Mutant of pre-fusion human metapneumovirus F protein and application thereof

Abstract

The invention relates to the technical field of biological products, in particular to a mutant of pre-fusion human metapneumovirus F protein and application of the mutant. The mutant is obtained by performing the following mutation on the basis of wild type pre-fusion human metapneumovirus F protein: amino acids at 86-112 sites or amino acids at 97-106 sites are replaced by flexible linkers. The application comprises the following steps: preparing a medicine for preventing or treating hMPV infection; preparing an immunogenic composition for inducing an organism to generate a neutralizing antibody aiming at the hMPV; preparing a reagent or a kit for detecting the hMPV antibody; screening or preparing an anti-hMPV antibody; and separating the hMPV specific B cells. The new pre-fusion human metapneumovirus F protein mutant is obtained through research and design, has remarkably better stability, expression level and affinity, can be used for hMPV diagnosis, antibody therapy and development of various vaccines, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, and in particular to a mutant fused with the F protein of a former human metapneumovirus and its applications. Background Technology

[0002] Human metapneumovirus (hMPV) is one of the important pathogens causing acute respiratory infections worldwide, belonging to the Paramyxoviridae family (…). Paramyxoviridae Epidemiological data shows that hMPV is one of the leading causes of severe lower respiratory tract infections in infants, the elderly, and immunocompromised individuals. Its clinical symptoms are similar to those of respiratory syncytial virus (RSV) infection, and it can lead to serious complications such as bronchitis and pneumonia. However, to date, there are no approved preventative vaccines or specific treatments for hMPV worldwide. Clinical treatment is mainly symptomatic, thus there is an urgent need to develop safe and effective prevention and control strategies.

[0003] Among the structural proteins of hMPV, the fusion protein (F protein) is the major transmembrane glycoprotein located on the surface of the viral envelope and belongs to type I viral fusion glycoproteins. The F protein plays a crucial role in viral infection, mediating the fusion of the viral envelope with the host cell membrane, thereby allowing viral genetic material to enter the host cell. During biosynthesis, the F protein is initially translated into an inactive single polypeptide precursor (F0), which is then cleaved by host cell proteases to form F2 and F1 subunits linked by disulfide bonds. These subunits further assemble to form a biologically active, metastable pre-fusion trimer.

[0004] Existing research indicates that the hMPV F protein exhibits high sequence conservation across different strains and contains major neutralizing epitopes, thus making it a highly promising vaccine target antigen. In particular, the type I fusion protein, stably in its pre-fusion conformation, can induce high-titer neutralizing antibodies, with significantly better immunogenicity than the post-fusion conformation.

[0005] Although the F protein is an ideal target for vaccine development, current technologies for vaccine development based on wild-type hMPV F protein still face the following significant technical bottlenecks: First, poor conformational stability. Wild-type hMPV F protein, in its pre-fusion conformation, exhibits thermodynamic metastable characteristics and is extremely unstable. During in vitro expression, purification, or formulation, this protein is highly susceptible to irreversible conformational changes, spontaneously transitioning from a highly immunogenic pre-fusion conformation to a less immunogenic post-fusion conformation. Studies have confirmed that the vast majority of potent neutralizing antibody epitopes are specifically present in the pre-fusion conformation; once conformational inversion occurs, these key epitopes are lost or masked, leading to a significant decrease in the ability to induce neutralizing antibodies, thus severely impacting the vaccine's immunoprotective efficacy. Second, low heterologous expression efficiency. Wild-type F protein exhibits low expression levels in commonly used heterologous expression systems such as CHO cells and 293F cells, making it difficult to obtain high-purity, high-yield recombinant proteins. This deficiency not only increases production costs but also fails to meet the demands of large-scale vaccine production and industrialization. Third, immunogenicity needs further improvement. Although the natural F protein can elicit a certain immune response, due to the epitope loss caused by the aforementioned conformational instability, it is often difficult to induce a sufficient titer of protective neutralizing antibodies using only the wild-type sequence.

[0006] In summary, how to obtain hMPV F protein mutants that can stably maintain the pre-fusion conformation, have high expression levels, and exhibit excellent immunogenicity through molecular design is a key technical problem that urgently needs to be solved in the current hMPV vaccine development field. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides a mutant fused with the F protein of a previous human metapneumovirus and its application.

[0008] In a first aspect, the present invention provides a mutant of human metapneumovirus F protein, which is obtained by the following mutation on the basis of wild-type human metapneumovirus F protein: amino acids 86-112 or 97-106 are replaced by a flexible linker.

[0009] Furthermore, it also includes: Mutate one or more of the amino acid sequences at positions 122, 125, 127, 129, 133, 134, 151, 168, 196, 258, 259, 260, 263, or 268 to C; Preferably, it also includes mutating the 122nd amino acid to C.

[0010] This invention defines the final form of amino acids, independent of the original sequence of wild-type human metapneumovirus F protein. Therefore, although existing wild-type human metapneumovirus F proteins may have several amino acid sequence differences, as long as the corresponding site contains the amino acid in the final form defined by this invention, it is within the scope of protection of this invention.

[0011] The amino acid sequence of the A2 or B2 wild-type hMPV F protein involved in this invention can be: It also includes at least one mutation from V / I122C, A125C, T127C, R129C, E133C, V134C, L151C, F168C, F196C, I258C, L259C, I260C, Y263C, or I268C, and preferably includes one or two of these mutations; Preferably, it also includes V / I122C.

