Respiratory syncytial virus F protein mutant and its application

Abstract

The present invention provides a respiratory syncytial virus F protein mutant and its application, and relates to the field of biotechnology. The respiratory syncytial virus F protein mutant contains at least one recombinant F2-F1 polypeptide, each recombinant F2-F1 polypeptide independently contains a mutated fragment of the F0 polypeptide, and the mutations include: (I) positions 104-144 are deleted and replaced by a connecting peptide; (II) 155C substitution, 289C substitution, 149C substitution, 458C substitution; 155C and 289C form a disulfide bond, 149C and 458C form a disulfide bond; and, (III) 373R substitution. The respiratory syncytial virus F protein mutant has excellent immunogenicity, antigenicity, stability and tolerance.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a respiratory syncytial virus F protein mutant and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Respiratory viral infections are one of the most important global public health burdens, resulting in millions of hospitalizations worldwide each year. Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in children under 2 years of age and adults over 65 years of age. Despite the serious harm of respiratory diseases caused by RSV infection, there is still a lack of effective means to prevent RSV.

[0004] The selection of mainstream vaccine antigens is mainly focused on F protein, especially F protein in prefusion conformation (PreF). Compared with F protein in postfusion conformation, F protein in prefusion conformation can expose more potential epitopes with neutralizing activity, such as epitope Φ that only exists in PreF. Antibodies produced against epitope Φ have excellent neutralizing activity. After binding to F protein in prefusion conformation, they can block viral membrane fusion mediated by conformational changes of F protein. At the same time, there is a certain degree of positive correlation between the purity of antigen trimer and antigen immunogenicity. Although there are currently two recombinant protein vaccines specifically for RSV on the market, the trimer production in the antigen preparation process is low, resulting in low yield.

[0005] Therefore, providing an RSV antigen with a higher trimer yield, thereby enhancing immunogenicity and improving stability, and a vaccine containing the same has become a technical problem that those skilled in the art urgently need to solve.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The object of the present invention is to provide a respiratory syncytial virus F protein mutant to improve the defects of existing RSV antigens.

[0008] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0009] In a first aspect, a respiratory syncytial virus F protein mutant is provided, the respiratory syncytial virus F protein mutant contains at least one recombinant F2-F1 polypeptide, each of the recombinant F2-F1 polypeptides independently contains a mutated fragment of the F0 polypeptide, and the mutation includes:

[0010] (I) Positions 104 to 144 were deleted and replaced by a connecting peptide;

[0011] (II) containing the following substitutions at positions for forming disulfide bonds: 155C substitution, 289C substitution, 149C substitution, 458C substitution; 155C and 289C form a disulfide bond, and 149C and 458C form a disulfide bond; and,

[0012] (III) 373R substitution;

[0013] The position corresponds to the amino acid sequence of the wild-type F0 polypeptide shown in SEQ ID NO.1.

[0014] In the second aspect, a biological material is also provided, including a polynucleotide, a vector or a cell; wherein the polynucleotide encodes the above-mentioned respiratory syncytial virus F protein mutant; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector or is capable of expressing the respiratory syncytial virus F protein mutant.

[0015] In the third aspect, the use of the respiratory syncytial virus F protein mutant described in the first aspect or the biomaterial described in the second aspect in the following (X1) to (X4) is also provided:

[0016] (X1) preparing antibodies that bind to respiratory syncytial virus antigen epitopes;

[0017] (X2) Preparation of vaccines for the prevention of respiratory syncytial virus infection;

[0018] (X3) Preparation of respiratory syncytial virus diagnostic products;

[0019] (X4) Detection of respiratory syncytial virus for non-diagnostic and non-therapeutic purposes.

[0020] In a fourth aspect, a vaccine for preventing respiratory syncytial virus infection is also provided, wherein the vaccine for preventing respiratory syncytial virus infection contains the respiratory syncytial virus F protein mutant described in the first aspect, or the biological material described in the second aspect.

[0021] In a fifth aspect, a respiratory syncytial virus diagnostic reagent or kit is also provided, wherein the respiratory syncytial virus diagnostic reagent or kit contains the respiratory syncytial virus F protein mutant described in the first aspect.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The respiratory syncytial virus (RSV) F protein mutant provided by the present invention is modified by specific amino acid mutations, so that the recombinant F2-F1 polypeptide is conducive to forming a high-purity trimer structure and achieving high expression in host cells. These mutants show good antigenicity and immunogenicity, and perform well in thermal stability, pH stability and osmotic pressure stability. The RSV F protein mutant provided by the present invention can induce the production of neutralizing antibodies during immunization, showing excellent immune protection effects. In summary, the RSV F protein mutant of the present invention performs well in multiple stability aspects, providing important technical support for vaccine development. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 The SDS-PAGE result of the K007 protein of Example 4, M1 is a marker, R represents a reducing band, and NR represents a non-reducing band;

[0026] Figure 2 The SDS-PAGE result of the protein K015 of Example 4, M1 is a marker, R represents a reducing band, and NR represents a non-reducing band;

[0027] Figure 3 The SDS-PAGE result of the K016 protein of Example 4, M1 is a marker, R represents a reducing band, and NR represents a non-reducing band;

[0028] Figure 4 This is the SDS-PAGE result of the DS-Cav1 protein of Example 4, M1 is a marker, R represents a reducing band, and NR represents a non-reducing band. DETAILED DESCRIPTION

[0029] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] As used herein, "and / or" is used to indicate that one or both of the described situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0031] In this document, unless otherwise specified, arbitrary numbering is used to distinguish one entity or behavior from another entity or behavior, and does not necessarily require or imply any actual relationship, order, or importance between these entities or behaviors, such as numbers (I) to (III), (I) to (IV), (A1) to (A5), (B1) to (B4) and (X1) to (X4).

[0032] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0033] Herein, the terms "comprise" or "comprising" are intended to include stated elements, integers or steps, but not to exclude any other elements, integers or steps.

