WO2024109611A1

WO2024109611A1 - Mutant respiratory syncytial virus pre-fusion f protein and use thereof

Info

Publication number: WO2024109611A1
Application number: PCT/CN2023/131875
Authority: WO
Inventors: 张建城; 陈晓雨; 易晓男; 黄玲玲; 阮宝阳; 刘林; 曹玉锋; 史力
Original assignee: 怡道生物科技(苏州)有限公司
Priority date: 2022-11-21
Filing date: 2023-11-15
Publication date: 2024-05-30
Also published as: CN116284266A; CN116284266B

Abstract

Provided is a mutant respiratory syncytial virus pre-fusion F protein, having the following mutations compared with a wild-type respiratory syncytial virus pre-fusion F protein: (a) at least one amino acid residue in an F1 subunit and/or an F2 subunit is substituted by cysteine, and (b) amino acid residues at positions 104-144 of the wild-type respiratory syncytial virus pre-fusion F protein are partially or completely substituted by a linker peptide, at least two lengths of the amino acid residues of the linker peptide being comprised. Compared with similar products, the mutant respiratory syncytial virus pre-fusion F protein has excellent neutralizing antibody binding activity and thermal stability, and is suitable for being developed into one of components of respiratory syncytial virus vaccines.

Description

Mutant respiratory syncytial virus prefusion F protein and its application

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to the Chinese patent application with application number 202211472876.7 filed with the State Intellectual Property Office of China on November 21, 2022, and entitled “Mutated respiratory syncytial virus prefusion F protein and its application”, the entire contents of which are incorporated by reference into this application.

Technical Field

The present disclosure relates to the field of bioengineering technology, and in particular to a mutant respiratory syncytial virus pre-fusion F protein and an application thereof.

Background technique

Respiratory syncytial virus (RSV) is an important pathogen that causes lower respiratory tract infections in infants, the elderly, and immunocompromised patients. In 2020, the number of children under 5 years old with severe RSV infection worldwide reached 34.6 million, of which 3 million were infected in China alone. Every year, approximately 3% to 7% of people over 60 years old are infected with RSV. The elderly are at higher risk of developing serious diseases due to decreased immunity and underlying diseases. The WHO estimates that 64 million children are infected with RSV worldwide each year, of which 150,000 children die from RSV infection, and 99% of deaths occur in low- and middle-income countries, causing a very serious global medical burden. It is estimated that the global direct medical costs related to RSV were 4.82 billion euros in 2017. RSV infection can lead to serious complications, including bronchiolitis or pneumonia, often accompanied by acute respiratory distress, requiring hospitalization. Natural RSV infection does not induce long-term immune protection and can be recurrent. Currently, there is only one licensed monoclonal antibody product (Palivizumab) used to reduce the incidence of severe diseases in high-risk newborns. There has been no effective RSV vaccine on the market for more than 30 years, and the development of a safe and effective RSV vaccine has been considered very challenging. Therefore, the respiratory syncytial virus vaccine has been listed by the WHO as one of the top priority vaccines for development worldwide.

Summary of the invention

The present disclosure provides a mutant respiratory syncytial virus prefusion F protein, wherein the mutant respiratory syncytial virus prefusion F protein has the following mutations compared to the wild-type respiratory syncytial virus prefusion F protein:

(a) at least one amino acid residue in the F1 subunit and/or the F2 subunit is substituted by cysteine, and the substitution results in a non-native disulfide bond between the F1 subunit and the F2 subunit of the respiratory syncytial virus prefusion F protein, wherein the non-native disulfide bond includes a disulfide bond other than Cys69-Cys212 and Cys37-Cys439 formed between the F1 subunit and the F2 subunit;

(b) amino acid residues 104 to 144 of the wild-type respiratory syncytial virus prefusion F protein are partially or completely replaced by a connecting peptide, wherein the amino acid residue length of the connecting peptide is at least 2;

The amino acid sequence of the wild-type respiratory syncytial virus pre-fusion F protein is shown in SEQ ID No.01.

Optionally, the cysteine substitution comprises at least one of S55C, T189C, P101C or V152C.

Optionally, the cysteine substitutions include S55C and T189C, and/or, P101C and V152C.

Optionally, amino acid residues 104 to 137 of the wild-type respiratory syncytial virus pre-fusion F protein are completely replaced by a connecting peptide.

Optionally, the amino acid sequence of the connecting peptide is GSGSGGSGSGRS.

Optionally, amino acid residues 104 to 144 of the wild-type respiratory syncytial virus pre-fusion F protein are all replaced by a connecting peptide, and the amino acid sequence of the connecting peptide is GSGSGRS or GS.

The present disclosure provides a biomaterial, the biomaterial comprising any one of the following (a) to (d):

(a) a nucleic acid molecule encoding the mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6;

(b) a recombinant vector containing the nucleic acid molecule described in (a), preferably, the original plasmid of the recombinant vector is pEE12.4;

(c) a transformed cell containing the nucleic acid molecule (a) or the recombinant vector (b); preferably, the host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells;

Further preferably, the mammalian cell is selected from Chinese hamster ovary cells, tumor cells, BHK cells or HEK293 cells;

(d) a recombinant virus containing the nucleic acid molecule described in (a) or the recombinant vector described in (b), preferably, the recombinant virus comprises an adenovirus, an adeno-associated virus, a vaccinia virus, a herpes virus or a retroviral vector.

The present disclosure provides a method for preparing the mutant respiratory syncytial virus pre-fusion F protein, which comprises culturing the aforementioned transformed cells or recombinant viruses, and inducing expression to obtain the mutant respiratory syncytial virus pre-fusion F protein.

Use of the aforementioned mutant respiratory syncytial virus prefusion F protein or the aforementioned biological material in any one of the following (a) to (c):

(a) Application in the preparation of respiratory syncytial virus-specific antibodies;

(b) preparing a medicament for preventing and/or treating respiratory syncytial virus infection, preferably, the medicament comprises a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine;

(c) preparing a diagnostic reagent for respiratory syncytial virus, optionally, the diagnostic reagent is used for diagnosing respiratory syncytial virus infection.

