RSV and HMPV fusion F proteins and uses thereof

Abstract

Provided are a truncated respiratory syncytial virus (RSV) F protein or a variant thereof, and a fusion protein comprising the same and a truncated body of human metapneumovirus (hMPV) F protein or a variant of the truncated body. Also provided are uses of the fusion protein as well as vaccines, immunogenic compositions, kits and pharmaceutical compositions comprising the same for the prevention and / or treatment of RSV and / or hMPV infection or diseases and / or symptoms caused by RSV and / or hMPV infection.

Description

RSV and hMPV fusion F proteins and uses thereof

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese patent application 202410796102.2, filed on June 19, 2024, the entire contents of which are incorporated herein by reference in their entirety. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular, the present application relates to a truncated respiratory syncytial virus (RSV) F protein or a variant thereof, and a fusion protein comprising a truncation of the RSV F protein or a variant of said truncation and a truncation of a human metapneumovirus (hMPV) F protein. The present application also relates to the use of said fusion protein and a vaccine, immunogenic composition, kit and pharmaceutical composition comprising the same for the prevention and / or treatment of RSV and / or hMPV infection or a disease and / or a symptom caused by RSV and / or hMPV infection. BACKGROUND

[0004] Respiratory syncytial virus (RSV) is one of the most important pathogens causing lower respiratory tract infection in infants and young children worldwide. In addition to infants and children, the elderly and immunosuppressed population are also at high risk of RSV infection, and the elderly often lead to obstructive pulmonary disease with cardiopulmonary complications. On May 4, 2023, GSK announced that the FDA has approved Arexvy for the prevention of lower respiratory tract disease caused by RSV infection in people aged 60 and older. This vaccine is the first RSV vaccine approved for the elderly worldwide, containing a recombinant pre-fusion conformational 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 disease caused by RSV infection in people aged 60 and older. ABRYSVO does not contain adjuvants and is composed of two Pre F proteins, which can provide maximum protection against RSV A and B subgroups infection, and its safety and effectiveness have been proven.

[0006] In addition to the two giants of multinational pharmaceutical companies, GSK and Pfizer, the RSV vaccine based on mRNA technology from the American company Moderna is also progressing rapidly. Currently, there is no RSV-related product approved in China.

[0007] Recent 20 years of research found that RSV F protein has pre-F and post-F conformation, and pre-F conformation can induce more effective neutralizing antibodies, which may be the key to solve the ERD reaction of inactivated vaccine. Therefore, the antigen based on the pre-F protein conformation becomes an important direction for RSV vaccine development.

[0008] At present, the pre-F conformation protein used by most manufacturers is derived from the work published by Jason S. McLellan et al. in SCIENCE in 2013. In order to prevent the molecule from rearranging into the post-F conformation, the team analyzed the molecular structure of the F protein in the pre-F conformation, analyzed the most effective neutralizing epitope of the F protein through structure, introduced two mutations (S155C and S290C) at the C-terminal end of the F protein to form a disulfide bond between them to increase the stability of the protein; In addition, modifications were also made at the two groove positions of the conformation (S190F and V207L) to fill them. The F protein modified by the above method is expressed by CHO cells and can be used to produce stable Pre F trimer vaccine (DS-Cav1). These modifications prevent the molecule from rearranging into the post-F conformation.

[0009] Human metapneumovirus (hMPV) was discovered in 2001, belonging to the family of pneumoviridae, and is one of the main pathogens causing acute lower respiratory tract infection. The first infection of hMPV usually occurs in children under 5 years old, and hMPV can be repeatedly infected, causing serious diseases in adults (especially in the elderly and immunodeficient patients). At present, there is no preventive vaccine or specific antiviral drug approved for hMPV.

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

[0011] In summary, in view of the blank of hMPV and RSV vaccine market, it is necessary to further develop vaccine products for RSV and / or hMPV. SUMMARY

[0012] During the preliminary investigation, the applicants found that the F protein of hMPV and RSV are highly similar in both function and structure. Both of them can cause respiratory tract disease, and the dominant epitopes of neutralizing antibodies are just in different domains of F protein. Therefore, the applicants proposed an idea that the key antigenic proteins (F proteins) of RSV and hMPV are chimerically modified to create a fusion F protein with double antigenic epitopes. Further, experiments confirmed that the fusion F protein can maintain the pre-F conformation and can induce neutralizing antibodies of hMPV and / or RSV.

[0013] Truncated respiratory syncytial virus (RSV) F protein or a variant thereof

[0014] Therefore, in a first aspect, the present application provides a truncated respiratory syncytial virus (RSV) F protein or a variant thereof, which has 50-60 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60) amino acid residues deleted from the N-terminus and / or 260-270 (e.g., 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270) amino acid residues deleted from the C-terminus compared to a wild-type RSV F protein.

[0015] In some embodiments, the truncated RSV F protein or a variant thereof of the present application retains at least one immunodominant epitope or a key fragment thereof of the pre-fusion conformation of F protein. In some embodiments, the truncated RSV F protein or a variant thereof of the present application is capable of binding to antibody D25.

[0016] In some embodiments, the truncated RSV F protein or a variant thereof of the present application is particularly suitable for constructing a fusion F protein. In some preferred embodiments, the truncated respiratory syncytial virus (RSV) F protein or a variant thereof is particularly suitable for constructing a fusion F protein with a fragment or a variant thereof derived from F protein of other viruses (e.g., hMPV).

[0017] In some embodiments, the fusion F protein constructed from the truncated RSV F protein or a variant thereof has increased stability compared to a wild-type RSV F protein. In some embodiments, the stability is measured by the binding of the fusion F protein to antibody D25.

[0018] In some embodiments, the fusion F protein constructed from the truncated RSV F protein or a variant thereof has increased stability compared to a wild-type RSV F protein. In some embodiments, the stability is measured by the binding of the fusion F protein to antibody D25.

[0019] In certain embodiments, the fusion F protein constructed from the truncated RSV F protein or variant thereof has a higher protein expression level compared to wild-type RSV F protein.

[0020] In certain embodiments, the fusion F protein constructed from the truncated RSV F protein or variant thereof has a higher pre-F protein fraction compared to wild-type RSV F protein. In certain embodiments, the fusion F protein comprises a pre-F conformation (pre-F) protein, a post-F conformation (post-F) protein, or a mixture of both. In certain embodiments, the pre-F protein fraction is measured by the ratio of antibody D25 and antibody palivizumab binding to the fusion F protein.

[0021] Stabilizing modifications

[0022] The truncated RSV F protein or variant thereof of the present application stabilizes the pre-F conformation by introducing one or more modifications, such as substitution (e.g., conservative substitution), deletion, and / or addition of one or more amino acids. It is understood that these stabilizing modifications do not adversely affect the immunologically dominant epitopes of the pre-F conformation of the F protein that are retained by the truncated RSV F protein or variant thereof of the present application or key fragments thereof.

[0023] A first stabilizing modification is to eliminate the furin cleavage site and / or the pep27 domain.

[0024] The furin cleavage site is located between the F2 domain and the Fl domain in the F0 precursor protein. One or both of the furin cleavage sites can be eliminated by deleting or replacing one or more amino acids of the furin cleavage site. In such embodiments, the F protein with the furin cleavage site deleted is able to avoid being cleaved into its constituent domains to maintain stability. In certain embodiments, one furin cleavage site is located at amino acid residues 105-109 of wild-type RSV F protein. In certain embodiments, the other furin cleavage site is located at amino acid residues 133-136 of wild-type RSV F protein. In certain embodiments, a sequence (e.g., a linker) can be used to replace the sequence of the furin cleavage site.

[0025] Thus, in certain embodiments, the truncated RSV F protein or variant thereof has at least one furin cleavage site deleted or has one or more amino acids of the furin cleavage site deleted compared to wild-type RSV F protein.

[0026] Similarly, the pep27 domain is located between the F2 domain and the Fl domain in the F0 precursor protein. The pep27 domain can be eliminated by deleting or replacing one or more amino acids of the pep27 domain. In certain embodiments, the pep27 domain is located at amino acid residues 110-136 of a wild-type RSV F protein.

[0027] In certain embodiments, the truncated RSV F protein or variant thereof lacks 1 or more (e.g., 1-5, 5-10, 10-15, 15-20, 20-27) amino acids of the pep27 domain as compared to a wild-type RSV F protein.

[0028] In certain embodiments, the furin cleavage site has a sequence as set forth in SEQ ID NO: 22.

[0029] In certain embodiments, the pep27 domain has a sequence as set forth in SEQ ID NO: 21.

[0030] In certain embodiments, the truncated RSV F protein lacks 54 amino acid residues at the N-terminus and 267 amino acid residues at the C-terminus as compared to a wild-type RSV F protein.

[0031] Another stabilizing modification is the introduction of an addition (insertion) of amino acids.

[0032] In certain embodiments, the variant of the truncated RSV F protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid additions as compared to the truncated RSV F protein.

[0033] In certain embodiments, the variant has increased stability as compared to the truncated RSV F protein; for example, increased stability of the variant itself, or of a fusion protein that it forms with another protein. In certain embodiments, the fusion protein is a chimeric F protein, and the amino acid addition is capable of stabilizing the pre-F conformation of the chimeric F protein.

[0034] In certain embodiments, the variant has higher protein expression as compared to the truncated RSV F protein.

[0035] In certain embodiments, the variant comprises an amino acid addition at a position selected from the group consisting of: between positions corresponding to positions 95 and 96, between positions corresponding to positions 147 and 148, between positions corresponding to positions 149 and 150, or any combination thereof, of a wild-type RSV F protein.

[0036] In certain embodiments, the sequence of the amino acids added between positions 95 and 96 is set forth in SEQ ID NO: 8, 9, or 23.

[0037] In certain embodiments, the sequence of the amino acids added between positions 147 and 148 is set forth in SEQ ID NO: 10.

[0038] In certain embodiments, the sequence of the amino acids added between positions 149 and 150 is set forth in SEQ ID NO: 11.

[0039] Another stabilizing modification is the introduction of a substitution (mutation) of an amino acid.

[0040] In certain embodiments, the truncated RSV F protein or variant thereof comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions compared to a wild-type RSV F protein.

[0041] In certain embodiments, the substitution is a conservative substitution.

[0042] In certain embodiments, the variant has one or more of the following selected from the group consisting of:

[0043] (1) has increased stability; for example, increased stability of the variant itself, or of a fusion protein it forms with another protein. In certain embodiments, the fusion protein is a chimeric F protein, and the amino acid addition stabilizes the pre-F conformation of the chimeric F protein;

[0044] (2) has higher protein expression;

[0045] In certain embodiments, the amino acid substitutions comprised by the variant are selected from the group consisting of (i) cavity-filling mutations, (ii) electrostatic mutations, (iii) engineered disulfide mutations, or any combination thereof.

[0046] Cavity-filling mutations

[0047] In certain embodiments, the variant comprises one or more cavity-filling mutations. In certain embodiments, such cavity-filling mutations contribute to the stability of the trimer in which they themselves or a fusion protein comprising them are formed. In this context, the term “cavity-filling mutation” refers to an amino acid residue of a protein that forms an internal cavity in the spatial structure being substituted with an amino acid residue that is capable of filling said internal cavity. Cavities formed by a protein can be identified by methods known in the art, such as by looking at the crystal structure of the protein or a fusion protein comprising 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]).

[0048] In certain embodiments, the cavity-filling mutation in a variant of the truncated RSV F protein of the present application is selected from S190F, V207L, or any combination thereof.

[0049] Engineered disulfide bond mutations

[0050] In certain embodiments, the variant comprises one or more engineered disulfide bond mutations. In this context, the term “engineered disulfide bond mutation” refers to a pair of amino acid residues in a protein being mutated to a pair of cysteine residues. The introduced pair of cysteine residues allows for the formation of a disulfide bond between the introduced cysteine residues, which is used to stabilize the conformation or oligomeric state of the protein, such as the prefusion conformation. Such residues can be identified by suitable methods known in the art, such as by looking at the crystal structure of the protein or a fusion protein comprising it, or by using computational protein design software.

