RSV and hmpv fusion f protein and use thereof

Abstract

Provided are an epitope or epitope peptide of a respiratory syncytial virus (RSV) F protein, and a fusion protein containing the epitope or epitope peptide and a truncated F protein of human metapneumovirus (hMPV) or a variant of the truncated protein. Further provided are the use of the fusion protein and of a vaccine, an immunogenic composition, a kit, and a pharmaceutical composition containing same in preventing and / or treating RSV and / or hMPV infection or diseases and / or symptoms caused by RSV and / or hMPV infection.

Description

RSV and hMPV fusion F protein and their applications

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application 202410796831.8, filed on June 19, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of biomedicine, and more specifically, to an epitope or epitope peptide of a respiratory syncytial virus (RSV) F protein, and a fusion protein comprising the epitope or epitope peptide and a truncated form or a variant of the truncated form of a human metapneumovirus (hMPV) F protein. This application also relates to the use of the fusion protein and vaccines, immunogenic compositions, kits, and pharmaceutical compositions comprising it for the prevention and / or treatment of RSV and / or hMPV infection or diseases and / or symptoms caused by RSV / or hMPV infection. Background Technology

[0004] Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infections in infants and young children worldwide. Besides infants and children, immunocompromised elderly individuals and those with weakened immune systems are also at high risk of RSV infection, often leading to obstructive pulmonary disease and cardiopulmonary complications in the elderly. On May 4, 2023, GlaxoSmithKline (GSK) announced that the FDA had approved Arexvy for the prevention of lower respiratory tract infections caused by RSV in individuals aged 60 and older. This vaccine is the world's first approved RSV vaccine for the elderly, containing a recombinant pre-fusion conformation RSV F glycoprotein antigen (RSV PreF3) and GSK's proprietary adjuvant AS01.

[0005] On June 6, Pfizer announced that the FDA has approved its RSV vaccine ABRYSVO for the prevention of lower respiratory tract infections caused by RSV in individuals aged 60 and older. ABRYSVO is adjuvant-free and consists of two PreF proteins, providing maximal protection against RSV A and B subgroups. Its safety and efficacy have been demonstrated.

[0006] Besides multinational pharmaceutical giants GlaxoSmithKline and Pfizer, Moderna's RSV vaccine, based on mRNA technology, is also progressing rapidly. Currently, no RSV-related products have been approved in China.

[0007] Research over the past 20 years has revealed that the RSV F protein possesses pre-fusion (pre-F) and post-fusion (post-F) conformations, with the pre-F conformation inducing more effective neutralizing antibodies. This may be key to addressing the ERD response generated by inactivated vaccines. Therefore, antigens based on the pre-F protein conformation have become an important direction for RSV vaccine development.

[0008] Currently, most manufacturers use Pre-F conformational proteins derived from the work published in SCIENCE by Jason S. McLellan et al. in 2013. To prevent the molecule from rearranging into the post-fusion conformation, the team resolved the molecular structure of the F protein in its pre-F conformation. Through structural analysis, they identified the most effective neutralizing epitopes of the F protein and introduced two mutations (S155C and S290C) at the C-terminus of the F protein to form disulfide bonds, increasing protein stability. Furthermore, they modified two grooves in the conformation (S190F and V207L) to fill them. The modified F protein, expressed in CHO cells, can be used to produce a stable Pre-F trimer vaccine (DS-Cav1), as these modifications prevent the molecule from rearranging into the post-fusion conformation.

[0009] Human metapneumovirus (hMPV), discovered in 2001, belongs to the family Pneumoviridae and the genus Metapneumovirus. It is one of the main pathogens causing acute lower respiratory tract infections. Primary infection with hMPV usually occurs in children under 5 years old. Recurrent hMPV infections can occur, causing severe illness in adults (especially the elderly and immunocompromised patients). Currently, there are no approved preventative vaccines or specific antiviral drugs for hMPV.

[0010] Similar to RSV, hMPV has three glycoproteins outside its envelope: adhesion protein (G), small hydrophobic protein (SH), and fusion protein F. Among them, F is essential for infection and is the only target protein of neutralizing antibodies. On the surface of mature particles, F exists in the form of a trimer. Each monomer is first synthesized into an inactive precursor protein F0, which is then enzymatically cleaved into two covalently linked subunits F1 and F2.

[0011] In summary, given the lack of vaccines for hMPV and RSV, further development of vaccine products targeting RSV and / or hMPV is needed. Summary of the Invention

[0012] During the preliminary research, the applicant discovered that the F proteins of hMPV and RSV are highly similar in function and structure. Both can cause respiratory infectious diseases, and their dominant epitopes for inducing neutralizing antibodies are located in different domains of the F protein. Therefore, the applicant proposed an idea: to chimericly modify the key antigenic proteins (F proteins) of RSV and hMPV to artificially create a fusion F protein with dual antigenic epitopes. Furthermore, experiments confirmed that this fusion F protein can maintain the pre-F conformation and induce neutralizing antibodies against hMPV and / or RSV.

[0013] Epitopes or epitope peptides of respiratory syncytial virus (RSV) F protein

[0014] Therefore, in a first aspect, this application provides an epitope or epitope peptide of a respiratory syncytial virus (RSV) F protein, comprising:

[0015] Amino acid residues corresponding to positions 60 to 70 (e.g., 61 to 70, 60 to 80, 60 to 88, 60 to 98) of the wild-type RSV F protein; and / or,

[0016] Amino acid residues corresponding to positions 194 to 209 (e.g., 194 to 228, 194 to 238, 194 to 242) of the wild-type RSV F protein.

[0017] In some embodiments, the epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein retains at least one immunodominant epitope or a key fragment thereof of the pre-fusion conformation of the F protein. In some embodiments, the polypeptide or protein containing the epitope or epitope peptide is capable of binding to any one or more monoclonal antibodies or antigen-binding fragments thereof selected from the following: D25, AM22, RSV-199, MPE8, and M1D2.

[0018] In some implementations, the epitope is a conformational epitope or a linear epitope.

[0019] In some embodiments, the epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein is particularly suitable for constructing a fusion F protein. In some preferred embodiments, the epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein is particularly suitable for constructing a fusion F protein with a fragment or variant of the F protein derived from other viruses (e.g., hMPV).

[0020] In some embodiments, the fusion F protein constructed from an epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein exhibits increased stability compared to the wild-type RSV F protein. In some embodiments, this stability is measured by the binding of the fusion F protein to antibody D25.

[0021] In some embodiments, the fusion F protein constructed from the epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein has a higher protein expression level compared to the wild-type RSV F protein.

[0022] In some embodiments, the fusion F protein constructed from an epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein has a higher pre-F protein content compared to the wild-type RSV F protein. In some embodiments, the fusion F protein comprises a pre-fusion conformation (pre-F) protein, a post-fusion conformation (post-F) protein, or a mixture of both. In some embodiments, the pre-F protein content is measured by the ratio of antibody D25 and antibody palivizumab bound to the fusion F protein.

[0023] In some embodiments, the epitope or epitope peptide comprises amino acid residues at positions 60 to 98 and 194 to 242 of the wild-type RSV F protein.

[0024] In some embodiments, the epitope or epitope peptide comprises amino acid residues at positions 61 to 70 and 194 to 209 of the wild-type RSV F protein.

[0025] In some embodiments, the epitope or epitope peptide comprises amino acid residues at positions 60 to 88 and 194 to 228 of the wild-type RSV F protein.

[0026] In some embodiments, the epitope or epitope peptide comprises amino acid residues at positions 60 to 242 of the wild-type RSV F protein.

[0027] In some embodiments, the epitope or epitope peptide comprises amino acid residues at positions 61 to 209 corresponding to wild-type RSV F protein.

[0028] In some embodiments, the epitope or epitope peptide comprises amino acid residues at positions 60 to 228 of the wild-type RSV F protein.

[0029] Fusion protein

[0030] In a second aspect, this application provides a fusion protein comprising a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues at positions 60 to 70 (e.g., 61 to 70, 60 to 80, 60 to 88, 60 to 98) corresponding to wild-type RSV F protein; and / or

[0031] A second truncated form or variant thereof of the RSV F protein contains amino acid residues at positions 194 to 209 (e.g., 194 to 228, 194 to 238, 194 to 242) of the wild-type RSV F protein;

[0032] The variant, compared to the wild-type RSV F protein, has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) amino acid substitutions (e.g., conserved substitutions), deletions and / or additions.

[0033] In some embodiments, it also comprises additional proteins or peptides. It is understood that these additional proteins or peptides will not adversely affect the activity and / or function of the epitope or epitope peptide.

[0034] Other proteins

[0035] In some embodiments, the additional protein is a portion of the RSV F protein or a variant thereof.

[0036] In some embodiments, the additional protein is an F protein derived from other viruses, or a portion thereof, or a variant thereof.

[0037] In some embodiments, the additional protein is a truncated form of wild-type human metapneumovirus (hMPV) F protein or a variant of the truncated form; wherein the variant, compared to the truncated form, has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid substitutions (e.g., conservative substitutions), deletions, or additions.

[0038] In some embodiments, the truncated form of the hMPV F protein of this application, or a variant thereof, retains at least one immunodominant epitope or a key fragment thereof of the pre-fusion conformation of the F protein.

[0039] In some embodiments, the fusion protein further comprises: a first truncated form of hMPV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, and / or a second truncated form of hMPV F protein or a variant thereof.

[0040] In some embodiments, the fusion protein comprises: a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 60 to 98 of the wild-type RSV F protein; a second truncated form of hMPV F protein or a variant thereof, comprising 50-80 (e.g., 50-60, 60-70, 70-80; e.g., 68) amino acid residues derived from the wild-type hMPV F protein; and a second truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 194 to 242 of the wild-type RSV F protein.

[0041] In some embodiments, the fusion protein comprises: a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 61 to 70 of the wild-type RSV F protein; a second truncated form of hMPV F protein or a variant thereof, comprising 80-120 (e.g., 80-90, 90-100, 100-110, or 110-120) amino acid residues derived from the wild-type or non-wild-type RSV F protein; and a second truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 194 to 209 of the wild-type RSV F protein.

[0042] In some embodiments, the fusion protein comprises: a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 60 to 88 of the wild-type RSV F protein; a second truncated form of hMPV F protein or a variant thereof, comprising 70-100 (e.g., 70-80, 80-90, 90-100; e.g., 85) amino acid residues derived from the wild-type or non-wild-type RSV F protein; and a second truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 194 to 228 of the wild-type RSV F protein.

[0043] In some embodiments, the second truncated form of the hMPV F protein or a variant thereof comprises amino acid residues corresponding to positions 60-100 (e.g., 60-63, 63-70, 70-78, 78-80, 80-85, 85-90, 90-95, 95-100) to 150-170 (e.g., 150-155, 155-160, 160-162, 162-165, 165-170) of the wild-type hMPV F protein.

[0044] In some embodiments, the second truncated form or a variant of the hMPV F protein comprises:

[0045] (1) The amino acid residues corresponding to positions 95 to 162 of the wild-type hMPV F protein;

[0046] (2) Amino acid residues corresponding to positions 63 to 162 of the wild-type hMPV F protein; or

[0047] (3) The amino acid residues corresponding to positions 78 to 162 of the wild-type hMPV F protein.

[0048] In some embodiments, the fusion protein comprises: a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 60 to 98 of the wild-type RSV F protein; a second truncated form of hMPV F protein or a variant thereof, comprising amino acid residues corresponding to positions 95 to 162 of the wild-type hMPV F protein; and a third truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 194 to 242 of the wild-type RSV F protein.

[0049] In some embodiments, the fusion protein comprises: a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 61 to 70 of the wild-type RSV F protein; a second truncated form of hMPV F protein or a variant thereof, comprising amino acid residues corresponding to positions 63 to 162 of the wild-type hMPV F protein; and a second truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 194 to 209 of the wild-type RSV F protein.

[0050] In some embodiments, the fusion protein comprises: a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 60 to 88 of the wild-type RSV F protein; a second truncated form of hMPV F protein or a variant thereof, comprising amino acid residues corresponding to positions 78 to 162 of the wild-type hMPV F protein; and a first truncated form of RSV F protein or a variant thereof, comprising amino acid residues corresponding to positions 194 to 228 of the wild-type RSV F protein.

[0051] Stabilization modification

[0052] The truncated form of the RSV F protein of this application, or a variant thereof, or a fusion protein thereof, stabilizes the pre-F conformation by introducing one or more modifications, such as substitution (e.g., conserved substitution), deletion, and / or addition of one or more amino acids. It is understood that these stabilizing modifications do not adversely affect the immunodominant epitope or key fragment thereof of the pre-fusion conformation of the F protein retained by the epitope or epitope peptide of this application.

[0053] One stabilizing modification is the introduction of amino acid substitutions (mutations).

[0054] In some embodiments, the variant, compared to the wild-type RSV F protein, contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions.

[0055] In some implementations, the permutation is a conservative permutation.

[0056] In some embodiments, the variant, compared to the wild-type RSV F protein, has one or more of the following characteristics:

[0057] (1) Increased stability; for example, increased stability of the variant itself, or of the fusion protein formed therewith with another protein. In some embodiments, the fusion protein is a chimeric F protein, and the amino acid addition is capable of stabilizing the pre-fusion conformation of the chimeric F protein;

[0058] (2) It has higher protein expression levels;

[0059] In some embodiments, the variant includes an amino acid substitution selected from (i) cavity-filling mutations, (ii) electrostatic mutations, (iii) engineered disulfide bond mutations, or any combination thereof.

[0060] Cavity filling mutation

[0061] In some embodiments, the variant comprises one or more cavity-filling mutations. In some embodiments, such cavity-filling mutations contribute to the stability of the fusion protein itself or that containing it in the formation of a trimer. Hereinafter, the term "cavity-filling mutation" refers to the substitution of amino acid residues that form an internal cavity in the spatial structure of a protein by amino acid residues capable of filling said internal cavity. The cavity formed by the protein can be identified by methods known in the art, such as by examining the crystal structure of the protein or a fusion protein containing it, or by using computational protein design software (such as BioLuminate™ [BioLuminate, Schrodinger LLC, New York, 2015], AlphaFold2 [DeepMind, London, UK, 2021], Amber [Assisted Model Building and Energy Refinement, AMBER Software Administrator University of California, San Francisco Dept. of Pharmaceutical Chemistry, 2023], and LAMMPS [Large-scale Atomic / Molecular Massively Parallel Simulator, Sandia National Laboratories, Albuquerque, NM 87185, USA, 2022]).

[0062] In some embodiments, the cavity-filling mutation in the variants of the truncated RSV F protein of this application is V207L.

