Recombinant RSV f protein and use thereof

Abstract

Provided are a recombinant respiratory syncytial virus (RSV) fusion (F) protein, and a fusion protein, vaccine, immunogenic composition, kit and pharmaceutical composition comprising same. Further provided is a use of the recombinant RSV F protein, fusion protein, vaccine, immunogenic composition, kit and pharmaceutical composition in preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection.

Description

Recombinant RSV F protein and its uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202410361533.6 filed on March 27, 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 biomedicine, and more specifically, to a recombinant respiratory syncytial virus (RSV) fusion (F) protein, as well as fusion proteins, vaccines, immunogenic compositions, kits, and pharmaceutical compositions comprising the same. The present application also relates to the use of the recombinant respiratory syncytial virus (RSV) fusion (F) protein, fusion proteins, vaccines, immunogenic compositions, kits, and pharmaceutical compositions for preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection. Background Art

[0004] Respiratory syncytial virus (RSV) is one of the most common pathogens causing lower respiratory tract infections in infants and young children worldwide. According to statistics, RSV infects 33 million children under the age of five globally each year, resulting in nearly 160,000 deaths. RSV infection does not provide lasting immunity, and recurrent infections can occur. Over 99% of children under the age of two have been infected with RSV at least once, and 70% of these patients are hospitalized for infection complications such as bronchiolitis, pneumonia, and asthma. In addition to infants and children, immunocompromised elderly and immunosuppressed individuals are also at high risk for RSV infection, which often leads to obstructive pulmonary disease and associated cardiopulmonary complications. Despite the significant global health and economic burden, there is currently no safe and effective vaccine for RSV.

[0005] RSV belongs to the genus Pneumovirus and the family Pneumoviridae. It is an enveloped, single-stranded, negative-sense RNA virus. Its genome is approximately 15.2 kb long and contains ten genes encoding eleven proteins. The adhesion glycoprotein G and membrane fusion protein F are located on the surface of the viral membrane. Compared to the G protein, the F protein is more conserved and is therefore considered the most important protective antigen of RSV.

[0006] The RSV F protein is a type I integral membrane protein. Its precursor protein, F0, consists of 574 amino acids. Three F0 segments form a trimer through hydrophobic interactions. During passage through the Golgi apparatus, the protein is cleaved by host furin proteases between amino acids 109 and 110 and between amino acids 136 and 137. This cleavage releases a short 27-amino acid peptide, P27. The remaining two segments, F2 and F1, are linked by two disulfide bonds to form the mature F protein, which is then displayed on the cell membrane or virion surface through budding. At this stage, the F protein is in a highly unstable, high-energy metastable state, termed pre-F. At the N-terminus of F1 lies a highly hydrophobic fusion peptide, FP, nestled within the hydrophobic cavity of the trimer. Triggered by currently unknown factors, the F1 N-terminus undergoes a series of dramatic structural changes. This process allows FP to insert into the cell membrane, aiding viral membrane fusion and thus infecting the cell. This also results in the pre-F structure transforming into a stable, low-energy post-F conformation. In comparison, the immune system produces stronger antibodies when encountering proteins in the pre-F conformation. However, using proteins in the pre-F conformation as antigens is difficult to maintain stability and can easily convert to the post-F conformation.

[0007] In 2013, McLellan et al. first obtained a stable pre-F protein through point mutation and named it DS-Cav1. Subsequently, several pre-F proteins were reported, including DS-Cav1-Cys-zipper, SC-TM, SC-DM, and SC-TM. Compared with post-F proteins, these pre-F proteins have been shown to stimulate significantly higher levels of neutralizing antibody titers. The pre-F protein of RSV can be divided into two parts: the "handle" region near the membrane end, including domain I and domain II, which is mainly composed of β-chain structure and ends at the C-terminal helix that enters the membrane; and a membrane-distal part, the RSV F "head" region, including domain III, which is mainly α-helix. The RSV F head contains at least two epitopes associated with neutralizing antibodies. Site Site I and site V are located at the top of the pre-F trimer and can be recognized by strong neutralizing antibodies including D25, AM22, 5C4 and hRSV90. Site II is located in the middle of the pre-F trimer and is the binding site of the commercial antibody palivizumab. Site I and site IV, which are also recognized by low-efficiency antibodies, are located in the stalk region of the pre-F protein. The stalk region of the pre-F protein accounts for 55% of the total surface area of ​​the trimer, which may be related to the high-neutralizing epitope site in the head region. or site II competition, resulting in a weakened antibody response against highly neutralized epitopes.

[0008] The main purpose of RSV vaccine research with F protein as the main protective antigen is to produce high- and medium-titer antibodies. Improving the immunogenicity of RSV vaccines with F protein as the protective antigen is of great significance to RSV vaccine development. Summary of the Invention

[0009] In order to solve the problems encountered in the development of RSV vaccines, the present application provides a recombinant RSV F protein to improve the stability and / or immunogenicity of the prefusion conformation of the F protein.

[0010] Recombinant RSV F protein

[0011] Therefore, in a first aspect, the present application provides a recombinant respiratory syncytial virus (RSV) fusion (F) protein, which comprises the F2 domain and the F1 domain of the wild-type RSV F protein; and, compared with the wild-type RSV F protein, further comprises at least one amino acid mutation that stabilizes the pre-fusion (pre-F) conformation of the RSV F protein.

[0012] In certain embodiments, the recombinant RSV F protein has increased stability compared to wild-type RSV F protein. In certain embodiments, the stability is measured by binding of the recombinant RSV F protein to antibody D25.

[0013] In certain embodiments, the recombinant RSV F protein has a higher pre-F protein ratio than the wild-type RSV F protein. In certain embodiments, the recombinant RSV F protein comprises a pre-fusion conformation (pre-F) protein, a post-fusion conformation (post-F) protein, or a mixture of the two. In certain embodiments, the pre-F protein ratio is measured by the ratio of antibody D25 and antibody palivizumab bound to the recombinant RSV F protein.

[0014] In certain embodiments, the recombinant RSV F protein has a higher protein expression level compared to the wild-type RSV F protein.

[0015] Stabilizing modification

[0016] The RSV F protein in the pre-fusion (pre-F) conformation is a soluble F protein, and the recombinant RSV F protein of the present application stabilizes the pre-F conformation by introducing one or more modifications, such as insertion, deletion and / or mutation of one or more amino acids. In certain embodiments, the recombinant RSV F protein retains at least one immunodominant epitope of the pre-fusion conformation of the F protein.

[0017] The first type of stabilizing modification is the introduction of amino acid insertions, deletions and / or mutations.

[0018] Therefore, in certain embodiments, the recombinant RSV F protein comprises one or more amino acid insertions, deletions, and / or mutations compared to the wild-type RSV F protein.

[0019] In certain embodiments, the recombinant RSV F protein comprises an amino acid mutation at one or more of the following positions: positions 35, 60, 79, 92, 189, 190, 227, 238, 298, 320, 326, 377, 405, 437, 481, and 496 corresponding to wild-type RSV F protein.

[0020] Another stabilizing modification is elimination of the furin cleavage site and / or the pep27 domain.

[0021] In some embodiments, the furin cleavage site is located between the F2 domain and the F1 domain in the F0 precursor protein. One or two furin cleavage sites can be eliminated by deleting or replacing one or more amino acids of the furin cleavage site. In this type of embodiment, the F albumen having lacked the furin cleavage site can avoid being cut into its constituent domain, to maintain stability. In certain embodiments, a furin cleavage site is located at the 105th-109th amino acid residue of wild RSV F albumen. In certain embodiments, another furin cleavage site is located at the 133rd-136th amino acid residue of wild RSV F albumen. In certain embodiments, the sequence of the furin cleavage site can be substituted with a certain section of sequence (such as connexon).

[0022] Therefore, in certain embodiments, the recombinant RSV F protein lacks at least one furin cleavage site, or lacks one or more amino acids in a furin cleavage site, compared to the wild-type RSV F protein.

[0023] Similarly, the pep27 domain is located between the F2 domain and the F1 domain in the F0 precursor protein. The pep27 domain can be eliminated by deleting or replacing one or more amino acids in the pep27 domain. In certain embodiments, the pep27 domain is located at amino acid residues 110-136 of the wild-type RSV F protein.

[0024] In certain embodiments, the recombinant RSV F protein lacks one or more (e.g., 1-5, 5-10, 10-15, 15-20, 20-27) amino acids of the pep27 domain compared to the wild-type RSV F protein.

[0025] Wild-type RSV F protein

[0026] Natural RSV F protein has shown higher sequence conservation between RSV different subtypes.For example, RSV subtype A and B share 90% sequence identity.Further, in RSV homologous subtypes, the sequence identity of F protein is higher, and in RSV subtype A or subtype B respectively, RSV F protein has about 98% sequence identity.In addition, the sequence of almost all identified RSV F proteins is composed of 574 amino acid lengths, and its length is usually only slightly different due to the length of the C-terminal cytoplasmic tail region.The sequence identity between these natural RSV F proteins is known in the art (see, for example, WO2014 / 160463).

[0027] In view of the conservativeness of RSV F protein sequence, those of ordinary skill in the art can easily compare the amino acid position between the sequence of different natural RSV F proteins, to identify the amino acid position of the corresponding RSV F protein between different RSV strains or the hypotype.For example, in nearly all natural RSV F proteins identified, the furin cleavage site is positioned at identical amino acid position.Therefore, the conservativeness of natural RSV F protein sequence between strain or hypotype enables it to be used as reference RSV F sequence to compare the amino acid at specific position in RSV F protein.

[0028] As used herein, when referring to the amino acid sequence of the wild-type RSV F protein, it is described using the sequence shown in SEQ ID NO: 1, 2 or 64. For example, the expression "position 298 of the wild-type RSV F protein" refers to the 298th amino acid residue of the protein shown in SEQ ID NO: 1, 2 or 64. However, those skilled in the art understand that the wild-type RSV F protein can have multiple versions, which have substantially the same primary structure (i.e., amino acid sequence) and higher-order structure (i.e., spatial structure), and substantially the same biological function, but they can still have slight differences in amino acid sequence from each other. Therefore, in this application, the wild-type RSV F protein is not limited to the protein shown in SEQ ID NO: 1, 2 or 64, but is intended to cover all known wild-type RSV F proteins. Therefore, in this application, the term "wild-type RSV F protein" should include various naturally occurring, biologically functional RSV F proteins, including, for example, the RSV F protein shown in SEQ ID NO: 1, 2 or 64 and naturally occurring variants thereof. Furthermore, when describing an amino acid position of the RSV F protein, it includes not only the specific amino acid position in SEQ ID NO: 1, 2, or 64, but also the amino acid position corresponding to the specific amino acid position in its natural variants. For example, the expression "position 298 of the wild-type RSV F protein" includes amino acid residue 298 of SEQ ID NO: 1, 2, or 64, as well as the corresponding amino acid position in its natural variants. According to the present application, the expression "corresponding amino acid position" refers to the amino acid position at the equivalent position in the compared sequences when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.

[0029] In certain embodiments, the wild-type RSV is a subtype A strain, a subtype B strain, or a strain derived from subtype A or subtype B.

[0030] In certain embodiments, the wild-type RSV is selected from strain A2, strain Ontario, strain Buenos Aires, or strain B18537.

[0031] In certain embodiments, the wild-type RSV F protein comprises or consists of a sequence selected from:

[0032] (i) the sequence shown in SEQ ID NO: 1, 2 or 64;

[0033] (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: 1, 2 or 64;

[0034] (iii) a sequence having 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: 1, 2, or 64.

[0035] Amino acid mutation

[0036] In certain embodiments, the recombinant RSV F protein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid mutations compared to wild-type RSV F protein.

[0037] In certain embodiments, the recombinant RSV F protein comprises an amino acid mutation at a position selected from the group consisting of: position 35, 189, 190, 227, 238, or 298 corresponding to a wild-type RSV F protein.

[0038] In certain embodiments, the recombinant RSV F protein comprises an amino acid mutation at position 298 corresponding to a wild-type RSV F protein; and one or more positions selected from the group consisting of positions 60, 79, 92, 189, 190, 227, 238, 320, 326, 377, 405, 437, 481, and 496 corresponding to a wild-type RSV F protein.

[0039] In certain embodiments, the amino acid at position 35 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is Y or F. In certain embodiments, the amino acid mutation at position 35 of the recombinant RSV F protein is an S to Y or F mutation.

