Truncated respiratory syncytial virus F proteins and uses thereof

Abstract

Fusion proteins and nucleic acid molecules comprising nucleotide sequences encoding the fusion proteins are provided. The present invention also relates to vaccines comprising the fusion proteins or nucleic acid molecules. Furthermore, the present invention also relates to methods for preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection by using the fusion proteins, nucleic acid molecules, and vaccines.

Description

[Technical Field]

[0001] This application relates to the field of biomedicine, and in particular, this application relates to fusion proteins and nucleic acid molecules comprising nucleotide sequences encoding the fusion proteins. This application also relates to vaccines comprising the fusion proteins or nucleic acid molecules. Furthermore, the present invention also relates to methods of using the fusion proteins, nucleic acid molecules, and vaccines for preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection. [Background technology]

[0002] Respiratory syncytial virus (RSV) is one of the most important pathogens causing lower respiratory tract infections in infants and children worldwide. Statistics show that 33 million children under the age of five are infected with RSV each year, resulting in nearly 160,000 deaths. RSV infection does not confer lasting immunity, and repeated infections are possible. More than 99% of children under the age of two have been infected with RSV at least once, and 70% of patients are hospitalized with infections complicated by bronchiolitis, pneumonia, and asthma. In addition to infants and children, immunocompromised elderly individuals are also at high risk for RSV infection. Elderly individuals often develop obstructive pulmonary disease accompanied by cardiopulmonary complications. Despite the serious illness and economic burden it causes worldwide, a safe and effective vaccine against RSV remains unavailable.

[0003] RSV is an enveloped, single-stranded, negative-sense RNA virus belonging to the genus Pneumovirus and family Pneumoviridae. The genome is approximately 15.2 kb long, transcribes 10 genes, and encodes a total of 11 proteins. Among these, the adhesion glycoprotein G and membrane fusion protein F are located on the surface of the viral membrane. The F protein is more highly conserved than the G protein and is therefore considered to be the most important protective antigen of RSV.

[0004] The RSV F protein belongs to the type I integral membrane proteins. Its precursor protein, F0, consists of 574 amino acids, and three F0 segments form a trimer through hydrophobic interactions. During transit through the Golgi apparatus, this is cleaved by the host's Furin protease between amino acids 109 and 110 and between amino acids 136 and 137. After cleavage, the F protein releases a short 27-amino acid peptide, P27. The remaining two segments, F2 and F1, are linked by two disulfide bonds to form the mature F protein, which is presented in a budding form on the cell membrane or the surface of the virion. At this stage, the F protein is in a high-energy metastable state, known as pre-F, and is highly unstable. The N-terminus of F1 contains the highly hydrophobic fusion peptide, FP, which is located in the hydrophobic cavity of the trimer. Triggered by currently unknown factors, the N-terminus of F1 undergoes a series of dramatic structural changes that insert FP into the cell membrane surface, helping the virus complete membrane fusion and infect cells, and also causing the pre-F structure to adopt a stable, low-energy post-F conformation.

[0005] In 2013, McLellan et al. obtained the first stable pre-F protein through point mutation, which they named DS-Cav1. Subsequently, several pre-F proteins, including DS-Cav1-Cys-zipper, SC-DM, and SC-TM, have been reported. These pre-F proteins have been shown to activate significantly higher levels of neutralizing antibodies compared to post-F proteins. The RSV pre-F protein can be divided into two parts: a membrane-proximal "handle" region containing domains I and II (which is primarily composed of β-strand structures and terminates in a C-terminal helix that enters the membrane), and a membrane-distal portion, i.e., the RSV F "head" region (which is primarily α-helix) containing domain III. The RSV F head contains at least two epitopes associated with neutralizing antibodies. Sites φ and V are located at the top of the pre-F trimer and can be recognized by potent neutralizing antibodies, including D25, AM22, 5C4, and hRSV90, respectively. Site II is located in the center of the pre-F trimer and is the binding site for the commercially available antibody palivizumab. Sites I and IV, which are also recognized by less efficient antibodies, are located in the handle region of the pre-F protein. The handle region of the pre-F protein accounts for 55% of the total surface area of ​​the trimer, which may compete with the highly neutralizing epitopes site φ or site V in the head region, potentially resulting in attenuation of the antibody response to these highly neutralizing epitopes. Summary of the Invention [Problem to be solved by the invention]

[0006] The main goal of research into RSV vaccines that use the F protein as the main protective antigen is to produce antibodies with high neutralizing titers, and improving the immunogenicity of RSV vaccines that use the F protein as the protective antigen is of great significance for the development of RSV vaccines. [Means for solving the problem]

[0007] Contents of the present invention Fusion proteins containing truncated portions In a first aspect, the present application provides a fusion protein comprising a first truncation, a second truncation, and a linker connecting the first truncation and the second truncation, Compared to the F2 protein of a wild respiratory syncytial virus (RSV), the first truncation is truncated by 5 to 25 amino acids (e.g., 5 to 8, 9 to 12, 13 to 16, 17 to 20, 21 to 25) at the N-terminus and 3 to 5 amino acids (e.g., 3, 4, 5) at the C-terminus of the F2 protein of a wild RSV; The second truncation provides a fusion protein in which, compared to the wild-RSV F1 protein, 7 to 9 (e.g., 7, 8, or 9) amino acids are truncated at the N-terminus of the wild-RSV F1 protein and 238 to 268 (e.g., 238 to 241, 242 to 245, 246 to 249, 250 to 253, 254 to 257, 258 to 261, 262 to 265, or 266 to 268) amino acids are truncated at the C-terminus.

[0008] In certain embodiments, the C-terminal truncation length of the second truncation falls within a certain range (i.e., a truncation of 238 to 268 amino acids), and the N-terminal truncation length of the first truncation falls within a certain range (i.e., a truncation of 5 to 25 amino acids), because within these ranges, the first and second truncations can still retain some protein secondary structures (e.g., β2 / β7, α1, and α4, etc.) that maintain conformational stability.

[0009] In some embodiments, the first truncation is truncated by 5 or 25 amino acids at the N-terminus and 4 amino acids at the C-terminus of the wild-type RSV F2 protein compared to the wild-type RSV F2 protein.

[0010] In some embodiments, the F2 protein of wild-type RSV has the amino acid sequence set forth in SEQ ID NO:13.

[0011] In some embodiments, the first truncation has the amino acid sequence set forth in SEQ ID NO:16 or SEQ ID NO:17.

[0012] In some embodiments, the second truncation is truncated by 8 or 9 amino acids at the N-terminus and 252 or 268 amino acids at the C-terminus of the wild-type RSV F1 protein compared to the wild-type RSV F1 protein.

[0013] In some embodiments, the F1 protein of wild-type RSV has the amino acid sequence set forth in SEQ ID NO:12.

[0014] In some embodiments, the second truncation has the amino acid sequence set forth in SEQ ID NO:14 or SEQ ID NO:15.

[0015] In some embodiments, the linker comprises at least one (eg, one, two) proline.

[0016] In some embodiments, the linker has between 3 and 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20) amino acids. In some embodiments, the linker has between 5 and 8 amino acids.

[0017] In some embodiments, the linker comprises multiple (eg, 2, 3, 4, 5, 6, 7) glycines and at least one (eg, 1, 2) proline.

[0018] In some embodiments, the linker has the sequence shown in SEQ ID NO:2.

[0019] In some embodiments, the first truncation is located at the N-terminus of the linker.

[0020] In some embodiments, the second truncation is located at the C-terminus of the linker.

[0021] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a first truncation, a linker, and a second truncation.

[0022] In some embodiments, the fusion protein has the amino acid sequence set forth in SEQ ID NO:3 or SEQ ID NO:4.

[0023] Fusion proteins containing truncation mutants In certain embodiments, the fusion protein further comprises one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions).

[0024] In certain embodiments, the first truncated form and / or the second truncated form of the fusion protein comprises one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions).

[0025] In certain embodiments, the sequence of the fusion protein has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to the sequence set forth in any one of SEQ ID NOs: 3 to 7.

[0026] In certain embodiments, the substitution is a conservative substitution.

[0027] In some embodiments, the sequence of the fusion protein has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO:4.

[0028] In some embodiments, compared to the sequence set forth in SEQ ID NO:4, the sequence of the fusion protein has an amino acid substitution at a position corresponding to position 29 of SEQ ID NO:4, and the sequence of the fusion protein also has an amino acid substitution at any one of positions corresponding to positions 47 to 214 of SEQ ID NO:4 (e.g., position 47, position 212, position 214, position 88, position 94, position 114, position 101).

[0029] In certain embodiments, compared to the sequence shown in SEQ ID NO: 4, the sequence of the fusion protein has an amino acid substitution at a position corresponding to position 114 of SEQ ID NO: 4, and the sequence of the fusion protein also has an amino acid substitution at any one of positions corresponding to positions 29 to 94 of SEQ ID NO: 4.

[0030] In certain embodiments, the sequence of the fusion protein, compared to the sequence set forth in SEQ ID NO:4, has one or more amino acid substitutions at positions corresponding to positions 29, 47, 212, 214, 88, 94, 114, and 101 of SEQ ID NO:4.

[0031] In certain embodiments, the sequence of the fusion protein, compared to the sequence shown in SEQ ID NO:4, has amino acid substitutions at positions corresponding to positions 29 and 212 of SEQ ID NO:4.

[0032] In certain embodiments, the sequence of the fusion protein, compared to the sequence shown in SEQ ID NO:4, has amino acid substitutions at positions corresponding to positions 29 and 214 of SEQ ID NO:4.