[0063] Disulfide bond mutation in engineering modification

[0064] In some embodiments, the variant comprises one or more engineered disulfide bond mutations. Hereinafter, the term "engineered disulfide bond mutation" refers to a protein in which a pair of amino acid residues is mutated to a pair of cysteine ​​residues. The introduced cysteine ​​residue pair allows for the formation of a disulfide bond between the introduced cysteine ​​residues, which serves to stabilize the conformation or oligomeric state of the protein, such as the pre-fusion conformation. Such residues can be identified by suitable methods known in the art, such as by examining the crystal structure of the protein or a fusion protein containing it, or by using computational protein design software.

[0065] electrostatic mutation

[0066] In some embodiments, the variants include one or more electrostatic mutations. In some embodiments, such electrostatic mutations contribute to the stability of the trimer formed by the variant itself or a fusion protein containing it. Hereinafter, the term "electrostatic mutation" refers to a mutation that reduces ionic repulsion between amino acid residues that are close to each other in the folded structure of a protein or increases ionic attraction between said amino acid residues. Since hydrogen bonding is a special case of ionic attraction, electrostatic mutations can increase hydrogen bonding between such close amino acid residues. Typically, introducing an electrostatic mutation will increase the Tm value of the protein or its fusion protein.

[0067] Unfavorable electrostatic interactions following the formation of a trimer configuration can be identified by methods known in the art, such as by examining the crystal structure of a protein in a trimer configuration or a fusion protein containing it, or by using computational protein design software.

[0068] In some embodiments, the truncated RSV F protein of this application has electrostatic mutations in its variants, such as S63N or S63D.

[0069] In some embodiments, the amino acid substitutions in the variant are selected from positions 61, 62, 63, 97, and / or 207 corresponding to wild-type RSV F protein. In some embodiments, the amino acid substitutions in the variant are selected from positions 61, 62, 63, 97, and / or 207 corresponding to the sequences shown in any one of SEQ ID NO: 1-4.

[0070] In some embodiments, the variant has amino acid V at position 61, corresponding to the wild-type RSV F protein.

[0071] In some implementations, the amino acid substitution at position 61 is an L-to-V substitution.

[0072] In some embodiments, the amino acid at position 62 corresponding to the wild-type RSV F protein is G.

[0073] In some implementations, the amino acid substitution at position 62 is an S-to-G substitution.

[0074] In some embodiments, the amino acid at position 63 corresponding to the wild-type RSV F protein is N.

[0075] In some implementations, the amino acid substitution at position 63 is an S-to-N or D substitution.

[0076] In some embodiments, the amino acid at position 97, corresponding to the wild-type RSV F protein, is E.

[0077] In some implementations, the amino acid substitution at position 97 is an M-to-E substitution.

[0078] In some embodiments, the variant has an amino acid L at position 207 corresponding to the wild-type RSV F protein.

[0079] In some implementations, the amino acid substitution at position 207 is a V-to-L substitution.

[0080] Wild-type RSV F protein

[0081] Natural RSV F proteins exhibit high sequence conservation across different RSV isoforms. For example, RSV isoforms A and B share 90% sequence identity. Furthermore, within RSV isoforms, the sequence identity of the F protein is even higher, with approximately 98% sequence identity in both RSV isoform A and isoform B. Moreover, almost all identified RSV F protein sequences consist of 574 amino acids in length, typically varying only slightly due to the length of the C-terminal cytoplasmic tail. This sequence identity among these natural RSV F proteins is known in the art (see, for example, WO2014 / 160463).

[0082] Given the conservation of RSV F protein sequences, those skilled in the art can readily compare the amino acid positions of different natural RSV F protein sequences to identify the corresponding amino acid positions of RSV F proteins from different RSV strains or subtypes. For example, in almost all identified natural RSV F proteins, the furin cleavage site is located at the same amino acid position. Therefore, the conservation of natural RSV F protein sequences among strains or subtypes allows them to be used as reference RSV F sequences for comparing amino acids at specific positions within RSV F proteins.

[0083] As used herein, when referring to the amino acid sequence of a wild-type RSV F protein, it is described using the sequence shown in any one of SEQ ID NO: 1-4. For example, the expression "position 63 of a wild-type RSV F protein" refers to the 63rd amino acid residue of the protein shown in any one of SEQ ID NO: 1-4. However, those skilled in the art will understand that wild-type RSV F proteins can have multiple versions that have substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), and substantially the same biological function, but may still have minor differences in their amino acid sequences. Therefore, in this application, wild-type RSV F protein is not limited to the protein shown in any one of SEQ ID NO: 1-4, but is intended to cover all known wild-type RSV F proteins. Therefore, in this application, the term "wild-type RSV F protein" should include various naturally occurring, biologically functional RSV F proteins, including, for example, the RSV F protein shown in any one of SEQ ID NO: 1-4 and its naturally occurring variants. Furthermore, when describing the amino acid positions of the RSV F protein, it includes not only the specific amino acid positions in any one of SEQ ID NO: 1-4, but also the corresponding amino acid positions in its natural variants. For example, the statement "position 63 of the wild-type RSV F protein" includes the 63rd amino acid residue in any one of SEQ ID NO: 1-4, and the corresponding amino acid position in its natural variant. According to this application, the statement "corresponding amino acid position" refers to the amino acid position at the equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0084] In some embodiments, the wild-type RSV is a strain of subtype A, a strain of subtype B, or a strain derived from subtype A or subtype B.

[0085] In some embodiments, the wild-type RSV is selected from strain A2, strain Ontario, strain Buenos Aires, or strain B18537.

[0086] In some embodiments, the wild-type RSV F protein comprises, or is composed of, sequences selected from, the following:

[0087] (i) The sequence shown in any one of SEQ ID NO: 1-4;

[0088] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in any one of SEQ ID NO: 1-4;

[0089] (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with any of the sequences shown in SEQ ID NO: 1-4.

[0090] Stabilization modification

[0091] Similar to the RSV F protein, truncated versions or variants of the hMPV F protein stabilize the pre-F conformation of the fusion protein by introducing one or more modifications, such as substitution (e.g., conserved substitution), deletion, and / or addition of one or more amino acids. It is understood that these stabilizing modifications do not adversely affect the immunodominant epitopes or key fragments of the pre-fusion conformation of the F protein retained by the truncated versions or variants of the hMPV F protein of this application.

[0092] One stabilizing modification is the introduction of amino acid substitutions (mutations).

[0093] In some embodiments, variants of the second truncated form of the hMPV F protein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions compared to the second truncated form of the hMPV F protein.

[0094] In some embodiments, the variant has increased stability compared to the truncated form; for example, increased stability of the variant itself, or of the fusion protein formed therewith with another protein. In some embodiments, the fusion protein is a fusion protein as described above.

[0095] In some implementations, the variant has a higher protein expression level compared to the truncated form.

[0096] In some embodiments, the variant includes an amino acid substitution selected from (i) cavity-filling mutations, (ii) electrostatic mutations, (iii) engineered disulfide bond mutations, or any combination thereof.

[0097] In some implementations, the specific definitions of cavity filling mutations, electrostatic mutations, and engineering-induced disulfide bond mutations are the same as those described above.

[0098] In some embodiments, the electrostatic discharge is selected from E453Q, N466S, I449V, or any combination thereof.

[0099] In some embodiments, the disulfide bond mutation of the engineering modification is selected from L110C, T127C, A140C, A147C, N153C, N322C, T365C, V463C, or any combination thereof.

[0100] In some embodiments, the amino acid substitutions in the variant are selected from positions 82, 100, 101, 110, 114, 127, 138, 140, 147, 153, 185, 219, 231, 232, 322, 365, 449, 453, 463, and / or 466 of the wild-type hMPV F protein. In some embodiments, the amino acid substitutions in the variant are selected from positions 82, 100, 101, 110, 114, 127, 138, 140, 147, 153, 185, 219, 231, 232, 322, 365, 449, 453, 463, and / or 466 of the sequence shown in SEQ ID NO:5.

[0101] In some embodiments, the amino acid at position 82 corresponding to the wild-type hMPV F protein is R.

[0102] In some implementations, the amino acid substitution at position 82 is a K-to-R substitution.

[0103] In some embodiments, the amino acid at position 100 of the variant, corresponding to the wild-type hMPV F protein, is R.

[0104] In some implementations, the amino acid substitution at position 100 is a Q-to-R substitution.

[0105] In some embodiments, the amino acid at position 101 corresponding to the wild-type hMPV F protein is R.

[0106] In some implementations, the amino acid substitution at position 101 is an S-to-R substitution.

[0107] In some embodiments, the amino acid at position 110 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0108] In some implementations, the amino acid substitution at position 110 is an L-to-C substitution.

[0109] In some embodiments, the amino acid at position 114 of the variant, corresponding to the wild-type hMPV F protein, is T.

[0110] In some implementations, the amino acid substitution at position 114 is an A-to-T substitution.

[0111] In some embodiments, the amino acid at position 127 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0112] In some implementations, the amino acid substitution at position 127 is a T-to-C substitution.

[0113] In some embodiments, the variant has an amino acid, K, at position 138 corresponding to the wild-type hMPV F protein.

[0114] In some implementations, the amino acid substitution at position 138 is an N-to-K substitution.

[0115] In some embodiments, the amino acid at position 140 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0116] In some implementations, the amino acid substitution at position 140 is an A-to-C substitution.

[0117] In some embodiments, the amino acid at position 147 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0118] In some implementations, the amino acid substitution at position 147 is an A-to-C substitution.

[0119] In some embodiments, the amino acid at position 153 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0120] In some implementations, the amino acid substitution at position 153 is an N-to-C substitution.

[0121] In some embodiments, the amino acid at position 185 of the variant, corresponding to the wild-type hMPV F protein, is P.

[0122] In some implementations, the amino acid substitution at position 185 is a D-to-P substitution.

[0123] In some embodiments, the variant has an amino acid, K, at position 219 corresponding to the wild-type hMPV F protein.

[0124] In some implementations, the amino acid substitution at position 219 is an L-to-K substitution.

[0125] In some embodiments, the variant has an amino acid I at position 231 corresponding to the wild-type hMPV F protein.

[0126] Preferably, the amino acid substitution at position 231 is a substitution from V to I.

[0127] In some embodiments, the variant has an amino acid S at position 232 corresponding to the wild-type hMPV F protein.

[0128] In some implementations, the amino acid substitution at position 232 is a P-to-S substitution.

[0129] In some embodiments, the amino acid at position 322 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0130] In some implementations, the amino acid substitution at position 322 is an N-to-C substitution.

[0131] In some embodiments, the amino acid at position 365 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0132] In some implementations, the amino acid substitution at position 365 is a T-to-C substitution.

[0133] In some embodiments, the variant has amino acid V at position 449, corresponding to the wild-type hMPV F protein.

[0134] In some implementations, the amino acid substitution at position 449 is a substitution from I to V.

[0135] In some embodiments, the amino acid at position 453 of the variant, corresponding to the wild-type hMPV F protein, is Q.

[0136] In some implementations, the amino acid substitution at position 453 is an E-to-Q substitution.

[0137] In some embodiments, the amino acid at position 463 of the variant, corresponding to the wild-type hMPV F protein, is C.

[0138] In some implementations, the amino acid substitution at position 463 is a V-to-C substitution.

[0139] In some embodiments, the amino acid at position 466 of the variant, corresponding to the wild-type hMPV F protein, is N.

[0140] In some implementations, the amino acid substitution at position 466 is an N-to-S substitution.

[0141] Wild-type hMPV F protein

[0142] The native hMPV F protein exhibits high sequence conservation across different hMPV subtypes. For example, hMPV subtypes A and B share 90% sequence identity. Furthermore, within the same hMPV subtype, the F protein shows even higher sequence identity, with approximately 98% sequence identity in either hMPV subtype A or subtype B. Exemplary hMPV F protein strains are summarized in Boivin et al. Emerg. Infect. Dis. 10: 1154-1157 (2004), which is incorporated herein by reference for the public disclosure of hMPV sequences.

[0143] Given the conservation of the hMPV F protein sequence, those skilled in the art can easily compare the amino acid positions between different natural hMPV F protein sequences to identify the corresponding amino acid positions of the hMPV F protein between different hMPV strains or subtypes. Therefore, the conservation of the natural hMPV F protein sequence among strains or subtypes allows it to be used as a reference hMPV F sequence for comparing amino acids at specific positions within the hMPV F protein.

[0144] As used herein, when referring to the amino acid sequence of wild-type hMPV F protein, it is described using the sequence shown in SEQ ID NO: 5. For example, the statement "position 322 of wild-type RSV F protein" refers to the 322nd amino acid residue of the protein shown in SEQ ID NO: 5. However, those skilled in the art will understand that wild-type hMPV F protein can have multiple versions that have substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), and substantially the same biological function, but may still have minor differences in amino acid sequence from one another. Therefore, in this application, wild-type hMPV F protein is not limited to the protein shown in SEQ ID NO: 5, but is intended to cover all known wild-type hMPV F proteins. Therefore, in this application, the term "wild-type hMPV F protein" should include various naturally occurring, biologically functional hMPV F proteins, including, for example, the RSV F protein shown in SEQ ID NO: 5 and its naturally occurring variants. Furthermore, when describing the amino acid positions of the hMPV F protein, it includes not only the specific amino acid position in SEQ ID NO: 5, but also the corresponding amino acid position in its natural variant. For example, the statement "position 322 of the wild-type hMPV F protein" includes the 322nd amino acid residue of SEQ ID NO: 5, and the corresponding amino acid position in its natural variant. According to this application, the statement "corresponding amino acid position" refers to the amino acid position at the equivalent position in the compared sequences when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage of identity.

[0145] In some implementations, the hMPV is a strain of subtype A, a strain of subtype B, or a strain derived from subtype A or subtype B.

[0146] In some embodiments, the hMPV is selected from strains ACJ53565.1, AHV79858.1, BBB35088.1, AHV79473.1, AAS22125.1, AUF72445.1 and ACJ53575.1.

[0147] In some embodiments, the wild-type hMPV F protein comprises, or is composed of, sequences selected from, the following:

[0148] (i) The sequence shown in SEQ ID NO:5;

[0149] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in SEQ ID NO: 5;

[0150] (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO: 5.

[0151] Other peptides

[0152] In some embodiments, as described above, the additional polypeptide is selected from: signal peptides, polymerization domains, tags, or any combination thereof.

[0153] signal peptide

[0154] To enhance protein production or secretion, the fusion protein of this application may contain a signal peptide.

[0155] In some embodiments, the signal peptide is a natural signal peptide of hMPV or a variant thereof, or a natural signal peptide of other organisms (e.g., RSV) or a variant thereof.

[0156] In some embodiments, the signal peptide is located at one end of the fusion protein (e.g., N-terminus, N-terminus).

[0157] In some embodiments, the signal peptide is located at the N-terminus of a first truncated form of the hMPV F protein or a variant thereof.

