A mutant of human metapneumovirus F protein and its application

Abstract

This application relates to the technical fields of virology, immunology, and biomedicine, specifically to a human metapneumovirus (HMPV) F protein mutant and its applications. The HMPV F protein mutant provided in this application has a stable pre-fusion conformation and high expression level, and can form a stable trimer, thus making it suitable for HMPV vaccine development. The F protein mutant provided in this application exhibits excellent immunogenicity, and when used in conjunction with the HA201 adjuvant complex (QS21+MPL liposomes), it can induce a superior immune response.

Description

Technical Field

[0001] This application relates to the fields of virology, immunology and biomedicine, and in particular to a human metapneumovirus F protein mutant and its applications. Background Technology

[0002] Human metapneumovirus (HMPV) is a respiratory virus belonging to the family Paramyxoviridae, subfamily Pneumovirinae, and genus Metapneumovirus. It is a single-stranded negative-sense RNA virus. Currently, HMPV infection treatment is mainly supportive, and no HMPV vaccine or specific drugs have been approved for marketing. Several vaccines are in clinical trials abroad, but there are no reports of HMPV vaccine development in China. Given the widespread prevalence of HMPV globally, the severe symptoms it causes in young children, the elderly, and immunocompromised individuals, and the lack of preventative measures against this virus, developing an HMPV vaccine is crucial for reducing the disease burden, especially considering the virus's unique ability to modulate host immunity. Therefore, natural HMPV infection often results in weak adaptive immunity, leading to reinfection.

[0003] However, traditional HMPV vaccine development methods are not applicable for various reasons. Studies have shown that in a rat model, primary immunization with a formalin-inactivated HMPV vaccine followed by a booster immunization, and then challenge with a homologous virus, resulted in increased or worsened levels of interstitial pneumonia and alveolitis in the vaccinated group, unlike the primary or secondary infection group. Therefore, the development of a novel, safe, effective, and long-term protective HMPV vaccine is urgently needed.

[0004] HMPV is classified into two genotypes, A and B, which are further subdivided into four subtypes: A1, A2, B1, and B2. Its genome is approximately 13 kb long and encodes nine proteins. Among these, the F protein is essential for membrane fusion and can mediate cell fusion without the involvement of adsorption proteins. In other words, the HMPV F protein can both adsorb onto cell receptors and activate the membrane fusion process it mediates, exhibiting a dual function of adsorption and fusion. The F protein plays a crucial role in inducing immune protection, high serum levels, and antibodies. Furthermore, the nucleotide sequence of the HMPV F protein is highly conserved, with up to 95% homology between the A and B types.

[0005] Therefore, the protective antibodies induced by the F protein can resist infections of different subtypes. Given the high immunogenicity and high conservation of the F protein, the HMPV F protein has become a major target antigen for candidate vaccines. Summary of the Invention

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

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

[0008] In a first aspect, this application provides a human metapneumovirus F protein mutant, employing the following technical solution:

[0009] A mutant human metapneumovirus F protein, wherein the mutant human metapneumovirus F protein has the amino acid sequence described in any one of SEQ ID No. 18-21.

[0010] The human metapneumovirus F protein mutant contains at least one mutation selected from the group consisting of:

[0011] Soluble expression is achieved by truncating the C-terminus;

[0012] Improve trimer stability by connecting trimerization domains in series at the C-terminus;

[0013] Adding a C-terminal tag facilitates protein purification;

[0014] Stabilize the pre-fusion conformation through point mutation;

[0015] By substituting the restriction site sequence, the efficiency of F0 enzyme cleavage into F2 and F1 subunits can be improved.

[0016] Optionally, the human metapneumovirus F protein mutant has the amino acid sequence described in SEQ ID No. 18.

[0017] Optionally, the human metapneumovirus F protein mutant has the amino acid sequence described in SEQ ID No. 19.

[0018] Optionally, the human metapneumovirus F protein mutant has the amino acid sequence described in SEQ ID No. 20.

[0019] Optionally, the human metapneumovirus F protein mutant has the amino acid sequence described in SEQ ID No. 21.

[0020] This application provides a method for preparing the above-mentioned human metapneumovirus F protein mutant, using the following technical solution:

[0021] A method for preparing the above-mentioned human metapneumovirus F protein mutant specifically includes the following steps:

[0022] S1 was cultured in host cells as described above under conditions suitable for human metapneumovirus F protein expression.

[0023] S2 collects the expression product and purifies it to obtain the human metapneumovirus F protein mutant.