[0051] In certain embodiments, the engineered disulfide bond mutation in a variant of the truncated RSV F protein of the present application is selected from S155C, S290C, or any combination thereof.

[0052] Electrostatic mutations

[0053] In certain embodiments, the variant comprises one or more electrostatic mutations. In certain embodiments, such electrostatic mutations contribute to the stability of the trimer in which it is or the fusion protein comprising it is formed. In the present context, the term "electrostatic mutation" refers to a mutation that is capable of reducing the ion repulsion or increasing the ion attraction between amino acid residues in a protein that are in close proximity to each other in the folded structure. Since hydrogen bonding is a special case of ion attraction, electrostatic mutations can increase hydrogen bonding between such proximal amino acid residues. Typically, the introduction of an electrostatic mutation will increase the Tm value of the protein or the fusion protein comprising it.

[0054] Unfavorable electrostatic interactions upon formation of the trimeric conformation can be identified by methods known in the art, such as by looking at the crystal structure of the protein or the fusion protein comprising it in the trimeric conformation, or by using computational protein design software.

[0055] In certain embodiments, the electrostatic mutations in the variants of the truncated RSV F proteins of the present application are selected from S63N, S105N, S275N, or any combination thereof.

[0056] In certain embodiments, the variant comprises an amino acid substitution at a position selected from the group consisting of: position 63, 105, 152, 155, 186, 190, 207, 266, 275, and / or 290, with respect to the wild-type RSV F protein. In certain embodiments, the variant comprises an amino acid substitution at a position selected from the group consisting of: position 63, 105, 152, 155, 186, 190, 207, 266, 275, and / or 290, with respect to the sequence set forth in any one of SEQ ID NOs: 1-4.

[0057] In certain embodiments, the amino acid at position 63 with respect to the wild-type RSV F protein is N.

[0058] In certain embodiments, the amino acid substitution at position 63 is a substitution of S to N.

[0059] In certain embodiments, the amino acid at position 105 with respect to the wild-type RSV F protein is N.

[0060] In certain embodiments, the amino acid substitution at position 105 is a substitution of S to N.

[0061] In certain embodiments, the amino acid at position 152 with respect to the wild-type RSV F protein is V.

[0062] In certain embodiments, the amino acid substitution at position 152 is a substitution of I to V.

[0063] In certain embodiments, the variant has an amino acid at a position corresponding to position 155 of the wild-type RSV F protein that is C.

[0064] In certain embodiments, the amino acid substitution at position 155 is a substitution of S to C.

[0065] In certain embodiments, the variant has an amino acid at a position corresponding to position 186 of the wild-type RSV F protein that is R.

[0066] In certain embodiments, the amino acid substitution at position 186 is a substitution of S to R.

[0067] In certain embodiments, the variant has an amino acid at a position corresponding to position 190 of the wild-type RSV F protein that is F.

[0068] In certain embodiments, the amino acid substitution at position 190 is a substitution of S to F.

[0069] In certain embodiments, the variant has an amino acid at a position corresponding to position 207 of the wild-type RSV F protein that is L.

[0070] In certain embodiments, the amino acid substitution at position 207 is a substitution of V to L.

[0071] In certain embodiments, the variant has an amino acid at a position corresponding to position 266 of the wild-type RSV F protein that is T.

[0072] In certain embodiments, the amino acid substitution at position 266 is a substitution of I to T.

[0073] In certain embodiments, the variant has an amino acid at a position corresponding to position 275 of the wild-type RSV F protein that is N.

[0074] In certain embodiments, the amino acid substitution at position 275 is a substitution of S to N.

[0075] In certain embodiments, the variant has an amino acid at a position corresponding to position 290 of the wild-type RSV F protein that is C.

[0076] In certain embodiments, the amino acid substitution at position 290 is a substitution of S to C.

[0077] Wild-type RSV F protein

[0078] The native RSV F proteins exhibit a high degree of sequence conservation between different subtypes of RSV. For example, RSV subtypes A and B share 90% sequence identity. Further, within a RSV homosubtype, the sequence identity of the F protein is even higher, with RSV F proteins having about 98% sequence identity within each of RSV subtype A or subtype B. Also, almost all identified RSV F proteins consist of 574 amino acids in length, with only minor differences in length often due to the length of the C-terminal cytoplasmic tail. The sequence identity between these native RSV F proteins is known in the art (see, e.g., WO 2014 / 160463).

[0079] In view of the conservation of the sequence of RSV F proteins, one of ordinary skill in the art can readily compare the amino acid positions between the sequences of different native RSV F proteins to identify the amino acid positions of the corresponding RSV F protein between different RSV strains or subtypes. For example, the furin cleavage site is located at the same amino acid position in almost all identified native RSV F proteins. Thus, the conservation of the native RSV F protein sequence across strains or subtypes enables it to be used as a reference RSV F sequence to compare the amino acids at a particular position in a RSV F protein.

[0080] As used herein, when referring to the amino acid sequence of a wild-type RSV F protein, it is described using the sequence set forth in any one of SEQ ID NOs: 1-4. For example, the expression "position 63 of the wild-type RSV F protein" refers to the 63rd amino acid residue of the protein set forth in any one of SEQ ID NOs: 1-4. However, one skilled in the art understands that there are multiple versions of wild-type RSV F proteins that have substantially identical primary structures (i.e., amino acid sequences) and higher order structures (i.e., spatial structures), as well as substantially identical biological functions, but can still have minor differences in the amino acid sequence from one another. Therefore, in the present application, wild-type RSV F protein is not limited to the protein set forth in any one of SEQ ID NOs: 1-4, but is intended to encompass all known wild-type RSV F proteins. Thus, in the present application, the term "wild-type RSV F protein" shall include various naturally-occurring, biologically functional RSV F proteins, including, for example, the RSV F protein set forth in any one of SEQ ID NOs: 1-4, as well as naturally-occurring variants thereof. Also, when describing the amino acid positions of a RSV F protein, it includes not only the particular amino acid position in any one of SEQ ID NOs: 1-4, but also the amino acid position corresponding to the particular amino acid position in a naturally-occurring variant thereof. For example, the expression "position 63 of the wild-type RSV F protein" includes the 63rd amino acid residue of any one of SEQ ID NOs: 1-4, as well as the corresponding amino acid position in a naturally-occurring variant thereof. According to the present application, the expression "corresponding amino acid position" refers to the amino acid position in a sequence being compared that is at an equivalent position when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percent identity.

[0081] In certain embodiments, the wild-type RSV is a strain of sub-type A, a strain of sub-type B, or is derived from a strain of sub-type A or sub-type B.

[0082] In certain embodiments, the wild-type RSV is selected from strain A2, strain Ontario, strain Buenos Aires, or strain B18537.

[0083] In certain embodiments, the wild-type RSV F protein comprises or consists of a sequence selected from the group consisting of:

[0084] (i) the sequence set forth in any one of SEQ ID NOs: 1-4;

[0085] (ii) a sequence which has one or more substitutions, deletions and / or additions of amino acids (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, deletions and / or additions of amino acids) compared to the sequence shown in any one of SEQ ID NOs: 1 -4;

[0086] (iii) a sequence which has at least 90% (e.g. 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity to the sequence shown in any one of SEQ ID NOs: 1 -4.

[0087] Fusion protein

[0088] In a second aspect, the present application provides a fusion protein comprising the truncated RSV F protein or variant thereof of the first aspect, and a further protein or polypeptide. It will be appreciated that these further proteins or polypeptides do not adversely affect the activity and / or function of the truncated RSV F protein or variant thereof.

[0089] Further protein

[0090] In certain embodiments, the further protein is an F protein or a portion thereof or a variant thereof derived from other viruses.

[0091] In certain embodiments, the further protein is located N-terminal and / or C-terminal to the truncated RSV F protein or variant thereof.

[0092] In certain embodiments, the further protein is a truncation of a wild-type human metapneumovirus (hMPV) F protein or a variant of the truncation; wherein the variant has one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14 or 15) substitutions (e.g. conservative substitutions), deletions or additions of amino acids compared to the truncation.

[0093] In certain embodiments, the truncation of a hMPV F protein or variant of the truncation of the present application retains at least one immunodominant epitope or key fragment thereof of the pre-fusion conformation of the F protein.

[0094] Wild-type hMPV F protein

[0095] The native hMPV F protein exhibits a high degree of sequence conservation among different subtypes of hMPV. For example, hMPV subtypes A and B share 90% sequence identity. Further, within a hMPV subtype, the sequence identity of the F protein is even higher, with hMPV F proteins having about 98% sequence identity within each of hMPV subtype A or subtype B. Exemplary strains of hMPV F protein are summarized in Boivin et al. Emerg. Infect. Dis. 10: 1154-1157 (2004), which summary is incorporated herein by reference for the disclosure of hMPV sequences.

[0096] In view of the conservation of the sequence of the hMPV F protein, one of ordinary skill in the art can readily compare the amino acid positions between the sequences of different native hMPV F proteins to identify the corresponding amino acid positions of the hMPV F protein between different hMPV strains or subtypes. Thus, the conservation of the native hMPV F protein sequence among strains or subtypes enables it to be used as a reference hMPV F sequence to compare the amino acids at a particular position in a hMPV F protein.

[0097] As used herein, when referring to the amino acid sequence of a wild-type hMPV F protein, it is described using the sequence set forth in SEQ ID NO: 5. For example, the expression "position 322 of the wild-type RSV F protein" refers to the 322nd amino acid residue of the protein set forth in SEQ ID NO: 5. However, those skilled in the art understand that there are multiple versions of wild-type hMPV F proteins that have substantially identical primary structures (i.e., amino acid sequences) and higher order structures (i.e., spatial structures), as well as substantially identical biological functions, but can still have minor differences in the amino acid sequence from one another. Therefore, in the present application, wild-type hMPV F protein is not limited to the protein set forth in SEQ ID NO: 5, but is intended to encompass all known wild-type hMPV F proteins. Thus, in the present application, the term "wild-type hMPV F protein" shall include various naturally-occurring, biologically functional hMPV F proteins, including, for example, the RSV F protein set forth in SEQ ID NO: 5, as well as naturally-occurring variants thereof. Also, when describing the amino acid positions of a hMPV F protein, it includes not only the specific amino acid position in SEQ ID NO: 5, but also the amino acid position corresponding to the specific amino acid position in its naturally-occurring variants. For example, the expression "position 322 of the wild-type hMPV F protein" includes the 322nd amino acid residue of SEQ ID NO: 5, as well as the corresponding amino acid position in its naturally-occurring variants. According to the present application, the expression "corresponding amino acid position" refers to the amino acid position at the equivalent position in the sequence being compared when the sequences are optimally aligned, i.e., when the sequences are aligned to obtain the highest percentage identity.

[0098] In certain embodiments, the hMPV is a strain of subtype A, a strain of subtype B, or is derived from a strain of subtype A or subtype B.

[0099] In certain embodiments, the hMPV is selected from the group consisting of strain ACJ53565.1, strain AHV79858.1, strain BBB35088.1, strain AHV79473.1, strain AAS22125.1, strain AUF72445.1, and strain ACJ53575.1.

[0100] In certain embodiments, the wild-type hMPV F protein comprises or consists of a sequence selected from the group consisting of:

[0101] (i) the sequence set forth in SEQ ID NO: 5;

[0102] (ii) a sequence that has one or more substitutions, deletions, and / or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base substitutions, deletions, and / or additions) of amino acids compared to the sequence set forth in SEQ ID NO: 5;

[0103] (iii) a sequence that has at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) sequence identity to the sequence set forth in SEQ ID NO: 5.

[0104] In certain embodiments, the fusion protein comprises: a first truncation of a hMPV F protein or a variant thereof, the truncated RSV F protein of the first aspect or a variant thereof, and a second truncation of a hMPV F protein or a variant thereof.

[0105] In certain embodiments, the first truncation of a hMPV F protein or a variant thereof corresponds to positions from 15-24 (e.g., position 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) to 40-50 (e.g., position 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) amino acid residues of a wild-type hMPV F protein.

