RSV F protein mutants

Abstract

The present invention discloses mutants of wild-type RSV F protein. The binding experiment of pre-fusion conformation trimer-specific monoclonal antibody AM14 shows that the mutant provided in the embodiment of the present disclosure can have strong binding activity with AM14 monoclonal antibody, while the wild-type RSV F protein before artificial mutation has no binding activity with AM14 monoclonal antibody. The results of accelerated stability test show that the mutant provided in the embodiment of the present disclosure is placed in a 37°C environment for 4 weeks, and the binding activity of AM14 monoclonal antibody has no obvious change. The results of animal immunization experiments show that compared with the wild-type RSV F protein before artificial mutation, the mutant provided in the embodiment of the present disclosure can induce the production of higher titers of neutralizing antibodies.

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceuticals, and in particular, to products such as mutants of RSV F protein, a method for stabilizing RSV F protein pre-fusion conformation trimers, and vaccines. Background Art

[0002] Respiratory syncytial virus (RSV) is a common virus that causes lung and respiratory tract infections, causing more than 30 million cases of lower respiratory tract infections worldwide each year, of which about 3 million people require hospitalization. RSV is extremely harmful to infants and the elderly, and can cause serious infections. It is the second leading cause of death in infants and the leading cause of morbidity and mortality from respiratory diseases in the elderly. Currently, there is no vaccine available for the prevention of RSV infection.

[0003] RSV is an enveloped negative-sense single-stranded RNA virus belonging to the genus Orthopneumovirus of the family Pneumoviridae. Its genome encodes 11 proteins, including 9 structural proteins and 2 non-structural proteins. The two transmembrane glycoproteins on the surface of the virus particles, namely the attachment protein G and the fusion protein F, play an important role in the initial stage of viral infection and are the main targets of neutralizing antibodies. RSV has two subtypes, namely subtype A and subtype B. Between the two subtypes, the G protein is quite different, while the F protein is more conservative, with a sequence homology of more than 90% (Ref: Harshbarger W, et al. PLoS Pathog., 2020, 16: e1008943.).

[0004] RSV F protein is a type I viral fusion protein that is responsible for the membrane fusion process between the virus and the host cell. The F protein is first translated into a single-chain inactive precursor protein F0 consisting of 574 amino acids in vivo. The signal peptide consisting of amino acids 1 to 25 at the N-terminus of the F0 protein is removed by a signal peptidase. In addition, there are two furin cleavage sites at amino acids 109 / 110 and 136 / 137 of F0. Through enzymatic cleavage by proteases, the amino acid polypeptides at positions 110 to 136 (called p27) are removed to generate F1 consisting of amino acids 137 to 574 and F2 consisting of amino acids 26 to 109. F1 and F2 are linked together by two disulfide bonds to form an F protein monomer. In the natural state, three F protein monomers are further assembled to form a homotrimer to exert their biological activity.

[0005] RSV F protein has two conformational states, namely pre-fusion conformation and post-fusion conformation, and the spatial structures of these two conformations have been resolved (Ref: McLellan JS, et al. Science, 2013, 340: 1113-1117. McLellan J.S., et al. J. Virol., 2011, 85: 7788-7796.). The pre-fusion conformation trimer is lollipop-shaped and can be vividly divided into a head domain away from the viral envelope end and a tail domain near the envelope end. During the virus infection of host cells, the refolding region 1 (refolding region 1, RR1) of the head region of the F protein and the refolding region 2 (refolding region 2, RR2) near the envelope end undergo significant conformational changes. RR1 transforms from a helix, random coil and β-sheet structure to a long helix structure and extends above the head domain. RR2 flips from the proximal end of the envelope to above the head domain at the distal end of the envelope and combines with RR1 to form a six-bundle helix structure. At this time, the F protein trimer transforms from a lollipop-shaped pre-fusion conformation to a cone-shaped post-fusion conformation, leading to the occurrence of the virus and host cell membrane fusion process (Ref: Krarup A., et al. Nat. Commun., 2015, 6: 8143).

[0006] Since F protein plays an important role in the early stage of RSV infection and is the main target of neutralizing antibodies in the human body, F protein is one of the main target antigens for vaccine development. However, most of the highly active neutralizing antibodies produced after human infection target the prefusion conformation (Ref: Battles MB, McLellan J.S. Nat. Rev. Microbiol., 2019, 17: 233-245). Many studies have successfully isolated and obtained high-affinity neutralizing antibodies that specifically bind to the prefusion conformation of F protein, such as AM14 monoclonal antibody that targets the interface between different monomers of the prefusion conformation trimer (Ref: Gilman MSA et al. PLoS Pathog., 2015, 11: e1005035.). However, the pre-fusion conformation of the F protein is an unstable state, which can easily spontaneously transform into a post-fusion conformation. Animal immunization experiments show that the neutralizing antibody titer induced by the F protein in the pre-fusion conformation is much higher than that in the post-fusion conformation (Ref: Battles MB, McLellan J.S. Nat. Rev. Microbiol., 2019, 17: 233-245; Krarup A. et al. Nat. Commun., 2015, 6: 8143). Therefore, stabilizing the RSV F protein in the pre-fusion conformation trimer state and preventing it from transforming to the post-fusion conformation can be used as a preferred antigen for vaccine development, providing an effective strategy for the development of RSV vaccines.