[0024] Furthermore, S1 includes:

[0025] S11 cloned the gene containing the human metapneumovirus F protein into the expression vector;

[0026] S12 transforms the expression vector obtained in S11 into genetically engineered cells;

[0027] S13 obtained a cell line that stably expresses human metapneumovirus F protein through cell pool screening;

[0028] S14 uses the cell line obtained from S13 for expression, and obtains cell culture supernatant containing human metapneumovirus F protein.

[0029] Furthermore, the genetically engineered cells are CHO cells.

[0030] Furthermore, the expression vector is a plasmid expression vector carrying the GS screening system and / or carrying the bleomycin resistance gene.

[0031] Secondly, this application provides a gene that expresses the aforementioned human metapneumovirus F protein mutant.

[0032] Thirdly, this application provides an expression vector. The expression vector includes the aforementioned gene.

[0033] Fourthly, this application provides a host cell. This host cell expresses the aforementioned gene or the aforementioned expression vector.

[0034] Fifthly, this application provides the use of the above-mentioned human metapneumovirus F protein mutant in the preparation of a vaccine for the prevention of HMPV infection.

[0035] Sixthly, this application provides a vaccine for preventing HMPV infection, employing the following technical solution:

[0036] A vaccine for the prevention of HMPV infection, said vaccine comprising the above-mentioned human metapneumovirus F protein mutant and a pharmaceutically acceptable vector or excipient.

[0037] This application also provides a method for preparing the above-mentioned vaccine for preventing HMPV infection, using the following technical solution:

[0038] A method for preparing a vaccine to prevent HMPV infection includes the following steps: packaging the purified F protein and adjuvant separately or mixing them thoroughly in a certain proportion.

[0039] Furthermore, the adjuvant can be the adjuvant system HA201.

[0040] Furthermore, the adjuvant system HA201 is a liposome mixture. This application has obtained a novel adjuvant system by adjusting the formulation and preparation method of the adjuvant system.

[0041] This application also provides a method for preparing a liposome mixture, using the following technical solution:

[0042] A method for preparing a liposome mixture includes the following steps: dissolving 3D-MLA, DOPC and cholesterol in 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 90-110 nm, adding QS-21, diluting, aseptically filtering and then dispensing to obtain the final product.

[0043] In summary, this application includes at least one of the following beneficial technical effects:

[0044] This application obtained an extracellular domain trimer protein of HMPV pre-fusion conformation that exposes more neutralizing antibody epitopes by substituting and deleting various amino acids in wild-type HMPV F protein. While maintaining a stable pre-fusion conformation, it ensures that it can induce effective neutralizing antibody response and antibody binding response to HMPV.

[0045] In addition, this application uses the F extracellular domain trimer protein of the pre-fusion conformation of HMPV (human metapneumovirus F protein mutant) as an immunogen adjuvant, which has a stronger immune induction effect than HMPV postF protein, improves humoral immune response and neutralizing antibody production, and enhances the immunogenicity of HMPV antigen.

[0046] The mutation methods disclosed in this application are applicable to other human HMPV viral strains; applicable to various vaccine forms that use HMPV F protein as an antigen, such as recombinant protein vaccines, nucleic acid vaccines, virus-like particle vaccines, and vector vaccines. Attached Figure Description

[0047] Figure 1 This is a structural diagram of the expression vector pGS in an embodiment of this application.

[0048] Figure 2 This is the screening result of a high-yield stable cell pool in the embodiments of this application.

[0049] Figure 3 It is a human metapneumovirus F protein mutant purified in the embodiments of this application.

[0050] Figure 4 This refers to the antibody titer in the embodiments of this application. Detailed Implementation

[0051] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0052] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0053] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0054] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0055] This application uses the full-length F protein sequence of HMPV A2 subtype (SEQ ID No. 1, 539 amino acids) as a basis to prepare mutants.

[0056] The human metapneumovirus F protein mutant provided in this application contains at least one mutation selected from the group consisting of:

[0057] Soluble expression is achieved by truncating the C-terminus: The transmembrane domain and the intracellular domain of the C-terminus 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. It can be named the extracellular domain of the HMPV F protein (SEQ ID No. 2), with a length of 485 amino acids.

[0058] The extracellular domain of the HMPV F protein includes an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where the HMPV F protein signal peptide sequence of residues 1-18 (SEQ ID No. 3) is hydrolyzed to form the mature F protein extracellular domain progenitor protein F0 (SEQ ID No. 4, 467 amino acids).

