Human metapneumovirus F protein mutant and application thereof

Abstract

The invention relates to the technical field of virology, immunology and biomedicine, in particular to a human metapneumovirus F protein mutant and application thereof. The human metapneumovirus F protein mutant provided by the invention has stable prefusion conformation and high expression quantity, and can form a stable trimer, so that the human metapneumovirus F protein mutant can be used for HMPV vaccine development. The F protein mutant provided by the invention has excellent immunogenicity, and can induce excellent immune response when being used together with an HA201 composite adjuvant (QS21 + MPL liposome).

Description

Technical Field

[0001] The present application relates to the technical fields of virology, immunology and biomedicine, and in particular to a human metapneumovirus F protein mutant and its application. Background Art

[0002] Human metapneumovirus (HMPV) is a respiratory virus belonging to the Paramyxoviridae, Pneumovirinae, and Metapneumovirus genus. It is a single-stranded negative-sense RNA virus. Currently, the treatment of HMPV infection is mainly supportive, and no HMPV vaccine or specific drug has been approved for marketing. Several vaccines are in the clinical trial stage abroad, and there are no reports on the development of HMPV vaccines in China. Given the widespread prevalence of HMPV around the world, the severe symptoms it causes in young children, the elderly, and people with immunodeficiency, and the lack of prevention for this virus. The development of an HMPV vaccine is crucial to reducing the burden of the disease, especially given that the virus has a unique ability to modulate host immunity, so that after natural HMPV infection, weak adaptive immunity is formed, leading to repeated infections.

[0003] However, for a variety of reasons, the traditional HMPV vaccine development method is not applicable. Studies have shown that in the cotton rat model, the use of formalin-inactivated HMPV vaccine for primary and booster immunization, followed by homologous virus challenge, showed that the levels of interstitial pneumonia and alveolitis in the vaccinated group were increased or aggravated, unlike the primary or secondary infection groups. Therefore, it is urgent to develop a new, safe, effective HMPV vaccine that can provide long-term protection.

[0004] HMPV is divided into two genotypes, A and B, which are further divided into four subtypes, A1, A2, B1 and B2. Its genome is about 13kb long and encodes 9 proteins. Among them, the F protein is essential for membrane fusion and can mediate cell fusion without the participation of adsorption proteins, that is, the F protein of HMPV can both adsorb cell receptors and activate the membrane fusion process mediated by it, and has the dual functions of adsorption and fusion. The F protein plays an important role in inducing immune protection, high levels of serum and antibodies. In addition, the nucleotide sequence of the HMPV F protein is highly conserved, and the homology between the A and B types can reach 95%.

[0005] Therefore, the protective antibodies induced by F protein can resist infection of different subtypes. In view of the high immunogenicity and high conservation of F protein, HMPV F protein has become the main target antigen of candidate vaccines. Summary of the invention

[0006] The present application provides a human metapneumovirus F protein mutant and its application.

[0007] The human metapneumovirus F protein mutant provided in this application has a stable pre-fusion conformation and high expression level, and can form a stable trimer, so it can be used for HMPV vaccine development. The F protein mutant provided in this application has excellent immunogenicity and can induce excellent immune response when used with HA201 composite adjuvant (QS-21+MPL liposome).

[0008] In the first aspect, the present application provides a human metapneumovirus F protein mutant, using the following technical solution: A human metapneumovirus F protein mutant, wherein the human metapneumovirus F protein mutant has an amino acid sequence as described in any one of SEQ ID No. 18-21.

[0009] The human metapneumovirus F protein mutant comprises at least one mutation selected from the following group: Soluble expression was achieved by C-terminal truncation; Improve trimer stability by tandem trimerization domains at the C-terminus; By adding a C-terminal tag, protein purification is facilitated; Stabilize the prefusion conformation through point mutations; By replacing the restriction site sequence, the efficiency of F0 enzymatic hydrolysis into F2 and F1 subunits can be improved.

[0010] Optionally, the human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No. 18.