The present disclosure provides a drug for preventing and/or treating respiratory syncytial virus infection, comprising the aforementioned mutant respiratory syncytial virus prefusion F protein, the aforementioned biological material or the respiratory syncytial virus-specific antibody described in (a);

Optionally, the drug includes a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.

The present disclosure provides a pharmaceutical composition, which comprises the mutant respiratory syncytial virus pre-fusion F protein, the biological material or the respiratory syncytial virus-specific antibody described in (a); and a pharmaceutically acceptable carrier.

The present disclosure provides a respiratory syncytial virus diagnostic reagent, comprising the aforementioned mutant respiratory syncytial virus pre-fusion F protein, or the aforementioned respiratory syncytial virus-specific antibody.

The present disclosure provides a method for preventing and/or treating respiratory syncytial virus infection, the method comprising administering a therapeutically effective amount of the aforementioned mutant respiratory syncytial virus prefusion F protein, the aforementioned biological material, or the aforementioned respiratory syncytial virus-specific antibody to a patient in need thereof;

Optionally, the mutant respiratory syncytial virus pre-fusion F protein, biological material or respiratory syncytial virus-specific antibody is administered in the form of a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.

The present disclosure provides a method for diagnosing respiratory syncytial virus infection, comprising the step of using the aforementioned mutant respiratory syncytial virus pre-fusion F protein, or the aforementioned respiratory syncytial virus-specific antibody.

The present disclosure provides the aforementioned mutant respiratory syncytial virus prefusion F protein, the aforementioned biological material, or the aforementioned respiratory syncytial virus-specific antibody for use in preventing and/or treating respiratory syncytial virus infection;

The present disclosure provides the aforementioned mutant respiratory syncytial virus pre-fusion F protein, or the aforementioned respiratory syncytial virus-specific antibody, for use in diagnosing respiratory syncytial virus infection.

BRIEF DESCRIPTION OF THE DRAWINGS

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

FIG1 is a plasmid map of the pEE12.4 expression vector used in the embodiments of the present disclosure;

FIG2 shows the binding of each mutant antigen to six different monoclonal antibodies (indirect ELISA);

FIG3 shows the binding of three connecting peptides to five monoclonal antibodies (indirect ELISA);

Figure 4 shows the reducing SDS-PAGE (left) and Western Blot (right) of the purified antigen;

Fig. 5 is a graph showing HPLC detection of the purified antigen;

FIG6 shows the binding of the dual-factor mutant antigen to the AM14 monoclonal antibody (indirect ELISA);

FIG. 7 is a turbidity point analysis graph of YD22-1 and SC-TM.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments.

Therefore, the following detailed description of the embodiments of the present disclosure provided in the accompanying drawings is not intended to limit the scope of the present disclosure claimed for protection, but merely represents selected embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present disclosure.

Respiratory Syncytial Virus (RSV) infection is mainly mediated by glycoproteins F and G. Adhesion protein (G) adheres to the host cell membrane, promoting the adsorption of the virus on the cell surface. Fusion protein (F) mediates the fusion of the viral envelope and the host cell membrane, allowing the virus to enter the cell. During viral fusion and entry, the F protein changes from a metastable pre-fusion conformation (pre-F) to a stable post-fusion conformation (post-F). The F protein sequence is relatively conserved among different subtypes and is an important antigenic target for RSV vaccine development. RSV F protein belongs to type I fusion protein, which mainly includes the following functional regions: signal peptide (1-26aa) located at the N-terminus of F protein, responsible for guiding F protein to the cell membrane surface; two furin cleavage sites, protease recognition sites mainly composed of basic amino acids, namely cleavage site 1 (KKRKRR, FCS1) and cleavage site 2 (RARR, FCS2); fusion peptide FP (137-146aa) composed of 19 amino acids, when the trimer of F protein is activated, the fusion peptide is responsible for inserting into the cell membrane of adjacent cells; heptad repeat region HRA (146-216aa) and HRB (460-514aa), During membrane fusion, HRA and HRB form a stable 6HB structure; the transmembrane region (514-574aa), the region where the F protein is embedded in the membrane; the CT region, the region where the F protein is located in the cytoplasm, which can interact with the viral M protein and is related to the packaging and budding of the virus.

The F protein is first synthesized as a protein precursor F0. During the cell fusion process, it is cleaved by furin to release a 27-amino acid polypeptide pep27 (109-127aa), forming F1 and F2 fragments connected by disulfide bonds, and transforming from an unstable pre-fusion conformation to a stable post-fusion conformation. Comparison of the RSV F protein structure before and after fusion showed that most of the secondary and tertiary structures were retained in the pre-fusion and post-fusion states. In contrast, the N-terminal and C-terminal regions of the F1 subunit showed obvious conformational changes. The fusion peptide and five secondary structural elements (α2, α3, α4 helices and β3, β4 folds) at the N-terminal end of the F1 subunit were rearranged and fused with the α5 helix to form a length > At the C-terminus of the F1 subunit, the only parallel strand (β22) spreads out, allowing the pre-fusion α10 helix to move toward the α5post helix to promote membrane fusion.

In recent years, as the conformational transition process of the F protein has been gradually revealed, the Φ epitope with more than 90% neutralizing activity in the prefusion conformation of the F protein has been discovered, and the mutation site modification used to stabilize the preF structure has also been confirmed. Stable preF protein mutants can activate more efficient neutralizing antibodies as vaccine antigens. Therefore, the development of stable prefusion conformation F protein antigens has become a new goal for RSV vaccine research and development. The stable prefusion conformation F protein vaccine developed based on structural biology has also entered the clinical stage.

The ideal design of a pre-fusion conformation F protein mutant should have the following characteristics: (1) Maintaining the pre-fusion conformation of the F protein trimer to obtain the correct neutralizing epitope; (2) High expression levels of the trimer protein to induce sufficient immune response and meet the needs of industrial manufacturing, that is, the F protein should have efficient expression and self-assembly capabilities; (3) Having a highly stabilized trimer conformation to continuously maintain the pre-fusion conformation, maintain the original or higher immune activity, and meet the needs of production preparations.