[0012] The mutation method described in this invention is a common mutation representation method in the art, such as V / I122C, which represents the mutation of amino acid V (valine) or I (isoleucine) at position 122 into C (cysteine).

[0013] Furthermore, it also includes at least one of the following mutations: (1) The 131st position mutates to P; (2) The 160th position mutates to F; (3) The 163rd position mutates to P; (4) The 185th position mutates to P; (5) The 459th position mutates to P; (6) The 73rd position mutates to W; (7) The 116th position mutates to H; (8) The 368th position mutates to N; (9) The 453rd position mutates to Q; Preferably, it also includes (1)-(5).

[0014] The amino acid sequence of the A2 or B2 wild-type hMPV F protein involved in this invention can be: It also includes at least one of the following mutations: E131P, T160F, R163P, D / A185P, A459P, L73W, A116H, H368N or E453Q; More preferably, it also includes: E131P, T160F, R163P, D185P, and A459P.

[0015] It also includes at least one of the following mutations: (1) The 123rd position mutates to C, and the 429th position mutates to C; (2) The 88th position mutates to C, and the 122nd position mutates to C; (3) The 127th position mutates to C, and the 151st position mutates to C; (4) The 119th position mutates to C, and the 428th position mutates to C; (5) The 115th position mutates to C, and the 375th position mutates to C; (6) The 84th position mutates to C, and the 249th position mutates to C; (7) The 140th position mutates to C, and the 147th position mutates to C; Preferably, it also includes a mutation at position 140 to C and a mutation at position 147 to C.

[0016] The amino acid sequence of the A2 or B2 wild-type hMPV F protein involved in this invention can be: (1) A123C and K429C; (2) Q88C and V / I122C; (3) T127C and L151C; (4) T119C and S428C; (5) A115C and L375C; (6) V84C and A249C; (7) A140C and A147C; Preferably, it also includes A140C and A147C.

[0017] Furthermore, it also includes at least one of the following mutations: (1) The 56th position mutates to F or M; (2) The 118th position mutates to M; (3) The 191st position mutates to M; (4) The 209th position mutates to E; (5) The 231st position mutates to I; (6) The 374th position mutates to M; (7) The 376th position mutates to T; (8) The 411th position mutates to M; (9) The 430th position mutates to Q or M; (10) The 449th position mutates to D; (11) The 453rd position mutates to P; (12) The 404th position mutates to P; (13) The 435th position mutates to E.

[0018] The amino acid sequence of the A2 or B2 wild-type hMPV F protein involved in this invention can be: It also includes at least one of the following mutations: E56F / M, V118M, V191M, D209E, V231I, A374M, S376T, T411M, V430Q / M, I449D, E453P, N404P or H435E.

[0019] Furthermore, it also includes the following mutations: Truncate the amino acid at any position between positions 485 and 489; Preferably, it is truncated at position 485.

[0020] Furthermore, it also includes the following mutations: Integrate trimerized structural domains at the C-end; Preferably, the trimerization domain is a T4 Fibritin trimerization domain.

[0021] The amino acid sequence of the T4 Fibritin trimerization domain described in this invention is: YIPEAPRDGQAYVRKDGEWVLLSTFL. The trimerization domain can be fused at the C-terminus via a flexible linker, such as SGGG or SAIG.

[0022] Furthermore, the flexible linker includes: PGCGSGGSG or GSGSGR; Preferably, amino acids at positions 86-112 are replaced by the flexible linker PGCGSGGSG, or amino acids at positions 97-106 are replaced by the flexible linker GSGSGR.

[0023] Furthermore, the wild-type human metapneumovirus F protein comprises the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.2.

[0024] The amino acid sequence shown in SEQ ID NO.1 (amino acid sequence of wild-type hMPV F protein A2 type): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAEKGNTGFIIVIILIAVLGSSMILVSIFIIIKKTKKPTGAPPELSGVTNNGFIPHS。

[0025] The amino acid sequence shown in SEQ ID NO.2 (amino acid sequence of wild-type hMPV F protein subtype B2): .

[0026] Based on SEQ ID NO.1, the amino acid sequence was truncated at position 485 and fused with a trimerized domain at the C-terminus via a flexible linker SGGG, resulting in the amino acid sequence shown in SEQ ID NO.3: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIR LESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGI LIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0027] Based on SEQ ID NO.1, the amino acid sequence was truncated at position 485 and fused with a trimerized domain at the C-terminus via a flexible linker SAIG ​​to obtain the amino acid sequence shown in SEQ ID NO.4: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGVAIAKTIR LESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGI LIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFENIENSQALVDQSNRILSSAESAIGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0028] Based on SEQ ID NO.2, the amino acid sequence was shortened at position 485 and fused with a trimerized domain at the C-terminus via a flexible linker SGGG, resulting in the amino acid sequence shown in SEQ ID NO.5: MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGIAIAKTIR LESEVNAIKGALKTTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGI LIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNKKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0029] Based on SEQ ID NO.2, the amino acid sequence was truncated at position 485 and fused with a trimerized domain at the C-terminus via a flexible linker SAIG ​​to obtain the amino acid sequence shown in SEQ ID NO.6: MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCTDGPSLIKTELDLTKSALRELKTVSADQLAREEQIENPRQSRFVLGAIALGVATAAAVTAGIAIAKTIR LESEVNAIKGALKTTNEAVSTLGNGVRVLATAVRELKEFVSKNLTSAINKNKCDIADLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSYMPTSAGQIKLMLENRAMVRRKGFGI LIGVYGSSVIYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNKKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKG VSCSIGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVALDQVFESIENSQALVDQSNKILNSAESAIGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0030] As a preferred embodiment, the present invention provides a mutant of human metapneumovirus F protein, the mutant comprising the amino acid sequence shown in SEQ ID NO.7-24.

