (6) the vaccine as described above; (7) the kit as described above; (8) the virus-like particles as described above.

[0092] (ii) Pharmaceutically acceptable carriers, excipients, buffers, adjuvants, or any combination thereof.

[0093] In some embodiments, the pharmaceutical composition may also contain additional active ingredients, such as additional vaccines, antiviral agents, and / or monoclonal antibodies.

[0094] In some preferred embodiments, the pharmaceutically acceptable carrier and / or excipient is selected from pH adjusters (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic, or nonionic surfactants such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0095] In some preferred embodiments, the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including petroleum-derived, animal-, plant-derived, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some preferred embodiments, the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

[0096] In some preferred embodiments, the pharmaceutically acceptable excipient may be selected from starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and any combination thereof.

[0097] In some preferred embodiments, the pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder (e.g., lyophilized powder), sustained-release formulation, etc.

[0098] The pharmaceutical compositions of the present invention can be administered by various suitable methods. Suitable methods of administration include, but are not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, oral, nasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In some preferred embodiments, the pharmaceutical compositions are formulated into pharmaceutical preparations suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans according to conventional procedures.

[0099] use

[0100] In another aspect, this application provides the use of the recombinant hMPV F protein of the first aspect, the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the vaccine as described above, the kit as described above, or the virus-like particles as described above, in the preparation of a pharmaceutical composition for inducing an immune response to hMPV in a subject.

[0101] In some embodiments, the immune response includes inducing the subject to produce antibodies against hMPV (e.g., neutralizing antibodies). In some embodiments, the subject is a mammal, such as a mouse or a human.

[0102] In another aspect, this application provides the use of the recombinant hMPV F protein of the first aspect, the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, the vector of the fourth aspect, the host cell of the fifth aspect, the vaccine as described above, the kit as described above, or the virus-like particle as described above, in the preparation of a pharmaceutical composition for the prevention and / or treatment of hMPV infection or disease caused by hMPV infection.

[0103] In some implementations, the subject is a mammal, such as a mouse or a human.

[0104] In some implementations, the illness caused by hMPV infection is a respiratory illness. In some implementations, the illness caused by hMPV infection is selected from the common cold, bronchitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0105] Terminology Definition

[0106] In this article, the term "human metapneumovirus (hMPV)" refers to a virus belonging to the genus Metapneumovirus in the family Pneumoviridae. hMPV comprises two main envelope glycoproteins: a fusion protein (F) and an attachment protein (G). Furthermore, the F protein of hMPV is a type I glycoprotein.

[0107] As used herein, the terms “wild,” “wild-type,” or “natural” are used interchangeably. When these terms are used to describe nucleic acid molecules, peptides, or proteins, they mean that the nucleic acid molecule, peptide, or protein exists in nature, is found in nature, and has not undergone any artificial modification or processing. As used herein, the F protein of wild-type human metapneumovirus (hMPV) refers to the naturally occurring, biologically active F protein.

[0108] As used herein, the term "mutation" refers to the presence of a missing, added, or substituted amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. In the specification (particularly the examples), the substitution of an amino acid at a specific position in the protein sequence is expressed as "(amino acid residue in wild-type protein) (amino acid position) (amino acid residue in engineered protein)". For example, "T365C" means that the threonine (T) residue at position 365 of the amino acid sequence of the reference protein is replaced by a cysteine ​​(C) residue.

[0109] As used in this article, the term "variant" refers to a nucleic acid or polypeptide that is different from a reference nucleic acid or polypeptide.

[0110] As used herein, the term "corresponding position" refers to the amino acid positions in the two sequences being compared that are at equivalent positions when performing an optimal alignment of the two sequences, i.e., when the two sequences are aligned to obtain the highest percentage identity. For example, the expression "corresponding to positions 110, 147, 185, 219, 231, 365, 368, and 463 of the wild-type hMPV F protein" means that, when performing an optimal alignment of a sequence with SEQ ID NO: 1, i.e., when a sequence is aligned with SEQ ID NO: 1 to obtain the highest percentage identity, the amino acid positions in the compared sequence that are at equivalent positions to positions 110, 147, 185, 219, 231, 365, 368, and 463 of SEQ ID NO: 1.

[0111] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48:443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0112] As used in this article, the term "C-terminal truncated by X amino acids" means that the X consecutive amino acids at the very end of the C-terminus are truncated. Similarly, the term "N-terminal truncated by X amino acids" means that the X consecutive amino acids at the very end of the N-terminus are truncated.

[0113] As used herein, the term "human EPM motif" refers to a short peptide sequence derived from a human protein that mediates the interaction between a target protein and the cell membrane. In some embodiments, it is typically 5–30 amino acids in length, contains at least three basic amino acid residues (such as lysine or arginine), and may include hydrophobic or amphiphilic amino acid regions. In some embodiments, the motif can bind to membrane phospholipids via electrostatic or hydrophobic interactions, thereby anchoring the fusion protein to the inner surface of the cell membrane. In this invention, the human EPM motif is used to drive the targeted localization of the antigen protein to the cell membrane.

[0114] As used herein, the term "linker peptide" or "GS linker" refers to a flexible linker peptide composed of repeating glycine (Gly, G) and serine (S) units. In some embodiments, its amino acid sequence is selected free from GGS. n (GGGS) n Or (G4S) n The group consists of n, where n is an integer from 1 to 10. The linker peptide has high backbone flexibility and hydrophilicity, and is used to link two or more functional domains in a fusion protein to reduce steric hindrance and maintain the independent folding and activity of each domain. In this invention, the GS linker is located between the human EPM motif and the EABR motif, or between the antigen protein and the self-assembling motif.