The present disclosure provides a mutant respiratory syncytial virus prefusion F protein, and it is expected that the mutant protein can have a stable prefusion conformation and high expression level, and can form a stable trimer, so that it can be used for the prevention, diagnosis and treatment of respiratory syncytial virus infection.

It should be noted that in all discussions herein, standard single-letter or three-letter codes for amino acids are used, and standard substitution notation is also used. It is understood that Cys69 herein means cysteine at position 69. S55C herein means that serine at position 55 is substituted with cysteine.

It should be noted that the "partial or complete replacement" means that the amino acid residues 104 to 144 of the wild-type respiratory syncytial virus pre-fusion F protein can be completely replaced or some fragments thereof can be partially replaced, including but not limited to "positions 104 to 110", "positions 104 to 120", "positions 104 to 130", "positions 104 to 137", "positions 110 to 130" or "positions 120 to 144".

In an alternative embodiment, the cysteine substitution comprises at least one of S55C, T189C, P101C or V152C.

In alternative embodiments, the cysteine substitutions include S55C and T189C, and/or, P101C and V152C.

It should be noted that the cysteine substitution includes S55C and T189C, which means that the two substitutions S55C and T189C exist at the same time, but it does not mean that the two substituted cysteines must form a disulfide bond connection. It is understandable that the two substituted cysteines can form a disulfide bond connection.

It should be noted that the cysteine substitution includes P101C and V152C, which means that the two substitutions P101C and V152C exist at the same time, but it does not mean that the two substituted cysteines must form a disulfide bond connection. It is understandable that the two substituted cysteines can form a disulfide bond connection.

In an optional embodiment, amino acid residues 104 to 137 of the wild-type respiratory syncytial virus prefusion F protein are completely replaced by a connecting peptide.

In an optional embodiment, the amino acid sequence of the connecting peptide is GSGSGGSGSGRS.

In an optional embodiment, amino acid residues 104 to 144 of the wild-type respiratory syncytial virus pre-fusion F protein are all replaced by a connecting peptide, and the amino acid sequence of the connecting peptide is GSGSGRS or GS.

The present disclosure provides a biomaterial, which comprises any one of the following (a) to (d):

(a) A nucleic acid molecule encoding the mutant respiratory syncytial virus pre-fusion F protein according to any one of claims 1 to 6.

(b) A recombinant vector containing the nucleic acid molecule described in (a), preferably, the original plasmid of the recombinant vector is pEE12.4.

(c) A transformed cell containing the nucleic acid molecule of (a) or the recombinant vector of (b), preferably, the host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells.

Further preferably, the mammalian cell is selected from Chinese hamster ovary cells (CHO), tumor cells, BHK cells or HEK293 cells.

The present disclosure provides a method for preparing the mutant respiratory syncytial virus pre-fusion F protein as described in any of the aforementioned embodiments, the preparation method comprising culturing the transformed cells or recombinant viruses as described in the aforementioned embodiments, and inducing expression to obtain the mutant respiratory syncytial virus pre-fusion F protein.

The present disclosure provides the use of the mutant respiratory syncytial virus pre-fusion F protein or biomaterial described in any one of the aforementioned embodiments in any one of the following (a) to (c):

(c) Preparation of respiratory syncytial virus diagnostic reagents.

The present disclosure provides a drug for preventing and/or treating respiratory syncytial virus infection, comprising the mutant respiratory syncytial virus pre-fusion F protein described in any one of the aforementioned embodiments, the aforementioned biological material, or the respiratory syncytial virus-specific antibody described in the aforementioned embodiments.

The present disclosure provides a respiratory syncytial virus diagnostic reagent, comprising the mutant respiratory syncytial virus pre-fusion F protein described in any one of the aforementioned embodiments, or the respiratory syncytial virus-specific antibody described in the aforementioned embodiments.

Preferably, the mutant respiratory syncytial virus prefusion F protein described in the present disclosure includes cysteine substitution mutations S55C and T189C and a mutant in which amino acid residues at positions 104 to 137 are replaced with GSGSGGSGSGRS, and is capable of becoming one of the components of a respiratory syncytial virus vaccine for prevention and/or treatment.

Preferably, the mutant respiratory syncytial virus prefusion F protein described in the present disclosure includes cysteine substitution mutations P101C and V152C, and is capable of becoming one of the components of a respiratory syncytial virus vaccine for prevention and/or treatment.

Preferably, the mutant respiratory syncytial virus prefusion F protein described in the present disclosure includes cysteine substitution mutations P101C and V152C and a mutant in which amino acid residues at positions 104 to 137 are replaced with GSGSGGSGSGRS, and is capable of becoming one of the components of a respiratory syncytial virus vaccine for prevention and/or treatment.

Preferably, the mutant respiratory syncytial virus prefusion F protein described in the present disclosure includes mutants of cysteine substitution mutations S55C, T189C, P101C and V152C, and is capable of becoming one of the components of a respiratory syncytial virus vaccine for prevention and/or treatment.

Preferably, the mutant respiratory syncytial virus prefusion F protein described in the present disclosure includes cysteine substitution mutations S55C, T189C, P101C and V152C, and a mutant in which the amino acid residues at positions 104 to 137 are replaced with GSGSGGSGSGRS, and is capable of becoming one of the components of a respiratory syncytial virus vaccine for prevention and/or treatment.

The position of the mutated amino acid residue in the present disclosure is shown in the amino acid sequence SEQ ID No. 01 of the wild-type respiratory syncytial virus prefusion F protein, which has three natural substitutions of P102A, I379V and M447V compared with the amino acid sequence of GenBank accession number: P03420. In addition, the amino acid sequence of other wild-type respiratory syncytial virus prefusion F protein can also be used as a template, including but not limited to directly using the amino acid sequence of GenBank accession number: P03420, or containing 1 or 2 natural substitutions on the basis of P03420.

The term "wild type" refers to a gene or gene product that is separated from a naturally occurring source. A wild-type gene is the most commonly observed gene in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of a gene. In contrast, the term "modified", "mutant" or "variant" refers to a gene or gene product that shows sequence modification (e.g., substitution, truncation or insertion), post-translational modification and/or functional properties (e.g., altered properties) compared to a wild-type gene or gene product. Note that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered properties compared to a wild-type gene or gene product. Methods for introducing or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced by arginine (R) by replacing the codon (ATG) for methionine at the relevant position in a polynucleotide encoding a mutant monomer. Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art.