[0034] Herein, "each... is independently selected from" is interchangeable with "... are independently selected from" and "... are independently selected from" and should be understood in a broad sense, referring to the range or options that each member of a group of variables or components can be independently selected, that is, the selection of each variable or component is independent and is not affected by the selection of other variables or components.

[0035] Herein, the term "F0 polypeptide", "F0 protein" or "F0" refers to the precursor polypeptide of RSV F protein, and the natural F0 polypeptide consists of a signal polypeptide sequence, an F1 polypeptide sequence, a pep27 polypeptide sequence and an F2 polypeptide sequence. With few exceptions, the F0 polypeptide of a known wild-type RSV strain consists of 574 amino acids.

[0036] Herein, the term "F1 polypeptide", "F1 protein" or "F1" refers to the polypeptide chain of the mature RSV F protein. The native F1 polypeptide includes approximate residues 137-574 of the RSV F0 precursor and consists of (from N-terminus to C-terminus) an extracellular region (approximate residues 137-524), a transmembrane domain (approximate residues 525-550) and a cytoplasmic domain (approximate residues 551-574).

[0037] Herein, the term "F2 polypeptide", "F2 polypeptide" or "F2" refers to the polypeptide chain of the mature RSV F protein. The native F2 polypeptide includes the approximate residues 26-109 of the RSV F0 precursor.

[0038] As used herein, the term "native" or "wild-type" protein, sequence or polypeptide refers to a naturally occurring protein, sequence or polypeptide that has not been artificially modified by selective mutation.

[0039] Herein, the terms "F0 polypeptide", "F1 polypeptide", and "F2 polypeptide" include polypeptides from natural sequences or modified polypeptides, artificial modifications include but are not limited to amino acid substitutions, insertions or deletions. The modifications are, for example, designed to stabilize the F mutant or enhance the immunogenicity of the F mutant. In the native RSV F protein, the F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form a F2-F1 heterodimer.

[0040] Herein, the terms "F protein", "F polypeptide" and "RSV F protein" refer to one or all of "F0 polypeptide", "F1 polypeptide" and "F2 polypeptide" in any state.

[0041] Herein, the term "pep27 polypeptide" or "pep27" refers to a 27 amino acid polypeptide that is cleaved from the F0 precursor during RSV F protein maturation. The sequence of pep27 is flanked by two furin cleavage sites that are cleaved by cellular proteases during F protein maturation to generate F1 and F2 polypeptides.

[0042] Herein, the term "DS-Cav1" refers to a form of RSV F protein having the amino acid sequence described in McLellan et al., Science, 342(6158), 592-598, 2013, the amino acid sequence of which is SEQ ID NO.5.

[0043] Herein, the term "D25" refers to the antibody described in WO 2008 / 147196 A2, which specifically recognizes the epitope Φ, and has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO.6 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO.7.

[0044] Herein, the term "AM14" refers to the antibody described in WO 2008 / 147196 A2, which recognizes a pre-fusion trimer-specific quaternary epitope, and has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO.8 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO.9.

[0045] As used herein, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and modified amino acids thereof, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0046] In this article, substitution is expressed as follows: "Substitution position X2" or "X1 substitution position X2", X1 represents the amino acid residue at the position before substitution, and X2 represents the amino acid residue after substitution; when "substitution position X2" is used, it means that the specific type of amino acid residue at the position before substitution is not limited. For example, 155C means that the 155th position is C after substitution, and A149C means that the 149th position is A before substitution and C after substitution.

[0047] Herein, unless otherwise specified, the amino acid positions of the RSV F0 polypeptide herein refer to the amino acid sequence of the RSV F0 protein shown in SEQ ID NO.1.

[0048] Natural RSV F protein shows significant sequence conservation between RSV subtypes. For example, RSV subtypes A and B share 90% sequence identity. Within RSV subtypes, F0 sequence identity is even higher; For example, in RSV A, B subtypes, RSV F0 precursor protein has about 98% sequence identity. Almost all identified RSV F0 precursor sequences are composed of 574 amino acid lengths, and the length is usually slightly different due to the length of the C-terminal cytoplasmic tail region. The sequence identity between various natural RSV F proteins is known in the art. In order to further illustrate the sequence conservation level of F protein, the non-shared amino acid residues between the F0 precursor polypeptide sequences from representative RSV A strains and RSV B strains are provided in Tables 1 and 2, respectively.

[0049] Table 1 Non-consensus amino acid residues among F protein sequences from selected RSV A strains.

[0050]

[0051] Table 2 Non-shared amino acid residues between F protein sequences from selected RSV B strains

[0052]

[0053] In view of the substantial conservatism of RSV F sequences, those of ordinary skill in the art can easily compare the amino acid positions between different natural RSV F sequences to identify the corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, between nearly all identified natural RSV F0 precursor proteins, the furin protease cleavage site falls in the same amino acid position. Therefore, the conservatism of natural RSV F protein sequences between strains and subtypes allows the use of reference RSV F sequences to compare the amino acids at specific positions in RSV F proteins.

[0054] Herein, unless otherwise specified, the amino acid positions of the RSV F0 protein herein are given with reference to the sequence of the F0 precursor polypeptide described in SEQ ID NO.1 (amino acid sequence of the full-length native F precursor polypeptide of RSV A2 strain; corresponding to GenInfo identifier GI 138251 and Swiss Prot identifier P03420). Thus, the substitution position corresponds to the position in the amino acid sequence of the wild-type F0 protein shown in SEQ ID NO.1.

[0055] However, it should be noted, and those skilled in the art will appreciate, that different RSV F0 sequences may have different numbering systems, for example, if additional amino acid residues are added or removed compared to SEQ ID NO.1. Therefore, it should be understood that when referring to a particular amino acid residue by its number, the description is not limited to the amino acid that is exactly at that numbered position when counting from a given amino acid sequence, but refers to the equivalent / corresponding amino acid residue in any and all RSV F sequences, even if the residue is not at the same exact numbered position, for example, if the RSV sequence is shorter or longer than SEQ ID NO.1, or has an insertion or deletion compared to SEQ ID NO.1.