[0115] As used herein, the term "EABR motif" refers to a short peptide sequence capable of actively recruiting the endosome sorting and transporting complex (ESCRT). In some embodiments, the motif, upon recognition by ESCRT components, initiates an ESCRT-mediated membrane budding process, thereby driving the fusion protein to co-form enveloped virus-like particles (eVLPs) with the cell membrane. In this invention, the EABR motif is located at the C-terminus of the intracellular segment of the antigen protein and is used to guide the self-assembly and release of the eVLP.

[0116] As used herein, the terms "multimerization domain" or "foldon" have the same meaning and are used interchangeably. It refers to an amino acid sequence capable of forming multimers. In some embodiments, it can promote the assembly of peptides or proteins into dimers, trimers, tetramers, or pentamers.

[0117] As used herein, the term "antigen" refers to a molecule that can be recognized by antibodies. Examples of antigens include those containing antigenic determinants, such as peptides, lipids, polysaccharides, and nucleic acids that are recognized by immune cells.

[0118] As used herein, the term "immune response" refers to the response of immune system cells, such as B cells, T cells, or monocytes, to a stimulus. An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen-specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some embodiments, the response is specific to a particular antigen (i.e., an "antigen-specific response"). If the antigen is derived from a pathogen, the antigen-specific response is a "pathogen-specific response." A "protective immune response" refers to an immune response that inhibits the harmful functions or activities of a pathogen, reduces pathogen infection, or alleviates symptoms (including death) arising from pathogen infection. In this document, immune response encompasses all of the above.

[0119] As used herein, the term “immunogenicity” refers to the ability of a substance, in the presence or absence of an adjuvant, to elicit, trigger, stimulate, or induce an immune response against a specific antigen in a human or animal.

[0120] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0121] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0122] As is known to those skilled in the art, codons exhibit degeneracy. That is, during protein translation, each amino acid can correspond to one or more codons, for example, up to six codons. Different species show significant differences in their use of degenerate codons encoding a particular amino acid, exhibiting different preferences. This preference phenomenon is known as "codon bias." Therefore, as used herein, the term "codon bias" refers to the situation where a species prefers to use certain specific codons to encode amino acids. Optimizing the sequence of nucleic acid molecules according to codon bias can be particularly advantageous in certain situations, for example, it may help improve the expression level of the protein encoded by the nucleic acid molecule. For example, when using E. coli (or human cells) to express a protein or fragment thereof, optimizing the nucleic acid sequence encoding the protein or fragment thereof against the codon bias of E. coli (or human cells) would be potentially advantageous.

[0123] As used herein, the term "virus-like particle (VLP)" is a multimeric particle whose structure may be similar to or dissimilar to that of a natural virus particle. In some embodiments, a VLP is a natural virus particle. In some embodiments, a VLP is a virus-like particle assembled from proteins. It has been demonstrated that some viral proteins (e.g., capsid proteins, surface proteins, envelope proteins) can spontaneously form VLPs after recombinant expression in a suitable expression system (e.g., RSV, HBV, HEV, HPV).

[0124] As used herein, the term "adjuvant" refers to an agent that enhances the production of an immune response in a nonspecific manner. Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) onto which antigens are adsorbed, as well as emulsions; said emulsions may include water-in-oil and oil-in-water (and their variants, including double emulsions and reversible emulsions), lipoglycosides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like receptor agonists (especially TLR2, TLR4, TLR7 / 8, and TLR9 agonists), and various combinations of the above components.

[0125] As used herein, the term “pharmaceutical acceptable” means something recognized in the pharmaceutical industry as suitable for use in animals, and particularly in humans. As used herein, the term “pharmaceutical acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters (including, but not limited to, phosphate buffers), surfactants (including, but not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including, but not limited to, sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0126] As used herein, pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including petroleum-derived, animal-, plant-based, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Physiological saline is a preferred carrier when administering pharmaceutical compositions intravenously. Saline solutions, as well as aqueous dextran and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions.

[0127] Pharmaceutically acceptable excipients, as used herein, may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also contain a wetting agent, or an emulsifier such as sodium hyaluronate, or a pH buffer. The pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0128] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cattle, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human.

[0129] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0130] Beneficial effects of the invention

[0131] Compared with wild-type hMPV F protein, the hMPV F protein or its fusion protein of this application has increased pre-F conformational stability, higher pre-F protein content, and higher protein expression level.

[0132] Furthermore, this application encapsulates the mRNA encoding the hMPV F protein with lipid nanoparticles (LNPs) to prepare a vaccine. After injection, the vaccine self-assembles in mice to generate eVLPs, which exhibit a highly dense surface display of the correctly structured Pre-F antigen. Compared with other vaccines, the vaccine of this application has stronger and more durable immunogenicity (e.g., significantly reduced viral titers in mouse lungs; significantly increased proportions of specific CD4⁺ and CD8⁺ T cells, indicating that it induces strong Th1-biased cellular immunity).

[0133] In summary, the hMPV F protein or its fusion protein or vaccine presented in this application have demonstrated good protective efficacy and safety, and are suitable for various vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and particulate vaccines.