[0011] Optionally, the human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No. 19.

[0012] Optionally, the human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No. 20.

[0013] Optionally, the human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No. 21.

[0014] The present application provides a method for preparing the above-mentioned human metapneumovirus F protein mutant, using the following technical scheme: A method for preparing the above-mentioned human metapneumovirus F protein mutant specifically comprises the following steps: S1 culturing the host cells described above under conditions suitable for the expression of human metapneumovirus F protein; S2 collects the expression products and purifies the expression products to obtain human metapneumovirus F protein mutants.

[0015] Further, the S1 includes: S11 cloned the gene containing human metapneumovirus F protein into an expression vector; S12 transforms the expression vector obtained in S11 into genetically engineered cells; S13 obtained a cell line that stably expressed human metapneumovirus F protein through screening of cell pools; S14 uses the cell line obtained in S13 for expression to obtain a cell culture supernatant containing human metapneumovirus F protein.

[0016] Furthermore, the genetically engineered cells are CHO cells.

[0017] Furthermore, the expression vector is a plasmid expression vector carrying a GS screening system and / or a bleomycin resistance gene.

[0018] In a second aspect, the present application provides a gene that expresses the human metapneumovirus F protein mutant.

[0019] In a third aspect, the present application provides an expression vector comprising the above gene.

[0020] In a fourth aspect, the present application provides a host cell that expresses the above gene or the above expression vector.

[0021] In a fifth aspect, the present application provides a use of the above-mentioned human metapneumovirus F protein mutant in the preparation of a vaccine for preventing HMPV infection.

[0022] In a sixth aspect, the present application provides a vaccine for preventing HMPV infection, using the following technical solution: A vaccine for preventing HMPV infection, comprising the human metapneumovirus F protein mutant and a pharmaceutically acceptable carrier or excipient.

[0023] The present application also provides a method for preparing the above-mentioned vaccine for preventing HMPV infection, using the following technical solution: A method for preparing a vaccine for preventing HMPV infection specifically comprises the following steps: separately packaging or fully mixing the purified F protein and adjuvant according to a certain proportion.

[0024] Furthermore, the adjuvant may be the adjuvant system HA201.

[0025] Furthermore, the adjuvant system HA201 is a liposome mixture. The present application obtains a new adjuvant system by adjusting the ratio and preparation method of the adjuvant system.

[0026] The present application also provides a method for preparing a liposome mixture, which adopts the following technical scheme: A method for preparing a liposome mixture specifically comprises the following steps: dissolving 3D-MLA, DOPC and cholesterol with isopropanol, evaporating and drying to form a liposome membrane, adding PBS buffer solution for hydration, homogenizing with a high-pressure homogenizer until the particle size reaches 90-110nm, adding QS-21, diluting, sterile filtering and then packaging to obtain the mixture.

[0027] In summary, the present application includes at least one of the following beneficial technical effects: The present application obtains an F extracellular domain trimer protein of the HMPV prefusion conformation capable of exposing more neutralizing antibody epitopes by replacing and deleting multiple amino acids in the wild-type HMPV F protein, while maintaining a stable prefusion conformation and ensuring that it can induce effective neutralizing antibody response and antibody binding response to HMPV.

[0028] In addition, the present application uses the F extracellular domain trimer protein in the pre-fusion conformation of HMPV (human metapneumovirus F protein mutant) as an immunogen combined with an adjuvant, which achieves a stronger immune induction efficacy than the HMPV postF protein, improves the humoral immune response and the production of neutralizing antibodies, and enhances the immunogenicity of the HMPV antigen.

[0029] The mutation method disclosed in the present application is applicable to other virus strains of human HMPV; it is applicable to various vaccine forms with HMPV F protein as antigen, such as recombinant protein vaccine, nucleic acid vaccine, virus-like particle vaccine and vector vaccine, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a structural diagram of the expression vector pGS in the example of the present application.

[0031] Figure 2 This is the screening result of the high-yield stable cell pool in the examples of the present application.