The various monoclonal antibodies described in the present disclosure are all obtained through genetic engineering technology, and high-purity antibodies are obtained after recombinant expression based on known antibody sequences and affinity chromatography for use as detection antibodies. There are a large number of neutralizing and non-neutralizing antigenic epitopes on the surface of the F protein, and there are 4 antigenic epitopes related to neutralizing activity (I, II, IV and Φ), of which I, II, and IV coexist in the pre-fusion and post-fusion conformations of the F protein. In the present disclosure, a variety of monoclonal antibodies with clear binding epitopes are used, which can be summarized as: D25 (the amino acid sequences of the light chain and heavy chain are shown in SEQ ID No.02 and SEQ ID No.03, respectively, and specifically recognize the pre-fusion conformation epitope Φ), AM14 (the amino acid sequences of the light chain and heavy chain are shown in SEQ ID No.04 and SEQ ID No.05, respectively, and specifically recognize the pre-fusion trimer conformation, and bind to epitopes Ⅳ and Ⅴ across monomers), MPE8 (the amino acid sequences of the light chain and heavy chain are shown in SEQ ID No.06 and SEQ ID No.07, respectively, and recognize epitopes Ⅱ-Ⅴ, and partially bind across monomers), 101F (the amino acid sequences of the light chain and The amino acid sequences of the light chain and heavy chain are shown in SEQ ID No.08 and SEQ ID No.09, respectively, recognizing epitope IV), Palivizumab (the amino acid sequences of the light chain and heavy chain are shown in SEQ ID No.10 and SEQ ID No.11, respectively, recognizing epitope II), Motavizumab (the amino acid sequences of the light chain and heavy chain are shown in SEQ ID No.12 and SEQ ID No.13, respectively, recognizing epitope II) and Nirsevimab (the amino acid sequences of the light chain and heavy chain are shown in SEQ ID No.14 and SEQ ID No.15, respectively, specifically recognizing the pre-fusion conformational epitope Φ).

A mutant respiratory syncytial virus prefusion F protein described in the present disclosure can be obtained by recombinant expression using genetic engineering technology, such as 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. Alternatively, the mutant protein described in the present disclosure 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. Alternatively, the mutant protein described in the present disclosure is delivered to the target host cell in the form of a naked or encapsulated nucleic acid vector, such as using linear RNA, circular RNA, dsDNA, cDNA, etc. Alternatively, the mutant protein is obtained in a commonly used or known form in this professional field.

A mutant respiratory syncytial virus pre-fusion F protein mutant form described in the present disclosure can also add other mutations on the basis of the mutant form described in the present disclosure to stabilize or improve the pre-fusion conformational activity or expression level, such as adding proline point mutations to obtain rigid fixation of the secondary/tertiary structure, adding amino acid point mutations with side chain groups to obtain cavity filling effects, adding hydrophobic amino acid point mutations to obtain hydrophobic effects, adding charged amino acid point mutations to obtain electrostatic strengthening or weakening effects, adding intermolecular disulfide bonds to obtain covalent connections between monomeric F protein molecules, adding heterologous tags to the C-terminus of the F protein to improve the stability and expression level of the F protein trimer, adding flexible amino acid chains at both ends of the F protein to form a Loop tandem structure, adding an amino acid chain with an α-helical structure to the C-terminus of the F protein to improve the stability of the F protein trimer, adding an amino acid chain with a structural change function, etc., or mutation methods or means commonly used or known in this professional field.

The mutant respiratory syncytial virus pre-fusion F protein described in the present disclosure has excellent neutralizing antibody binding activity and thermal stability compared with similar products, and is suitable for development as one of the components of the respiratory syncytial virus vaccine.

In conjunction with the accompanying drawings, some embodiments of the present disclosure are described in detail below. In the absence of conflict, the following embodiments and features in the embodiments can be combined with each other.

Example 1: Disulfide bond mutation design and activity screening of conformationally stable respiratory syncytial virus prefusion F protein

By studying the spatial structure of the pre-fusion F protein of respiratory syncytial virus, amino acid mutations that can stabilize its pre-fusion conformation were designed according to the principles of structural biology. Using the amino acid sequence of the pre-fusion F protein of wild-type respiratory syncytial virus SEQ ID No.01 or the amino acid sequence of the F protein of other wild-type virus strains as a template, intramolecular disulfide bond mutations were added to enhance the structural stability between the pre-fusion F1 subunit and the F2 subunit or within the F1 to prevent the conformational transition of the HRA and HRB regions of the heptapeptide repeat region, while maintaining or enhancing the binding activity of important sites such as the Φ epitope, thereby improving the pre-fusion F protein-specific immunogenicity of the mutant antigen. In addition, in order to achieve soluble expression of the F protein, the transmembrane region 514 to 574aa at the C-terminus of the F protein was replaced with "SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID No.47)" during plasmid construction. This fragment is a soluble T4Fibritin trimerization domain to maintain the trimer structure of the F protein. The disulfide bond mutations designed according to the above objectives are shown in Table 1, and the detailed sequences are shown in SEQ ID No.16 to No.29.