[0056] As used herein, the term "identity" percentage refers to the degree to which the amino acids of two polypeptides are identical at equivalent positions when the two sequences are optimally aligned. The comparison of amino acid sequence identity percentages can be performed in various ways known in the art, such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW or CLUSTAL OMEGA software known in the art. One skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required to achieve maximum alignment over the entire length of the comparison sequence.

[0057] Herein, the term "conservative amino acid substitution" refers to replacing one amino acid residue with another amino acid residue that is similar in physicochemical properties, and after the replacement, the properties and functions of the entire polypeptide or protein are unchanged or almost unchanged. Conservative amino acid substitution is well known to those of ordinary skill in the art. The following are examples of amino acids that are considered conservative substitutions: A, S and T are mutually substituted; D and E are mutually substituted; N and Q are mutually substituted; R and K are mutually substituted; I, L, M and V are mutually substituted; and F, Y and W are mutually substituted.

[0058] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, and nucleic acid molecules include ribonucleotides and / or deoxyribonucleotides. Examples of nucleic acid molecules include, but are not limited to, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleotide bases. When a nucleic acid molecule encodes a protein or polypeptide, the encoding may optionally encode a sense strand or an antisense strand. Nucleic acid molecules may be naturally occurring, synthetic, recombinant, or any combination thereof. "Nucleic acid molecules," "nucleic acids," and "polynucleotides" may be used interchangeably.

[0059] Herein, the term "vector" refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and the genetic element can be expressed, such as producing a protein, RNA or DNA encoded by the genetic element, or replicating the genetic element. The vector can be used to transform, transduce or transfect a host cell so that the genetic element it carries is expressed in the host cell. For example, vectors include: plasmids, episomal plasmids, minicircle DNA, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC), phages such as lambda phages or M13 phages, and animal viruses, etc. The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site. The vector may also include components that assist it in entering the cell, including, but not limited to, viral particles, liposomes or protein shells. The vector may be an expression vector or a cloning vector. In some embodiments, the vectors (e.g., expression vectors) provided by the present disclosure contain a nucleic acid sequence encoding an antibody or an antigen-binding fragment thereof as described in the present disclosure, at least one promoter (e.g., SV40, CMV, EF-1α) operably linked to the nucleic acid sequence, and at least one selection marker.

[0060] In a first aspect, a respiratory syncytial virus F protein mutant is provided, the respiratory syncytial virus F protein mutant contains at least one recombinant F2-F1 polypeptide, each of the recombinant F2-F1 polypeptides independently contains a mutated fragment of the F0 polypeptide, and the mutation includes:

[0061] (I) Positions 104 to 144 were deleted and replaced by a connecting peptide;

[0062] (II) containing the following substitutions at positions for forming disulfide bonds: 155C substitution, 289C substitution, 149C substitution, 458C substitution; 155C and 289C form a disulfide bond, and 149C and 458C form a disulfide bond; and,

[0063] (III) 373R substitution;

[0064] The position corresponds to the amino acid sequence of the wild-type F0 polypeptide shown in SEQ ID NO.1.

[0065] In alternative embodiments, the 155C substitution is an S155C substitution; and / or, the 289C substitution is an M289C substitution; and / or, the 149C substitution is an A149C substitution; and / or, the 458C substitution is a Y458C substitution.

[0066] In an optional embodiment, the respiratory syncytial virus F protein mutant is a multimer containing at least two or at least three recombinant F2-F1 polypeptides, preferably a multimer containing three recombinant F2-F1 polypeptides.

[0067] In an optional embodiment, the respiratory syncytial virus F protein mutant is a trimer, and the recombinant F2-F1 polypeptide forms a trimer through the multimerization domain of T4 Fibritin or the multimerization domain of GCN4.

[0068] In an optional embodiment, the amino acid sequence of the multimerization domain of T4 Fibritin is as shown in SEQ ID NO.14.

[0069] In an optional embodiment, the amino acid sequence of the multimerization domain of GCN4 is as shown in SEQ ID NO.15.

[0070] In an optional embodiment, the mutation further comprises an amino acid substitution that fills the cavity, and the amino acid substitution that fills the cavity is selected from (A1) or (A2) or (A3):

[0071] (A1) at least one of 190F, 190I, 207L, 54H, 296I and 488W;

[0072] (A2) 190F and 207L replaced;

[0073] (A3) 54H, 296I and 488W substitutions.

[0074] In an optional embodiment, the mutation further comprises an amino acid substitution of an electrostatic mutation, and the amino acid substitution of the electrostatic mutation is selected from (B1) or (B2) or (B3):

[0075] (B1) at least one of 46G, 465Q, 92D, 486S, 486H, 487Q and 489H;

[0076] (B2) 46G, 465Q, and 92D substitutions;

[0077] (B3) 486H, 487Q and 489H replaced.

[0078] In an optional embodiment, each of the recombinant F2-F1 polypeptides independently contains the following structure from the N-terminus to the C-terminus:

[0079] [F2 polypeptide fragment]-[connecting peptide 1]-[F1 polypeptide fragment]-[connecting peptide 2]-[multimerization domain];

[0080] The F2 polypeptide fragment is positions 26 to 103 of the F0 polypeptide;

[0081] The F1 polypeptide fragment is from position 145 to (510-524) of the F0 polypeptide position, for example, it can be but is not limited to positions 145 to 510, 145 to 513, 145 to 515, 145 to 518, 145 to 520, 145 to 521, 145 to 522 or 145 to 524 of the F0 protein position, preferably positions 145 to 524.

