Protein of RSV, PIV and / or MPV, preparation method and application of protein in preparation of combined vaccine

Abstract

The invention relates to a protein comprising a protein M and / or a protein P. The protein can form a multimeric structure and is used for preparing a vaccine alone or in combination, the vaccine can stimulate a body to generate neutralizing antibodies against HMPV and / or HPIV and has broad spectrum, meanwhile, the protein has physical stability, and under physical extreme conditions, the protein can be used for preparing a neutralizing antibody against HMPV and / or HPIV. And good immunogenicity can also be maintained. When used together with an RSV vaccine, the vaccine has no mutual inhibition effect, and the immunogenicity of the vaccine can be improved.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to an RSV, PIV and / or MPV protein, its preparation method, and its application in the preparation of combined vaccines. Background Technology

[0002] Human Respiratory Syncytial Virus (HRSV) was first discovered in 1955. It belongs to the family Paramyxoviridae, subfamily Pneumovirinae, and genus Pneumovirus. Based on the sequence of its G protein, it can be divided into two subtypes, A and B. RSSV is a non-segmented, negative-sense RNA virus. Its encoded proteins include fusion proteins (F). The stable pre-fusion (pre-F) conformation of its F protein defines the viral epitope and can induce highly neutralizing antibodies.

[0003] HRSV is a viral pathogen that causes respiratory tract infection (RTI). Infection primarily causes lower respiratory tract symptoms, with a significant proportion of patients experiencing severe symptoms (such as bronchiolitis and pneumonia), requiring hospitalization and having a high mortality rate. RSV can be transmitted through person-to-person contact, inhalation via coughing or sneezing, or contact with contaminated objects. It primarily infects the epithelial cells of the nasal cavity and the large and small airways of the lungs, and may also infect alveolar macrophages and other types of lung cells, causing cell fusion to form syncytia.

[0004] Human metapneumovirus (HMPV) is a newly discovered human respiratory pathogen that emerged in 2001. According to statistics from 2018, more than 14 million children under the age of five were infected with HMPV, with over 600,000 hospitalized and more than 16,000 dying. Children, the elderly, and other immunocompromised individuals are the main groups infected by HMPV. Similar to RSV, the stable pre-fusion (pre-F) conformation of the metapneumovirus fusion (F) protein defines the viral epitope, which can induce highly neutralizing antibodies.

[0005] Human parainfluenza virus (HPIV) primarily causes acute respiratory infections in children under 3 years old, with a clinical incidence rate second only to respiratory syncytial virus (RSV). More than half of these cases are caused by HPIV type 3 (HPIV-3 or HPIV3), posing a significant threat to the health and lives of newborns and infants. Currently, there are no effective treatments or preventative vaccines for HPIV-3 infection, and most vaccines designed for HPIV type 3 are live attenuated vaccines. The first step in HPIV-3 infection of host cells is the binding of the HN protein on the viral particle to the sialic acid receptor on the host cell membrane protein. Then, the F protein mediates the fusion of the viral envelope with the host cell membrane, facilitating viral entry into the host cell. The F protein must be cleaved by host cell hydrolases into subunits F1 and F2, linked by two disulfide bonds, before it acquires fusion activity.

[0006] There are currently no suitable vaccines for the three common viruses that cause respiratory infections. Given the significant overlap in the infected populations of HRSV, HMPV, and HPIV-3, and the substantial overlap in their infection window periods, it is necessary to develop a combined vaccine against HRSV, HMPV, and / or HPIV-3. Summary of the Invention

[0007] To overcome the above-mentioned deficiencies, this invention provides a protein comprising HRSV, HMPV, and / or HPIV-3 proteins, a preparation method thereof, and its application in the preparation of single or combined vaccines. Specifically, In a first aspect, the present invention provides a protein comprising protein M and / or protein P, wherein, The protein M includes the F protein of human metapneumovirus (HMPV); The protein P includes the F protein of human parainfluenza virus (HPIV3); The F protein mutant of the HMPV has mutations at positions 116, 128, 230 and / or 269 relative to the wild-type F protein. The HPIV3 F protein mutant has mutations relative to the wild-type F protein, including at positions 94, 143, 148, 172, 197, 294, 390, and / or 423.

[0008] Preferably, the protein further includes protein R, which includes the F protein of Human Respiratory Syncytial Virus (HRSV).

[0009] Preferably, the protein R, protein M and / or protein P further include a signal peptide.

[0010] Preferably, the F protein in protein R, protein M and / or protein P is directly or indirectly linked to antibody Fc structural fragments, foldon domains, ferritin, lumazine synthase (LS), virus-like particles (VLP), dihydrothioacetyltransferase (E2p), I53-50 nanoparticles, etc., to form a multimeric structure.

[0011] More preferably, the indirect connection includes connection via a linker peptide, and even more preferably, the linker peptide includes GGSGG (SEQ ID No. 13), SGSGGGSG (SEQ ID No. 14), GSGGGGSG (SEQ ID No. 15), and GGGGSGGGGGSGGG (SEQ ID No. 16).

[0012] Preferably, the ferritin is derived from any ferritin, such as ferritin from anaerobic and aerobic microorganisms, archaea, bacteria, higher animals and plants. More preferably, the ferritin is derived from insect ferritin or Helicobacter pylori-ferritin (HPF).

[0013] Since the stable pre-fusion (pre-F) conformation of the F protein determines the viral epitope, the F protein below is sometimes also referred to as pre-F.

[0014] Preferred, The HMPV F protein mutant also has mutations at positions 183, 436, 437 and / or 441 relative to the wild-type F protein. The HPIV F protein mutant, relative to the wild-type F protein, also includes mutations at positions 489-491. Preferably, the mutation includes substitution, deletion, addition, etc.