[0106] In certain embodiments, the first truncation of a hMPV F protein or a variant thereof corresponds to positions from 19 to 45 amino acid residues of a wild-type hMPV F protein.

[0107] In certain embodiments, the second truncation of a hMPV F protein or a variant thereof corresponds to positions from 272-281 (e.g., position 272, 273, 274, 275, 276, 277, 278, 279, 280, or 281) to 485-495 (e.g., position 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, or 495) amino acid residues of a wild-type hMPV F protein.

[0108] In certain embodiments, the second truncation of a hMPV F protein or a variant thereof corresponds to positions from 277 to 490 amino acid residues of a wild-type hMPV F protein.

[0109] Stabilizing modifications

[0110] A truncation of the hMPV F protein or a variant of the truncation stabilizes the pre-F conformation of the fusion protein by introducing one or more modifications, such as substitution (e.g., conservative substitution), deletion, and / or addition of one or more amino acids. It is understood that these stabilizing modifications do not adversely affect the immunologically dominant epitopes of the pre-fusion conformation of the F protein, or key fragments thereof, that are retained by the truncation of the hMPV F protein or a variant of the truncation of the present application.

[0111] One stabilizing modification is the introduction of a substitution (mutation) of an amino acid.

[0112] In certain embodiments, a variant of a second truncation of the F protein of hMPV comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions compared to the second truncation of the F protein of hMPV.

[0113] In certain embodiments, the variant has increased stability compared to the truncation; e.g., increased stability of the variant itself, or of a fusion protein that it forms with another protein. In certain embodiments, the fusion protein is a fusion protein as previously described.

[0114] In certain embodiments, the variant has higher protein expression compared to the truncation.

[0115] In certain embodiments, the amino acid substitutions comprised by the variant are selected from the group consisting of (i) cavity-filling mutations, (ii) electrostatic mutations, (iii) engineered disulfide mutations, or any combination thereof.

[0116] In certain embodiments, the cavity-filling mutations, electrostatic mutations, and engineered disulfide mutations are as defined above.

[0117] In certain embodiments, the electrostatic mutations are selected from the group consisting of E453Q, N466S, I449V, or any combination thereof.

[0118] In certain embodiments, the engineered disulfide mutations are selected from the group consisting of N322C, T365C, V463C, or any combination thereof.

[0119] In certain embodiments, the positions of the amino acid substitutions comprised by the variant are selected from the group consisting of positions corresponding to positions 322, 365, 449, 453, 463, and / or 466 of the wild-type hMPV F protein. In certain embodiments, the positions of the amino acid substitutions comprised by the variant are selected from the group consisting of positions corresponding to positions 322, 365, 449, 453, 463, and / or 466 of the sequence set forth in SEQ ID NO: 5.

[0120] In certain embodiments, the variant has a C at the position corresponding to amino acid 322 of the wild-type hMPV F protein.

[0121] In certain embodiments, the amino acid substitution at position 322 is a substitution of N to C.

[0122] In certain embodiments, the variant has a C at the position corresponding to amino acid 365 of the wild-type hMPV F protein.

[0123] In certain embodiments, the amino acid substitution at position 365 is a substitution of T to C.

[0124] In certain embodiments, the variant has a V at the position corresponding to amino acid 449 of the wild-type hMPV F protein.

[0125] In certain embodiments, the amino acid substitution at position 449 is a substitution of I to V.

[0126] In certain embodiments, the variant has a Q at the position corresponding to amino acid 453 of the wild-type hMPV F protein.

[0127] In certain embodiments, the amino acid substitution at position 453 is a substitution of E to Q.

[0128] In certain embodiments, the variant has a C at the position corresponding to amino acid 463 of the wild-type hMPV F protein.

[0129] In certain embodiments, the amino acid substitution at position 463 is a substitution of V to C.

[0130] In certain embodiments, the variant has a S at the position corresponding to amino acid 466 of the wild-type hMPV F protein.

[0131] In certain embodiments, the amino acid substitution at position 466 is a substitution of N to S.

[0132] Another stabilizing modification is the addition (insertion) of amino acids.

[0133] In certain embodiments, the variant of the truncation of the hMPV F protein has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid additions compared to the truncation of the hMPV F protein.

[0134] Additional polypeptides

[0135] In certain embodiments, the fusion protein as described previously, the additional polypeptide is selected from the group consisting of: a signal peptide, a multimerization domain, a tag, or any combination thereof.

[0136] Signal peptide

[0137] To enhance the production or secretion of the protein, the fusion protein of the present application can comprise a signal peptide.

[0138] In certain embodiments, the signal peptide is a native signal peptide of hMPV or a variant thereof, or a native signal peptide derived from other organisms (e.g., RSV) or a variant thereof.

[0139] In certain embodiments, the signal peptide is located at one end (e.g., N-terminus) of the fusion protein.

[0140] In certain embodiments, the signal peptide is located at the N-terminus of the first truncation of hMPV F protein or a variant thereof.

[0141] In certain embodiments, the signal peptide is connected to the first truncation of hMPV F protein or a variant thereof with or without a first linker peptide.

[0142] In certain embodiments, the signal peptide has a sequence as set forth in SEQ ID NO: 12.

[0143] Tag

[0144] In certain embodiments, the fusion protein of the present application can be connected with a tag to facilitate the expression, detection, tracking and / or purification of the protein of the present application. Such epitope tags are well known to those skilled in the art, examples of which 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 tracking).

[0145] In certain embodiments, the tag is selected from the group consisting of a polyhistidine tag, an antigen or epitope tag, an enzyme tag, or any combination thereof.

[0146] In certain embodiments, the tag is located at the end (e.g., C-terminus) of the fusion protein.

[0147] In certain embodiments, the tag is located at the C-terminus of the multimerization domain.

[0148] In certain exemplary embodiments, the tag is a polyhistidine tag, i.e., 6xHis-tag (HHHHHH). In certain exemplary embodiments, the tag is connected to the polyhistidine tag through a restriction site.

[0149] Multimerization domain

[0150] The fusion protein provided herein can be linked to a multimerization domain to facilitate the formation of multimers (e.g., dimers, trimers, tetramers, pentamers) of the recombinant fusion protein.

[0151] Exogenous multimerization domains that can facilitate the formation of stable multimers of soluble proteins are known in the art. In particular, examples of multimerization domains that can be linked to the fusion protein of the present application include, but are not limited to:

[0152] (1) GCN4 leucine zippers (see Harbury et al. 1993 Science 262: 1401-1407 for specific sequences and information);

[0153] (2) trimerization motif from pulmonary surfactant protein (see Hoppe et al. 1994 FEB S Lett 344: 191-195 for specific sequences and information);

[0154] (3) collagen (see McAlinden et al. 2003 Biol Chem 278: 42200-42207 for specific sequences and information); and

[0155] (4) bacteriophage T4 fibritin foldon (see Miroshnikov et al. 1998 Protein Eng 11: 329-414 for specific sequences and information).

[0156] Typically, the multimerization domain is linked to the C-terminus or C-terminus of the fusion protein. It can be directly joined to the fusion protein, or linked to the fusion protein via a linker, such as an amino acid linker, e.g., a sequence of GG, GS, or SAIG. The linker can also be a longer linker (e.g., a sequence that includes the repeat sequence of GG). In some embodiments, the fusion protein can further comprise a protease cleavage site for removing the multimerization domain from the fusion protein.

[0157] In certain embodiments, the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or a pentamerization domain.

[0158] In certain embodiments, the multimerization domain is derived from T4 bacteriophage fibritin. In certain embodiments, the multimerization domain has a sequence as set forth in SEQ ID NO: 6.

[0159] In certain embodiments, the multimerization domain is a leucine zipper or an isoleucine zipper. In certain embodiments, the multimerization domain has a sequence as set forth in SEQ ID NO: 7.

[0160] In certain embodiments, the multimerization domain is derived from a lung surfactant protein.

[0161] In certain embodiments, the multimerization domain is located at the C-terminus of the F1 domain.

[0162] In certain embodiments, the multimerization domain is linked to the second truncation of the hMPV F protein or the variant thereof, with or without a second linker peptide.

[0163] In certain embodiments, the second linker peptide has a sequence as set forth in SEQ ID NO: 24.

[0164] In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: the first truncation of the hMPV F protein or the variant thereof, the truncated RSV F protein or the variant thereof as described in the first aspect, and the second truncation of the hMPV F protein or the variant thereof.

[0165] In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: the first truncation of the hMPV F protein or the variant thereof, the truncated RSV F protein or the variant thereof as described in the first aspect, and the second truncation of the hMPV F protein or the variant thereof, a multimerization domain.

[0166] In certain embodiments, the fusion protein comprises, in order from N-terminus to C-terminus: a signal peptide, the first truncation of the hMPV F protein or the variant thereof, the truncated RSV F protein or the variant thereof as described in the first aspect, and the second truncation of the hMPV F protein or the variant thereof, a multimerization domain; optionally, the multimerization domain is linked to the second truncation of the hMPV F protein or the variant thereof, with or without a second linker peptide.

[0167] Fusion proteins CRM1 and CRM2

[0168] In certain embodiments, the fusion protein comprises:

[0169] (1) the first truncation of the hMPV F protein corresponding to positions 19 to 45 amino acid residues of the wild-type hMPV F protein;

[0170] (2) the variant of the truncated RSV F protein having a deletion of 54 amino acid residues at the N-terminus and a deletion of 267 amino acid residues at the C-terminus compared to the wild-type RSV F protein;

[0171] and which further comprises an amino acid addition at a position corresponding to between positions 95 and 96 and between positions 147 and 148 of the wild-type RSV F protein; in certain embodiments, the sequence of the amino acids added between positions 95 and 96 is set forth in SEQ ID NO: 8 or SEQ ID NO: 23; in certain embodiments, the sequence of the amino acids added between positions 147 and 148 is set forth in SEQ ID NO: 10;

[0172] and which further comprises an amino acid substitution at a position corresponding to positions 63, 105, 152, 155, 190, 207, 275, and 290 of the wild-type RSV F protein; preferably, the amino acid substitution is selected from S63N, S105N, 1152V, S155C, S190F, V207L, S275N, and S290C;

[0173] and,

[0174] (3) a variant of a second truncation of a hMPV F protein corresponding to positions 277 to 490 amino acid residues of a wild-type hMPV F protein;

[0175] and which further comprises an amino acid substitution at a position corresponding to positions 322, 365, 449, 453, 463, and 466 of the wild-type F protein of hMPV; preferably, the amino acid substitution is selected from N322C, T365C, 1449V, E453Q, V463C, and N466S.

[0176] In certain embodiments, the first truncation of a hMPV F protein has a sequence set forth in SEQ ID NO: 13. In certain embodiments, the variant of a truncated RSV F protein has a sequence set forth in SEQ ID NO: 15 or 16. In certain embodiments, the variant of a second truncation of a hMPV F protein has a sequence set forth in SEQ ID NO: 14.

[0177] Fusion protein CRM3

[0178] In certain embodiments, the fusion protein comprises:

[0179] (1) a first truncation of a hMPV F protein corresponding to positions 19 to 45 amino acid residues of a wild-type hMPV F protein;

[0180] (2) a variant of a truncated RSV F protein which has a deletion of 54 amino acid residues at the N-terminus and a deletion of 267 amino acid residues at the C-terminus compared to a wild-type RSV F protein;

[0181] and further comprising an amino acid substitution at a position corresponding to position 63, 105, 152, 155, 186, 190, 207, 266, 275, and 290 of a wild-type RSV F protein; in certain embodiments, the amino acid substitution is selected from the group consisting of S63N, S105N, 1152V, S155C, S186R, S190F, V207L, 1266T, S275N, and S290C;

[0182] and,

[0183] (3) a variant of a second truncation of a hMPV F protein corresponding to positions 277 to 490 of a wild-type hMPV F protein;

[0184] and further comprising an amino acid substitution at a position corresponding to position 322, 365, 449, 453, 463, and 466 of a wild-type F protein of hMPV; in certain embodiments, the amino acid substitution is selected from the group consisting of N322C, T365C, 1449V, E453Q, V463C, and N466S.