[0032] Figure 3 It is the human metapneumovirus F protein mutant purified in the examples of the present application.

[0033] Figure 4 It is the neutralizing antibody titer in the examples of this application. DETAILED DESCRIPTION

[0034] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are only used for the purpose of describing specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by ordinary technicians in the field to which the terms belong.

[0035] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of this application, unless otherwise specified, the meaning of "plurality" is two or more.

[0036] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] In the present application, the terms "comprise" or "include" are open expressions, that is, including the contents specified in the present application but not excluding other contents.

[0038] The present application prepares mutants based on the full-length F protein sequence of HMPV A2 subtype (SEQ ID No. 1, 539 amino acids).

[0039] The human metapneumovirus F protein mutant provided in the present application comprises at least one mutation selected from the following groups: Soluble expression is achieved through C-terminal truncation: the transmembrane domain and the C-terminal intracellular domain of the HMPV F protein are removed, and the truncated extracellular domain sequence is expressed as a single polypeptide precursor in the host cell through gene recombination, which can be named HMPV F protein extracellular domain (SEQ ID No. 2), with a length of 485 amino acids.

[0040] The HMPV F protein ectodomain includes an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where the 1-18 residues of the HMPV F protein signal peptide sequence (SEQ ID No. 3) are hydrolyzed and removed to form the mature F protein ectodomain proprotein F0 (SEQ ID No. 4, 467 amino acids).

[0041] Improving trimer stability by tandemly connecting trimerization domains: The recombinant F protein extracellular domain has weak trimerization ability due to the lack of a transmembrane domain and a C-terminal intracellular domain. By adding a trimerization sequence to its C-terminus, such as the C-terminal trimerization domain foldon of T4 fibrin (SEQ ID No. 5) or the long α-helical trimerization domain LAH of the stem domain of influenza virus hemagglutinin (HA) (SEQ ID No. 6), the tandem expression of the trimerization domain and the target protein can not only form a stable trimer structure and improve the stability of the protein, but also increase the particle size of the antigen and improve the immunogenicity of the antigen.

[0042] Add C-terminal tags to facilitate protein purification: HRV 3C restriction site (SEQ ID No. 7), His (SEQ ID No. 8) and strepII (SEQ ID No. 9) purification tags were added to facilitate protein purification. The sequences of the recombinant F protein extracellular domain trimer with purification tags are F0-Fd (SEQ ID No. 10) and F0-LAH (SEQ ID No. 11).

[0043] Stabilize the pre-fusion conformation by point mutation: The recombinant F protein extracellular domain trimer is unstable, and its pre-fusion conformation undergoes a dramatic structural rearrangement to convert to a post-fusion conformation. By introducing multiple amino acid substitutions, the F protein extracellular domain trimer is stably "locked" in the pre-fusion conformation to improve its solubility, stability, expression and immunogenicity. The amino acid substitutions introduced into the F protein extracellular domain are: V155P, A185P, A140C, A147C. The HR1 region is stabilized by two proline substitutions, and a new disulfide bond is formed by two cysteine ​​substitutions to prevent the recombinant F protein from transforming to the postF conformation. The pre-fusion conformation of the F protein extracellular domain preF (SEQ ID No. 12) and the C-terminal tandem trimerization domain form a stable pre-fusion conformation of the F protein extracellular domain trimer preFF0-Fd (SEQ ID No. 13) and preFF0-LAH (SEQ ID No. 14).