[0015] More preferably, The F protein mutant of the HMPV has mutations of V116L, E128D, L183R, R230N, K269Q, Q437C and / or D441C relative to the wild-type F protein. The HPIV F protein mutant has K94R, E143D, K148R, V172I, L197R, R294N, V390L, E423N and / or deletion of positions 489-491 relative to the wild-type F protein. More preferably, the wild-type F protein of the HMPV includes the protein shown in positions 1-470 of SEQ ID No. 2.

[0016] The HPIV F protein mutant, relative to the wild-type F protein, includes positions 1-491 of SEQ ID No. 5. More preferably, the amino acid sequence of protein R includes (A1) The amino acid sequence shown in positions 1-475 of SEQ ID No. 1, preferably the amino acid sequence shown in SEQ ID No. 1. (A2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (A1); (A3) and any one of (A1)-(A2) that has more than 80% identity and the same function.

[0017] More preferably, the protein M comprises: (B1) The amino acid sequence shown in positions 1-471 of SEQ ID No. 3 or positions 1-471 of SEQ ID No. 4, preferably, the amino acid sequence shown in either SEQ ID No. 3 or 4. (B2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (B1); (B3) and any one of (B1)-(B2) that has more than 80% identity and the same function.

[0018] More preferably, the protein P comprises: (C1) The amino acid sequence shown in positions 1-488 of SEQ ID No. 6, preferably the amino acid sequence shown in SEQ ID No. 6. (C2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (C1); (C3) and any one of (C1)-(C2) that has more than 80% identity and the same function.

[0019] In this article, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0020] In this document, the 80% or more of identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0021] The protein comprises a combination of proteins, the combination of proteins including, 1) Protein M and Protein P; 2) Protein M and Protein R 3) Protein P and Protein R; 4) Protein M, Protein P and Protein R.

[0022] Preferably, the protein further includes a tag protein.

[0023] The tagged protein refers to a polypeptide or protein that is fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0024] More preferably, the protein self-assembles into nanoparticles after being expressed in cells, and even more preferably, into trimers.

[0025] In a second aspect, the present invention provides a nucleotide molecule that encodes any of the aforementioned proteins R, M, or P.

[0026] Preferably, the nucleotide molecule comprises DNA and / or RNA, such as recombinant DNA, or mRNA.

[0027] More preferably, the nucleotide molecule comprises: (D1), positions 1-1425 of SEQ ID No: 7, positions 1-1413 of SEQ ID No: 9, positions 1-1413 of SEQ ID No: 10, and / or positions 1-1464 of SEQ ID No: 12, preferably, the sequences shown in SEQ ID No. 7, 9, 10, and / or 12. The complementary, degenerate, or transcribed sequences of (D2) and (D1), (D3) A DNA molecule or mRNA that has more than 75% identity with the DNA molecule or mRNA defined by (D1) or (D2) and encodes the corresponding protein in the protein.

[0028] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein described above using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that possess 75% or higher identity to the nucleotide sequence encoding the protein described above, as long as they encode the protein and have the same function, are derived from and equivalent to the nucleotide sequence of this invention.

[0029] The term "identity" refers to the sequence similarity to the compared nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence shown in SEQ ID No: 2 or 4 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0030] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0031] In a third aspect, the present invention provides a carrier comprising any of the nucleotide molecules described above.

[0032] Preferably, the vector further includes regulatory factors, such as promoters that initiate transcription of the aforementioned polypeptide or protein-coding gene sequence, and may also include terminators that terminate transcription of the aforementioned polypeptide or protein-coding gene sequence. Furthermore, the vector may also include enhancer sequences.

[0033] The vectors described herein refer to vectors capable of delivering exogenous DNA, mRNA, or target genes into host cells for amplification and expression. These vectors can be cloning vectors or expression vectors, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, and viral vectors (such as retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, etc.). In one or more embodiments of this invention, the vector is a pUC57 vector and / or a PKS001 (pKS001) vector.

[0034] In a fourth aspect, the present invention provides a host cell comprising any of the aforementioned nucleotide molecules or carriers.

[0035] The host cell (also called the recipient cell) described herein may be a plant cell or an animal cell. The term "host cell" can be understood not only to refer to a specific recipient cell, but also to the offspring of such a cell, which may not necessarily be identical to the original parent cell due to natural, accidental, or intentional mutations and / or alterations, but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (Vero cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human kidney epithelial cell lines or their derivatives, such as HEK293, 293T / 17, etc., human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), amphibian cells (e.g., Xenopus laevis cells or Andrias cells), etc. The host cells can be, but are not limited to, davidianus cells, fish cells (e.g., grass carp, carp, rainbow trout, or catfish cells), insect cells (e.g., Sf21 cells or Sf-9 cells), etc. In one or more embodiments of the present invention, the host cells are CHO-K1 cells or 293T cells.

[0036] Preferably, the host cell can also be a microorganism, which may be bacteria, fungi, actinomycetes, protozoa, algae, or viruses. The bacteria may originate from, but are not limited to, species such as *Escherichia sp.*, *Erwinia sp.*, *Agrobacterium sp.*, *Flavobacterium sp.*, *Alcaligenes sp.*, *Pseudomonas sp.*, and *Bacillus sp.*, for example, *Escherichia coli*, *Bacillus subtilis*, or *Bacillus pumilus*. In one or more embodiments of the present invention, the microorganism is a TOP10 competent cell.

[0037] More preferably, the host cell cannot develop into an animal or plant species.

[0038] In a fifth aspect, the present invention provides a vaccine comprising any of the aforementioned proteins, nucleotide molecules, vectors, host cells, or antibodies.

[0039] In a sixth aspect, the present invention provides a combination vaccine comprising a combination of proteins, wherein the combination of proteins comprises any two or three proteins of protein M, protein P, and protein R. The protein M includes the F protein of human metapneumovirus (HMPV); The protein P includes the F protein of human parainfluenza virus (HPIV3); The protein R includes the F protein of Human Respiratory Syncytial Virus (HRSV); Preferably, the combination of proteins includes: 1) Protein M and Protein P; 2) Protein M and Protein R 3) Protein P and Protein R; 4) Protein M, Protein P, and Protein R, Preferably, the proteins M, P, and R are as defined in the first aspect.