Table 1. Disulfide bond mutation antigen design

(2) By genetic engineering technology, the mutant antigen nucleotide sequence described in Table 1 was inserted into the pEE 12.4 expression vector (see Figure 1 for the plasmid map, and the nucleotide sequence is shown in SEQ ID No. 30) and then the whole plasmid was synthesized. The mutant plasmid was transfected into HEK 293T cells cultured in six-well plates using TransIT-X2 ^TM Dynamic Delivery System (Mirus, MIR6003) or Lipofectamine ^TM 3000 (Invitrogen, L3000001) transfection kit. After continuous culture for 72 hours, the supernatant culture fluid was collected for Western Blot protein immunoblotting to confirm the expression of the target protein, the primary antibody Motavizumab, the secondary antibody Direct-Blot ^TM HRP anti-human IgG1Fc Antibody (BioLegend, 410603). The mutant plasmids confirmed to be expressed normally were transiently transferred to 200 mL of ExpiCHO-S ^TM (Gibco, A29127) suspension cell culture medium using the ExpiCHO ^TM expression system kit (Gibco, A29133) or other methods, and the supernatant culture medium was collected by centrifugation after continuous culture for 2 to 3 days. The target protein was purified by ion exchange chromatography and/or ceramic hydroxyapatite chromatography and/or gel molecular sieve chromatography, and the expression of each mutant plasmid was finally detected and counted. The results are shown in Table 2, and DS-Cav1 (SEQ ID No. 31), SC-TM (SEQ ID No. 32) and pXCS852 (SEQ ID No. 33) were used as positive controls. Among them, YD03, YD06, YD07, YD12, YD13, YD16, and YD22 mutant antigens were expressed normally.

Among them, "DS-Cav1" is the first pre-fusion conformation F protein developed by the team of the National Institute of Allergy and Infectious Diseases (NIAID) of the United States. The intramolecular disulfide bond is obtained by S155C and S290C point mutations in the F1 fragment, and S190F and V207L point mutations are added to enhance the binding activity of the key neutralizing epitope (DOI: 10.1126/science.1243283). "pXCS852" is a mutant "pXCS852" mentioned in the embodiment of Pfizer's public patent (CN201680075615.8). On the basis of retaining pep27, S55C and L188C intramolecular disulfide bonds, as well as T54H and D486S point mutations are added to enhance the stability and epitope activity of the pre-fusion conformation.

Table 2. Expression of mutant antigens

(3) The purified mutant antigens were further subjected to epitope activity detection, and six monoclonal antibodies D25, AM14, MPE8, Palivizumab, Motavizumab and Nirsevimab (the light chain and heavy chain sequences are described in any one of SEQ ID No. 02-05, respectively, as described above) were used as primary antibodies to detect the binding of each mutant antigen. The operation method is simply described as follows: the purified mutant antigens were diluted to 1.1 μg/well or 3.3 μg/well, 3-fold gradient dilution to 7 concentration points, and the antigens were coated in a 96-well plate using a coating buffer. After 2 hours, the blocking solution was added overnight, and the primary antibody and the secondary antibody Direct-Blot ^TM HRP anti-human IgG1Fc Antibody were added in sequence for incubation and TMB color development (Solarbio, PR1210) was performed. The absorption at 450 nm was detected by an enzyme reader, and the data were counted and analyzed (as shown in Figure 2), and DS-Cav1 and SC-TM were used as positive controls. In comparison, YD12 and YD22 have strong binding to D25, YD16 has binding to D25, YD03, YD06, YD07 and YD13 have weak binding to D25; YD12 and YD22 have strong binding to AM14, and the other antigens have weak binding; YD12 and YD22 have strong binding to MPE8, YD03 and YD16 have binding to MPE8, YD06, YD07 and YD13 have weak binding to MPE8; YD12 has strong binding to Palivizu Mab binding was the strongest, YD07 had the weakest binding to Palivizumab, and the rest of the antigens were bound; each antigen was bound to Motavizumab; YD12 and YD22 had strong binding to Nirsevimab, YD16 had binding to Nirsevimab, and YD03, YD06, YD07 and YD13 had weak binding to Nirsevimab. Because Nirsevimab and D25 belong to the same Φ epitope, the binding trends were consistent. In summary, the ELISA results showed that YD12 and YD22 had better binding activity with each monoclonal antibody, among which AM14 had significantly better binding activity than DS-Cav1 and SC-TM in the positive control group, showing excellent quaternary structure stability.

(4) The non-labeled biomolecular analyzer GatorPrime Plus (Gator Bio) was used to detect the binding ability of each mutant antigen with seven different monoclonal antibodies D25, AM14, MPE8, 101F, Palivizumab, Motavizumab and Nirsevimab. The operation method is simply described as follows: After the seven different monoclonal antibodies are immobilized with HFC (Anti-Human IgG Fc, Gator Bio) optical probes, they are bound to different mutant antigen proteins until saturation, and the displacement signal (nm) is monitored after the probes are transferred to the dissociation buffer. The dissociation constant value (K _off ) of each mutant antigen is calculated by the equipment data processing software as shown in Table 3. DS-Cav1, SC-TM and pXCS852 were used as positive controls. It can be seen that YD03, YD12 and YD22 have very good binding ability with the seven monoclonal antibodies, reflecting that the mutated disulfide bond can stabilize the pre-fusion F protein conformation and maintain the original epitope activity. In addition, compared with DS-Cav1 and SC-TM, the epitope activities of pXCS852 were relatively weak, and its binding activity with D25 and AM14 was weaker than that with YD12 and YD22.

Table 3. Affinity test of mutant antigens and seven different monoclonal antibodies

*The values in the table are dissociation constants (K _off )×10 ⁵ , in 1/s; **NB means no binding signal was detected; ***ND means no dissociation signal was detected

Example 2: Design of connecting peptides and activity screening of respiratory syncytial virus prefusion F protein

(1) The natural wild-type F protein first forms the F0 precursor protein and then releases a polypeptide pep27 composed of 27 amino acids after being cleaved by furin, forming F1 and F2 fragments connected by two pairs of disulfide bonds. The three F0 monomers are self-assembled after enzyme cleavage to form a pre-fusion trimer conformation in a metastable state. This process is orderly and complex. Factors such as cell metabolic state and F0 precursor protein expression will directly affect the final pre-fusion F protein trimer yield and conformation. The present disclosure replaces pep27 with a shorter connecting peptide and knocks out the two enzyme cleavage sites FCS1 and FCS2 at the same time, thereby increasing the F0 precursor expression and the yield of the pre-fusion F trimer. More importantly, the replacement of the connecting peptide can lock the connection between the FP fusion region at the N-terminus of the F1 fragment and the F2 fragment, thereby achieving the purpose of further stabilizing the pre-fusion conformation. At the same time, the selection of the connecting peptide sequence will affect the charge and hydrophobicity near the region, thereby affecting the epitope activity of the pre-fusion F protein. According to the principles of structural biology, the present invention uses bioinformatics analysis and protein structure prediction methods to design three replacement connecting peptides, which are named GS12, GRS12 and Q4, respectively. The specific sequences are shown in Table 4. All mutated connecting peptides were modified using the amino acid sequence SEQ ID No.01 of the wild-type respiratory syncytial virus prefusion F protein as a template, and then inserted into the plasmid pEE12.4 (SEQ ID No.30) for full plasmid synthesis. In addition, in order to achieve soluble expression of F protein, the transmembrane region 514 to 574aa at the C-terminus of F protein was replaced with "SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFL" during plasmid construction. This fragment is a soluble T4Fibritin trimerization domain to maintain the trimer structure of F protein.