[0059] By tandemly adding trimerizing domains, the stability of the trimer can be improved: The extracellular domain of the recombinant F protein lacks a transmembrane domain and a C-terminal intracellular domain, resulting in weak trimerizing ability. Adding trimerizing sequences to its C-terminus, such as the C-terminal trimerizing domain foldon of T4 fibrin (SEQ ID No. 5) or the long α-helical trimerizing domain LAH of the stem domain of influenza virus hemagglutinin (HA) (SEQ ID No. 6), not only forms a stable trimer structure and improves protein stability after the trimerizing domain is tandemly expressed with the target protein, but also increases the particle size of the antigen and improves its immunogenicity.

[0060] Adding 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 are added to facilitate protein purification. The extracellular domain trimer sequences of the recombinant F protein with purification tags are F0-Fd (SEQ ID No. 10) and F0-LAH (SEQ ID No. 11).

[0061] By point mutation, the pre-fusion conformation is stabilized: the recombinant F protein extracellular domain trimer is conformationally unstable, and its pre-fusion conformation undergoes drastic structural rearrangement to transform into the post-fusion conformation. By introducing multiple amino acid substitutions, the F protein extracellular domain trimer is stably "locked" into 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, and A147C. The HR1 region is stabilized by two proline substitutions, while two cysteine ​​substitutions form new disulfide bonds, preventing the recombinant F protein from transitioning to the postF conformation. The pre-fusion conformation F protein extracellular domain preF (SEQ ID No. 12) has a C-terminal tandem trimeric domain, forming stable pre-fusion conformation F protein extracellular domain trimers preFF0-Fd (SEQ ID No. 13) and preFF0-LAH (SEQ ID No. 14).

[0062] The F0 protein is enzymatically hydrolyzed into disulfide-linked F2 and F1 subunits. The native HMPV F protein cleavage site sequence is an RQSR sequence of residues 99-102. In cell culture, trypsin can be added to cleave the protein at this site, but incomplete cleavage leads to poor homogeneity of the recombinant protein. The C-terminal sequence of the HMPV F protein F2 polypeptide chain, residues 91-102 (SEQ ID No. 15), is replaced with a glycine and serine linker peptide sequence (SEQ ID No. 16). This linker peptide directly links the F2 and F1 subunits, improving the homogeneity of the recombinant F protein. The resulting single-chain pre-fusion conformation F protein extracellular domain trimer sequences are: preF-SC-Fd (SEQ ID No. 18) and preF-SC-LAH (SEQ ID No. 19).

[0063] Furthermore, the C-terminal 91-102 residue sequence of the HMPV F protein F2 polypeptide chain (SEQ ID No. 15) can be replaced with the RSV F protein P27 polypeptide sequence (SEQ ID No. 17) to improve the enzyme digestion efficiency and thus enhance the uniformity of the recombinant F protein. The resulting F protein extracellular domain trimer sequences in the pre-fusion conformation formed by enzyme digestion are: preF-P27-Fd (SEQ ID No. 20) and preF-P27-LAH (SEQ ID No. 21).

[0064] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0065] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0066] The present application will be further described in detail below with reference to the embodiments and test results. Example Example 1

[0067] This embodiment provides codon optimization and whole-genome synthesis of the HMPV F protein.

[0068] The gene for the HMPV F protein was optimized using codons: commonly used restriction enzyme sites were avoided; based on the codon preference in CHO cells, high-frequency codons were used to replace low-frequency synonymous codons, and rare codons were controlled; the GC content in the sequence was controlled between 40% and 60% to improve the transcription efficiency of mRNA, while avoiding the influence of high GC content on the secondary structure of mRNA, which in turn affects the translation efficiency. That is, it was expressed as a single polypeptide precursor in the host cell through gene recombination, which can be named the pre-fusion conformation F protein extracellular domain trimer (SEQ ID No. 21).

[0069] A signal peptide sequence was added to the 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 PmlHI restriction site were added downstream of the sequence to perform whole-genome nucleotide sequence synthesis. Example 2

[0070] This embodiment provides the construction of the HMPV F protein expression plasmid.