[0040] The proteins M, P, and R are combined in a mass ratio of (1-5):(1-5):(1-5).

[0041] Preferably, the combined vaccine comprises a combination of nucleotide molecules, a combination of vectors, or a combination of host cells.

[0042] Accordingly, the combination of nucleotide molecules includes a combination of nucleotide molecules encoding any of the proteins described above.

[0043] The combination of vectors includes a combination of vectors for any of the above-mentioned nucleotide molecules. Different nucleotide molecules can be introduced into the same vector, or they can be introduced into the same or different vectors respectively.

[0044] The combination of host cells includes any of the above-mentioned nucleotide molecules or vectors in host cells. Different nucleotide molecules or vectors can be expressed simultaneously in one host cell or can be introduced into different or the same host cells for expression.

[0045] Preferably, the vaccine or combination vaccine further includes an adjuvant.

[0046] More preferably, the adjuvant may be a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the antigen co-inoculated with it. The adjuvants described herein may be those known to those skilled in the art, including but not limited to: plant adjuvants (such as alkylamines, phenolic compounds, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharides, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte clone stimulating factor, leukocyte cytokines IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), and emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0047] As is well known to those skilled in the art, in order to enhance the immunogenicity of antigen proteins, in addition to adding compounds with immunomodulatory effects as adjuvants, gene combinations can be adjusted to express them into particulate structures; or they can be aggregated in vitro and encapsulated in liposomes or microspheres.

[0048] Preferably, the vaccine or combination vaccine also includes a vaccine delivery system.

[0049] The vaccine delivery system described herein can be a substance capable of carrying antigens to the body's immune system, where they can be stored and exert their antigenic effects for an extended period. The vaccine delivery system described herein can be an aluminum salt gel adjuvant vaccine delivery system, an emulsion adjuvant vaccine delivery system, a liposome adjuvant vaccine delivery system, or a nano-adjuvant vaccine delivery system.

[0050] Furthermore, the vaccine or combination vaccine may also include one or more pharmaceutically acceptable carriers.

[0051] The pharmaceutically acceptable carrier may be a diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, adsorbent, surfactant, or lubricant, but is not limited thereto.

[0052] The vaccine or combination vaccine for preventing infection described in this invention may be an intramuscular liquid injection, an intravenous liquid injection, an intranasal liquid injection, an intradermal liquid injection, or a subcutaneous liquid injection.

[0053] In a seventh aspect, the present invention provides a method for preparing the above-mentioned protein, vaccine or combination vaccine, the method comprising introducing the nucleotide molecules or vector encoding the above-mentioned protein R, protein M and protein P into a host cell and culturing the host cell.

[0054] Preferably, the nucleotide molecules encoding proteins R, M, and P are introduced into the same vector or different vectors.

[0055] Preferably, the preparation method includes screening for monoclonal cell lines with high and stable expression levels.

[0056] More preferably, the screening method includes screening methods conventional in the art, such as adding screening reagents, comparing the biological activity of clonal pools using ELISA, etc.

[0057] In an eighth aspect, the present invention provides an application of the above-described protein, nucleotide molecule, vector host cell, vaccine, or combination vaccine, said application comprising any of the following: (1) Use in the preparation of products for the prevention and / or treatment of diseases caused by HRSV, HMPV and / or HPIV viral infections; (2) Use in the preparation of products for inducing immune responses to HRSV, HMPV and / or HPIV viral antigens; (3) Use in the prevention and / or treatment of diseases caused by HRSV, HMPV and / or HPIV viral infections; (4) Use in inducing immune responses to HRSV, HMPV and / or HPIV viral antigens.

[0058] The products described in this article may be reagents, drugs, or vaccines.

[0059] The products described in (1) and (2) may be vaccines or antibodies against HRSV, HMPV and HPIV viruses, and the antibodies include full-length antibodies or antigen-binding fragments (such as Fab fragments, Fv fragments, Fab′ fragments, F(ab′)2 fragments, single-chain antibodies (ScFv), nanobodies (single-domain antibodies), bispecific antibodies or minimum recognition units (MRUs), etc., but not limited to these).

[0060] Furthermore, the HRSV, HMPV, and / or HPIV antibodies may be neutralizing antibodies that specifically bind to the F protein of HRSV, HMPV, and / or HPIV. The neutralizing antibodies may be high-titer neutralizing antibodies against multiple prevalent strains of HRSV, HMPV, and / or HPIV.

[0061] In the above applications or antibodies, the diseases caused by HRSV, HMPV and / or HPIV virus infections may include respiratory system infections, digestive system infections, cardiovascular system infections, and / or nervous system infections.

[0062] Preferably, the respiratory infection may include respiratory tract infection and / or lung infection.

[0063] Preferably, the digestive system infection may include intestinal disease, loss of appetite, nausea, vomiting, abdominal pain and / or diarrhea.

[0064] More preferably, the respiratory infection may include severe acute respiratory syndrome, hypoxic respiratory failure, sepsis, septic shock, nasopharyngitis, rhinitis, pharyngitis, tracheitis and / or bronchitis.

[0065] More preferably, the lung infection may include pneumonia and / or lung injury.

[0066] Preferred products for the prevention and / or treatment of diseases caused by HRSV, HMPV and / or HPIV viral infections include vaccines.

[0067] Preferably, the HRSV includes two subtypes, A and / or B.

[0068] Preferably, the HPIV virus includes HPIV types 1-4, and more preferably, the HPIV virus includes HPIV type 3 (HPIV-3, or HPIV3).

[0069] In a ninth aspect, the present invention also provides a method for generating an immune response, the method comprising administering any of the above-described vaccines or combination vaccines to a subject.