[0082] In an optional embodiment, the structure [F2 polypeptide fragment]-[connecting peptide 1]-[F1 polypeptide fragment]-[connecting peptide 2]-[multimerization domain] also satisfies one or more of the following (i) to (iv):

[0083] (i) the multimerization domain is a trimerization domain;

[0084] (ii) the multimerization domain is selected from the multimerization domain of T4 Fibritin or the multimerization domain of GCN4;

[0085] (iii) Connector 1 is selected from GS, GG or SAIG;

[0086] (iv) Connector peptide 2 is selected from SAIG.

[0087] In an optional embodiment, the respiratory syncytial virus F protein mutant is also connected to a thrombin recognition sequence and / or at least one purification tag. The purification tag is selected from conventional purification tags known in the art, and exemplary purification tags include but are not limited to histidine tags, histidine tags, glutathione sulfhydryl transferase tags, maltose binding protein tags, FLAG tags, streptomycin tags or Avi tags.

[0088] The amino acid sequence of the F0 polypeptide used as the basis for mutation in the respiratory syncytial virus F protein mutant provided in the first aspect can be selected from the amino acid sequence of the wild-type F0 polypeptide of each subtype known in the art, or the amino acid sequence obtained by modifying these wild-type amino acid sequences. The amino acid sequences of many kinds of natural RSV F proteins from different RSV subtypes and the nucleic acid sequences encoding such proteins are known in the art. For example, the sequences of several subtype A and B RSV F0 precursor proteins are shown in SEQ ID NO.1 (strain A2), SEQ ID NO.2 (strain B18537), SEQ ID NO.3 (strain Ontario), SEQ ID NO.4 (strain Buenos Aires), SEQ ID NO.10 (strain ON 1) and SEQ ID NO.11 (strain BA 9).

[0089] In an optional embodiment, the amino acid sequence of the F0 polypeptide is selected from the sequence shown in SEQ ID NO.1, 2, 3, 4, 10, 11 or 12.

[0090] In an optional embodiment, the amino acid sequence of the F0 polypeptide is selected from the sequences shown in SEQ ID NO.1, 2, 3 and 4.

[0091] In an optional embodiment, the amino acid sequence of the F0 polypeptide is selected from SEQ ID NO.1 or SEQ ID NO.12, preferably SEQ ID NO.12.

[0092] In an optional embodiment, the amino acid sequence of each of the recombinant F2-F1 polypeptides is independently selected from the sequences shown in SEQ ID NOs. 24 to 33.

[0093] The respiratory syncytial virus F protein mutant provided in the first aspect can be prepared by an optional method known in the art. Genetic engineering technology is optionally used to obtain recombinant expression, such as by Chinese hamster ovary cells (CHO), tumor cell lines, BHK cells, HEK293 cell lines and other mammalian cells or bacteria, yeast, fungi, insect cells, etc., or transgenic animals, or transgenic plants. Recombinant expression. Optionally, the respiratory syncytial virus F protein mutation is delivered to the target host cell in the form of a viral vector, such as adenovirus (human and chimpanzee), adeno-associated virus, vaccinia virus, herpes virus, retroviral vector, etc. It is optionally used to deliver the respiratory syncytial virus F protein to the target host cell in the form of a naked or encapsulated nucleic acid vector, for example, using linear RNA, circular RNA, dsDNA, cDNA, etc. The respiratory syncytial virus F protein mutant is optionally obtained using a commonly used or known form in this professional field.

[0094] In the second aspect, a biological material is also provided, including a polynucleotide, a vector or a cell; wherein the polynucleotide encodes the above-mentioned respiratory syncytial virus F protein mutant; the vector carries the polynucleotide; the cell carries the polynucleotide, or contains the vector or is capable of expressing the respiratory syncytial virus F protein mutant.

[0095] In the third aspect, the use of the respiratory syncytial virus F protein mutant described in the first aspect or the biomaterial described in the second aspect in the following (X1) to (X4) is also provided:

[0096] (X1) preparing antibodies that bind to respiratory syncytial virus antigen epitopes;

[0097] (X2) Preparation of vaccines for the prevention of respiratory syncytial virus infection;

[0098] (X3) Preparation of respiratory syncytial virus diagnostic products;

[0099] (X4) Detection of respiratory syncytial virus for non-diagnostic and non-therapeutic purposes.

[0100] In a fourth aspect, a vaccine for preventing respiratory syncytial virus infection is also provided, wherein the vaccine for preventing respiratory syncytial virus infection contains the respiratory syncytial virus F protein mutant described in the first aspect, or the biological material described in the second aspect.

[0101] In an optional embodiment, the vaccine further comprises optional adjuvants acceptable in the art, including but not limited to adjuvants, such as aluminum salts (such as aluminum phosphate, aluminum hydroxide or potassium aluminum sulfate), oil emulsion adjuvants (such as Freund's adjuvant, white oil Span adjuvant, MF-59, ISA 206, ISA 720, Adjuvant-65 or SAF), nucleotide adjuvants (CpG ODN, dsRNA or IL-12 DNA); stabilizers, such as sugars (such as lactose or sucrose), amino acids (such as glycine), gelatin and proteins (such as recombinant human albumin); surfactants; and diluents, such as one or more of water.

[0102] In an optional embodiment, the prevention of respiratory syncytial virus infection includes preventing RSV subtype A and / or subtype B.

[0103] In an optional embodiment, the vaccine for preventing respiratory syncytial virus infection is a recombinant protein vaccine containing the respiratory syncytial virus F protein mutant described in the first aspect.

[0104] In an optional embodiment, the vaccine for preventing respiratory syncytial virus infection is a nucleic acid vaccine containing DNA encoding the respiratory syncytial virus F protein mutant or a vector integrated with the DNA encoding the respiratory syncytial virus F protein mutant.

[0105] In an optional embodiment, the vaccine for preventing respiratory syncytial virus infection is an RNA vaccine containing RNA encoding the respiratory syncytial virus F protein mutant, such as mRNA or circular RNA.

