Modified respiratory syncytial virus F proteins and uses thereof

Abstract

The invention provides a respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation selected from the group consisting of: (1) introducing one or more amino acid mutations into the RSV F protein for filling a hydrophobic cavity; (2) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein; and (3) removing a p27 peptide fragment in the RSV F protein. As a recombinant protein antigen, the RSV F protein is high in stability and easy to store and transport, the expression quantity is increased, the yield is easy to increase, and the modified RSV F protein is higher in immunogenicity and can generate a better immune protection effect.

Description

Technical Field

[0001] The present invention relates to the fields of medicine and immunology, and in particular to a modified respiratory syncytial virus F protein and its use in preventing and treating respiratory syncytial virus infection. Background Art

[0002] Respiratory syncytial virus (RSV) belongs to the genus Pneumovirus of the family Paramyxoviridae. It has only one serotype, which is divided into two subtypes, A and B. RSV mainly encodes 10 proteins, namely three transmembrane proteins, fusion protein (F), adhesion protein (G) and small hydrophobic protein (SH), two matrix proteins M1 and M2, three proteins that bind to viral RNA to form nucleocapsids (N, P and L), and two non-structural proteins (NS1 and NS2). Among them, the G and F surface proteins play an important role in the adsorption and fusion process of RSV, respectively, and are the most important antigenic sites for the production of neutralizing antibodies, and are also the main targets for inducing immunogenicity and antiviral activity in the body.

[0003] The virus is highly contagious, and the source of infection is secretions or contaminants containing the virus. It is mainly transmitted through droplets and direct contact. It is more common in newborns and infants under 6 months old, with high fever, rhinitis, pharyngitis and laryngitis, and later manifests as bronchiolitis and pneumonia. A few sick children may have complications such as otitis media, pleurisy and myocarditis. After infection, adults and older children mainly show upper respiratory tract infections. There is currently no commercial vaccine for RSV. High-income countries such as the United States use the neutralizing monoclonal antibody Palivizumab for immunization prevention, which is used to a certain extent to prevent RSV disease in extremely premature infants or patients with congenital heart disease. However, the high cost of monthly medication makes it difficult to use in low-income countries. Therefore, the development of an effective RSV vaccine is particularly critical for the prevention and control of the disease.

[0004] Since the antibodies caused by F protein have stronger neutralizing effect than those caused by G protein and weaker variability than the latter, the current development of RSV candidate vaccines mainly targets the highly conserved F protein. However, RSV F protein has two conformations. The prefusion conformation (PreF) is metastable. When mediating membrane fusion or spontaneous triggering, the prefusion conformation undergoes irreversible rearrangement to a non-functional postfusion conformation. Before infecting human cells, it is in the first conformation. During the infection process, it transforms into the second conformation. Although about 50% of the surface is shared before and after fusion (referring to sites I to IV), the antigenic sites most sensitive to neutralization are only located in the prefusion conformation (referring to sites V and Φ). The antibody response stimulated by F protein in the second conformation is not strong. Therefore, modifying the F protein to stabilize it in the prefusion conformation is the main strategy for developing vaccines. In 2023, the FDA approved two RSV vaccines, GSK's "Arexvy" and Pfizer's "Abrysvo", for the prevention of RSV infection in people over 60 years old. Both of the above vaccines are recombinant protein vaccines with modified F protein as the main antigen.

[0005] At present, the research and development of RSV vaccines in China is still in its infancy. In view of the harm caused by RSV, the development of an effective RSV vaccine is of great significance to the prevention and control of RSV in my country. Summary of the invention

[0006] The present invention provides a modified respiratory syncytial virus F protein and its use in preventing and treating respiratory syncytial virus infection. In addition, the present invention also provides a nucleic acid sequence encoding the above F protein, a vector containing the above nucleic acid sequence and a corresponding host cell, an immunogenic composition, a vaccine composition, a pharmaceutical composition, a kit involving the above F protein, and a method and use of the above F protein in preventing and treating respiratory syncytial virus infection.

[0007] In a first aspect, the present invention provides a respiratory syncytial virus (RSV) F protein, comprising at least one modification or mutation selected from the following for increasing the stability of the RSV F protein pre-fusion conformation:

[0008] (1) introducing one or more amino acid mutations into the RSV F protein to fill the hydrophobic cavity;

[0009] (2) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein;

[0010] (3) Remove the p27 peptide from RSV F protein.

[0011] In some embodiments, the amino acid mutation for filling the hydrophobic cavity comprises an amino acid substitution at an amino acid position selected from the group consisting of: asparagine 42 residue, valine 65 residue, leucine 71 residue, leucine 205 residue, methionine 264 residue, isoleucine 267 residue, isoleucine 192 residue and / or serine 165 residue.

[0012] In some embodiments, amino acid mutations for filling the hydrophobic cavity include: N42L, N42F, N42Y, N42W, N42H, V65Y, V65F, L71I, L71Y, L205F, L205Y, M264F, M264Y, I267L, I267F, I267Y, I192L, S165I, S165F, S165Y and / or S165L.

[0013] In some embodiments, the amino acid mutation for electrostatic engineering comprises an amino acid substitution at the amino acid position of aspartic acid 461 residue and / or glutamic acid 462 residue.

[0014] In some embodiments, amino acid mutations for electrostatic modification include: D461S, D461N, D461Q, D461E, D461G, D461A, D461K, D461R, D461H, E462Q, E462S, E462N, E462D, E462G, E462A, E462K, E462R, and E462H.

[0015] In some embodiments, the RSV F protein comprises the following mutation combinations: N42L, V65Y, M264F, D461S, S165I.

[0016] In some embodiments, the p27 peptide segment of the RSV F protein is replaced with a linker.

[0017] In some preferred embodiments, the linker is a flexible polypeptide.