[0070] In the above method, administering the vaccine or combination vaccine to the subject can induce an immune response against HRSV, HMPV, and / or HPIV viruses. This immune response can be a cellular immune response, a humoral immune response, or a combination of both.

[0071] The cellular immune response may include B cell immune response and T cell immune response.

[0072] The subjects described in this article may be humans or non-human animals.

[0073] Furthermore, the non-human animal may be a non-human mammal.

[0074] The non-human mammal may be any one of the following, but is not limited to: mouse, rat, guinea pig, hamster, pig, dog, sheep, monkey, rabbit, cat, cow, horse.

[0075] The subjects mentioned in this article include, but are not limited to, healthy subjects, symptomatic infected subjects, asymptomatic infected subjects, or recovered subjects (subjects who have recovered after infection).

[0076] The administration methods described herein include, but are not limited to, intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral or nasal spray, or nebulized inhalation.

[0077] In summary, compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a vaccine against HRSV, HMPV, and / or HPIV that can be used in combination. Whether used alone or in combination, the vaccine produces an effective neutralizing protective titer when used alone, and when used in combination, the vaccine produces a neutralizing protective titer that is no less than that produced by immunization alone, thus achieving a "non-inferiority" immunization effect. At the same time, the above-mentioned vaccines are safe.

[0078] Given the significant overlap among the infected populations and the substantial overlap in their infection window periods, the combined vaccine of this invention improves the prevention and control efficiency of respiratory infectious diseases, expands the vaccine's applicable population, reduces the number of vaccinations, and reduces anxiety, pain, and adverse reactions after vaccination.

[0079] The vaccine of this invention is in the form of nanoparticles, which better presents the antigen on the outer surface, improving the immunogenicity of the antigen. At the same time, it has physical stability, maintaining more than 75% of the original untreated protein under extreme conditions for 1 hour, such as pH 3.8, pH 10, and temperature 70°C. Attached Figure Description

[0080] Figure 1SDS-PAGE electrophoresis results of ferritin-PreF fusion protein: In the top image, from left to right, the second lanes of the top three images are MP39-PreF-NP, MP68-PreF-NP, and MP67-PreF-NP, respectively. In the bottom image, the second and third lanes are PI12-PreF-NP and PI15-PreF-NP, respectively.

[0081] Figure 2 Electron micrographs of ferritin-PreF fusion protein nanoparticles, from left to right, representing ferritin-PreF fusion proteins PI15-PreF-NP and MP67-PreF-NP, respectively. Detailed Implementation

[0082] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the examples are listed below; unless otherwise specified, all others are commercially available.

[0084] Example 1: Design, preparation and purification of ferritin-PreF fusion protein This invention is based on the formation of a ferritin-PreF fusion protein from PreF proteins of different viral origins and Helicobacter pylori ferritin, which, after expression, self-assembles into 24-mer nanoparticles. Specifically, I. Sequence of Ferritin-PreF fusion protein and its encoding gene 1. RSV-PreF-B-NP, as shown in SEQ ID No.1, is one of the fusion proteins formed by the PreF mutant protein of RSV and ferritin.

[0085] 2. MP39-PreF-NP, as shown in SEQ ID No. 2, is a fusion protein formed by the PreF wild-type protein of MPV and ferritin. 3. MP68-PreF-NP, as shown in SEQ ID No. 3, is one of the fusion proteins formed by the PreF mutant protein of MPV and ferritin.

[0086] 4. MP67-PreF-NP, as shown in SEQ ID No. 4, is one of the fusion proteins formed by the PreF mutant protein of MPV and ferritin.

[0087] 5. PI12-PreF-NP, as shown in SEQ ID No. 5, is a fusion protein formed by the PreF wild-type protein of PIV and ferritin.

[0088] 6. PI15-PreF-NP, as shown in SEQ ID No. 6, is one of the fusion proteins formed by the PreF mutant protein of PIV and ferritin.

[0089] 7. The RSV-PreF-B-NP encoding gene sequence is shown in SEQ ID No. 7.

[0090] 8. The coding gene sequence of MP39-PreF-NP is shown in SEQ ID No. 8.

[0091] 9. The coding gene sequence of MP68-PreF-NP is shown in SEQ ID No. 9.

[0092] 10. The coding gene sequence of MP67-PreF-NP is shown in SEQ ID No. 10.

[0093] 11. The coding gene sequence of PI12-PreF-NP is shown in SEQ ID No. 11.

[0094] 12. The coding gene sequence of PI15-PreF-NP is shown in SEQ ID No. 12.

[0095] II. Preparation of Ferritin-PreF Fusion Protein 1. Construction of recombinant plasmids The Pre-F mutant protein and optimized ferritin were fused using a linker (SGSGGGSG (SEQ ID No. 14), GSGGGGSG (SEQ ID No. 15), or GGGGSGGGGGSGGG (SEQ ID No. 16)) to prepare a ferritin-PreF fusion protein. The Pre-F mutant protein was located at the N-terminus of the ferritin-PreF fusion protein, and the optimized ferritin was located at the C-terminus. Plasmids containing the encoding gene sequences of each of the aforementioned ferritin-PreF fusion proteins were synthesized by Nanjing Genscript Biotech Co., Ltd.

[0096] 2. Expression of ferritin-PreF fusion protein Electroporation and expression were performed in CHO K1Q cells (Kangsheng Biopharmaceutical Co., Ltd., catalog number A14101), and cell lines with high expression were screened. The specific steps are as follows: The recombinant plasmid pKS001-Ferritin-preF was transfected into CHO K1Q cells using the EBXP-F1 electroporator from Suzhou Yida Biotechnology Co., Ltd. The specific electroporation steps are as follows: 1. Remove the buffer, cell culture medium, and D-PBS 30 minutes before electroporation and allow them to return to room temperature.

[0097] 2. Cell collection and counting: After the cells are evenly suspended, they are placed in centrifuge tubes for counting.