[0033] In certain embodiments, the sequence of the fusion protein, compared to the sequence shown in SEQ ID NO:4, has amino acid substitutions at positions corresponding to positions 29 and 114 of SEQ ID NO:4.

[0034] In certain embodiments, the sequence of the fusion protein, compared to the sequence set forth in SEQ ID NO:4, has amino acid substitutions at positions corresponding to positions 29, 47, 101, and 214 of SEQ ID NO:4.

[0035] In certain embodiments, the sequence of the fusion protein, compared to the sequence set forth in SEQ ID NO:4, has amino acid substitutions at positions corresponding to positions 88, 101, 114, and 214 of SEQ ID NO:4.

[0036] In certain embodiments, the sequence of the fusion protein, compared to the sequence shown in SEQ ID NO:4, has amino acid substitutions at positions corresponding to positions 94 and 114 of SEQ ID NO:4.

[0037] In certain embodiments, the substitution at position 29 is a substitution of isoleucine I with leucine L.

[0038] In certain embodiments, the substitution at position 212 is a substitution of valine V with methionine M.

[0039] In certain embodiments, the substitution at position 214 is a substitution of alanine A with leucine L.

[0040] In certain embodiments, the substitution at position 94 is a substitution of valine V with isoleucine I.

[0041] In certain embodiments, the substitution at position 47 is a substitution of alanine A for methionine M.

[0042] In certain embodiments, the substitution at position 114 is a substitution of isoleucine I for tyrosine Y.

[0043] In certain embodiments, the substitution at position 101 is a substitution of valine V with isoleucine I.

[0044] In certain embodiments, the substitution at position 214 is a substitution of alanine A with leucine L.

[0045] In certain embodiments, the substitution at position 88 is a substitution of the amino acid leucine L with alanine A.

[0046] In certain embodiments, the fusion protein has an amino acid sequence set forth in any one of SEQ ID NOs:28 to 34.

[0047] In some embodiments, the fusion protein further comprises a signal peptide, a multimerization motif (eg, a trimerization motif), and / or a membrane anchor region.

[0048] In some embodiments, the membrane anchor region has the amino acid sequence set forth in SEQ ID NO:8.

[0049] In some embodiments, the signal peptide has the amino acid sequence set forth in SEQ ID NO:9.

[0050] In some embodiments, the multimerization motif has the amino acid sequence set forth in SEQ ID NO:21 or SEQ ID NO:22.

[0051] In some embodiments, the signal peptide is located at the N-terminus of the first truncation, hi some embodiments, the signal peptide is linked to the first truncation via a first linker peptide.

[0052] In some embodiments, the membrane anchoring region is located at the C-terminus of the second truncated form, hi some embodiments, the membrane anchoring region is linked to the second truncated form via a second linker peptide.

[0053] In some embodiments, the multimerization motif is located at the C-terminus of the second truncated form, hi some embodiments, the multimerization motif is linked to the second truncated form via a second linker peptide.

[0054] In some embodiments, the first linker peptide and the second linker peptide each independently comprise: (G m S) n (wherein m is an integer selected from 1 to 6, and n is an integer selected from 1 to 6). In some embodiments, m is 3, 4, or 5. In some embodiments, n is 1 or 2. In some embodiments, the first linker peptide and the second linker peptide have the sequence set forth in SEQ ID NO:18.

[0055] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a signal peptide, a first linker peptide, a first truncation, a linker, a second truncation, a second linker peptide, and a membrane anchor region.

[0056] In certain embodiments, the fusion protein has the sequence set forth in SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:45, or SEQ ID NO:46.

[0057] On the other hand, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the above-mentioned fusion protein.

[0058] In certain embodiments, the nucleotide sequence is codon-optimized according to the codon preferences of the host cell or is not codon-optimized.

[0059] On the other hand, the present application provides a vector comprising the above-mentioned nucleic acid molecule.

[0060] In certain embodiments, the vector is a viral vector.

[0061] In certain embodiments, the viral vector is selected from the group consisting of an influenza virus vector, an enterovirus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papovavirus vector.

[0062] On the other hand, the present application provides a host cell comprising the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, or the above-mentioned vector.

[0063] In certain embodiments, the host cell is selected from the group consisting of a prokaryotic cell (eg, an Escherichia coli cell) and a eukaryotic cell.

[0064] In some embodiments, the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell.

[0065] In some embodiments, the fusion protein comprises a membrane anchor region and is displayed on the surface of the plasma membrane of the host cell.

[0066] On the other hand, the present application provides a method for expressing or producing the above-mentioned fusion protein, which comprises culturing the above-mentioned host cell under conditions that allow protein expression, and optionally recovering or purifying the expressed fusion protein.

[0067] On the other hand, the present application provides a kit comprising an antigen component and a carrier component capable of presenting the antigen component, the antigen component comprises (i) the fusion protein described above, (ii) the nucleic acid molecule described above, and / or (iii) an mRNA product transcribed from the nucleic acid molecule described above; Kits are provided in which the carrier component is selected from the group consisting of nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers, and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), multimerization motifs, virus-like particles (VLPs), or any combination thereof.

[0068] In some embodiments, the antigen component and the carrier component in the kit are provided separately or in the form of a complex formed therefrom.

[0069] In certain embodiments, the antigen component is a monomer or multimer (eg, dimer, trimer, tetramer, pentamer).

[0070] In certain embodiments, the multimerization motif and / or VLP in the kit is provided in the form of a protein or a nucleic acid molecule comprising a nucleotide sequence encoding the protein.

[0071] In certain embodiments, the multimerization motif has the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:22.

[0072] In certain embodiments, the VLPs are assembled from proteins derived from RSV, hepatitis E virus (HEV), hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0073] On the other hand, the present application relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and / or additive and the following items (1) to (4): (1) the above fusion protein, (2) the nucleic acid molecule described above; (3) the above vector, (4) the host cell described above; and one or more selected from The present invention provides a pharmaceutical composition comprising:

[0074] In another aspect, the present application provides a method for producing a vaccine against respiratory syncytial virus (RSV), comprising providing the kit or pharmaceutical composition described above and formulating it into a pharmaceutically acceptable vaccine.

[0075] In another aspect, the present application provides a vaccine comprising the fusion protein described above, or the nucleic acid molecule described above, or the mRNA product transcribed from the nucleic acid molecule described above, or the vector described above.

[0076] In certain embodiments, a vaccine is prepared from the kit or pharmaceutical composition described above.

[0077] In certain embodiments, the vaccine further comprises an adjuvant.

[0078] In certain embodiments, the vaccines described above can be administered to a subject, such as a human subject. The total dose of fusion protein in a single dose of the vaccine can be, for example, about 0.01 μg to about 10 mg, e.g., 1 μg to 1 mg, e.g., 10 μg to 100 μg. Determining the dose to be administered can be determined empirically, and determining the dose is routine for one of skill in the art.

[0079] Meanwhile, the present application provides a method for inducing antibodies against RSV, comprising administering (e.g., injecting) an effective amount of the fusion protein, or the nucleic acid molecule, or the vector, or the host cell, or the pharmaceutical composition, or the vaccine to a cell in vitro or to a subject in vivo.

[0080] In certain embodiments, the antibody is a neutralizing antibody.

[0081] In certain embodiments, administration includes parenteral administration, such as intradermal administration, intramuscular administration, subcutaneous administration, transdermal administration, or mucosal administration, such as intranasal administration, oral administration, etc. In certain embodiments, the composition is administered by intramuscular injection.

[0082] Additionally, the fusion proteins of the present invention can be used as diagnostic tools to detect the immune status of a subject, for example, by determining the presence in the subject's serum of antibodies capable of binding to the fusion protein.

[0083] Therefore, the present application also provides an in vitro method for detecting whether a subject is infected with RSV, the method comprising contacting a biological sample obtained from the subject with the above-described fusion protein and detecting the presence of a complex formed by the fusion protein and an antibody from the biological sample.

[0084] In certain embodiments, the subject is a mammal, eg, a mouse, a human.

[0085] In certain embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, or any combination thereof.

[0086] On the other hand, the present application provides a method for screening a candidate drug capable of inhibiting RSV infection in a cell, the method comprising contacting a host cell with the candidate drug before, simultaneously with, or after contacting the host cell with the above-mentioned fusion protein, or the above-mentioned vector, or the above-mentioned pharmaceutical composition, or the above-mentioned vaccine.

[0087] Meanwhile, the present application provides the use of the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned pharmaceutical composition, or the above-mentioned vaccine in the manufacture of a kit for inducing an immune response against RSV in a subject.

[0088] In certain embodiments, the immune response comprises inducing the subject to produce antibodies against RSV.

[0089] In certain embodiments, the antibody is a neutralizing antibody.

[0090] In certain embodiments, the subject is a mammal, eg, a mouse, a human.

[0091] Meanwhile, the present application provides the use of the above-mentioned fusion protein, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned host cell, or the above-mentioned pharmaceutical composition, or the above-mentioned vaccine in the manufacture of a kit for preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection.