[0031] The first step of this invention involves stabilization design and mutation introduction design to obtain the mutants shown in SEQ ID NO. 7-17, all of which show significantly increased expression levels. The mutation modes of these mutants (compared to wild-type human metapneumovirus F protein) are as follows: (1) Amino acids at positions 86-112 or 97-106 are replaced by flexible linkers.

[0032] (2) It also includes one or two of V122C, A125C, T127C, R129C, E133C, V134C, L151C, F168C, F196C, I258C, L259C, I260C, Y263C or I268C.

[0033] (3) One or more of E131P, T160F, R163P, D185P, A459P, L73W, A116H, H368N or E453Q.

[0034] (4) Truncate any amino acid position between positions 480 and 489.

[0035] (5) Integrate the trimerized structural domain at the C end.

[0036] The expression levels of these mutants were particularly high in MFM-06 and MFM-68, with MFM-06 showing the highest level. Furthermore, the MFM-06 mutant also exhibited stronger binding activity.

[0037] The amino acid sequence shown in SEQ ID NO.7 (MFM-06, which, in addition to SEQ ID NO.3, also includes V122C, E131P, T160F, R163P, D185P, and A459P, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSPGCGSGGSGATAAAVTAGCAIAKTIRLPSEVTAIKNALK TTNEAVSTLGNGVRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVPLDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0038] The amino acid sequence shown in SEQ ID NO.8 (MFM-68, which, in addition to SEQ ID NO.3, also includes V122C, L73W, A116H, D185P, N368H, and E454Q, with segments 97-106 replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSPGCLAREEQIEGSGSGRAIALGVATAHAVTAGCAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0039] The amino acid sequence shown in SEQ ID NO.9 (MFM-71, which also includes A125C, I260C, L73W, A116H, D185P, N368H, E454Q based on SEQ ID NO.3, and residues 97 - 106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAICKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILCGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0040] The amino acid sequence shown in SEQ ID NO.10 (MFM-72, further including A125C, I258C, L73W, A116H, D185P, H368N, E454Q based on SEQ ID NO.3, and residues 97-106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAICKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGCLIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0041] The amino acid sequence shown in SEQ ID NO.11 (MFM-73, which further includes A125C, I259C, L73W, A116H, D185P, H368N, E454Q based on SEQ ID NO.3, and residues 97-106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAICKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGICIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0042] The amino acid sequence shown in SEQ ID NO. 12 (MFM-74, further including V134C, Y263C, L73W, A116H, D185P, H368N, E454Q based on SEQ ID NO. 3, and residues 97-106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESECTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVCGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0043] The amino acid sequence shown in SEQ ID NO.13 (MFM-75, which also includes V134C, I268C, L73W, A116H, D185P, H368N, E454Q based on SEQ ID NO.3, and residues 97-106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESECTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVCYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0044] The amino acid sequence shown in SEQ ID NO. 14 (MFM-76, further including E133C, L151C, L73W, A116H, D185P, H368N, E454Q based on SEQ ID NO. 3, and residues 97-106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESCVTAIKNALKTTNEAVSTCGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0045] The amino acid sequence shown in SEQ ID NO.15 (MFM-77, which also includes T127C, L151C, L73W, A116H, D185P, H368N, E454Q based on SEQ ID NO.3, and residues 97-106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKCIRLESEVTAIKNALKTTNEAVSTCGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0046] The amino acid sequence as set forth in SEQ ID NO. 16 (MFM-78, further including F168C, F196C, L73W, A116H, D185P, H368N, E454Q on the basis of SEQ ID NO. 3, and residues 97-106 are replaced by the flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDCVSKNLTRAINKNKCDIPDLKMAVSFSQCNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL。

[0047] The amino acid sequence as set forth in SEQ ID NO.17 (MFM-79, further including R129C, I260C, L73W, A116H, D185P, H368N, E454Q on the basis of SEQ ID NO.3, and residues 97-106 are replaced by flexible linker GSGSGR): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDWTKSALRELKTVSADQLAREEQIEGSGSGRAIALGVATAHAVTAGVAIAKTICLES EVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILC GVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGV SCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPQDQFNVALDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0048] Based on the design concept of MFM-06, this invention further introduces mutations, as shown in (1)-(7) above, especially the MFM-06-C1 expression level is increased the most by introducing (7) A140C and A147C.

[0049] The amino acid sequence shown in SEQ ID NO.18 (MFM-06-C1, which, in addition to SEQ ID NO.3, also includes V122C, E131P, A140C, A147C, T160F, R163P, D185P, and A459P, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVFNLTCSDGPSLIKTELDLTKSALRELKTVSPGCGSGGSGATAAAVTAGCAIAKTIRLPSEVTAIKNCLK TTNECVSTLGNGVRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPEDQFNVPLDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0050] The amino acid sequence shown in SEQ ID NO.19 (MFM-06-DF-C2, which is a truncated version of SEQ ID NO.1 at position 489, and also includes T69Y, V84C, A117H, V122C, E131P, R163P, D185P, E209D, V231I, A249C, H368N, A459P, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG, L473W, D475R, Q476K, S477F, N478D, R479E, A484I): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDLTKSALRELKTCSPGCGSGGSGATAAHVTAGCAIAKTIRLPSEVTA IKNALKTTNEAVSTLGNGVRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSENAGITPAISLDLMTDAELARAISNMPTSAGQIKLMLENRCMVRRK GFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISM VALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPPDQFNVALDQVFENIENSQAWVRKFDEILSSIEKGNT.