[0040] In certain embodiments, the amino acid at position 298 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is I, H, M, P, or L. In certain embodiments, the amino acid mutation at position 298 of the recombinant RSV F protein is an A to I, H, M, P, or L mutation.

[0041] In certain embodiments, the amino acid at position 238 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L, I, T, or V. In certain embodiments, the amino acid mutation at position 238 of the recombinant RSV F protein is an S to L, I, T, or V mutation.

[0042] In certain embodiments, the amino acid at position 189 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L, I, or V. In certain embodiments, the amino acid mutation at position 189 of the recombinant RSV F protein is a T to L, I, or V mutation.

[0043] In certain embodiments, the amino acid at position 190 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is I, L, M, V, or Y. In certain embodiments, the amino acid mutation at position 190 of the recombinant RSV F protein is an S to I, L, M, V, or Y mutation.

[0044] In certain embodiments, the amino acid at position 227 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L. In certain embodiments, the amino acid mutation at position 227 of the recombinant RSV F protein is an N to L mutation.

[0045] In certain embodiments, the amino acid at position 320 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is A, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y. In certain embodiments, the amino acid mutation at position 320 of the recombinant RSV F protein is a mutation from P to A, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y.

[0046] In certain embodiments, the amino acid at position 326 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is D, E, or P. In certain embodiments, the amino acid mutation at position 326 of the recombinant RSV F protein is a T to D, E, or P mutation.

[0047] In certain embodiments, the amino acid at position 377 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is P or Q. In certain embodiments, the amino acid mutation at position 377 of the recombinant RSV F protein is an S to P or Q mutation.

[0048] In certain embodiments, the amino acid at position 405 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is V. In certain embodiments, the amino acid mutation at position 405 of the recombinant RSV F protein is an S to V mutation.

[0049] In certain embodiments, the amino acid at position 60 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is F or Y. In certain embodiments, the amino acid mutation at position 60 of the recombinant RSV F protein is an E to F or Y mutation.

[0050] In certain embodiments, the amino acid at position 437 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is R or Y. In certain embodiments, the amino acid mutation at position 437 of the recombinant RSV F protein is an N to R or Y mutation.

[0051] In certain embodiments, the amino acid at position 481 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is R. In certain embodiments, the amino acid mutation at position 481 of the recombinant RSV F protein is an L to R mutation.

[0052] In certain embodiments, the amino acid at position 496 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is F or Y. In certain embodiments, the amino acid mutation at position 496 of the recombinant RSV F protein is an N to F or Y mutation.

[0053] In certain embodiments, the amino acid at position 92 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is T, V, M, or I. In certain embodiments, the amino acid mutation at position 92 of the recombinant RSV F protein is an E to T, V, M, or I mutation.

[0054] In certain embodiments, the amino acid at position 79 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L. In certain embodiments, the amino acid mutation at position 79 of the recombinant RSV F protein is an I to L mutation.

[0055] F2 domain and F1 domain

[0056] The F1 territory of the recombinant RSV F protein of the present application can have the length identical with the total length F1 territory of wild-type RSV F protein.It is understandable that it can also comprise the fragment in the total length F1 territory of RSV F protein, as long as the pre-F conformation of stabilization is maintained.For example, the F1 territory of the recombinant RSV F protein can be from the C-terminal deletion 1,2,3,4,5,6,7,8,9,10 or up to 60 amino acid residues in total length F1 territory, perhaps, from the N-terminal deletion 1,2,3,4,5,6,7,8,9,10 or up to 60 amino acid residues in total length F1 territory. In certain embodiments, the full-length F1 domain of the RSV F protein corresponds to positions 137-574 of the wild-type RSV F protein and includes (from N-terminus to C-terminus) the extracellular domain (ED) (positions 137-524), the transmembrane domain (TM) (positions 525-550), and the cytoplasmic tail (CT) (positions 551-574).

[0057] In certain embodiments, the F1 domain of the recombinant RSV F protein of the present application lacks all or part of the cytoplasmic tail region. In other embodiments, the F1 domain of the recombinant RSV F protein lacks the cytoplasmic tail region and all or part of the transmembrane domain. Generally, for the recombinant RSV F protein intended to be connected to the multimerization domain (i.e., foldon), all or part of the 514th to 574th amino acid residues can be missing. Therefore, in some specific embodiments, the F1 domain of the recombinant RSV F protein does not include the transmembrane domain and / or the cytoplasmic tail region of the wild-type RSV F protein. In some specific embodiments, the F1 domain of the recombinant RSV F protein does not include the 514th to 574th amino acid residues of the wild-type RSV F protein.

[0058] In some embodiments, the F2 domain of the recombinant RSV F protein of the present invention can have the same length as the total length F2 domain of wild-type RSV F protein.It is understandable that it can also comprise the fragment of the total length F2 domain of RSV F protein, as long as the pre-F conformation of stabilization is maintained.For example, the F2 domain of the recombinant RSV F protein can be from the C-terminal deletion 1,2,3,4,5,6,7,8,9,10 or up to 30 amino acid residues in the total length F2 domain.Or, from the N-terminal deletion 1,2,3,4,5,6,7,8,9,10 or up to 30 amino acid residues in the total length F2 domain.In certain embodiments, the total length F2 domain of RSV F protein corresponds to the 1-109 position of wild-type RSV F protein.

[0059] In certain embodiments, there is no furin cleavage site and / or pep27 domain between the F2 domain and the F1 domain.

[0060] In certain embodiments, the F2 domain comprises the entire F2 domain of a wild-type RSV F protein (eg, a portion corresponding to positions 1-109 of a wild-type RSV F protein) or a fragment thereof.

[0061] In certain embodiments, the F2 domain comprises a portion corresponding to positions 1-95, 5-95, 10-95, 1-100, 5-100, 10-100, 1-105, 5-105, 10-105, 1-110, 5-110, or 10-110 of a wild-type RSV F protein.

[0062] In certain embodiments, the F2 domain comprises a portion corresponding to positions 1-105 of the wild-type RSV F protein.

[0063] In certain embodiments, the F2 domain is deleted from the N-terminus and / or C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues compared to the F2 domain of the wild-type RSV F protein.

[0064] In certain embodiments, the F2 domain has 5 or 25 amino acids deleted from the N-terminus and 4 amino acids deleted from the C-terminus compared to the F2 domain of the wild-type RSV F protein.

[0065] In certain embodiments, the F1 domain comprises the entire F1 domain of a wild-type RSV F protein (eg, a portion corresponding to positions 137-574 of a wild-type RSV F protein) or a fragment thereof.

[0066] In certain embodiments, the F1 domain comprises the extracellular domain, transmembrane domain, and / or cytoplasmic tail of a wild-type RSV F protein.

[0067] In certain embodiments, the F1 domain comprises a portion corresponding to positions 135-503, 140-503, 145-503, 135-508, 140-508, 145-508, 135-513, 140-513, 145-513, 135-518, 140-518, or 145-518 of a wild-type RSV F protein.

[0068] In certain embodiments, the F1 domain comprises a portion corresponding to positions 145-513 of the wild-type RSV F protein.

[0069] In certain embodiments, the F1 domain is deleted from the N-terminus and / or C-terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acid residues (e.g., 30-50, 50-100, 100-150, 150-200, 200-250, 250-300) compared to the F1 domain of the wild-type RSV F protein.

[0070] In certain embodiments, the F1 domain is deleted from the N-terminus by 8 amino acids and from the C-terminus by 252 or 268 amino acids compared to the F1 domain of the wild-type RSV F protein.

[0071] Connector

[0072] The F2 domain and the F1 domain as described above may be linked by a disulfide bond, or may be linked by a peptide bond or a linker. The linker of the present application may be any suitable linker that links two polypeptides together, and the specific sequences of these linkers include, but are not limited to, G, GG, GGG, GS, SAIG, GGPG, GPGGG, GGGPG, EAAAK, and PAAAK.

[0073] Therefore, in certain embodiments, the recombinant RSV F protein further comprises a linker.

[0074] In certain embodiments, the linker is located at the C-terminus of the F2 domain.

[0075] In certain embodiments, the linker is located at the N-terminus of the F1 domain.

[0076] In certain embodiments, the linker has 3-20 amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20).

[0077] In certain embodiments, the linker comprises at least 1 glycine (e.g., 1, 2, 3, 4, 5, 6, 7).

[0078] In certain embodiments, the linker further comprises at least 1 (eg, 1, 2, 3) proline.

[0079] In certain embodiments, the linker comprises or consists of a sequence selected from the group consisting of: a sequence shown in any one of SEQ ID NOs: 54-59.

[0080] Fusion protein

[0081] In a second aspect, the application provides a fusion protein comprising the recombinant RSV F protein of the first aspect, wherein the fusion protein further comprises another protein or polypeptide. It is understood that these other proteins or polypeptides do not adversely affect the activity and / or function of the recombinant RSV F protein.

[0082] In certain embodiments, the additional protein or polypeptide is selected from the group consisting of: a signal peptide, a multimerization domain, a tag, or any combination thereof.

[0083] signal peptide

[0084] In order to enhance protein production or secretion, the fusion protein of the present application may comprise a signal peptide.

[0085] In certain embodiments, the signal peptide is a native signal peptide of RSV or a variant thereof, or a native signal peptide or a variant thereof derived from other organisms.

[0086] In certain embodiments, the signal peptide is located at one end (eg, the N-terminus) of the fusion protein.

[0087] In certain embodiments, the signal peptide is located at the N-terminus of the F2 domain.

[0088] In certain embodiments, the signal peptide is linked to the F2 domain with or without a first linker peptide.

[0089] Multimerization domain

[0090] The recombinant RSV F proteins provided herein can be linked to a multimerization domain to promote the formation of multimers (eg, dimers, trimers, tetramers, pentamers) by the recombinant RSV F protein.

[0091] Exogenous multimerization domains that can promote the formation of stable multimers of soluble proteins are known in the art. Specifically, examples of multimerization domains that can be linked to the recombinant RSV F protein of the present application include, but are not limited to:

[0092] (1) GCN4 leucine zipper (for specific sequence and information, see Harbury et al. 1993 Science 262: 1401-1407);

[0093] (2) a trimerization motif from the pulmonary surfactant protein (for specific sequence and information, please see Hoppe et al. 1994 FEB S Lett 344: 191-195);

[0094] (3) collagen (for specific sequence and information, see McAlinden et al. 2003 Biol Chem 278: 42200-42207); and

[0095] (4) Bacteriophage T4 fibritin fold (for specific sequence and information, please see Miroshnikov et al. 1998 Protein Eng 11: 329-414).

[0096] Typically, the multimerization domain is connected to the C-terminus of the F1 domain. It can be directly joined to the F1 domain, or connected to the F1 domain via a joint (such as an amino acid joint, for example, the sequence GG, GS or SAIG). The joint can also be a longer joint (for example, a sequence comprising repeats of the sequence GG). In some embodiments, a protease cleavage site can also be included between the F1 domain and the multimerization domain for removing the multimerization domain from the recombinant RSV F protein.

[0097] In certain embodiments, the multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or a pentamerization domain.

[0098] In certain embodiments, the multimerization domain is derived from T4 fibritin.

[0099] In certain embodiments, the multimerization domain has the sequence shown in SEQ ID NO:39.

[0100] In certain embodiments, the multimerization domain is a leucine zipper or an isoleucine zipper.

[0101] In certain embodiments, the multimerization domain has the sequence shown in SEQ ID NO:60.

[0102] In certain embodiments, the multimerization domain is derived from a lung surfactant protein.

[0103] In certain embodiments, the multimerization domain is located at the C-terminus of the F1 domain.

[0104] In certain embodiments, the multimerization domain is linked to the F1 domain with or without a second linker peptide.

[0105] In certain embodiments, the second connecting peptide has the sequence shown in SEQ ID NO:61.

[0106] Label

[0107] In certain embodiments, the recombinant RSV F protein of the present invention can be connected to a tag for expression, detection, tracing and / or purification of the protein of the present invention. 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 tracing).

[0108] In certain embodiments, the tag is selected from a polyhistidine tag, an antigen or epitope tag, an enzyme tag, or any combination thereof.

[0109] In certain embodiments, the tag is located at the terminus (eg, the C-terminus) of the fusion protein.

[0110] In certain embodiments, the tag is located at the C-terminus of the F1 domain or the multimerization domain.