[0098] 3. Centrifugation: Take the required culture medium and place the cells into a new centrifuge tube, then place it in a centrifuge (Suzhou Guofei Laboratory Instruments Co., Ltd., item number: TDL-5A) and centrifuge at 1000 rpm for 5 min.

[0099] 4. DPBS washing: Discard the supernatant culture medium, obtain the desired cells, add 1 mL of D-PBS (Thermo Fisher Gibco, catalog number: 2334304) to resuspend the cells, and centrifuge at 1000 rpm for 5 min.

[0100] 5. Mix DNA, cells, and buffer: Discard D-PBS, add the required amount of electroporation buffer (Suzhou Yida Biotechnology Co., Ltd., catalog number: H10305) and 10ug of plasmid, and gently pipette to mix.

[0101] 6. Electroporation: Add the cell suspension containing plasmids to the H1 electroporation cuvette (Suzhou Yida Biotechnology Co., Ltd., catalog number: H10201) at a ratio of 200 μl + DNA volume / cuvette. Insert the electroporation cuvette into the base and perform electroporation according to the electroporation conditions shown in Table 1.

[0102] Table 1 Electrostatic Transfer Conditions

[0103] 7. Add the electroporated cells to a T25 flask (Wuxi Nais Life Science & Technology Co., Ltd., catalog number: 707003) containing 10 mL of CD04 medium (Kangsheng Biopharmaceutical Co., Ltd., catalog number: A11004) and incubate for 48 hours.

[0104] The specific steps for culturing and screening cell clones are as follows: Cells were sampled from the T25 flasks and their viability was monitored using a cell counter (Sanofi, model: Countess II FL). When the viability was above 70%, 10,000 cells per well were seeded into 96-well plates and cultured in CD04 medium containing 25 mM MSX (Sigma, catalog number: M5379-1G). Positive clones were selected using ELISA. The cells were then expanded to 125 mL shake flasks (Wuxi Nice Life Science Co., Ltd., catalog number: 781011). After approximately 5-7 days of culture in the 125 mL shake flasks, when the viability decreased to between 50-80%, the supernatant was collected for ELISA detection.

[0105] The ELISA detection method is as follows: The supernatant was diluted 10-fold, 100-fold, 1000-fold, and 10000-fold and coated. A 1500-fold diluted F protein antibody (Pujian Biotechnology (Wuhan) Co., Ltd., catalog number: 62814) was used as the primary antibody, and goat anti-human IgG-HRP (Solepro, catalog number SE101-1ml) was used as the secondary antibody. Signal readings were performed using a microplate reader (Shanghai Kehua, catalog number: RD-SH-012), and the sample with the strongest signal was selected as the highest expression sample. The supernatant from the highest expression sample was then purified.

[0106] III. Purification of Ferritin-PreF Fusion Protein Following the method described in the literature "Flexible RSV Prefusogenic Fusion Glycoprotein Exposes Multiple Neutralizing Epitopes that May Collectively Contribute to Protective Immunity," the supernatant of the expression cell line culture medium was purified using Capto Lentil Lectin (Cytiva, catalog number: 17548902), Q Sepharose FF (Cytiva, catalog number: 17051060), Capto Core 400 (Cytiva, catalog number: 17372402), and Superose 6 prepgrade (Cytiva, catalog number: 10321079). The specific purification steps were as follows: the selected cell supernatant culture medium was centrifuged at 8000 rpm for 20 minutes, filtered through a 0.45 μm filter membrane (Jinteng, catalog number: JTSF 025013 / 014), yielding approximately 100 mL of solution. Equilibration buffer was then added to a final volume of 200 mL. Equilibrate the QFF column with equilibration buffer, load the sample using an A1 pump at a flow rate of 1.5 mL / min. After loading, wash with equilibration buffer until the absorbance returns to and stabilizes at the pre-loading level. Elute with a gradient of elution buffer (20 mM Tris, 0.5 M NaCl, pH 8.5) at a flow rate of 2 mL / min, 0-100% B, for 50 min. Collect the elution peak. Concentrate the supernatant 5-10 times and pass it through a Superose 6 prep grade column at a flow rate of 1 mL / min. Collect the sample with the absorption peak to obtain the ferritin-PreF fusion protein solution, which is then concentrated for SDS-PAGE and Western blot analysis.

[0107] The specific steps for SDS-PAGE analysis are as follows: Add 20 μL of 5× protein loading solution to 80 μL of ferritin-PreF fusion protein solution, incubate at 95℃ for 10 min, and then centrifuge. Take 15 μL of the supernatant for SDS-PAGE analysis, and observe protein expression after staining.

[0108] For example, the SDS-PAGE electrophoresis results of the ferritin-PreF fusion protein solution are as follows: Figure 1 As shown.

[0109] The results showed that the above recombinant plasmids were successfully expressed in CHO K1Q cells to obtain the target protein of approximately 74KD, which was in line with the expected size. The bands of the fusion proteins MP39-PreF-NP, MP68-PreF-NP, MP67-PreF-NP, PI12-PreF-NP, and PI15-PreF-NP were in line with the expected size.

[0110] Figure 1 In the middle, from left to right, the second lanes in the top three images are MP39-PreF-NP, MP68-PreF-NP, and MP67-PreF-NP, respectively. In the bottom image, the second and third lanes are PI12-PreF-NP and PI15-PreF-NP, respectively.