[0158] In some embodiments, the signal peptide is linked to a first truncated form or a variant of the hMPV F protein, with or without a first linker peptide.

[0159] In some embodiments, the signal peptide has a sequence as shown in SEQ ID NO:17.

[0160] Label

[0161] In some embodiments, the fusion protein of the present invention can be linked to a tag to facilitate the expression, detection, tracing, and / or purification of the protein. Such epitope tags are well known to those skilled in the art, and examples include, but are not limited to, His, V5, FLAG, HA, Myc, VSV-G, Trx, etc., and those skilled in the art know how to select a suitable epitope tag according to the desired purpose (e.g., purification, detection, or tracing).

[0162] In some embodiments, the tag is selected from multihistidine tags, antigen or epitope tags, enzyme tags, or any combination thereof.

[0163] In some embodiments, the tag is located at the end (e.g., the C-terminus) of the fusion protein.

[0164] In some implementations, the tag is located at the C-terminus of the polymerization domain.

[0165] In some exemplary embodiments, the tag is a multihistidine tag, i.e., a 6×His-tag (HHHHHH). In some exemplary embodiments, the tag is linked to the multihistidine tag via an enzyme cleavage site.

[0166] Multi-domain

[0167] The fusion protein provided in this application can be linked to a multimerization domain to promote the formation of multimers (e.g., dimers, trimers, tetramers, pentamers) from the recombinant fusion protein.

[0168] Exogenous multimerizing domains capable of promoting the formation of stable polymers from soluble proteins are known in the art. Specifically, examples of multimerizing domains that can be linked to the fusion protein of this application include, but are not limited to:

[0169] (1) GCN4 leucine zipper (for specific sequence and information, please refer to Harbury et al., 1993 Science 262:1401-1407);

[0170] (2) Trimerization motifs from lung surfactant proteins (for specific sequences and information, please refer to Hoppe et al. 1994 FEB S Lett 344:191-195);

[0171] (3) Collagen (for specific sequences and information, please refer to McAlinden et al. 2003 Biol Chem 278:42200-42207); and

[0172] (4) Phage T4 fibritin fold (for specific sequence and information, please refer to Miroshnikov et al. 1998 Protein Eng 11:329-414).

[0173] Typically, the multimerizing domain is attached to the C-terminus or C-terminus of the fusion protein. It can bind directly to the fusion protein or be attached via a linker (such as an amino acid linker, e.g., the sequence GG, GS, or SAIG). The linker can also be a longer linker (e.g., a sequence including the repeating sequence GG). In some embodiments, the fusion protein may also include a protease cleavage site for removing the multimerizing domain from the fusion protein.

[0174] In some implementations, the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or a pentamerization domain.

[0175] In some embodiments, the polymerizing domain is derived from T4 phage fibrin. In some embodiments, the polymerizing domain has a sequence as shown in SEQ ID NO:6.

[0176] In some embodiments, the polymerizing domain is a leucine zipper or an isoleucine zipper. In some embodiments, the polymerizing domain has a sequence as shown in SEQ ID NO:7.

[0177] In some implementations, the polymerization domain is derived from lung surfactant proteins.

[0178] In some implementations, the polymerization domain is located at the C-terminus of the F1 domain.

[0179] In some embodiments, the polymerization domain is linked to a second truncated form or a variant of the hMPV F protein, with or without a linker peptide.

[0180] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, and a second truncated form of RSV F protein or a variant thereof.

[0181] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a first truncated form of hMPV F protein or a variant thereof, a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, a second truncated form of RSV F protein or a variant thereof, and a third truncated form of hMPV F protein or a variant thereof.

[0182] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a first truncated form of hMPV F protein or a variant thereof, a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, a second truncated form of RSV F protein or a variant thereof, and a third truncated form of hMPV F protein or a variant thereof, and a polymerizing domain.

[0183] In some embodiments, the fusion protein comprises, from the N-terminus to the C-terminus, a signal peptide, a first truncated form of hMPV F protein or a variant thereof, a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, a second truncated form of RSV F protein or a variant thereof, and a third truncated form of hMPV F protein or a variant thereof, and a polymerization domain.

[0184] Another stabilizing modification is the introduction of amino acid additions (insertions).

[0185] The fusion protein of this application stabilizes the pre-F conformation by introducing one or more modifications, such as the addition of one or more amino acids (e.g., an artificially designed linker peptide), between a truncated form of the RSV F protein or a variant thereof and a truncated form of the hMPV F protein or a variant thereof. It is understood that these stabilizing modifications do not adversely affect the immunodominant epitopes or key fragments of the pre-fusion conformation of the F protein retained by the fusion protein of this application.

[0186] In some embodiments, the first truncated form of the hMPV F protein or a variant thereof and the first truncated form of the RSV F protein or a variant thereof are linked by or not through a first linker peptide. In some embodiments, the first linker peptide has the sequence shown in SEQ ID NO:14.

[0187] In some embodiments, a first truncated form of the RSV F protein or a variant thereof and a second truncated form of the hMPV F protein or a variant thereof are linked by or without a second linker peptide; in some embodiments, the second linker peptide has a sequence as shown in SEQ ID NO:16.

[0188] In some embodiments, the second truncated form of the hMPV F protein or a variant thereof and the second truncated form of the RSV F protein or a variant thereof are linked by or without a third linker peptide. In some embodiments, the third linker peptide has a sequence as shown in SEQ ID NO:15 or 35.

[0189] fusion protein CRM4

[0190] In some embodiments, the fusion protein comprises:

[0191] (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein;

[0192] (2) The first truncated form of RSV F protein or a variant thereof, which corresponds to the positions of amino acid residues 60 to 98 of wild-type RSV F protein;

[0193] Furthermore, it also includes amino acid substitutions at positions corresponding to positions 63 and 97 of the wild-type RSV F protein. In some embodiments, the amino acid substitutions are selected from S63N and M97E;

[0194] (3) A second truncated form of hMPV F protein or a variant thereof, corresponding to amino acid residues from position 95 to position 162 of wild-type hMPV F protein;

[0195] Furthermore, it also includes amino acid substitutions at the following positions: positions 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C;

[0196] (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 242 of the wild-type RSV F protein;

[0197] Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from V207L;

[0198] (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 213 to 490 of wild-type hMPV F protein;

[0199] Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S.

[0200] In some embodiments, the fusion protein has one or more features selected from the following:

[0201] (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:26; (3) The second truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:20; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:30; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:23.

[0202] Fusion protein CRM5

[0203] In some embodiments, the fusion protein comprises:

[0204] (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 52 of wild-type hMPV F protein;

[0205] (2) The first truncated form of RSV F protein or a variant thereof, which corresponds to the positions of amino acid residues 61 to 70 of wild-type RSV F protein;

[0206] Furthermore, it also includes an amino acid substitution at the position corresponding to position 63 of the wild-type RSV F protein; in some embodiments, the amino acid substitution is selected from S63N;

[0207] (3) A second truncated form of hMPV F protein or a variant thereof, corresponding to amino acid residues 63 to 162 of wild-type hMPV F protein;

[0208] Furthermore, it also includes amino acid substitutions at the following positions: positions 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C;

[0209] (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 209 of the wild-type RSV F protein;

[0210] Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; in some embodiments, the amino acid substitution is selected from V207L;

[0211] (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 180 to 490 of wild-type hMPV F protein;

[0212] Furthermore, it also includes amino acid substitutions at the following positions: corresponding to positions 185, 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from D185P, L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S.

[0213] In some embodiments, the fusion protein has one or more features selected from the following:

[0214] (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:19; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:30; (3) The second truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:23; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:33; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:26.

[0215] Fusion protein CRM6

[0216] In some embodiments, the fusion protein comprises:

[0217] (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein;

[0218] (2) The first truncated form of RSV F protein or a variant thereof, which corresponds to the positions of amino acid residues 60 to 98 of wild-type RSV F protein;

[0219] Furthermore, it also includes amino acid substitutions at positions corresponding to positions 63 and 97 of the wild-type RSV F protein; in some embodiments, the amino acid substitutions are selected from S63N and M97E.

[0220] (3) A second truncated form of hMPV F protein or a variant thereof, corresponding to amino acid residues from position 95 to position 162 of wild-type hMPV F protein;

[0221] Furthermore, it also includes amino acid substitutions at the following positions: positions 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C;

[0222] (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 242 of the wild-type RSV F protein;

[0223] Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from V207L;

[0224] (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 213 to 490 of wild-type hMPV F protein;

[0225] Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; in some embodiments, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S;

[0226] Furthermore, the first truncated form of the hMPV F protein or a variant thereof and the first truncated form of the RSV F protein or a variant thereof are linked by or without a first linker peptide; in some embodiments, the first linker peptide has a sequence as shown in SEQ ID NO:14;

[0227] Furthermore, the second truncated form of the hMPV F protein or a variant thereof and the second truncated form of the RSV F protein or a variant thereof are linked by or without a third linker peptide; in some embodiments, the third linker peptide has a sequence as shown in SEQ ID NO:15.

[0228] In some embodiments, the fusion protein has one or more features selected from the following:

[0229] (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:26; (3) The second truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:20; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:30; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:23.

[0230] Fusion protein CRM7 / 8

[0231] In some embodiments, the fusion protein comprises:

[0232] (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein;

[0233] (2) The first truncated form of RSV F protein or a variant thereof, corresponding to the positions of amino acid residues 60 to 88 of wild-type RSV F protein;

[0234] Furthermore, it also includes an amino acid substitution at the position corresponding to position 63 of the wild-type RSV F protein; in some embodiments, the amino acid substitution is selected from S63N;

[0235] (3) The second truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues from position 78 to position 162 of wild-type hMPV F protein;

[0236] Furthermore, it also includes amino acid substitutions at the following positions: positions 82, 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; in some embodiments, the amino acid substitutions are selected from K82R, Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C;

[0237] (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 228 of the wild-type RSV F protein;

[0238] Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; in some embodiments, the amino acid substitution is selected from V207L;

[0239] (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 199 to 490 of wild-type hMPV F protein;

[0240] Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; in some embodiments, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S.

[0241] In some embodiments, a first truncated form of the RSV F protein or a variant thereof and a second truncated form of the hMPV F protein or a variant thereof are linked, with or without a second linker peptide. In some embodiments, the second linker peptide has the form SEQ ID NO:16.

[0242] In some embodiments, the fusion protein has one or more features selected from the following:

[0243] (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:27; (3) The second truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:21; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:31; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:24.

[0244] Fusion protein CRM9

[0245] In some embodiments, the fusion protein comprises:

[0246] (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein;

[0247] (2) The first truncated form of RSV F protein or a variant thereof, corresponding to the positions of amino acid residues 61 to 88 of wild-type RSV F protein;

[0248] Furthermore, it also includes amino acid substitutions at positions corresponding to positions 61, 62, and 63 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from L61V, S62G, and S63D.

[0249] (3) The second truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues from position 78 to position 162 of wild-type hMPV F protein;

[0250] Furthermore, it also includes amino acid substitutions at the following positions: positions 82, 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; in some embodiments, the amino acid substitutions are selected from K82R, Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C;

[0251] (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 228 of the wild-type RSV F protein;

[0252] Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; in some embodiments, the amino acid substitution is selected from V207L;

[0253] (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 199 to 490 of wild-type hMPV F protein;

[0254] Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; in some embodiments, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S.

[0255] In some embodiments, the second truncated form of the hMPV F protein or a variant thereof and the second truncated form of the RSV F protein or a variant thereof are linked by or without a third linker peptide. In some embodiments, the third linker peptide has the sequence shown in SEQ ID NO:35.

[0256] In some embodiments, the fusion protein has one or more of the following features: (1) a first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) a first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:28; (3) a second truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:21; (4) a second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:31; (5) a third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:24.

[0257] In some embodiments, the fusion protein has:

[0258] (i) The sequence shown in any one of SEQ ID NO: 8-13;

[0259] (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in any of SEQ ID NO:8-13;

[0260] (iii) A sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with any of the sequences shown in SEQ ID NO: 8-13;

[0261] In some embodiments, the fusion protein is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture of both.

[0262] In some embodiments, the fusion protein is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixture of both.

[0263] Nucleic acid molecules

[0264] It is readily understood that the nucleic acid molecules can be used to clone or express the epitopes or epitope peptides or fusion proteins of the present invention. In some cases, to improve efficiency, the nucleotide sequence of the nucleic acid molecules can be codon-optimized according to cell preferences.

[0265] Therefore, in a third aspect, this application provides a nucleic acid molecule comprising a nucleotide sequence encoding the epitope or epitope peptide described in the first aspect or the fusion protein described in the second aspect.

[0266] In some implementations, the nucleotide sequence may or may not be codon-optimized based on the host cell's codon preference.

[0267] mRNA

[0268] The nucleic acid molecules of this application may include DNA, cDNA, and RNA sequences. In some embodiments, the nucleic acid molecule is DNA, or an RNA (mRNA) product transcribed from said DNA, or a mixture of both.

[0269] In some embodiments, the nucleic acid molecule comprises mRNA encoding an open reading frame (ORF) of the epitope or epitope peptide described in the first aspect or the fusion protein described in the second aspect.

[0270] In some embodiments, the mRNA has chemical modifications (e.g., Cap1 capping, 1-methyl-pseudouridine modification, 1-ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine).

[0271] In some embodiments, the modification comprises a chemical modification. These chemical modifications can confer increased stability and low immunogenicity to the mRNA, thereby promoting protein expression. For example, N1-methyl-pseudouridylidine is superior to several other nucleoside modifications in terms of translational ability. In some embodiments, the mRNA molecule used herein may replace uracil with pseudouracil, such as a 1-methyl-3'-pseudouridylyl base.

[0272] In some implementations, the mRNA may include a 5' cap, a 5' UTR element, a codon-optimized or unoptimized open reading frame, a 3' UTR element, and a poly A sequence and / or a polyadenylation signal.

[0273] In some embodiments, the mRNA has a sequence as shown in any one of SEQ ID NO:36-42.

[0274] Nucleic acid molecules, whether natural or modified, can be delivered in delivery media such as lipid nanoparticles. Lipid nanoparticles may comprise one or more nucleic acid molecules in a weight ratio of about 5:1 to about 1:100 to the weight of the lipid nanoparticles. In some embodiments, the weight ratio of nucleic acid molecules to lipid nanoparticles is about 5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any value derived therefrom.

[0275] carrier

[0276] Vectors for expressing the epitopes, epitope peptides, or fusion proteins of this application in insect or mammalian cells are well known in the art. These vectors can be cloning vectors or expression vectors. In some preferred embodiments, the vectors of the present invention can be, for example, plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and viral vectors, etc. Viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40).

[0277] In some preferred embodiments, the vectors of this application are capable of expressing or used to express the epitopes, epitope peptides, or fusion proteins of the present invention. In some preferred embodiments, the vectors of the present invention are capable of expressing or used to express the epitopes, epitope peptides, or fusion proteins of the present invention in a subject (e.g., a mammal, such as a human).