[0134] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0135] Figure 1The expression levels of two Pre-F protein mutants were detected by indirect immunofluorescence. The left side shows representative fluorescence microscopy images, and the right side shows the quantitative fluorescence analysis results obtained using a high-content imaging system. Statistical analysis was performed using two-way ANOVA; significance was indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0136] Figure 2 The expression levels of five Pre-F protein constructs were detected using indirect immunofluorescence. The top image shows a representative fluorescence microscope image, and the bottom image shows the quantitative fluorescence analysis results obtained using a high-content imaging system. Statistical analysis was performed using two-way ANOVA; significance was indicated by **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0137] Figure 3 Schematic diagram of hMPV Pre-F-EABR antigen construct and mRNA vaccine design. The pre-fusion stabilized F protein uses the hMPV A2 strain F protein sequence. Key domains include: signal peptide (SP), TMPRSS2 protease cleavage site modification, fusion peptide, and transmembrane domain (TM). Pre-fusion stabilization mutations are marked with solid circles (•). T4-fibrin foldon domain mediates trimerization. Other marked regions include the endocytosis repressive motif (EPM) and the ESCRT and ALIX binding regions (EABR).

[0138] Figure 4 Agarose gel electrophoresis analysis of hMPV mRNA and self-replicating RNA (saRNA) transcripts. Figure 4 A represents the agarose gel electrophoresis results of mRNA obtained after in vitro transcription based on a traditional linear mRNA backbone construct; Figure 4 B represents the agarose gel electrophoresis results of mRNA obtained after in vitro transcription based on the self-replicating mRNA (saRNA) backbone construct.

[0139] Figure 5 Immunofluorescence assay was used to detect the expression of hMPV Pre-F-EABR antigen in BHK-21 cells. The top image shows a representative fluorescence microscopy image, and the bottom image shows the quantitative fluorescence analysis results obtained using a high-content imaging system. Statistical analysis was performed using one-way or two-way ANOVA; significance was indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0140] Figure 6Characterization results of mRNA-LNP. Among them, Figure 6 A: Physicochemical properties of mRNA-LNP, including Z-mean particle size and polydispersity index (PI) as determined by dynamic light scattering (DLS); data are expressed as mean ± standard deviation (SD) (n=3); Figure 6 B: Encapsulation efficiency (EE) as determined by the RiboGreen RNA kit; data are expressed as mean ± standard deviation (SD) (n=3); Figure 6 C: LNP particle size distribution curve obtained by DLS technology; Figure 6 D: Results of mRNA encapsulation efficiency evaluated by agarose gel electrophoresis after Triton X-100 treatment; Figure 6 E: After transfecting HEK293T cells with the prepared mRNA-LNP, the expression of Pre-F antigen in the cells was detected by indirect immunofluorescence.

[0141] Figure 7 hMPV mRNA-LNP vaccination induced a dose-dependent immune response in a mouse model. The NT50 value (the reciprocal of the serum dilution required to achieve 50% neutralizing titer) was determined by fitting a four-parameter sigmoid curve. All data are expressed as geometric mean titer ± 95% confidence interval, with the dashed line indicating the limit of detection. Statistical significance was assessed using a two-tailed unpaired t-test (**p < 0.01, ****p < 0.0001).

[0142] Figure 8 Comparison of Pre-F and Post-F specific IgG antibody responses induced by hMPV mRNA-LNP vaccine immunization in BALB / c mice. Figure 8 A: Pre-F specific binding antibody titer measured by ELISA on day 21 after primary immunization; Figure 8 B: Pre-F specific binding antibody titer measured after booster immunization (day 42); Figure 8 C: Post-F specific binding antibody titer measured after booster immunization. Data are expressed as geometric mean titer (GMT) ± 95% confidence interval (CI), with the dashed line representing the limit of detection. Statistical analysis was performed using one-way ANOVA; significance was indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0143] Figure 9Serum hMPV pre-F specific IgG subclass levels and Th1 / Th2 immune response bias analysis. Data are expressed as geometric mean titer (GMT) ± 95% confidence interval (CI), with the dashed line representing the limit of detection. Statistical analysis was performed using one-way ANOVA; significance was indicated by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0144] Figure 10 Serum neutralizing antibody titers induced in mice immunized with hMPV mRNA-LNP vaccine (targeting hMPV A2 and B subtypes). Among them, the neutralizing antibody titer (NT) 50 The titer (defined as the reciprocal of the serum dilution factor required to achieve 50% neutralizing activity) was calculated using a four-parameter logistic regression curve. Data are expressed as geometric mean titer (GMT) ± 95% confidence interval (CI), with the dashed line indicating the limit of detection. Statistical significance was assessed using a two-tailed unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0145] Figure 11 Antigen-specific T-cell immune responses induced in mice immunized with hMPV mRNA-LNP vaccine (flow cytometry analysis). Figure 11 A: Percentage of CD4⁺ T cells that produce IFN-γ, TNF-α, IL-2, or IL-4; Figure 11 B: Expression of corresponding cytokines in CD8⁺T cells. Statistical significance was determined using a two-tailed unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

[0146] Figure 12 The protective effect of hMPV mRNA-LNP vaccine immunization against challenge in mice (lung viral titer and RNA copy number). Figure 12 A: Viral titer in the lungs as detected by plaque assay; Figure 12 B: Lung viral RNA copy number detected by qPCR. Statistical significance was determined using a two-tailed unpaired t-test (*p < 0.05, **p < 0.01, ***p < 0.001).

[0147] Sequence information

[0148] Information on some sequences involved in this invention is provided in the table below. Detailed Implementation

[0149] The invention will now be described in the following non-limiting embodiments.