[0111] IV. ELISA Identification of Fusion Proteins The specific steps of ELISA analysis are as follows: 200 ng, 20 ng, 2 ng, and 0.2 ng of purified proteins (MP39-PreF-NP, MP68-PreF-NP, MP67-PreF-NP, PI12-PreF-NP, and PI15-PreF-NP) per well were used for coating. For MPV, Anti-hMPV fusion protein antibody [EPR29365-579] (ABCAM, catalog number ab320652) was used as the primary antibody, and rabbit secondary antibody (ABCAM, catalog number ab6721) was used as the secondary antibody. For PIV, Anti-PIV type 3 F fusion protein antibody (Creative Diagnostics, Catalog # CABT-NS1550) was used as the primary antibody, and mouse secondary antibody (CellSignaling Technology, catalog number 7076S) was used as the secondary antibody. Signal readings were performed using a microplate reader (Shanghai Kehua, catalog number RD-SH-012). The purified proteins all exhibited good specific ELISA binding, as confirmed by specific ELISA. The results are shown in Table 2. Table 2. ELISA binding activity of each fusion protein

[0112] Example 2: Immunogenicity study of ferritin-PreF fusion protein I. Immunity 1. Experimental Materials and Methods Experimental materials: 6-8 week old female Balb / c mice (Speford (Beijing) Biotechnology Co., Ltd., catalog number: B201-02).

[0113] Experimental methods: Sixty-four 6-8 week old Balb / c mice were randomly divided into 8 groups of 8 mice each. The treatment methods for each group were as follows: The fusion proteins MP39-PreF-NP, MP68-PreF-NP, MP67-PreF-NP, PI12-PreF-NP, and PI15-PreF-NP were administered via intramuscular injection into the thigh twice, on day 0 and day 21, respectively. Each injection consisted of 1 μg of ferritin-PreF fusion protein, 50 μg of aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05), and 100 μL of PBS buffer (Solarbio, catalog number P1020).

[0114] II. Mouse serum CPE neutralization test The TCID50 of HMPV virus type A (described in the literature: Human Metapneumovirus Laboratory Methods for Isolation Propagation and PlaqueTitration) cultured in DMEM medium with 10% bovine serum using Vero cells was 2.81E+07.

[0115] LLC-MK2 cells grown in T75 flasks were washed twice with 1×PBS. 0.25% trypsin was added, and the cells were incubated at 37°C for 2 min, then resuspended and centrifuged at 4000 rpm for 5 min. After resuspending the cells in culture medium, they were counted and evenly seeded into 96-well cell culture plates, allowing them to adhere for 1 hour. The plates were then incubated at 37°C with 5% CO2 for 24 hours. The HMPV virus strain was removed from -80°C, thawed in a 37°C water bath, diluted to 200 TCID50, and placed on ice. Eight serum samples from each group of mice were selected and diluted with 1×PBS. Starting with a 40-fold dilution, the serum was serially diluted 3-fold to 29160-fold, mixed with an equal volume of 200 TCID50 virus solution, and incubated at 37°C for 1 hour. Each mouse serum sample was used in triplicate. Afterwards, the serum and virus mixture was aspirated, and 200 μL of a capping layer (MEM + 2% serum + 1% penicillin antibody + 1.5% methylcellulose) was added. The cells were then incubated at 37°C for 25 hours. The cells were gently inverted and the capping layer was removed. 100 μL of tissue fixative was added to each well and incubated at room temperature for 30 minutes. After discarding the tissue fixative, to improve cell permeability, 100 μL of 1% TRITON X-100 dissolved in PBS was added to each well and incubated at room temperature for 30 minutes. After discarding the permeabilization solution, 10% BSA was added to PBS for blocking and incubation for 1 hour. After discarding the blocking solution, 100 μL of a 1:500 diluted primary antibody Anti-hMPV fusion protein antibody [EPR29365-579] (ABCAM, catalog number ab320652) was added to each well and incubated overnight at 4°C. After washing the plate three times with 1*PBST, add 100 μL of secondary antibody (goat anti-rabbit IgG, 1:500, Invitrogen, catalog number A-11008) to each well and incubate at 37°C for 45 minutes. After washing the plate five times with 1*PBST, image and analyze using a CQ1 analyzer.

[0116] The results are shown in Table 3. The results indicate that the neutralizing protective efficacy of MP68-PreF-NP and MP67-PreF-NP proteins in this invention is significantly higher than that of wild-type MP39-PreF-NP.

[0117] Table 3 Neutralizing titers of HMPV series fusion proteins

[0118] The TCID50 of PIV type 3 virus cultured in Hep-2 cells in DMEM medium with 10% bovine serum was 2.34E+07 (Reference: Functional and structural basis of human parainfluenza virus type 3 neutralization with human monoclonal antibodies). Vero-E6 cells grown in T75 flasks were washed twice with 1*PBS, 0.25% trypsin was added, and the cells were incubated at 37°C for 2 min, then resuspended and centrifuged at 4000 rpm for 5 min. After resuspending the cells in culture medium, the cells were counted, and Vero-E6 cells were evenly seeded in 96-well cell culture plates and allowed to adhere for 1 hour. The plates were then incubated at 37°C in a 5% CO2 cell culture incubator for 24 hours.

[0119] After removing the HPIV3 virus from -80°C, it was thawed in a 37°C water bath, diluted 40-fold, and placed on ice. Eight serum samples from each of the above groups were selected and diluted with 1XPBS. Starting with a 40-fold dilution, the samples were serially diluted 3-fold to 29-160-fold, mixed with an equal volume of 200 TCID50 virus solution, and incubated at 37°C for 1 hour. 200 μL of the mixture was seeded into each well of the corresponding cell plate, with three replicates per mouse serum sample. The serum and virus mixture was then discarded, and 200 μL of a capping layer (DMEM + 2% serum + 1% penicillin antibody + 1.5% methylcellulose) was added. The plate was incubated at 37°C for 25 hours. The plate was then gently inverted to remove the capping layer, and 100 μL of tissue fixative was added to each well, incubating at room temperature for 30 minutes. After discarding the tissue fixative, to improve cell permeability, 100 μL of 1% TRITON X-100 dissolved in PBS was added to each well, and the plate was incubated at room temperature for 30 minutes. After discarding the permeabilization buffer, block and incubate with PBS containing 10% BSA for 1 hour. After discarding the blocking buffer, add 100 μl of primary antibody (Anti-PIV type 3 F protein Monoclonal Antibody, Creative Diagnostics, Catalog # CABT-NS1550, 1:500 dilution) to each well and incubate overnight at 4°C. After washing three times with 1*PBST, add 100 μl of secondary antibody (HRP-labeled goat anti-mouse IgG, Invitrogen, catalog number A-11008) to each well and incubate at 37°C for 45 minutes. After washing five times with 1*PBST, image and analyze using a CQ1 analyzer.