[0071] A cloning vector containing the complete gene synthesis sequence was transformed into DH5α competent bacteria, followed by large-scale amplification. After plasmid extraction, the cloning vector was double-digested with restriction endonucleases BamHI and PmlHI, and the expression vector pGS (e.g., ...) was also double-digested with restriction endonucleases BamHI and PmlHI. Figure 1 (As shown). The HMPV F protein gene portion of the double-digested cloning vector was recovered by gel digestion, and the backbone portion of the expression vector was recovered by gel digestion. Both were ligated with T4 enzyme and transformed into DH5α competent bacteria. The bacteria were screened by plating on ampicillin-containing plates. Positive colonies were picked, amplified, and plasmids were extracted. These were then identified by double digestion with BamHI and Pml. Correct recombinant expression vectors were verified by sequencing. Example 3

[0072] This embodiment provides a process for screening high-yield, stable cell pools. Specific implementation steps.

[0073] (1) Identify the correct recombinant expression vector, extract a large amount of plasmid after enrichment, digest the recombinant expression vector with Pvu I single enzyme, recover the linearized vector by gel digestion, filter and sterilize before use.

[0074] (2) CHO cells are passaged more than twice after revival and the cell viability is greater than 95% when they are used for electroporation.

[0075] (3) In a clean bench, add 0.6 ml of cell suspension (approximately 0.5 × 10⁻⁶ ml) to a 4 mm electroporation cup. 7(1 cell), 50 μL of linearized recombinant expression vector (approximately 40 μg), and electroporation conditions were set at 300 V and 950 μF. After electroporation, the cells were transferred to a T-25 culture flask containing 5 ml of CD CHO Fusion medium (with 6 mM Glun) for resuscitation. The culture flask was then transferred to an incubator at 37°C and 8% CO2 concentration and incubated statically for 24 hours.

[0076] (4) Resuspend the cell suspension in Minipool medium (20% CD CHO Fusion and 80% CHO Cloning Medium) at a ratio of 200 μL per well and 5 × 10⁶ cells per well. 3 Cells were added at a density of 1,000 cells per well into 96-well plates. The culture plates were then transferred to an incubator at 37°C, 80% humidity, and 8% carbon dioxide concentration for static incubation.

[0077] (5) Observe the growth status of cells during the culture process. On day 10, add 50 μL of CD CHOFusion medium to each well.

[0078] (6) After 19 days of culture in 96-well plates, 192 minipools were selected based on confluence for ELISA detection (using recombinant HMPV01 antibody; the HMPV01 antibody light chain follows the amino acid sequence described in SEQ ID No. 22, and the HMPV01 antibody heavy chain follows the amino acid sequence described in SEQ ID No. 23). The top 120 minipools were selected based on the detection results and transferred to 24-well plates for 3 days of culture before ELISA detection. The top 30 minipools were selected based on the detection results and transferred to 6-well plates for 4 days of culture before ELISA detection. The top 15 cells were selected based on the detection results and transferred to SF125 cells for 3 days of culture before ELISA detection. Finally, the cells from the top 8 expression pools were cryopreserved. (e.g.) Figure 2 (As shown). Example 4

[0079] This embodiment provides fed-batch culture of stable cells.

[0080] Following the above screening, the top 8 stable cell groups were selected for fed-batch (FB) culture, as shown in Table 1. During FB culture, viable cell density (VCD), cell viability (Via), glucose (Glu), and lactic acid (Lac) were recorded. After 14 days of FB culture, the peak viable cell density of stable cells ranged from 41.9 × 10⁻⁶ cells / year. 6 Up to 52.5×10 6 The cell count is between 90% and 99% at harvest.

[0081] Table 1 Minipool FB Culture Protocol

[0082] Example 5

[0083] This embodiment provides HMPV F protein purification and stability testing.

[0084] The FB culture supernatant was collected by centrifugation and filtered through a 0.45 μm vacuum filter to remove cell debris and other impurities. The filtered FB culture supernatant was purified by affinity chromatography and anion exchange chromatography to obtain F proteins with high purity. RP-HPLC and SEC-HPLC analysis showed that 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 were all above 90%, and the trimer content was all above 95%. All three preF proteins were stable at 2-8℃ (as shown in Tables 2 and 3).

[0085] The purified preF-P27-Fd (SEQ ID No. 20) showed a single 58 kDa band on non-reducing SDS-PAGE, and bands of the F1 and F2 subunits were visible on reduced SDS-PAGE, indicating relatively complete enzyme digestion. No protein aggregation or degradation was observed after incubation at 4℃ or 37℃ for 3 days or at 50℃ for 1 day, indicating good stability. Figure 3 (As shown).

[0086] Table 2. RP-HPLC results of three F proteins after incubation at 2-8℃ for 5 days.