[0185] In certain embodiments, the first truncation of a hMPV F protein has a sequence as set forth in SEQ ID NO: 13. In certain embodiments, the variant of a truncated RSV F protein has a sequence as set forth in SEQ ID NO: 17. In certain embodiments, the variant of a second truncation of a hMPV F protein has a sequence as set forth in SEQ ID NO: 14.

[0186] In certain embodiments, the fusion protein has:

[0187] (i) a sequence as set forth in any one of SEQ ID NOs: 18-20;

[0188] (ii) a sequence that has one or more substitutions, deletions, and / or additions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 base substitutions, deletions, and / or additions) of amino acids compared to a sequence as set forth in any one of SEQ ID NOs: 18-20;

[0189] (iii) a sequence that has at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% sequence identity to a sequence as set forth in any one of SEQ ID NOs: 18-20.

[0190] In certain embodiments, the fusion protein is in the form of a multimer (e.g., a dimer, a trimer, a tetramer), in the form of a monomer, or a mixture of both.

[0191] In certain embodiments, the fusion protein is in a pre-F conformation, a post-F conformation, or a mixture of both.

[0192] Nucleic acid molecule

[0193] It will be readily appreciated that the nucleic acid molecule can be used to clone or express the truncated RSV F protein or variant thereof or fusion protein of the application. In certain cases, to improve efficiency, the nucleotide sequence of the nucleic acid molecule can be codon optimized according to cell preference.

[0194] Thus, in a third aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the truncated RSV F protein or variant thereof of the first aspect or the fusion protein of the second aspect.

[0195] In certain embodiments, the nucleotide sequence is codon optimized or not optimized according to codon bias of the host cell.

[0196] mRNA

[0197] The nucleic acid molecule of the present application can include DNA, cDNA and RNA sequences. In certain embodiments, the nucleic acid molecule is DNA, or is an RNA (mRNA) product transcribed from the DNA, or a mixture of both.

[0198] In certain embodiments, the nucleic acid molecule comprises an mRNA encoding an open reading frame (ORF) of the truncated RSV F protein or variant thereof of the first aspect or the fusion protein of the second aspect.

[0199] In certain embodiments, the mRNA has chemical modifications (e.g., Cap1 capping modification, 1 -methyl-pseudouridine modification, 1 -ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine).

[0200] In certain embodiments, the modification comprises a chemical modification. These chemical modifications can confer increased stability and low immunogenicity to the mRNA, thereby facilitating expression of the protein. For example, N1 -methyl-pseudouridine is superior to several other nucleoside modifications in terms of translational capacity. In some embodiments, the mRNA molecule used herein can replace uracil with pseudouracil, such as 1 -methyl-3’-pseudouridylyl base.

[0201] In certain embodiments, the mRNA can comprise a 5’ cap, a 5’ UTR element, an open reading frame codon optimized or not, a 3’ UTR element, and a poly A sequence and / or a polyadenylation signal.

[0202] In certain embodiments, the mRNA has a sequence as set forth in any one of SEQ ID NOs: 25-27.

[0203] For nucleic acid molecules, whether natural or modified, can be delivered in a delivery vehicle such as a lipid nanoparticle. The lipid nanoparticle can comprise one or more nucleic acid molecules in a weight ratio to the lipid nanoparticle of about 5: 1 to about 1 : 100. In some embodiments, the weight ratio of nucleic acid molecules to lipid nanoparticle 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 derivable therein.

[0204] Vectors

[0205] Vectors for expressing the truncated RSV F proteins or variants or fusion proteins thereof of the present application in insect or mammalian cells are well known in the art. These vectors can be cloning vectors or expression vectors. In certain preferred embodiments, the vectors of the present application can be, for example, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1 -derived artificial chromosomes (PACs); bacteriophages such as lambda phage or M13 phage; and viral vectors, among others. Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40).

[0206] In certain preferred embodiments, the vectors of the present application are capable of expressing or are used to express the truncated RSV F proteins or variants or fusion proteins thereof of the present application. In certain preferred embodiments, the vectors of the present application are capable of or are used to express the truncated RSV F proteins or variants or fusion proteins thereof of the present application in vivo in a subject (e.g., a mammal, such as a human).

[0207] The vectors of the present application can contain a variety of elements, including but not limited to one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as transcriptional control elements (e.g., promoters, enhancers, terminators) and / or one or more translational signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell.

[0208] In a fourth aspect, the present application provides a vector comprising the nucleic acid molecule of the third aspect.

[0209] In certain embodiments, the vector is a viral vector.

[0210] In certain embodiments, the viral vector is selected from an influenza virus vector, a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, a herpes virus vector, a poxvirus vector, a baculovirus vector, a papillomavirus vector, or a papovavirus vector.

[0211] Host cell

[0212] The truncated RSV F protein or variant thereof or fusion protein provided herein can be prepared by conventional methods known in the art, such as by expressing the host cell in a recombinant host cell using a suitable vector, including, for example, insect cells, mammalian cells, avian cells, bacteria, and yeast cells. Examples of insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, 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 in, for example, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Baculovirus avian cell expression systems are also known to those skilled in the art and described in, for example, 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 in the art and described in, for example, Yeast Genetic Engineering (Barr et al., eds., 1989) Butterworths, London.

[0213] In a fifth aspect, the present application provides a host cell comprising the truncated RSV F protein or variant thereof of the first aspect or the fusion protein of the second aspect or the nucleic acid molecule of the third aspect or the vector of the fourth aspect.

[0214] In certain embodiments, the host cell is selected from a prokaryotic cell (e.g., an E. coli cell), a eukaryotic cell.

[0215] In certain embodiments, the eukaryotic cell is a mammalian cell, for example, a mouse cell, a human cell.

[0216] In certain embodiments, the truncated RSV F protein or variant fusion protein thereof is displayed on the surface of the cell membrane of the host cell.

[0217] Methods of preparation

[0218] Methods for expressing and purifying the truncated RSV F protein or variant thereof of the first aspect or the fusion protein of the second aspect of the present application are common in the art and can be found, inter alia, in 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 Ed., John Wiley & Sons, Inc., 999.

[0219] Accordingly, in a sixth aspect, the present application provides a method of expressing or producing the truncated RSV F protein or variant thereof of the first aspect or the fusion protein of the second aspect, the method comprising culturing the host cell of the fifth aspect under conditions that allow expression of the protein, and optionally, recovering or purifying the truncated RSV F protein or variant thereof or fusion protein expressed thereby.

[0220] Vaccine

[0221] The vaccine provided by the present application is not limited to a protein form, a nucleic acid form, or a mixture of both. Also, the nucleic acid can be selected from DNA, cDNA, RNA (e.g., mRNA), or any combination thereof.

[0222] In a seventh aspect, the present application provides a vaccine comprising one or more selected from the following (1) to (4):

[0223] (1) the truncated RSV F protein or variant thereof of the first aspect;

[0224] (2) the fusion protein of the second aspect;

[0225] (3) the nucleic acid molecule of the third aspect;

[0226] (4) the vector of the fourth aspect.

[0227] In certain embodiments, the vaccine further comprises an adjuvant and / or a buffer.

[0228] In certain embodiments, the adjuvant is selected from a metal salt, 3-D-mono-phosphoryl lipid A (MPL), saponin, an oil and water emulsion, a liposome, a nanoparticle (e.g., LPN), or any combination thereof.

[0229] In certain embodiments, the fusion protein in the vaccine is in the form of a multimer (e.g., dimer, trimer, tetramer), a monomer, or a mixture.

[0230] In certain embodiments, the fusion protein in the vaccine is in the pre-F conformation, the post-F conformation, or a mixture.

[0231] The vaccines provided herein can be implemented using standard routes of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration. The subject can be given a single administration, as well as one or more booster administrations. If a booster vaccination is performed, this booster vaccination will typically be administered to the same individual between 1 week and 10 years, e.g., between 2 weeks and 6 months, after the first administration to the individual (which in such cases is referred to as the "prime vaccination").

[0232] The vaccines provided herein can also be used with one or more other vaccines. For example, in adults, it can be used with an influenza vaccine, Prevnar, a tetanus vaccine, a diphtheria vaccine, and a pertussis vaccine. For children, the vaccines provided herein can be used with any other vaccine indicated for pediatric patients.

[0233] Immunogenic composition

[0234] In an eighth aspect, the present application provides an immunogenic composition comprising:

[0235] (i) a first immunogenic component, wherein the first immunogenic component is selected from one or more of the following (1) to (4):

[0236] (1) the truncated RSV F protein or variant thereof of the first aspect;

[0237] (2) the fusion protein of the second aspect;

[0238] (3) the nucleic acid molecule of the third aspect;

[0239] (4) the vector of the fourth aspect; and,

[0240] (ii) a second immunogenic component.

[0241] In certain embodiments, the first and second immunogenic components are each independently selected from a protein component, a nucleic acid component, or any combination thereof.

[0242] In certain embodiments, the first immunogenic component and the second immunogenic component are formulated for simultaneous or separate administration.

[0243] In certain embodiments, the immunogenic composition further comprises a plurality of immunogenic components (e.g., a third immunogenic component, a fourth immunogenic component, a fifth immunogenic component).

[0244] Kit

[0245] In a ninth aspect, the present application provides a kit comprising an immunogen component selected from one or more of the following (1) to (4): (1) the truncated RSV F protein or variant thereof of the first aspect; (2) the fusion protein of the second aspect; (3) the nucleic acid molecule of the third aspect; (4) the vector of the fourth aspect.

[0246] In certain embodiments, the kit further comprises a carrier component capable of displaying the immunogen component.

[0247] In certain embodiments, the carrier component is selected from the group consisting of: a nanomaterial (e.g., a lipid nanoparticle, a protein nanoparticle, a polymeric nanoparticle, an inorganic nanocarrier, and a biomimetic nanoparticle), an outer membrane vesicle (OMV) of a bacterium, a multimerization scaffold, a virus-like particle (VLP), or any combination thereof.

[0248] In certain embodiments, the immunogen component and the carrier component in the kit are provided separately or as a complex.

[0249] In certain embodiments, the immunogen component is in the form of a multimer (e.g., a dimer, a trimer, a tetramer), a monomer, or a mixture.

[0250] In certain embodiments, the immunogen component in the kit is provided in the form of a protein or a nucleic acid.

[0251] In certain embodiments, the carrier component in the kit is provided in the form of a protein or a nucleic acid.

[0252] In certain embodiments, the VLP is assembled from proteins obtained from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0253] Pharmaceutical composition

[0254] In a tenth aspect, the present application provides a pharmaceutical composition comprising:

[0255] (i) any one or more selected from the following (1) to (8):

[0256] (1) the truncated RSV F protein or variant thereof of the first aspect;

[0257] (2) the fusion protein of the second aspect;

[0258] (3) the nucleic acid molecule of the third aspect;

[0259] (4) the vector of the fourth aspect;

[0260] (5) the host cell of the fifth aspect;

[0261] (6) the vaccine of the seventh aspect;

[0262] (7) the immunogenic composition of the eighth aspect;

[0263] (8) the kit of the ninth aspect; and

[0264] (ii) a pharmaceutically acceptable carrier and / or excipient, a buffer, an adjuvant, or any combination thereof.

[0265] In certain preferred embodiments, the pharmaceutically acceptable carrier and / or excipient is selected from the group consisting of a pH adjusting agent (including but not limited to phosphate buffer), a surfactant (including but not limited to a cationic, anionic or non-ionic surfactant such as Tween-80), an adjuvant, an ionic strength enhancer (including but not limited to sodium chloride), a diluent, an excipient, a medium for containing or administering a therapeutic agent, and any combination thereof.

[0266] In certain preferred embodiments, the pharmaceutically acceptable carrier can be a sterile liquid such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. In certain preferred embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

[0267] In certain preferred embodiments, the pharmaceutically acceptable excipient can be selected from the group consisting of starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skim milk, glycerol, propylene, glycol, water, ethanol, and any combination thereof.