[0120] The results are shown in Table 4. The results indicate that the neutralizing potency of PI15-PreF-NP in this invention is significantly higher than that of PI12-PreF-NP.

[0121] Table 4 Neutralizing titers of HPIV series fusion proteins

[0122] The preparation methods and immunogenicity of RSV-PreF-B-NP and RSV-PreF-B-NP separately can be referenced simultaneously to the patent with publication number CN115850396A, entitled "An RSV Nanoparticle Vaccine and Its Preparation Method and Application", which is incorporated herein by reference in its entirety as a part of this application.

[0123] Example 3: Nanoparticle morphology analysis of ferritin-PreF fusion protein The purified products of the exemplary ferritin-PreF fusion proteins PI15-PreF-NP and MP67-PreF-NP prepared in Example 1 were negatively stained. The specific negative staining procedure was as follows: The ultrathin carbon membrane was pre-vacuumed for 3 min using a Harrick Basic PlasmaCleaner PDC-32G-2 instrument, followed by glow discharge at medium setting for 30 s, and then removed. A 4 μm sample was pipetted onto the carbon membrane, placed horizontally for 1 min, and then blotted dry with filter paper. Then, 7 μm of 2% uranium acetate was added, placed for 1 min, blotted dry with filter paper, and after several minutes, the negatively stained purified sample was observed under a transmission electron microscope (TEM) using a FEI Tecnai Arctica TEM D683.

[0124] Exemplary results are as follows Figure 2 As shown, the results indicate that the purified products of ferritin-PreF fusion proteins PI15-PreF-NP and MP67-PreF-NP can be observed as regular nanoparticles (from left to right) under electron microscopy. Transmission electron microscopy analysis also revealed clear nanoparticle morphology and good particle integrity.

[0125] Example 4: Stability test of ferritin-PreF fusion protein To verify the stability of the ferritin-PreF fusion protein prepared in this invention, purified ferritin-PreF fusion proteins PI12-PreF-NP, PI15-PreF-NP, MP39-PreF-NP, MP67-PreF-NP, and MP68-PreF-NP were selected for physical stability (physical environmental challenge) tests. The specific steps are as follows: Five solutions of 40 μg / μL ferritin-PreF fusion proteins PI15-PreF-NP and MP67-PreF-NP, as well as serially diluted solutions of ferritin-PreF fusion proteins PI15-PreF-NP and MP67-PreF-NP, were placed in environments of pH 5.0 (25℃), pH 3.8 (25℃), pH 10 (25℃), 50℃ (pH 5.0), and 70℃ (pH 5.0) for 1 hour, respectively, and were analyzed by ELISA using the same method as cell clone screening.

[0126] The results are shown in Table 5-9. The results indicate that the antigen-binding activity of the protein remained above 75% of that of the original untreated protein after treatment with different pH and temperature conditions. This demonstrates that the protein possesses sufficient physical stability.

[0127] Table 5. Antigen binding activity of PI12-PreF-NP after treatment at different pH and temperature.

[0128] Table 6. Antigen binding activity of PI15-PreF-NP after treatment at different pH and temperature.

[0129] Table 7. Antigen binding activity of MP39-PreF-NP after treatment at different pH and temperature.

[0130] Table 8. Antigen binding activity of MP67-PreF-NP after treatment at different pH and temperature.

[0131] Table 9. Antigen binding activity of MP68-PreF-NP after treatment at different pH and temperature.

[0132] Example 5: Immunogenicity Study of the Triple Fusion Protein Investigating the immunogenicity of mice immunized with three proteins: RSV-PreF-B-NP, PI15-PreF-NP, and MP67-PreF-NP. I. Immunity 1. Experimental Materials and Methods Experimental materials: 6-8 week old female Balb / c mice (Speford (Beijing) Biotechnology Co., Ltd., catalog number: B201-02).

[0133] Experimental methods: Sixty-four 6-8 week old Balb / c mice were randomly divided into 8 groups of 8 mice each. The treatment methods for each group were as follows: The fusion proteins RSV-PreF-B-NP, PI15-PreF-NP, and MP67-PreF-NP (Program 1) were grouped according to Table 10 below and administered twice via intramuscular injection into the thigh on day 0 and day 21, using 50 μg aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05) and 100 μL PBS buffer (Solarbio, catalog number P1020). Serum samples were collected 14 days after the second immunization.

[0134] Table 10 Grouping of each immunization group

[0135] Simultaneously, the three proteins RSV-PreF-B-NP, PI12-PreF-NP, and MP68-PreF-NP (Scheme 2) were grouped according to Table 11 below and administered twice via intramuscular injection in the thigh on day 0 and day 21, using 50 μg aluminum hydroxide adjuvant (Croda, model: AJV3012 / 0250 / VP05) and 100 μL PBS buffer (Solarbio, catalog number P1020). Serum samples were collected 14 days after the second immunization.

[0136] Table 11 Grouping of each immunization group

[0137] II. Mouse serum CPE neutralization test Based on the neutralization method described above, neutralization titer analyses were performed on RSV, MPV, and PIV respectively, and the neutralization titers are shown in Tables 12 and 13 below.

[0138] Table 12 Neutralizing titers in each immunization group of Scheme 1

[0139] Table 13 Neutralizing titers in each immunization group of Scheme 2

[0140] Data showed that in combined immunization with two or three of the three proteins RSV-PreF-B-NP, PI15-PreF-NP, MP67-PreF-NP (Option 1) or RSV-PreF-B-NP, PI12-PreF-NP, MP68-PreF-NP (Option 2), neutralizing protective titers were produced that were no lower than those produced by immunization alone or that were not significantly different from those produced by immunization alone, with Option 1 being superior to Option 2.