[0018] In some more preferred embodiments, the above-mentioned flexible polypeptide is selected from (GS)n, (GGSG)n, (GGGGS)n, (GGGSGG)n, (GGGSGGG)n, (EA)n, (EAK)n, (EAKA)n, (AAAAK)n, (EAAAK)n, (LEAAK)n, wherein n is an integer from 1 to 4.

[0019] In some more preferred embodiments, the above-mentioned flexible polypeptide is selected from the amino acid sequences shown in SEQ ID NOs: 7-50.

[0020] In some embodiments, the RSV F protein comprises an amino acid sequence having 80% or more identity with SEQ ID NO: 2, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity.

[0021] In some embodiments, the N-terminus of the RSV F protein is connected to a signal peptide.

[0022] In some preferred embodiments, the signal peptide is selected from a wild-type RSV F protein signal peptide or a non-RSV F protein signal peptide.

[0023] In some preferred embodiments, the signal peptide comprises an amino acid sequence selected from SEQ ID NO: 3, 4, and 5.

[0024] In some preferred embodiments, the C-terminus of the RSV F protein is connected to a trimerization tag.

[0025] In some preferred embodiments, the trimerization tag comprises an amino acid sequence as shown in SEQ ID NO: 6 or any variant thereof.

[0026] In a second aspect, the present invention provides a nucleic acid molecule encoding the RSV F protein of any aspect described above.

[0027] In a third aspect, the present invention provides a vector comprising the above nucleic acid molecule.

[0028] In some embodiments, the above-mentioned vector is an expression vector.

[0029] In a fourth aspect, the present invention provides a host cell comprising the above-mentioned vector.

[0030] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0031] In some preferred embodiments, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0032] In some preferred embodiments, the mammalian cells are selected from CHO cells, HEK293 cells or COS cells.

[0033] In a fifth aspect, the present invention provides an immunogenic composition comprising the RSV F protein of any of the above aspects.

[0034] In a sixth aspect, the present invention provides a vaccine composition comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[0035] In a seventh aspect, the present invention provides a kit for immunizing a subject against viral infection, comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[0036] In some preferred embodiments, the above-mentioned viral infection is RSV infection.

[0037] In some preferred embodiments, the subject is a human subject.

[0038] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the RSV F protein of any of the above aspects and a pharmaceutically acceptable carrier.

[0039] In some preferred embodiments, the above-mentioned pharmaceutical composition further comprises other therapeutic agents.

[0040] In a ninth aspect, the present invention provides a method for generating an immune response against a viral infection, comprising administering the RSV F protein of any of the above aspects in a pharmaceutically acceptable formulation to a subject.

[0041] In some preferred embodiments, the above-mentioned pharmaceutically acceptable formulation comprises an adjuvant.

[0042] In a tenth aspect, the present invention provides use of the above RSV F protein in the preparation of a medicament for preventing or treating a disease or condition associated with respiratory syncytial virus (RSV) infection.

[0043] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[0044] In an eleventh aspect, the present invention provides a method for preventing or treating a disease associated with respiratory syncytial virus (RSV) infection, comprising administering an effective amount of the RSV F protein of any one of the above aspects in a pharmaceutically acceptable formulation to a subject in need thereof.

[0045] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[0046] Compared with the prior art, the RSV F protein involved in the present invention has high stability as a recombinant protein antigen, is easy to store and transport, has an improved expression level, and is easy to achieve an increase in yield. In addition, the modified RSV F protein has higher immunogenicity and can produce better immune protection effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the specification and, together with the description, serve to explain the principles of the specification.

[0048] Figure 1 The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of selected pre-fusion F protein mutants pXCS847 and CL-073 under reducing conditions are shown.

[0049] Figure 2 Shown are the thermal stabilities of the pre-fusion F protein mutants pXCS847 and CL-073 analyzed by differential scanning fluorimetry (DSF).

[0050] Figure 3 The results of neutralizing antibody responses of mice immunized with Post-F, pXCS847, and CL-073 are shown.

[0051] Figure 4 Graph showing mouse body weight changes for protection against challenge provided by RSV F protein mutant antigens in mice.

[0052] Figure 5 Virus copies in mouse lung tissues showing protection from challenge provided by RSV F protein mutant antigens in mice.

[0053] Figure 6 Shown are mouse lung tissue live virus titers for protection against challenge provided by RSV F protein mutant antigens in mice. DETAILED DESCRIPTION

[0054] I. Definitions

[0055] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0056] Provided herein is a respiratory syncytial virus fusion (RSV F) protein and its uses. In specific aspects, provided herein is a RSV F protein modified or mutated (e.g., amino acid point mutation, introduction of disulfide bond). In specific aspects, provided herein is a RSV F protein comprising modification or mutation (e.g., introduction of disulfide bond, filling hydrophobic cavity, electrostatic mutation) for increasing RSV F protein fusion before conformational stability, compared with wild-type RSV F protein without modification or mutation, modified RSV F protein can better maintain conformation before fusion, increase stability, be conducive to storage and transportation.

[0057] The modified or mutated RSV F protein provided by the present invention is derived from RSV subtype A or RSV subtype B. In specific aspects, the RSV subtype A includes RSV A2 strain, RSV Long strain, RSV ON1 strain or RSV Ontario strain; the RSV subtype B includes RSV B1 strain, RSV BA9 strain, RSV Buenos strain or 18537 strain.

[0058] As used herein and unless otherwise specified, the term "about" or "approximately" means within plus or minus 10% of a given value or range. Where an integer is required, the term means within plus or minus 10% of a given value or range, rounded up or down to the nearest integer.

[0059] With respect to protein or polypeptide sequences, the phrase "substantially identical" is understood to mean a polypeptide sequence that exhibits at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the term is understood to mean a nucleotide sequence that exhibits at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence.