[0092] In certain embodiments, the subject is a mammal, e.g., a mouse, a human. In certain embodiments, the disease or condition caused by RSV infection is selected from bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0093] Definition of Terms As used herein, the terms "respiratory syncytial virus" and "RSV" have the same meaning and are viruses belonging to the Pneumoviridae family and the Pneumovirus genus. The RSV genome is approximately 15 Kb long, contains 10 genes, and encodes 11 proteins, including eight structural proteins (F, G, M2-1, M2-2, SH, N, P, L) and three nonstructural proteins (NS1, NS2, NS3). Among them, the fusion protein (F) and the attachment protein (G) are the two major envelope glycoproteins. The F protein is a type I glycoprotein. After being cleaved into F1 and F2 by cellular proteases, the F protein retains biological activity and can fuse the viral envelope with the host cell membrane to form a multinucleated giant cell. The sequence of the F protein can be obtained from a public database (e.g., the GenBank database). In certain embodiments, the amino acid sequence of a wild-type F protein is set forth in SEQ ID NO: 1.

[0094] As used herein, the terms "wild" and "natural" are used interchangeably. When these terms are used to describe a nucleic acid molecule, a polypeptide, or a protein, they indicate that the nucleic acid molecule, polypeptide, or protein exists in nature, is found in nature, and has not been artificially modified or engineered. As used herein, the F1 / F2 protein of wild respiratory syncytial virus (RSV) refers to a naturally occurring, biologically active F1 / F2 protein. Those skilled in the art can easily obtain the amino acid sequences of the F1 protein and the F2 protein from various public databases (e.g., the GenBank database). For example, the amino acid sequence of the F2 protein of wild RSV can be set forth in SEQ ID NO: 13. The amino acid sequence of the F1 protein of wild RSV can be set forth in SEQ ID NO: 12.

[0095] As used herein, the term "truncated by X amino acids at the C-terminus" means that the most distal contiguous X amino acids at the C-terminus have been truncated. Similarly, the term "truncated by X amino acids at the N-terminus" means that the most distal contiguous X amino acids at the N-terminus have been truncated.

[0096] As used herein, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, thereby allowing the genetic material elements carried by the vector to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Vectors may contain various elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain an origin of replication site.

[0097] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including, but not limited to, a prokaryotic cell such as Escherichia coli or Bacillus subtilis, a fungal cell such as a yeast cell or an Aspergillus cell, an insect cell such as an S2 Drosophila cell or an Sf9 cell, or an animal cell such as a fibroblast, a CHO cell, a COS cell, an NSO cell, a HeLa cell, a BHK cell, an HEK293 cell, or a human cell.

[0098] As known to those skilled in the art, codons are degenerate. That is, during protein translation, each amino acid can correspond to one or more codons, for example, up to six codons. Different species have significant differences and preferences in the use of degenerate codons to encode a particular amino acid. This preference phenomenon is called "codon preference." Therefore, as used herein, the term "codon preference" refers to a situation in which a species prefers to use a particular codon to encode an amino acid. Optimizing the sequence of a nucleic acid molecule according to codon preference can be particularly advantageous in some cases, and can help, for example, to increase the expression level of a protein encoded by the nucleic acid molecule. For example, when expressing a protein or a fragment thereof using E. coli (or human cells), it can be advantageous to optimize the nucleic acid sequence encoding the protein or a fragment thereof to match the codon preference of E. coli (or human cells).

[0099] As used herein, the term "virus-like particle (VLP)" refers to a polymeric particle whose structure is similar to or different from a naturally occurring virus particle. In some embodiments, a VLP is a naturally occurring virus particle. In some embodiments, a VLP is a virus-like particle assembled from proteins. It has been shown that proteins (e.g., capsid proteins, surface proteins, envelope proteins) of some viruses (e.g., RSV, HBV, HEV, HPV) can spontaneously form VLPs after recombinant expression in an appropriate expression system.

[0100] As used herein, the term "pharmaceutically acceptable" means that it is recognized in the pharmaceutical field that it can be used in animals, particularly humans. As used herein, the term "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, and are well known in the art (see, for example, Remington's Pharmaceutical Sciences (ed. by Gennaro AR), 19th ed., PA: Mack Publishing Company, 1995), including, but not limited to, pH adjusting agents (including, but not limited to, phosphate buffers), surfactants (including, but not limited to, cationic surfactants, anionic surfactants, or nonionic surfactants such as Tween-80), adjuvants, ionic strength enhancers (including, but not limited to, sodium chloride), diluents, excipients, vehicles for containing or administering therapeutic agents, and any combination thereof.

[0101] As used herein, pharmaceutically acceptable carriers can be sterile liquids such as water and oils, including those derived from petroleum, animals, or plants, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, physiological saline is a preferred carrier. Physiological saline solution, as well as aqueous dextrose and aqueous glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions.

[0102] As used herein, pharmaceutically acceptable additives include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, milk powder, glycerol, propylene glycol, ethylene glycol, water, ethanol, etc. If necessary, the pharmaceutical composition may contain a wetting agent, an emulsifier such as sodium hyaluronate, or a pH buffering agent. The pharmaceutical composition may take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0103] As used herein, the term "subject" refers to a mammal, including, but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human.

[0104] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired result. For example, an effective amount for disease prevention refers to an amount sufficient to prevent, inhibit, or delay the onset of a disease, and an effective amount for disease treatment refers to an amount sufficient to cure or at least partially prevent a disease and its complications in a patient already suffering from the disease. Determining such effective amounts is within the skill of one of ordinary skill in the art. The effective amount for therapeutic use will depend, for example, on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the mode of administration of the drug, and other treatments administered at the same time.

[0105] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include the replacement of an amino acid residue with an amino acid residue having a similar side chain, such as a substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical properties including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32: 1180-1187 (1993), Kobayashi et al., Protein Eng. 12(10): 879-884 (1999), and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997), which are incorporated herein by reference).

[0106] The 20 conventional amino acids referred to herein are written according to convention. See, for example, Immunology - A Synthesis (2nd ed., ES Golub and DR Gren (eds.), Sinauer Associates, Sunderland, Mass. (1991)) (incorporated herein by reference). In the present invention, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0107] Beneficial effects of the invention The fusion protein of the present application contains a unique truncated F1 and F2 truncated RSV protein. The truncated fusion protein has the ability to induce specific antibodies comparable to the full-length pre-F protein and can also induce significantly higher neutralizing antibody titers than the full-length pre-F protein. The presence of a linker in the fusion protein also serves to improve the stability of the pre-F-specific epitope region Φ. Furthermore, the resulting fusion protein is mutated, and the mutated fusion protein has stronger affinity for antibodies, higher thermal stability, and stronger immunogenicity.

[0108] In summary, the fusion protein of the present application exhibits good protection and safety, and is suitable for various forms of vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and granulated vaccines. Therefore, the fusion protein of the present application has great potential for inducing an immune response against RSV in a subject and preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection.

[0109] Hereinafter, the embodiments of the present invention will be described in detail in conjunction with drawings and examples, but those skilled in the art will understand that the following drawings and examples are used only to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following drawings and detailed description of the preferred embodiments. [Brief explanation of the drawings]

[0110] [Figure 1] FIG. 1 shows the results of expression of site II and site φ, which are highly neutralizing antibody epitopes, after transfection of F proteins containing various linker peptides into 293t cells. [Figure 2] FIG. 1 shows a schematic diagram of the structure of the wild-type F protein Fwt and the F protein truncated LC2. [Figure 3] FIG. 1 shows a schematic diagram of the structure of F protein truncated LC2 predicted by AlphaFold2. [Figure 4] FIG. 1 shows the expression of F protein truncations detected by Western blot. The first lane is the marker, the second lane is pre-F protein SC-TM, and the third lane is F protein truncation LC2. [Figure 5] FIG. 5 shows the binding of F protein truncated LC2 to the site II-specific antibody mota (Panel A in FIG. 5) and the site φ-specific antibodies D25 and AM22 (Panels B and C in FIG. 5), as detected by immunofluorescence. [Figure 6] FIG. 1 shows serum binding antibody titers and serum antibody typing in mice induced by an mRNA vaccine encoding an F protein truncation. [Figure 7] FIG. 1 shows the results of neutralizing antibody titers in mice induced by an mRNA vaccine encoding an F protein truncation. [Figure 8]FIG. 1 shows a schematic diagram of the construction of the wild-type F protein Fwt and the mutated F protein truncated LC2A, where the wild-type F protein Fwt comprises a signal peptide, F2, F1, a membrane anchor region, and a cytoplasmic region, and the mutated F protein truncated LC2A comprises a signal peptide, a first linker peptide, a mutated first truncated form, a linker, a mutated second truncated form, a second linker peptide, and a multimerization motif. [Figure 9] This figure shows the results of polyacrylamide gel electrophoresis of the control protein SC-TM and the mutated F protein truncations, where the first lane is the marker, the second lane is LC2A, and the third lane is SC-TM. [Figure 10] FIG. 1 shows binding of three antibodies to the F protein of SC-TM and seven mutated F protein truncations. [Figure 11] This figure shows the affinity results of SC-TM and seven mutated F protein truncations with two antibodies against F protein detected by Biacore, the thermal stability results of the proteins detected by DSF, and the thermal stability results of the epitopes detected by ELISA. [Figure 12] 12 shows antibody titers in the serum of mice immunized with SC-TM and seven mutated F protein truncations, where panel A in FIG. 12 shows antibody titers binding to pre-F, and panel B in FIG. 12 shows antibody titers binding to post-F. DETAILED DESCRIPTION OF THE INVENTION

[0111] Sequence information Some sequence information relevant to the present invention is shown in Table 1 below.

[0112] [Table 1-1]

[0113] [Table 1-2]

[0114] [Table 1-3]

[0115] [Table 1-4]

[0116] [Table 1-5]

[0117] [Table 1-6]

[0118] [Table 1-7]

[0119] [Table 1-8]

[0120] [Table 1-9]

[0121] [Table 1-10]

[0122] Specific Models for Implementing the Invention The invention will now be described with reference to the following examples, which are intended to illustrate (but not limit) the invention.