[0111] In certain embodiments, the tag is linked to the F1 domain or the multimerization domain with or without a third connecting peptide.

[0112] In certain exemplary embodiments, the tag is a polyhistidine tag, i.e., a 6×His-tag (HHHHHH). In certain exemplary embodiments, the tag is connected to the polyhistidine tag via an enzyme cleavage site.

[0113] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, an F2 domain, a linker, and an F1 domain.

[0114] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, an F2 domain, a linker, an F1 domain, and a multimerization domain. In certain embodiments, the multimerization domain is connected to the F1 domain with or without a second connecting peptide.

[0115] In certain embodiments, the fusion protein comprises, from N-terminus to C-terminus, an F2 domain, a linker, an F1 domain, a second connecting peptide, and a multimerization domain. In certain embodiments, the C-terminus of the fusion protein may or may not contain a tag. In certain embodiments, the N-terminus of the fusion protein may or may not contain a signal peptide.

[0116] In certain embodiments, the fusion protein has:

[0117] (i) a sequence shown in any one of SEQ ID NOs: 4-38;

[0118] (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 NOs: 4-38;

[0119] (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 to the sequence shown in any one of SEQ ID NOs: 4-38.

[0120] In certain embodiments, the fusion protein is in the form of a multimer (eg, dimer, trimer, tetramer), a monomer, or a mixture of the two.

[0121] In certain embodiments, the fusion protein is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixture of the two.

[0122] Nucleic acid molecules

[0123] It is easy to understand that the nucleic acid molecules can be used to clone or express recombinant RSV F protein or fusion protein of the present invention.In some cases, in order to improve efficiency, the nucleotide sequence of the nucleic acid molecule can be codon optimized according to cell preference.

[0124] Therefore, in a third aspect, the present application provides a nucleic acid molecule comprising a nucleotide sequence encoding the recombinant RSV F protein of the first aspect or the fusion protein of the second aspect.

[0125] In certain embodiments, the nucleotide sequence is codon-optimized or non-optimized according to the codon preference of the host cell.

[0126] In certain embodiments, the nucleic acid molecule is DNA, or the RNA (mRNA) product transcribed from the DNA, or a mixture of the two.

[0127] The nucleic acid molecules of the present application can include DNA, cDNA and RNA sequences. The application also encompasses nucleic acid molecules encoding the individual F2 domains or F1 domains of the recombinant RSV F protein described in the first aspect. The nucleic acid molecules can be incorporated into vectors, such as expression vectors.

[0128] carrier

[0129] The vectors for expressing the recombinant RSV F protein or fusion protein 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 vector of the present invention can be, for example, a plasmid; a phagemid; a cosmid; an artificial chromosome, such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or an artificial chromosome (PAC) derived from P1; a bacteriophage such as lambda phage or M13 phage; and a viral vector, etc. Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40).

[0130] In certain preferred embodiments, the vector of the present application can express or be used to express recombinant RSV F protein of the present invention or fusion protein.In certain preferred embodiments, the vector of the present invention can or be used to express recombinant RSV F protein of the present invention or fusion protein in subject (such as mammal, such as human) body.

[0131] The vectors of the present application may 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 transcription control elements (e.g., promoters, enhancers, terminators) and / or one or more translation signals; and a signal sequence or leader sequence for targeting to the secretory pathway in a selected host cell.

[0132] In a fourth aspect, the present application provides a vector comprising the nucleic acid molecule described in the third aspect.

[0133] In certain embodiments, the vector is a viral vector.

[0134] 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 papillomatovirus vector.

[0135] host cells

[0136] The recombinant RSV F protein provided herein can be prepared by conventional methods known in the art, such as by using suitable vector expression host cells including, for example, insect cells, mammalian cells, avian cells, bacteria and yeast cells in recombinant host cells. The example of insect cells includes, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells. The example of mammalian cells includes 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 embryo 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 are described in, for example, Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Avian cell expression systems are also known to those skilled in the art and are described, for example, in U.S. Patent Nos. 5,340,740; 5,656,479; 5,830,510; 6,114,168 and 6,500,668. Similarly, bacterial and mammalian cell expression systems are also known in the art and are described, for example, in Yeast Genetic Engineering (Barr et al., ed., 1989) Butterworths, London.

[0137] In a fifth aspect, the present application provides a host cell comprising the recombinant RSV F protein of the first aspect, the fusion protein of the second aspect, the nucleic acid molecule of the third aspect, or the vector of the fourth aspect.

[0138] In certain embodiments, the host cell is selected from a prokaryotic cell (eg, an Escherichia coli cell) or a eukaryotic cell.

[0139] In certain embodiments, the eukaryotic cell is a mammalian cell, eg, a mouse cell, a human cell.

[0140] In certain embodiments, the recombinant RSV F protein or fusion protein is displayed on the surface of the cell membrane of the host cell.

[0141] Preparation method

[0142] The recombinant RSV F protein or fusion protein of the present application can be prepared using any method known in the art, and is not limited to a specific expression method (e.g., recombinant expression). The method for expressing and purifying the recombinant RSV F protein described in the first aspect of the application or the fusion protein described in the second aspect is common in the art, and specifically can be found in the following references: Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 200; and Ausubel et al. Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons, Inc., 999.

[0143] Therefore, in a sixth aspect, the present application provides a method for expressing or producing the recombinant RSV F protein 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 permitting protein expression, and optionally recovering or purifying the recombinant RSV F protein or fusion protein expressed therefrom.

[0144] vaccine

[0145] The vaccine provided herein is not limited to protein form, nucleic acid form, or a mixture of the two. Furthermore, the nucleic acid can be selected from DNA, cDNA, RNA, or any combination thereof.

[0146] In a seventh aspect, the present application provides a vaccine comprising one or more selected from the following (1) to (4):

[0147] (1) the recombinant RSV F protein described in the first aspect;

[0148] (2) the fusion protein described in the second aspect;

[0149] (3) the nucleic acid molecule described in the third aspect;

[0150] (4) The carrier described in the fourth aspect.

[0151] In certain embodiments, the vaccine further comprises an adjuvant and / or a buffer.

[0152] In certain embodiments, the adjuvant is selected from a metal salt, 3-D-monophosphoryl lipid A (MPL), saponin, an oil and water emulsion, a liposome, a nanoparticle, or any combination thereof.

[0153] In certain embodiments, the fusion protein in the vaccine is in the form of a multimer (eg, dimer, trimer, tetramer), a monomer, or a mixed form.

[0154] In certain embodiments, the recombinant RSV F protein or fusion protein in the vaccine is in a prefusion conformation (pre-F), a postfusion conformation (post-F), or a mixed form.

[0155] The vaccine provided herein can be implemented using a standard route of administration. Non-limiting embodiments include parenteral administration, such as intradermal, intramuscular, subcutaneous, transdermal, mucosal or oral administration. Single administration or one or more booster administrations can be given to the subject. If booster vaccination is performed, this booster vaccination will typically be administered to the same individual between 1 week and 10 years, for example, between 2 weeks and 6 months after the first administration to the individual (which is referred to as "primary vaccination" in this type of situation).

[0156] The vaccines provided herein can also be used in combination with one or more other vaccines. For example, in adults, they can be used in combination with influenza vaccine, Prevnar, tetanus vaccine, diphtheria vaccine, and pertussis vaccine. For children, the vaccines provided herein can be used in combination with any other vaccines indicated for use in pediatric patients.

[0157] Immunogenic composition

[0158] In an eighth aspect, the present application provides an immunogenic composition comprising:

[0159] (i) a first immunogenic component, wherein the first immunogenic component is selected from one or more of the following (1) to (4):

[0160] (1) the recombinant RSV F protein described in the first aspect;

[0161] (2) the fusion protein described in the second aspect;

[0162] (3) the nucleic acid molecule described in the third aspect;

[0163] (4) the vector according to the fourth aspect; and

[0164] (ii) a second immunogenic component.

[0165] In certain embodiments, the first immunogenic component and the second immunogenic component are each independently selected from a protein component, a nucleic acid component, or any combination thereof.

[0166] In certain embodiments, the first immunogenic component and the second immunogenic component are formulated for simultaneous or separate administration.

[0167] In certain embodiments, the immunogenic composition further comprises more immunogenic components (eg, a third immunogenic component, a fourth immunogenic component, a fifth immunogenic component).

[0168] In certain embodiments, the second immunogenic component is an immunogen derived from an additional strain of RSV.

[0169] For example, the first immunogenic component is an immunogen derived from a strain of RSV subgroup A, and the second immunogenic component is an immunogen derived from a strain of RSV subgroup B. In certain embodiments, the recombinant RSV F proteins comprised by the first and second immunogenic components can comprise the same or different amino acid mutations.

[0170] In certain embodiments, the second immunogenic component is an immunogen derived from another protein of RSV.

[0171] For example, the first immunogenic component is an immunogen derived from RSV F protein, and the second immunogenic component is an immunogen derived from RSV attachment protein (G) protein or RSV small hydrophobin (SH) protein. In certain embodiments, the recombinant RSV F protein contained in the first immunogenic component and the second immunogenic component may contain the same or different amino acid mutations.

[0172] In certain embodiments, the second immunogenic component is an immunogen derived from a pathogenic organism other than RSV.

[0173] For example, the first immunogenic component is an immunogen derived from RSV, and the second immunogenic component is an immunogen derived from a virus different from RSV (e.g., hepatitis B virus (HBV), parainfluenza virus (PIV), poliovirus, influenza virus). In certain embodiments, the recombinant RSV F protein included in the first and second immunogenic components may include the same or different amino acid mutations.

[0174] Reagent test kit

[0175] In a ninth aspect, the present application provides a kit comprising an immunogenic component, wherein the immunogenic component is selected from one or more of the following (1) to (4): (1) the recombinant RSV F protein described in the first aspect; (2) the fusion protein described in the second aspect; (3) the nucleic acid molecule described in the third aspect; (4) the vector described in the fourth aspect.

[0176] In certain embodiments, the kit further comprises a carrier component capable of displaying the immunogenic component.

[0177] In certain embodiments, the carrier component is selected from: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), a polymerized base, a virus-like particle (VLP), or any combination thereof.

[0178] In certain embodiments, the immunogen component and the carrier component of the kit are provided separately or as a complex.

[0179] In certain embodiments, the immunogenic components are in multimeric (eg, dimer, trimer, tetramer) form, monomeric form, or in mixed form.

[0180] In certain embodiments, the immunogenic components of the kit are provided in the form of proteins or nucleic acids.

[0181] In certain embodiments, the vector components of the kit are provided in the form of proteins or nucleic acids.

[0182] In certain embodiments, the VLP is assembled from proteins obtained from RSV, hepatitis B virus (HBV), human papillomavirus (HPV), or human immunodeficiency virus (HIV).

[0183] Pharmaceutical composition

[0184] In a tenth aspect, the present application provides a pharmaceutical composition comprising:

[0185] (i) Any one or more selected from the following (1) to (8):

[0186] (1) the recombinant RSV F protein described in the first aspect;

[0187] (2) the fusion protein described in the second aspect;

[0188] (3) the nucleic acid molecule described in the third aspect;

[0189] (4) the vector described in the fourth aspect;

[0190] (5) the host cell according to the fifth aspect;

[0191] (6) The vaccine according to the seventh aspect;

[0192] (7) The immunogenic composition of the eighth aspect;

[0193] (8) The kit according to the ninth aspect; and

[0194] (ii) a pharmaceutically acceptable carrier and / or excipient, a buffer, an adjuvant, or any combination thereof.

[0195] In certain preferred embodiments, the pharmaceutically acceptable carrier and / or excipient is selected from a pH adjuster (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.

[0196] In certain preferred embodiments, the pharmaceutically acceptable carrier can be a sterile liquid, such as water and oils, including oils of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. In certain preferred embodiments, the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerol, and any combination thereof.

[0197] In certain preferred embodiments, the pharmaceutically acceptable excipient can be selected from starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, milk powder, glycerol, propylene, ethylene glycol, water, ethanol, and any combination thereof.

[0198] In certain preferred embodiments, the pharmaceutical composition can take the form of a solution, suspension, emulsion, tablet, pill, capsule, powder (eg, lyophilized powder), sustained-release formulation, or the like.