[0060] The term "respiratory syncytial virus" or "RSV" is a single-stranded negative-strand RNA virus belonging to the family Paramyxoviridae and the genus Pneumovirus. Because the virus can cause cultured cells to produce unique cell fusion, it is named respiratory syncytial virus (RSV). According to the difference in surface antigens, RSV can be divided into two subtypes, RSV-A and RSV-B. The virus is transmitted through air droplets and close contact. Respiratory syncytial virus contains 10 genes, encoding 11 proteins, of which surface glycoprotein fusion protein (F protein) and attachment protein (G protein) are the most important viral antigens that stimulate the body to produce protective antibodies. Because G protein differs greatly between subtypes, and F protein is highly conserved between subtypes, and F protein directly mediates the fusion of virus and cell, the penetration of virus and the formation of syncytium, it is the main target protein that stimulates the body to produce protective antibodies. Herein, "respiratory syncytial virus" or "RSV" refers to any respiratory syncytial virus or RSV molecule known to those skilled in the art. For example, RSV can include any of the above-mentioned subtypes, such as RSV can be from mammals, such as RSV can be from humans.

[0061] The terms "RSV F protein", "F protein" or "RSV fusion protein" refer to a polypeptide or protein having all or part of the amino acid sequence of an RSV fusion protein polypeptide. The F protein is an N-glycosylated type I transmembrane protein with a total length of 574 amino acids, which has a signal peptide cleaved at the N-terminus and a membrane anchor near the C-terminus. The amino acid sequence of the F protein is provided in GenBank under accession number AAX23994. The F protein has a pre-fusion conformation (Pre-F) and a post-fusion conformation (Post-F) structure on the surface of the virion. Pre-F exists as a trimer, which undergoes conformational changes, causing the hydrophobic fusion peptide to be inserted into the host cell membrane, and then refolding the F protein into a stable, elongated post-fusion conformation (Post-F), resulting in fusion of the virus and host cell membranes. The term "p27 polypeptide" or "pep27" refers to a 27-amino acid polypeptide that is excised from its precursor during the maturation of the RSV F protein. The sequence of the p27 polypeptide is flanked by two furin cleavage sites, which are cleaved by cellular proteases during maturation of the F protein to generate F1 and F2 polypeptides. Therefore, it should be understood that the multiple peptide segments formed when the p27 peptide segment in the RSV F protein is removed are also included in the meaning of the term "RSV F protein".

[0062] As used herein, the terms "protein", "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified (conservatively substituted) variants thereof.

[0063] As used herein, the terms sequence "identity," "identity," or "homology" have their art-recognized meanings, and the percentage of sequence identity between two nucleic acids or polypeptides or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule. Although there are many methods for measuring the identity between two polynucleotides or polypeptides, the term "identity" is well known to those of skill in the art.

[0064] As used herein, the term "variant" refers to an amino acid sequence corresponding to a reference sequence that contains at least one amino acid difference (substitution, insertion or deletion). In certain embodiments, a "variant" has a high degree of amino acid sequence homology and / or conservative amino acid substitutions, deletions and / or insertions compared to a reference sequence. In some embodiments, a variant has no more than 75, 50, 40, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acid differences compared to a reference sequence. In some embodiments, a variant has at least 70%, at least 75%, at least 78%, at least 80%, at least 82%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity compared to a reference sequence.

[0065] As used herein, the term "substitution" variant is a variant in which at least one amino acid residue in the native sequence is removed and inserted into the same position by a different amino acid. The substitution may be single, in which only one amino acid is substituted in the molecule; or multiple, in which two or more amino acids are substituted in the same molecule. Multiple substitutions may be located at consecutive sites. Similarly, an amino acid may be substituted by multiple residues, wherein such variants include both substitutions and insertions. An "insertion" variant is a variant in which one or more amino acids are inserted into an amino acid at a specific position adjacent to a natural sequence. Adjacent amino acids means connected to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is a variant in which one or more amino acids in the native amino acid sequence are removed. Typically, a deletion variant has one or two amino acids deleted in a specific region of its molecule.

[0066] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) usually linked together by a phosphodiester bond. As used herein, the terms "polynucleotide" and "nucleic acid molecule" are intended to include DNA molecules and RNA molecules, which may be single-stranded or double-stranded, and may be cDNA. The term also includes codon-optimized nucleic acid molecules.

[0067] As used herein, the term "conservative substitutions" or "conservative sequence modifications" of a sequence refers to nucleotide and amino acid sequence modifications that do not eliminate the binding of an antibody encoded by a nucleotide sequence or containing an amino acid sequence to an antigen. These conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing described herein by standard techniques known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are already defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Methods for identifying conservative substitutions of nucleotides and amino acids that do not abolish antigen binding are well known in the art.

[0068] Exemplary amino acids that can be conservatively substituted are shown in Table 1 below:

[0069] Table 1 Examples of conservative amino acid substitutions

[0070]

[0071]

[0072] As used herein, the term "expression" refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assay, and Bradford protein assay.

[0073] As used herein, the term "host cell" is a cell that is used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express polypeptides encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid. The host cell can be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.

[0074] As used herein, the term "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into an appropriate host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells, for amplification of nucleic acids or for expression / display of polypeptides encoded by nucleic acids. Vectors are usually kept free, but can be designed to integrate genes or parts thereof into chromosomes of the genome. Artificial chromosome vectors are also contemplated, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles are well known to those skilled in the art.

[0075] As used herein, vectors also include “viral vectors” or “viral vectors.” Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer (as a vehicle or shuttle) the exogenous genes into cells.