The “SC-TM” structure acquired a prefusion conformation with higher neutralizing activity by means of three point mutations (N67I, S215P, E487Q) and replacement of the pep27 region with a linker peptide (Janssen Pharmaceuticals, DOI: 10.1038/ncomms9143).

Table 4. Amino acid sequence and plasmid information of the connecting peptide

(2) The synthesized plasmid was transiently transferred into 200 mL of ExpiCHO-S ^TM suspension cell culture medium using the ExpiCHO ^TM expression system kit or other methods. After continuous culture for 2 to 3 days, the supernatant culture medium was collected by centrifugation. The purified antigen was diluted to a specific protein concentration, and the antigen was coated in a 96-well plate using a coating buffer. After 2 hours, a blocking solution was added overnight. The primary antibody and the secondary antibody Direct-Blot ^TM HRP anti-human IgG1Fc Antibody were added in sequence for incubation. TMB color was developed, and the absorption at 450 nm was detected by an enzyme reader. After data processing, it was shown in Figure 3. SC-TM was used as a positive control. From the results, it can be observed that among the three different replacement linker peptides, the GRS12 linker peptide mutant antigen has better binding activity with five different monoclonal antibodies (D25, AM14, MPE8, 101F and Motavizumab) compared with GS12 and Q4. At the same time, there was no significant difference in the expression level between the GRS12 mutant antigen and SC-TM. This shows that the GRS12 linker peptide has the potential to increase expression and maintain the pre-fusion conformation.

Example 3: Dual-factor mutation design and activity screening of conformationally stable respiratory syncytial virus prefusion F protein

On the basis of intramolecular disulfide bond mutation screening and connecting peptide screening, the present disclosure combines the preferred results obtained by two different strategies for screening, i.e., dual-factor mutation screening. According to the characteristics of the pre-fusion F protein, the present disclosure aims to obtain a mutant antigen with a pre-fusion conformation, high expression ability, high epitope binding activity and stability, thereby developing a recombinant protein with the ability to prevent or treat RSV infection. The intramolecular disulfide bond mutations selected S55C-T189C (YD03), P101C-V152C (YD12) and two pairs of disulfide bonds mutated simultaneously (YD22), and the GRS12 connecting peptide mutation was added to the above three disulfide bond mutations to form YD03-1 (SEQ ID No. 42), YD12-1 (SEQ ID No. 43) and YD22-1 (SEQ ID No. 44). At the same time, in order to further investigate the effect of the length of the connecting peptide on the pre-fusion conformation, two connecting peptide mutations of different lengths, GSGSGRS and GS, were added using YD12 as a template to form YD12-2 (SEQ ID No.45) and YD12-3 (SEQ ID No.46). The information of all mutant plasmids is shown in Table 5.

Table 5. Antigen information of dual-factor mutation screening

The same method as described in Example 2, the synthesized plasmid was transiently transferred to 200mL volume of ExpiCHO-S ^TM suspension cell culture medium using ExpiCHO ^TM expression system kit or other methods, and the supernatant culture medium was collected by centrifugation after continuous culture for 2-3 days. The purified antigen protein was subjected to reducing SDS-PAGE electrophoresis (SurePAGE ^TM , Genscript) to detect the molecular weight and purity of the antigen monomer, Western Blot protein immunoblotting to detect the antigen (primary antibody: Motavizumab, secondary antibody: Direct-Blot ^TM HRP anti-human IgG1 Fc Antibody), ECL chemiluminescence ultrasensitive color development (Biyuntian, P0018AM), and DS-Cav1 and SC-TM were used as positive controls, and the results are shown in Figure 4. The molecular weights of the 8 mutant antigen monomers expressed were consistent with the theory, and the SDS-PAGE and Western Blot bands were carefully compared to confirm that the miscellaneous bands of the protein electrophoresis part belonged to the antigen-related bands, and the purity of each mutant antigen met the requirements of subsequent experiments.

The concentration of each purified protein was detected using Pierce ^TM BCA protein quantification kit (Thermo, 23225) and the antigen expression was calculated. The results are shown in Table 6. It can be observed that the addition of the linker peptide mutation significantly increased the expression of the disulfide bond mutant antigen, among which the expression of YD03-1 increased by 2.64 times compared with YD03, the expression of YD12-1 increased by 6.37 times compared with YD12, and the expression of YD22-1 increased by 8 times compared with YD22. The expression of YD12-1, YD12-2 and YD12-3 was 18.06 times, 14.28 times and 17.04 times higher than that of DS-Cav1, respectively, and the expression of YD12-1, YD12-2 and YD12-3 was 2.49 times, 1.97 times and 2.35 times higher than that of SC-TM, respectively, with significant high expression characteristics.

Table 6. Expression of dual-factor mutant antigens

The purity and molecular weight of the purified antigens were detected by size exclusion chromatography. The detection equipment was an Agilent 1260 high performance liquid chromatograph, the chromatographic column used was Agilent Advance Bio SEC 300A (7.8×300mm), the mobile phase selected was an ammonium formate buffer system, the flow rate was controlled at 0.5-1.0 mL/min, and the DAD detector monitored the OD280 signal. The results are shown in Figure 5. According to the principle of size exclusion chromatography, the molecular sizes of the mutant antigens can be analyzed from large to small, in the order of YD03-1, YD03, YD12-2, YD12-3, YD22, YD12, DS-Cav1, YD12-1, YD22-1, and SC-TM. The chromatographic detection of each purified antigen was a single protein peak, which met the purity requirements.