[0199] The pharmaceutical compositions of the present invention can be administered in various suitable ways. Suitable modes of administration include, but are not limited to, parenteral administration, such as intravenous, intradermal, subcutaneous, oral, intranasal (e.g., inhalation), transdermal (e.g., topical), transmucosal, and rectal administration. In certain preferred embodiments, the pharmaceutical compositions are formulated into pharmaceutical preparations suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans according to conventional procedures.

[0200] Typically, the pharmaceutical composition for injection (e.g., intravenous administration, e.g., by push injection or continuous infusion) is sterile, isotonic. If desired, such pharmaceutical composition can also include a solubilizing agent and a local anesthetic such as ergotamine to alleviate pain at the injection site. In addition, the pharmaceutical composition for injection can also contain a preservative. In certain preferred embodiments, the pharmaceutical composition for injection can also exist in unit dosage form (e.g., stored in an ampoule or in a multidose container).

[0201] In certain embodiments, the pharmaceutical compositions of the present invention may comprise additional active ingredients, for example, additional vaccines, antiviral agents and / or monoclonal antibodies.

[0202] In certain embodiments, the additional vaccine, antiviral agent and / or monoclonal antibody is an additional vaccine, antiviral agent and / or monoclonal antibody directed against RSV; or is a vaccine, antiviral agent and / or monoclonal antibody directed against a pathogenic organism other than RSV.

[0203] In certain embodiments, the recombinant RSV F protein 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 and the additional active ingredient can be administered simultaneously, separately, or sequentially.

[0204] use

[0205] On the other hand, the present application provides the use of the recombinant RSV F protein 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 in preparing a pharmaceutical composition for inducing an immune response to RSV in a subject.

[0206] In certain embodiments, the immune response includes inducing the subject to produce antibodies (eg, neutralizing antibodies) against RSV.

[0207] In certain embodiments, the subject is a mammal, eg, a mouse, a human.

[0208] In certain embodiments, the subject is 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.

[0209] On the other hand, the present application provides the use of the recombinant RSV F protein 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 in preparing a pharmaceutical composition for preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection.

[0210] In certain embodiments, the subject is a mammal, eg, a mouse, a human.

[0211] In certain embodiments, the subject is 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.

[0212] In certain embodiments, the diseases and symptoms caused by RSV infection are selected from bronchitis, pneumonia, asthma, obstructive pulmonary disease, and cardiopulmonary complications.

[0213] method

[0214] On the other hand, the present application provides a method for inducing antibodies against RSV, comprising administering an effective amount of the recombinant RSV F protein 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 in cells in vitro or in a subject.

[0215] In certain embodiments, the mode of administration includes intradermal, intramuscular, subcutaneous, transdermal, mucosal, or oral administration.

[0216] In certain embodiments, the subject is a mammal, eg, a mouse, a human.

[0217] In certain embodiments, the subject is 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.

[0218] In another aspect, the present application provides a method for detecting the presence of RSV infection in a subject in vitro, the method comprising: contacting a biological sample obtained from the subject with the recombinant RSV F protein of the first aspect, or the fusion protein of the second aspect; and detecting the presence of a complex formed by the recombinant RSV F protein or the fusion protein and an antibody.

[0219] In certain embodiments, the subject is a mammal, eg, a mouse, a human.

[0220] In certain embodiments, the subject is 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.

[0221] In certain embodiments, the biological sample is selected from whole blood, serum, plasma, or any combination thereof.

[0222] On the other hand, the present application provides a method for screening candidate drugs that can inhibit RSV infection of cells, the method comprising contacting the host cell with the candidate drug before, simultaneously or after contacting the recombinant RSV F protein described in the first aspect or the fusion protein described in the second aspect with the host cell.

[0223] Definition of terms

[0224] As used herein, the term "respiratory syncytial virus (RSV)" refers to a virus of the genus Pneumovirus, family Pneumoviridae. The RSV genome is approximately 15 kilobases (Kb) long 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). Fusion protein (F) and attachment protein (G) are the two main envelope glycoproteins. The F protein is a type I glycoprotein that can be cleaved by cellular proteases into F1 and F2 polypeptides.

[0225] RSV strains exist in subtype A and subtype B, and the amino acid sequences of their F proteins are about 90% homologous. A variety of RSV strains have been isolated so far and the amino acid sequences of their F proteins have been reported. For example, an example sequence of the F protein of subtype A is provided in SEQ ID NO: 1 (A2 strain; GenBank GI: QGW56794.1; Swiss Prot P03420), and an example sequence of the F protein of subtype B is provided in SEQ ID NO: 2 (18537 strain; GenBank GI: 138250; Swiss Prot P13843) or SEQ ID NO: 64. SEQ ID NO: 1 and SEQ ID NO: 2 are both sequences of 574 amino acids. In this article, RSV covers naturally occurring RSV and RSV produced by genetic drift, artificial synthesis and / or recombination, and also includes variants derived from naturally occurring RSV.

[0226] As used herein, the term "F protein" or "fusion protein" or "F protein polypeptide" or "fusion protein polypeptide" refers to a polypeptide or protein having all or part of the amino acid sequence of an RSV fusion protein. The term "G protein" as used herein refers to a polypeptide or protein having all or part of the amino acid sequence of an RSV adhesion protein. The sequences of many RSV fusion proteins and adhesion proteins have been reported. WO 2008 / 114149 discloses exemplary F and G protein variants (e.g., naturally occurring variants) that can be obtained.

[0227] As used herein, the terms "wild," "wild-type," or "native" are used interchangeably. When these terms are used to describe a nucleic acid molecule, polypeptide, or protein, they indicate that the nucleic acid molecule, polypeptide, or protein exists in nature, is found in nature, and has not been modified or processed by humans. As used herein, wild-type respiratory syncytial virus (RSV) F protein refers to a naturally occurring, biologically active F protein.

[0228] As used herein, the term "F0 polypeptide (F0)" or "F0 precursor" refers to the precursor polypeptide of the RSV F protein. The F0 polypeptide of the RSV strain consists of 574 amino acids. In vivo, F0 oligomerizes in the endoplasmic reticulum and is proteolytically processed by furin at two conserved furin consensus sequences (furin cleavage sites), i.e., RARR (SEQ ID NO: 62) and RKRR (SEQ ID NO: 63), to produce an oligomer consisting of two disulfide-linked fragments. The smaller of these fragments is F2 and is derived from the N-terminal portion of the F0 precursor. The larger is F1 and is derived from the C-terminal portion of the F0 precursor.

[0229] As used herein, the terms "Fi polypeptide (F1)" or "F1 domain" are used interchangeably and refer to the polypeptide chain of a mature RSV F protein. Natural Fi polypeptides include approximately 137-574 amino acids of the RSV F0 precursor and are comprised of (from N-terminus to C-terminus) an extracellular domain (ED) (position 137-524), a transmembrane domain (TM) (position 525-550), and a cytoplasmic tail (CT) (position 551-574). As used herein, "Fi polypeptide (F1)" or "F1 domain" encompasses natural Fi polypeptides or fragments thereof, and Fi polypeptides including modifications (e.g., one or more amino acid substitutions, insertions, or deletions) from natural Fi polypeptide sequences. In certain embodiments, the modification is a modification that stabilizes the immunogenicity of the recombinant RSV F protein or enhances the immunogenicity of the recombinant RSV F protein.

[0230] As used herein, the terms "F2 polypeptide (F2)" or "F2 domain" are used interchangeably to refer to the polypeptide chain of a mature RSV F protein. A native F2 polypeptide includes amino acids approximately 1-109 of the RSV F0 precursor. As used herein, "F2 polypeptide (F2)" or "F2 domain" encompasses native F2 polypeptides or fragments thereof, as well as F2 polypeptides including modifications (e.g., one or more amino acid substitutions, insertions, or deletions) from native F2 polypeptide sequences. In certain embodiments, the modification is a modification that stabilizes the recombinant RSV F protein or enhances the immunogenicity of the recombinant RSV F protein.

[0231] As used herein, the terms "pep27 polypeptide" or "pep27 domain" are used interchangeably and refer to a 27-amino acid polypeptide that is cleaved from the F0 precursor during maturation of the RSV F protein. The sequence of the pep27 domain is flanked by two furin cleavage sites that are cleaved by cellular proteases during maturation of the F protein to generate the F1 polypeptide and the F2 polypeptide.

[0232] As used herein, the terms "multimerization domain," "multimerization motif," "foldon," "folding domain," or "foldon" have the same meaning and are used interchangeably. They refer to amino acid sequences that are capable of forming multimers. In certain embodiments, they are capable of promoting the assembly of polypeptides or proteins into dimers, trimers, tetramers, or pentamers.

[0233] As used herein, the term "immunogenicity" refers to the ability of a substance to elicit, trigger, stimulate or induce an immune response in a human or animal against a specific antigen, in the presence or absence of an adjuvant.

[0234] As used herein, the term "immunogen" or "immunogenic component" refers to a substance with immunogenicity. In certain embodiments, the symptoms or diseases caused by a pathogen are prevented (or alleviated or improved) by suppressing the replication of a pathogen (such as RSV) after the subject is exposed to the pathogen. In this article, "immunogen" or "immunogenic component" should be understood as encompassing a substance (e.g., a vaccine) intended for administering to a subject or subject population to induce a protective or palliative immune response against RSV.

[0235] As used herein, the term "immunogenic composition" refers to a composition of matter suitable for administering (e.g., in an experimental setting) to a human or animal subject that can elicit a specific immune response, e.g., a specific immune response to a pathogen such as RSV. Therefore, the immunogenic composition comprises one or more antigens (e.g., polypeptide antigens) or antigenic epitopes. The immunogenic composition can also include one or more other compositions that can elicit or enhance an immune response, such as an excipient, a carrier, and / or an adjuvant.

[0236] As used herein, the term "immune response" refers to the response of immune system cells such as B cells, T cells, or monocytes to stimuli. 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 to a particular antigen (i.e., an "antigen-specific response"). If the antigen is derived from a pathogen, the antigen-specific response is a "pathogen-specific response." A "protective immune response" refers to an immune response that suppresses the harmful functions or activity of a pathogen, reduces pathogen infection, or reduces symptoms (including death) of a pathogen infection. In this article, immune response encompasses all of the above.

[0237] As used herein, the term "adjuvant" refers to an agent that enhances the production of an immune response in a nonspecific manner. Common adjuvants include suspensions of minerals (alum, aluminum hydroxide, aluminum phosphate) onto which antigens are adsorbed, and emulsions; the emulsions may include water-in-oil, and oil-in-water (and their variants, including double emulsions and reversible emulsions), liposaccharides, 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 ingredients.

[0238] As used herein, the term "mutation" refers to an amino acid residue that is missing, added, or substituted in the amino acid sequence of a protein or polypeptide compared to the amino acid sequence of a reference protein or polypeptide. In the specification (particularly in the Examples), a specific positional amino acid substitution in a protein sequence is described using the following format: "(amino acid residue in the wild-type protein)(amino acid position)(amino acid residue in the engineered protein)". For example, "A298L" refers to a substitution of an alanine (A) residue at position 298 of the amino acid sequence of a reference protein by a leucine (L) residue.

[0239] As used herein, the term "corresponding positions" refers to amino acid positions that are at equivalent positions in the two sequences being compared when the two sequences are optimally aligned, i.e., when the sequences are aligned for the highest percent identity. For example, the expression "corresponding to positions 35, 60, 79, 92, 189, 190, 227, 238, 298, 320, 326, 377, 405, 437, 481, and 496 of SEQ ID NO: 1" refers to amino acid positions that are at equivalent positions to positions 35, 60, 79, 92, 189, 190, 227, 238, 298, 320, 326, 377, 405, 437, 481, and 496 of SEQ ID NO: 1 when the sequences are optimally aligned, i.e., when the sequences are aligned for the highest percent identity.

[0240] As used herein, the term "identity" refers to the match between two polypeptides or between two nucleic acids. When a position in both sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences have 60% identity. For example, the DNA sequences CTGACT and CAGGTT share 50% identity (3 out of 6 positions match). Typically, two sequences are compared when they are aligned for maximum identity. Such an alignment can be achieved, for example, by using the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453, which can be conveniently performed using a computer program such as the Align program (DNAstar, Inc.). The percent 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)), which has been incorporated 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 percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J Mol Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossum 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0241] 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 antigenic determinants, such as those recognized by immune cells.

[0242] As used herein, the term "D25" refers to the antibody described in WO 2008 / 147196 A2, which has a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:40 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:41.