[0076] As used herein, the term "expression vector" includes vectors capable of expressing DNA, which is operably linked to regulatory sequences such as promoter regions that can affect the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more replication origins, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, expression vectors refer to recombinant DNA or RNA constructs, such as plasmids, phages, recombinant viruses or other vectors, which, when introduced into appropriate host cells, result in the expression of cloned DNA. Suitable expression vectors are well known to those skilled in the art, and include expression vectors that are replicable in eukaryotic cells and / or prokaryotic cells and expression vectors that remain free or are integrated into the host cell genome.

[0077] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable formulation" refers to one or more non-toxic materials that do not interfere with the biological activity of the active ingredient administered with the therapeutic agent, including but not limited to buffers, preservatives, compatible carriers, diluents, adjuvants (e.g., Freund's adjuvant (complete and incomplete)), excipients, vehicles, and optionally other additives or encapsulating materials. Pharmaceutical carriers suitable for use in the present invention can be conventional pharmaceutical preparation excipients; and compositions and formulations suitable for delivering the disclosed neutralizing antibodies.

[0078] The term "vaccine" or "vaccine composition" refers to a composition comprising at least one immunogenic composition that induces an immune response in an animal.

[0079] As used herein, the term "treating" an individual suffering from a disease or condition means that the individual's symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment and / or cure. Prevention refers to preventing potential diseases and / or preventing symptoms from worsening or disease progression. Treatment also includes any pharmaceutical use of any antibody or antigen-binding fragment thereof provided and any composition provided herein.

[0080] As used herein, the term "therapeutically effective amount" or "therapeutically effective dose" refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or disorder.

[0081] As used herein, the term "prophylactically effective amount" or "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, e.g., preventing or delaying the onset or recurrence of a disease or symptom, reducing the likelihood of the onset or recurrence of a disease or symptom. A complete prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective dose may be administered in one or more administrations.

[0082] As used herein, the term "patient" or "subject" refers to a mammal, such as a human.

[0083] As used herein, "pXCS847"; "pXCS847 replacement", a genetic modification of the RSV F protein comprising 4 amino acid replacements (T78C, I23C, S65I, D461S). See WO 2017 / 109629, the contents of which are incorporated herein by reference.

[0084] As used herein, "D25" or "D25 antibody" describes a neutralizing antibody that specifically binds to the prefusion RSV F peptide. This antibody is described in U.S. Patent Application Publication No. US2010 / 0239593, the entire contents of which are incorporated herein by reference.

[0085] As used herein, "Motavizumab" or "Motavizumab antibody" describes a neutralizing antibody that specifically binds to the RSV F peptide. This antibody is described in U.S. Patent Application Publication No. US2010 / 053558, the entire contents of which are incorporated herein by reference.

[0086] II. Detailed description of specific implementation scheme

[0087] The modified or mutated RSV F protein provided by the present invention is derived from RSV subtype A or RSV subtype B.

[0088] In some embodiments, the RSV subtype A includes RSV A2 strain (SEQ ID NO:51), RSV Long strain (SEQ ID NO:52), RSV ON1 strain (SEQ ID NO:53) or RSV Ontario strain (SEQ ID NO:54); the RSV subtype B includes RSV B1 strain (SEQ ID NO:55), RSV BA9 strain (SEQ ID NO:56), RSV Buenos strain (SEQ ID NO:57) or 18537 strain (SEQ ID NO:58).

[0089] In some preferred embodiments, the RSV subtype A is RSV A2 strain (SEQ ID NO: 51).

[0090] In some more preferred embodiments, the modified or mutated RSV F protein provided by the present invention is derived from the RSVA2 strain, and the wild-type RSV F protein carries three natural mutations (P77A, I354V and M422V) for increasing the expression amount, and its amino acid sequence is shown in SEQ ID NO: 1.

[0091] In one aspect, the present invention provides a respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation selected from the following for increasing the stability of the RSV F protein prefusion conformation:

[0092] (1) introducing one or more amino acid mutations into the RSV F protein to fill the hydrophobic cavity;

[0093] (2) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein;

[0094] (3) Remove the p27 peptide from RSV F protein.

[0095] In some embodiments, the amino acid mutation for filling the hydrophobic cavity comprises an amino acid substitution at an amino acid position selected from the group consisting of: asparagine 42 residue, valine 65 residue, leucine 71 residue, leucine 205 residue, methionine 264 residue, isoleucine 267 residue, isoleucine 192 residue and / or serine 165 residue.

[0096] In some embodiments, amino acid mutations for filling the hydrophobic cavity include: N42L, N42F, N42Y, N42W, N42H, V65Y, V65F, L71I, L71Y, L205F, L205Y, M264F, M264Y, I267L, I267F, I267Y, I192L, S165I, S165F, S165Y and / or S165L.

[0097] In some embodiments, the amino acid mutation for electrostatic engineering comprises an amino acid substitution at the amino acid position of aspartic acid 461 residue and / or glutamic acid 462 residue.

[0098] In some embodiments, amino acid mutations for electrostatic modification include: D461S, D461N, D461Q, D461E, D461G, D461A, D461K, D461R, D461H, E462Q, E462S, E462N, E462D, E462G, E462A, E462K, E462R, and E462H.

[0099] In some embodiments, the RSV F protein comprises the following mutation combinations: N42L, V65Y, M264F, D461S, S165I.

[0100] In some embodiments, the p27 peptide segment (SEQ ID NO: 59) of the RSV F protein described above is replaced with a linker.

[0101] In some embodiments, when the p27 peptide segment is replaced with a linker, some of the residues upstream and downstream of the p27 peptide segment may be replaced together.

[0102] In some embodiments, when the p27 peptide segment is replaced by a linker, the p27 peptide segment may be partially or completely replaced by a linker.

[0103] In some preferred embodiments, when the p27 peptide segment is replaced with a linker, the starting point of the linker connection is any site between amino acid residue positions 75-84 of the RSV F protein, and the end point is any site between amino acid residue positions 112-125 of the RSV F protein.