Similar to the method described in Example 1, the binding of each mutant antigen to seven different monoclonal antibodies, D25, AM14, MPE8, 101F, Palivizumab, Motavizumab and Nirsevimab, was detected using a non-labeled biomolecular analyzer, Gator Prime Plus (Gator Bio). The operation method is simply described as follows: first, the mutant antigen to be tested is labeled with biotin, and the mutant antigen is solidified with SA (Streptavidin, Gator Bio) optical probe, and then the intermolecular affinity is detected with different monoclonal antibodies. The affinity constant of each mutant antigen is calculated by the equipment data processing software. The results are shown in Table 7. DS-Cav1 and SC-TM are used as positive controls. Among them, by comparing YD03 and YD03-1, YD12 and YD12-1, YD22 and YD22-1, it can be observed that the addition of the GRS12 connecting peptide mutation enhances the affinity of the above antigens with D25, AM14 and MPE8. By comparing the affinity of YD12-1, YD12-2 and YD12-3 with D25, AM14 and MPE8, it can be observed that the length of the connecting peptide affects the affinity. It is particularly noteworthy that the affinity of D25 (recognition of Φ site, key neutralization epitope), AM14 (specific recognition of trimer quaternary structure) and MPE8 (recognition of quaternary structure) of YD22-1 and YD22 was enhanced by at least 1000 times, 20 times and 1000 times respectively, proving that the GRS12 connecting peptide mutation has the ability to significantly enhance the pre-fusion conformation and also increased the expression level by 8 times. The affinity of each monoclonal antibody of YD22-1 and SC-TM is at the same level and significantly better than DS-Cav1. YD22-1 has the potential to prevent or treat RSV infection.

Table 7. Affinity test of dual-factor mutant antigens and seven different monoclonal antibodies

*The values in the table are affinity constants (KD), in nM; **NB means no binding signal was detected

To further investigate the enhancement effect of the double-factor mutant antigen on the pre-fusion conformation, the purified antigen was diluted to a specific protein concentration, and the antigen was coated in a 96-well plate using a coating buffer. After 2 hours, the blocking solution was added overnight, and the primary antibody AM14 and the secondary antibody Direct-Blot ^TM HRP anti-human IgG1Fc Antibody were added in sequence for incubation. TMB color was developed, and the absorption at 450nm was detected by an enzyme reader. After data processing, it is shown in Figure 6, and DS-Cav1 and SC-TM were used as positive controls. Consistent with the trend of the affinity test results, YD22-1 showed excellent binding ability to the pre-fusion trimer conformation, which was significantly better than SC-TM and other antigens, and at the same time, it was significantly improved compared to YD22, proving that the GRS12 linker peptide has the effect of enhancing the pre-fusion conformation.

Example 4: Evaluation of thermal stability of conformationally stabilized respiratory syncytial virus prefusion F protein

(1) Differential scanning fluorescence (DSF) was used to detect the melting point temperature of each double-factor mutant antigen to investigate the difference in thermal stability. The operation method is simply described as follows: SYPRO ^TM Orange (Thermo, S6650) fluorescent dye was added to the sample to be tested at a ratio of 1:400, and the changes in the fluorescence signal were monitored using a CFX96Touch fluorescent quantitative PCR instrument (Bio-Rad). The parameters were set to the FRET channel, +0.5°C/30s, 25°C~90°C, and the first melting temperature Tm ₁ of each mutant antigen was calculated by the equipment data processing software as shown in Table 8. According to the principle of differential scanning fluorescence, the Tm ₁ value represents the thermal stability of the trimer conformation before fusion, and the larger the Tm ₁ value, the more stable the trimer conformation. It can be seen that compared with SC-TM (Tm ₁ = 60.5°C), the thermal stability of the mutant antigens in the present disclosure is enhanced, among which YD22-1 (Tm ₁ = 69.5°C) has the best thermal stability. By comparing the Tm ₁ values of YD03-1 and YD03, and YD12-1 and YD12, it can be observed that the addition of the GRS12 linker peptide mutation enhanced or maintained the thermal stability of the mutant antigen.

Table 8. Melting temperature (Tm ₁ ) of mutant antigens

(2) The turbidity points of the mutant antigen YD22-1 and the control antigen SC-TM were determined by gradually increasing the temperature of the protein sample while monitoring the light scattering signal (the turbidity point refers to the temperature at which the sample begins to become turbid under heating conditions). The concentration of the sample to be tested was adjusted to 500 μg/mL, the sample volume was 500 μL, and the sample was measured every time the temperature was increased by 5°C. The signal at OD350nm was detected using a SpectraMax M3 multi-function microplate reader (Molecular Devices). The results are shown in Figure 7. The measured turbidity point of YD22-1 was around 85.0°C, while the turbidity point of SC-TM was around 75.0°C. The turbidity point of YD22-1 was nearly 10.0°C higher than that of SC-TM, and YD22-1 showed excellent thermal stability.

To further investigate the pre-fusion conformational stability of mutant antigen YD22-1 under heat stress, the protein concentration of YD22-1 and control antigen SC-TM was adjusted to 500 μg/mL, the sample volume was 100 μL, 50 μL was placed in a ProFlex ^TM PCR instrument (Applied Biosystems) for heat treatment at 60°C for 1 hour (after heat treatment), and the remaining 50 μL sample was temporarily stored at 2-8°C (before heat treatment). After the samples before and after heat treatment were diluted to a specific protein concentration, the antigen was coated in a 96-well plate using a coating buffer, and a blocking solution was added overnight after 2 hours. The primary antibody (AM14 or D25) and the secondary antibody Direct-Blot ^TM HRP anti-human IgG1Fc Antibody were added in sequence for incubation and TMB color development was performed. The absorption at OD450nm was detected by a microplate reader. The ratio of the values before and after heat treatment was the relative binding activity change between the antigen and the monoclonal antibody. The results are shown in Table 9. It can be seen that YD22-1 can still maintain most of the specific trimeric antibody binding activity (AM14 relative activity 0.83) after heat treatment at 60℃ for 1 hour, which is significantly better than the positive control SC-TM (AM14 relative activity 0.65), indicating that the pre-fusion trimeric conformation of YD22-1 is more stable. At the same time, the pre-fusion conformation epitope Φ binding activity (D25) of YD22-1 and SC-TM remains unchanged before and after heat treatment.