[0051] The amino acid sequence shown in SEQ ID NO. 20 (MFM-06-DF-C3, which is a shortened version of SEQ ID NO. 1, trunculated at position 489, and also includes T69Y, A117H, V122C, E131P, V155C, R163P, D185P, E209D, V231I, A249C, H368N, N395C, A459P, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG, L473W, D475R, Q476K, S477F, N478D, R479E, A484I): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKYELDLTKSALRELKTVSPGCGSGGSGATAAHVTAGCAIAKTIRLPSEVTA IKNALKTTNEAVSTLGNGCRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSENAGITPAISLDLMTDAELARAISNMPTSAGQIKLMLENRAMVRRK GFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISM VALSPLGALVACYKGVSCSIGSCRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPPDQFNVALDQVFENIENSQAWVRKFDEILSSIEKGNT.

[0052] Based on the design concept of MFM-06-C1, this invention further introduces one or more of E56F / M, V118M, V191M, D209E, V231I, A374M, S376T, T411M, V430Q / M, I449D, E453P, N404P, or H435E to obtain multiple mutants as shown in SEQ ID NO. 21-25. These mutants exhibit similar affinity (ability to bind antibodies) and thermostability to MFM-06-C1, and also show good binding ability to antibodies against Ø epitopes, III epitopes, and II-V epitopes. Among these mutants, MFM-106 shows the highest expression level.

[0053] The amino acid sequence shown in SEQ ID NO.21 (MFM-106, which, in addition to SEQ ID NO.4, also includes V122C, E131P, A140C, A147C, T160F, R163P, D185P, D209E, V231I, E453P, A459P, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGWVFNLTCSDGPSLIKTELDLTKSALRELKTVSPGCGSGGSGATAAAVTAGCAIAKTIRLPSEVTAIKNCLK TTNECVSTLGNGVRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSENAGITPAISLDLMTDAELARAISNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPPDQFNVPLDQVFENIENSQALVDQSNKILNSAESAIGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0054] The amino acid sequence shown in SEQ ID NO.22 (MFM-117, which, in addition to SEQ ID NO.3, also includes E56F, V122C, E131P, A140C, A147C, T160F, R163P, D185P, V430Q, I449D, A459P, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVFNLTCSDGPSLIKTELDLTKSALRELKTVSPGCGSGGSGATAAAVTAGCAIAKTIRLPSEVTAIKNCLK TTNECVSTLGNGVRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPIFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRHPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKQEGEQHVIKGRPVSSSFDPDKFPEDQFNVPLDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0055] The amino acid sequence shown in SEQ ID NO.23 (MFM-122, which, in addition to SEQ ID NO.3, also includes V122C, E131P, A140C, A147C, T160F, R163P, D185P, N404P, H435E, and A459P, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVFNLTCSDGPSLIKTELDLTKSALRELKTVSPGCGSGGSGATAAAVTAGCAIAKTIRLPSEVTAIKNCLK TTNECVSTLGNGVRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAVSFSQFNRRFLNVVRQFSDNAGITPAISLDLMTDAELARAVSNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPLFGVIDTPCWIVKAAPSCSGKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLPKGCSYITNQDADTVTIDNTVYQLSKVEGEQEVIKGRPVSSSFDPIKFPEDQFNVPLDQVFENIENSQALVDQSNRILSSAESGGGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0056] The amino acid sequence shown in SEQ ID NO. 24 (MFM-128, which, in addition to SEQ ID NO. 4, also includes V118M, V122C, E131P, V146M, A140C, A147C, T160F, R163P, D185P, V191M, D209E, V231I, H368N, A374M, S376T, T411M, V430M, E453P, A459P, R479K, S482N, with amino acids 86-112 replaced by the flexible linker PGCGSGGSG): MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVMNLTCSDGPSLIKTELDLTKSALRELKTVSPGCGSGGSGATAAAMTAGCAIAKTIRLPSEVTAIKNCLK TTNMCVSTLGNGVRVLAFAVPELKDFVSKNLTRAINKNKCDIPDLKMAMSFSQFNRRFLNVVRQFSENAGITPAISLDLMTDAELARAISNMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPIFGVIDTPCWIVKAAPLCSEKKGNYACFLRMDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRNPISMVMLTPLGALVACYKGVSCS IGSNRVGIIKQLNKGCSYIMNQDADTVTIDNTVYQLSKMEGEQHVIKGRPVSSSFDPIKFPPDQFNVPLDQVFEMIENSQALVDQSNKILNSAESAIGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0057] The amino acid sequence shown in SEQ ID NO.25 (MFM-135, which is a truncated version of SEQ ID NO.6 at position 485, and also includes V118M, V122C, E131P, V146M, A140C, A147C, T160F, R163P, V191M, D209E, V231I, H368N, A374M, S376T, T411M, V430M, E453P, A459P, R479K, S482N, with amino acids at positions 86-112 replaced by the flexible linker PGCGSGGSG): MSWKVMIIISLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVMNLTCTDGPSLIKTELDLTKSALRELKTVSPGCGSGGSGATAAAMTAGCAIAKTIRLPSEVNAIKGCLK TTNECVSTLGNGVRVLAFAVPELKEFVSKNLTSAINKNKCDIADLKMAMSFSQFNRRFLNVVRQFSENAGITPAISLDLMTDAELARAISYMPTSAGQIKLMLENRAMVRRKGFGILIGVYGSSV IYMVQLPIFGVIDTPCWIIKAAPSCSEKDGNYACLLREDQGWYCKNAGSTVYYPNKKDCETRGDHVFCDTAAGINVAEQSRECNINISTTNYPCKVSTGRNPISMVALSPLGALVACYKGVSCS IGSNRVGIIKQLPKGCSYIMNQDADTVTIDNTVYQLSKMEGEQHVIKGRPVSSSFDPIKFPPDQFNVPLDQVFEMIENSQALVDQSNKILNSAESAIGYIPEAPRDGQAYVRKDGEWVLLSTFL.