[0104] In some preferred embodiments, the linker is a flexible polypeptide.

[0105] In some preferred embodiments, the above-mentioned flexible polypeptide is selected from (GS)n, (GGSG)n, (GGGGS)n, (GGGSGG)n, (GGGSGGG)n, (EA)n, (EAK)n, (EAKA)n, (AAAAK)n, (EAAAK)n, (LEAAK)n, wherein n is an integer from 1 to 4.

[0106] In some preferred embodiments, the above-mentioned flexible polypeptide is selected from the amino acid sequences shown in Table 2 below:

[0107] Table 2 Optional flexible polypeptide sequences

[0108]

[0109]

[0110] In some embodiments, the RSV F protein comprises an amino acid sequence having 80% or more identity with SEQ ID NO: 2, preferably an amino acid sequence having 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity, and more preferably an amino acid sequence having 98% or more identity.

[0111] In some embodiments, the N-terminus of the RSV F protein is connected to a signal peptide.

[0112] In some preferred embodiments, the signal peptide is selected from a wild-type RSV F protein signal peptide or a non-RSV F protein signal peptide.

[0113] In some preferred embodiments, the signal peptide comprises an amino acid sequence selected from SEQ ID NO: 3, 4, and 5.

[0114] In some preferred embodiments, the RSV F protein sequence does not contain a signal peptide, and its N-terminus may be connected to a RSV A2 subtype F protein signal peptide as shown in SEQ ID NO: 3 or any variant thereof, or may not be connected to a signal peptide.

[0115] In some preferred embodiments, the RSV F protein sequence does not contain a signal peptide, and its N-terminus may be connected to another signal peptide, which optionally has one or more amino acid mutations, substitutions, insertions or deletions compared to the RSV F protein signal peptide shown in SEQ ID NO: 3.

[0116] In some preferred embodiments, the RSV F protein sequence does not contain a signal peptide, and its N-terminus may be connected to a non-RSV F protein signal peptide, for example, an amino acid sequence as shown in SEQ ID NO: 4, 5 or any variant thereof or other common signal peptide sequences.

[0117] In some embodiments, a trimerization tag is linked to the C-terminus of the RSV F protein.

[0118] In some preferred embodiments, the trimerization tag comprises an amino acid sequence as shown in SEQ ID NO: 6 or any variant thereof.

[0119] In some preferred embodiments, the trimerization tag may be other short peptides that are connected to the C-terminus of the RSV F protein and form a trimer in vitro after expression.

[0120] In a second aspect, the present invention provides a nucleic acid molecule encoding the RSV F protein of any aspect described above.

[0121] In a third aspect, the present invention provides a vector comprising the above nucleic acid molecule.

[0122] In some embodiments, the above-mentioned vector is an expression vector.

[0123] In a fourth aspect, the present invention provides a host cell comprising the above-mentioned vector.

[0124] In some embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0125] In some preferred embodiments, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0126] In some preferred embodiments, the mammalian cells are selected from CHO cells, HEK293 cells or COS cells.

[0127] In a fifth aspect, the present invention provides an immunogenic composition comprising the RSV F protein of any of the above aspects.

[0128] In a sixth aspect, the present invention provides a vaccine composition comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[0129] In a seventh aspect, the present invention provides a kit for immunizing a subject against viral infection, comprising the RSV F protein, nucleic acid molecule, vector and / or host cell according to any one of the above aspects.

[0130] In some preferred embodiments, the above-mentioned viral infection is RSV infection.

[0131] In some preferred embodiments, the subject is a human subject.

[0132] In an eighth aspect, the present invention provides a pharmaceutical composition comprising the RSV F protein of any of the above aspects and a pharmaceutically acceptable carrier.

[0133] In some preferred embodiments, the above-mentioned pharmaceutical composition further comprises other therapeutic agents.

[0134] In a ninth aspect, the present invention provides a method for generating an immune response against a viral infection, comprising administering the RSV F protein of any of the above aspects in a pharmaceutically acceptable formulation to a subject.

[0135] In some preferred embodiments, the above-mentioned pharmaceutically acceptable formulation comprises an adjuvant.

[0136] In a tenth aspect, the present invention provides use of the above RSV F protein in the preparation of a medicament for preventing or treating a disease or condition associated with respiratory syncytial virus (RSV) infection.

[0137] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[0138] In an eleventh aspect, the present invention provides a method for preventing or treating a disease associated with respiratory syncytial virus (RSV) infection, comprising administering an effective amount of the RSV F protein of any one of the above aspects in a pharmaceutically acceptable formulation to a subject in need thereof.

[0139] In some preferred embodiments, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

[0140] In a twelfth aspect, the present invention provides a RSV F protein for preventing or treating diseases associated with respiratory syncytial virus (RSV) infection, wherein the RSV F protein is as described in any of the above aspects.

[0141] For purposes of clarity and concise description, features are described herein as part of the same or separate embodiments, however, it will be understood that the scope of the invention may include embodiments having a combination of all or some of the described features.

[0142] Example

[0143] Example 1: Design and construction of RSV F protein mutants

[0144] 1. Design ideas for RSV F protein mutants

[0145] The mutants were first designed with single points or single pairs of the extracellular domain of the wild-type RSV F protein of the A2 strain with naturally occurring substitutions P77A, I354V and M422V (SEQ ID NO: 1) as the parent, including the introduction of disulfide bonds, filling of hydrophobic cavities and electrostatic mutation. Subsequently, based on the ELISA data results, the three transformation methods (introduction of disulfide bonds, filling of hydrophobic cavities, electrostatic mutation) were internally combined, the best combination in each method was selected, and finally the best combination of the three transformation methods was selected for combination.