[0106] In a fifth aspect, a respiratory syncytial virus diagnostic reagent or kit is also provided, wherein the respiratory syncytial virus diagnostic reagent or kit contains the respiratory syncytial virus F protein mutant described in the first aspect.

[0107] In an optional embodiment, the reagent or kit may also optionally include reagents and / or consumables used in the art for detection reactions and well known to those skilled in the art, including but not limited to one or more of buffer reagents, salts, secondary antibodies, chromogenic substrates, blocking solutions, washing solutions, solvents, eluents, coupling agents, negative controls, positive controls, standards, quality control products, and markers.

[0108] In an optional embodiment, the reagent or kit can be used in a general immunoassay method acceptable in the art, including but not limited to immunofluorescence staining, flow sorting, immunoblotting, immunohistochemistry, ELISA, immunochromatography or immunomagnetic beads. A person skilled in the art can prepare the reagent or other reagents in the kit according to the corresponding detection means, and the present invention is not limited to this.

[0109] In an optional embodiment, the RSV F protein mutant in the reagent or kit is used to detect whether anti-RSV antigen antibodies are present in a sample, so as to detect whether a subject is infected with RSV.

[0110] In an optional embodiment, the respiratory syncytial virus F protein mutant in the reagent or kit is used as a standard and / or quality control product.

[0111] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0112] Preparation Example 1

[0113] This preparation example provides a preparation method for RSV Pre-F protein mutants, including: designing the DNA sequence of the target RSV Pre-F protein mutant, performing sequence optimization and DNA synthesis. The complete sequence is cloned into the pcDNA3.1 vector. The plasmid is extracted. CHO-S cells are cultured at 37°C and 5% CO2. An appropriate concentration of cells is inoculated one day before transfection. On the day of transfection, the plasmid is mixed with the transfection agent and added to the cells. About 24 hours after transfection, DMEM+10% FBS is added to the cells. After 5 days of culture, the cell supernatant is centrifuged and filtered. AmMag TM Ni Magnetic Beads was used to purify the filtered cell supernatant for the first time. The binding buffer and elution buffer were 25 mM Tris-HCl, 300 mM NaCl (pH=8.0) and 25 mM Tris-HCl, 300 mM NaCl, 500 mM Imidazole (pH=8.0), respectively, and dialyzed with PBS (pH=7.2). HiLoad 16 / 600 Superdex 200pg 120ml was used for the second purification, and dialyzed with PBS (pH=7.2) (if one-step purification was performed, this step was omitted). The product was tested for content and / or SDS-PAGE.

[0114] Processing example

[0115] Treatment example 1: Temperature stress related treatment

[0116] Incubate the protein at 60°C for 1 hour or at 4°C for 2 weeks or perform 10 freeze-thaw cycles.

[0117] Treatment Example 2: High and Low pH Stress Related Treatments

[0118] The pH of the protein mutant (pH = 7.2) was adjusted to 3.5 with citric acid (pH = 1.4), incubated at room temperature for 1 hour, and then the pH was adjusted back to 7.2 with glycine / sodium hydroxide buffer (pH = 12).

[0119] The pH of the protein mutant (pH = 7.2) was adjusted to pH 10 with glycine / sodium hydroxide buffer (pH = 12), incubated at room temperature for 1 hour, and then adjusted back to pH 7.2 with citric acid (pH = 1.4).

[0120] Treatment Example 3: High and Low Osmotic Stress Treatment

[0121] The protein mutant (original osmotic pressure of 137 mM) was diluted with distilled water to reduce its osmotic pressure to 10 mM, incubated at room temperature for 1 hour, and then the osmotic pressure was adjusted back to 137 mM with NaCl solution.

[0122] MgCl2 was added to the protein mutant solution (original osmotic pressure was 137 mM) to raise the osmotic pressure of the protein solution to 3 M. The solution was incubated at room temperature for 1 hour, and then the osmotic pressure was adjusted back to 137 mM with PBS buffer.

[0123] Treatment Example 4: Immunization of mice

[0124] BALB / c mice, 6-8 weeks old, weighing 16-20 g, 10 mice / group, were injected intramuscularly with 50 μL of 10 μg protein mutant + 50 μg Al(OH)3 on D0 and D21. Blood was collected on D35 and serum was separated.

[0125] Test example

[0126] Test Example 1: Antigenicity Detection of RSV Pre-F Protein Mutants

[0127] The antigenicity of RSV Pre-F protein mutants was detected by ELISA.

[0128] The specific detection method is:

[0129] Dilute the target antibody to 2.0ug / mL with CBS buffer, add 100μL to each well of the ELISA plate, and adsorb it to the solid phase on the ELISA plate overnight at 4℃. Wash the plate the next day, add 300μL of 2% BSA to each well, and block at 37℃ for 1.5h. Add 1:400 diluted RSV Pre-F protein mutant to the plate wells, add 100μL to each well, and incubate at 37℃ for 1.0h. Add HRP secondary antibody, 100μL to each well, and incubate at 37℃ for 1.0h. Add 100μL of TMB colorimetric substrate to each well and incubate at 37℃ for 20min. Stop with 2N sulfuric acid stop solution and read at dual wavelengths of 450nm and 630nm using an ELISA reader.

[0130] Test Example 2: Antigenicity Detection of RSV Pre-F Protein Mutants

[0131] Antigenicity detection was performed using ForteBio Octet.