[0058] Furthermore, the amino acid sequence of the reported closed-state MPV-2c positive control involved in this invention is shown in SEQ ID NO.26: MSWKVVIIFSLLITPQHGLKESYLEESCSTITEGYLSVLRTGWYTNVFTLEVGDVENLTCSDGPSLIKTELDLTKSALRELKTVSADQLRRRRELPRFMNYTLNNAKKTNVTLSKKRKRRFVLGAIAL GRATAAAVTAGVAIAKTIRLESEVTAIKNALKTTNEAVSTLGNGVRVLATAVRELKDFVSKNLTRAINKNKCDIDDLKMAVSFSQFNRRFLNVVRQFSENAGITPAISLDLMTDAELARAISNMPTSAG QIKLMLENRAMVRRKGFGILIGVYGSSVIYMVQLPIFGVIDTPCWIVKAAPSCSEKKGNYACLLREDQGWYCQNAGSTVYYPNEKDCETRGDHVFCDTAAGINVAEQSKECNINISTTNYPCKVSTGRN PISMVALSPLGALVACYKGVSCSIGSNRVGIIKQLNKGCSYITNQDADTVTIDNTVYQLSKVEGEQHVIKGRPVSSSFDPIKFPPDQFNVALDQVFENIENSQAWVRKFDEILSSIEKGNTGGSEPEA.

[0059] In a second aspect, the present invention provides a recombinant protein multimer comprising: amino acid sequences at positions 26-85 and 113-485 of the aforementioned mutant, or amino acid sequences at positions 26-96 and 107-485. Preferably, it also includes the trimerization domain mentioned in the aforementioned mutant.

[0060] Thirdly, the present invention provides a nucleic acid for encoding the aforementioned mutant or the aforementioned recombinant protein polymer.

[0061] Fourthly, the present invention provides a biological material comprising the aforementioned nucleic acid; said biological material is an expression cassette, vector, cell, or recombinant viral particle.

[0062] The expression cassette of this invention includes a promoter, a coding sequence (e.g., a nucleotide sequence corresponding to the aforementioned nucleic acid), and a termination signal for terminating the transcription process (e.g., including a terminator and a polyadenylation signal). It can also guide the cell to add a poly(A) tail to the end of the mRNA to increase mRNA stability and translation efficiency. Common examples include SV40polyA and BGH polyA. Furthermore, various elements can be added to the expression cassette, such as enhancers, introns, Kozak sequences, Shine-Dalgarno sequences, or selectable marker genes.

[0063] The vectors described in this invention include: plasmid vectors (extrachromosomal circular DNA molecules derived from bacteria or yeast), viral vectors (modified viruses that have had their pathogenicity and self-replication capabilities removed, but retain their ability to efficiently infect cells and deliver genetic material into cells), bacteriophage vectors, or artificial chromosome vectors (e.g., bacterial artificial chromosome BAC or yeast artificial chromosome YAC).

[0064] The transgenic cells described in this invention are cells whose genetic material has undergone stable artificial alterations, such as the introduction of the nucleic acids provided in this application. The transgenic cells described in this invention include animal cells, plant cells, or microbial cells, wherein the animal and plant cells do not have the potential to develop into a complete individual (and do not belong to any animal or plant species).

[0065] The recombinant viral particles described in this invention are in the form of virus-like particles, where a protein coat (viral capsid) encapsulates genetic material (such as the aforementioned nucleic acid). For example, the recombinant viral particles are prepared by transfecting the aforementioned viral vector and other helper plasmids into a packaging cell line (such as HEK293T cells), which will complete the expression and assembly of viral proteins and recombinant genes to obtain complete recombinant viral particles.

[0066] Those skilled in the art, having access to the nucleic acids disclosed in this application, are fully aware of the preparation methods of the aforementioned expression cassettes, vectors, transgenic cells, and recombinant viral particles based on existing technology. There are no technical obstacles involved, and therefore, expression cassettes, vectors, transgenic cells, and recombinant viral particles containing the aforementioned gene mutants are also within the scope of this invention.

[0067] Fifthly, the present invention provides a kit comprising the aforementioned mutant, or the aforementioned recombinant protein polymer, or the aforementioned nucleic acid, or the aforementioned biological material.