[0141] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been provided, it should be understood that further modifications can be made to the invention.

[0142] In summary, based on the principles of this invention, this application is intended to include any modifications, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A protein, characterized in that, The proteins include protein M and / or protein P, wherein, The protein M includes a mutant of the F protein of human metapneumovirus (HMPV); The protein P includes a mutant of the F protein of human parainfluenza virus (HPIV); The F protein mutant of the HMPV has mutations at positions 116, 128, 230 and / or 269 relative to the wild-type F protein. The HPIV F protein mutant has mutations relative to the wild-type F protein, including at positions 94, 143, 148, 172, 197, 294, 390, and / or 423.

2. The protein according to claim 1, characterized in that, The protein also includes protein R, which includes the F protein of Human Respiratory Syncytial Virus (HRSV). Preferably, the protein R, protein M, and / or protein P further include a signal peptide. More preferably, the F protein in protein R, protein M and / or protein P is directly or indirectly linked to antibody Fc structural fragments, foldon domains, ferritin, lumazine synthase (LS), virus-like particles (VLP), dihydrothioacetyltransferase (E2p), I53-50 nanoparticles, etc., to form a multimeric structure.

3. The protein according to any one of claims 1-2, characterized in that, The HMPV F protein mutant also has mutations at positions 183, 436, 437 and / or 441 relative to the wild-type F protein. The HPIV F protein mutant, relative to the wild-type F protein, also includes mutations at positions 489-491. Preferably, the mutation includes The F protein mutant of the HMPV has mutations of V116L, E128D, L183R, R230N, K269Q, Q437C and / or D441C relative to the wild-type F protein. The HPIV F protein mutant has K94R, E143D, K148R, V172I, L197R, R294N, V390L, E423N and / or deletion of positions 489-491 relative to the wild-type F protein. More preferably, The amino acid sequence of protein R includes (A1) The amino acid sequence shown in positions 1-475 of SEQ ID No. 1, preferably the amino acid sequence shown in SEQ ID No.

1. (A2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (A1); (A3) and any one of (A1)-(A2) that has more than 80% identity and the same function; The protein M includes: (B1) The amino acid sequence shown in positions 1-471 of SEQ ID No. 3 or positions 1-471 of SEQ ID No. 4, preferably, the amino acid sequence shown in either SEQ ID No. 3 or 4. (B2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (B1); (B3) is a protein that shares more than 80% identity with and has the same function as any of (B1)-(B2). And / or, The protein P includes: (C1) The amino acid sequence shown in positions 1-488 of SEQ ID No. 6, preferably the amino acid sequence shown in SEQ ID No.

6. (C2) A protein with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of (C1); (C3) and any one of (C1)-(C2) that has more than 80% identity and the same function.

4. A nucleotide molecule, characterized in that, The nucleotide molecule encodes the protein according to any one of claims 1-3; Preferably, the nucleotide molecule comprises DNA and / or RNA; more preferably, the nucleotide molecule comprises: (D1), positions 1-1425 of SEQ ID No: 7, positions 1-1413 of SEQ ID No: 9, positions 1-1413 of SEQ ID No: 10, and / or positions 1-1464 of SEQ ID No: 12, preferably, the sequences shown in SEQ ID No. 7, 9, 10, and / or 12. The complementary, degenerate, or transcribed sequences of (D2) and (D1), (D3) A DNA molecule or mRNA that has more than 75% identity with the DNA molecule or mRNA defined by (D1) or (D2) and encodes the corresponding protein in the protein.

5. A carrier, characterized in that, The carrier comprises the nucleotide molecule of claim 4.

6. A host cell, characterized in that, The host cell comprises the nucleotide molecule of claim 4 or the vector of claim 5.

7. A vaccine, characterized in that, The vaccine comprises the protein of any one of claims 1-3, the nucleotide molecule of claim 4, the vector of claim 5, or the host cell of claim 6. Preferably, the vaccine further comprises an adjuvant.

8. A combination vaccine, characterized in that, The combined vaccine comprises a combination of proteins, wherein the combination of proteins includes any two or three of proteins M, P, and R. The protein M includes the F protein of human metapneumovirus (HMPV); The protein P includes the F protein of human parainfluenza virus (HPIV3); The protein R includes the F protein of Human Respiratory Syncytial Virus (HRSV); Preferably, the combination of proteins includes: 1) Protein M and Protein P; 2) Protein M and Protein R 3) Protein P and Protein R; 4) Protein M, Protein P, and Protein R; Preferably, the combined vaccine comprises a combination of proteins according to any one of claims 1-3, a combination of nucleotide molecules according to claim 4, a combination of vectors according to claim 5, or a combination of host cells according to claim 6; Preferably, the combined vaccine also includes an adjuvant.

9. A method for preparing the protein according to any one of claims 1-3, the vaccine according to claim 7, or the combination vaccine according to claim 8, characterized in that, The preparation method includes introducing the nucleotide molecule of claim 4 or the vector of claim 5 into a host cell and culturing the host cell.

10. The application of any one of the proteins of claims 1-3, the nucleotide molecule of claim 4, the vector of claim 5, the host cell of claim 6, the vaccine of claim 7, or the combination vaccine of claim 8, wherein the application comprises any one of the following: (1) Use in the preparation of products for the prevention and / or treatment of diseases caused by HRSV, HMPV and / or HPIV viral infections; (2) Use in the preparation of products for inducing immune responses to HRSV, HMPV and / or HPIV viral antigens; (3) Use in the prevention and / or treatment of diseases caused by HRSV, HMPV and / or HPIV viral infections; (4) Use in inducing immune responses to HRSV, HMPV and / or HPIV viral antigens.

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