[0123] Unless otherwise indicated, the experiments and methods described in the examples were essentially performed according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention may be obtained from sources such as "MOLECULAR CLONING: A LABORATORY MANUAL," 2nd Edition, by Sambrook, Fritsch, and Maniatis (1989); "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY" (F.M. Ausubel et al., eds., (1987)); "METHODS IN ENZYMOLOGY series" (Academic Publishing Company); "PCR 2: A PRACTICAL APPROACH" (M.J. MacPherson, B.D. Hames, and G.R. Taylor, eds., (1995)); and "ANIMAL CELL CULTURE" (R.I. Freshney (ed.) (1987).

[0124] Furthermore, unless specific conditions are specified in the examples, these were carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment used without a manufacturer designation were commercially available conventional products. Those skilled in the art will appreciate that the examples are provided to illustrate the present invention and are not intended to limit the scope of protection claimed in the present invention. All disclosures and other references cited herein are hereby incorporated by reference in their entirety. [Example]

[0125] Example 1. Screening experiment for flexible linker peptides of F protein truncations During the conformational change of the RSV F protein, the release of the fusion peptide within the hydrophobic cavity is a crucial triggering event. To help stabilize the fusion peptide within the hydrophobic cavity and the pre-F conformation, flexible linker peptides of various lengths were introduced into the C-terminus of F2 (SEQ ID NO: 12) and the N-terminus of F1 (SEQ ID NO: 11). A total of six flexible linker peptides were designed, and their amino acid sequences are shown in SEQ ID NOs: 23, 2, 24, 25, 26, and 27, respectively. The nucleotide sequences of the F protein containing the flexible linker peptides were submitted to a company (Shanghai Shenggong Biotechnology Co., Ltd.) for humanization codon optimization, and HindIII and xBaI restriction sites were introduced at the 5' and 3' ends, respectively, before being inserted into the pcDNA3.1 eukaryotic expression vector. Recombinant F protein plasmids containing the flexible linker peptides were constructed and designated LA, LB, LC, LD, LE, and LF, respectively. Immunofluorescence was used to detect the expression of highly neutralizing antibody epitopes, site II and site φ, of the F protein containing the flexible linker peptide.

[0126] 1) On the first night, 293T cells were plated in a 96-well plate at 3 x 10 cells per well. 4 were inoculated individually. 2) The next day, when cell confluence reached 85%, plasmid transfection was performed. 5 μL of opti-MEM culture medium was taken and added to a 96-well U-bottom plate. 0.2 μg of plasmid and 0.3 μL of P3000 were added, vortexed to mix, and marked as A. Another 5 μL of opti-MEM culture medium was taken and added to a 96-well U-bottom plate. 0.3 μL of Lipofectamine 3000 was added, vortexed to mix, and marked as B. The mixture in tube A was added to tube B, vortexed to mix, and left for 15 minutes. The solution was then added to the 293T cell-coated plate, gently shaken, and placed in a cell culture incubator for incubation. 3) 24 hours after transfection, 100 μL of 4% formaldehyde solution was added to each well to fix the cells at room temperature for 30 minutes. 4) After discarding the cell culture medium and formaldehyde solution, the cells were washed three times with PBS for 5 minutes each time. 5) 100 μL of PBS containing 2% non-fat dry milk was added to each well, and blocking was carried out at room temperature for 2 hours. 6) After washing once with PBS, antibodies capable of recognizing different epitopes (site II: motavizumab, site φ: AM22, D25) were added and incubated at room temperature for 1 hour. 7) Washing was performed three times with PBS. 8) 100 μL of Alexa Fluor 568 (1:2000 dilution) (manufacturer: Thermo, catalog number: A-11013) fluorescent secondary antibody was added to each well and incubated at room temperature for 1 hour. 9) Washing was performed three times with PBS. 10) 100 μL of the nuclear dye DAPI (manufacturer: Invitrogen, catalog number: D1306) was added to each well, and staining was carried out for 10 minutes. 11) Washing was performed three times with PBS. 12) Photographs were taken using a high-content imaging system and quantitative analysis was performed.

[0127] Figure 1 shows the expression of the highly neutralizing antibody epitopes, site II and site φ, after transfection of 293t cells with F proteins containing various linker peptides. The results showed that F proteins containing the flexible linker peptides LA and LC retained the highest expression of site φ.

[0128] Example 2. Design of F protein truncations The amino acid sequence of the RSV A2 fusion protein was obtained from the GenBank database (GenBank ID: FJ614814.1) and wtThe amino acid sequence of is shown in SEQ ID NO: 1. The amino acid sequences of the F protein truncations were characterized in that the N- and C-termini of the F2 and F1 peptide chains were shortened to obtain truncated F2 and truncated F1, and truncated F2 was linked to truncated F1 via a flexible linker peptide containing proline to help stabilize the conformation of pre-F. The C-terminus of truncated F1 was linked to various presentation motifs via a flexible linker peptide. A total of 10 F protein truncations were constructed as follows: (1) The amino acids at positions 31 to 105 of SEQ ID NO: 1 were linked to the amino acids at positions 145 to 322 of SEQ ID NO: 2 via a flexible linker peptide containing proline. The F protein truncated form had a total length of 314 amino acids and was designated LC1, and its amino acid sequence is shown in SEQ ID NO: 3. LC1 retained epitopes at sites φ, V, III, and II. (2) The amino acids at positions 51 to 105 of SEQ ID NO: 1 were linked to the amino acids at positions 145 to 306 via a flexible linker peptide (SEQ ID NO: 2) containing proline. The F protein truncated form had a total length of 284 amino acids and was designated LC2, whose amino acid sequence is shown in SEQ ID NO: 4. LC2 retained the epitopes at sites φ, V, and II. (3) The amino acids at positions 56 to 105 of SEQ ID NO: 1 were linked to the amino acids at positions 145 to 306 via a flexible linker peptide (SEQ ID NO: 2) containing proline. The F protein truncated form had a total length of 280 amino acids and was designated LC3, whose amino acid sequence is shown in SEQ ID NO: 5. LC3 retained the epitopes at sites φ and II. (4) The amino acids at positions 51 to 105 of SEQ ID NO: 1 were linked to the amino acids at positions 145 to 297 via a flexible linker peptide (SEQ ID NO: 2) containing proline. The F protein truncated form had a total length of 276 amino acids and was designated LC4, whose amino acid sequence is shown in SEQ ID NO: 6. LC4 retained the epitopes at sites φ and II. (5) The amino acids at positions 56 to 105 of SEQ ID NO: 1 were linked to the amino acids at positions 145 to 297 via a flexible linker peptide (SEQ ID NO: 2) containing proline. The F protein truncated form had a total length of 271 amino acids and was designated LC5, whose amino acid sequence is shown in SEQ ID NO: 7. LC5 retained the epitope at site II.

[0129] Example 3. Construction of recombinant plasmids expressing truncated F protein The nucleotide sequences encoding the five F protein truncations were individually linked to the nucleotide sequence encoding the F protein cytoplasmic segment (amino acid sequence shown in SEQ ID NO: 8) via a nucleotide sequence encoding a flexible linker peptide. A nucleotide sequence encoding a signal peptide (amino acid sequence shown in SEQ ID NO: 9) was inserted at the 5' end of each of the nucleotide sequences of the truncations. The nucleotide sequences of the F protein truncations were submitted to a company (Shanghai Shenggong Biotechnology Co., Ltd.) for humanization codon optimization, and HindIII and xBaI restriction sites were introduced at the 5' and 3' ends, respectively. The truncated sequences were then inserted into the pcDNA3.1 eukaryotic expression vector to construct recombinant plasmids containing the target genes of the F protein truncations. Figure 2 shows a schematic diagram of the F protein truncation LC2. Figure 3 shows a schematic diagram of the structure of the F protein truncations predicted by AlphaFold2. Additionally, as a control, a full-length F protein SC-TM recombinant plasmid with a pre-F structure was constructed according to the above method (the sequence of the SC-TM protein was obtained from GenBank: 5C6B_F). It was synthesized by a company (Shanghai Shenggong Biotechnology Co., Ltd.). Plasmids that were successfully sequenced were subjected to large-scale plasmid extraction and then single-digested with the restriction enzyme XbaI. The results of the single-digestion were identified by capillary electrophoresis. The identified plasmids were detected by spectrophotometer at OD260 and OD280 to determine their DNA concentration and purity. The plasmids were stored at -20°C.