[0278] The vector of this application may contain a variety of elements, including but not limited to one or more of the following: origin of replication; optional marker gene; one or more expression control elements, such as transcription control elements (e.g., promoter, enhancer, terminator) and / or one or more translation signals; and signal sequences or leader sequences for targeting the secretion pathway in selected host cells.

[0279] In a fourth aspect, this application provides a carrier comprising the nucleic acid molecules described in the third aspect.

[0280] In some implementations, the vector is a viral vector.

[0281] In some embodiments, the viral vector is selected from: influenza virus vector, reverse transcriptase virus vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, poxvirus vector, baculovirus vector, papillomavirus vector, or papillomavirus vector.

[0282] host cells

[0283] The epitopes or epitope peptides or fusion proteins provided in this application can be prepared by conventional methods known in the art, such as by expression in recombinant host cells using suitable vectors. Host cells include, for example, insect cells, mammalian cells, avian cells, bacterial cells, and yeast cells. Examples of insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, and Schneider S2 cells. Examples of mammalian cells include Chinese hamster ovary (CHO) cells, human embryonic kidney cells (HEK293 or Expi 293 cells), NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. Avian cells include, for example, chicken embryonic stem cells, chicken embryonic fibroblasts, chicken embryonic germ cells, quail fibroblasts, and duck cells. Insect cell expression systems, such as baculovirus vector systems, are known to those skilled in the art and described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Avian cell expression systems are also known to those skilled in the art and described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168 and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known to those skilled in the art and described, for example, in Yeast Genetic Engineering (edited by Barr et al., 1989), Butterworth, London.

[0284] In a fifth aspect, this application provides a host cell comprising the epitope or epitope peptide described in the first aspect, the fusion protein described in the second aspect, the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect.

[0285] In some embodiments, the host cell is selected from prokaryotic cells (e.g., Escherichia coli cells) or eukaryotic cells. In some embodiments, the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell. In some embodiments, the epitope or epitope peptide or fusion protein is displayed on the surface of the cell membrane of the host cell.

[0286] Preparation method

[0287] The methods used for expressing and purifying the epitopes or epitope peptides described in the first aspect of this application or the fusion proteins described in the second aspect are common in the art and can be found in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 200; and Ausubel et al., Short Protocols in Molecular Biology, 4th Edition, John Wiley & Sons, Inc., 999.

[0288] Therefore, in a sixth aspect, this application provides a method for expressing or generating the epitope or epitope peptide described in the first aspect or the fusion protein described in the second aspect, the method comprising culturing the host cell described in the fifth aspect under conditions that allow protein expression, and optionally, recovering or purifying the expressed epitope or epitope peptide or fusion protein.

[0289] vaccine

[0290] The vaccine provided in this application is not limited to protein form, nucleic acid form, or a mixture of both. Furthermore, the nucleic acid can be selected from DNA, cDNA, RNA (e.g., mRNA), or any combination thereof.

[0291] In a seventh aspect, this application provides a vaccine comprising one or more of the following (1) to (4):

[0292] (1) The epitope or epitope peptide described in the first aspect;

[0293] (2) The fusion protein described in the second aspect;

[0294] (3) The nucleic acid molecules described in the third aspect;

[0295] (4) The carrier described in the fourth aspect.

[0296] In some implementations, the vaccine also contains adjuvants and / or buffer solutions.

[0297] In some embodiments, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles (e.g., LPN), or any combination thereof.

[0298] In some embodiments, the fusion protein in the vaccine is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture.

[0299] In some embodiments, the fusion protein in the vaccine is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixture thereof.

[0300] The vaccine provided in this application can be administered using standard routes of administration. Non-limiting administration methods include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration. A single dose can be given to the subject, or one or more booster doses. If a booster vaccination is performed, it is typically administered to the same individual at a time between 1 week and 10 years after the first dose (referred to in such cases as the "primitive vaccination"), for example, between 2 weeks and 6 months.

[0301] The vaccine provided in this application can also be used in conjunction with one or more other vaccines. For example, in adults, it can be used with influenza vaccines, Prevnar vaccines, tetanus vaccines, diphtheria vaccines, and pertussis vaccines. In children, the vaccine provided in this application can be used in conjunction with any other vaccines indicated for pediatric patients.

[0302] Immunogenic Compositions

[0303] In an eighth aspect, this application provides an immunogenic composition comprising:

[0304] (i) a first immunogenic component, wherein the first immunogenic component is selected from one or more of the following (1) to (4):

[0305] (1) The epitope or epitope peptide described in the first aspect;

[0306] (2) The fusion protein described in the second aspect;

[0307] (3) The nucleic acid molecules described in the third aspect;

[0308] (4) The carrier described in the fourth aspect; and,

[0309] (ii) Second immunogenic component.

[0310] In some embodiments, the first immunogenic component and the second immunogenic component are each independently selected from protein components, nucleic acid components, or any combination thereof.

[0311] In some embodiments, the first immunogenic component and the second immunogenic component are formulated to be administered simultaneously or separately.

[0312] In some embodiments, the immunogenic composition further comprises more immunogenic components (e.g., a third immunogenic component, a fourth immunogenic component, a fifth immunogenic component).

[0313] Reagent test kit

[0314] In a ninth aspect, this application provides a kit comprising an immunogen component selected from one or more of the following (1) to (4): (1) the epitope or epitope peptide described in the first aspect; (2) the fusion protein described in the second aspect; (3) the nucleic acid molecule described in the third aspect; and (4) the carrier described in the fourth aspect.

[0315] In some embodiments, the kit further includes a carrier component capable of displaying the immunogen component.

[0316] In some embodiments, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized pedestals, virus-like particles (VLPs), or any combination thereof.

[0317] In some embodiments, the immunogen and carrier components in the kit are provided separately or as a complex.

[0318] In some embodiments, the immunogen component is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture.

[0319] In some implementations, the immunogenic components in the kit are provided in the form of proteins or nucleic acids.

[0320] In some embodiments, the carrier component in the kit is provided in the form of a protein or nucleic acid.

[0321] In some embodiments, the VLP is assembled from proteins derived from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0322] Pharmaceutical Composition

[0323] In a tenth aspect, this application provides a pharmaceutical composition comprising:

[0324] (i) Selected from any one or more of the following (1) to (8):

[0325] (1) The epitope or epitope peptide described in the first aspect;

[0326] (2) The fusion protein described in the second aspect;

[0327] (3) The nucleic acid molecules described in the third aspect;

[0328] (4) The carrier described in the fourth aspect;

[0329] (5) The host cell described in the fifth aspect;

[0330] (6) The vaccine described in the seventh aspect;

[0331] (7) The immunogenic composition described in aspect eight;

[0332] (8) The reagent kit described in aspect nine; and

[0333] (ii) Pharmaceutically acceptable carriers and / or excipients, buffers, adjuvants, or any combination thereof.

[0334] In some preferred embodiments, the pharmaceutically acceptable carrier and / or excipient is selected from pH adjusters (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic, or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0335] In some preferred embodiments, the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including petroleum-derived, animal-, plant-derived, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In some preferred embodiments, the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

[0336] In some preferred embodiments, the pharmaceutically acceptable excipient may be selected from starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and any combination thereof.

[0337] In some preferred embodiments, the pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder (e.g., lyophilized powder), sustained-release formulation, etc.

[0338] The pharmaceutical compositions of the present invention can be administered by various suitable methods. Suitable methods of administration include, but are not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, oral, nasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In some preferred embodiments, the pharmaceutical compositions are formulated into pharmaceutical preparations suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans according to conventional procedures.

[0339] Typically, pharmaceutical compositions intended for injection (e.g., intravenous administration, such as by bolus or continuous infusion) are sterile and isotonic. If desired, such pharmaceutical compositions may also contain solubilizers and local anesthetics such as ergotamine to reduce pain at the injection site. Furthermore, pharmaceutical compositions intended for injection may contain preservatives. In some preferred embodiments, pharmaceutical compositions intended for injection may also be available in unit dose form (e.g., stored in ampoules or in multi-dose containers).

[0340] In some embodiments, the pharmaceutical compositions of the present invention may contain additional active ingredients, such as additional vaccines, antiviral agents, and / or monoclonal antibodies.

[0341] In some embodiments, the additional vaccine, antiviral agent, and / or monoclonal antibody is an additional vaccine, antiviral agent, and / or monoclonal antibody against RSV and / or hMPV; or a vaccine, antiviral agent, and / or monoclonal antibody against pathogenic organisms other than RSV and / or hMPV.

[0342] In some embodiments, the truncated epitope or epitope peptide described in the first aspect, or the fusion protein described in the second aspect, or the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect, or the host cell described in the fifth aspect, or the vaccine described in the seventh aspect, or the immunogenic composition described in the eighth aspect, or the kit described in the ninth aspect, may be administered simultaneously, separately, or sequentially with the other active ingredients.

[0343] use

[0344] In another aspect, this application provides the use of the epitope or epitope peptide described in the first aspect, or the fusion protein described in the second aspect, or the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect, or the host cell described in the fifth aspect, or the vaccine described in the seventh aspect, or the immunogenic composition described in the eighth aspect, or the kit described in the ninth aspect, in the preparation of a pharmaceutical composition for inducing an immune response in a subject to RSV and / or hMPV.

[0345] In some implementations, the immune response includes inducing the subject to produce antibodies (e.g., neutralizing antibodies) against RSV and / or hMPV.

[0346] In some implementations, the subject is a mammal, such as a mouse or a human.

[0347] In some implementations, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women carrying a fetus.

[0348] In another aspect, this application provides the use of the epitope or epitope peptide described in the first aspect, or the fusion protein described in the second aspect, or the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect, or the host cell described in the fifth aspect, or the vaccine described in the seventh aspect, or the immunogenic composition described in the eighth aspect, or the kit described in the ninth aspect, in the preparation of a pharmaceutical composition for the prevention and / or treatment of RSV and / or hMPV infection or diseases and / or symptoms caused by RSV and / or hMPV infection.

[0349] In some implementations, the subject is a mammal, such as a mouse or a human.

[0350] In some implementations, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women carrying a fetus.

[0351] In some implementations, the illness caused by RSV and / or hMPV infection is a respiratory illness (e.g., a lower respiratory tract illness).

[0352] In some implementations, the diseases and symptoms caused by RSV and / or hMPV infection are selected from bronchitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0353] method

[0354] In another aspect, this application provides a method for inducing antibodies against RSV and / or hMPV, the method comprising administering an effective amount of the epitope or epitope peptide described in the first aspect, or the fusion protein described in the second aspect, or the nucleic acid molecule described in the third aspect, or the vector described in the fourth aspect, or the host cell described in the fifth aspect, or the vaccine described in the seventh aspect, or the immunogenic composition described in the eighth aspect, or the kit described in the ninth aspect, either in vitro or in a subject.

[0355] In some embodiments, the administration method includes intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration.

[0356] In some implementations, the subject is a mammal, such as a mouse or a human.

[0357] In some implementations, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women carrying a fetus.

[0358] In another aspect, this application provides a method for detecting in vitro whether a subject is infected with RSV and / or hMPV, the method comprising: contacting a biological sample obtained from the subject with the epitope or epitope peptide described in the first aspect, or the fusion protein described in the second aspect; and detecting the presence of a complex formed by the epitope or epitope peptide or the fusion protein and an antibody.

[0359] In some implementations, the subject is a mammal, such as a mouse or a human.

[0360] In some implementations, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women carrying a fetus.

[0361] In some embodiments, the biological sample is selected from whole blood, serum, plasma, or any combination thereof.

[0362] On the other hand, this application provides a method for screening candidate drugs capable of inhibiting RSV and / or hMPV infection of cells, the method comprising contacting the host cells with the candidate drug before, simultaneously with or after contacting the epitope or epitope peptide described in the first aspect or the fusion protein described in the second aspect with the host cells.

[0363] Terminology Definition

[0364] In this article, the term "respiratory syncytial virus (RSV)" refers to a virus belonging to the genus Pneumovirus in the family Pneumoviridae. The RSV genome is approximately 15 kb in length, containing 10 genes encoding 11 proteins, including 8 structural proteins (F, G, M2-1, M2-2, SH, N, P, L) and 3 non-structural proteins (NS1, NS2, NS3). Among these, the fusion protein (F) and the attachment protein (G) are two major envelope glycoproteins. The F protein is a type I glycoprotein that can be cleaved by cellular proteases into F1 and F2 polypeptides.

[0365] RSV strains exist in subtypes A and B, and their F protein amino acid sequences share approximately 90% homology. To date, multiple RSV strains have been isolated and their F protein amino acid sequences have been reported. For example, an example sequence of the F protein of subtype A is provided in SEQ ID NO:1 (A2 strain; GenBank GI: QGW56794.1; Swiss Prot P03420), and an example sequence of the F protein of subtype B is provided in SEQ ID NO:2 (18537 strain; GenBank GI: 138250; Swiss Prot P13843). Both SEQ ID NO:1 and SEQ ID NO:2 are 574 amino acid sequences. In this document, RSV encompasses naturally occurring RSV as well as RSV produced through genetic drift, artificial synthesis, and / or recombinant processes, and includes variants derived from naturally occurring RSV.

[0366] In this article, the term "human metapneumovirus (hMPV)" refers to a virus belonging to the genus Metapneumovirus in the family Pneumoviridae. Similar to RSV, hMPV comprises two main envelope glycoproteins: a fusion protein (F) and an attachment protein (G). Furthermore, like RSV, the hMPV F protein is a type I glycoprotein, first translated into a single polypeptide precursor (F0), which is then cleaved by cellular proteases into F1 and F2 polypeptides.

[0367] As used herein, the terms “F protein” or “fusion protein” or “F protein polypeptide” or “fusion protein polypeptide” refer to a polypeptide or protein of all or part of the amino acid sequence of the fusion protein in RSV or hMPV, or to a polypeptide or protein of all or part of the amino acid sequence of the protein resulting from the fusion of RSV and hMPV.

[0368] As used herein, the terms “wild,” “wildtype,” or “natural” are used interchangeably. When these terms are used to describe nucleic acid molecules, polypeptides, or proteins, they mean that the nucleic acid molecule, polypeptide, or protein exists in nature, is found in nature, and has not undergone any artificial modification or processing. As used herein, the F protein of wild-type respiratory syncytial virus (RSV) refers to a naturally occurring, biologically active F protein. As used herein, the F protein of wild-type human metapneumovirus (hMPV) refers to a naturally occurring, biologically active F protein.