[0007] However, so far, the development of vaccines targeting the RSV F protein prefusion conformation trimer still cannot meet the extensive clinical needs. Therefore, finding a new RSV F protein mutant that stabilizes the RSV F protein in the prefusion conformation trimer state and can induce the production of excellent neutralizing antibody titers has become an urgent problem to be solved. Summary of the invention

[0008] Technical issues solved:

[0009] One aspect disclosed in the present invention is to provide a new mutant of RSV F protein and a new strategy for stabilizing the trimer of RSV F protein before fusion, in view of the lack of new RSV F protein mutants that can stably maintain the trimer state before fusion and can induce the production of excellent neutralizing antibody titers in the prior art.

[0010] Specifically, in the disclosed embodiment, by comparing the structure of the conformation before and after the fusion of RSV F protein, other conformational change regions coupled with the RR1 conformational transition are identified in the head domain of RSV F protein, and then mutations are introduced in these regions to stabilize its conformation, and the conformational transition of RR1 is prevented by coupling regulation, and / or mutations are directly introduced in the interaction region involved in RR1 to prevent its conformational transition. Based on this strategy, a variety of mutation schemes are provided in the disclosed embodiment to stabilize RSV F protein in the conformational trimer state before fusion. At the same time, in the disclosed embodiment, mutations are also introduced in the tail domain to further enhance the stability of the conformational trimer before the fusion of RSV F protein, thereby solving the above problems.

[0011] The technical solution provided by this disclosure:

[0012] A mutant of a wild-type RSV F protein, wherein the mutant has at least one amino acid mutation site compared to the wild-type RSV F protein before artificial mutation, and the amino acid mutation site includes:

[0013] (a) any position between amino acids 73 to 96; or

[0014] (b) any position between amino acids 216 to 240; or

[0015] (c) amino acid at position 141, amino acid at position 190, amino acid at position 193, amino acid at position 195, amino acid at position 199 or amino acid at position 373.

[0016] In the embodiments disclosed herein, based on the spatial structural comparison analysis of the RSV F protein before and after fusion, mutations are introduced into the head domain to prevent the conformational transition of RR1 and stabilize the F protein in the pre-fusion conformational trimer state. Binding experiments with the pre-fusion conformational trimer-specific monoclonal antibody AM14 show that the mutants provided in the embodiments disclosed herein can have a certain binding activity with the AM14 monoclonal antibody, while the wild-type RSV F protein before artificial mutation has no binding activity with the AM14 monoclonal antibody. Palivizumab The binding experiment shows that the mutant provided in the embodiment of the present disclosure and the wild-type RSV F protein before artificial mutation have strong binding activity with palivizumab. The above experimental results show that the introduced mutation successfully stabilizes the RSV F protein in the pre-fusion conformation trimer state and folds well. Therefore, any combination of the technical solutions provided in the above-mentioned embodiments of the present disclosure can solve the problems in the prior art and achieve the same or similar technical effects.

[0017] In the embodiments of the present disclosure, the amino acids 73 to 96 and 216 to 240 are located in the head domain of the trimer of the wild-type RSV F protein before fusion (e.g. Figure 1A α1 helix and α5 helix (as shown) Figure 2 The head domain is defined in the present invention for the purpose of describing the structure of the RSV F protein. We define the amino acids 1 to 485 as the head domain, and the amino acids 486 to the C-terminus of the F protein as the tail domain. The approximate spatial positions of the head domain and the tail domain are shown in FIG. Figure 1A As shown. In an embodiment of the present disclosure, the above-mentioned α1 helix or α5 helix refers to the first or fifth α helix starting from the N-terminus in the head domain, which is approximately located at amino acids 73 to 96 and 216 to 240 in the primary structure of the RSV F protein. Based on the research results disclosed in the present disclosure, it can be considered that any amino acid mutation in the α1 helix or α5 helix of the head domain of the RSV F protein pre-fusion conformation trimer will stabilize the RSV F protein in the pre-fusion conformation trimer state. Therefore, the 73rd to 96th amino acids and the 216th to 240th amino acids described in the present disclosure may exist one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid position offsets, which are also considered to be included in the protection scope of the present disclosure.