[0150] Those skilled in the art will understand that the embodiments are described by way of example only and are not intended to limit the scope of protection claimed in this application. Unless otherwise specified, the experimental methods in the embodiments are conventional methods. Where specific conditions are not specified in the embodiments, they are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0151] Example 1: hMPV Pre-F-EABR antigen design, mRNA construction and in vitro expression validation

[0152] Antigen sequence design:

[0153] I. Modification of Pre-F protein

[0154] Based on the hMPV A1 subtype F protein (NCBI reference sequence YP_009513268.1, SEQ ID NO: 1), the intracellular region of the Pre-F protein was truncated (Pre-F-∆CT) to enhance membrane expression. The C-terminus was truncated by 50 amino acids consecutively, and the truncated Pre-F sequence is shown in SEQ ID NO: 2.

[0155] Then, the truncated Pre-F was stabilized in its pre-fusion conformation by site-directed mutagenesis. Specifically, based on SEQ ID NO: 2, one or more mutations that stabilize the pre-F conformation were introduced, specifically the following two combinations of mutations: (1) H368N, D185P; and (2) L110C, A147C, D185P, L219K, V231I, T365C, H368N, V463C. The sequence containing the (2) mutation combination is shown in sequence SEQ ID NO: 3.

[0156] The expression level of Pre-F protein was detected using three different F protein antibodies via indirect immunofluorescence assay. Figure 1 Specifically, it will encode the full-length wild-type hMPV F protein and its two mutant Pre-F mRNAs (of which, Figure 1 V1-2 in the above corresponds to the mutation combination in (2); Figure 1V1 in the above (1) corresponds to the mutation combination) and was transfected into BHK-21 cells using Lipo8000 transfection reagent. 24 h after transfection, indirect immunofluorescence detection was performed using three monoclonal antibodies against F protein (MPE8, ADI61026 and DS7) to analyze the expression level and distribution of Pre-F protein in cells and on the cell membrane surface. The results showed that the protein expression of the mutant containing 8 mutations was significantly better than that of the wild type and the mutant containing two mutations. Therefore, the Pre-F construct containing 8 mutations (L110C, A147C, D185P, L219K, V231I, T365C, H368N, V463C) was finally selected for subsequent experiments.

[0157] Furthermore, based on the aforementioned SEQ ID NO: 3 mutant, the TMPRSS2 protease cleavage site (RQSR) was modified to construct five additional constructs, named Modification #1 to Modification #5, respectively. The sequences of the five modified constructs are shown in SEQ ID NO: 4-8, respectively.

[0158] Pre-F protein expression level was detected using an F protein antibody via indirect immunofluorescence assay. Figure 2 The detection method was the same as described above. BHK-21 cells were transfected with mRNA for 24 h, and then stained with the hMPV Pre-F protein-specific monoclonal antibody (ADI-61026). The results showed that the protein expression level of the construct containing the "RRRR" modification was significantly higher than that of other constructs. Finally, the Pre-F construct shown in SEQ ID NO: 4 (i.e., containing the "RRRR" modification) was selected for subsequent experiments.

[0159] II. Fusion of Pre-F proteins

[0160] Furthermore, based on the Pre-F construct (SEQ ID NO: 4) containing the mutation site and the “RRRR” modification, it was combined with different combinations of the coding sequences of the Foldon trimer domain (SEQ ID NO: 10), the transmembrane domain TM (SEQ ID NO: 11), the human EPM motif (SEQ ID NO: 13), the “GS” linker coding sequence (SEQ ID NO: 14), and the preferred EABR motif (SEQ ID NO: 15) to construct... Figure 3 The following are the constructors in the code:

[0161] (1) Pre-F-tm (SEQ ID NO: 16), which comprises, in sequence: Pre-F construct (SEQ ID NO: 4) and transmembrane domain TM (SEQ ID NO: 11);

[0162] (2) Pre-FF-tm (SEQ ID NO: 17), which comprises, in sequence: Pre-F construct (SEQ ID NO: 4), Foldon trimerization domain (SEQ ID NO: 10), and transmembrane domain TM (SEQ ID NO: 11);

[0163] (3) Pre-F-tm-EABR (SEQ ID NO: 9), which sequentially includes: Pre-F construct (SEQ ID NO: 4), transmembrane domain TM (SEQ ID NO: 11), human EPM motif (SEQ ID NO: 13) coding sequence, “GS” linker coding sequence (SEQ ID NO: 14), and EABR motif (SEQ ID NO: 15);

[0164] (4) Pre-FF-tm-EABR (SEQ ID NO: 12), which comprises, in sequence: Pre-F construct (SEQ ID NO: 4), Foldon trimer domain (SEQ ID NO: 10), transmembrane domain TM (SEQ ID NO: 11), human EPM motif (SEQ ID NO: 13) coding sequence, “GS” linker coding sequence (SEQ ID NO: 14), and EABR motif (SEQ ID NO: 15).

[0165] mRNA plasmid construction and preparation: The coding nucleic acid sequences of the four final constructs were cloned into in vitro transcription plasmids containing the T7 promoter. After linearization of the recombinant plasmids, the linearized DNA template was prepared by PCR amplification using PhantaFlash Master Mix (Vazyme #P510-03). The product was purified by gel extraction using a GeneJET gel extraction kit (Thermo Fisher Scientific #K0692). The in vitro transcription of mRNA was carried out under the catalysis of T7 RNA polymerase. The reaction system included Enzyme Mix (Hzymes Biotech #HBP000331-2), a mixture of ribonucleoside triphosphates (100 mM ATP, GTP, CTP, and N1-methyl-pseudouridine triphosphate; Henovcom #HN3004), and the Cap1 analog LZCap. ®(Henovcom #HN3004). After transcription, the DNA template was degraded using DNase I (NEB #M0303L), and the mRNA was purified by LiCl precipitation. The purified mRNA was dissolved in enzyme-free water (Beyotime #ST876), and its concentration was determined spectrophotometrically. The integrity of the mRNA was detected by agarose gel electrophoresis. All mRNA samples were stored at −80°C.