[0369] As used herein, the term "F0 polypeptide (F0)" or "F0 precursor" refers to the precursor polypeptide of the RSV or hMPV F protein. The RSV strain's F0 polypeptide consists of 574 amino acids. In vivo, F0 oligomerizes in the endoplasmic reticulum and is proteasically processed by furin at two conserved furin proteases (furin cleavage sites), namely RARR (SEQ ID NO: 62) and RKRR (SEQ ID NO: 63), to produce an oligomer consisting of two disulfide-linked fragments. The smaller of these fragments is F2, which originates from the N-terminal portion of the F0 precursor. The larger fragment is F1, which originates from the C-terminal portion of the F0 precursor.

[0370] As used herein, the terms “pep27 polypeptide” or “pep27 domain” are used interchangeably, referring to a polypeptide consisting of 27 amino acids cleaved from the F0 precursor during RSV F protein maturation. The pep27 domain is flanked by two furin cleavage sites, which are cleaved by cellular proteases during F protein maturation to generate F1 and F2 polypeptides.

[0371] As used herein, the terms “multimerizing domain,” “multimerizing motif,” “foldon,” “fold domain,” or “foldon” have the same meaning and are used interchangeably. They refer to amino acid sequences capable of forming multimers. In some embodiments, they can facilitate the assembly of peptides or proteins into dimers, trimers, tetramers, or pentamers.

[0372] In this document, the term "epitope" refers to a specific set of atoms or amino acids on an antigen that an antibody binds to. Epitopes can be linear or conformational. A "linear epitope" is formed from a continuous amino acid sequence from an antigen and interacts with the antibody according to its primary structure. A "conformational epitope," on the other hand, is formed from discontinuous portions of the antigen's amino acid sequence and interacts with the antibody according to the antigen's 3D structure. Typically, epitopes are five or six amino acids long or longer. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope within the antigen. In some embodiments, a conformational epitope may be found on one form of the protein (e.g., the pre-fusion conformation of the RSV F protein) but not on another form of the protein (e.g., the post-fusion conformation of the RSV F protein).

[0373] As used herein, the term “immunogenicity” refers to the ability of a substance, in the presence or absence of an adjuvant, to elicit, trigger, stimulate, or induce an immune response against a specific antigen in a human or animal.

[0374] As used herein, the term "immunogen" or "immunogen component" refers to a substance that is immunogenic. In some embodiments, symptoms or illness caused by a pathogen are prevented (or mitigated or improved) by inhibiting the replication of the pathogen (e.g., RSV and / or hMPV) after a subject has been exposed to the pathogen. In this document, "immunogen" or "immunogen component" should be understood to encompass substances (e.g., vaccines) intended for administration to a subject or group of subjects to elicit a protective or palliative immune response against RSV and / or hMPV.

[0375] As used herein, the term "immunogenic composition" refers to a composition of substances suitable for administration to human or animal subjects (e.g., in an experimental setting) that can elicit a specific immune response, such as a specific immune response against pathogens such as RSV and / or hMPV. Therefore, an immunogenic composition contains one or more antigens (e.g., peptide antigens) or antigenic epitopes. An immunogenic composition may also contain one or more other components capable of eliciting or enhancing an immune response, such as excipients, carriers, and / or adjuvants.

[0376] As used herein, the term "immune response" refers to the response of immune system cells, such as B cells, T cells, or monocytes, to a stimulus. An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen-specific neutralizing antibodies. An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some embodiments, the response is specific to a particular antigen (i.e., an "antigen-specific response"). If the antigen is derived from a pathogen, the antigen-specific response is a "pathogen-specific response." A "protective immune response" refers to an immune response that inhibits the harmful functions or activities of a pathogen, reduces pathogen infection, or alleviates symptoms (including death) arising from pathogen infection. In this document, immune response encompasses all of the above.

[0377] As used herein, the term "adjuvant" refers to an agent that enhances the production of an immune response in a nonspecific manner. Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) onto which antigens are adsorbed, as well as emulsions; said emulsions may include water-in-oil and oil-in-water (and their variants, including double emulsions and reversible emulsions), lipoglycosides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like receptor agonists (especially TLR2, TLR4, TLR7 / 8, and TLR9 agonists), and various combinations of the above components.

[0378] As used herein, the term "mutation" refers to the presence of a missing, added, or substituted amino acid residue in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. In the specification (particularly the examples), the substitution of an amino acid at a specific position in the protein sequence is expressed as "(amino acid residue in wild-type protein)(amino acid position)(amino acid residue in engineered protein)". For example, "V207L" means that the valine (V) residue at position 207 of the amino acid sequence of the reference protein is replaced by a leucine (L) residue.

[0379] As used in this article, the term "variant" refers to a nucleic acid or polypeptide that is different from a reference nucleic acid or polypeptide.

[0380] As used herein, the term "corresponding position" refers to the amino acid position at an equivalent position in the two sequences being compared when performing an optimal alignment, i.e., when the two sequences are aligned to obtain the highest percentage identity. For example, the expression "corresponding to positions 61, 62, 63, 97, and / or 207 in the sequence shown in SEQ ID NO:4" means, when performing an optimal alignment of a sequence with SEQ ID NO:4, i.e., when a sequence is aligned with SEQ ID NO:4 to obtain the highest percentage identity, the amino acid position in the compared sequence that is equivalent to the 61, 62, 63, 97, and / or 207 positions corresponding to SEQ ID NO:4.

[0381] As used herein, the term "identity" refers to the sequence matching between two polypeptides or two nucleic acids. Two compared sequences are identical at a position when the same base or amino acid monomeric subunit occupies the same location (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine). The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (three out of six positions match). Typically, two sequences are compared to produce the maximum identity. Such comparisons can be made using methods readily available, for example, computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453. The percentage identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl Biosci., 4:11-17 (1988)) integrated into the ALIGN program (version 2.0), which uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percentage identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (J MoI Biol. 48:444-453 (1970)) in the GAP program integrated into the GCG software package (available at www.gcg.com), which uses a Blossum 62 matrix or a PAM250 matrix, along with gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0382] As used herein, the term "antigen" refers to a molecule that can be recognized by antibodies. Examples of antigens include those containing antigenic determinants, such as peptides, lipids, polysaccharides, and nucleic acids that are recognized by immune cells.

[0383] As used herein, the term "D25" refers to a specific binding site for RSV. Antibodies at the site, the sequences of which are provided exemplary in WO 2008 / 147196 A2.

[0384] As used in this article, the term "Palivizuma" refers to palivizumab.

[0385] As used in this article, the term "C-terminal truncated by X amino acids" means that the X consecutive amino acids at the very end of the C-terminus are truncated. Similarly, the term "N-terminal truncated by X amino acids" means that the X consecutive amino acids at the very end of the N-terminus are truncated.

[0386] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); 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, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0387] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0388] As is known to those skilled in the art, codons exhibit degeneracy. That is, during protein translation, each amino acid can correspond to one or more codons, for example, up to six codons. Different species show significant differences in their use of degenerate codons encoding a particular amino acid, exhibiting different preferences. This preference phenomenon is known as "codon bias." Therefore, as used herein, the term "codon bias" refers to the situation where a species prefers to use certain specific codons to encode amino acids. Optimizing the sequence of nucleic acid molecules according to codon bias can be particularly advantageous in certain situations, for example, it may help improve the expression level of the protein encoded by the nucleic acid molecule. For example, when using E. coli (or human cells) to express a protein or fragment thereof, optimizing the nucleic acid sequence encoding the protein or fragment thereof against the codon bias of E. coli (or human cells) would be potentially advantageous.

[0389] As used herein, the term "virus-like particle (VLP)" is a multimeric particle whose structure may be similar to or dissimilar to that of a natural virus particle. In some embodiments, a VLP is a natural virus particle. In some embodiments, a VLP is a virus-like particle assembled from proteins. It has been demonstrated that some viral proteins (e.g., capsid proteins, surface proteins, envelope proteins) can spontaneously form VLPs after recombinant expression in a suitable expression system (e.g., RSV, HBV, HEV, HPV).

[0390] As used herein, the term “pharmaceutical acceptable” means something recognized in the pharmaceutical industry as suitable for use in animals, and particularly in humans. As used herein, the term “pharmaceutical acceptable carrier and / or excipient” means a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters (including, but not limited to, phosphate buffers), surfactants (including, but not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including, but not limited to, sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0391] As used herein, pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including petroleum-derived, animal-, plant-based, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Physiological saline is a preferred carrier when administering pharmaceutical compositions intravenously. Saline solutions, as well as aqueous dextran and glycerol solutions, can also be used as liquid carriers, particularly for injectable solutions.

[0392] Pharmaceutically acceptable excipients, as used herein, may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also contain a wetting agent, or an emulsifier such as sodium hyaluronate, or a pH buffer. The pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0393] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cattle, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human.

[0394] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for disease prevention is an amount sufficient to prevent, stop, or delay the onset of disease; an effective amount for disease treatment is an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and other concurrent treatments, etc.

[0395] Beneficial effects of the invention

[0396] The epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein of this application is particularly suitable for constructing fusion F proteins with fragments or variants of F proteins derived from other viruses (e.g., hMPV). Furthermore, the RSV F protein epitope or epitope peptide of this application was used to construct a fusion protein with a truncated form of the human metapneumovirus (hMPV) F protein or a variant of said truncated form. The fusion protein and its corresponding mRNA exhibit increased stability, a higher pre-F protein content, higher protein expression levels, and / or better thermal stability compared to wild-type RSV and / or hMPV F proteins. Moreover, the fusion protein of this application also possesses good immunogenicity and can induce neutralizing antibodies against RSV and / or hMPV F.

[0397] In summary, the fusion protein of this application exhibits good protective and safety profiles and is suitable for various vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and particulate vaccines. Therefore, the fusion protein of this application has significant potential in inducing immune responses to RSV and / or hMPV in subjects, and in preventing and / or treating RSV and / or hMPV infection or diseases and / or symptoms caused by RSV and / or hMPV infection.

[0398] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings and examples. However, those skilled in the art will understand that the following drawings and examples are for illustrative purposes only and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art from the following detailed description of the drawings and preferred embodiments. Attached Figure Description

[0399] Figure 1 shows the SDS-PAGE results of CRM4, where M1: protein marker; R: under reducing conditions; NR: under non-reducing conditions.

[0400] Figure 2 shows the SDS-PAGE results of CRM5, where M1: protein marker; R: under reducing conditions; NR: under non-reducing conditions.

[0401] Figure 3 shows the SDS-PAGE results of CRM6, where M1: protein marker; R: under reducing conditions; NR: under non-reducing conditions.

[0402] Figure 4 shows the SDS-PAGE results of CRM7, where M1: protein marker; R: under reducing conditions; NR: under non-reducing conditions.

[0403] Figure 5 shows the SDS-PAGE results of CRM8, where M1: protein marker; R: under reducing conditions; NR: under non-reducing conditions.

[0404] Figure 6 shows the SDS-PAGE results of CRM9, where M1: protein marker; R: under reducing conditions; NR: under non-reducing conditions.

[0405] Figure 7 shows the Western blot results of CRM5, where M2: protein marker; R: under reducing conditions; NR: under non-reducing conditions; P: multi-tag protein control.

[0406] Figure 8 shows the Western blot results of CRM9, where M2: protein marker; R: under reducing conditions; NR: under non-reducing conditions; P: multi-tag protein control.

[0407] Figure 9 shows the HPLC results for CRM5.

[0408] Figure 10 shows the HPLC results for CRM9.

[0409] Figure 11 shows the results of serum neutralizing antibody detection in mice induced by CRM4-9 on day 35.

[0410] Figure 12 shows the results of viral load detection in mouse lung tissue after CRM4-9 treatment.

[0411] Sequence information

[0412] Information on some of the sequences involved in this invention is provided in Table 1 below.

[0413] Table 1: Sequence Description Detailed Implementation

[0414] The invention will now be described with reference to the following embodiments, which are intended to illustrate the invention (and not limit it).

[0415] Unless otherwise specified, the experiments and methods described in the embodiments are performed in accordance with conventional methods well known in the art and described in various references. For example, conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in this invention can be found in Sambrook, Fritsch, and Maniatis, *Molecular Cloning: A Laboratory Manual*, 2nd edition (1989); *Current Protocols in Molecular Biology* (edited by FM. Ausubel et al., (1987)); the *Methods in Enzymology* series (academic publishing company): *PCR 2: A PRACTICAL APPROACH* (edited by MJ. MacPherson, BD. Hames, and GR. Taylor, (1995)); and *Animal Cell Culture*. CULTURE (edited by R.R. Freshney (1987)).

[0416] Furthermore, unless specific conditions are specified in the examples, conventional conditions or conditions recommended by the manufacturer should be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products. Those skilled in the art will understand that the examples are described by way of illustration and are not intended to limit the scope of protection claimed by the invention. All disclosures and other references mentioned herein are incorporated herein by reference in their entirety.

[0417] Example 1. Design of RSV-hMPV chimeric antigen

[0418] Based on wild-type RSV F protein (SEQ ID NO:4) and wild-type hMPV F protein (SEQ ID NO:5), this embodiment designed six different RSV-hMPV chimeric antigens, named CRM4-CRM9 respectively. The specific amino acid sequences are shown in Table 1 (SEQ ID NO:8 to SEQ ID NO:33). The specific design concept is as follows:

[0419] Using the Amber software package, spatial vector alignment revealed a high structural similarity between the F protein of hMPV and RSV. Therefore, the hMPV and RSV F proteins were chimeric, artificially creating an RSV Φ epitope on hMPV. Analysis of the crystal structure of the RSV-neutralizing antibody complex (5W23\5UDC\5TOK) confirmed that RSV primarily binds to the neutralizing antibody via three discontinuous α-helices. The main neutralizing epitopes are located in amino acid regions 60-100 and 190-240 of RSV, while amino acid regions 301-574 form a relatively independent domain unrelated to antibody binding. Two independent sequences (60-100aa and 190-240aa) containing neutralizing epitopes were extracted separately. Then, using techniques such as MSA feature extraction, deep neural network structure prediction, and potential energy assessment (mainly applying the relevant functions of software packages such as Amber and Large-scale Atomic / Molecular Massively Parallel Simulator), it was determined that the two truncated sequences composed of amino acids 60-98 and 194-242 could retain their structural features and immunogenicity to the greatest extent. Therefore, these two sequences were used as the transplanted sequences to generate CRM4. CRM5-CRM9 were further iteratively optimized based on CRM4.

[0420] Analysis using structural prediction software such as alphafold2 revealed potential spatial and conformational conflicts between some sites in the transplanted RSV epitope region and the hMPV backbone protein. Therefore, a linker sequence was introduced to mitigate these conflicts. Amino acids A, T, V, R, E, and K were introduced near three discontinuous α-helical sequences, and the final linker positions, amino acid numbers, and composition were determined through molecular modeling and energy function evaluation. Based on the same reasons and methods, incompatible amino acid sites in the RSV epitope region were mutated using methods such as cavity filling, electrostatic mutation, and engineered disulfide bonds to stabilize the conformation. Structural analysis of the hMPV backbone protein further optimized the RSV epitope region structure by introducing cavity filling, electrostatic mutation, and engineered disulfide bonds.