[0068] Sixthly, the present invention provides the use of the aforementioned mutant or the aforementioned recombinant protein multimer in any of the following: (1) To prepare drugs for the prevention or treatment of hMPV infection; (2) Preparation of an immunogenic composition that induces the body to produce neutralizing antibodies against hMPV; (3) Prepare reagents or kits for detecting hMPV antibodies; (4) Screening or preparing anti-hMPV antibodies; (5) Isolate hMPV-specific B cells; Preferably, the drug is a vaccine.

[0069] In a seventh aspect, the present invention provides the use of the aforementioned nucleic acid or the aforementioned biological material in the preparation of mRNA or DNA vaccines for the prevention or treatment of hMPV infection.

[0070] The present invention has the following beneficial effects: This invention designs and screens mutants that fuse with the F protein of human metapneumovirus, significantly improving their stability. When stored under different conditions for more than 28 days, their binding ability with neutralizing antibodies does not decrease significantly. At the same time, it increases their expression level, up to about 1800 times, to meet the needs of large-scale vaccine production. In addition, the proportion of correct conformation is significantly increased, and the binding activity with antibodies against Ø epitope, III epitope and II-V epitope is significantly improved.

[0071] The mutant of the pre-fusion human metapneumovirus F protein provided by this invention can be used to develop various types of hMPV vaccines (such as subunit vaccines, viral vector vaccines, mRNA vaccines, etc.), diagnostic reagents (detecting various specific antibodies against the pre-fusion human metapneumovirus F protein), and antibody drugs (neutralizing antibodies targeting the pre-fusion conformational epitope). Attached Figure Description

[0072] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0073] Figure 1 The binding ability of MFM-06 and MFM-68 antibodies provided in Example 1 of this invention is compared with that of hMPV wild-type WT.

[0074] Figure 2 The results show the comparison of antibody binding ability based on the MFM-06 optimized mutant provided in Example 2 of this invention.

[0075] Figure 3 The results show the comparison of antibody binding ability and thermal stability based on the optimized mutant MFM-06-C1 provided in Example 3 of this invention.

[0076] Figure 4 The SPR detection results for the affinity of MFM-06-C1 with different epitope antibodies provided in Example 5 of this invention are shown.

[0077] Figure 5 The storage stability results of MFM-06-C1 and different epitope antibodies provided in Example 6 of this invention are shown.

[0078] Figure 6 The storage stability results of MFM-106 and different epitope antibodies provided in Example 6 of this invention.

[0079] Figure 7 The storage stability results of MFM-117 and different epitope antibodies provided in Example 6 of this invention.

[0080] Figure 8 The storage stability results of MFM-122 and different epitope antibodies provided in Example 6 of this invention are shown.

[0081] Figure 9 The storage stability results of MFM-128 and different epitope antibodies provided in Example 6 of this invention.

[0082] Figure 10 This invention provides a comparison of the binding ability of MFM-128, MFM-135 antibodies against different epitopes of the positive control antigen MFM-130 (MPV2c) provided in Example 7 of the present invention. Detailed Implementation

[0083] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0084] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0085] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0086] In the following examples, the mutation positions of all mutants are referenced to the amino acid sequence shown in SEQ ID NO.1.

[0087] Example 1: Design and screening of hMPV preF protein mutants In this embodiment, the amino acid sequence (SEQ ID NO.1) of wild-type hMPV F protein was used as a template. By evaluating the spatial conformation of the F protein before hMPV fusion, a non-natural disulfide bond mutation was introduced to obtain an F protein mutant with high expression level and correct pre-fusion conformation.

[0088] To achieve this objective, the present invention attempts to enhance the conformational correctness of the F protein before fusion and maintain the binding activity of key neutralizing epitopes by designing internal disulfide bonds in F1 and inter-F1 disulfide bonds between F2 and F1 respectively.

[0089] The mutation sites mentioned above include: (1) Valine (V) at position 122 is mutated to cysteine ​​(C); (2) Alanine (A) at position 125 is mutated to cysteine ​​(C); (3) Threonine (T) at position 127 is mutated to cysteine ​​(C); (4) Arginine (R) at position 129 is mutated to cysteine ​​(C); (5) The glutamic acid at position 133 (E) is mutated to cysteine ​​(C); (6) Valine (V) at position 134 is mutated to cysteine ​​(C); (7) Leucine (L) at position 151 is mutated to cysteine ​​(C); (8) The phenylalanine (F) at position 168 is mutated to cysteine ​​(C); (9) The phenylalanine (F) at position 196 is mutated to cysteine ​​(C). (10) The isoleucine (I) at position 258 is mutated to cysteine ​​(C); (11) Leucine (L) at position 259 is mutated to cysteine ​​(C); (12) The isoleucine (I) at position 260 is mutated to cysteine ​​(C); (13) Tyrosine (Y) at position 263 is mutated to cysteine ​​(C); (14) Tyrosine (F) at position 268 is mutated to cysteine ​​(C); Meanwhile, to increase the correctness of the pre-fusion conformation of the F protein, the above-mentioned mutation design was combined with L73W, A116H, E131P, R163P, D185P, N368H, E453Q, A459P, and T160F in different combinations. Furthermore, F2 and F1 were linked by a linker, and a soluble T4 Fibritin trimerizing domain was added at position 485 of the transmembrane region at the C-terminus of the F protein to increase the stability of the F protein trimer structure. The mutants designed according to the above objectives are shown in Table 1.