[0132] The specific detection method is:

[0133] The HIS1K biosensor was incubated in 1×PBS, 0.02% Tween-20, 0.1% BSA for 60 seconds and the baseline was adjusted. The protein mutant was diluted to 10 μg / mL with 1×PBS, 0.02% Tween-20, 0.1% BSA and incubated with the sensor for a period of time until the amount of solidification on the sensor reached 1.0 nm. The biosensor was run in 1×PBS, 0.02% Tween-20, 0.1% BSA for 60 seconds and the baseline was adjusted. The target antibody was diluted to 6 concentration ranges (200~6.25 nM) with 1×PBS, 0.02% Tween-20, 0.1% BSA, a zero concentration reference group was set, and incubated with the sensor for 60 seconds, during which the protein mutant on the sensor can bind to the target antibody. The sensor was incubated in 1×PBS, 0.02% Tween-20, 0.1% BSA for 120 seconds, during which the antibody dissociated from the sensor. Calculate the response signal value using analysis software.

[0134] Test Example 3: Structural stability of RSV Pre-F protein mutants

[0135] The specific detection method is:

[0136] Uncle instrument was used to characterize the structural thermal stability of purified RSV Pre-F protein mutants. 0.2-0.5 mg / mL RSV Pre-F protein mutants in 1×PBS (pH=7.2) were scanned from 10℃ to 80℃ at 90℃ / hour, and the Tm values ​​were analyzed using UNcleClient.

[0137] Test Example 4: Immunogenicity of RSV F protein mutants

[0138] Detect serum neutralizing antibody IC50.

[0139] The specific detection method is:

[0140] Take HEp-2 cells in the logarithmic growth phase, digest and count them, and then add 100 μL / well to a 37℃, 5% CO2 incubator for overnight culture. Take serum, inactivate at 56℃ for 30min, dilute 20 times with DMEM medium, and then dilute in 3-fold gradients to 8 gradients with a dilution of 20~43740 times. Thaw the virus solution, add an equal volume of serum diluent (neutralizing solution) according to its titer, and place it in a 37℃, 5% CO2 incubator for neutralization and incubation for 1 h. Take a 96-well cell plate, wash it twice with PBS, add neutralizing solution, place it in a 37℃, 5% CO2 incubator, and culture it for 24 h. Add 100 μL of 4% paraformaldehyde fixative, fix it at -20℃ for 30 min, wash, block with PBST, incubate with anti-F protein primary antibody, wash, incubate with HRP secondary antibody, wash, develop with Trueblue, and read with Elispot instrument. The calculation method for the neutralizing antibody titer is: inhibition rate = [1-(number of sample spots-number of cell control spots) / (number of virus control spots-number of cell control spots)] × 100%; with the sample dilution multiple as the X-axis and the inhibition rate as the Y-axis, GraphPad prism software was used for four-parameter fitting to calculate the IC50 value of the sample.

[0141] Example 1

[0142] 1. Design of backbone sequence: amino acid residues 1-513 of the natural RSV A2 F0 precursor polypeptide described in SEQ ID NO.12 with naturally occurring P102A, I379V and M447V substitutions relative to SEQ ID NO.1 were intercepted, and the amino acid at position 513 was connected to the T4 Fibritin (GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQID NO.14) or GCN4 (EDKIEEILSKIYHIENEIARIKKLIGEA, SEQ ID NO.15) trimerization domain through a SAIG ​​(SEQ ID NO.13) linker, and the trimerization structure was then connected to the thrombin recognition sequence and purification tag (His tag) through a GG linker, and the purification tag was then connected to the streptomycin tag II through a GS linker (the amino acid sequence from the thrombin recognition sequence to the streptomycin tag II is: LVPRGSHHHHHHGS WSHPQFEK, SEQ ID NO.16).

[0143] 2. Candidate site screening: The secondary structure of the sequence SEQ ID NO.1 was retrieved (Swiss Prot identifier P03420), and the positions of the four pairs of disulfide bond substitutions S55C-L188C, T103C-I148C, S155C-S290C, and A149C-Y458C in the secondary structure of the sequence SEQ ID NO.1 were analyzed. S55 was located at the central symmetric position of the β-chain structure of amino acids 51 to 60 (GWYTSVITIE, SEQ ID NO.17), L188 was located in the helical structure of amino acids 160 to 203 (LEGEVNKIKSALLSTNKAVVSLSNGVSVLTS KVLDLKNYIDKQL, SEQ ID NO.18); T103 was located in the linear structure after the turn structure of amino acids 100 to 102 (TNNRARR, SEQ ID NO.19), I148 is located between the helical structure of amino acids 145-147 and the helical structure of amino acids 149-158; S155 is located in the helical structure of amino acids 149-158 (ASGVAVSKVL, SEQ ID NO.20), S290 is located at the central symmetric position of the β-chain structure of amino acids 286-293 (YSIMSIIK, SEQ ID NO.21); A149 is located at the head of the helical structure of amino acids 149-158 (ASGVAVSKVL, SEQ ID NO.22), and Y458 is located at the tail of the β-chain structure of amino acids 455-458 (TLYY, SEQ ID NO.23). Therefore, the substitutions that form disulfide bonds and stabilize the pre-fusion conformation may also include: V56C-L188C, N104C-I148C, N105C-I148C, S155C-M289C. Therefore, the above four groups of substitutions that form disulfide bonds and stabilize the pre-fusion conformation were selected as candidate mutation sites.

[0144] 3. Based on the backbone sequence of step 1, further engineering modification was performed according to Table 3 to obtain amino acid sequences CDK001-CDK002, CDK027, K003-K010. The difference between sequences CDK027, K007-K010 and CDK002, K003-K006 is that amino acids 104-144 are removed, amino acids 103 and 145 are connected with GS linkers, and L373R substitution and A149C-Y458C substitution are performed. The amino acid sequence of K007 in Table 3 is shown in SEQ ID NO.24.