Table 9. Changes in relative activity of mutant antigens and monoclonal antibodies after heat treatment (60°C, 1 hour)

*The format of the values in the table is: relative activity before and after heat treatment (OD450 value after heat treatment/OD450 value before heat treatment)

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure 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 disclosure.

Industrial Applicability

The mutant respiratory syncytial virus prefusion F protein provided in the present disclosure has the following mutations compared to the wild-type respiratory syncytial virus prefusion F protein: (a) at least one amino acid residue in the F1 subunit and/or the F2 subunit is replaced by cysteine, (b) the amino acid residues 104 to 144 of the wild-type respiratory syncytial virus prefusion F protein are partially or completely replaced by a connecting peptide, and the amino acid residue length of the connecting peptide is at least 2. A mutant respiratory syncytial virus prefusion F protein described in the present disclosure has excellent neutralizing antibody binding activity and thermal stability compared to similar products, is suitable for development as one of the components of a respiratory syncytial virus vaccine, and has excellent industrial applicability.

Claims

A mutant respiratory syncytial virus prefusion F protein, characterized in that the mutant respiratory syncytial virus prefusion F protein has the following mutations compared to the wild-type respiratory syncytial virus prefusion F protein:

(a) at least one amino acid residue in the F1 subunit and/or the F2 subunit is substituted by cysteine, and the substitution results in a non-native disulfide bond between the F1 subunit and the F2 subunit of the respiratory syncytial virus prefusion F protein, wherein the non-native disulfide bond includes a disulfide bond other than Cys69-Cys212 and Cys37-Cys439 formed between the F1 subunit and the F2 subunit;

(b) amino acid residues 104 to 144 of the wild-type respiratory syncytial virus prefusion F protein are partially or completely replaced by a connecting peptide, wherein the amino acid residue length of the connecting peptide is at least 2;

The amino acid sequence of the wild-type respiratory syncytial virus pre-fusion F protein is shown in SEQ ID No.01.
The mutant respiratory syncytial virus prefusion F protein according to claim 1, characterized in that the cysteine substitution comprises at least one of S55C, T189C, P101C or V152C.
The mutant respiratory syncytial virus prefusion F protein according to claim 2, characterized in that the cysteine substitutions include S55C and T189C, and/or, P101C and V152C.
The mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 3, characterized in that amino acid residues 104 to 137 of the wild-type respiratory syncytial virus prefusion F protein are all replaced by a connecting peptide.
The mutant respiratory syncytial virus prefusion F protein according to claim 4, characterized in that the amino acid sequence of the connecting peptide is GSGSGGSGSGRS.
The mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 3, characterized in that amino acid residues 104 to 144 of the wild-type respiratory syncytial virus prefusion F protein are all replaced by a connecting peptide, and the amino acid sequence of the connecting peptide is GSGSGRS or GS.
The biomaterial is characterized in that the biomaterial comprises any one of the following (a) to (d):

(a) a nucleic acid molecule encoding the mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6;

(b) a recombinant vector containing the nucleic acid molecule described in (a), preferably, the original plasmid of the recombinant vector is pEE12.4;

(c) a transformed cell containing the nucleic acid molecule (a) or the recombinant vector (b), preferably, the host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells;

Further preferably, the mammalian cell is selected from Chinese hamster ovary cells, tumor cells, BHK cells or HEK293 cells;

(d) a recombinant virus containing the nucleic acid molecule described in (a) or the recombinant vector described in (b), preferably, the recombinant virus comprises an adenovirus, an adeno-associated virus, a vaccinia virus, a herpes virus or a retroviral vector.
The method for preparing the mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6 is characterized in that the transformed cells or recombinant viruses described in claim 7 are cultured and induced to express to obtain the mutant respiratory syncytial virus prefusion F protein.
Use of the mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6 or the biomaterial according to claim 7 in any one of the following (a) to (c):

(a) Application in the preparation of respiratory syncytial virus-specific antibodies;

(b) preparing a medicament for preventing and/or treating respiratory syncytial virus infection, preferably, the medicament comprises a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine;

(c) preparing a diagnostic reagent for respiratory syncytial virus, optionally, the diagnostic reagent is used for diagnosing respiratory syncytial virus infection.
A drug for preventing and/or treating respiratory syncytial virus infection, characterized in that it comprises the mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6, the biological material according to claim 7, or the respiratory syncytial virus-specific antibody according to claim 9 (a);

Optionally, the drug includes a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.
A respiratory syncytial virus diagnostic reagent, characterized in that it contains the mutant respiratory syncytial virus pre-fusion F protein as described in any one of claims 1 to 6, or the respiratory syncytial virus-specific antibody as described in claim 9 (a).
A method for preventing and/or treating respiratory syncytial virus infection, the method comprising administering to a patient in need thereof a therapeutically effective amount of the mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6, the biological material according to claim 7, or the respiratory syncytial virus-specific antibody according to claim 9 (a);

Optionally, the mutant respiratory syncytial virus pre-fusion F protein, biological material or respiratory syncytial virus-specific antibody is administered in the form of a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.
A method for diagnosing respiratory syncytial virus infection, the method comprising the step of using the mutant respiratory syncytial virus prefusion F protein as described in any one of claims 1 to 6, or the respiratory syncytial virus-specific antibody as described in claim 9 (a).
The mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6, the biological material according to claim 7, or the respiratory syncytial virus-specific antibody according to claim 9 (a), for preventing and/or treating respiratory syncytial virus infection;

Optionally, the mutant respiratory syncytial virus pre-fusion F protein, biological material or respiratory syncytial virus-specific antibody is administered in the form of a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.
The mutant respiratory syncytial virus prefusion F protein according to any one of claims 1 to 6, or the respiratory syncytial virus-specific antibody according to claim 9 (a), is used for diagnosing respiratory syncytial virus infection.