[0268] In certain preferred embodiments, the pharmaceutical composition can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, e.g., lyophilized powders, sustained release formulations and the like.

[0269] The pharmaceutical compositions of the application can be administered by a variety of suitable means. Suitable means of administration include, but are not limited to, parenteral administration, e.g., intravenous, intradermal, subcutaneous, oral, nasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In certain preferred embodiments, the pharmaceutical compositions are formulated in accordance with routine procedures as pharmaceutical formulations adapted to the appropriate mode of administration.

[0270] Generally, the pharmaceutical compositions for injection (e.g., intravenous administration, e.g., by bolus injection or continuous infusion) are sterile and isotonic. If desired, such pharmaceutical compositions can also include solubilizers and local anesthetics such as procaine to lessen pain at the site of the injection. In addition, the pharmaceutical compositions for injection can also contain preservatives. In certain preferred embodiments, the pharmaceutical compositions for injection can also be presented in unit dosage form (e.g., in ampoules or in multi-dose containers).

[0271] In certain embodiments, the pharmaceutical compositions of the application can comprise additional active ingredients, e.g., additional vaccines, antiviral agents, and / or monoclonal antibodies.

[0272] In certain embodiments, the additional vaccines, antiviral agents, and / or monoclonal antibodies are additional vaccines, antiviral agents, and / or monoclonal antibodies against RSV and / or hMPV; or are vaccines, antiviral agents, and / or monoclonal antibodies against pathogenic organisms other than RSV and / or hMPV.

[0273] In certain embodiments, the truncated RSV F protein or variant thereof of the first aspect, or the fusion protein of the second aspect, or the nucleic acid molecule of the third aspect, or the vector of the fourth aspect, or the host cell of the fifth aspect, or the vaccine of the seventh aspect, or the immunogenic composition of the eighth aspect, or the kit of the ninth aspect, can be administered simultaneously, separately or sequentially with the additional active ingredient.

[0274] Use

[0275] In another aspect, the application provides the use of the truncated RSV F protein or variant thereof of the first aspect, or the fusion protein of the second aspect, or the nucleic acid molecule of the third aspect, or the vector of the fourth aspect, or the host cell of the fifth aspect, or the vaccine of the seventh aspect, or the immunogenic composition of the eighth aspect, or the kit of the ninth aspect, for the manufacture of a pharmaceutical composition for inducing an immune response in a subject against RSV and / or hMPV.

[0276] In certain embodiments, the immune response comprises inducing the subject to produce antibodies (e.g., neutralizing antibodies) against RSV and / or hMPV.

[0277] In certain embodiments, the subject is a mammal, e.g., a mouse, a human.

[0278] In certain embodiments, the subject is selected from the group consisting of an infant (e.g., no more than 1 year old), a child (e.g., 1 to 6 years old), an adolescent (e.g., 7 to 17 years old), an adult (e.g., 18 to 60 years old), a senior (e.g., 60 or older), and a pregnant woman carrying a fetus.

[0279] In another aspect, the present application provides the use of the truncated RSV F protein of the first aspect or a variant thereof, or the fusion protein of the second aspect, or the nucleic acid molecule of the third aspect, or the vector of the fourth aspect, or the host cell of the fifth aspect, or the vaccine of the seventh aspect, or the immunogenic composition of the eighth aspect, or the kit of the ninth aspect, in the manufacture of a pharmaceutical composition for the prevention and / or treatment of RSV and / or hMPV infection or a disease and / or a symptom caused by RSV and / or hMPV infection.

[0280] In certain embodiments, the subject is a mammal, e.g., a mouse, a human.

[0281] In certain embodiments, the subject is selected from the group consisting of an infant (e.g., no more than 1 year old), a child (e.g., 1 to 6 years old), an adolescent (e.g., 7 to 17 years old), an adult (e.g., 18 to 60 years old), a senior (e.g., 60 or older), and a pregnant woman carrying a fetus.

[0282] In certain embodiments, the disease caused by RSV and / or hMPV infection is a respiratory disease (e.g., a lower respiratory tract disease).

[0283] In certain embodiments, the disease and symptom caused by RSV and / or hMPV infection is selected from the group consisting of bronchitis, pneumonia, asthma, obstructive lung disease, and cardiorespiratory complications.

[0284] Methods

[0285] In another aspect, the present application provides a method of inducing an antibody against RSV and / or hMPV, the method comprising administering an effective amount of the truncated RSV F protein of the first aspect or a variant thereof, or the fusion protein of the second aspect, or the nucleic acid molecule of the third aspect, or the vector of the fourth aspect, or the host cell of the fifth aspect, or the vaccine of the seventh aspect, or the immunogenic composition of the eighth aspect, or the kit of the ninth aspect, in a cell in vitro or in a subject in vivo.

[0286] In certain embodiments, the manner of administration comprises intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration.

[0287] In certain embodiments, the subject is a mammal, e.g., a mouse, a human.

[0288] In certain embodiments, the subject is selected from the group consisting of an infant (e.g., no more than 1 year old), a child (e.g., 1 to 6 years old), an adolescent (e.g., 7 to 17 years old), an adult (e.g., 18 to 60 years old), an elderly person (e.g., older than 60), and a pregnant woman carrying a fetus.

[0289] In another aspect, the present application provides a method for detecting the presence or absence of RSV and / or hMPV infection in a subject in vitro, the method comprising: contacting a biological sample obtained from the subject with the truncated RSV F protein or variant thereof of the first aspect, or the fusion protein of the second aspect; and detecting the presence or absence of a complex formed by the truncated RSV F protein or variant thereof or the fusion protein and an antibody.

[0290] In certain embodiments, the subject is a mammal, e.g., a mouse, a human.

[0291] In certain embodiments, the subject is selected from the group consisting of an infant (e.g., no more than 1 year old), a child (e.g., 1 to 6 years old), an adolescent (e.g., 7 to 17 years old), an adult (e.g., 18 to 60 years old), an elderly person (e.g., older than 60), and a pregnant woman carrying a fetus.

[0292] In certain embodiments, the biological sample is selected from the group consisting of whole blood, serum, plasma, or any combination thereof.

[0293] In another aspect, the present application provides a method of screening a candidate drug capable of inhibiting RSV and / or hMPV infection of a cell, the method comprising contacting a host cell with the candidate drug before, simultaneously with, or after contacting the host cell with the truncated RSV F protein or variant thereof of the first aspect, or the fusion protein of the second aspect.

[0294] Definitions of terms

[0295] In the present context, the term "respiratory syncytial virus (RSV)" is a virus of the family Pneumoviridae, genus Pneumovirus. The genome of RSV is about 15 Kb in length and contains 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 them, the fusion protein (F) and the attachment protein (G) are two major envelope glycoproteins. The F protein is a type I glycoprotein and can be cleaved by cellular proteases into F1 and F2 polypeptides.

[0296] There are A and B subtypes of RSV strains, and the F proteins of which are about 90% homologous in amino acid sequence. So far, a number of 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 the A subtype 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 the B subtype 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 sequences of 574 amino acids. In the present context, RSV encompasses naturally occurring RSV as well as RSV produced by genetic drift, artificial synthesis and / or recombination, and also includes variants derived from naturally occurring RSV.

[0297] In the present context, the term "human metapneumovirus (hMPV)" is a virus of the family Pneumoviridae, genus Metapneumovirus. Similarly to RSV, hMPV contains the fusion protein (F) and the attachment protein (G) as two major envelope glycoproteins. Also similarly to RSV, the F protein of hMPV is a type I glycoprotein and is first translated as a single polypeptide precursor (F0) and then can be cleaved by cellular proteases into F1 and F2 polypeptides.

[0298] In the present context, the term "F protein" or "fusion protein" or "F protein polypeptide" or "fusion protein polypeptide" refers to a polypeptide or protein of all or part of the amino acid sequence of the fusion protein (Fusion Protein) in RSV or hMPV, or to a polypeptide or protein of all or part of the amino acid sequence of the protein after fusion of RSV and hMPV.

[0299] As used herein, the terms "wild-type," "wild," or "native" are used interchangeably. When these terms are used to describe a nucleic acid molecule, polypeptide, or protein, it means that the nucleic acid molecule, polypeptide, or protein exists in nature, is found in nature, and has not been subjected to any modification or processing by man. As used herein, a wild-type F protein of respiratory syncytial virus (RSV) refers to a naturally occurring, biologically active F protein. As used herein, a wild-type F protein of human metapneumovirus (hMPV) refers to a naturally occurring, biologically active F protein.

[0300] As used herein, the term "F0 polypeptide (F0)" or "F0 precursor" refers to the precursor polypeptide of the RSV or hMPV F protein. The F0 polypeptide of RSV strain A2 consists of 574 amino acids. In vivo, F0 is oligomerized in the endoplasmic reticulum and proteolytically processed by furin proteases at two conserved furin consensus sequences (furin cleavage sites), RARR (SEQ ID NO: 62) and RKRR (SEQ ID NO: 63) to generate an oligomer consisting of two disulfide-linked fragments. The smaller of these fragments is F2 and is derived from the N-terminal portion of the F0 precursor. The larger is Fl and is derived from the C-terminal portion of the F0 precursor.

[0301] As used herein, the terms "pep27 polypeptide" or "pep27 domain" are used interchangeably and refer to the 27 amino acid polypeptide excised from the F0 precursor during RSV F protein maturation. The sequence of the pep27 domain is flanked by two furin cleavage sites that are cleaved by cellular proteases during F protein maturation to generate the Fl polypeptide and the F2 polypeptide.

[0302] As used herein, the terms "multimerization domain," "multimerization motif," "foldon," "folding domain," or "foldon" have the same meaning and are used interchangeably. It refers to an amino acid sequence that is capable of forming a multimer. In certain embodiments, it is capable of facilitating the assembly of a polypeptide or protein into a dimer, trimer, tetramer, or pentamer.

[0303] The term "epitope" refers herein to a specific group of atoms or amino acids on an antigen that is bound by an antibody. An epitope can be a linear epitope or a conformational epitope. Linear epitopes are formed by contiguous amino acid sequences from an antigen and interact with an antibody according to its primary structure. Conformational epitopes, on the other hand, are composed of non-contiguous parts of an antigen's amino acid sequence and interact with an antibody according to the 3D structure of the antigen. Typically, an epitope is five or six amino acids in length or longer. Two antibodies can bind to the same epitope within an antigen if they exhibit competitive binding to the antigen.

[0304] As used herein, the term "immunogenic" refers to the ability of a substance to elicit, provoke, stimulate, or induce an immune response in a human or animal against a particular antigen, in the presence or absence of an adjuvant.

[0305] As used herein, the term "immunogen" or "immunogen component" refers to a substance that is immunogenic. In certain embodiments, the immunogen prevents (or lessens or ameliorates) symptoms or disease caused by a pathogen (e.g., RSV and / or hMPV) by inhibiting replication of the pathogen after the subject has been exposed to the pathogen. In this context, "immunogen" or "immunogen component" is understood to encompass a substance (e.g., a vaccine) intended for administration to a subject or a population of subjects to elicit a protective or palliative immune response against RSV and / or hMPV.

[0306] As used herein, the term "immunogenic composition" refers to a composition of substances suitable for administration to a human or animal subject (e.g., in an experimental context) that is capable of eliciting a specific immune response, e.g., a specific immune response against a pathogen such as RSV and / or hMPV. Thus, an immunogenic composition comprises one or more antigens (e.g., polypeptide antigens) or antigenic epitopes. An immunogenic composition can also comprise one or more additional components capable of eliciting or enhancing an immune response, such as excipients, carriers, and / or adjuvants.

[0307] 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 that 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 for 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 a deleterious function or activity of a pathogen, lessens a pathogen infection, or lessens symptoms (including death) resulting from a pathogen infection. In this context, an immune response encompasses all of the above.

[0308] As used herein, the term "adjuvant" refers to an agent that enhances the production of an immune response in a non-specific manner. Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) and emulsions to which antigens are adsorbed; the emulsions can include water-in-oil, and oil-in-water (and variants thereof, including double and reverse emulsions), glycolipids, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides), liposomes, Toll-like receptor agonists (particularly TLR2, TLR4, TLR7 / 8, and TLR9 agonists), and various combinations of the above.