[0146] 2. Construction of RSV F protein mutants

[0147] The wild-type RSV F protein expression vector of A2 strain was synthesized by GenScript Biotech Co., Ltd., and the expression vector backbone was pcDNA3.1(+). The point mutation primers were designed using an online tool (https: / / crm.vazyme.com / cetool / singlepoint.html), and SEQ ID NO:1 was used as a template to perform PCR according to standard molecular biology techniques to introduce the desired amino acid mutation. After the PCR product was transformed into competent DH5α, it was cultured at 37°C overnight, and a single colony was picked for sequencing the next day. After sequencing was correct, the bacterial solution was expanded to 15 mL, and the endotoxin-free plasmid was extracted according to the instructions of EndoFree Mini Plasmid KitⅡ (Cat:#DP118-02), and the expression vector of RSV F protein mutant was obtained. RSV F protein mutant CL-073 is based on the wild-type RSV F protein shown in SEQID NO:1, and N42L, V65Y, M264F, D461S, and S165I mutations were introduced.

[0148] Example 2: ELISA detection of antigenic properties of RSV F protein mutants

[0149] (1) Transfection of mutant expression plasmid

[0150] According to Nulen PlusTrans TM Transfection kit (Cat: CT801) instructions Transfect the mutant expression vector into a 96-well plate with 293T cells. The steps are as follows: Plate the 293T cells one day in advance, and observe the cells grow to a density of 80-100% the next day before transfection. Dilute 0.5μg expression vector and 0.9μL transfection reagent with 25μL opti-men medium, then mix the two to form a transfection complex, incubate at room temperature for 20min, and then drop into the 96-well plate. After culturing in a 37℃ carbon dioxide incubator for 72h, aspirate the supernatant for subsequent detection.

[0151] (2) ELISA detection of mutant antigen characteristics

[0152] The coating antibodies used were D25 and Motavizumab, the former recognizes the Φ site, and the latter recognizes the Ⅱ site on the RSV F protein.

[0153] Coating: ELISA coating solution was used to dilute antibody D25 and Motavizumab to concentrations of 3 μg / mL and 1 μg / mL, respectively, and added to a 96-well plate, 100 μL per well, sealed, and coated overnight at 4°C;

[0154] Washing: discard the liquid in the wells and pat dry with absorbent paper. Add 300 μL PBST to each well, let stand for 5 minutes, spin dry, pat dry the ELISA plate, and repeat three times;

[0155] Blocking: Prepare 2% BSA solution with PBST, 200 μL per well to block the ELISA plate, and block at 37°C for 2 h;

[0156] Washing: Same as above;

[0157] Antigen incubation: After transfection, the supernatant was diluted with PBST 1:5 and 100 μL was added to each ELISA plate. At the same time, a PBS negative control well was set up and incubated at 37°C for 2 h.

[0158] Washing: Same as above;

[0159] Primary antibody incubation: add 100 μL HRP conjugated mouse anti-His tag antibody (1:5000 dilution) and incubate at 37°C for 1 h;

[0160] Washing: Same as above;

[0161] Color development: Add 100 μL TMB substrate to each well and incubate at room temperature in the dark for 15 min;

[0162] Termination: Add 50 μL ELISA reaction stop solution to each well to terminate the reaction.

[0163] Detection: Enable the microplate reader and read the OD value at a wavelength of 450 nm.

[0164] Analysis: The higher the ratio of the OD value of the sample well to the wild-type OD value, the better the binding effect between the sample and the corresponding antibody.

[0165] Example 3: Stability of RSV F protein mutant crude culture supernatant

[0166] The stability of RSV F protein mutants designed by heat stress test experimental assessment.During heat stress test, the crude culture supernatant of the mutant designed was incubated at 55 ℃ or 60 ℃ for 1 hour and detected with anti-RSV F protein monoclonal antibody Motavizumab and pre-fusion specific monoclonal antibody D25 in ELISA assay.The ratio (OD450 ratio) of the antibody reactivity of the sample of heat stress compared to the sample of no stress is defined as stress resistance parameter.It is expected that more stable mutants have higher stress resistance.

[0167] The heat shock stability results of CL-073 are presented in Table 3. CL-073 was subjected to heat stress and probed with two monoclonal antibodies, Motavizumab and D25. The results showed that CL-073 remained stable after heat shock at 55°C or 60°C for 1 hour, and the reactivity with Motavizumab and D25 remained at a high level, comparable to the positive reference pXCS847.

[0168] Table 3 Heat shock stability of mutants

[0169]

[0170] Example 4: Expression and purification of RSV F protein mutants

[0171] HEK293F cells were cultured in 1L cell shake flasks at 37°C and 5% CO2. 6 When cells / mL, transfection reagent PEI was used and transfection was carried out according to the amount of plasmid: transfection reagent = 1:3. Five days after transfection, the cell supernatant was recovered by centrifugation at 5000g for 20 minutes. The recovered cell supernatant was added to a Ni-NTA affinity chromatography column that had been equilibrated with PBS pH 7.4 for enrichment, and then washed with PBS pH7.4, 20mM imidazole, and eluted with PBS pH 7.4, 200mM imidazole. The eluted fusion protein was concentrated using an ultrafiltration tube, and after concentration, it was further purified by gel filtration chromatography, and the buffer environment was PBS solution. The protein obtained in the above process was identified by SDS-PAGE for its purity ( Figure 1 ), it can be seen that CL-073 and pXCS847 are both cut into F1 polypeptide and F2 polypeptide. It should be noted that the use of nickel column affinity chromatography to purify the fusion protein depends on the tag carried by the fusion protein. In other embodiments, other purification methods can also be selected by replacing the tag. The crude culture supernatant and purified protein were used for in vitro and in vivo assays described herein.