[0421] Example 2. Preparation of chimeric antigen protein samples

[0422] We commissioned Nanjing GenScript Biotech Co., Ltd. to prepare protein samples of chimeric molecules using CHO cells. Testing revealed expression of CRM4-9. Among them, CRM5 and CRM9 showed better expression, so we commissioned GenScript to amplify their expression and purified them using a two-step chromatography process involving nickel column and molecular sieve.

[0423] The SDS-PAGE results of CRM4 to CRM9 are shown in Figures 1 to 6, where M1: protein marker; R: under reducing conditions; NR: under non-reducing conditions.

[0424] The Western blot results of CRM5 and CRM9 are shown in Figures 7 and 8, where M2: protein marker; R: under reducing conditions; NR: under non-reducing conditions; P: multi-tag protein control.

[0425] The HPLC results for CRM5 and CRM9 are shown in Figures 9 and 10.

[0426] Example 3. Preparation of chimeric antigen molecule mRNA samples

[0427] Meanwhile, Nanjing Genscript Biotech Co., Ltd. was commissioned to prepare mRNA-LNP samples of the RSV-hMPV chimeric antigen. To ensure the reliability of the mRNA samples, the most widely accepted molecular design and delivery system on the market was used, including Cap1 capping modification, N1-methyl-pseudouridine base modification, and the same LNP delivery system as Moderna's COVID-19 vaccine. The mRNA sequences corresponding to CRM4-9 (SEQ ID NO:36 to SEQ ID NO:41) all sequentially contain a T7 promoter sequence, a 5' UTR, a CDS sequence (with an added stop codon), a 3' UTR sequence, and a polyA sequence (100 A's).

[0428] The main preparation process of mRNA vaccines involves adding a UTR sequence to the 5' end and a polyA tail and UTR sequence to the 3' end of a codon-optimized chimeric protein sequence, followed by insertion into a plasmid. The plasmid is amplified and extracted using *E. coli* and then linearized with the restriction endonuclease BspQI. Using the linearized plasmid as a template, mRNA is obtained through in vitro transcription using T7 RNA polymerase. The transcription template is digested with DNase I, and the mRNA is purified using the NEB-Monarch RNA Cleanup Kits. The purified mRNA is dissolved in acidic RNase-free sodium acetate buffer to obtain an mRNA solution. SM-102, DSPC, cholesterol, and PEG2000-DMG are dissolved in anhydrous ethanol and mixed at a molar ratio of 50:10:38.5:1.5 to obtain a lipid mixture solution. The mRNA and lipid mixture solution is encapsulated using a microfluidic chip, dialyzed into PBS solution, and 10% sucrose is added as a cryoprotectant to obtain the mRNA vaccine.

[0429] Example 4. Detection of in vitro activity of chimeric proteins by nickel plate ELISA

[0430] Using DS-Cav1 protein (McLellan JS et al. Science. 2013) as a positive control for RSV PreF (i.e., Con1 protein in Table 3), the specific binding RSV proteins listed in Table 2 were utilized. Six antibodies—D25 and AM22 antibodies targeting specific sites, RSV-199, MPE8, and M1D2 cross-antibodies against RSV and hMPV, and DS7 antibody against hMPV—were used as primary antibodies to detect CRM5 and CRM9 proteins captured by nickel plates. The reaction of these six antibodies against four different protein concentrations was investigated. The experimental results are shown in Table 3, and the procedure is as follows:

[0431] (1) Rinsing: Use an automatic plate washing machine to wash the nickel plate three times with 1×PBST and spin dry.

[0432] (2) Sealing: Add 200 μl of 5% skim milk to each well and seal overnight at 4°C.

[0433] (3) Incubation of antigens: Dilute each antigen to 10 μg / ml with 1×PBS, and perform 5-fold dilutions at four gradients (10, 2, 0.4, and 0.08 μg / ml, respectively). The diluent is 1×PBS (negative control). Add 100 μl to each well and incubate at 37°C for 1 hour.

[0434] (4) Rinsing: Use an automatic plate washer with 1×PBST to rinse five times and spin dry.

[0435] (5) Incubation of primary antibodies: Dilute the primary antibodies (D25, AM22, RSV-199, MPE8, M1D2 and DS7) with 2% skim milk to a concentration of 1 μg / ml, 100 μl per well, and incubate at 37℃ for 1 hour. Detect these six antibodies for each antigen.

[0436] (6) Rinsing: Rinse the microplate 5 times with 1×PBST using a plate washer, and then spin dry.

[0437] (7) Incubation of secondary antibody: Dilute the corresponding secondary antibody (GAH-HRP) with 1×ED at a ratio of 1:5000 and add 100 μl to each well. Incubate at 37°C for 1 hour.

[0438] (8) Rinsing: Rinse the microplate 5 times with 1×PBST using a plate washer, and then spin dry.

[0439] (9) Color development: Mix equal volumes of color developer A and color developer B, 100 μl / well, and incubate at 37°C for 10 minutes.

[0440] (10) Termination: The reaction was terminated by adding 50 μl of the termination solution per well.

[0441] (11) Plate reading: Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance at a wavelength of 450 nm.

[0442] Table 2 Antibody Information

[0443] The results of the nickel plate ELISA assay are shown in Table 3. The results show that the D25 and AM22 antibodies all reacted to the three proteins DS-Cav1, CRM5, and CRM9, indicating that CRM5 and CRM9 do indeed retain RSV. Sites. Cross-antibodies RSV-199 and MPE8 reacted to DS-Cav1, CRM5, and CRM9 proteins, while M1D2 reacted to CRM5 and CRM9 proteins. The antibody DS7 against hMPV showed good reactivity to CRM5 and CRM9 proteins.

[0444] Table 3. Results of nickel plate ELISA detection of chimeric proteins

[0445] Example 5. Immunization and challenge of chimeric protein mRNA vaccine in mice

[0446] We commissioned Jiangsu Kemaqi Biotechnology Co., Ltd. to conduct mouse immunization and challenge protection experiments with the chimeric protein mRNA vaccine. The experimental animals were 5-6 week old Balb / c mice purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., and housed in the ABSL2 animal facility of Jiangsu Kemaqi Biotechnology Co., Ltd.

[0447] The entire experimental procedure was as follows: initial immunization on day 0, booster immunization on day 21 (with blood samples collected on days -1, 14, and 35), RSV A2 strain challenge test on day 49, and euthanasia and gross autopsy on day 54. Experimental groups are shown in Table 4. PBS solution was used as a negative control, and commercially available GSK RSV vaccines Arexvy, DS-Cav1 (McLellan JS et al. Science. 2013) recombinant protein vaccine, and mRNA vaccine were used as positive controls. Mice were anesthetized with isoflurane before challenge, and then infected with RSV A2 strain (1.4 × 10⁻⁶) via intranasal drip in the ABSL-2 laboratory. 5 PFU / animal, 80μL / animal).

[0448] Table 4 Information on immunization samples from animal experiments using chimeric protein mRNA vaccines

[0449] Example 6. Detection of neutralizing antibody titers in mouse serum

[0450] (1) Mouse serum was inactivated by incubation at 56°C for 30 minutes and then serially diluted with serum-free medium at dilutions of 10, 30, 90, 270, 810 and 2430.

[0451] (2) Dilute RSV-A2 virus to 60 PFU / μL with serum-free medium;

[0452] (3) Mix the serially diluted mouse serum with the virus solution in equal volumes and incubate at 37°C for 1 hour;

[0453] (4) Transfer the serum-virus mixture to a 96-well plate containing a monolayer of Hep2 cells and incubate at 37°C and 5% CO2 for 2 hours. Then, remove the supernatant, add cell maintenance medium, and incubate at 37°C and 5% CO2 for 48 hours.

[0454] (5) After 48 hours of culture, cells were incubated with 4% paraformaldehyde solution at room temperature in the dark for 30 minutes, then blocked with 5% BSA solution at room temperature for 1 hour. Cells were then incubated with RSV-F mouse monoclonal antibody as the primary antibody and HRP-labeled goat anti-mouse antibody as the secondary antibody for color development. Finally, OD was measured. 450 .

[0455] The experimental results are shown in Figure 11. Compared with the negative control group, all CRM4-CRM9 groups induced the production of neutralizing antibodies. Furthermore, the neutralizing antibody titers in all CRM4-CRM9 groups were significantly increased (* indicates P < 0.05 compared with the PBS blank control group, ** indicates P < 0.01 compared with the PBS blank control group, and *** indicates P < 0.001 compared with the PBS blank control group), indicating that the chimeric protein of this application can effectively induce the production of RSV neutralizing antibodies.

[0456] Example 7. Detection of viral load in mouse lung tissue

[0457] (1) Take lung tissue from the euthanized mice in Example 5, weigh 50 mg of lung tissue, and flash freeze in liquid nitrogen. Add 1 mL of Trizol, and homogenize the tissue with a homogenizer until there are no obvious particles. Let it stand at room temperature for 10 minutes.

[0458] (2) Add 200 μL of chloroform, shake thoroughly to mix, and let stand at room temperature for 3-5 min. Centrifuge at 12000 rpm for 10 min at 4℃, and transfer 400 μL of the supernatant to a clean 1.5 mL EP tube;

[0459] (3) Add 400 μL of isopropanol and mix well. Let stand at room temperature for 10 min. Then centrifuge at 12000 rpm for 10 min at 4 °C. Discard the supernatant and keep the white precipitate at the bottom.

[0460] (4) Wash the white precipitate twice with 500 μL of 75% ethanol. After removing the remaining liquid with a 10 μL pipette, leave the cap open and allow it to dry at room temperature for 5–10 min.

[0461] (5) Add an appropriate amount of DEPC water to fully dissolve the RNA, place the EP tube on ice, measure the mRNA concentration, and perform reverse transcription according to Table 5.

[0462] Table 5. mRNA reverse transcription system and reaction conditions for viral load detection.

[0463] Using the reverse transcription product as a template, qPCR detection was performed. A standard curve was plotted using the CT value and the known viral titer value to calculate the viral load in the sample to be tested.

[0464] The viral load results are shown in Figure 12. Compared with the negative control PBS, the viral load in the lungs of animals in the CRM5 and CRM6 groups was significantly reduced (p<0.05), indicating that these two molecules have the potential to be developed into a preventive vaccine for RSV.

[0465] Example 8. Detection of neutralizing antibody titers in mouse serum

[0466] A second round of mouse immunization was performed, and the neutralizing activity of serum against hMPV was measured. The sample groups are as follows:

[0467] The specific steps for mouse immunization and serum testing are as follows:

[0468] The experimental animals were 5-6 week old Balb / c mice housed in the ABSL2 animal facility. The experimental procedure consisted of initial immunization on day 0, booster immunization on day 21, and blood collection on day 35. PBS solution was used as a negative control, and the commercially available GSK RSV vaccine Arexvy was used as a positive control. The titer of hMPV neutralizing antibodies was detected using a micro-neutralization method, the specific procedure of which was as follows:

[0469] (1) Prepare 96-well cell culture plates one day before the experiment, with a cell density of 1.5 × 10⁶ cells / well. 4 Cells / well, cultured overnight;

[0470] (2) Dilute the virus solution to 1.5 × 10⁻⁶. 4 FFU / ml. Mix 40 μl of virus solution with the serum diluted sample and incubate at 34°C and 5% CO2 for 1 hour;

[0471] (3) Discard the culture medium in the 96-well cell plate and wash once with 100 μl of PBS. Transfer 60 μl of the incubated virus-serum mixture to the cell plate and centrifuge at 1500 rpm and 34°C for 2 h;

[0472] (4) After centrifugation, discard the liquid, add 0.4% CMC (serum-free, containing TPCK), 100 μl / well, and incubate at 34℃ and 5% CO2 for 48 h;

[0473] (5) Add 100 μl / well of 4% PFA and fix at room temperature for 30 min.

[0474] (6) Gently wash the cell plate three times with PBS, add 50 μl / well of a solution containing 0.2% Triton-X100 prepared with 1% BSA, incubate at room temperature for 30 min, then gently wash three times with PBST, 200 μl / well.

[0475] (7) Add hMPV-specific antibody prepared with 1% BSA, 50 μl / well, and incubate at 37°C for 1 h;

[0476] (8) Wash 3 times with PBST, add HRP-Goat Anti-rabbit IgG (H+L) at 50 μl / well in the dark, and incubate at 37°C for 1 h.

[0477] Wash 3 times with PBST, add TrueBlue for color development in the dark, and add water to stop the color development after 10 minutes.

[0478] The results are as follows:

[0479] Experimental results showed that the chimeric protein and its corresponding mRNA of this application could induce neutralizing antibodies against hMPV F. Among them, the neutralizing antibody titer produced after immunizing mice with CRM5 protein and its corresponding mRNA was significantly higher than that of other samples, especially higher than that of the commercially available GSK RSV vaccine Arexvy.

[0480] Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and variations can be made to the details based on all the published teachings, and all such changes are within the scope of protection of the invention. The entire scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. An epitope or epitope peptide of the respiratory syncytial virus (RSV) F protein, comprising: Amino acid residues corresponding to positions 60 to 70 (e.g., 61 to 70, 60 to 80, 60 to 88, 60 to 98) of the wild-type RSV F protein; and / or, Amino acid residues corresponding to positions 194 to 209 (e.g., 194 to 228, 194 to 238, 194 to 242) of the wild-type RSV F protein; Preferably, the polypeptide or protein containing the epitope or epitope peptide is capable of binding to any one or more monoclonal antibodies or antigen-binding fragments thereof selected from the following: D25, AM22, RSV-199, MPE8, and M1D2.

2. The epitope or epitope peptide of claim 1, comprising: (1) The amino acid residues corresponding to positions 60 to 98 and 194 to 242 of the wild-type RSV F protein; (2) Amino acid residues corresponding to positions 61 to 70 and 194 to 209 of the wild-type RSV F protein; or (3) Amino acid residues corresponding to positions 60 to 88 and 194 to 228 of the wild-type RSV F protein; Preferably, the epitope or epitope peptide comprises: (1) The amino acid residues corresponding to positions 60 to 242 of the wild-type RSV F protein; (2) Amino acid residues corresponding to positions 61 to 209 of the wild-type RSV F protein; or (3) The amino acid residues corresponding to positions 60 to 228 of the wild-type RSV F protein.