[0309] As used herein, the term "mutation" refers to an amino acid residue comprising a deletion, addition, or substitution in the amino acid sequence of a protein or polypeptide as compared to the amino acid sequence of a reference protein or polypeptide. In the specification, particularly in the examples, a substitution of an amino acid at a particular position in a protein sequence is denoted using the format "(amino acid residue in wild-type protein) (amino acid position) (amino acid residue in engineered protein)." For example, "V207L" refers to a substitution of a valine (V) residue at position 207 of the amino acid sequence of a reference protein with a leucine (L) residue.

[0310] As used herein, the term "variant" refers to a nucleic acid or polypeptide that is different from a reference nucleic acid or polypeptide.

[0311] As used herein, the term "corresponding position" refers to an amino acid position in two sequences that are at an equivalent position when the two sequences are optimally aligned, i.e., when the two sequences are aligned to obtain the highest percent identity. For example, the expression "corresponding to positions 63, 105, 152, 155, 186, 190, 207, 266, 275, and / or 290 of SEQ ID NO: 4" refers to an amino acid position in a sequence being compared that is at an equivalent position to position 63, 105, 152, 155, 186, 190, 207, 266, 275, and / or 290 of SEQ ID NO: 4 when the sequence is optimally aligned with SEQ ID NO: 4, i.e., when the sequence is aligned with SEQ ID NO: 4 to obtain the highest percent identity.

[0312] As used herein, the term "identity" is used in reference to the match of sequences between two polypeptides or between two nucleic acids. When a position in each of two sequences being compared is occupied by the same base or amino acid monomer subunit (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), then the molecules are identical at that position. The "percentage of identity" between two sequences is a function of the number of matching positions shared by the sequences divided by the number of positions compared x 100. For example, if two sequences have 6 of their 10 positions matched, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions are matched). Typically, the comparison is made over the full length of the sequences being compared. Such a comparison can be conveniently performed by the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, using, for example, the computer program ALIGN, which is available by computer program from DNAstar, Inc. The percentage of 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)) as integrated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) as implemented in the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a gap length weight of 1, 2, 3, 4, 5, or 6.

[0313] As used herein, the term "antigen" refers to a molecule that can be recognized by an antibody. Examples of antigens include polypeptides, lipids, polysaccharides, and nucleic acids that contain an antigenic determinant, such as recognized by an immune cell.

[0314] As used herein, the term "D25" refers to an antibody that can specifically bind to the RSV site, the sequence of which is exemplarily provided in WO 2008 / 147196 A2.

[0315] As used herein, the term "Palivizuma" refers to palivizumab.

[0316] As used herein, the term "C-terminal truncation of X amino acids" refers to the C-terminal most consecutive X amino acids being truncated. Similarly, the term "N-terminal truncation of X amino acids" refers to the N-terminal most consecutive X amino acids being truncated.

[0317] As used herein, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When the vector is capable of directing the expression of the inserted polynucleotide, the vector is referred to as an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection and allows for expression of the genetic material elements it carries in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 -derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papova viruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to, promoter sequences, transcriptional initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, a vector can contain a replication origin.

[0318] As used herein, the term "host cell" refers to a cell that can be used for the introduction of a vector and includes, but is not limited to, prokaryotic cells such as E. 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 fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0319] As known to those skilled in the art, there is degeneracy in codons. That is, during translation of a protein, each amino acid can correspond to one or more codons, for example, up to six codons. Different species vary greatly in their use of degenerate codons to encode a given amino acid, with different preferences. This preference is known as "codon bias". Thus, as used herein, the term "codon bias" refers to the preference of a species to use certain specific codons to encode an amino acid. It can be particularly advantageous to optimize the sequence of a nucleic acid molecule according to the codon bias in certain situations, for example, it can help to increase the expression level of a protein encoded by the nucleic acid molecule. For example, when using E. coli (or human cells) to express a protein or fragment thereof, it would be potentially advantageous to optimize the nucleic acid sequence encoding the protein or fragment thereof according to the codon bias of E. coli (or human cells).

[0320] As used herein, the term "virus-like particle (VLP)" is a multimeric particle that is similar or dissimilar in structure to a native viral particle. In certain embodiments, the VLP is a native viral particle. In certain embodiments, the VLP is a virus-like particle assembled from proteins. It has been demonstrated that proteins (e.g., capsid proteins, surface proteins, envelope proteins) of some viruses (e.g., RSV, HBV, HEV, HPV) can spontaneously form VLPs upon recombinant expression in an appropriate expression system.

[0321] As used herein, the term "pharmaceutically acceptable" means that which is approved by a regulatory agency of the Federal or a state government of the United States to be used in animals, and more particularly in humans, with general recognition as safe, effective and non- harmful. As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is compatible, pharmacologically and / or physiologically, with the subject and the active ingredient, is well-tolerated by the subject, and is well-recognized in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusting agents (including, but not limited to, phosphate buffers), surfactants (including, but not limited to, cationic, anionic or non-ionic 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.

[0322] As used herein, a pharmaceutically acceptable carrier can be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Physiological saline is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

[0323] As used herein, a pharmaceutically acceptable excipient can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dairy products, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical compositions, if desired, can also contain wetting agents, or emulsifiers such as sodium lauryl sulfate, or a pH buffering agent. The pharmaceutical compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.

[0324] As used herein, the term "subject" refers to a mammal, including, but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, and the like. Preferably, the subject is a human.

[0325] As used herein, the term "effective amount" refers to an amount that is sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactically effective amount refers to an amount that is sufficient to prevent, retard, or delay the onset of a disease; a therapeutically effective amount refers to an amount that is sufficient to cure or at least partially arrest the disease and its complications in an already afflicted patient. Determining such an effective amount is well within the capability of those skilled in the art. For example, an amount effective for therapeutic purposes will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient, e.g., age, weight, and gender, the mode of administration of the drug, and other therapies that the patient may be undergoing, and the like.

[0326] Advantages of the Invention

[0327] The truncated respiratory syncytial virus (RSV) F protein or variant thereof of the present application is particularly suitable for constructing a fusion F protein with fragments or variants thereof derived from F proteins of other viruses, e.g., hMPV. Further, the truncated RSV F protein or variant thereof of the present application is constructed into a fusion protein with truncations or variants of the truncations of the human metapneumovirus (hMPV) F protein. The fusion protein has increased stability, higher pre-F protein ratio, higher protein expression, and / or better thermal stability compared to wild-type RSV and / or hMPV F protein. Moreover, the fusion protein of the present application and its corresponding mRNA also have better immunogenicity, which is capable of inducing neutralizing antibodies against RSV and / or hMPV F.

[0328] In summary, the fusion protein of the present application exhibits good protection and safety, and is suitable for various forms of vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and particulate vaccines, etc. Therefore, the fusion protein of the present application has great potential in inducing an immune response against RSV and / or hMPV in a subject, and preventing and / or treating RSV and / or hMPV infection or diseases and / or symptoms caused by RSV and / or hMPV infection.

[0329] Embodiments of the present application will be described in detail with reference to the attached drawings and examples, but it will be understood that the drawings and examples are merely intended to illustrate the present application and not to limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments, with reference to the attached drawings. BRIEF DESCRIPTION OF DRAWINGS

[0330] Figure 1 is the SDS-PAGE result of CRM1, wherein M1: Protein Marker; R: under reducing condition; NR: under non-reducing condition.

[0331] Figure 2 is the SDS-PAGE result of CRM2, wherein M1: Protein Marker; R: under reducing condition; NR: under non-reducing condition.

[0332] Figure 3 is the SDS-PAGE result of CRM3, wherein M1: Protein Marker; R: under reducing condition; NR: under non-reducing condition.

[0333] Figure 4 is the Western blot result of CRM1, wherein M2: Protein Marker; R: under reducing condition; NR: under non-reducing condition; P: multi-tag protein control.

[0334] Figure 5 is the HPLC result of CRM1.

[0335] Figure 6 is the result of detecting serum neutralizing antibody of mice induced by CRM1-3 on day 35.

[0336] Figure 7 is the result of detecting virus load of lung tissue of mice treated by CRM1-3.

[0337] Sequence information

[0338] The information of part of sequences involved in the present application is provided in Table 1 below.

[0339] Table 1: Description of sequences DETAILED DESCRIPTION

[0340] The present application will now be described with reference to the following examples, which are intended to illustrate the present application (but not to limit the present application).

[0341] Unless specifically indicated otherwise, the experiments and methods described in the examples were performed generally in accordance with conventional methods well known in the art and described in various references. For example, the general techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, among others, used in the present application are those known and practiced by those in the art, generally in accordance with the teachings of Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M. J. MacPherson, B. D. Hames and G. R. Taylor eds., (1995)), and ANIMAL CELL CULTURE (R. I. Freshney ed., (1987)).

[0342] In addition, unless otherwise specified, the examples were performed under conventional conditions or under conditions recommended by the manufacturer. The reagents or instruments used, when not specified, are conventional products available on the market. The person skilled in the art knows that the examples describe the application by way of example and are not intended to limit the scope of the application as claimed. All the publications and other references mentioned herein are incorporated by reference in their entirety.

[0343] Example 1. Design of RSV-hMPV chimeric antigens

[0344] Three different RSV-hMPV chimeric antigens were designed in this example, which were named CRM1-3, respectively, and the specific amino acid sequences are shown in Table 1, and the specific design ideas are as follows:

[0345] The alignment of the F proteins of hMPV and RSV based on the space vector using the Amber software package found that the F proteins of hMPV and RSV were highly consistent in structure, so it was prepared to perform chimeric modification on the F proteins of hMPV and RSV, and to artificially create a Φ epitope of RSV on hMPV.

[0346] By analyzing the crystal structure of RSV and neutralizing antibody complex (5W23\5UDC\5TOK), it can be determined that RSV mainly forms an epitope region that specifically binds to neutralizing antibodies through three discontinuous alpha-helices. The main neutralizing epitope is distributed in the 50-310 amino acid region of RSV, while the 301-574 amino acid region forms a relatively independent domain unrelated to antibody binding. The sequence containing the neutralizing epitope (50-310 aa) is extracted alone, and techniques such as MSA feature extraction, deep neural network structure prediction, and potential energy evaluation (mainly using the functions of software packages such as Amber, Amber, Large-scale Atomic / Molecular Massively Parallel Simulator) are used to determine that the truncated body composed of amino acids 54-307 can maximize the retention of its structural characteristics and immunogenicity. Therefore, this sequence is used as the transplanted sequence.

[0347] Through analysis by structure prediction software such as alphafold2, some positions in the transplanted RSV epitope region may have spatial and conformational conflicts with the hMPV backbone protein, so linker sequences are introduced to alleviate the conflicts. By introducing amino acids such as A, T, V, R, and E near the three discontinuous alpha-helix sequences, and through molecular modeling, energy function evaluation, and other methods, the final linker position and the number and composition of amino acids are determined. For the same reason and method, the amino acid sites in the RSV epitope region that are not suitable are mutated, including introducing cavity filling, electrostatic mutation, and engineered disulfide bonds to stabilize the conformation. Through the analysis of the structure of the hMPV backbone protein, to further stabilize the structure of the RSV epitope region, the hMPV 82-89 amino acid sequence KTVSADQL is inserted into the RSV sequence to extend its alpha-helix structure.

[0348] Example 2. Preparation of chimeric antigen molecule protein samples

[0349] The chimeric molecule protein samples were prepared by Nanjing Kingsrui Biotechnology Co., Ltd. using CHO cells, and the test found that CRM1-3 were all expressed. Among them, the expression effect of CRM1 was better, so the company was commissioned to expand the expression, and nickel column and molecular sieve two-step chromatography purification were used.

[0350] The SDS-PAGE results of CRM1-3 are shown in Figures 1-3, respectively. Among them, M1: protein Marker; R: under reducing conditions; NR: under non-reducing conditions.