[0172] Example 5: Study on the stability of purified RSV F protein mutants

[0173] The purified RSV F protein mutants need to be subjected to multiple treatments to evaluate their stability, including 5 freeze-thaw cycles, storage at 4°C and 25°C for 3 days, shaking at 4°C and 25°C for 3 days, and heat stress at 40°C and 50°C for 1 hour. The treated proteins were evaluated for stability by gel filtration chromatography and SDS-PAGE electrophoresis. CL-073 had comparable effects to the positive reference pXCS847, and no significant changes were observed after the above treatments.

[0174] pXCS847 and CL-073 were analyzed by differential scanning fluorescence (DSF) to evaluate their thermal stability. The protein sample was diluted with PBS pH 7.4 to a final concentration of 1 mg / ml, and the Sypro@orange dye was diluted with ultrapure water to a concentration of 50× for standby use. The reaction system was: 3 μl of diluted protein sample, 3 μl of diluted dye and 24 μl of PBS pH 7.4. After the reaction system was prepared as required, it was detected using a fluorescent quantitative PCR instrument (Bio-Rad CFX96 Touch). The PCR instrument reaction program used a melting curve module and HEX channel detection. The control system started to heat up from 20°C and increased by 1°C every 1min until the system reached 80°C. During this period, the instrument collected the changes in fluorescence intensity with temperature according to the default parameters. The instrument has its own data processing function. The temperature corresponding to the minimum negative value of the first derivative of fluorescence intensity to temperature is the Tm value. The Tm values ​​of CL-073 and pXCS847 were both about 61°C ( Figure 2 ), indicating that the thermal stability of the two is comparable.

[0175] Example 6: Immunogenicity studies

[0176] Female SPF BALB / c mice aged 6-8 weeks were purchased from Jicui Pharmaceutical Co., Ltd., with 5 mice in each group. On day 0 and day 21, 50 μl of 50 μg aluminum hydroxide adjuvant ( Cat: #AJV3012)'s Post-F antigen (3μg, Cat: #RSF-V52H6), CL-073 antigen (10μg, 3μg and 0.3μg) and pXCS847 antigen (10μg, 3μg and 0.3μg). Blood was collected by removing the eyeball 14 days after the second immunization (5 weeks after the first immunization) to separate serum for virus neutralization experiment or ELISA experiment.

[0177] Table 4 Mouse immunization scheme

[0178] Group antigen Dosage (μg) 1 Aluminum hydroxide control group NA 2 Post-F 3 3 pXCS847 10 4 pXCS847 3 5 pXCS847 0.3 6 CL-073 10 7 CL-073 3 8 CL-073 0.3

[0179] The neutralizing titres (NT) of immune serum samples were determined by cytopathic effect method.

[0180] (1) The day before, HEp-2 cells (Cat: ATCC#CCL-23) were cultured at 1.5×10 4 / well were inoculated into 96-well plates.

[0181] (2) On the second day, the test sample and the control sample were diluted three times at the highest dilution ratio of 1:20, with 2 replicate wells for each dilution, for a total of 6 dilutions.

[0182] (3) In a BSL-2 laboratory, RSV A2 virus suspension with a multiplicity of infection (MOI) of 0.2 was added to each well and incubated at 37°C for 1 h.

[0183] (4) Aspirate and discard the cell culture supernatant, add 150 μL of serum-virus mixture to each well, and then place in a constant temperature and humidity incubator at 37°C and 5% CO2.

[0184] (5) Observe and record the cell lesions under a microscope every 24 hours.

[0185] (6) After 72 hours of culture, the cytopathic rate was read using a full-view cell analyzer (Celigo Image Cytometer, Celigo) and the NT95 value was calculated.

[0186] The results showed that after two immunizations in mice, all tested mutants triggered neutralizing antibody responses, among which CL-073 had a good inhibitory effect on RSV A2 strain at the cellular level. The neutralization effect and NT95 value are shown in Figure 3 .from Figure 3 It can be seen that under the same dose immunization conditions, CL-073 neutralizes RSV A2 activity better than the control pXCS847. In summary, for mutant CL0-73, the antibody titer is always high at three antigen doses, indicating that this mutant is a more immunogenic form of the stable RSV prefusion F glycoprotein.

[0187] Example 7: Protection against RSV attack

[0188] Female SPF BALB / c mice aged 6-8 weeks were purchased from Jicui Pharmaceutical Co., Ltd., with 5 mice in each group. On day 0 and day 21, 50 μl of 50 μg aluminum hydroxide adjuvant ( Cat:#AJV3012)'s Post-F antigen (3 μg, Cat:#RSF-V52H6), CL-073 antigen (10 μg, 3 μg and 0.3 μg) and pXCS847 antigen (10 μg, 3 μg and 0.3 μg).

[0189] Table 5 Mouse immunization scheme

[0190] Group antigen Dosage (μg) 1 Aluminum hydroxide control group NA 2 Post-F 3 3 pXCS847 10 4 pXCS847 3 5 pXCS847 0.3 6 CL-073 10 7 CL-073 3 8 CL-073 0.3

[0191] BALB / c mice were infected by intranasal drops 14 days after booster immunization, and general clinical observations were performed once a day after infection, including the mice's mobility and survival status; eyes and hair; death and any observable clinical manifestations.

[0192] (1) 12-week-old female BALB / c mice that had been immunized for 35 days were lightly anesthetized with isoflurane.

[0193] (2) Each mouse was infected intranasally with 50 μL of 1×10 6 PFU of RSV A2 (stored in the BSL-2 laboratory of Guangzhou National Laboratory).

[0194] (3) The mice were weighed on the day of challenge (i.e., D0) and daily thereafter (D1, D2, D3, and D4).

[0195] (4) Detect the viral load and titer in lung tissue.

[0196] On the morning of D4, 4 days after infection, mice were euthanized, half of the right lung was taken, weighed and added with 1 mL of sterile PBS to fully homogenize; qRT / PCR was used to detect the viral load (Fusion gene); and the FFA method was used to determine the viral titer.