3. A fusion protein comprising: A first truncated form or a variant thereof of the RSV F protein, comprising amino acid residues at positions 60 to 70 (e.g., 61 to 70, 60 to 80, 60 to 88, 60 to 98) of the wild-type RSV F protein; and / or A second truncated form or variant thereof of the RSV F protein contains amino acid residues at positions 194 to 209 (e.g., 194 to 228, 194 to 238, 194 to 242) of the wild-type RSV F protein; in, The variant, compared to the wild-type RSV F protein, has one or more (e.g., conserved substitutions), deletions, and / or additions of amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

4. The fusion protein of claim 3, further comprising additional proteins or polypeptides; Preferably, the additional protein is a portion of the RSV F protein or a variant thereof; Preferably, the additional protein is an F protein derived from other viruses, or a portion thereof, or a variant thereof; Preferably, the additional protein is a truncated form of wild-type human metapneumovirus (hMPV) F protein or a variant of the truncated form; in, The variant, compared to the truncated form, has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) amino acid substitutions (e.g., conservative substitutions), deletions, or additions. Preferably, the fusion protein further comprises: a first truncated form of hMPV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, and / or a second truncated form of hMPV F protein or a variant thereof.

5. The fusion protein of claim 3 or 4, comprising any one of the following: (1) A first truncated form of RSV F protein or a variant thereof, which contains amino acid residues at positions 60 to 98 corresponding to wild-type RSV F protein; A second truncated form or variant thereof of the hMPV F protein, comprising 50-80 (e.g., 50-60, 60-70, 70-80; e.g., 68) amino acid residues derived from the wild-type hMPV F protein; and, A second truncated form or a variant thereof of the RSV F protein, comprising amino acid residues corresponding to positions 194 to 242 of the wild-type RSV F protein; (2) The first truncated form of RSV F protein or a variant thereof, which contains amino acid residues corresponding to positions 61 to 70 of wild-type RSV F protein; A second truncated form or variant thereof of the hMPV F protein, comprising 80-120 (e.g., 80-90, 90-100, 100-110, or 110-120) amino acid residues derived from wild-type or non-wild-type RSV F protein; and, A second truncated form or variant thereof of RSV F protein, comprising amino acid residues corresponding to positions 194 to 209 of wild-type RSV F protein; (3) A first truncated form of RSV F protein or a variant thereof, which contains amino acid residues at positions 60 to 88 corresponding to wild-type RSV F protein; A second truncated form or variant thereof of the hMPV F protein, comprising 70-100 (e.g., 70-80, 80-90, 90-100; e.g., 85) amino acid residues derived from wild-type or non-wild-type RSV F protein; and, A second truncated form or variant thereof of the RSV F protein, which contains amino acid residues corresponding to positions 194 to 228 of the wild-type RSV F protein.

6. The fusion protein described in claim 4 or 5, wherein, The second truncated form of the hMPV F protein or a variant thereof contains amino acid residues corresponding to positions 60-100 (e.g., 60-63, 63-70, 70-78, 78-80, 80-85, 85-90, 90-95, 95-100) to 150-170 (e.g., 150-155, 155-160, 160-162, 162-165, 165-170) of the wild-type hMPV F protein; Preferably, the second truncated form or a variant of the hMPV F protein comprises: (1) The amino acid residues corresponding to positions 95 to 162 of the wild-type hMPV F protein; (2) Amino acid residues corresponding to positions 63 to 162 of the wild-type hMPV F protein; or (3) The amino acid residues corresponding to positions 78 to 162 of the wild-type hMPV F protein.

7. The fusion protein of claim 5 or 6, comprising: (1) A first truncated form of RSV F protein or a variant thereof, which contains amino acid residues at positions 60 to 98 corresponding to wild-type RSV F protein; A second truncated form or variant thereof of the hMPV F protein, comprising amino acid residues corresponding to positions 95 to 162 of the wild-type hMPV F protein; and, A third truncated form of RSV F protein or a variant thereof, which contains amino acid residues corresponding to positions 194 to 242 of the wild-type RSV F protein; (2) The first truncated form of RSV F protein or a variant thereof, which contains amino acid residues corresponding to positions 61 to 70 of wild-type RSV F protein; A second truncated form or variant thereof of the hMPV F protein, comprising amino acid residues corresponding to positions 63 to 162 of the wild-type hMPV F protein; and, A second truncated form or a variant thereof of the RSV F protein, comprising amino acid residues corresponding to positions 194 to 209 of the wild-type RSV F protein; or (3) A first truncated form of RSV F protein or a variant thereof, which contains amino acid residues at positions 60 to 88 corresponding to wild-type RSV F protein; A second truncated form or variant thereof of the hMPV F protein, comprising amino acid residues corresponding to positions 78 to 162 of the wild-type hMPV F protein; The first truncated form or variant thereof of the RSV F protein contains amino acid residues corresponding to positions 194 to 228 of the wild-type RSV F protein.

8. The fusion protein according to any one of claims 3-7, wherein the fusion protein further comprises: a first truncated form of the hMPV F protein or a variant thereof, wherein, The first truncated form of the hMPV F protein or a variant thereof corresponds to the positions of amino acid residues from positions 16-24 (e.g., positions 16, 17, 18, 19, 20, 21, 22, 23, or 24) to positions 48-58 (e.g., positions 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 58) of the wild-type hMPV F protein; The variant, compared to the truncated form, has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) amino acid substitutions (e.g., conservative substitutions), deletions or additions; Preferably, the first truncated form of the hMPV F protein or a variant thereof corresponds to the position of amino acid residues from position 19 to position 51 or 52 of the wild-type hMPV F protein.

9. The fusion protein according to any one of claims 3-8, wherein the fusion protein further comprises: a third truncated form of the hMPV F protein or a variant thereof, wherein, The third truncated form of the hMPV F protein or its variants correspond to the positions of amino acid residues from positions 170-220 (e.g., positions 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, or 220) to positions 485-495 (e.g., positions 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, or 495) of the wild-type hMPV F protein; The variant, compared to the truncated form, has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) amino acid substitutions (e.g., conservative substitutions), deletions or additions; Preferably, the third truncated form of the hMPV F protein or a variant thereof corresponds to the positions of amino acid residues 180, 199, or 213 to 490 of the wild-type hMPV F protein.

10. The fusion protein according to any one of claims 1-7, wherein, The wild-type RSV is a strain of subtype A, a strain of subtype B, or a strain derived from subtype A or subtype B. Preferably, the wild-type RSV is selected from strain A2, strain Ontario, strain Buenos Aires, or strain B18537. Preferably, the wild-type RSV F protein comprises, or is composed of, sequences selected from, the following: (i) The sequence shown in any one of SEQ ID NO: 1-4; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in any one of SEQ ID NO: 1-4; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with any of the sequences shown in SEQ ID NO: 1-4.

11. The fusion protein according to any one of claims 3-10, wherein the variant, compared with the wild-type RSV F protein, further comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions; Preferably, the amino acid substitutions included in the variant are selected from (i) cavity-filling mutations, (ii) electrostatic mutations, (iii) engineered disulfide bond mutations, or any combination thereof; Preferably, the cavity filling abruptly changes to V207L; Preferably, the electrostatic mutation is S63N or S63D.

12. The fusion protein of claim 11, wherein the amino acid substitutions are selected from positions 61, 62, 63, 97 and / or 207 corresponding to the wild-type RSV F protein; Preferably, the variant has one or more features selected from the following: (1) The amino acid V at position 61 corresponding to the wild-type RSV F protein in the variant is V; Preferably, the amino acid substitution at position 61 is an L to V substitution; (2) The amino acid at position 62 corresponding to the wild-type RSV F protein in the variant is G; Preferably, the amino acid substitution at position 62 is an S-to-G substitution; (3) The amino acid at position 63 corresponding to the wild-type RSV F protein in the variant is N; Preferably, the amino acid substitution at position 63 is an S-to-N or D substitution; (4) The amino acid at position 97 of the variant, corresponding to the wild-type RSV F protein, is E; Preferably, the amino acid substitution at position 97 is an M-to-E substitution; (5) The amino acid at position 207 of the variant, corresponding to the wild-type RSV F protein, is L; Preferably, the amino acid substitution at position 207 is a V-to-L substitution.

13. The fusion protein according to any one of claims 3-12, wherein, The wild-type hMPV is a strain of subtype A, a strain of subtype B, or a strain derived from subtype A or subtype B. Preferably, the hMPV is selected from strains ACJ53565.1, AHV79858.1, BBB35088.1, AHV79473.1, AAS22125.1, AUF72445.1 and ACJ53575.1; Preferably, the wild-type hMPV F protein comprises, or is composed of, sequences selected from, the following sequences. composition: (i) The sequence shown in SEQ ID NO:5; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in SEQ ID NO: 5; (iii) A sequence having at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity with the sequence shown in SEQ ID NO:

5.

14. The fusion protein according to any one of claims 3-13, wherein, Compared to the wild-type hMPV F protein, the variant also contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) amino acid substitutions; Preferably, the amino acid substitutions included in the variant are selected from (i) cavity-filling mutations, (ii) electrostatic mutations, (iii) engineered disulfide bond mutations, or any combination thereof; Preferably, the electrostatic mutation is selected from E453Q, N466S, I449V, or any combination thereof; Preferably, the disulfide bond mutation of the engineering modification is selected from L110C, T127C, A140C, A147C, N153C, N322C, T365C, V463C, or any combination thereof.

15. The fusion protein of claim 14, wherein the variant comprises amino acid substitutions selected from positions 82, 100, 101, 110, 114, 127, 138, 140, 147, 153, 185, 219, 231, 232, 322, 365, 449, 453, 463 and / or 466 corresponding to wild-type hMPV F protein; Preferably, the variant has one or more features selected from the following: (1) The amino acid at position 82 of the variant corresponding to the wild-type hMPV F protein is R; Preferably, the amino acid substitution at position 82 is a K-to-R substitution; (2) The amino acid at position 100 corresponding to the wild-type hMPV F protein in the variant is R; preferably, the amino acid substitution at position 100 is a substitution from Q to R; (3) The amino acid at position 101 corresponding to the wild-type hMPV F protein in the variant is R; preferably, the amino acid substitution at position 101 is a substitution from S to R; (4) The amino acid at position 110 of the variant corresponding to the wild-type hMPV F protein is C; preferably, the amino acid substitution at position 110 is an L to C substitution; (5) The amino acid at position 114 of the variant corresponding to the wild-type hMPV F protein is T; preferably, the amino acid substitution at position 114 is a substitution from A to T; (6) The amino acid at position 127 of the variant corresponding to the wild-type hMPV F protein is C; preferably, the amino acid substitution at position 127 is a substitution from T to C; (7) The amino acid at position 138 of the variant corresponding to the wild-type hMPV F protein is K; preferably, the amino acid substitution at position 138 is an N-to-K substitution; (8) The amino acid at position 140 of the variant corresponding to the wild-type hMPV F protein is C; preferably, the amino acid substitution at position 140 is a substitution from A to C; (9) The amino acid at position 147 of the variant corresponding to the wild-type hMPV F protein is C; preferably, the amino acid substitution at position 147 is a substitution from A to C; (10) The amino acid at position 153 corresponding to the wild-type hMPV F protein in the variant is C; preferably, the amino acid substitution at position 153 is an N-to-C substitution; (11) The amino acid at position 185 of the variant corresponding to the wild-type hMPV F protein is P; preferably, the amino acid substitution at position 185 is a substitution from D to P; (12) The amino acid at position 219 of the variant corresponding to the wild-type hMPV F protein is K; preferably, the amino acid substitution at position 219 is a substitution from L to K; (13) The amino acid at position 231 corresponding to the wild-type hMPV F protein in the variant is I; preferably, the amino acid substitution at position 231 is a substitution from V to I; (14) The amino acid at position 232 corresponding to the wild-type hMPV F protein in the variant is S; preferably, the amino acid substitution at position 232 is a substitution from P to S; (15) The amino acid at position 322 corresponding to the wild-type hMPV F protein in the variant is C; preferably, the amino acid substitution at position 322 is an N-to-C substitution; (16) The amino acid at position 365 of the variant corresponding to the wild-type hMPV F protein is C; preferably, the amino acid substitution at position 453 is a T-to-C substitution; (17) The amino acid at position 449 of the variant corresponding to the wild-type hMPV F protein is V; preferably, the amino acid substitution at position 322 is a substitution from I to V; (18) The amino acid at position 453 corresponding to the wild-type hMPV F protein in the variant is Q; preferably, the amino acid substitution at position 453 is a substitution from E to Q; (19) The amino acid at position 463 corresponding to the wild-type hMPV F protein in the variant is C; preferably, the amino acid substitution at position 463 is a substitution from V to C; (20) The amino acid at position 466 of the variant, corresponding to the wild-type hMPV F protein, is N; Preferably, the amino acid substitution at position 466 is an N-to-S substitution.

16. The fusion protein according to any one of claims 4-15, wherein the additional polypeptide is selected from: signal peptide, polymerization domain, tag, or any combination thereof; Preferably, the signal peptide has one or more of the following characteristics: (1) The signal peptide is a natural signal peptide of hMPV or a variant thereof, or a natural signal peptide of other organisms (e.g., RSV) or a variant thereof; (2) The signal peptide is located at one end (e.g., the N-terminus) of the fusion protein; (3) The signal peptide is located at the N-terminus of the first truncated form of the hMPV F protein or a variant thereof; (4) The signal peptide is linked to a first truncated form or a variant thereof of the hMPV F protein, with or without the first linker peptide; (5) The signal peptide has the sequence shown in SEQ ID NO:17; Preferably, the label has one or more features selected from the following: (1) The tag is selected from multiple histidine tags, antigen or epitope tags, enzyme tags, or any combination thereof; (2) The tag is located at one end of the fusion protein (e.g., the C-terminus); (3) The label is located at the C end of the polymerization domain.

17. The fusion protein of claim 16, wherein, The multimerization domain has one or more of the following characteristics: (1) The multimerging domain is a dimerizing domain, a trimerizing domain, a tetramerizing domain, or a pentamerizing domain; (2) The polymerization domain is derived from T4 phage fibrin; Preferably, the multimerizing domain has a sequence as shown in SEQ ID NO:6; (3) The polymerization domain is a leucine zipper or an isoleucine zipper; Preferably, the multimerizing domain has a sequence as shown in SEQ ID NO:7; (4) The polymerization domain is derived from lung surfactant protein; (5) The polymerization domain is located at the C-terminus of the fusion protein.

18. The fusion protein according to any one of claims 3-17, wherein, The fusion protein comprises, from N-terminus to C-terminus, the following: a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, and a second truncated form of RSV F protein or a variant thereof; Preferably, the fusion protein comprises, from the N-terminus to the C-terminus, the following components in sequence: a first truncated form of hMPV F protein or a variant thereof, a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, a second truncated form of RSV F protein or a variant thereof, and a third truncated form of hMPV F protein or a variant thereof; Preferably, the fusion protein comprises, from the N-terminus to the C-terminus, the following components in sequence: a first truncated form of hMPV F protein or a variant thereof, a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, a second truncated form of RSV F protein or a variant thereof, and a third truncated form of hMPV F protein or a variant thereof, and a polymerization domain; Preferably, the fusion protein comprises, from the N-terminus to the C-terminus, a signal peptide, a first truncated form of hMPV F protein or a variant thereof, a first truncated form of RSV F protein or a variant thereof, a second truncated form of hMPV F protein or a variant thereof, a second truncated form of RSV F protein or a variant thereof, and a third truncated form of hMPV F protein or a variant thereof, and a polymerizing domain.