[0243] As used herein, the term "Palivizuma" refers to palivizumab having a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO:42 and a light chain variable domain comprising the amino acid sequence of SEQ ID NO:43.

[0244] As used herein, the term "C-terminal truncation of X amino acids" means that the most consecutive X amino acids at the C-terminus are truncated. Similarly, the term "N-terminal truncation of X amino acids" means that the most consecutive X amino acids at the N-terminus are truncated.

[0245] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material elements it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as 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, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an initiation of replication site.

[0246] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

[0247] As is known to those skilled in the art, codons have degeneracy. That is, during the translation of a protein, each amino acid may correspond to one or more codons, for example, up to six codons. Different species have great differences in the use of degenerate codons to encode a certain amino acid, and have different preferences. This preference phenomenon is called "codon preference". Therefore, as used herein, the term "codon preference" refers to the situation in which a species prefers to use certain specific codons to encode an amino acid. Optimizing the sequence of a nucleic acid molecule based on codon preference is particularly advantageous in some cases, for example, it may help to improve the expression level of the protein encoded by the nucleic acid molecule. For example, when using Escherichia coli (or human cells) to express a protein or its fragment, it would be potentially advantageous to optimize the nucleic acid sequence encoding the protein or its fragment based on the codon preference of Escherichia coli (or human cells).

[0248] As used herein, the term "virus-like particle (VLP) " is a polymeric particle whose structure is similar or dissimilar to natural virus particles. In certain embodiments, VLP is a natural virus particle. In certain embodiments, VLP is a virus-like particle assembled into by proteins. It has been confirmed that the proteins (e.g., capsid proteins, surface proteins, envelope proteins) of some viruses (e.g., RSV, HBV, HEV, HPV) can spontaneously form VLPs after recombinant expression in a suitable expression system.

[0249] As used herein, the term "pharmaceutically acceptable" means that it is generally recognized in the pharmaceutical field that it can be used in animals, particularly in humans. As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH regulators (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic or nonionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and any combination thereof.

[0250] As used herein, pharmaceutically acceptable carriers can be sterile liquids such as water and oils, including those derived from petroleum, animals, plants, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, physiological saline is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions.

[0251] As used herein, pharmaceutically acceptable excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also include a wetting agent, or an emulsifier such as sodium hyaluronate, or a pH buffer. The pharmaceutical composition may be in the form of a solution, suspension, emulsion, tablet, pill, capsule, powder, sustained-release formulation, etc.

[0252] As used herein, the term "subject" refers to mammals, including but not limited to humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, etc. Preferably, the subject is a human.

[0253] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, a prophylactic effective amount is an amount sufficient to prevent, arrest, or delay the onset of a disease; a therapeutic effective amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered treatments.

[0254] Advantageous Effects of the Invention

[0255] The recombinant RSV F protein of the present application comprises the amino acid mutation of the pre-fusion (pre-F) conformation of stabilizing the RSV F protein, therefore, it has increased stability compared with wild-type RSV F protein. Moreover, it also has higher pre-F protein proportion, higher protein expression amount and better thermostability compared with wild-type RSV F protein. Further, the recombinant RSV F protein of the present application also has good immunogenicity, can induce neutralizing antibodies.

[0256] In summary, the recombinant RSV F protein of the present application demonstrates good protection and safety, and is suitable for various vaccine platforms, such as nucleic acid vaccines, recombinant protein vaccines, viral vector vaccines, and particulate vaccines. Therefore, the recombinant RSV F protein of the present application has great potential for inducing an immune response to RSV in subjects, and for preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0257] FIG1 exemplarily illustrates the predicted structure of a recombinant RSV F protein of the present application.

[0258] FIG2 exemplarily lists the SDS-PAGE results of nRSVF79 protein and nRSVF116 protein, wherein M: protein marker; reduction: under reducing conditions; non-reduction: under non-reducing conditions.

[0259] FIG3 exemplarily lists the thermal stability test results of 7 recombinant RSV F proteins of the present application.

[0260] FIG4 shows the titers of specific antibodies produced after mice were immunized with the 10 recombinant RSV F proteins of the present application and the control proteins pre-F and post-F proteins.

[0261] FIG5 shows the titers of neutralizing antibodies produced after mice were immunized with the 10 recombinant RSV F proteins of the present application and the control proteins pre-F and post-F proteins.

[0262] Below in conjunction with embodiment, embodiment of the present invention is described in detail, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, rather than to limit the scope of the invention. According to the following detailed description of preferred embodiments, various objects and advantages of the present invention will become apparent to those skilled in the art.

[0263] Sequence information

[0264] Information on the partial sequences involved in the present invention is provided in Table 1 below.

[0265] Table 1: Description of sequences DETAILED DESCRIPTION

[0266] The invention will now be described with reference to the following examples which are intended to illustrate the invention but not to limit it.

[0267] Unless otherwise indicated, the experiments and procedures described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA used in the present invention can be found in Sambrook, Fritsch, and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (FM Ausubel et al., eds., (1987)); METHODS IN ENZYMOLOGY series (Academic Press): PCR 2: A PRACTICAL APPROACH (MJ MacPherson, BD Hames, and GR Taylor, eds. (1995)); and ANIMAL CELL CULTURE. CULTURE) (RI Freshney, ed. (1987)).

[0268] In addition, if specific conditions are not specified in the examples, the experiments were performed under conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially. It is understood that the examples describe the present invention by way of example and are not intended to limit the scope of the present invention. All publications and other references mentioned herein are incorporated herein by reference in their entirety.

[0269] Example 1. Design of recombinant RSV F protein

[0270] The present embodiment provides the scheme of multiple design and preparation recombinant RSV F protein.These recombinant RSV F proteins comprise the trimeric folding domain foldon of the F2 fragment, F1 fragment and T4 bacteriophage fibrin of RSV F protein.These recombinant RSV F proteins also comprise the connector connecting F2 fragment and F1 fragment, and can comprise one or more amino acid mutations (for example, the mutation enhancing expression amount and / or stability). The application has prepared 116 recombinant RSV F proteins in total, and these recombinant RSV F proteins prepared are successively named as nRSVF1, nRSVF2, ... nRSVF116. In addition, the structure of these proteins has also been predicted by software, and it is found that the structure of these proteins is relatively similar, and wherein, Fig. 1 exemplarily lists the structure diagram of one protein prediction.

[0271] In short, the design and preparation of recombinant RSV F protein in this application refers to the amino acid sequences shown in SEQ ID NO:3 and SEQ ID NO:1. The amino acid residues 1 to 513 in SEQ ID NO:3 are identical to the amino acid residues 1 to 513 of the F0 precursor polypeptide of wild-type RSV A2 (see SEQ ID NO:1). Compared with SEQ ID NO:1, SEQ ID NO:3 removes the transmembrane and intracellular regions and is connected to the trimerization foldon domain and His tag through SAIG ​​and GS connecting peptides. Each recombinant RSV F protein consists of an F2 polypeptide, an F1 polypeptide and an intermediate connector. Among them, the F2 polypeptide includes amino acids 26 to 105 of SEQ ID NO:3, and the F1 polypeptide includes amino acids 137 to 513 of SEQ ID NO:3 (excluding specially noted introduced mutations or truncations). The signal peptide (amino acids 1-26 of SEQ ID NO: 3) and pep27 (amino acids 110-136 of SEQ ID NO: 3) were deleted during expression or when a linker was used. The plasmid construction and expression and purification procedures for these recombinant RSV F proteins are described in Examples 2 and 3.

[0272] The specific design and screening process of the recombinant RSV F protein is as follows:

[0273] 1) Design and screening of linkers between F2 and F1

[0274] By introducing a covalent linker between the C-terminus of F2 and the N-terminus of F1, the release of the fusion peptide can be prevented, thereby promoting the formation and stabilization of the pre-F conformation. Specifically, 8 recombinant RSV F proteins were synthesized, including:

[0275] nRSVF0 is the F0 precursor polypeptide of wild-type RSV A2 without the linker (SEQ ID NO: 1);

[0276] nRSV F1 is a recombinant RSV F protein comprising the F2 segment (amino acids 1-105 of SEQ ID NO: 1), a linker (SEQ ID NO: 54), and the F1 segment (amino acids 137-513 of SEQ ID NO: 1);

[0277] nRSV F2 is a recombinant RSV F protein comprising the F2 segment (amino acids 1-105 of SEQ ID NO: 1), a linker (SEQ ID NO: 55), and the F1 segment (amino acids 137-513 of SEQ ID NO: 1);

[0278] nRSVF3 is a recombinant RSV F protein comprising the F2 segment (amino acids 1-105 of SEQ ID NO: 1), a linker (SEQ ID NO: 56), and the F1 segment (amino acids 137-513 of SEQ ID NO: 1);

[0279] nRSVF4 is a recombinant RSV F protein comprising the F2 segment (amino acids 1-105 of SEQ ID NO: 1), a linker (SEQ ID NO: 57), and the F1 segment (amino acids 137-513 of SEQ ID NO: 1);

[0280] nRSVF5 is a recombinant RSV F protein comprising the F2 segment (amino acids 1-105 of SEQ ID NO: 1), a linker (SEQ ID NO: 58), and the F1 segment (amino acids 110-513 of SEQ ID NO: 1);

[0281] nRSVF6 is a recombinant RSV F protein comprising the F2 segment (amino acids 1-105 of SEQ ID NO: 1), a connector (SEQ ID NO: 58), and an F1 segment (amino acids 110-513 of SEQ ID NO: 1); wherein, compared to nRSVF5, nRSVF6 lacks the N-terminal six amino acids of the hydrophobic fusion peptide (FP) (amino acids 140-145 of SEQ ID NO: 1, FLLGVG);

[0282] nRSVF7 is a recombinant RSV F protein comprising the F2 segment (amino acids 1-105 of SEQ ID NO: 1), a linker (SEQ ID NO: 59), and the F1 segment (amino acids 145-513 of SEQ ID NO: 1);

[0283] As shown in Table 2, this embodiment attempts several different linkers. These recombinant RSV F proteins containing linkers are mainly evaluated by three indicators in Table 2: the first indicator is to characterize the expression amount by evaluating the binding strength of the recombinant RSV F protein and the co-reactive antibody palivizumab (expression amount) of the F protein. The higher the value, the higher the expression amount. The second indicator is to characterize whether the recombinant RSV F protein retains the conformation of pre-F by evaluating the binding strength of the recombinant RSV F protein and the pre-F protein-specific antibody D25. The higher the value, the more pre-F is retained. The third indicator is to calculate the relative proportion of pre-F protein in the total F protein by calculating the ratio of D25 to palivizumab (i.e., D / P). The higher the value, the higher the representative proportion. Wherein, the VH sequence of antibody D25 is shown in SEQ ID NO:40, and the VL sequence is shown in SEQ ID NO:41; the VH sequence of antibody palivizumab is shown in SEQ ID NO:42, and the VL sequence is shown in SEQ ID NO:43; the specific evaluation process is described in Example 4.

[0284] Compared to F0 (SEQ ID NO:3), the relative proportions of pre-F proteins in several recombinant RSV F proteins containing different connectors are increased. The expression of nRSVF6 can be suitable with F0, and pre-F accounts for a significant improvement. Although the expression of nRSVF7 is lower than F0, it is still at a higher level, and its pre-F accounts for a very effective improvement. In summary, the connector design scheme in nRSVF6 (SEQ ID NO:4) and nRSVF7 (SEQ ID NO:5) is a more advantageous scheme.

[0285] Table 2. Results of recombinant RSV F protein

[0286] 2) Introducing point mutations to increase expression and pre-F protein ratio

[0287] As can be seen from the above-described embodiments, the pre-F proportion of nRSVF7 is higher, which is a promising connector transformation scheme, but its expression still has room for optimization. Therefore, on the basis of the F2 fragment, connector and F1 fragment included in nRSVF7, by introducing point mutations, usually to attempt to change the expression of protein. The present embodiment further designs a variety of mutation schemes to optimize the expression of recombinant RSV F protein, and the relative position of the mutation site of these recombinant RSV F proteins is based on SEQ ID NO:1. The specifically synthesized recombinant RSV F protein and the mutation they comprise are as shown in Table 3. The effect of the recombinant RSV F protein carrying point mutations is evaluated by three indices of expression, the binding strength (D25) of pre-F specific antibodies and pre-F accounting for (D / P).