[0166] In vitro expression validation: BHK-21 or HEK293T cells were seeded in 12-well plates and cultured overnight at 37°C until transfection, at which point cell confluence reached 70%–90%. Routine transfection procedures were as follows: Following the manufacturer's instructions, mRNA was compounded with Lipo8000 transfection reagent (Beyotime #C0533) in Opti-MEM I low-serum medium (Gibco #31985070) to prepare the transfection complex; if LNP-encapsulated mRNA was used, the particles were directly added to the culture wells. Immunofluorescence staining was performed 24 hours later. To detect hMPV F protein expression on the cell surface, cells were fixed with 4% paraformaldehyde 24 hours after transfection, washed with PBS, and incubated at 37°C for 1 hour with (ADI-61026) (1 µg / mL) and the universal antibody for F protein (DS7) (1 µg / mL). After washing three times with PBS, the sample was incubated for 1 hour with Alexa Fluor 488-labeled goat anti-human IgG antibody (Thermo Fisher #A48276, 1:2000 dilution). Finally, after washing with PBS, fluorescence imaging analysis was performed using the Operetta CLS high-content analysis system (PerkinElmer).

[0167] like Figure 5 As shown, all prepared mRNAs were well expressed in cells. Furthermore, the constructs containing transmembrane domains (Pre-F-tm, Pre-FF-tm, Pre-F-tm-EABR, and Pre-FF-tm-EABR) all showed significant enrichment on the cell membrane. Among them, based on both fluorescence imaging and fluorescence intensity results, Pre-F-tm-EABR and Pre-FF-tm-EABR exhibited the most significant membrane localization, suggesting that the antigens were effectively expressed and successfully localized to the cell membrane structure. Simultaneously, Western blotting and nanoparticle tracking analysis of the cell supernatant confirmed the release of eVLP particles.

[0168] Example 2: Preparation of hMPV Pre-F-EABR mRNA-LNP vaccine

[0169] The synthesized mRNA was encapsulated using LNP encapsulation with a microfluidic mixing technique (RNACure). The mRNA solution and lipid solution were mixed at a volume ratio of 3:1 at a total flow rate of 12 mL / min. The mRNA was dissolved in 50 mM citrate buffer (pH 4.0) to a final concentration of 150 μg / mL; the lipid mixture was dissolved in anhydrous ethanol to a concentration of 8 mM. The lipid system consisted of SM-102 ionizable lipids (AVT, Shanghai, #O02010), distearate phosphatidylcholine (DSPC; AVT #S01005), cholesterol (AVT #57-88-5), and polyethylene glycol 2000-dimyristoylglycerol (PEG2000-DMG; AVT #O02005) in a molar ratio of 50:38.5:10:1.5.

[0170] The mixed LNP suspension was dialyzed with PBS to remove ethanol and replace the buffer system. Subsequently, the hydrodynamic particle size and polydispersity index (PDI) of LNP were determined using a dynamic light scattering system (Malvern Nano-ZS zetasizer); RNA encapsulation efficiency and concentration were quantified using the Quant-iT RiboGreen fluorescence kit (Thermo Fisher Scientific). All experimental procedures were strictly performed according to the manufacturer's instructions.

[0171] like Figure 6 As shown, the prepared mRNA-LNPs have uniform hydrodynamic particle size, mainly distributed between 60–80 nm, and a polydispersity index (PDI) ≤ 0.15, indicating that the particles have good uniformity. Figure 6 A, C). Furthermore, mRNA content was quantified using the RiboGreen method, and combined with agarose gel electrophoresis analysis of Triton X-100 treated and untreated samples, the results showed that the encapsulation efficiency of all mRNA-LNPs was higher than 90%. Figure 6 (B, D). The prepared mRNA-LNPs were further transfected into HEK293T cells, and indirect immunofluorescence results showed that the antigen was well expressed within the cells. The above comprehensive characterization results indicate that all prepared hMPV mRNA-LNPs were successfully constructed and possess good physicochemical properties and biological activity.

[0172] Example 3: Immunogenicity evaluation of hMPV mRNA-LNP vaccine in mouse model

[0173] Immunization schedule: Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 5. They were administered different doses (0.2 μg, 1 μg, 5 μg) of Pre-F-tm-EABR mRNA-LNP vaccine, Pre-F-tm mRNA-LNP vaccine, or GFP mRNA-LNP (as a negative control) via intramuscular injection. A primari-boost immunization schedule was used at days 0 and 21.

[0174] Humoral immunity assessment: Blood samples were collected 2 weeks after booster immunization. Serum hMPV Pre-F binding antibody levels and neutralizing antibody titers against hMPV A2 strain were detected by ELISA.

[0175] Female BALB / c mice (n = 5 per group) were intramuscularly injected with 0.2, 1, or 5 μg of two hMPV mRNA-LNP vaccines (Pre-F-tm and Pre-F-tm-EABR), respectively, and immunized according to the primary-boost immunization schedule (day 0 and day 21). Serum was collected after booster immunization, and the titer of hMPV Pre-F protein-specific IgG antibodies was measured by ELISA. The neutralizing activity of the serum against the hMPV A2 strain was assessed using a virus neutralization assay.