[0044] The F0 protein is converted into disulfide-bonded F2 and F1 subunits by enzymatic hydrolysis. The natural HMPV F protease cleavage site sequence is the RQSR sequence of residues 99-102. In cell culture, trypsin can be added to achieve protein cleavage at this cleavage site, but incomplete enzymatic cleavage will result in poor uniformity of the recombinant protein. The C-terminal 91-102 residue sequence of the F2 polypeptide chain of the HMPV F protein (SEQ ID No. 15) was replaced with a connecting peptide sequence of glycine and serine (SEQID No. 16), and the connecting peptide was used to directly connect the F2 and F1 subunits to improve the uniformity of the recombinant F protein. The sequences of the F protein extracellular domain trimer in the pre-fusion conformation of the single-chain form are: preF-SC-Fd (SEQ ID No. 18), preF-SC-LAH (SEQ ID No. 19) In addition, the C-terminal 91-102 residue sequence of the F2 polypeptide chain of the HMPV F protein (SEQ ID No. 15) can be replaced with the RSV F protein P27 polypeptide sequence (SEQ ID No. 17) to improve the efficiency of enzyme cleavage and thus improve the uniformity of the recombinant F protein. The sequences of the F protein extracellular domain trimer in the pre-fusion conformation of the enzyme cleavage form are: preF-P27-Fd (SEQ ID No. 20), preF-P27-LAH (SEQ ID No. 21).

[0045] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without making creative work belong to the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application, and cannot be interpreted as limiting the present application.

[0046] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0047] The present application is further described in detail below in conjunction with the embodiments and test results. Example Example 1

[0048] This example provides HMPV F protein codon optimization and full gene synthesis.

[0049] The gene of the HMPV F protein was codon optimized by avoiding commonly used restriction sites; according to the codon preference in CHO cells, the codons with low frequency were replaced by codons with high frequency to control the rare codons; the GC content in the sequence was controlled to be 40% to 60% to improve the transcription efficiency of mRNA, while avoiding the high GC content affecting the secondary structure of mRNA and further affecting the translation efficiency, that is, it was expressed as a single polypeptide precursor in the host cell through gene recombination, which can be named as the F protein extracellular domain trimer in the pre-fusion conformation (SEQ ID No. 21).

[0050] A signal peptide sequence was added in front of the optimized sequence; a BamHⅠ restriction site and a Kozak sequence were introduced upstream of the sequence, and a stop codon and a PmlⅠ restriction site were added downstream of the sequence to perform whole genome synthesis of the nucleotide sequence. Example 2

[0051] This example provides the construction of an HMPV F protein expression plasmid.

[0052] The cloning vector containing the complete gene synthesis sequence was transformed into DH5α competent bacteria, and then amplified in large quantities. After the plasmid was extracted, the cloning vector was double-digested with restriction endonucleases BamHⅠ and PmlⅠ, and the expression vector pGS (such as Figure 1 As shown). The HMPV F protein gene part was recovered by gel cutting of the cloning vector with double enzyme digestion, and the backbone part was recovered by gel cutting of the expression vector. The two were connected with T4 enzyme and transformed into DH5α competent bacteria, and the plates containing ampicillin resistance were coated for screening. The positive colonies were amplified and plasmids were extracted, and then identified by double enzyme digestion with BamHI and Pml, and the correct recombinant expression vector was sequenced and verified. Example 3

[0053] This example provides a process for screening a high-yield stable cell pool.

[0054] (1) Identify the correct recombinant expression vector, extract a large amount of plasmid after bacterial proliferation, cut the recombinant expression vector with Pvu I, recover the linearized vector after enzyme cutting by gel cutting, filter and sterilize it for later use.

[0055] (2) After CHO cells are revived, they are subcultured for more than two times and used for electroporation when the cell viability is greater than 95%.

[0056] (3) In a clean bench, add 0.6 ml of cell suspension (about 0.5 × 10 7cells), 50ul of the linearized recombinant expression vector (about 40μg), and set the electroporation conditions to 300V and 950μF. After electroporation, transfer the cells to a T-25 culture flask containing 5ml CD CHO Fusion (supplemented with 6mM Gln) medium and resuspend them. Transfer the culture flask to a 37℃, 8% carbon dioxide concentration incubator and culture for 24 hours.

[0057] (4) Resuspend the cell suspension in Minipool medium (20% CD CHO Fusion and 80% CHO Cloning Medium) at a rate of 200 μl per well and 5 × 10 3 The cells were added to a 96-well plate at a density of 10 cells / well. The culture plate was then transferred to an incubator at 37°C, 80% humidity, and 8% carbon dioxide concentration for static culture.