[0288] The results showed that mutations in nRSVF8, nRSVF11, nRSVF26, and nRSVF31 significantly increased the expression of recombinant RSV F protein, while nRSVF14 slightly increased the expression.

[0289] Further, in order to improve the proportion of pre-F, on the basis of the mutation site corresponding to nRSVF8 (SEQ ID NO:6), nRSVF11 (SEQ ID NO:7), nRSVF14 (SEQ ID NO:8), nRSVF26 (SEQ ID NO:9) and nRSVF31 (SEQ ID NO:10), other amino acid whose mutation schemes were attempted.Specific mutation scheme and result are as shown in Table 4, and most of the expansion mutations based on nRSVF26 and nRSVF11 can make expression amount obtain and significantly improve, and prove that the mutation site included by these two recombinant RSV F proteins has a larger impact on expression amount.To sum up, nRSVF31, nRSVF61 (SEQ ID NO:11), nRSVF66 (SEQ ID NO:12) and nRSVF67 (SEQ ID NO:13) these several recombinant RSV F proteins retain the advantage of expression amount and pre-F proportion simultaneously.

[0290] Table 3. Results of recombinant RSV F protein

[0291] Table 4. Results of recombinant RSV F protein

[0292] 3) Combination mutation and stability testing

[0293] In addition to protein expression and pre-F ratio, stability is also a very important indicator for evaluating the quality of mutant proteins. Since the mutation of nRSVF61 significantly improves the expression level and its pre-F ratio is high, the recombinant RSV F protein is further added based on the recombinant RSV F protein, and the freeze-thaw stability and temperature stability of the mutated recombinant RSV F protein are tested. The stability of the recombinant RSV F protein is evaluated by the retention binding rate of D25 after ten freeze-thaw cycles or after treatment at 60°C for 1 hour. The higher the retention binding rate, the better the stability. The specific evaluation process of stability is described in detail in Example 5.

[0294] As shown in Table 5, the recombinant RSV F protein with combined mutations retained a higher freeze-thaw stability as a whole, especially nRSVF71 (SEQ ID NO: 14), nRSVF78, nRSVF79 (SEQ ID NO: 21), nRSVF80 (SEQ ID NO: 22), nRSVF82 (SEQ ID NO: 23), nRSVF83 (SEQ ID NO: 24), nRSVF105, and nRSVF109 to nRSVF116. After freezing and thawing ten times, the retention binding rate of D25 of these recombinant RSV F proteins was still greater than 0.9, showing good freeze-thaw stability. The temperature stability was weaker than the freeze-thaw stability as a whole. After some recombinant RSV F proteins were heat treated at 60°C for one hour, the retention binding rate of D25 was less than 0.5. However, the D25 retention binding rate of nRSVF73 to nRSVF78 (SEQ ID NO: 15-20), nRSVF94 (SEQ ID NO: 25), nRSVF96 (SEQ ID NO: 26), nRSVF100 (SEQ ID NO: 27), nRSVF105 (SEQ ID NO: 28), nRSVF106 (SEQ ID NO: 29) and nRSVF116 (SEQ ID NO: 37) is still greater than 0.8, showing good thermal stability. nRSVF78, nRSVF105 and nRSVF116 also retain high freeze-thaw stability and thermal stability.

[0295] Table 5. Results of recombinant RSV F protein

[0296] 4) Evaluation of trimer pre-F ratio

[0297] Although the degree of binding to D25 can indicate the proportion of pre-F, some monomeric pre-F can also bind well to D25. It is unclear whether this monomeric pre-F will impair the immunogenicity of the recombinant RSV F protein. The binding effect of the recombinant RSV F protein and the trimeric-specific antibody AM14 (the VH sequence of antibody AM14 is shown in SEQ ID NO: 44, and the VL sequence is shown in SEQ ID NO: 45) was further evaluated. The specific evaluation process is described in Example 4.

[0298] The results are shown in Table 6. Some recombinant RSV F proteins had no binding to AM14 at all (<0.1), but nRSVF79 showed an extremely high ability to bind to AM14 (>3). In addition, nRSVF72 (SEQ ID NO: 38), nRSVF74, nRSVF78 to nRSVF80, nRSVF82, nRSVF82, nRSVF83 and nRSVF112 to nRSVF116 also had certain binding to AM14 (>0.2, <1).

[0299] Table 6. Results of recombinant RSV F protein

[0300] Example 2. Construction of recombinant RSV F protein (F protein mutant) expression vector

[0301] This example is the specific experimental operation steps implemented in Example 1, and the relevant experimental results are reflected in Example 1.

[0302] The amino acid sequence of the precursor (nRSVF0) of the recombinant RSV F protein (hereinafter also referred to as the F protein mutant) is shown in SEQ ID NO: 3, which includes a signal peptide (amino acid residues 1-25), an F2 polypeptide chain (amino acid residues 26-109), pep27 (amino acid residues 110-136), an F1 polypeptide chain (amino acid residues 137-513), a foldon domain (amino acid residues 518-544), a thrombin cleavage site (amino acid residues 547-552), a His purification tag (amino acid residues 553-558), and a flexible linker sequence (amino acid residues 514-517, 545, and 546).

[0303] Submit the amino acid sequence of SEQ ID NO: 3 to Genscript TM The synthesized gene fragment was inserted into the commercially available expression vector pcDNA3.1, which contains a CMV promoter and ampicillin resistance. Lightning Site-Directed Mutagenesis Kits (Agilent, 210519) were used to replace, insert, or delete nucleotide sequences to generate various F protein mutants. The target sequences of the F protein mutants were confirmed by DNA sequencing. The plasmids used for transfection were extracted using an endotoxin-free plasmid extraction kit (TianGen, DP120), and DNA concentration and purity were determined by spectrophotometric measurement of OD260 and OD280. Plasmids were stored at -20°C. All commercial reagents were used according to the manufacturer's instructions.

[0304] Example 3. Expression and purification of recombinant RSV F protein (F protein mutant)

[0305] This example is the specific experimental operation steps implemented in Example 1, and the relevant experimental results are reflected in Example 1. Figure 2 exemplifies the SDS-PAGE results of nRSVF79 protein and nRSVF116 protein, where M: protein marker; reduction: under reducing conditions; non-reduction: under non-reducing conditions.

[0306] Prepare ExpiCHOTM suspension cells in the logarithmic growth phase and culture them in a cell shaker at 125 rpm, 37°C, 8% CO2 to a density of 6×10 6 Cells / mL, with a viability of >98%. Place 25 mL of cells into a new cell culture flask as a transfection system. Tube A: 1 mL ExpiCHO™ Expresssion Medium containing 25 μg of plasmid; Tube B: 1 mL ExpiCHO™ Expresssion Medium containing 80 μL of transfection reagent from the ExpiFectamine™ CHO Transfection Kit (Thermo Scientific; Catalog No. A29129). Mix Tubes A and B and let stand at room temperature for 2 minutes. After 2 minutes, pour the mixture into 25 mL of the prepared transfection system. Incubate in a shaker at 125 rpm, 37°C, 8% CO2 for 18-22 hours. Add 150 μL of enhancer and 4 mL of supplement from the ExpiFectamine™ CHO Transfection Kit to each flask and incubate in a shaker at 125 rpm, 32°C, 5% CO2 for 8-15 days. After incubation, centrifuge at 4000 rpm at 4°C for 10 minutes and collect the supernatant.

[0307] Filter the supernatant using a 0.22 μm filter. Turn on the AKTA instrument and rinse pipe A and pipe B with solution A (200 mM sodium phosphate dodecahydrate) and solution B (100 mM citric acid monohydrate), respectively, and then install the protein A column. Use solution A to equilibrate the protein A column at a flow rate of 8 mL / min for more than 15 minutes. After the UV value, pH value, and conductivity detected by the instrument are stable, proceed to the next step. Load the sample at a flow rate of 6-10 ml / min. The UV value will then rise. This peak is the penetration peak. Continue to wash the column with solution A and collect the penetration peak sample for testing. After the pH value no longer changes, add solution B at a flow rate of 6-10 ml / min. The pH value will then drop and the UV value will rise. This peak is the elution peak. The antibody is mainly present in the elution peak. Collect the elution peak sample for testing. Equilibrate the column with solution A, then fill the pipe and protein A column with 20% ethanol, remove the column, and store at 4°C. The samples of the penetration peak and the elution peak were purified and their concentrations were determined by UV spectrometry.

[0308] Example 4. Expression level and epitope characteristics analysis of recombinant RSV F protein (F protein mutant)

[0309] This example is the specific experimental operation steps implemented in Example 1, and the relevant experimental results are reflected in Example 1.

[0310] Five days after transfection, a portion of the transfection supernatant containing the F protein mutant plasmid was collected and centrifuged at low speed to remove cell debris. Binding of the expression supernatant to palivizumab, D25, or AM14 (sequence) was then assayed using a Nickel-Coated Plate (PIERCE, 15442). The nickel-coated plate was washed once with PBST (20mM PB7.4, 150mM NaCl, 0.1% Tween 20). The expression supernatant was diluted fivefold with blank cell culture supernatant, and 100 μL was added to the nickel-coated plate and incubated at 37°C for 60 minutes. The detection antibody was diluted with 2% skim milk powder (Wandashan) to a concentration of 2.5 μg / mL. The nickel-coated plate was washed five times with PBST, and 100 μL of the diluted detection antibody was added to each well and incubated at 37°C for 60 minutes. The nickel-ion-coated plate was washed five times with PBST, and 100 μl of horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Abcam, ab97225) was added to each well. The plate was incubated at 37°C for 30 minutes. After the enzyme labeling step, the plate was washed five times with PBST, and 50 μl of TMB colorimetric reagent (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) was added to each well. The plate was incubated at 37°C for 15 minutes. After the color development step, 50 μl of stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) was added to each well of the plate, and the OD450 / 630 value of each well was measured on a microplate reader. The expression level was represented by the absorbance value of palivizumab, the content of pre-F was represented by the absorbance value of D25, the content of trimer was represented by the absorbance value of AM14, and the D / P ratio was obtained by dividing the absorbance value of D25 by the absorbance value of palivizumab.

[0311] Example 5. Stability analysis of recombinant RSV F protein (F protein mutant)

[0312] This example is the specific experimental operation steps implemented in Example 1, and the relevant experimental results are reflected in Example 1.

[0313] Five days after transfection, a portion of the transfection supernatant containing the F protein mutant plasmid was collected and centrifuged at low speed to remove cell debris. The supernatant was then freeze-thawed or heat-treated. For the freeze-thaw test, the transfection supernatant was frozen in a mixture of dry ice and alcohol, then rapidly thawed in a 37°C water bath, repeating this step 10 times. For the thermal stability test, the supernatant was placed in a 60°C water bath for one hour. An untreated sample was placed on ice at 4°C as a control. D25 retention was determined by adding pre- and post-treatment cell supernatants to palivizumab-coated plates. Specifically, palivizumab at a concentration of 2.5 μg / mL was added to a blank ELISA plate at a volume of 100 μL per well. The plate was incubated at 37°C for 60 minutes and washed five times with PBST. 200 μL of 5% skim milk powder was added and the plate was blocked at 37°C for 2 hours. After the blocking reaction, the wells were washed five times with PBST, and 100 μl of biotinylated D25 antibody was added to each well and incubated at 37°C for 60 minutes. After the reaction, the wells were washed five times with PBST, and 100 μl of poly-HRP streptavidin (PIERCE, N200) was added to each well and incubated at 37°C for 1 hour. After the reaction, the plate was washed five times with PBST, and 50 μl of TMB colorimetric reagent (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to each well and incubated at 37°C for 15 minutes. After the color development step, 50 μl of stop solution (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to each well of the plate, and the OD450 / 630 value of each well was measured on a microplate reader. The retained binding rate of D25 was determined by comparing the absorbance of the supernatant after freeze-thaw or heat treatment to the absorbance of the supernatant before treatment.

[0314] Example 6. Affinity analysis of recombinant RSV F protein (mutated F protein) and antibody disc

[0315] The binding affinities of the F protein mutants to the F protein-specific antibodies palivizumab, D25, AM14, and hRSV90 were determined using surface plasmon resonance (SPR). Histidine-tagged F protein mutants were immobilized at approximately 100 nM on an NTA sensor chip (Cytiva) using a Biacore-8K (Cytiva) instrument. Antibodies were then added in a gradient dilution range (200 to 0.78 nM). RU data were fitted to a 1:1 binding model using Biacore™ Insight software.