[0176] Figure 7 The hMPV mRNA-LNP vaccine induced a dose-dependent immune response in a mouse model. Furthermore, compared to the negative control, both the Pre-F-tm-EABR mRNA-LNP vaccine and the Pre-F-tm mRNA-LNP vaccine induced higher titers of specific IgG antibodies in mice at low doses (e.g., 0.2 μg), demonstrating their good immunogenicity.

[0177] Example 4: Immunogenicity evaluation of hMPV mRNA-LNP vaccine in mouse model

[0178] Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 5-10. They were administered any of the following vaccines via intramuscular injection: GFP mRNA-LNP (as a negative control), F(WT) (as a positive control), PreF-F (as a positive control), PreF-F, PreF-tm, PreF-tm-EABR, or PreF-F-tm-EABR mRNA-LNP vaccine as described in this application. Serum samples were collected to detect the hMPV Pre-F and Post-F specific IgG antibody responses in the mice. Female BALB / c mice (n = 5 per group) underwent primary-boost immunization by intramuscular injection of 5 μg mRNA-LNP vaccine on day 0 and day 21, respectively. Serum samples were collected on day 21 (before booster immunization) and day 42.

[0179] Figure 8 The results of hMPV Pre-F and Post-F specific IgG antibody responses in BALB / c mice after immunization with mRNA-LNPs are shown. Figure 8 A: Pre-F specific binding antibody titer measured by ELISA on day 21 after primary immunization; Figure 8 B: Pre-F specific binding antibody titer measured after booster immunization (day 42); Figure 8 C: Post-F specific binding antibody titer measured after booster immunization.

[0180] Figure 9 To enhance the analysis of hMPV Pre-F specific IgG subclass levels and Th1 / Th2 immune response bias in serum after immunization, the titers of anti-hMPV Pre-F protein IgG1 and IgG2a antibodies were measured by ELISA, and the IgG2a / IgG1 ratio was calculated to assess the Th1 or Th2 bias of the immune response.

[0181] Figure 10 The results show the neutralizing antibody response in BALB / c mice after immunization with hMPV mRNA-LNPs. Female BALB / c mice (n = 10 per group) were immunized intramuscularly with 5 μg of mRNA-LNPs according to the primary-booster protocol, and the neutralizing activity of serum against hMPV-A2 and hMPV-B strains was measured.

[0182] In summary, as Figure 8 , Figure 9As shown, compared with mRNA vaccines encoding wild-type hMPV F protein (F(WT)), the four mRNA-LNP vaccines encoding Pre-F antigens designed in this study were able to induce significantly higher levels of Pre-F specific antibody responses. Among them, the Pre-F-tm-EABR and Pre-FF-tm-EABR vaccines induced significantly higher Pre-F specific antibody titers in all dose groups. Simultaneously, the Pre-F / Post-F antibody ratio induced by these two vaccines was significantly increased, indicating that they effectively preserved the Pre-F conformational epitope.

[0183] Furthermore, the vaccine-induced Th1 / Th2 immune polarization was assessed by detecting the levels of Pre-F specific IgG1 and IgG2a antibody subclasses in serum after booster immunization. The results showed that all mRNA vaccines could induce high titers of Pre-F specific IgG1 and IgG2a binding antibodies, with an IgG2a / IgG1 ratio close to 1.0, suggesting that the vaccines could induce a relatively balanced Th1 / Th2 immune response.

[0184] The neutralizing activity of immune serum against hMPV A2 and B subtypes was further detected by a micro-neutralization assay. Figure 10 As shown, the neutralizing antibody titers (NT) induced by Pre-F-tm-EABR and Pre-FF-tm-EABR vaccines 50 The levels were significantly higher than those in the control group, and the levels showed good cross-neutralization ability.

[0185] Cellular Immunoassay: Female BALB / c mice (n = 6 per group) were vaccinated with mRNA-LNP vaccine according to a primary-booster immunization schedule. Mice were sacrificed on day 42 post-immunization, and spleen cells were isolated. Subsequently, spleen cells were stimulated in vitro using a peptide library containing the hMPV A2 strain F protein. This peptide library covers the full-length sequence of the hMPV F protein and consists of 15 amino acids with overlapping regions between adjacent peptides. The final stimulation concentration was 1 μg / mL (purchased from SinoBiological). After stimulation, the level of cellular immune response, including the expression of IFN-γ, TNF-α, IL-2, and IL-4 in CD4⁺ and CD8⁺ T cells, was detected by intracellular cytokine staining (ICS) combined with flow cytometry. The GFP mRNA-LNP immunization group served as the control group.

[0186] The results are as follows Figure 11As shown, compared with the GFP mRNA-LNP control group, all mRNA vaccine immunization groups were able to induce significant CD4⁺ (… Figure 11 A) and CD8⁺ Figure 11 B) T-cell immune response, characterized by significantly increased expression levels of IFN-γ, TNF-α, and IL-2. Specifically, the Pre-F-tm-EABR and Pre-FF-tm-EABR vaccine immunization groups showed generally higher IFN-γ, TNF-α, and IL-2 expression levels induced in CD4⁺ and CD8⁺ T cells. Furthermore, IL-4 expression levels induced by all vaccine groups were low, and no significant differences were observed between the different vaccine groups. These results indicate that the mRNA-LNP vaccine constructed in this embodiment can induce a significant T-cell immune response, exhibiting immune response characteristics dominated by Th1-type cytokines.