[0351] The Western blot results of CRM1 are shown in Figure 4, where M2: protein Marker; R: under reducing conditions; NR: under non-reducing conditions; P: multi-tag protein control.

[0352] HPLC results of CRM1 are shown in Figure 5.

[0353] Example 3. Preparation of mRNA samples of chimeric antigen molecules

[0354] Meanwhile, the mRNA-LNP samples of RSV-hMPV chimeric antigens were prepared by Nanjing Kingsray Biotech Co., Ltd. To ensure the reliability of the mRNA samples, the mRNA samples used the highest recognized molecular design and delivery system on the market, including Cap1 capping modification, N1-methyl-pseudouridine modified bases, and the same LNP delivery system as the Moderna COVID-19 vaccine. Among them, the mRNA sequences corresponding to CRM1-3 each contain a T7 promoter sequence, a 5' UTR, a CDS sequence (with an additional stop codon), a 3' UTR sequence, and a polyA (100 A).

[0355] The main preparation process of the mRNA vaccine is to add UTR sequences at the 5' end and polyA tails and UTR sequences at the 3' end to the codon-optimized chimeric protein sequence, and then insert it into a plasmid. The plasmid is linearized by restriction enzyme BspQI after being amplified and extracted by E. coli. Using the linearized plasmid as a template, T7 RNA polymerase is used for in vitro transcription to obtain mRNA. The transcription template is digested with DNase I, and the mRNA is purified using the NEB-Monarch RNA Cleanup Kits column purification kit. The purified mRNA is dissolved in an 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 in a molar ratio of 50:10:38.5:1.5 to obtain a lipid mixture solution. The mRNA and lipid mixture solution are encapsulated by a microfluidic chip, dialyzed into a PBS solution after encapsulation, and 10% sucrose is added as a cryoprotectant to obtain an mRNA vaccine.

[0356] Example 4. Mouse immunization and challenge of chimeric protein mRNA vaccine

[0357] The Jiangsu Komaqi Biotechnology Co., Ltd. was commissioned to conduct mouse immunization and challenge protection tests of chimeric protein mRNA vaccines. The test animals were 5-6-week-old Balb / c mice purchased from Zhejiang Vantolliva Experimental Animal Technology Co., Ltd. and were raised in the ABSL2 animal house of Jiangsu Komaqi Biotechnology Co., Ltd.

[0358] The whole test procedure is first immunization on day 0 and booster immunization on day 21, wherein blood is collected on day -1, day 14 and day 35, RSV A2 strain challenge test is performed on day 49, euthanasia is performed on day 54 and gross autopsy is performed. The test groups are shown in Table 2, with PBS solution as negative control, with marketed GSK RSV product Arexvy, DS-Cav1 (McLellan JS et al. Science. 2013) recombinant protein vaccine and mRNA vaccine as positive control. After isoflurane anesthesia of mice at challenge, RSV A2 strain (1.4 x 10 5 PFU per mouse, 80 μL per mouse) is used to infect mice in an ABSL-2 laboratory by nasal drop method.

[0359] Table 2 Information of immunization samples in chimeric protein mRNA vaccine animal test

[0360] Example 5. Detection of mouse serum neutralizing antibody titer

[0361] (1) Mouse serum is inactivated by incubation at 56°C for 30 minutes, and then gradient dilution is performed with serum-free medium, and the dilution degrees are 10, 30, 90, 270, 810 and 2430 respectively;

[0362] (2) RSV-A2 virus is diluted with serum-free medium to 60 PFU / μL;

[0363] (3) The gradient-diluted mouse serum is mixed with the virus solution in equal volume, and incubated at 37°C for 1 hour;

[0364] (4) The serum-virus mixture is transferred to a 96-well plate coated with a monolayer of Hep2 cells, and incubated at 37°C in a 5% CO2 incubator for 2 hours, then the supernatant is aspirated and cell maintenance solution is added, and the plate is incubated at 37°C in a 5% CO2 incubator for 48 hours;

[0365] (5) After 48 hours of culture, the cells are incubated with 4% paraformaldehyde solution at room temperature for 30 minutes, then blocked with 5% BSA solution at room temperature for 1 hour, and then incubated with RSV-F mouse monoclonal antibody as primary antibody and HRP-labeled goat anti-mouse antibody as secondary antibody for color development, and finally the OD 450 .

[0366] The test results are shown in Figure 6. Compared with the negative control group, the CRM1-CRM3 groups all induced the production of neutralizing antibodies. Moreover, the neutralizing antibody titers of the CRM1 and CRM3 groups were significantly increased (* represents P<0.05 compared with the PBS blank control group, ** represents P<0.01 compared with the PBS blank control group, and *** represents P<0.001 compared with the PBS blank control group), indicating that the chimeric protein of the present patent can effectively induce the production of RSV neutralizing antibodies.

[0367] Example 6. Detection of viral load in lung tissue of mice

[0368] (1) Take the lung tissue of the euthanized mice in Example 4, weigh 50 mg of lung tissue, and freeze in liquid nitrogen. Add 1 mL of Trizol and grind the tissue with a homogenizer until there are no obvious particles. Let stand at room temperature for 10 minutes;

[0369] (2) Add 200 μL of chloroform, mix well, and let stand at room temperature for 3-5 min. Centrifuge at 4°C, 12000 rpm for 10 min, and transfer 400 μL of the upper layer to a clean 1.5 mL EP tube;

[0370] (3) Add 400 μL of isopropanol and mix well. Let stand at room temperature for 10 min, then centrifuge at 4°C, 12000 rpm for 10 min, discard the supernatant, and retain the white precipitate at the bottom;

[0371] (4) Wash the white precipitate with 500 μL of 75% ethanol twice. After the residual liquid is absorbed with a 10 μL pipette, open the cap and dry at room temperature for 5-10 min;

[0372] (5) Add an appropriate amount of DEPC water to dissolve the RNA completely. Place the EP tube on ice, measure the mRNA concentration, and perform reverse transcription according to Table 3;

[0373] Table 3 mRNA reverse transcription system and reaction conditions for viral load detection

[0374] Use the reverse transcription product as a template for qPCR detection. Use the CT value and the known virus titer value to draw a standard curve, and calculate the viral load in the sample to be tested.

[0375] The viral load results are shown in Figure 7. Compared with the negative control PBS, the lung viral load of the CRM1 group animals was significantly reduced (p<0.05), indicating that this molecule has the potential to be developed as an RSV prophylactic vaccine.

[0376] Example 7. Detection of serum hMPV neutralizing antibody titers in mice

[0377] Perform the second round of mouse immunization and detect the neutralizing activity of the serum against hMPV. The sample groups are as follows:

[0378] The specific steps for mouse immunization and serum detection are as follows:

[0379] The test animals are 5-6 week old Balb / c mice, which are bred in the ABSL2 animal house. The whole test procedure is first immunization on day 0, and booster immunization on day 21, in which blood sampling is performed on day 35. PBS solution is used as negative control, and the marketed GSK RSV commercial vaccine Arexvy is used as positive control. The hMPV neutralizing antibody titer is detected by micro-neutralization method, and the specific operation is as follows:

[0380] (1) Prepare 96-well cell culture plates one day before the experiment, and the cell density is 1.5×10 4 cells / well, and culture overnight;

[0381] (2) Dilute the virus solution to 1.5×10 4 FFU / ml. Take 40 μl of virus solution and mix with serum dilution sample, and incubate at 34℃, 5% CO2 environment for 1h;

[0382] (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 for 2h at 34℃;

[0383] (4) After centrifugation, discard the liquid, and add 0.4% CMC (serum-free, containing TPCK), 100 μl / well, and incubate at 34℃, 5% CO2 environment for 48h;

[0384] (5) Add 100 μl / well of 4% PFA and fix at room temperature for 30 min.

[0385] (6) Gently wash the cell plate with PBS for 3 times, add 1% BSA containing 0.2% Triton-X100 solution, 50 μl / well, and incubate at room temperature for 30 min, then gently wash with PBST for 3 times, 200 μl / well;

[0386] (7) Add hMPV specific antibody prepared with 1% BSA, 50 μl / well, and incubate at 37℃ for 1h;

[0387] (8) Wash with PBST for 3 times, and add HRP-Goat Anti-rabbit IgG (H+L) under light protection, 50 μl / well, and incubate at 37℃ for 1h.

[0388] (9) Wash with PBST for 3 times, and add TrueBlue under light protection, and terminate the color development after 10 min by adding water.

[0389] The detection results are as follows:

[0390] The experimental results show that the chimeric protein and the corresponding mRNA of the application can induce neutralizing antibodies against hMPV F. Among them, the neutralizing antibody titer produced by the CRM1 protein is significantly higher than that of other samples, especially higher than the marketed GSK RSV product Arexvy.

[0391] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details without departing from the spirit and scope of the present application in accordance with all the teachings of the present disclosure. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A truncated respiratory syncytial virus (RSV) F protein or a variant thereof, which, compared with the wild-type RSV F protein, has 50-60 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60) amino acid residues missing at the N-terminus and / or 260-270 (e.g., 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270) amino acid residues missing at the C-terminus; in, The variant, compared to the truncated 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.

2. The truncated RSV F protein or a variant thereof as described in claim 1, wherein, compared with the wild-type RSV F protein, it lacks at least one furin cleavage site, or lacks one or more amino acids in the furin cleavage site; and / or, lacks one or more (e.g., 1-5, 5-10, 10-15, 15-20, 20-27) amino acids of the pep27 domain; Preferably, the furin cleavage site has the sequence shown in SEQ ID NO:22; Preferably, the pep27 field has a sequence as shown in SEQ ID NO:21; Preferably, the truncated RSV F protein, compared with the wild-type RSV F protein, has 54 amino acid residues missing at the N-terminus and 267 amino acid residues missing at the C-terminus.

3. The truncated RSV F protein or a variant thereof as described in claim 1 or 2, 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.

4. The truncated RSV F protein or a variant thereof according to any one of claims 1-3, wherein the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid additions compared to the truncated RSV F protein; Preferably, the variant, compared to the truncated RSV F protein, has one or more of the following characteristics: (1) It has increased stability; for example, by increasing the stability of the variant itself or the fusion protein formed therewith with another protein; preferably, 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. (2) It has higher protein expression levels; Preferably, the amino acid addition position of the variant is selected from: between positions 95 and 96, between positions 147 and 148, between positions 149 and 150 of the wild-type RSV F protein, or any combination thereof; Preferably, the sequence of the amino acid added between positions 95 and 96 is as shown in SEQ ID NO:8, 9 or 23; Preferably, the sequence of the amino acid added between positions 147 and 148 is as shown in SEQ ID NO:10; Preferably, the sequence of the amino acid added between positions 149 and 150 is as shown in SEQ ID NO:

11.

5. The truncated RSV F protein or a variant thereof according to any one of claims 1-4, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions compared to the wild-type RSV F protein; Preferably, the variant, compared to the truncated RSV F protein, has one or more of the following characteristics: (1) It has increased stability; for example, by increasing the stability of the variant itself or the fusion protein formed therewith with another protein; preferably, 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. (2) It has higher protein expression levels; 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 mutation is selected from S190F, V207L, or any combination thereof; Preferably, the electrostatic mutation is selected from S63N, S105N, S275N, or any combination thereof; Preferably, the disulfide bond mutation in the engineering modification is selected from S155C, S290C, or any combination thereof.