[0197] Infection was carried out by intranasal drip, and the mice were weighed once a day after infection. The results showed that after two immunizations in mice, the tested mutants all had a protective effect against RSV-attacked mice, and the weight of mice immunized with CL-073 recovered faster than that of the control group ( Figure 4 ).from Figure 5 As can be seen in the figure, the viral copies in the lung tissues of mice immunized with CL-073 were significantly lower than those in the control group. Figure 6 As can be seen in the figure, no live virus was detected in the lung tissue of mice immunized with the CL-073 group. In summary, for the mutant CL-073, the effect of protecting mice was always good at three antigen doses, indicating that this mutant is a more immunogenic form of the stabilized RSV prefusion F glycoprotein.

[0198] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A respiratory syncytial virus (RSV) F protein comprising at least one modification or mutation selected from the following that increases the stability of the RSV F protein prefusion conformation: (1) introducing one or more amino acid mutations into the RSV F protein to fill the hydrophobic cavity; (2) introducing one or more amino acid mutations for electrostatic modification into the RSV F protein; (3) Remove the p27 peptide from RSV F protein.

2. RSV F protein according to claim 1, wherein, The amino acid mutation for filling the hydrophobic cavity comprises an amino acid substitution at an amino acid position selected from the group consisting of: asparagine 42 residue, valine 65 residue, leucine 71 residue, leucine 205 residue, methionine 264 residue, isoleucine 267 residue, isoleucine 192 residue and / or serine 165 residue.

3. The RSV F protein according to any one of claims 1 or 2, wherein The amino acid mutations used to fill the hydrophobic cavity include: N42L, N42F, N42Y, N42W, N42H, V65Y, V65F, L71I, L71Y, L205F, L205Y, M264F, M264Y, I267L, I267F, I267Y, I192L, S165I, S165F, S165Y and / or S165L.

4. The RSV F protein according to any one of claims 1 to 3, wherein The amino acid mutation for electrostatic modification comprises an amino acid substitution at the amino acid position of aspartic acid 461 residue and / or glutamic acid 462 residue.

5. The RSV F protein according to any one of claims 1 to 4, wherein The amino acid mutations used for electrostatic modification include: D461S, D461N, D461Q, D461E, D461G, D461A, D461K, D461R, D461H, E462Q, E462S, E462N, E462D, E462G, E462A, E462K, E462R, and E462H.

6. The RSV F protein according to any one of claims 1 to 5, wherein The RSV F protein comprises the mutation combination: N42L, V65Y, M264F, D461S, S165I.

7. The RSV F protein according to any one of claims 1 to 6, wherein The p27 peptide segment of the RSV F protein is replaced by a linker; Preferably, the linker is a flexible polypeptide; Preferably, the flexible polypeptide is selected from (GS)n, (GGSG)n, (GGGGS)n, (GGGSGG)n, (GGGSGGG)n, (EA)n, (EAK)n, (EAKA)n, (AAAAK)n, (EAAAK)n, (LEAAK)n, wherein n is an integer from 1 to 4; More preferably, the flexible polypeptide is selected from the amino acid sequences shown in SEQ ID NOs: 7-50.

8. The RSV F protein according to any one of claims 1 to 7, wherein The RSV F protein comprises an amino acid sequence that is 80% or more identical to SEQ ID NO: 2, preferably an amino acid sequence that is 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical, and more preferably an amino acid sequence that is 98% or more identical to SEQ ID NO:

2.

9. The RSV F protein according to any one of claims 1 to 8, wherein The N-terminus of the RSV F protein is connected to a signal peptide; Preferably, the signal peptide is selected from a wild-type RSV F protein signal peptide or a non-RSV F protein signal peptide; More preferably, the signal peptide comprises an amino acid sequence selected from SEQ ID NO: 3, 4, 5 or any variant thereof.

10. The RSV F protein according to any one of claims 1 to 9, wherein The C-terminus of the RSV F protein is connected to a trimerization tag; Preferably, the trimerization tag comprises an amino acid sequence as shown in SEQ ID NO: 6 or any variant thereof.

11. A nucleic acid molecule encoding the RSV F protein of any one of claims 1-10.

12. A vector comprising the nucleic acid molecule of claim 11; Preferably, the vector is an expression vector.

13. A host cell comprising the vector of claim 12; Preferably, the host cell is a prokaryotic cell or a eukaryotic cell; Preferably, the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof; Preferably, the mammalian cell is selected from CHO cells, HEK293 cells or COS cells.

14. An immunogenic composition comprising the RSV F protein of any one of claims 1-10.

15. A vaccine composition comprising the RSV F protein of any one of claims 1-10, the nucleic acid molecule of claim 11, the vector of claim 12 and / or the host cell of claim 13.

16. A kit for immunizing a subject against viral infection, comprising the RSV F protein of any one of claims 1 to 10, the nucleic acid molecule of claim 11, the vector of claim 12 and / or the host cell of claim 13; Preferably, the viral infection is RSV infection; Preferably, the subject is a human subject.

17. A pharmaceutical composition comprising the RSV F protein of any one of claims 1-10 and a pharmaceutically acceptable carrier; Preferably, the pharmaceutical composition further comprises other therapeutic agents.

18. A method for generating an immune response against a viral infection, comprising administering the RSV F protein of any one of claims 1-10 in a pharmaceutically acceptable formulation to a subject; Preferably, the pharmaceutically acceptable formulation comprises an adjuvant.

19. Use of the RSV F protein according to any one of claims 1 to 10 in the preparation of a medicament for preventing or treating a disease or condition associated with respiratory syncytial virus (RSV) infection; Preferably, the respiratory syncytial virus (RSV) is selected from one or more of type A RSV virus and type B RSV virus.

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