[0316] Affinity results for several exemplary F protein mutants are shown in Table 7. These F protein mutants containing single or double point mutations exhibit nanomolar or picomolar affinities for the co-reactive antibody palivizumab, demonstrating that the mutants are properly folded and expressed. Furthermore, these mutants also exhibited good affinity for the pre-F-specific antibodies D25, AM14, and hRSV90, demonstrating that the introduction of these single or double point mutations, or any combination thereof, can have similar or even stronger effects in stabilizing the pre-F structure.

[0317] Table 7. Affinity analysis of recombinant RSV F protein and antibodies

[0318] Example 7. Testing the thermal stability of recombinant RSV F protein (F protein mutant) by differential scanning fluorimetry

[0319] The thermal stability of F protein mutants was analyzed using differential scanning fluorimetry (DSF). The test protein sample was diluted to a concentration of 1 mg / mL in sterile PBS. Eight two-fold dilutions were performed to determine the appropriate sample concentration. A blank PBS control was used. SYPRO Orange (Sigma-Aldrich, catalog number S5692-500UL) was diluted with deionized water to a final concentration of 50X. 45 μL of the diluted test protein sample was added to a Bio-Rad PCR plate, followed by 5 μL of 50X SYPRO Orange. The system was placed in a quantitative fluorescence PCR instrument (Bio-Rad). The excitation and emission wavelengths were adjusted to the corresponding wavelengths of the fluorescent dye. The temperature was ramped from 0°C to 100°C at a rate of 0.5°C / 10 s. The data were processed using BIO-RAD manager, and the curve of fluorescence intensity versus temperature was plotted. The Tm value of the protein sample was calculated by fitting the Boltzmann transport equation (BTE).

[0320] The experimental results are shown in Figure 3. Several exemplary F protein mutants have similar decomposition temperatures, with Tm values ​​between 56°C and 60°C. Among them, the Tm temperatures of nRSVF61 and nRSVF116 reached 57.8°C and 59.3°C, respectively. This shows that the introduction of these single-point mutations or double-point mutations, or any combination, can make the F protein have good thermal stability.

[0321] Example 8. Immunogenicity Analysis of Recombinant RSV F Protein (F Protein Mutants)

[0322] (1) Mouse immunization experiment

[0323] The animal husbandry and experimental procedures were carried out in accordance with the procedures established by the Laboratory Animal Use and Management Committee of Xiamen University. The experimental animals were BALB / c female mice, 6-8 weeks old, purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and housed at the Laboratory Animal Center of Xiamen University. The immunization protocol consisted of mixing 10 F protein mutants and control proteins, pre-F and post-F proteins, with aluminum adjuvant in a volume ratio of 1:1 for immunization of mice. The pre-F protein and post-F protein for immunization were constructed as follows: the sequences of the pre-F and post-F proteins were downloaded from GenBank (GenBank ID: LY628284.1, GenBank ID: 6APB_A), the nucleotide sequences encoding the two proteins were cloned into the pcDNA3.1 eukaryotic expression vector, expressed using the CHO eukaryotic expression system, and purified by affinity chromatography to obtain the pre-F protein (SEQ ID NO: 52) and post-F protein (SEQ ID NO: 53), following the same procedures as in Examples 2 and 3. Each group consisted of six mice, immunized by unilateral thigh intramuscular injection at a dose of 2 μg protein per mouse. Fourteen days after the two injections, orbital blood was collected and serum was isolated. Serum samples were inactivated at 56°C for 30 minutes and stored at 4°C until use.

[0324] (2) Evaluation of Pre-F specific antibody titer in serum

[0325] The method for constructing the pre-F protein used for coating is described above. On the first night, dilute the pre-F protein with PBS and coat a 96-well plate at 100 ng / well at 4°C overnight. Discard the coating protein solution and wash once with PBST. Add 200 μl / well of blocking solution containing 5% fetal bovine serum and incubate at 37°C for 2 hours. Discard the blocking solution and wash once with PBST. Add serially diluted mouse serum (200-fold in the first well, 5-fold in the next well) at 100 μl / well and incubate at 37°C for 1 hour. Discard the serum dilution solution and wash five times with PBST. Add 1:5000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Abcam, ab97265) at 100 μl / well and incubate at 37°C for 1 hour. Discard the secondary antibody and wash five times with PBST. Add 100 μl / well of colorimetric solution and develop for 10 minutes at room temperature in the dark. Add 50 μl / well of stop solution and measure the absorbance at OD450 / 630 using a microplate reader. The endpoint titer is determined by 3 times the reading in the negative wells.

[0326] The experimental results, shown in Figure 4, show that F protein mutants containing single or double point mutations all induced pre-F binding antibody titers comparable to those of the pre-F control protein, and the pre-F binding antibody titers of these mutants were approximately 100-fold higher than those of the post-F protein. This indicates that the introduction of single or double point mutations can maintain the F protein in the pre-F conformation, which is also stable in animals.

[0327] (2) Serum neutralizing antibody titer assessment

[0328] Gradient dilution of serum: 10-fold in the first well, 4-fold gradient dilution for a total of 10 gradients. Then add an equal volume of rRSV-mkatushka2 (MOI = 0.1), mix and incubate at 37°C for 1 hour. Transfer the serum virus mixture to a 96-well plate with a monolayer of HeLa cells at a volume of 100 μl / well and incubate at 37°C. At the same time, set up a virus control well without serum as a negative control. After 24 hours of culture, use SpectraMax Paradigm Multi-Mode Microplate Reader to read the plate. The highest serum dilution factor with a mean fluorescence intensity lower than 50% of the negative control well is used as the neutralizing antibody titer of the serum.

[0329] The experimental results, shown in Figure 5, show that the introduction of some stabilizing single-point mutations resulted in average neutralizing antibody titers of ~200, approximately five times higher than the post-F control protein. When double-point mutations were introduced, neutralizing antibody titers for most mutants reached over 500, demonstrating that combined mutations have a greater advantage in stabilizing the pre-F protein conformation in animals. These immunogens incorporating single-point or double-point mutations exhibited robust immune responses and may provide strong candidate antigens for the development of recombinant RSV protein vaccines.

[0330] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A recombinant respiratory syncytial virus (RSV) fusion (F) protein comprising the F2 and F1 domains of a wild-type RSV F protein; and further comprising at least one amino acid mutation that stabilizes the pre-fusion (pre-F) conformation of the RSV F protein compared to the wild-type RSV F protein.

2. The recombinant RSV F protein of claim 1, which has one or more characteristics selected from the following, compared to wild-type RSV F protein: (1) having increased stability; preferably, the stability is measured by binding of the recombinant RSV F protein to antibody D25; (2) have a higher proportion of pre-F protein; Preferably, the recombinant RSV F protein comprises a prefusion conformation (pre-F) protein, a postfusion conformation (post-F) protein, or a mixture of the two; Preferably, the proportion of pre-F protein is measured by the ratio of antibody D25 and antibody palivizumab binding to the recombinant RSV F protein; (3) have higher protein expression; (4) Contains one or more amino acid insertions, deletions, and / or mutations; (5) the positions of the amino acid mutations contained in the recombinant RSV F protein are selected from any one or more of the following: positions 35, 60, 79, 92, 189, 190, 227, 238, 298, 320, 326, 377, 405, 437, 481, and 496 corresponding to wild-type RSV F protein; (6) lack of at least one furin cleavage site, or lack of one or more amino acids in the furin cleavage site; (7) Deletion of one or more (e.g., 1-5, 5-10, 10-15, 15-20, 20-27) amino acids in the pep27 domain.

3. The recombinant RSV F protein of claim 1 or 2, wherein The wild-type RSV is a subtype A strain, a subtype B strain, 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 a sequence selected from the group consisting of: composition: (i) the sequence shown in SEQ ID NO: 1, 2 or 64; (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: 1, 2 or 64; (iii) a sequence having 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: 1, 2, or 64.

4. The recombinant RSV F protein of any one of claims 1-3, comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid mutations compared to wild-type RSV F protein; Preferably, the position of the amino acid mutation contained in the recombinant RSV F protein is selected from: positions corresponding to position 35, 189, 190, 227, 238 or 298 of the wild-type RSV F protein; Preferably, the recombinant RSV F protein comprises amino acid mutations at positions corresponding to position 298 of the wild-type RSV F protein; and one or more positions selected from the group consisting of positions 60, 79, 92, 189, 190, 227, 238, 320, 326, 377, 405, 437, 481, and 496 of the wild-type RSV F protein.

5. The recombinant RSV F protein of any one of claims 1-4, having one or more characteristics selected from the group consisting of: (1) The amino acid at position 35 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is Y or F; Preferably, the amino acid mutation at position 35 is a mutation from S to Y or F; (2) the amino acid at position 298 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is I, H, M, P, or L; Preferably, the amino acid mutation at position 298 is a mutation from A to I, H, M, P or L; (3) the amino acid at position 238 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L, I, T, or V; Preferably, the amino acid mutation at position 238 is a mutation from S to L, I, T or V; (4) the amino acid at position 189 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L, I, or V; Preferably, the amino acid mutation at position 189 is a mutation from T to L, I or V; (5) the amino acid at position 190 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is I, L, M, V, or Y; Preferably, the amino acid mutation at position 190 is a mutation from S to I, L, M, V or Y; (6) the amino acid at position 227 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L; Preferably, the amino acid mutation at position 227 is an N to L mutation; (7) the amino acid at position 320 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is A, F, H, I, K, L, M, N, Q, R, S, T, V, W, or Y; Preferably, the amino acid mutation at position 320 is a mutation from P to A, F, H, I, K, L, M, N, Q, R, S, T, V, W or Y; (8) The amino acid at position 326 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is D, E, or P; Preferably, the amino acid mutation at position 326 is a mutation from T to D, E or P; (9) The amino acid at position 377 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is P or Q; Preferably, the amino acid mutation at position 377 is a mutation from S to P or Q; (10) The amino acid at position 405 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is V; Preferably, the amino acid mutation at position 405 is an S to V mutation; (11) The amino acid at position 60 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is F or Y; Preferably, the amino acid mutation at position 60 is a mutation from E to F or Y; (12) The amino acid at position 437 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is R or Y; Preferably, the amino acid mutation at position 437 is a mutation from N to R or Y; (13) the amino acid at position 481 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is R; Preferably, the amino acid mutation at position 481 is an L to R mutation; (14) The amino acid at position 496 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is F or Y; Preferably, the amino acid mutation at position 496 is a mutation from N to F or Y; (15) The amino acid at position 92 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is T, V, M, or I; Preferably, the amino acid mutation at position 92 is a mutation from E to T, V, M or I; (16) The amino acid at position 79 of the recombinant RSV F protein corresponding to the wild-type RSV F protein is L; Preferably, the amino acid mutation at position 79 is an I to L mutation.

6. The recombinant RSV F protein of any one of claims 1-5, wherein There is no furin cleavage site and / or pep27 domain between the F2 domain and the F1 domain; Preferably, the F2 domain has one or more characteristics selected from the following: (1) the F2 domain comprises the complete F2 domain of the wild-type RSV F protein (e.g., a portion corresponding to positions 1-109 of the wild-type RSV F protein) or a fragment thereof; (2) the F2 domain comprises a portion corresponding to positions 1-95, 5-95, 10-95, 1-100, 5-100, 10-100, 1-105, 5-105, 10-105, 1-110, 5-110, or 10-110 of the wild-type RSV F protein; (3) the F2 domain comprises a portion corresponding to positions 1-105 of the wild-type RSV F protein; (4) the F2 domain lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues from the N-terminus and / or C-terminus compared to the F2 domain of the wild-type RSV F protein; For example, the F2 domain is deleted from the N-terminus by 5 or 25 amino acids and from the C-terminus by 4 amino acids compared to the F2 domain of the wild-type RSV F protein; Preferably, the F1 domain has one or more characteristics selected from the following: (1) the F1 domain comprises the complete F1 domain of the wild-type RSV F protein (e.g., a portion corresponding to positions 137-574 of the wild-type RSV F protein) or a fragment thereof; (2) the F1 domain comprises the extracellular domain, transmembrane domain, and / or cytoplasmic tail of the wild-type RSV F protein; (3) the F1 domain comprises a portion corresponding to positions 135-503, 140-503, 145-503, 135-508, 140-508, 145-508, 135-513, 140-513, 145-513, 135-518, 140-518, or 145-518 of the wild-type RSV F protein; (4) the F1 domain comprises a portion corresponding to positions 145-513 of the wild-type RSV F protein; (5) the F1 domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acid residues (e.g., 30-50, 50-100, 100-150, 150-200, 200-250, 250-300) from the N-terminus and / or C-terminus compared to the F1 domain of the wild-type RSV F protein; For example, the F1 domain is deleted from the N-terminus by 8 amino acids and from the C-terminus by 252 or 268 amino acids compared to the F1 domain of the wild-type RSV F protein.