[0058] (5) During the culture process, observe the growth status of the cells. On the 10th day, add 50ul of CD CHOFusion medium to each well.

[0059] (6) After 19 days of culture in 96-well plates, 192 Minipools were selected based on the confluence for ELISA testing (using recombinant HMPV01 antibodies for testing, the light chain of HMPV01 antibodies has an amino acid sequence as described in SEQ ID No. 22, and the heavy chain of HMPV01 antibodies has an amino acid sequence as described in SEQ ID No. 23). The Top120 Minipools were selected based on the test results and transferred to 24-well plates for culture. After 3 days of culture in 24-well plates, ELISA testing was performed. The Top30 Minipools were selected based on the test results and transferred to 6-well plates for culture. After 4 days of culture in 6-well plates, ELISA testing was performed. The Top15 Minipools were selected based on the test results and transferred to SF125 for culture. After 3 days of culture, ELISA testing was performed. Finally, the cells in the Top8 cell pools with the highest expression were selected for cryopreservation. (As Figure 2 as shown). Example 4

[0060] This example provides fed-batch culture of stably transfected cells.

[0061] After the above screening, the cells of the top8 stable cell pool were obtained for fed batch culture (FB). The FB scheme is shown in Table 1. During the FB culture process, the viable cell density (VCD), cell viability (Via), glucose content (Glu) and lactic acid content (Lac) were recorded. After 14 days of FB culture, the peak viable cell density of the stable cells ranged from 41.9×10 6 Up to 52.5×10 6 cells / ml; when harvested, the cell viability was maintained in the range of 90% to 99%.

[0062] Table 1 Minipool FB culture scheme Example 5

[0063] This example provides HMPV F protein purification and stability testing.

[0064] The FB culture supernatant was collected by centrifugation and filtered with a 0.45um vacuum filter to remove impurities such as cell debris. The filtered FB culture supernatant was purified by affinity chromatography and anion chromatography to obtain a high-purity F protein. The purity of preF F0-Fd (SEQ ID No. 13), preF-SC-Fd (SEQ ID No. 18) and preF-P27-Fd (SEQ ID No. 20) proteins was above 90% by RP-HPLC and SEC-HPLC, and the proportion of trimers was above 95%. The three preF proteins were stable at 2-8°C (as shown in Tables 2 and 3).

[0065] Purified preF-P27-Fd (SEQ ID No. 20) showed a single band of 58 kDa under non-reducing SDS-PAGE, and F1 subunit and F2 subunit bands were visible under reducing SDS-PAGE, indicating that the enzyme cleavage was relatively complete. No protein aggregation or degradation was observed after being placed at 4°C or 37°C for 3 days or at 50°C for 1 day, indicating that its stability was good (e.g. Figure 3 as shown).

[0066] Table 2 RP-HPLC results of three F proteins stored at 2-8℃ for 5 days

[0067] Table 3 SEC-HPLC results of three F proteins stored at 2-8℃ for 5 days Example 6

[0068] This example provides the process of vaccine formulation.

[0069] The F protein with a purity of more than 90% obtained in Example 5 was mixed with the liposome mixture HA201 to obtain a recombinant HMPV vaccine.

[0070] The liposome mixture HA201 is prepared by dissolving 40-60 mg 3D-MLA, 800-1200 mg DOPC and 200-300 mg cholesterol in 20-30 mL isopropanol, and after evaporation and drying to form a liposome membrane, adding 40-60 ml PBS buffer solution for hydration, homogenizing with a high-pressure homogenizer until the particle size reaches 90-120 nm, adding 40-60 mg QS-21, and after dilution, sterile filtering and subpackaging are performed. Example 7

[0071] This example provides the process of animal immunization.