[0090] Table 1. Design of truncated pre-F protein mutants

[0091] Using standard molecular biology techniques, the truncated nucleic acid sequence of the pre-F protein mutant shown in Table 1 was inserted into the pEE12.4 expression vector sequence for full plasmid synthesis. The plasmid was then transfected into 50 mL of CHO cells using PEI, and after 7 days of continuous culture, the supernatant was collected, purified by affinity chromatography, and expressed as OD200. 280 Total protein content was measured, and the expression level of each protein was calculated. The results are shown in Table 2. The results indicate that the expression levels of all mutants were higher than those of the wild type, with MFM-06 and MFM-68 showing the highest yields.

[0092] Table 2. Expression of preF protein mutants

[0093] This invention further employs ELISA to evaluate the binding affinity of each antigen and antibody. The binding affinity to each mutant was detected using monoclonal antibodies ADI61026 targeting the Ø epitope and MPE8 targeting the III epitope as primary antibodies. Purified mutants were diluted to 100 μg / well, serially diluted 3-fold, and the antigens were coated with coating buffer into 96-well plates. Primary antibodies and HRP anti-human IgG1 Fc Antibody were added sequentially, followed by incubation and TMB development. The absorbance at 450 nm was measured using a microplate reader, and the data were statistically analyzed. Results are as follows: Figure 1 As shown, the mutant MFM-06 exhibits strong binding activity with various monoclonal antibodies, superior to MFM-68 and wild-type WT. Therefore, MFM-06 can be used as a base sequence for further optimization.

[0094] Example 2 Design and screening of hMPV preF protein mutants To obtain mutants with better stability and yield, this invention screened new combinations of mutation sites based on the mutant MFM-06 obtained in Example 1, and further evaluated the yield and stability of the mutants. The mutants designed according to the above objectives are shown in Table 3.

[0095] Table 3. Mutation design of truncated preF protein

[0096] In this example, the plasmid synthesis, transfection, and fermentation culture of the truncated preF protein mutants were the same as in Example 1. The expression levels of each mutant after purification are shown in Table 4. The results showed that the mutant MFM-06-C1 had the highest expression level, reaching 25 μg / mL. The binding ability of each mutant to the antibody was evaluated as follows: Figure 2 The results showed that MFM-06-C1 had a better binding ability to the antibody than other mutants.

[0097] Table 4. Expression of the truncated preF protein mutant

[0098] Example 3: Design and screening of hMPV preF protein mutants To obtain mutants with better stability and yield, this invention optimizes the MFM-06-C1 model and further evaluates the yield and stability of the mutants. The mutant design is shown in Table 5.

[0099] Table 5. Design of truncated preF protein mutations

[0100] In this embodiment, the plasmid synthesis, transfection, and fermentation culture of the truncated preF protein mutant were the same as in Example 1. The binding affinity and thermostability of each mutant to the antibody were evaluated as follows: Figure 3 The results showed that the binding ability and thermal stability of each mutant to the antibody were comparable to those of MFM-06-C1.

[0101] Example 4: Construction and expression of stable cell pools for each mutant In this invention, plasmids expressing MFM-06-C1, MFM106, MFM117, MFM122, and MFM128 were mixed with transfection reagent PEI at a ratio of 1:3. After standing at room temperature for 15-20 minutes, the complexes were added dropwise to a prepared CHO cell suspension. 24-48 hours after transfection, cell status was observed, and three pressure screenings were performed to obtain a stable cell pool. The cell pool was cultured for 14 days, and the supernatant was harvested and purified. The protein expression levels after purification are shown in Table 6.

[0102] Table 6. Expression of truncated preF protein mutants

[0103] Example 5: Detection of antigen-antibody affinity using surface plasmon resonance technology In this embodiment, surface plasmon resonance (SPR) technology is used for kinetic analysis.

[0104] First, the Protein A chip was installed in the system, and the system was started with running buffer at a constant flow rate of 10 μL / min until a stable baseline was obtained. Subsequently, diluted antibody solution was injected at the same flow rate for capture, with a binding time of 1 minute, to increase the capture amount (expressed as RU value) by 50-200 RU, forming a stable post-capture baseline as the starting point for subsequent analyses.

[0105] Next, binding and dissociation analysis was performed: the antigen solution (MFM-06-C1) was injected at a constant flow rate of 30 μL / min for 120 seconds, and the rise in RU value was monitored in real time to reflect the binding process of antigen and antibody; then, the flow buffer was switched back, and dissociation was carried out for 300 seconds, with the decrease in RU value monitored to reflect the dissociation process of antigen. The regeneration phase involved injecting a pH 2.5 glycine regeneration solution at a flow rate of 30 μL / min for 30 seconds to dissociate the antigen-antibody complex, causing the RU value to drop sharply to near the baseline. Subsequently, the surface was washed with the flow buffer to stabilize the baseline and restore a clean Protein A surface, preparing for the next cycle.

[0106] Finally, the binding rate (ka), dissociation rate (kd), and affinity constant (KD) of the antigen were obtained through data analysis. The experimental results are shown in [Figure number missing]. Figure 4 See Table 7. The results show that all three antibodies can specifically bind to the antigen MFM-06-C1 and have a high affinity level.