[0187] Example 5: Evaluation of the protective effect of hMPV mRNA-LNP vaccine against viral challenge

[0188] Female BALB / c mice aged 6-8 weeks were randomly divided into groups of 5-10. They were administered one of the following vaccines via intramuscular injection: GFP mRNA-LNP (as a negative control), F(WT) (as a positive control), PreF-F (as a positive control), or the PreF-F, PreF-tm, PreF-tm-EABR, or PreF-F-tm-EABR mRNA-LNP vaccines described in this application. Three weeks after booster immunization, both immunized and control mice were challenged with live hMPV A2 virus. Specifically, after completing the immunization schedule, female BALB / c mice (n = 6 per group) were inoculated with hMPV A2 virus via intranasal drop at a dose of approximately 5 × 10⁻⁶. 5 PFU / mouse. Mice were sacrificed on day 4 post-challenge and lung tissue samples were collected. The viral load in the lung tissue was quantitatively detected to assess the protective effect of the vaccine.

[0189] Clinical observation: After viral challenge, mice in each group were observed and their weight changes were recorded daily. The results showed that mice immunized with Pre-F-tm-EABR and Pre-FF-tm-EABR vaccines experienced only a slight decrease in weight, which recovered quickly; while mice in the unimmunized control group or the conventional vaccine group experienced a more significant and sustained decrease in weight.

[0190] Viral load detection: On day 4 post-challenge, mice were sacrificed, and lung and nasal turbinate tissues were collected. One portion of the tissue was homogenized, and the viral titer was determined using a focus forming assay (FFA). RNA was extracted from the other portion, and the viral gene copy number was quantified using qPCR.

[0191] The results are as follows Figure 12 As shown, viral titers in mouse lung tissue were determined by FFA. Compared with the GFP control group, all vaccine groups (including the mRNA vaccine encoding wild-type hMPV F protein (F(WT)))) showed good protective effects.

[0192] Further viral gene copy number analysis results showed that the viral gene copy numbers in the lung tissue of mice immunized with Pre-F-tm, Pre-FF-tm, Pre-F-tm-EABR, and Pre-FF-tm-EABR vaccines were significantly reduced. Compared with the mRNA vaccine encoding wild-type hMPV F protein (F(WT)) and the Pre-F mRNA vaccine, they all showed stronger viral inhibition effects, and the viral titer in the mouse lungs was further reduced by about 1-2 orders of magnitude compared with the positive control group. Overall, the Pre-F-tm-EABR and Pre-FF-tm-EABR vaccines constructed in this application can significantly reduce the viral titer in the mouse lungs, demonstrating excellent protective effects.

[0193] Pathological analysis: H&E staining of lung tissue sections showed that the degree of lesions such as lung inflammation and alveolar septal thickening in mice immunized with Pre-F-tm-EABR and Pre-FF-tm-EABR was significantly reduced.

[0194] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. A recombinant human metapneumovirus (hMPV) fusion (F) protein, which, compared with the wild-type hMPV F protein, contains one or more amino acid mutations that stabilize the pre-fusion (pre-F) conformation of the hMPV F protein; wherein The amino acid mutations contained in the recombinant hMPV F protein are selected from any one or more of the following positions: positions 110, 147, 185, 219, 231, 365, 368 and 463 corresponding to the wild-type hMPV F protein.

2. The recombinant hMPV F protein of claim 1, wherein the recombinant hMPV F protein lacks the extracellular domain or a portion thereof of the wild-type hMPV F protein; Preferably, the recombinant hMPV F protein has 10-30, 30-50, 50-70, 70-100, or more amino acids shortened from the C-terminus of the wild-type hMPV F protein; Preferably, the recombinant hMPV F protein is truncated by 50 amino acids at the C-terminus of the wild-type hMPV F protein.

3. The recombinant hMPV F protein according to claim 1 or 2, wherein the protease cleavage site of the recombinant hMPV F protein contains a mutation of one or more amino acids; Preferably, the mutation of the amino acid corresponds to positions 99 to 102 of the wild-type hMPV F protein; Preferably, the amino acid mutations in the protease cleavage site of the recombinant hMPV F protein are located at positions 100 and 101, corresponding to the wild-type hMPV F protein.

4. The recombinant hMPV F protein of any one of claims 1-3, wherein, The wild-type hMPV is a strain of subtype A, a strain of subtype B, or a strain derived from subtype A or subtype B. Preferably, the wild-type hMPV F protein comprises, or is composed of, sequences selected from, the following: (i) the sequence shown in SEQ ID NO: 1; or (ii) a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO:

1.

5. The recombinant hMPV F protein according to any one of claims 1-4, wherein the amino acid mutations in the recombinant hMPV F protein are located at positions 110, 147, 185, 219, 231, 365, 368, and 463 corresponding to the wild-type hMPV F protein; Preferably, the amino acid mutations contained in the recombinant hMPV F protein are: L110C, A147C, D185P, L219K, V231I, T365C, H368N, and V463C. Preferably, the sequence of the recombinant hMPV F protein is shown in SEQ ID NO:

3.

6. The recombinant hMPV F protein of any one of claims 1-5, wherein, The amino acid mutations contained in the protease cleavage site of the recombinant hMPV F protein are: Q100R and S101R; Preferably, the sequence of the recombinant hMPV F protein is shown in SEQ ID NO:

9.

7. A fusion protein comprising the recombinant hMPV F protein according to any one of claims 1-6, wherein the fusion protein further comprises additional proteins or polypeptides; Preferably, the additional protein or polypeptide is selected from: human-derived EPM motif, linker peptide, EABR motif (ESCRT-Activating Budding Region motif), signal peptide, polymerization domain, tag, transmembrane domain TM, or any combination thereof.