[0087]

[0088] Table 3. SEC-HPLC results of three F proteins after incubation at 2-8℃ for 5 days.

[0089] Example 6

[0090] This embodiment provides the process for vaccine preparation.

[0091] The recombinant HMPV vaccine was obtained by mixing the F protein with a purity of over 90% obtained in Example 5 with the liposome mixture HA201.

[0092] The liposome mixture HA201 is prepared by dissolving 40-60 mg of 3D-MLA, 800-1200 mg of DOPC and 200-300 mg of cholesterol in 20-30 mL of isopropanol, evaporating and drying to form a liposome membrane, adding 40-60 mL of PBS buffer solution for hydration, homogenizing with a high-pressure homogenizer until the particle size reaches 90-120 nm, adding 40-60 mg of QS-21, diluting, aseptically filtering and then dispensing. Example 7

[0093] This embodiment provides the process of animal immunization.

[0094] Female BALB / c mice aged 6-8 weeks were immunized and randomly divided into groups of 6 mice each: 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 grouping and numbering are shown in Table 4.

[0095] Table 4. Grouping and numbering of immunized mice

[0096]

[0097] Animals in the negative control group were given PBS buffer; animals in the test group were given 10 μg of the target protein mixed with 25 μL of HA201 adjuvant. The vaccine from Example 6 was diluted to a concentration of 100 μg / ml of F protein for animal experiments, and the injection volume was 100 μL / animal.

[0098] The immunization method was intramuscular injection in the hind limb, with each mouse receiving two immunizations at a 3-week interval. Two weeks after the second immunization, blood was collected from each group of mice, and the collected serum was stored at -80℃ for subsequent neutralizing antibody detection. Example 8

[0099] This embodiment provides an immunogenicity assessment. The method is neutralizing antibody detection. The specific steps are as follows:

[0100] (1) The immunized serum was incubated in a water bath at 56°C for 30 min;

[0101] (2) The inactivated serum samples were started at appropriate folds and serially diluted 4-fold in 96-well plates with DMEM medium containing 2% FBS, for a total of 6 folds, with 4 replicates per sample;

[0102] (3) Add 120 PFU of HMPV A2 and HMPV B strains to the wells respectively, mix well and incubate at 37°C and 5% CO2 for 1 h;

[0103] (4) Transfer the virus-serum mixture to a 96-well plate pre-inoculated with Vero cells for adsorption. Add covering material and incubate at 37°C in a 5% CO2 incubator.

[0104] (5) After fixation with 4% paraformaldehyde at room temperature, Triton X-100 was added for permeabilization. The mixture was blocked at 37°C, and Anti-HMPVF protein primary antibody was added and incubated at 37°C. HRP-labeled goat anti-human IgG was added and incubated at 37°C; TrueBlue chromogenic solution was added until the spots were clear.

[0105] (6) Record the number of plaques in each well and calculate the neutralizing antibody titer using the Karber method.

[0106] Test results as follows Figure 4 As shown.

[0107] like Figure 4 The results showed that, compared with the negative control PBS group, the serum of mice in each experimental group two weeks after the second immunization could induce higher levels of neutralizing antibodies, and the serum of mice in each experimental group three weeks after the second immunization could efficiently neutralize the replication of type A and type B wild-type live viruses in in vitro cells.

[0108] There was no significant difference in the levels of neutralizing antibodies induced by the three HMPV preF proteins in this application, but the levels of neutralizing antibodies induced by them were all significantly higher than those induced by the HMPV postF protein (SEQ ID No. 2).

[0109] In summary, the HMPV F protein mutant provided in this application can induce a higher level of neutralizing antibody titer compared to HMPV postF, and when used as a vaccine product, it can induce cross-protection against both strains A and B.

[0110] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A human metapneumovirus F protein mutant, characterized in that, The human metapneumovirus F protein mutant has the amino acid sequence described in SEQ ID No.

20.

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

3. An expression carrier, characterized in that, The expression vector comprises the gene as described in claim 2.

4. A host cell, characterized in that, The host cell expresses the gene of claim 2 or the expression vector of claim 3.

5. The use of the human metapneumovirus F protein mutant of claim 1 in the preparation of a vaccine for the prevention of HMPV infection.

6. A vaccine for preventing HMPV infection, characterized in that, The vaccine comprises the human metapneumovirus F protein mutant as described in claim 1, and a pharmaceutically acceptable vector or excipient.

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