6. The truncated RSV F protein of claim 5 or a variant thereof, wherein the amino acid substitutions contained in the variant are selected from positions 63, 105, 152, 155, 186, 190, 207, 266, 275 and / or 290 corresponding to the wild-type RSV F protein; Preferably, the variant has one or more features selected from the following: (1) 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 substitution; (2) The amino acid at position 105 of the variant, corresponding to the wild-type RSV F protein, is N; Preferably, the amino acid substitution at position 105 is an S-to-N substitution; (3) The amino acid at position 152 of the variant, corresponding to the wild-type RSV F protein, is V; Preferably, the amino acid substitution at position 152 is a substitution from I to V; (4) The amino acid at position 155 of the variant, corresponding to the wild-type RSV F protein, is C; Preferably, the amino acid substitution at position 155 is an S-to-C substitution; (5) The amino acid at position 186 of the variant, corresponding to the wild-type RSV F protein, is R; Preferably, the amino acid substitution at position 186 is an S-to-R substitution; (6) The amino acid at position 190 of the variant corresponding to the wild-type RSV F protein is F; Preferably, the amino acid substitution at position 190 is an S-to-F substitution; (7) 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; (8) The amino acid at position 266 of the variant, corresponding to the wild-type RSV F protein, is T; Preferably, the amino acid substitution at position 266 is a substitution from I to T; (9) The amino acid at position 275 of the variant, corresponding to the wild-type RSV F protein, is N; Preferably, the amino acid substitution at position 275 is an S-to-N substitution; (10) The amino acid at position 290 of the variant corresponding to the wild-type RSV F protein is C; Preferably, the amino acid substitution at position 290 is an S-to-C substitution.

7. A fusion protein comprising the truncated RSV F protein or a variant thereof as described in any one of claims 1-6, and additional proteins or polypeptides; 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 located at the N-terminus and / or C-terminus of the truncated RSV F protein or a variant thereof.

8. The fusion protein of claim 7, wherein 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 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.

9. The fusion protein of claim 7 or 8, wherein the fusion protein comprises: a first truncated form of hMPV F protein or a variant thereof, a truncated RSV F protein or a variant thereof of any one of claims 1-6, and a second truncated form of hMPV F protein or a variant thereof; Preferably, the first truncated form of the hMPV F protein or a variant thereof corresponds to the positions of amino acid residues from positions 15-24 (e.g., positions 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24) to positions 40-50 (e.g., positions 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) of the wild-type hMPV F protein; Preferably, the first truncated form of the hMPV F protein or a variant thereof corresponds to the positions of amino acid residues 19 to 45 of the wild-type hMPV F protein; Preferably, the second truncated form of the hMPV F protein or a variant thereof corresponds to the positions of amino acid residues from positions 272-281 (e.g., positions 272, 273, 274, 275, 276, 277, 278, 279, 280, or 281) to 485-495 (e.g., positions 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, or 495) of the wild-type hMPV F protein; Preferably, the second truncated form of the hMPV F protein or a variant thereof corresponds to the positions of amino acid residues 277 to 490 of the wild-type hMPV F protein.

10. The fusion protein of claim 9, wherein a variant of the second truncated form of the F protein of hMPV comprises, compared to the second truncated form of the F protein of hMPV, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid substitutions; Preferably, the variant, compared to the truncated form, has one or more of the following characteristics: (1) It has increased stability; for example, increased stability of the variant itself, or of the fusion protein formed therefrom with another protein; preferably, the fusion protein is the fusion protein according to any one of claims 7-9; (2) It has higher protein expression levels; 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 in the engineering modification is selected from N322C, T365C, V463C, or any combination thereof.

11. The fusion protein of claim 10, wherein the amino acid substitutions in the variant are selected from positions 322, 365, 449, 453, 463 and / or 466 corresponding to wild-type hMPV F protein; Preferably, it has one or more features selected from the following: (1) The amino acid at position 322 of the variant, corresponding to the wild-type hMPV F protein, is C; Preferably, the amino acid substitution at position 322 is an N-to-C substitution; (2) 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 365 is a T-to-C substitution; (3) 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 449 is a substitution from I to V; (4) The amino acid at position 453 of the variant, corresponding to the wild-type hMPV F protein, is Q; Preferably, the amino acid substitution at position 453 is an E-to-Q substitution; (5) The amino acid at position 463 of the variant, corresponding to the wild-type hMPV F protein, is C; Preferably, the amino acid substitution at position 463 is a V-to-C substitution; (6) The amino acid at position 466 of the variant corresponding to the wild-type hMPV F protein is S; Preferably, the amino acid substitution at position 466 is an N-to-S substitution.

12. The fusion protein according to any one of claims 7-11, 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:12; 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.

13. The fusion protein of claim 12, 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; (6) The polymerization domain is linked to a second truncated form or a variant thereof of the hMPV F protein, with or without the second linker peptide; Preferably, the second linker peptide has the sequence shown in SEQ ID NO:

24.

14. The fusion protein according to any one of claims 7-13, wherein, The fusion protein comprises, from N-terminus to C-terminus, the following: a first truncated form of hMPV F protein or a variant thereof, a truncated RSV F protein or a variant thereof as described in any one of claims 1-6, and a second 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 in sequence: a first truncated form of hMPV F protein or a variant thereof, a truncated RSV F protein or a variant thereof as described in any one of claims 1-6, and a second truncated form of hMPV F protein or a variant thereof, and a polymerizing 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 truncated RSV F protein or a variant thereof as described in any one of claims 1-6, and a second truncated form of hMPV F protein or a variant thereof, and a polymerizing domain; optionally, the polymerizing domain is linked to the second truncated form of hMPV F protein or a variant thereof, with or without a second linker peptide.

15. The fusion protein of claim 14, wherein, The fusion protein comprises: (1) The first truncated form of the hMPV F protein, which corresponds to the positions of amino acid residues 19 to 45 of the wild-type hMPV F protein; (2) A truncated variant of RSV F protein, which, compared with wild-type RSV F protein, has 54 amino acid residues missing at the N-terminus and 267 amino acid residues missing at the C-terminus. Furthermore, it also includes the addition of amino acids at the following positions: between positions 95 and 96 and between positions 147 and 148 corresponding to the wild-type RSV F protein; preferably, the sequence of the amino acids added between positions 95 and 96 is as shown in SEQ ID NO:8 or SEQ ID NO:23; preferably, the sequence of the amino acids added between positions 147 and 148 is as shown in SEQ ID NO:10; Furthermore, it also includes amino acid substitutions at the following positions: positions 63, 105, 152, 155, 190, 207, 275, and 290 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from S63N, S105N, I152V, S155C, S190F, V207L, S275N, and S290C; and, (3) A variant of the second truncated form of the hMPV F protein, which corresponds to the positions of amino acid residues 277 to 490 of the wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 322, 365, 449, 453, 463, and 466 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from N322C, T365C, I449V, E453Q, V463C, and N466S; Preferably, the first truncated form of the hMPV F protein has the sequence shown in SEQ ID NO:13; Preferably, the variant of the truncated RSV F protein has a sequence as shown in SEQ ID NO:15 or 16; Preferably, the variant of the second truncated form of the hMPV F protein has the sequence shown in SEQ ID NO:

14.

16. The fusion protein of claim 14, wherein, The fusion protein comprises: (1) The first truncated form of the hMPV F protein, which corresponds to the positions of amino acid residues 19 to 45 of the wild-type hMPV F protein; (2) A truncated variant of RSV F protein, which, compared with wild-type RSV F protein, has 54 amino acid residues missing at the N-terminus and 267 amino acid residues missing at the C-terminus. Furthermore, it also includes amino acid substitutions at the following positions: positions 63, 105, 152, 155, 186, 190, 207, 266, 275, and 290 of the wild-type RSV F protein; preferably, the amino acid substitutions are selected from S63N, S105N, I152V, S155C, S186R, S190F, V207L, I266T, S275N, and S290C; and, (3) A variant of the second truncated form of the hMPV F protein, which corresponds to the positions of amino acid residues 277 to 490 of the wild-type hMPV F protein; Furthermore, it also includes amino acid substitutions at the following positions: positions 322, 365, 449, 453, 463, and 466 of the wild-type F protein corresponding to hMPV; preferably, the amino acid substitutions are selected from N322C, T365C, I449V, E453Q, V463C, and N466S; Preferably, the first truncated form of the hMPV F protein has the sequence shown in SEQ ID NO:13; Preferably, the variant of the truncated RSV F protein has the sequence shown in SEQ ID NO:17; Preferably, the variant of the second truncated form of the hMPV F protein has the sequence shown in SEQ ID NO:

14.

17. The fusion protein according to any one of claims 14-16, wherein, The fusion protein has the following characteristics: (i) The sequence shown in any one of SEQ ID NO: 18-20; (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:18-20; (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: 18-20; 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.

18. A nucleic acid molecule comprising a nucleotide sequence encoding a truncated RSV F protein or a variant thereof as described in any one of claims 1-6 or a fusion protein as described in any one of claims 7-17; 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 an mRNA encoding an open reading frame (ORF) of the truncated RSV F protein or a variant thereof as described in any one of claims 1-6 or the fusion protein as described in any one of claims 7-17; 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:25-27.

19. A carrier comprising the nucleic acid molecule of claim 18; 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.

20. A host cell comprising the truncated RSV F protein of any one of claims 1-6 or a variant thereof, or the fusion protein of any one of claims 7-17, or the vector of claim 19; 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 truncated RSV F protein or its variants or fusion proteins are displayed on the surface of the host cell's cell membrane.

21. A method for expressing or producing a truncated RSV F protein or a variant thereof as claimed in any one of claims 1-6 or a fusion protein as claimed in any one of claims 7-17, the method comprising culturing a host cell as claimed in claim 20 under conditions that allow protein expression, and optionally, recovering or purifying the truncated RSV F protein or a variant thereof or a fusion protein expressed therein.

22. A vaccine comprising one or more of the following (1) to (4): (1) The truncated RSV F protein or a variant thereof as described in any one of claims 1-6; (2) The fusion protein according to any one of claims 7-17; (3) The nucleic acid molecule according to claim 18; (4) The carrier according to claim 19; 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.

23. 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 truncated RSV F protein or a variant thereof as described in any one of claims 1-6; (2) The fusion protein according to any one of claims 7-17; (3) The nucleic acid molecule according to claim 18; (4) The carrier according to claim 19; and, (ii) Second immunogenic component.

24. A kit comprising an immunogen component selected from one or more of the following (1) to (4): (1) a truncated RSV F protein or a variant thereof as claimed in any one of claims 1-6; (2) a fusion protein as claimed in any one of claims 7-17; (3) a nucleic acid molecule as claimed in claim 18; (4) a vector as claimed in claim 19; 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).

25. A pharmaceutical composition comprising: (i) Selected from any one or more of the following (1) to (8): (1) The truncated RSV F protein or a variant thereof as described in any one of claims 1-6; (2) The fusion protein according to any one of claims 7-17; (3) The nucleic acid molecule according to claim 18; (4) The carrier according to claim 19; (5) The host cell according to claim 20; (6) The vaccine according to claim 22; (7) The immunogenic composition according to claim 23; (8) The kit according to claim 24; 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.

26. Use of the truncated RSV F protein or a variant thereof as described in any one of claims 1-6, or the fusion protein as described in any one of claims 7-17, or the nucleic acid molecule as described in claim 18, or the vector as described in claim 19, or the host cell as described in claim 20, or the vaccine as described in claim 22, or the immunogenic composition as described in claim 23, or the kit as described in claim 24 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.

27. Use of the truncated RSV F protein or a variant thereof as described in any one of claims 1-6, or the fusion protein as described in any one of claims 7-17, or the nucleic acid molecule as described in claim 18, or the vector as described in claim 19, or the host cell as described in claim 20, or the vaccine as described in claim 22, or the immunogenic composition as described in claim 23, or the kit as described in claim 24, 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; 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.

28. A method for inducing antibodies against RSV and / or hMPV, the method comprising administering, in vitro or in vivo, an effective amount of the truncated RSV F protein or a variant thereof as described in any one of claims 1-6, or the fusion protein as described in any one of claims 7-17, or the nucleic acid molecule as described in claim 18, or the vector as described in claim 19, or the host cell as described in claim 20, or the vaccine as described in claim 22, or the immunogenic composition as described in claim 23, or the kit as described in claim 24; 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.

29. 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 truncated RSV F protein or a variant thereof as described in any one of claims 1-6 or the fusion protein as described in any one of claims 7-17; and detect the presence of a complex formed by the truncated RSV F protein or a variant thereof or the 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.

30. 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 truncated RSV F protein of any one of claims 1-6 or a variant thereof or a fusion protein of any one of claims 7-17 with the host cells.

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