19. The fusion protein of claim 18, having one or more of the following characteristics: (1) The first truncated form of hMPV F protein or a variant thereof and the first truncated form of RSV F protein or a variant thereof are linked by or without a first linker peptide; Preferably, the first linker peptide has the sequence shown in SEQ ID NO:14; (2) The first truncated form of RSV F protein or a variant thereof and the second truncated form of hMPV F protein or a variant thereof are linked by or without a second linker peptide; Preferably, the second linker peptide has the sequence shown in SEQ ID NO:16; (3) The second truncated form of hMPV F protein or a variant thereof and the second truncated form of RSV F protein or a variant thereof are linked by or without a third linker peptide; Preferably, the third linker peptide has a sequence as shown in SEQ ID NO:15 or 35.

20. The fusion protein of claim 18 or 19, wherein, The fusion protein comprises: (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein; (2) The first truncated form of RSV F protein or a variant thereof, which corresponds to the positions of amino acid residues 60 to 98 of wild-type RSV F protein; Furthermore, it also includes amino acid substitutions at positions corresponding to positions 63 and 97 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from S63N and M97E. (3) A second truncated form of hMPV F protein or a variant thereof, corresponding to amino acid residues from position 95 to position 162 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C; (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 242 of the wild-type RSV F protein; Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from V207L; (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 213 to 490 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S; Preferably, the fusion protein has one or more of the following characteristics: (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:26; (3) The second truncated form or variant thereof of the hMPV F protein has the sequence shown in SEQ ID NO:20; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:30; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:

23.

21. The fusion protein of claim 18 or 19, wherein, The fusion protein comprises: (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 52 of wild-type hMPV F protein; (2) The first truncated form of RSV F protein or a variant thereof, which corresponds to the positions of amino acid residues 61 to 70 of wild-type RSV F protein; Furthermore, it also includes an amino acid substitution at the position corresponding to the 63rd position of the wild-type RSV F protein; preferably, the amino acid substitution is selected from S63N; (3) A second truncated form of hMPV F protein or a variant thereof, corresponding to amino acid residues 63 to 162 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C; (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 209 of the wild-type RSV F protein; Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from V207L; (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 180 to 490 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: corresponding to positions 185, 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from D185P, L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S; Preferably, the fusion protein has one or more of the following characteristics: (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:19; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:30; (3) The second truncated form or variant thereof of the hMPV F protein has the sequence shown in SEQ ID NO:23; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:33; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:

26.

22. The fusion protein of claim 18 or 19, wherein, The fusion protein comprises: (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein; (2) The first truncated form of RSV F protein or a variant thereof, which corresponds to the positions of amino acid residues 60 to 98 of wild-type RSV F protein; Furthermore, it also includes amino acid substitutions at positions corresponding to positions 63 and 97 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from S63N and M97E. (3) A second truncated form of hMPV F protein or a variant thereof, corresponding to amino acid residues from position 95 to position 162 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C; (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 242 of the wild-type RSV F protein; Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from V207L; (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 213 to 490 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S; Furthermore, the first truncated form of the hMPV F protein or a variant thereof and the first truncated form of the RSV F protein or a variant thereof are linked by or without a first linker peptide; preferably, the first linker peptide has the sequence shown in SEQ ID NO:14; Furthermore, the second truncated form of the hMPV F protein or a variant thereof and the second truncated form of the RSV F protein or a variant thereof are linked by or without a third linker peptide; preferably, the third linker peptide has the sequence shown in SEQ ID NO:15; Preferably, the fusion protein has one or more of the following characteristics: (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:26; (3) The second truncated form or variant thereof of the hMPV F protein has the sequence shown in SEQ ID NO:20; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:30; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:

23.

23. The fusion protein of claim 18 or 19, wherein, The fusion protein comprises: (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein; (2) The first truncated form of RSV F protein or a variant thereof, corresponding to the positions of amino acid residues 60 to 88 of wild-type RSV F protein; Furthermore, it also includes an amino acid substitution at the position corresponding to the 63rd position of the wild-type RSV F protein; preferably, the amino acid substitution is selected from S63N; (3) The second truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues from position 78 to position 162 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 82, 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from K82R, Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C; (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 228 of the wild-type RSV F protein; Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from V207L; (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 199 to 490 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S; Preferably, the first truncated form of RSV F protein or a variant thereof and the second truncated form of hMPV F protein or a variant thereof are linked by or without a second linker peptide; preferably, the second linker peptide has the form SEQ ID NO:16; Preferably, the fusion protein has one or more of the following characteristics: (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:27; (3) The second truncated form or variant thereof of the hMPV F protein has the sequence shown in SEQ ID NO:21; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:31; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:

24.

24. The fusion protein of claim 18 or 19, wherein, The fusion protein comprises: (1) The first truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 19 to 51 of wild-type hMPV F protein; (2) The first truncated form of RSV F protein or a variant thereof, corresponding to the positions of amino acid residues 61 to 88 of wild-type RSV F protein; Furthermore, it also includes amino acid substitutions at positions corresponding to positions 61, 62, and 63 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from L61V, S62G, and S63D. (3) The second truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues from position 78 to position 162 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 82, 100, 101, 110, 114, 127, 138, 140, 147, and 153 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from K82R, Q100R, S101R, L110C, A114T, T127C, N138K, A140C, A147C, and N153C; (4) A second truncated form of RSV F protein or a variant thereof, corresponding to amino acid residues 194 to 228 of the wild-type RSV F protein; Furthermore, it also includes an amino acid substitution at the position corresponding to position 207 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from V207L; (5) The third truncated form of hMPV F protein or a variant thereof, which corresponds to the positions of amino acid residues 199 to 490 of wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 219, 231, 232, 322, 365, 449, 453, 463, and 466 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from L219K, V231I, P232S, N322C, T365C, I449V, E453Q, V463C, and N466S; Preferably, the second truncated form of the hMPV F protein or a variant thereof and the second truncated form of the RSV F protein or a variant thereof are linked by or without a third linker peptide; preferably, the third linker peptide has the sequence shown in SEQ ID NO:35; Preferably, the fusion protein has one or more of the following characteristics: (1) The first truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:18; (2) The first truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:28; (3) The second truncated form or variant thereof of the hMPV F protein has the sequence shown in SEQ ID NO:21; (4) The second truncated form of the RSV F protein or a variant thereof has the sequence shown in SEQ ID NO:31; (5) The third truncated form of the hMPV F protein or a variant thereof has the sequence shown in SEQ ID NO:

24.

25. The fusion protein according to any one of claims 18-24, wherein, The fusion protein has the following characteristics: (i) The sequence shown in any one of SEQ ID NO: 8-13; (ii) A sequence having one or more amino acid substitutions, deletions and / or additions (e.g., substitutions, deletions and / or additions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases) compared to the sequence shown in any of SEQ ID NO:8-13; (iii) A sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity with any of the sequences shown in SEQ ID NO: 8-13; Preferably, the fusion protein is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture of both; Preferably, the fusion protein is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixture of both.

26. A nucleic acid molecule comprising a nucleotide sequence encoding an epitope or epitope peptide as described in claim 1 or 2, or a fusion protein as described in any one of claims 3-25; Preferably, the nucleotide sequence is codon-optimized or not optimized according to the codon preference of the host cell; Preferably, the nucleic acid molecule is DNA, or an RNA (mRNA) product transcribed from the DNA, or a mixture of both; Preferably, the nucleic acid molecule comprises mRNA encoding an open reading frame (ORF) of the epitope or epitope peptide of claim 1 or 2 or the fusion protein of any one of claims 3-25; Preferably, the mRNA has chemical modifications (e.g., Cap1 capping modification, 1-methyl-pseudouridine modification, 1-ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine); Preferably, the mRNA has a sequence as shown in any one of SEQ ID NO:36-42.

27. A vector comprising the nucleic acid molecule of claim 26; Preferably, the vector is a viral vector; Preferably, the viral vector is selected from: influenza virus vector, reverse transcriptase virus vector, adenovirus vector, adeno-associated virus vector, herpesvirus vector, poxvirus vector, baculovirus vector, papillomavirus vector, or papillomavirus vector.

28. A host cell comprising the epitope or epitope peptide of claim 1 or 2, or the fusion protein of any one of claims 3-25, or the vector of claim 27; Preferably, the host cell is selected from prokaryotic cells (e.g., Escherichia coli cells) or eukaryotic cells; Preferably, the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell; Preferably, the epitope, epitope peptide, or fusion protein is displayed on the surface of the host cell's cell membrane.

29. A method for expressing or producing an epitope or epitope peptide as claimed in claim 1 or 2 or a fusion protein as claimed in any one of claims 3-25, the method comprising culturing a host cell as claimed in claim 28 under conditions that allow protein expression, and optionally, recovering or purifying the expressed epitope or epitope peptide or fusion protein.

30. A vaccine comprising one or more of the following (1) to (4): (1) The epitope or epitope peptide according to claim 1 or 2; (2) The fusion protein according to any one of claims 3-25; (3) The nucleic acid molecule according to claim 26; (4) The carrier according to claim 27; Preferably, the vaccine further comprises an adjuvant and / or a buffer solution; Preferably, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponins, oil and water emulsions, liposomes, nanoparticles (e.g., LPN), or any combination thereof; Preferably, the fusion protein in the vaccine is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture thereof; Preferably, the fusion protein in the vaccine is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixture thereof.

31. An immunogenic composition comprising: (i) The first immunogenic component, wherein, The first immunogenic component is selected from one or more of the following (1) to (4): (1) The epitope or epitope peptide according to claim 1 or 2; (2) The fusion protein according to any one of claims 3-25; (3) The nucleic acid molecule according to claim 26; (4) The carrier according to claim 27; and, (ii) Second immunogenic component.

32. A kit comprising an immunogen component selected from one or more of the following (1) to (4): (1) an epitope or epitope peptide according to claim 1 or 2; (2) a fusion protein according to any one of claims 3-25; (3) a nucleic acid molecule according to claim 26; (4) a vector according to claim 27; Preferably, the kit further comprises a carrier component capable of displaying the immunogen component; Preferably, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized pedicles, virus-like particles (VLPs), or any combination thereof; Preferably, the immunogen component and carrier component in the kit are provided separately or as a complex; Preferably, the immunogen component is in the form of a polymer (e.g., a dimer, trimer, or tetramer), a monomer, or a mixture; Preferably, the immunogen components in the kit are provided in the form of proteins or nucleic acids; Preferably, the carrier component in the kit is provided in the form of protein or nucleic acid; Preferably, the VLP is assembled from proteins obtained from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

33. A pharmaceutical composition comprising: (i) Selected from any one or more of the following (1) to (8): (1) The epitope or epitope peptide according to claim 1 or 2; (2) The fusion protein according to any one of claims 3-25; (3) The nucleic acid molecule according to claim 26; (4) The carrier according to claim 27; (5) The host cell according to claim 28; (6) The vaccine according to claim 30; (7) The immunogenic composition according to claim 31; (8) The kit according to claim 32; and (ii) Pharmaceutically acceptable carriers, excipients, buffers, adjuvants, or any combination thereof; Preferably, the pharmaceutical composition may also contain additional active ingredients; for example, additional vaccines, antiviral agents, and / or monoclonal antibodies; Preferably, the additional vaccine, antiviral agent, and / or monoclonal antibody is an additional vaccine, antiviral agent, and / or monoclonal antibody against RSV and / or hMPV; or a vaccine, antiviral agent, and / or monoclonal antibody against pathogenic organisms other than RSV and / or hMPV.

34. Use of the epitope or epitope peptide of claim 1 or 2, or the fusion protein of any one of claims 3-25, or the nucleic acid molecule of claim 26, or the vector of claim 27, or the host cell of claim 28, or the vaccine of claim 30, or the immunogenic composition of claim 31, or the kit of claim 32 in the preparation of a pharmaceutical composition, wherein the pharmaceutical composition is used to induce an immune response in a subject to RSV and / or hMPV; Preferably, the immune response includes inducing the subject to produce antibodies (e.g., neutralizing antibodies) against RSV and / or hMPV; Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women with fetuses.

35. The use of the epitope or epitope peptide of claim 1 or 2, or the fusion protein of any one of claims 3-25, or the nucleic acid molecule of claim 26, or the vector of claim 27, or the host cell of claim 28, or the vaccine of claim 30, or the immunogenic composition of claim 31, or the kit of claim 32 in the preparation of a pharmaceutical composition, said pharmaceutical composition for the prevention and / or treatment of RSV and / or hMPV infection or diseases and / or symptoms caused by RSV and / or hMPV infection; Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women with fetuses; Preferably, the disease caused by RSV and / or hMPV infection is a respiratory disease (e.g., a lower respiratory tract disease); Preferably, the diseases and symptoms caused by RSV and / or hMPV infection are selected from bronchitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

36. A method for inducing antibodies against RSV and / or hMPV, the method comprising administering, in vitro in cells or in a subject, an effective amount of the epitope or epitope peptide of claim 1 or 2, or the fusion protein of any one of claims 3-25, or the nucleic acid molecule of claim 26, or the vector of claim 27, or the host cell of claim 28, or the vaccine of claim 30, or the immunogenic composition of claim 31, or the kit of claim 32; Preferably, the method of application includes intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration; Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women with fetuses.

37. A method for detecting the presence of RSV and / or hMPV infection in a subject in vitro, the method comprising: Contact a biological sample obtained from the subject with the epitope or epitope peptide of claim 1 or 2 or the fusion protein of any one of claims 3-25; and detect the presence of a complex formed by the epitope or epitope peptide or fusion protein and an antibody. Preferably, the subject is a mammal, such as a mouse or a human; Preferably, the subjects are selected from infants (e.g., no more than 1 year old), children (e.g., 1 to 6 years old), adolescents (e.g., 7 to 17 years old), adults (e.g., 18 to 60 years old), the elderly (e.g., over 60 years old) and pregnant women with fetuses; Preferably, the biological sample is selected from whole blood, serum, plasma, or any combination thereof.

38. A method for screening candidate drugs capable of inhibiting RSV and / or hMPV infection of cells, the method comprising contacting the host cells with the candidate drug before, simultaneously with or after contacting the epitope or epitope peptide of claim 1 or 2 or the fusion protein of any one of claims 3-25 with the host cells.

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