[0130] Example 4: Detection of recombinant plasmid expression by Western blot 1) On the first night, seed 293T cells into a 6-well plate at 8 x 10 cells per well. 5 were inoculated individually. 2) On day 2, when cell confluence reached 85%, plasmid transfection was performed. 125 μL of opti-MEM (manufacturer: Gibco, catalog number: 31985-070) culture medium was collected and added to a 1.5 mL centrifuge tube. 2 μg of plasmid and 4 μL of P3000 were added, vortexed to mix, and marked as A. Another 125 μL of opti-MEM culture medium was collected and added to another 1.5 mL centrifuge tube. 4 μL of Lipofectamine 3000 (manufacturer: Invitrogen, catalog number: L3000015) was added, vortexed to mix, and marked as B. The mixture in tube A was added to tube B, vortexed to mix, and left for 15 minutes. The solution was then added to a 6-well plate containing 293T cells, gently shaken, and placed in a cell culture incubator for incubation. 3) On day 3, the culture medium was discarded and the cells were washed twice with pre-chilled PBS. 4) 150 μL of RIPA lysis buffer containing protease inhibitors was added, and the cells were lysed on ice for 30 minutes. 5) Centrifugation was carried out at 12000 rpm and 4°C for 20 minutes, and the supernatant was collected. 6) The protein concentration of the supernatant was determined using a BCA quantitative reagent (manufacturer: PIERCE, catalog number: 23225). 7) The amount of sample loaded for electrophoresis was 20 μg per well, and β-mercaptoethanol and loading buffer were added to the sample, followed by heat denaturation at 100° C. for 10 minutes. 8) A commercially available 12% precast gel was added, and electrophoresis was carried out at a voltage of 180 V for 40 minutes. 9) After electrophoresis, proteins were transferred to an NC membrane using a wet transfer method. 10) Blocking was performed for 2 hours with a PBS solution containing 5% non-fat dry milk. 11) Washing was performed once with PBST. 12) The human monoclonal antibody motavizumab was diluted 1:2000 with 2% non-fat dry milk, and the NC membrane was transferred to the diluted antibody and incubated on a shaker at room temperature for 2 hours. 13) Washing was performed three times with PBST for 5 minutes each time. 14) GAH-HRP was diluted with PBS containing 2% non-fat dry milk, and the NC membrane was transferred to the diluted secondary antibody and incubated on a shaker at room temperature for 1 hour. 15) Washing was performed three times with PBST for 5 minutes each time. 16) A color-developing substrate was added, a photograph was taken, and detection was performed using a WB imaging system.

[0131] As shown in Figure 4, the expression of F protein truncations on 293t cells was analyzed by Western blot. The detection antibody was motavizumab. The leftmost channel in this figure was a 180 kD protein marker. SC-TM was a control pre-F protein, and LC2 was the F protein truncation described in this invention.

[0132] Example 5: Immunofluorescence detection of epitope integrity of F protein truncations 1) On the first night, 293T cells were plated in a 96-well plate at 3 x 10 cells per well. 4 were inoculated individually. 2) The next day, when cell confluence reached 85%, plasmid transfection was performed. 5 μL of opti-MEM culture medium was taken and added to a 96-well U-bottom plate. 0.2 μg of plasmid and 0.3 μL of P3000 were added, vortexed to mix, and marked as A. Another 5 μL of opti-MEM culture medium was taken and added to a 96-well U-bottom plate. 0.3 μL of Lipofectamine 3000 was added, vortexed to mix, and marked as B. The mixture in tube A was added to tube B, vortexed to mix, and left for 15 minutes. The solution was then added to the plate containing 293T cells, gently shaken, and placed in a cell culture incubator for incubation. 3) 24 hours after transfection, 100 μL of 4% formaldehyde solution was added to each well to fix the cells for 30 minutes at room temperature. 4) The cell culture medium and formaldehyde solution were discarded, and the cells were washed three times with PBS for 5 minutes each time. 5) 100 μL of PBS containing 2% non-fat dry milk was added to each well, and blocking was carried out at room temperature for 2 hours. 6) After washing once with PBS, antibodies capable of recognizing different epitopes (site II: motavizumab, site φ: AM22, D25) were added and incubated at room temperature for 1 hour. 7) Washing was performed three times with PBS. 8) 100 μL of Alexa Fluor 568 (1:2000 dilution) (manufacturer: Thermo, catalog number: A-11013) fluorescent secondary antibody was added to each well and incubated at room temperature for 1 hour. 9) Washing was performed three times with PBS. 10) 100 μL of the nuclear dye DAPI (manufacturer: Invitrogen, catalog number: D1306) was added to each well, and staining was carried out for 10 minutes. 11) Washing was performed three times with PBS. 12) Photographs were taken using a high-content imaging system and quantitative analysis was performed.

[0133] As shown in Figure 5, immunofluorescence demonstrated the expression of site II and site φ, highly neutralizing antibody epitopes, of the full-length pre-F protein SC-TM and the F protein truncated LC2 after transfection into 293t cells.

[0134] Example 6. Construction of mRNA vaccine of truncated F protein RNA transcription-related elements were constructed in an in vitro transcription plasmid containing the F protein truncated LC2 and full-length pre-F protein SC-TM according to the method described in the publication by Andrew J. Bett et al. (PMID: 32128257), and the plasmid was extracted and identified according to the method described in Example 1. After large-scale plasmid extraction, linearization was performed using the restriction endonuclease BspQ1. Transcription was performed using a T7 in vitro transcription kit (manufacturer: Nanjing Nuoweizan Biotechnology Co., Ltd., catalog number: DD4202) to obtain capped mRNA. The transcription template was digested with DNase I. The mRNA was purified by precipitation with lithium chloride (manufacturer: Invitrogen, catalog number: AM9480).

[0135] The purified mRNA was dissolved in an acidic sodium citrate buffer solution for later use. Preparation of lipid nanoparticles (LNP): Cationic lipid: DSPC: cholesterol: DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5 was dissolved in ethanol and mixed. The mixed solution of mRNA and lipid was encapsulated at a flow ratio of 3:1. After encapsulation, it was dialyzed into a PBS solution to obtain the mRNA vaccine.

[0136] Example 7. Immunization with F protein truncated mRNA vaccine Animal feeding and experimental procedures were carried out in accordance with the procedures established by the Experimental Animal Use and Management Committee of Xiamen University. Experimental animals were 6- to 8-week-old BALB / c female mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and housed at the Xiamen University Experimental Animal Center.

[0137] 1) Purchased mice were randomly divided into groups of four. 5 μg of mRNA-LNP was injected into the quadriceps femoris muscle of each mouse. The control group was injected with PBS containing no mRNA-LNP. 2) A second booster immunization was administered two weeks after the first immunization, for a total of two immunizations. 3) 14 days after the booster immunization, blood was collected from the orbit and serum was separated and analyzed.

[0138] Example 8. Serum antibody titer and typing detection 1) The pre-F and post-F proteins for coating were constructed as follows: The sequences of the pre-F and post-F proteins were downloaded from GenBank (GenBank ID: LY628284.1, GenBank ID: 6APB_A), and the nucleotide sequences encoding the two proteins were cloned into the pcDNA3.1 eukaryotic expression vector, expressed using the CHO eukaryotic expression system, and purified by affinity chromatography to obtain the pre-F and post-F proteins. 2) On the first night, pre-F protein (SC-TM) and post-F protein were diluted with PBS and coated onto a 96-well plate at an input of 100 ng per well overnight at 4°C. 3) The protein coating solution was discarded and the plate was washed once with PBST. 4) 200 μL of 5% fetal bovine serum blocking solution was added per well, and the plate was incubated at 37° C. for 2 hours. 5) The blocking solution was discarded and the plate was washed once with PBST. 6) Gradient diluted mouse serum (200-fold in the first well, 5-fold gradient) was added at 100 μL per well and incubated at 37°C for 1 hour. 7) The diluted serum solution was discarded, and the plate was washed five times with PBST. 8) Horseradish peroxidase-labeled goat anti-mouse IgG antibody, IgG1 antibody, and IgG2a antibody (manufacturer: Abcam, catalog number: ab97265) diluted at 1:5000 were added at 100 μL per well, and the plate was incubated at 37°C for 1 hour. 9) Discard the secondary antibody and wash five times with PBST. 10) 100 μL of color-developing solution was added per well, and color development was carried out at room temperature in the dark for 10 minutes. 11) Stop solution was added at 50 μL per well, and the absorbance at 450 nm was detected using an ELISA reader. 12) The criteria for endpoint titer was 3 times the reading of a negative well.

[0139] The experimental results are shown in Figure 6. Panel A in Figure 6 showed that the F protein truncated LC2 of the present application induced a high level of pre-F binding antibody titer, comparable to that of SC-TM (i.e., full-length pre-F protein). Panel B in Figure 6 showed that immunization in the form of an mRNA vaccine induced a higher level of IgG2a antibody, indicating Th-1 bias.

[0140] Example 9. Detection of neutralizing antibody titers 1) Serum isolated after orbital bleeding was incubated at 56°C for 30 minutes to inactivate complement. 2) Gradient dilution of serum: 10-fold dilution in the first well, followed by 4-fold gradient dilutions over a total of 10 gradients. 3) An equal volume of rRSV-mkatushka2 (MOI = 0.1) was added, mixed, and incubated at 37°C for 1 hour. 4) The serum and virus mixture was transferred to a 96-well plate coated with a monolayer of HeLa cells at a volume of 100 μL per well and incubated at 37°C. 5) After 24 hours of incubation, the plate was read using a SpectraMax Paradigm multimode microplate reader.

[0141] The experimental results are shown in Figure 7. The experimental results showed that compared to SC-TM (i.e., the full-length pre-F protein), the F protein truncated LC2 of the present application induced a neutralizing antibody titer significantly higher than that of SC-TM.

[0142] In summary, compared with the full-length F protein, the F protein truncations of the present invention induced higher levels of pre-F-specific antibodies and significantly higher neutralizing antibody titers, exhibited good protection and safety, and were suitable for various vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and granulated vaccines.