7. The recombinant RSV F protein of any one of claims 1-6, wherein The recombinant RSV F protein further comprises a linker; Preferably, the linker is located at the C-terminus of the F2 domain; Preferably, the linker is located at the N-terminus of the F1 domain; Preferably, the linker has 3-20 amino acids (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 15, 20); Preferably, the linker comprises at least 1 glycine (e.g., 1, 2, 3, 4, 5, 6, 7); Preferably, the linker further comprises at least 1 (e.g., 1, 2, 3) proline; Preferably, the linker comprises a sequence selected from the following, or consists of a sequence selected from the following: a sequence shown in any one of SEQ ID NOs: 54-59.

8. A fusion protein comprising the recombinant RSV F protein of any one of claims 1-7, further comprising another protein or polypeptide; Preferably, the additional protein or polypeptide is selected from: a signal peptide, a multimerization domain, a tag, or any combination thereof.

9. The fusion protein according to claim 8, wherein The signal peptide has one or more characteristics selected from the following: (1) The signal peptide is the natural signal peptide of RSV or a variant thereof, or a natural signal peptide or a variant thereof derived from other organisms; (2) the signal peptide is located at one end (e.g., N-terminus) of the fusion protein; (3) the signal peptide is located at the N-terminus of the F2 domain; (4) The signal peptide is connected to the F2 domain through or without the first connecting peptide.

10. The fusion protein according to claim 8 or 9, wherein The multimerization domain has one or more characteristics selected from the group consisting of: (1) The multimerization domain is a dimerization domain, a trimerization domain, a tetramerization domain, or a pentamerization domain; (2) The multimerization domain is derived from T4 fibritin; Preferably, the multimerization domain has the sequence shown in SEQ ID NO: 39; (3) The multimerization domain is a leucine zipper or an isoleucine zipper; Preferably, the multimerization domain has the sequence shown in SEQ ID NO: 60; (4) the multimerization domain is derived from lung surfactant protein; (5) the multimerization domain is located at the C-terminus of the F1 domain; (6) the multimerization domain is connected to the F1 domain with or without a second connecting peptide; Preferably, the second connecting peptide has the sequence shown in SEQ ID NO:

61.

11. The fusion protein according to any one of claims 8 to 10, wherein The label has one or more characteristics selected from the following: (1) The tag is selected from a polyhistidine tag, an antigen or epitope tag, an enzyme tag, or any combination thereof; (2) the tag is located at the end (e.g., C-terminus) of the fusion protein; (3) the tag is located at the C-terminus of the F1 domain or the multimerization domain; (4) The tag is connected to the F1 domain or the multimerization domain through or without a third connecting peptide.

12. The fusion protein according to any one of claims 8 to 11, wherein The fusion protein comprises, from N-terminus to C-terminus, an F2 domain, a linker, and an F1 domain; Preferably, the fusion protein comprises, from N-terminus to C-terminus, an F2 domain, a linker, an F1 domain, and a multimerization domain; optionally, the multimerization domain is connected to the F1 domain with or without a second connecting peptide; Preferably, the fusion protein comprises, from N-terminus to C-terminus, an F2 domain, a linker, an F1 domain, a second connecting peptide, and a multimerization domain; optionally, the C-terminus of the fusion protein comprises or does not comprise a tag; optionally, the N-terminus of the fusion protein comprises or does not comprise a signal peptide; Preferably, the fusion protein has: (i) a sequence shown in any one of SEQ ID NOs: 4-38; (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 NOs: 4-38; (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 to the sequence shown in any one of SEQ ID NOs: 4-38; Preferably, the fusion protein is in the form of a multimer (e.g., a dimer, a trimer, a tetramer), a monomer, or a mixture of the two; Preferably, the fusion protein is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixture of the two.

13. A nucleic acid molecule comprising a nucleotide sequence encoding the recombinant RSV F protein of any one of claims 1-7 or the fusion protein of any one of claims 8-12; Preferably, the nucleotide sequence is codon-optimized or not codon-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 the two.

14. A vector comprising the nucleic acid molecule according to claim 13; Preferably, the vector is a viral vector; Preferably, the viral vector is selected from: influenza virus vector, retrovirus vector, adenovirus vector, adeno-associated virus vector, herpes virus vector, poxvirus vector, baculovirus vector, papillomavirus vector, or papillomasovavirus vector.

15. A host cell comprising the recombinant RSV F protein of any one of claims 1-7, the fusion protein of any one of claims 8-12, the nucleic acid molecule of claim 13, or the vector of claim 14; Preferably, the host cell is selected from prokaryotic cells (such as Escherichia coli cells), eukaryotic cells; Preferably, the eukaryotic cell is a mammalian cell, such as a mouse cell or a human cell; Preferably, the recombinant RSV F protein or fusion protein is displayed on the surface of the cell membrane of the host cell.

16. A method for expressing or producing the recombinant RSV F protein described in any one of claims 1-7 or the fusion protein described in any one of claims 8-12, said method comprising, under conditions that allow protein expression, cultivating the host cell described in claim 15, and optionally, reclaiming or purifying its expressed recombinant RSV F protein or fusion protein.

17. A vaccine comprising one or more selected from the following (1) to (4): (1) The recombinant RSV F protein of any one of claims 1 to 7; (2) The fusion protein according to any one of claims 8 to 12; (3) The nucleic acid molecule according to claim 13; (4) The vector according to claim 14; Preferably, the vaccine further comprises an adjuvant and / or a buffer; Preferably, the adjuvant is selected from metal salts, 3-D-monophosphoryl lipid A (MPL), saponin, oil and water emulsions, liposomes, nanoparticles, or any combination thereof; Preferably, the fusion protein in the vaccine is in the form of a multimer (e.g., a dimer, a trimer, a tetramer), a monomer, or a mixed form; Preferably, the recombinant RSV F protein or fusion protein in the vaccine is in a pre-fusion conformation (pre-F), a post-fusion conformation (post-F), or a mixed form.

18. An immunogenic composition comprising: (i) a first immunogenic component, wherein The first immunogenic component is selected from one or more of the following (1) to (4): (1) The recombinant RSV F protein of any one of claims 1 to 7; (2) The fusion protein according to any one of claims 8 to 12; (3) The nucleic acid molecule according to claim 13; (4) The vector according to claim 14; and (ii) a second immunogenic component.

19. The immunogenic composition according to claim 18, wherein The immunogenic composition has one or more characteristics selected from the group consisting of: (1) the first immunogenic component and the second immunogenic component are each independently selected from a protein component, a nucleic acid component, or any combination thereof; (2) the second immunogenic component is an immunogen derived from another strain of RSV; For example, the first immunogenic component is an immunogen derived from a strain of RSV subtype A, and the second immunogenic component is an immunogen derived from a strain of RSV subtype B; (3) the second immunogenic component is an immunogen derived from other proteins of RSV; For example, the first immunogenic component is an immunogen derived from the RSV F protein, and the second immunogenic component is an immunogen derived from the RSV attachment (G) protein or the RSV small hydrophobin (SH) protein; (4) the second immunogenic component is an immunogen derived from a pathogenic organism different from RSV; For example, the first immunogenic component is an immunogen derived from RSV, and the second immunogenic component is an immunogen derived from a virus different from RSV (e.g., hepatitis B virus (HBV), parainfluenza virus (PIV), poliovirus, influenza virus); (5) the first immunogenic component and the second immunogenic component are formulated for simultaneous or separate administration; (6) The immunogenic composition further comprises more immunogenic components (eg, a third immunogenic component, a fourth immunogenic component and / or a fifth immunogenic component).

20. A kit comprising an immunogenic component selected from one or more of the following (1) to (4): (1) the recombinant RSV F protein of any one of claims 1-7; (2) the fusion protein of any one of claims 8-12; (3) the nucleic acid molecule of claim 13; (4) the vector of claim 14; Preferably, the kit further comprises a carrier component capable of displaying the immunogenic component; Preferably, the carrier component is selected from the group consisting of: nanomaterials (e.g., lipid nanoparticles, protein nanoparticles, polymer nanoparticles, inorganic nanocarriers and biomimetic nanoparticles), bacterial outer membrane vesicles (OMVs), polymerized bases, virus-like particles (VLPs), or any combination thereof; Preferably, the immunogen component and the carrier component in the kit are provided separately or in the form of a complex; Preferably, the immunogenic components are in the form of polymers (e.g., dimers, trimers, tetramers), monomers, or mixed forms; Preferably, the immunogen component in the kit is provided in the form of protein or nucleic acid; Preferably, the vector 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).

21. A pharmaceutical composition comprising: (i) Any one or more selected from the following (1) to (8): (1) The recombinant RSV F protein of any one of claims 1 to 7; (2) The fusion protein according to any one of claims 8 to 12; (3) The nucleic acid molecule according to claim 13; (4) The vector according to claim 14; (5) The host cell according to claim 15; (6) The vaccine according to claim 17; (7) The immunogenic composition according to claim 18 or 19; (8) The kit according to claim 20; and (ii) a pharmaceutically acceptable carrier, excipient, buffer, adjuvant, or any combination thereof; Preferably, the pharmaceutical composition may further comprise 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 directed against RSV; or a vaccine, antiviral agent and / or monoclonal antibody directed against a pathogenic organism other than RSV.

22. Use of the recombinant RSV F protein of any one of claims 1-7, or the fusion protein of any one of claims 8-12, or the nucleic acid molecule of claim 13, or the vector of claim 14, or the host cell of claim 15, or the vaccine of claim 17, or the immunogenic composition of claim 18 or 19, or the kit of claim 20 in preparing a pharmaceutical composition for inducing an immune response to RSV in a subject; Preferably, the immune response includes inducing the subject to produce antibodies (e.g., neutralizing antibodies) against RSV; Preferably, the subject is a mammal, for example, a mouse or a human; Preferably, the subject is 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.

23. Use of the recombinant RSV F protein of any one of claims 1-7, or the fusion protein of any one of claims 8-12, or the nucleic acid molecule of claim 13, or the vector of claim 14, or the host cell of claim 15, or the vaccine of claim 17, or the immunogenic composition of claim 18 or 19, or the kit of claim 20 in the preparation of a pharmaceutical composition for preventing and / or treating RSV infection or diseases and / or symptoms caused by RSV infection; Preferably, the subject is a mammal, for example, a mouse or a human; Preferably, the subject is 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), elderly people (e.g., over 60 years old) and pregnant women with fetuses; Preferably, the diseases and symptoms caused by RSV infection are selected from bronchitis, pneumonia, asthma, obstructive pulmonary disease and cardiopulmonary complications.

24. A method of inducing antibodies against RSV, the method comprising administering an effective amount of the recombinant RSV F protein of any one of claims 1-7, or the fusion protein of any one of claims 8-12, or the nucleic acid molecule of claim 13, or the vector of claim 14, or the host cell of claim 15, or the vaccine of claim 17, or the immunogenic composition of claim 18 or 19, or the kit of claim 20, in cells in vitro or in vivo in a subject; Preferably, the administration method includes intradermal, intramuscular, subcutaneous, transdermal, mucosal or oral administration; Preferably, the subject is a mammal, for example, a mouse or a human; Preferably, the subject is 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.

25. A method for detecting in vitro whether a subject has RSV infection, the method comprising: contacting a biological sample obtained from the subject with the recombinant RSV F protein of any one of claims 1-7 or the fusion protein of any one of claims 8-12; and detecting the presence of a complex formed by the recombinant RSV F protein or the fusion protein and an antibody; Preferably, the subject is a mammal, for example, a mouse or a human; Preferably, the subject is 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), elderly people (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.

26. A method for screening a candidate drug capable of inhibiting RSV infection of cells, the method comprising contacting a host cell with the candidate drug before, simultaneously with, or after contacting the recombinant RSV F protein of any one of claims 1-7 or the fusion protein of any one of claims 8-12 with the host cell.

Images