[0145] Table 3 Sequence characteristics of sequences CDK001-CDK002, CDK027, K003-K010

[0146]

[0147] According to the method of Preparation Example 1, the amino acid sequences CDK001-CDK002, CDK027, and K003-K010 were expressed and purified in one step in a 30 ml culture system, and the protein expression was detected. According to the method of Detection Example 1, the antigenicity of the sequences CDK001-CDK002, CDK027, and K003-K010 to D25 and AM14 was detected. The test results are shown in Table 4:

[0148] Table 4 Protein expression and antigenicity of mutants CDK001-CDK002, CDK027, K003-K010

[0149]

[0150] Combined with Table 4, among K003-K006, only K003 had higher antigenicity to D25 and AM14 than CDK002; the expression level of K006 was too low, and the antigenicity to D25 and AM14 was not detected. After further removal of amino acids 104-144, amino acids 103 and 145 were connected with a GS linker, and L373R and A149C-Y458C substitutions were performed, K008-K009 removed N104C and N105C engineering designs during the removal of 104-144, and 148C randomly formed disulfide bonds with other cysteines in the sequence, resulting in unstable structure, and the yield was further reduced relative to K004-K005, and the antigenicity to D25 and AM14 did not change much. K007 has a higher yield than K003, further improved antigenicity to D25 and AM14, and has better antigenicity than CDK027, and a yield more than twice that of CDK027.

[0151] Example 2

[0152] Using sequence K007 as the skeleton, further engineering transformation was performed according to Table 5 to obtain amino acid sequences K011-K014. The difference between sequence K011 and K007 is 103-GG-145, the difference between sequence K012 and K007 is 103-SAIG-145, the difference between sequence K013 and K007 is that the length of F0 protein truncation is 1-524, and the difference between sequence K014 and K007 is that the trimerization domain is GCN4. The amino acid sequences of K011~014 in Table 5 are shown in SEQ ID NO.25~28 respectively.

[0153] Table 5 Sequence characteristics of sequences K011-K014

[0154]

[0155] According to the method of Preparation Example 1, the amino acid sequence K011-K014 was expressed and purified in one step in a 30 ml culture system, and the protein expression was detected. According to the method of Detection Example 1, the antigenicity of the sequence K011-K014 to D25 and AM14 was detected. The test results are shown in Table 6:

[0156] Table 6 Protein expression and antigenicity of sequences K007, K011-K014

[0157]

[0158] Combined with Table 6, the expression level and antigenicity of sequence K007 to D25 and AM14 were not much different from those of sequences K011-K014.

[0159] Example 3

[0160] The expression proteins of sequences CDK002, K003, K007, K011-K014, and CDK027 were subjected to temperature stress-related treatment according to the method of Treatment Example 1, and then the antigenicity to D25 and AM14 was detected according to the method of Detection Example 1, and the activity of the treated proteins was compared with that of the fresh proteins. The activity ratio is defined as the stability of the protein, and it is expected that a more stable protein has a higher activity ratio. The test results are shown in Table 7.

[0161] Table 7 Stability of sequences CDK002, K003, K007, K011-K014, CDK027

[0162]

[0163] Combined with Table 7 , the stability of sequence K007 after 10 freeze-thaw cycles at 4°C (2 weeks), 60°C (1 hour), was better than that of sequences CDK002, K003, and CDK027, and was not much different from sequences K011-K014.

[0164] Example 4

[0165] Based on the backbone sequence of Example 1, further engineering modification was performed according to Table 8 to obtain amino acid sequences, and amino acid sequences K015-K016 and DS-Cav1 were obtained. The difference between K015 and K016 relative to K003 and K007 is that the substitution S190F-V207L ​​for filling the cavity is further added. The truncated length of the F0 protein of K015-K016 and DS-Cav1 is 1-513, and the trimerization domain is T4 Fibritin. The amino acid sequence of K016 in Table 8 is shown in SEQ ID NO.29.

[0166] Table 8 Sequence characteristics of sequences K015-K016 and DS-Cav1

[0167]

[0168] According to the method of Preparation Example 1, the amino acid sequences K007, K015-K016 and DS-Cav1 were expressed and purified in two steps in a 100 ml culture system, and the main peak was collected for SDS-PAGE detection. The reducing band of the trimer protein is expected to be located between the 50-75 band of the marker, and the non-reducing band of the trimer protein is expected to be located between the 150-250 band of the marker. The results are as follows Figure 1~Figure 4 As shown, K015 and DS-Cav1 cannot express a trimer structure with high purity under the culture system and two-step purification conditions of Preparation Example 1, while K007 and K016 can express a trimer structure with high purity.

[0169] The response signal values ​​of sequences K007, K015-K016 and DS-Cav1 expression proteins to D25 and AM14 were detected by the method of detection example 2. The higher the response signal value, the higher the binding activity of the expected protein to the antibody. The sequences K007, K015-K016 and DS-Cav1 expression proteins were treated by the methods of treatment examples 2 and 3, and the antigenicity to D25 and AM14 was detected again by the method of detection example 1, and the activity of the treated proteins was compared with that of the fresh proteins. The activity ratio is defined as the stability of the protein, and it is expected that a more stable protein has a higher activity ratio. The Tm values ​​of sequences K007, K015-K016 and DS-Cav1 expression proteins were detected by the method of detection example 3. The higher the Tm value, the better the thermal stability of the expected protein. The results are shown in Table 9.

[0170] Table 9 Response signal values, activity ratios under different treatment conditions and Tm values ​​of sequences DS-Cav1, K007, K015 and K016.

[0171]

[0172] Combined with Table 9, the antigenicity, pH tolerance, osmotic pressure and thermal stability of K007 and K016 are better than those of DS-Cav1, and K015 is not much different from DS-Cav1. Compared with K007, K016 has further improved antigenicity and stability after cavity filling modification.

[0173] Example 5

[0174] Based on the backbone sequence of Example 1, further engineering modification was performed according to Table 10 to obtain amino acid sequences DS-Cav2 and K017-K020. The F0 protein truncation lengths of DS-Cav2 and K017-K020 were both 1-513, and the trimerization domains were all T4 Fibritin. The amino acid sequences of K017~K020 in Table 10 are shown in SEQ ID NO.30~33, respectively.