[0143] Example 10. Design of mutated F protein truncations In this example, the F protein truncated LC2 (SEQ ID NO: 4) obtained in the above example was used as a basis and further mutated to generate a total of seven mutants, which are specifically described below: (1) In SEQ ID NO: 4, the amino acid I at position 29 was mutated to L, and the amino acid V at position 212 was mutated to M. The mutated F protein truncation was named LC2A, and its amino acid sequence is shown in SEQ ID NO: 28. (2) In SEQ ID NO: 4, the amino acid I at position 29 was mutated to L, and the amino acid A at position 214 was mutated to L. The mutated F protein truncation was named LC2B, and its amino acid sequence is shown in SEQ ID NO: 29. (3) In SEQ ID NO: 4, the amino acid I at position 29 was mutated to L, and the amino acid V at position 94 was mutated to I. The mutated F protein truncation was named LC2C, and its amino acid sequence is shown in SEQ ID NO: 30. (4) In SEQ ID NO: 4, the amino acid M at position 47 was mutated to A, and the amino acid Y at position 114 was mutated to I. The mutated F protein truncation was named LC2D, and its amino acid sequence is shown in SEQ ID NO: 31. (5) In SEQ ID NO: 4, the amino acid I at position 29 was mutated to L, the amino acid M at position 47 was mutated to A, the amino acid V at position 101 was mutated to I, and the amino acid A at position 214 was mutated to L. The mutated F protein truncation was named LC2E, and its amino acid sequence is shown in SEQ ID NO: 32. (6) In SEQ ID NO: 4, the amino acid L at position 88 was mutated to A, the amino acid V at position 101 was mutated to I, the amino acid Y at position 114 was mutated to I, and the amino acid A at position 214 was mutated to L. The mutated F protein truncation was named LC2F, and its amino acid sequence is shown in SEQ ID NO: 33. (7) In SEQ ID NO: 4, the amino acid V at position 94 was mutated to I, and the amino acid Y at position 114 was mutated to I. The mutated F protein truncation was named LC2G, and its amino acid sequence is shown in SEQ ID NO: 34.

[0144] Example 11. Construction of recombinant plasmids expressing truncated F protein The nucleotide sequences encoding the seven mutated F protein truncations were individually linked to a nucleotide sequence encoding the trimerization motif, Foldon (the amino acid sequence of which is shown in SEQ ID NO: 21), via a nucleotide sequence encoding GSGS amino acids. A nucleotide sequence encoding a signal peptide (SEQ ID NO: 9) was inserted at the 5' end of the nucleotide sequences encoding the seven mutated F protein truncations to construct fusion expression genes. The nucleotide sequences of the mutated F protein truncations were submitted to Shanghai Shenggong Biotechnology Co., Ltd. for humanization codon optimization, and HindIII and xBaI restriction sites were introduced at the 5' and 3' ends, respectively. The nucleotide sequences were then inserted into the pcDNA3.1 eukaryotic expression vector to construct recombinant plasmids containing the target genes of the F protein truncations. Furthermore, as a control, a full-length F protein SC-TM recombinant plasmid with a pre-fusion structure was constructed according to the above method (the sequence of the SC-TM protein was obtained from GenBank: 5C6B_F, SEQ ID NO: 35). This was synthesized by Shanghai Shenggong. Figure 8 shows the wild-type F protein F wt Schematic connection diagrams of the control F protein SC-TM and the mutated F protein truncated LC2A are shown.

[0145] Plasmid powder containing the target gene was dissolved in 10 μL of double-distilled water, and 1 μL of the solution was added to 100 μL of DH5α competent Escherichia coli cells. The sample was incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, and then incubated on ice for 2 minutes. The resulting Escherichia coli cells were then added to LB medium and plated onto ampicillin-resistant agarose plates. After colony growth, single clones were picked, and plasmids were extracted using an endotoxin-free plasmid extraction kit (manufacturer: Tiangen, catalog number: DP117) for later use.

[0146] Example 12. Protein expression and purification ExpiCHO™ suspension cells in logarithmic growth phase were prepared, with 6 x 10 cells per mL. 6 The cells were cultured on a cell shaker at 125 rpm, 37°C, and 8% CO2 until the cells reached a density of 100 cells and a viability of over 98%. 25 mL of cells were harvested and placed into a new cell culture flask as the transfection system. Tube A: 1 mL of ExpiCHO™ Expression medium containing 25 μg of plasmid; Tube B: 1 mL of ExpiCHO™ Expression medium containing 80 μL of transfection reagent from the ExpiFectamine™ CHO Transfection Kit (manufacturer: Thermo Scientific, catalog number: A29129). The medium from Tube A was mixed with the medium from Tube B and left at room temperature for 2 minutes. The mixture was then poured into the 25 mL of prepared transfection cell system. The cells were cultured on a cell shaker at 125 rpm, 37°C, and 8% CO2 for 18 to 22 hours. 150 μL of enhancer and 4 mL of additive from the ExpiFectamine™ CHO transfection kit were added to each flask and cultured on a cell shaker at 125 rpm, 32°C, and 5% CO for 8 to 15 days. After the culture was completed, the cells were centrifuged at 4°C, 4000 rpm for 10 minutes, and the cell supernatant was collected.

[0147] The supernatant was filtered using a 0.22 μm filter. The AKTA instrument was turned on, and flow paths A and B were first rinsed with solution A (200 mM sodium hydrogen phosphate dodecahydrate) and solution B (100 mM citric acid monohydrate), followed by the installation of the Protein A column. The Protein A column was equilibrated with solution A at a flow rate of 8 mL / min for over 15 minutes. The UV value, pH value, and conductivity detected by the instrument were stabilized before proceeding to the next step. When the sample was loaded at a flow rate of 6 mL / min to 10 mL / min, the UV value increased. This peak was the breakthrough peak, and the column was continuously washed with solution A. A sample of the breakthrough peak was collected for detection. When the pH value no longer changed, solution B was injected at a flow rate of 6 mL / min to 10 mL / min. The pH then decreased and the UV value increased. This peak was the elution peak, and the antibody was primarily present in the elution peak. A sample of the elution peak was collected for detection. The column was equilibrated with solution A, and then the tubing and Protein A column were loaded with 20% ethanol. The column was then removed and stored at 4°C. Samples of the breakthrough peak and elution peak were purified and subjected to identification by SDS-PAGE (the samples were boiled in water for 5 minutes to open the disulfide bond between the heavy and light chains of the antibody (see "Molecular Cloning: A Laboratory Manual," 3rd Edition)). The purified monoclonal antibody was dialyzed overnight against 20 mM PBS buffer, its concentration was determined by ultraviolet spectroscopy or BCA, and then it was dispensed into 1.5 ml tubes and stored at -20°C for later use.

[0148] Example 13. Polyacrylamide gel electrophoresis On the first night, 293T cells were plated in a 6-well plate at 8 x 10 cells per well. 5The cells were inoculated at 1000 x 1000 cells / well. On day 2, when the cells reached 85% confluence, plasmid transfection was performed. 125 μL of opti-MEM (manufacturer: Gibco, catalog number 31985-070) medium was collected and added to a 1.5 mL centrifuge tube. 2 μg of plasmid and 4 μL of P3000 were added, vortexed to mix, and marked as A. Another 125 μL of opti-MEM medium was collected and added to another 1.5 mL centrifuge tube. 4 μL of Lipofectamine 3000 (manufacturer: Invitrogen, catalog number: L3000015) was added, vortexed to mix, and marked as B. The mixture in tube A was added to tube B, vortexed to mix, and left for 15 minutes. The solution was then added to a 6-well plate containing 293T cells, gently shaken, and placed in a cell culture incubator for incubation. On day 3, the culture medium was discarded and the cells were washed twice by adding pre-chilled PBS. 150 μL of RIPA lysis buffer containing protease inhibitors was added, and the cells were lysed on ice for 30 minutes. After centrifugation at 12,000 rpm and 4°C for 20 minutes, the supernatant was collected. The protein concentration of the supernatant was determined using a BCA quantification reagent (manufacturer: PIERCE, catalog number: 23225). The sample was loaded at 20 μg per well for electrophoresis. β-mercaptoethanol and loading buffer were added to the sample, and heat denaturation was performed at 100°C for 10 minutes. A commercially available 12% precast gel was added, and electrophoresis was performed at 180 V for 40 minutes. After electrophoresis, the SDS-PAGE gel was stained overnight with 0.25% Coomassie Brilliant Blue (Sigma). It was then destained using destaining buffer (300 ml of methanol, 100 ml of acetic acid, and 600 ml of double-distilled water).

[0149] Analysis of the results: The molecular weight of the control protein SC-TM was approximately 60 kDa, while the molecular weight of the mutated F protein truncation was smaller at approximately 30 kDa. Figure 9 shows the results of polyacrylamide gel electrophoresis of SC-TM and LC2A.