[0072] Female BALB / C mice aged 6-8 weeks were selected for immunization and randomly divided into groups, 6 mice per group, including negative control group, preF F0-Fd (SEQ ID No. 13) + HA201 group, preF-SC-Fd (SEQ ID No. 18) + HA201 group, preF-P27-Fd (SEQ ID No. 20) + HA201 group and postF (SEQ ID No. 2) + HA201 group. The specific groups and numbers are shown in Table 4.

[0073] Table 4 Grouping and numbering of immunized mice

[0074] The animals in the negative control group were given PBS buffer; the test group was given 10 μg of the target protein and 25 μL of HA201 adjuvant. The vaccine of Example 6 was diluted to contain 100 μg / ml of F protein for animal experiment, and the injection volume was 100 uL / animal.

[0075] The immunization method was 2 times of hind limb intramuscular injection per mouse, with an interval of 3 weeks. Blood was collected from each group of mice two weeks after the second immunization, and the collected serum was stored at -80°C for subsequent neutralizing antibody detection. Example 8

[0076] This example provides an immunogenicity assessment. The method is neutralizing antibody detection. The specific steps are: (1) Incubate the immunized serum in a 56°C water bath for 30 min; (2) The inactivated serum samples were diluted 4-fold in a 96-well plate using DMEM medium containing 2% FBS at an appropriate multiple, for a total of 6 gradients, with 4 replicate wells for each sample; (3) Add 120 PFU of HMPV A2 and HMPV B strains to each well, mix well, and incubate at 37°C, 5% CO2 for 1 h. (4) Transfer the virus and serum mixture to a 96-well plate pre-inoculated with Vero cells for adsorption. Add a cover and place in a 37°C, 5% CO2 incubator for incubation; (5) Fix with 4% paraformaldehyde at room temperature and then add Triton X-100 for permeabilization. Block at 37°C, add Anti-HMPVF protein primary antibody, and incubate at 37°C. Add HRP-labeled goat anti-human IgG and incubate at 37°C; add TrueBlue colorimetric solution until the spots are clear; (6) Record the number of plaques in each well and calculate the neutralizing antibody titer using the Karber method.

[0077] Test results such as Figure 4 shown.

[0078] like Figure 4 The results showed that compared with the negative control PBS group, the sera of mice in each experimental group two weeks after the second immunization could induce higher levels of neutralizing antibodies, and the sera of mice in each experimental group three weeks after the second immunization could effectively neutralize the replication of type A and type B wild-type live viruses in in vitro cells.

[0079] There was no significant difference in the neutralizing antibody levels induced by the three HMPV preF proteins of the present application, but the neutralizing antibodies induced by them were significantly higher than those of the HMPV postF protein (SEQ ID No. 2).

[0080] In summary, the HMPV F protein mutant provided in the present application can induce a higher level of neutralizing antibody titer than HMPV postF, and when used as a vaccine product, it can induce cross-protection against both A and B strains.

[0081] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A human metapneumovirus F protein mutant, characterized in that: The human metapneumovirus F protein mutant is an amino acid sequence described in any one of SEQ ID Nos. 18-21.

2. The human metapneumovirus F protein mutant according to claim 1, characterized in that The human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No.

18.

3. The human metapneumovirus F protein mutant according to claim 1, characterized in that: The human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No.

19.

4. The human metapneumovirus F protein mutant according to claim 1, characterized in that The human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No.

20.

5. The human metapneumovirus F protein mutant according to claim 1, characterized in that: The human metapneumovirus F protein mutant is the amino acid sequence described in SEQ ID No.

21.

6. A gene, characterized in that The gene expresses the human metapneumovirus F protein mutant described in claim 1.

7. An expression vector, characterized in that: The expression vector comprises the gene according to claim 6.

8. A host cell, characterized in that The host cell expresses the gene according to claim 6 or the expression vector according to claim 7.

9. Use of the human metapneumovirus F protein mutant according to claim 1 in the preparation of a vaccine for preventing HMPV infection.

10. A vaccine for preventing HMPV infection, characterized in that: The vaccine comprises the human metapneumovirus F protein mutant according to claim 1, and a pharmaceutically acceptable carrier or excipient.

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