[0175] Table 10 Sequence characteristics of sequences DS-Cav2, K017-K020

[0176]

[0177] According to the method of Preparation Example 1, the sequences DS-Cav1, DS-Cav2, K007, and K016-K020 were expressed and purified in two steps in a 100 ml culture system, and the main peak was collected. According to the method of Treatment Example 4, mice were immunized with proteins expressed by the sequences DS-Cav1, DS-Cav2, K007, and K016-K020, and serum was collected. The grouping method is shown in Table 11.

[0178] Table 11 Immunogrouping of sequences DS-Cav1, DS-Cav2, K007, K016-K020

[0179]

[0180] The IC50 of the neutralizing antibodies of the sera of Groups 1 to 8 against RSV A2 strain was tested in the manner of Test Example 4. The results are shown in Table 12.

[0181] Table 12 Geometric mean IC50 of neutralizing antibodies of sequences DS-Cav1, DS-Cav2, K007, K016-K020

[0182]

[0183] Combined with Table 12, the geometric mean IC50 of neutralizing antibody in K007 immune serum is higher than that in DS-Cav1 and not much different from that in DS-Cav2, and the geometric mean IC50 of neutralizing antibody in K016-K020 immune serum is higher than that in DS-Cav1 and DS-Cav2. Compared with K007, K016, and K020, the geometric mean IC50 of neutralizing antibody in immune serum is further improved after cavity filling and electrostatic mutation modification.

[0184] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention 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 replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A respiratory syncytial virus F protein mutant, characterized in that: The respiratory syncytial virus F protein mutant is a trimer, composed of three recombinant F2-F1 polypeptides, and the structure from the N-terminus to the C-terminus of the recombinant F2-F1 polypeptide is as follows: [F2 polypeptide fragment]-[connecting peptide 1]-[F1 polypeptide fragment]-[connecting peptide 2]-[multimerization domain]; The sequence of [F2 polypeptide fragment]-[connecting peptide 1]-[F1 polypeptide fragment] is obtained by mutating the amino acid sequence of the F0 polypeptide shown in SEQ ID NO.12 at positions 1 to 513 or 1 to 524, wherein the mutation is: (I) Positions 104 to 144 were deleted and replaced by connecting peptide 1; (II) substitutions at the following positions for forming disulfide bonds: 155C substitution, 289C substitution, 149C substitution, 458C substitution; 155C and 289C form a disulfide bond, and 149C and 458C form a disulfide bond; and, (III) 373R substitution.

2. A respiratory syncytial virus F protein mutant, characterized in that: The respiratory syncytial virus F protein mutant is a trimer, composed of three recombinant F2-F1 polypeptides, and the structure from the N-terminus to the C-terminus of the recombinant F2-F1 polypeptide is as follows: [F2 polypeptide fragment]-[connecting peptide 1]-[F1 polypeptide fragment]-[connecting peptide 2]-[multimerization domain]; The sequence of [F2 polypeptide fragment]-[connecting peptide 1]-[F1 polypeptide fragment] is obtained by mutating the amino acid sequence of the F0 polypeptide shown in SEQ ID NO.12 at positions 1 to 513 or 1 to 524, wherein the mutation is: (I) Positions 104 to 144 were deleted and replaced by connecting peptide 1; (II) Substitutions at the following positions for forming disulfide bonds: 155C substitution, 289C substitution, 149C substitution, 458C substitution; 155C and 289C form a disulfide bond, and 149C and 458C form a disulfide bond; (III) 373R substitution; and, (IV) Amino acid substitutions that fill cavities and / or amino acid substitutions that cause electrostatic mutations; The cavity-filling amino acid substitution is selected from (A1) or (A2) or (A3): (A1) at least one of 190F, 190I, 207L, 54H, 296I and 488W; (A2) 190F and 207L replaced; (A3) 54H, 296I and 488W substitutions; The amino acid substitution of the electrostatic mutation is selected from (B1) or (B2) or (B3): (B1) at least one of 46G, 465Q, 92D, 486S, 486H, 487Q and 489H; (B2) 46G, 465Q, and 92D substitutions; (B3) 486H, 487Q and 489H replaced.

3. The respiratory syncytial virus F protein mutant according to claim 1 or 2, characterized in that: And at least one or more of the following (i) to (iii) are met: (i) the multimerization domain is selected from the multimerization domain of T4 Fibritin or the multimerization domain of GCN4; (ii) connecting peptide 1 is selected from GS, GG or SAIG; (iii) Connector peptide 2 is selected from SAIG.

4. A polynucleotide, characterized in that The polynucleotide encodes the respiratory syncytial virus F protein mutant according to any one of claims 1 to 3.

5. A carrier, characterized in that The vector carries the polynucleotide according to claim 4.

6. A cell, characterized in that The cell carries the polynucleotide according to claim 4, or contains the vector according to claim 5, or expresses the respiratory syncytial virus F protein mutant according to any one of claims 1 to 3.

7. Use of the respiratory syncytial virus F protein mutant according to any one of claims 1 to 3, or the polynucleotide according to claim 4, or the vector according to claim 5, or the cell according to claim 6 in the following (X1) to (X4): (X1) preparing antibodies that bind to respiratory syncytial virus antigen epitopes; (X2) Preparation of vaccines for the prevention of respiratory syncytial virus infection; (X3) Preparation of respiratory syncytial virus diagnostic products; (X4) Detection of respiratory syncytial virus for non-diagnostic and non-therapeutic purposes.

8. A vaccine for preventing respiratory syncytial virus infection, characterized in that: Containing the respiratory syncytial virus F protein mutant according to any one of claims 1 to 3, or the polynucleotide according to claim 4, or the vector according to claim 5, or the cell according to claim 6.

9. A respiratory syncytial virus diagnostic reagent or kit, characterized in that: Contains the respiratory syncytial virus F protein mutant according to any one of claims 1 to 3.

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