[0150] Example 14. Analysis of epitope characteristics of mutated F protein truncations SC-TM and the seven mutants were diluted with PBS buffer to prepare a coating solution with a final concentration of 1 μg / mL. 100 μL of the diluted coating solution was added to a 96-well ELISA plate and coated overnight at 4°C. The plate was washed once with PBST washing solution (20 mM PB(7.4), 150 mM NaCl, 0.1% Tween 20) and spun dry. 200 μL of blocking solution (containing 20% ​​bovine calf serum, 1% casein, and 20 mM NaHPO / NaHPO buffer solution (pH 7.4)) was then added to each well. Blocking was performed at 37°C for 2 hours, after which the blocking solution was discarded. After drying, the plate was packed in an aluminum foil bag and stored at 4°C for later use. F protein-specific monoclonal antibodies MOTA, D25, and hRSV90, which recognize different epitopes (the VH and VL sequences of the three antibodies are shown in SEQ ID NOs: 36 to 41 in Table 1, respectively, where the three antibodies have the same heavy chain constant region with the specific sequence shown in SEQ ID NO: 42, and the three antibodies have the same light chain constant region with the specific sequence shown in SEQ ID NO: 43), were selected and each was subjected to a 5-fold gradient dilution with PBS solution over a total of eight gradients, starting from an initial concentration of 100 μg / ml. The coated ELISA plate was then loaded with 100 μl of diluted antibody sample per well and incubated in a 37°C incubator for 60 minutes. The ELISA plate was washed five times with PBST washing solution (20 mM PB(7.4), 150 mM NaCl, 0.1% Tween 20), and 100 μl of horseradish peroxidase (HRP)-labeled goat anti-human IgG reaction solution was added per well. The plate was then incubated at 37°C for 30 minutes. After the enzyme-labeling reaction was completed, the ELISA plate was washed five times with PBST washing solution (20 mM PB(7.4), 150 mM NaCl, 0.1% Tween 20). 50 μl of TMB colorimeter (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) was added per well. The plate was then incubated at 37°C for 15 minutes.After the color reaction process was completed, 50 μl of stop solution (purchased from Beijing Wantai Biological Pharmaceutical Co., Ltd.) was added to each well of the ELISA plate, and the OD450 / 630 value of each well was detected by a microplate reader.

[0151] Results analysis: The results showed that the binding ability of some mutated F protein truncations to the test antibodies was better than that of SC-TM, and that the binding ability of LC2A, LC2B, and LC2E, in particular, to the highly neutralizing antibody D25 and hRSV90 was significantly improved (Figure 10).

[0152] Example 15. Analysis of the affinity of mutated F protein truncations with antibodies The binding affinity of the mutated F protein truncations to the F protein-specific antibody D25 and hRSV90 was determined by surface plasmon resonance (SPR) analysis. Histidine-tagged mutated F protein truncations were immobilized at approximately 100 nM on an NTA sensor chip (Cytiva) using a Biacore-8K (Cytiva). Serially diluted antibodies (with concentrations ranging from 200 nM to 0.78 nM) were added. The RU data were fitted to a 1:1 binding model using Biacore™ Insight software.

[0153] Results analysis: The results showed that the affinity of some mutated F protein truncations with the test antibody was comparable to that of SC-TM, with the affinity of LC2A with D25 being 3.85-fold higher than that of SC-TM, the affinity of LC2B with D25 being 1.25-fold higher than that of SC-TM, and the affinity of LC2B with hRSV90 being 3.85-fold higher than that of SC-TM (Figure 11).

[0154] Example 16. Analysis of thermostability of mutated F protein truncations (1) Differential scanning fluorimetry (DSF) was used to analyze the thermal stability of SC-TM and seven mutated F protein truncations. The test protein samples were first diluted to a concentration of 1 mg / mL in sterile PBS solution, then subjected to a two-fold gradient dilution across a total of eight gradients to determine the appropriate sample concentration. Blank PBS was used as a control. SYPRO Orange dye (manufacturer: SIGMA-ALDRICH, catalog number: S5692-500UL) was diluted with deionized water to a final concentration of 50x. 45 μL of the diluted test protein sample was taken and added to a Bio-Rad PCR plate, followed by the addition of 5 μL of 50x SYPRO Orange dye. The system was then placed in a fluorescent quantitative PCR instrument (BIO-RAD). The excitation and emission wavelengths were adjusted to the corresponding wavelengths of the fluorescent dye, the heating rate was set to 0.5°C / 10 s, and the temperature range was 0°C to 100°C. The data were processed using BIO-RAD manager, and the fluorescence intensity versus temperature curves were plotted and fitted to the Boltzmann transport equation (BTE) to calculate the Tm values ​​of the protein samples.

[0155] Results analysis: The results showed that there was no significant difference in the thermal stability of the seven mutated F protein truncations compared with the control protein preF, and among them, the Tm value of LC2A was slightly higher than that of SC-TM (58.4°C vs. 59.3°C) (Figure 11).

[0156] (2) The epitope thermal stability of SC-TM and seven mutated F protein truncations was analyzed by enzyme-linked immunosorbent assay (ELISA). The test proteins were diluted to 10 μg / mL using PBS solution and incubated at 25°C, 50°C, 70°C, and 90°C for 1 hour, respectively, and then immediately cooled on ice. The coating and detection processes were the same as those described in Example 4.

[0157] Results analysis: The results showed that the binding of the control protein SC-TM and the seven mutated F protein truncations to D25 could still be detected at 70°C and 90°C, and the binding of the seven mutants was better than that of SC-TM (Figure 11).

[0158] Example 17. Analysis of immunogenicity of mutated F protein truncations (1) Immunization experiments in mice Animal feeding and experimental procedures were carried out in accordance with the procedures established by the Experimental Animal Use and Management Committee of Xiamen University. Experimental animals were 6- to 8-week-old BALB / c female mice purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and housed at the Xiamen University Experimental Animal Center. The immunization scheme consisted of immunizing mice with SC-TM and the seven mutated F protein truncations individually mixed with aluminum adjuvant at a 1:1 volume ratio. Each group consisted of five mice, immunized via unilateral thigh muscle injection, and the immunization dose was 20 μg of protein per mouse. Immunization was performed with two injections, and orbital blood was collected from the mice on day 7 after the booster immunization. Serum was separated, and serum samples were inactivated at 56°C for 30 minutes and stored at 4°C for later use.

[0159] (2) Evaluation of serum pre-F protein and post-F protein specific antibody titers The pre-F protein used for coating was SC-TM, and the sequence of the post-F protein was downloaded from GenBank (GenBank ID: 6APB_A, SEQ ID NO: 44). The nucleotide sequence encoding the post-F protein was cloned into the pcDNA3.1 eukaryotic expression vector and expressed using the ExpiCHO™ system. The expression process was the same as that described in Example 3. The protein coating and blocking processes were the same as those described in Example 4. The serum was subjected to a 5-fold gradient dilution starting from 200-fold in the first well over a total of 8 gradients. The diluted serum was added to a blocked 96-well plate at a volume of 100 μL per well and incubated at 37°C for 1 hour. The serum was discarded, and the plate was washed five times with PBST and spun dry. A horseradish peroxidase-labeled goat anti-mouse IgG antibody (manufacturer: Abcam, catalog number: ab97265) diluted 1:5000 was added at a volume of 100 μL per well and incubated at 37°C for 1 hour. The secondary antibody was discarded, and the plate was washed five times with PBST. A color development solution was added at a volume of 100 μL per well, and the color was developed at room temperature in the dark for 10 minutes. A stop solution was added at a volume of 50 μL per well, and the absorbance at 450 nm was detected using a microplate reader. The criterion for endpoint titer was three times the reading of the negative well.

[0160] Results analysis: The results showed that all seven mutated F protein truncations were able to induce antibody responses biased toward pre-F, and that the pre-F binding antibody titers induced by LC2A, LC2B, LC2C, LC2D, and LC2F were comparable to those of the full-length pre-F protein SC-TM (Figure 12).

[0161] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various changes and modifications can be made to the details based on all the teachings disclosed, and these modifications fall within the scope of protection of the present invention. The entirety of the present invention is defined by the appended claims and any equivalents thereof.

Claims

1. A fusion protein comprising a first truncated form, a second truncated form, and a linker connecting the first truncated form and the second truncated form, Compared to the F2 protein of a wild respiratory syncytial virus (RSV), the first truncated form is truncated by 5 to 25 amino acids (e.g., 5 to 8, 9 to 12, 13 to 16, 17 to 20, 21 to 25) at the N-terminus and 3, 4, or 5 amino acids at the C-terminus of the F2 protein of the wild RSV; The second truncated form is a fusion protein in which, compared to the wild-type RSV F1 protein, the wild-type RSV F1 protein is truncated by 7, 8, or 9 amino acids at the N-terminus and 238 to 268 amino acids (e.g., 238 to 241, 242 to 245, 246 to 249, 250 to 253, 254 to 257, 258 to 261, 262 to 265, 266 to 268) at the C-terminus.

2. Compared to the wild-type RSV F2 protein, the first truncation is truncated by 5 or 25 amino acids at the N-terminus and 4 amino acids at the C-terminus of the wild-type RSV F2 protein; Preferably, the wild RSV F2 protein has the amino acid sequence set forth in SEQ ID NO: 13, The fusion protein of claim 1 , wherein the first truncated form preferably has the amino acid sequence shown in SEQ ID NO: 16 or SEQ ID NO:

17.

3. Compared to the F1 protein of wild-type RSV, the second truncation is truncated by 8 or 9 amino acids at the N-terminus and 252 or 268 amino acids at the C-terminus of the F1 protein of wild-type RSV; Preferably, the wild RSV F1 protein has the amino acid sequence set forth in SEQ ID NO: 12, 3. The fusion protein of claim 1 or 2, wherein the second truncated form preferably has the amino acid sequence shown in SEQ ID NO: 14 or SEQ ID NO:

15.

4. the linker comprises at least one (e.g., one or two) proline; Preferably, the linker has 3 to 20 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20) amino acids, more preferably, the linker has 5 to 8 amino acids; Preferably, the linker comprises a plurality of (e.g., 2, 3, 4, 5, 6, 7) glycines and at least one (e.g., 1, 2, 3) proline; Preferably, the linker has the sequence shown in SEQ ID NO: 2, Preferably, the first truncation is located at the N-terminus of the linker; Preferably, the second truncation is located at the C-terminus of the linker; Preferably, the fusion protein comprises, from N-terminus to C-terminus, the first truncation, the linker, and the second truncation; The fusion protein according to any one of claims 1 to 3, wherein the fusion protein preferably has the amino acid sequence shown in SEQ ID NO:3 or SEQ ID NO:

4.