8. The fusion protein of claim 7, comprising, from the N-terminus to the C-terminus, the following: (a) The recombinant hMPV F protein according to any one of claims 1-6, with a transmembrane domain TM; (b) The recombinant hMPV F protein according to any one of claims 1-6, with a polymerizing domain and a transmembrane domain TM; (c) The recombinant hMPV F protein according to any one of claims 1-6, comprising the transmembrane domain TM, the human EPM motif, the linker peptide, and the EABR motif; or, (d) The recombinant hMPV F protein according to any one of claims 1-6, including the polymerization domain, transmembrane domain TM, human EPM motif, linker peptide, and EABR motif; Optionally, the N-terminus of the recombinant hMPV F protein is also linked to a signal peptide.

9. The fusion protein of claim 7 or 8, having one or more of the following features: (1) The multimerization domain is a trimerization domain; Preferably, the trimerization domain has a sequence as shown in SEQ ID NO: 10; (2) The human EPM motif has a sequence as shown in SEQ ID NO: 13; (3) The linker peptide comprises at least one glycine (e.g., 1, 2, 3, 4, 5, 6, 7) and at least one (e.g., 1, 2, 3) proline; Preferably, the linker peptide has the sequence shown in SEQ ID NO: 14; (4) The EABR motif has a sequence as shown in SEQ ID NO: 15; (5) The transmembrane domain TM has the sequence shown in SEQ ID NO: 11; Preferably, the fusion protein has a sequence as shown in SEQ ID NO: 12, 9, 17 or 16.

10. A nucleic acid molecule comprising a nucleotide sequence encoding the recombinant hMPV F protein of any one of claims 1-6 or the fusion protein of any one of claims 7-9; Preferably, the nucleic acid molecule is DNA, or an RNA (mRNA) product transcribed from the DNA, or a mixture of both.

11. A vector comprising the nucleic acid molecule of claim 10; Preferably, the vector is an expression vector or a cloning vector.

12. A host cell comprising the recombinant hMPV F protein of any one of claims 1-6, the fusion protein of any one of claims 7-9, the nucleic acid molecule of claim 10, or the vector of claim 11; Preferably, the host cell is selected from prokaryotic cells (e.g., Escherichia coli cells) or eukaryotic cells; Preferably, the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell.

13. A vaccine comprising one or more of the following (1) to (4): (1) The recombinant hMPV F protein according to any one of claims 1-6; (2) The fusion protein according to any one of claims 7-9; (3) The nucleic acid molecule according to claim 10; (4) The carrier according to claim 11; Preferably, the nucleic acid may be selected from DNA, cDNA, RNA (e.g., mRNA), or any combination thereof; Preferably, the vaccine further comprises an adjuvant and / or a buffer solution; Preferably, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles (e.g., lipid nanoparticles), or any combination thereof.

14. A kit comprising an immunogen component selected from one or more of the following (1) to (4): (1) the recombinant hMPV F protein of any one of claims 1-6; (2) the fusion protein of any one of claims 7-9; (3) the nucleic acid molecule of claim 10; (4) the vector of claim 11; Preferably, the kit further comprises a carrier component capable of displaying the immunogen component; Preferably, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized substrates, virus-like particles (VLPs), or any combination thereof; Preferably, the immunogen component and carrier component in the kit are provided separately or as a complex; Preferably, the immunogen components in the kit are provided in the form of proteins or nucleic acids; Preferably, the carrier component in the kit is provided in the form of protein or nucleic acid; Preferably, the VLP is assembled from proteins obtained from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

15. A virus-like particle (VLP) comprising: the recombinant hMPV F protein of any one of claims 1-6 or the fusion protein of any one of claims 7-9; and a carrier component displaying the immunogenic component; Preferably, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized substrates, or any combination thereof; Preferably, the VLP is an enveloped virus-like particle (eVLP).

16. A pharmaceutical composition comprising: (i) Selected from any one or more of the following (1) to (8): (1) The recombinant hMPV F protein according to any one of claims 1-6; (2) The fusion protein according to any one of claims 7-9; (3) The nucleic acid molecule according to claim 10; (4) The carrier according to claim 11; (5) The host cell according to claim 12; (6) The vaccine according to claim 13; (7) The kit according to claim 14; (8) The virus-like particles of claim 15; and (ii) Pharmaceutically acceptable carriers, excipients, buffers, adjuvants, or any combination thereof; Preferably, the pharmaceutical composition may also contain additional active ingredients; for example, additional vaccines, antiviral agents and / or monoclonal antibodies.

17. Use of the recombinant hMPV F protein of any one of claims 1-6, or the fusion protein of any one of claims 7-9, or the nucleic acid molecule of claim 10, or the vector of claim 11, or the host cell of claim 12, or the vaccine of claim 13, or the kit of claim 14, or the virus-like particle of claim 15, in the preparation of a pharmaceutical composition for inducing an immune response to hMPV in a subject; Preferably, the immune response includes inducing the subject to produce antibodies against hMPV (e.g., neutralizing antibodies). Preferably, the subject is a mammal, such as a mouse or a human.

18. Use of the recombinant hMPV F protein of any one of claims 1-6, or the fusion protein of any one of claims 7-9, or the nucleic acid molecule of claim 10, or the vector of claim 11, or the host cell of claim 12, or the vaccine of claim 13, or the kit of claim 14, or the virus-like particle of claim 15, in the preparation of a pharmaceutical composition for the prevention and / or treatment of hMPV infection or disease caused by hMPV infection; Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the disease caused by hMPV infection is a respiratory disease; Preferably, the diseases caused by hMPV infection are selected from the common cold, bronchitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

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