[0107] Table 7. Binding kinetics and affinity parameters of each antibody with antigen MFM-06-C1

[0108] Example 6: Stability evaluation of mutants expressed in the stable cell In this embodiment, the mutant antigens MFM-06-C1, MFM106, MFM117, MFM122, and MFM128 were stored at -60℃, 2-8℃, 25℃, and 40℃ for 28 days, respectively. The binding ability of each antigen was evaluated by ELISA using antibodies ADI61026 (targeting the Ø epitope), MPV364 (targeting the III epitope), and MPV467 (targeting the V epitope). The results are as follows: Figures 5-9 As shown, with -60℃ storage as a control, the binding ability of each mutant to the antibody did not decrease significantly after storage at 2-8℃, 25℃ and 40℃. After long-term storage in a wide temperature range, each mutant could still maintain the spatial structure integrity of the key neutralizing epitopes (Ø, III and V, etc.) in the pre-fusion conformation, and the pre-fusion structural stability was excellent, meeting the storage requirements for vaccine research and development and industrialization.

[0109] Example 7: Comparison of antibody binding ability between MPV-2c in the closed state and the closed state. MPV-2c is a reported closed-state trimer structure. In this embodiment, the antigenicity differences of different mutants were evaluated by comparing the binding ability of MFM-128 (A2), MFM-135 (B2) and MPV2c to antibodies ADI61026 (targeting the Ø epitope), MPV364 (targeting the III epitope) and MPV467 (targeting the V epitope).

[0110] The results are as follows Figure 10 As shown, the antibodies targeting different epitopes exhibited comparable binding abilities to the three antigens, with no significant differences, indicating that the mutant constructed in this invention has no significant structural or antigenic differences from the closed-state MPV-2c.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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; and these 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 the present invention.

Claims

1. A mutant of the human metapneumovirus F protein, characterized in that, The mutant was obtained by the following mutation on the wild-type human metapneumovirus F protein: amino acids 86-112 or 97-106 were replaced by a flexible linker.

2. The mutant according to claim 1, characterized in that, Also includes: Mutate one or more of the amino acid sequences at positions 122, 125, 127, 129, 133, 134, 151, 168, 196, 258, 259, 260, 263, or 268 to C.

3. The mutant according to claim 1 or 2, characterized in that, It also includes at least one of the following mutations: (1) The 131st position mutates to P; (2) The 160th position mutates to F; (3) The 163rd position mutates to P; (4) The 185th position mutates to P; (5) The 459th position mutates to P; (6) The 73rd position mutates to W; (7) The 116th position mutates to H; (8) The 368th position mutates to N; (9) The 453rd position mutates to Q.

4. The mutant according to claim 1 or 2, characterized in that, It also includes at least one of the following mutations: (1) The 123rd position mutates to C, and the 429th position mutates to C; (2) The 88th position mutates to C, and the 122nd position mutates to C; (3) The 127th position mutates to C, and the 151st position mutates to C; (4) The 119th position mutates to C, and the 428th position mutates to C; (5) The 115th position mutates to C, and the 375th position mutates to C; (6) The 84th position mutates to C, and the 249th position mutates to C; (7) The 140th position mutates to C, and the 147th position mutates to C.

5. The mutant according to claim 1 or 2, characterized in that, It also includes at least one of the following mutations: (1) The 56th position mutates to F or M; (2) The 118th position mutates to M; (3) The 191st position mutates to M; (4) The 209th position mutates to E; (5) The 231st position mutates to I; (6) The 374th position mutates to M; (7) The 376th position mutates to T; (8) The 411th position mutates to M; (9) The 430th position mutates to Q or M; (10) The 449th position mutates to D; (11) The 453rd position mutates to P; (12) The 404th position mutates to P; (13) The 435th position mutates to E.

6. The mutant according to claim 1 or 2, characterized in that, It also includes the following mutations: It is truncated at any amino acid position between positions 480 and 489.

7. The mutant according to claim 1 or 2, characterized in that, It also includes the following mutations: The C-end integrates a trimerized structural domain.

8. The mutant according to claim 1 or 2, characterized in that, Amino acids at positions 86-112 are replaced by the flexible linker PGCGSGGSG, or amino acids at positions 97-106 are replaced by the flexible linker GSGSGR.

9. The mutant according to claim 1 or 2, characterized in that, The wild-type human metapneumovirus F protein includes the amino acid sequence shown in SEQ ID NO.1 or SEQ ID NO.

2.

10. A recombinant protein polymer, characterized in that, include: The amino acid sequence of the mutant according to any one of claims 1-9, specifically positions 26-85 and 113-485, or positions 26-96 and 107-485.

11. A nucleic acid, characterized in that, The nucleic acid is used to encode the mutant according to any one of claims 1-9, or the recombinant protein polymer according to claim 10.

12. A biomaterial, characterized in that, include: The nucleic acid as described in claim 11; the biological material is an expression cassette, vector, cell, or recombinant viral particle.

13. A reagent kit, characterized in that, This includes the mutant according to any one of claims 1-9, the recombinant protein polymer according to claim 10, the nucleic acid according to claim 11, or the biological material according to claim 12.

14. The use of the mutant according to any one of claims 1-9, or the recombinant protein multimer according to claim 10, in any of the following: (1) To prepare drugs for the prevention or treatment of hMPV infection; (2) Preparation of an immunogenic composition that induces the body to produce neutralizing antibodies against hMPV; (3) Prepare reagents or kits for detecting hMPV antibodies; (4) Screening or preparing anti-hMPV antibodies; (5) Isolate hMPV-specific B cells.

15. The use of the nucleic acid of claim 11 or the biological material of claim 12 in the preparation of an mRNA vaccine or DNA vaccine for the prevention or treatment of hMPV infection.

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