5. the fusion protein further comprises one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions); Preferably, the first truncated form and / or the second truncated form of the fusion protein comprises one or more amino acid substitutions, deletions or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions or additions), Preferably, the sequence of the fusion protein has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to the sequence set forth in any one of SEQ ID NOs: 3 to 7; The fusion protein according to any one of claims 1 to 4, wherein the substitution is preferably a conservative substitution.

6. the sequence of the fusion protein has one or more amino acid substitutions, deletions, or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, or additions) compared to the sequence set forth in SEQ ID NO:4; Preferably, compared to the sequence shown in SEQ ID NO: 4, the sequence of the fusion protein has an amino acid substitution at a position corresponding to position 29 of SEQ ID NO: 4, and the sequence of the fusion protein also has an amino acid substitution at any one of positions corresponding to positions 47 to 214 of SEQ ID NO: 4; Preferably, compared to the sequence shown in SEQ ID NO: 4, the sequence of the fusion protein has an amino acid substitution at a position corresponding to position 114 of SEQ ID NO: 4, and the sequence of the fusion protein also has an amino acid substitution at any one of positions corresponding to positions 29 to 94 of SEQ ID NO: 4; Preferably, the sequence of the fusion protein, compared to the sequence shown in SEQ ID NO: 4, has one or more amino acid substitutions at positions corresponding to positions 29, 47, 212, 214, 88, 94, 114, and 101 of SEQ ID NO: 4; Preferably, the fusion protein has one or more characteristics selected from the following: (1) compared to the sequence shown in SEQ ID NO: 4, the sequence of the fusion protein has amino acid substitutions at positions corresponding to positions 29 and 212 of SEQ ID NO: 4; (2) compared to the sequence shown in SEQ ID NO: 4, the sequence of the fusion protein has amino acid substitutions at positions corresponding to positions 29 and 214 of SEQ ID NO: 4; (3) compared to the sequence shown in SEQ ID NO: 4, the sequence of the fusion protein has amino acid substitutions at positions corresponding to positions 29 and 114 of SEQ ID NO: 4; (4) compared to the sequence set forth in SEQ ID NO: 4, the sequence of the fusion protein has amino acid substitutions at positions corresponding to positions 29, 47, 101, and 214 of SEQ ID NO: 4; (5) Compared to the sequence set forth in SEQ ID NO: 4, the sequence of the fusion protein has amino acid substitutions at positions corresponding to positions 88, 101, 114, and 214 of SEQ ID NO: 4; (6) Compared to the sequence shown in SEQ ID NO: 4, the sequence of the fusion protein has amino acid substitutions at positions corresponding to positions 94 and 114 of SEQ ID NO: 4; The fusion protein of claim 5, having the following structure:

7. The fusion protein may have one or more characteristics selected from the following: (1) the substitution at position 29 is a replacement of isoleucine I with leucine L; (2) the substitution at position 212 is a substitution of valine V with methionine M; (3) the substitution at position 214 is a substitution of alanine A with leucine L; (4) the substitution at position 94 is a substitution of valine V with isoleucine I; (5) the substitution at position 47 is a substitution of alanine A for methionine M; (6) the substitution at position 114 is a substitution of tyrosine Y with isoleucine I; (7) the substitution at position 101 is a substitution of valine V with isoleucine I; (8) the substitution at position 214 is a substitution of alanine A with leucine L; (9) the substitution at position 88 is a substitution of the amino acid leucine L with alanine A; and The fusion protein according to claim 6, wherein the fusion protein preferably has an amino acid sequence as set forth in any one of SEQ ID NOs: 28 to 34.

8. the fusion protein further comprises a signal peptide, a multimerization motif (e.g., a trimerization motif), and / or a membrane anchor region; Preferably, the membrane anchor region has the amino acid sequence shown in SEQ ID NO: 8, Preferably, the signal peptide has the amino acid sequence shown in SEQ ID NO: 9, Preferably, the multimerization motif has the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, Preferably, the signal peptide is located at the N-terminus of the first truncation, and preferably, the signal peptide is linked to the first truncation via a first linker peptide; Preferably, the membrane anchor region is located at the C-terminus of the second truncation, and preferably, the membrane anchor region is linked to the second truncation via a second linker peptide; Preferably, the multimerization motif is located at the C-terminus of the second truncation, and preferably, the multimerization motif is linked to the second truncation via a second linker peptide; Preferably, the first linker peptide and the second linker peptide each independently have the structure: (G ｍ S) ｎ wherein m is an integer selected from 1 to 6 and n is an integer selected from 1 to 6, preferably m is 3, 4, or 5, and preferably n is 1 or 2, and preferably the first linker peptide and the second linker peptide have the sequence shown in SEQ ID NO: 18; Preferably, the fusion protein comprises, from N-terminus to C-terminus, the signal peptide, the first linker peptide, the first truncation, the linker, the second truncation, the second linker peptide, and the membrane anchor region; The fusion protein according to any one of claims 1 to 7, wherein the fusion protein preferably has the sequence shown in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 45 or SEQ ID NO:

46.

9. A nucleic acid molecule comprising a nucleotide sequence encoding the fusion protein of any one of claims 1 to 8, Preferably, the nucleotide sequence is codon-optimized according to the codon preference of the host cell or is not codon-optimized.

10. A vector comprising the nucleic acid molecule of claim 9, Preferably, the vector is a viral vector, Preferably, the viral vector is selected from the group consisting of an influenza virus vector, an enterovirus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papovavirus vector.

11. A host cell comprising a fusion protein according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 9, or a vector according to claim 10, Preferably, the host cell is selected from the group consisting of a prokaryotic cell (e.g., an Escherichia coli cell) and a eukaryotic cell; Preferably, the eukaryotic cell is a mammalian cell such as a mouse cell or a human cell, Preferably, the fusion protein is displayed on the surface of the cell membrane of the host cell.

12. 12. A method of expressing or producing a fusion protein according to any one of claims 1 to 8, comprising culturing a host cell according to claim 11 under conditions allowing protein expression, and optionally recovering or purifying the expressed fusion protein.

13. A kit comprising an antigen component and a carrier component capable of presenting said antigen component, The antigen component is (i) a fusion protein according to any one of claims 1 to 8, (ii) a nucleic acid molecule according to claim 9, and / or (iii) comprising an mRNA product transcribed from the nucleic acid molecule of claim 9, the carrier component is selected from the group consisting of nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers, and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), multimerization motifs, virus-like particles (VLPs), or any combination thereof; Preferably, the antigen component and the carrier component in the kit are provided separately or in the form of a complex formed therefrom; Preferably, the antigen component is a monomer or multimer (e.g., dimer, trimer, tetramer, pentamer); Preferably, said multimerization motif and / or said VLP in said kit is provided in the form of a protein or a nucleic acid molecule, Preferably, the multimerization motif has the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22, Preferably, the VLPs are assembled from proteins derived from RSV, hepatitis E virus (HEV), hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

14. A pharmaceutically acceptable carrier and / or additive and the following items (1) to (4): (1) The fusion protein according to any one of claims 1 to 8, (2) The nucleic acid molecule according to claim 9. (3) The vector according to claim 10. (4) The host cell according to claim 11. and one or more selected from A pharmaceutical composition comprising:

15. A vaccine comprising a fusion protein according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 9, or an mRNA product transcribed from the nucleic acid molecule according to claim 9, or a vector according to claim 10, Preferably, the vaccine is prepared by a kit according to claim 13, Preferably, the vaccine further comprises an adjuvant.

16. A method for inducing antibodies against RSV, comprising administering (e.g., injecting) an effective amount of a fusion protein according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 9, or a vector according to claim 10, or a host cell according to claim 11, or a pharmaceutical composition according to claim 14, or a vaccine according to claim 15 to a cell in vitro or to a subject; Preferably, the antibody is a neutralizing antibody.

17. 10. A method for detecting the presence of an RSV infection in a subject in vitro, comprising contacting a biological sample obtained from the subject with the fusion protein of any one of claims 1 to 8, and detecting the presence of a complex formed by the fusion protein and an antibody from the biological sample, Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the biological sample is selected from the group consisting of whole blood, serum, plasma, or any combination thereof.

18. A method for screening a candidate drug capable of inhibiting RSV infection in a cell, the method comprising contacting a host cell with the candidate drug before, simultaneously with, or after contacting the host cell with a fusion protein described in any one of claims 1 to 8, or a vector described in claim 10, or a pharmaceutical composition described in claim 14, or a vaccine described in claim 15.

19. 16. Use of a fusion protein according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 9, or a vector according to claim 10, or a host cell according to claim 11, or a pharmaceutical composition according to claim 14, or a vaccine according to claim 15, in the manufacture of a kit for inducing an immune response against RSV in a subject, comprising: Preferably, the immune response comprises inducing the subject to produce antibodies against RSV; Preferably, the antibody is a neutralizing antibody, Preferably, the subject is a mammal, such as a mouse or a human.

20. 16. Use of a fusion protein according to any one of claims 1 to 8, or a nucleic acid molecule according to claim 9, or a vector according to claim 10, or a host cell according to claim 11, or a pharmaceutical composition according to claim 14, or a vaccine according to claim 15, in the manufacture of a kit for preventing and / or treating an RSV infection or a disease and / or symptom caused by an RSV infection, comprising: Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the diseases and symptoms caused by RSV infection are selected from the group consisting of bronchiolitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

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