[0018] In the embodiments of the present disclosure, the above amino acid position numbers are all based on the wild-type RSV F protein as a reference, and are calculated starting from the first amino acid in its amino acid sequence. For example, the 73rd amino acid, i.e., from the wild-type RSV F protein sequence M The calculation starts from the first amino acid methionine of ELPILKA.

[0019] In the embodiments of the present disclosure, the artificial mutation generally refers to the replacement, deletion or insertion of amino acid residues in the wild-type RSV F protein. However, in some embodiments, the artificial mutation only refers to the replacement of amino acid residues. The mutants described in the present disclosure are obtained by the above-mentioned artificial mutations.

[0020] In some other embodiments, the mutant has multiple amino acid mutation sites compared to the wild-type RSV F protein before artificial mutation, and the multiple amino acid mutation sites may include:

[0021] (a) at least two positions selected from any of amino acids 73 to 96; or

[0022] (b) at least two positions selected from any of amino acids 216 to 240; or

[0023] (c) at least one arbitrary position selected from amino acids 73 to 96, and at least one arbitrary position selected from amino acids 216 to 240.

[0024] Further, in some other embodiments, the mutant has multiple amino acid mutation sites compared to the wild-type RSV F protein before artificial mutation, and the multiple amino acid mutation sites may include any one of the following (a), (b) or (c) and a combination of (d):

[0025] (a) at least two positions selected from any of amino acids 73 to 96;

[0026] (b) at least two positions selected from any of amino acids 216 to 240;

[0027] (c) at least one selected from any position among amino acids 73 to 96, and at least one selected from any position among amino acids 216 to 240;

[0028] (d) at least one site selected from L141, S190, L193, L195, I199 or L373.

[0029] In some embodiments, the plurality can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more.

[0030] In some embodiments, the above-mentioned amino acid mutation site can be: at least one selected from the 79th amino acid, the 82nd amino acid, the 92nd amino acid, the 93rd amino acid, the 96th amino acid, the 141st amino acid, the 190th amino acid, the 193rd amino acid, the 195th amino acid, the 199th amino acid, the 220th amino acid, the 224th amino acid, the 227th amino acid, the 230th amino acid, the 234th amino acid, the 238th amino acid or the 373rd amino acid.

[0031] Furthermore, in some other embodiments, the amino acid mutation sites may include the following:

[0032] (1) amino acid at position 92; or

[0033] (2) amino acid at position 96; or

[0034] (3) amino acid 227; or

[0035] (4) amino acid 230; or

[0036] (5) amino acid 238; or

[0037] (6) amino acid 227 and amino acid 230; or

[0038] (7) amino acid at position 82 and amino acid at position 227; or

[0039] (8) amino acid at position 89 and amino acid at position 227; or

[0040] (9) amino acid at position 92 and amino acid at position 227; or

[0041] (10) amino acid at position 92 and amino acid at position 230; or

[0042] (11) amino acid at position 96 and amino acid at position 227; or

[0043] (12) amino acid at position 96 and amino acid at position 230; or

[0044] (13) amino acid at position 96 and amino acid at position 238; or

[0045] (14) amino acid at position 93 and amino acid at position 227; or

[0046] (15) amino acid at position 89 and amino acid at position 230; or

[0047] (16) amino acid at position 82 and amino acid at position 230; or

[0048] (17) amino acid at position 93 and amino acid at position 230; or

[0049] (18) amino acid 227 and amino acid 238; or

[0050] (19) amino acid 230 and amino acid 238; or

[0051] (20) amino acid 227 and amino acid 234; or

[0052] (21) amino acid at position 82 and amino acid at position 234; or

[0053] (22) amino acid 193 and amino acid 195; or

[0054] (23) amino acid 190, amino acid 193 and amino acid 195; or

[0055] (24) amino acid 193 and amino acid 199; or

[0056] (25) amino acid 141 and amino acid 373; or

[0057] (26) amino acid at position 82 and amino acid at position 224; or

[0058] (27) amino acid at position 92 and amino acid at position 238; or

[0059] (28) amino acid 79 and amino acid 220; or

[0060] (29) amino acid 97 and amino acid 291; or

[0061] (30) amino acid at position 93 and amino acid at position 234; or

[0062] (31) amino acid at position 92 and amino acid at position 234; or

[0063] (32) amino acid 193, amino acid 227, and amino acid 230; or

[0064] (33) amino acid 190, amino acid 193 and amino acid 230; or

[0065] (34) amino acid 193 and amino acid 230; or

[0066] (35) amino acid 190, amino acid 193, amino acid 227, and amino acid 230; or

[0067] (36) amino acid at position 93, amino acid at position 230, and amino acid at position 234; or

[0068] (37) amino acid at position 93, amino acid at position 193, amino acid at position 230, and amino acid at position 234; or

[0069] (38) amino acid at position 93, amino acid at position 190, amino acid at position 193, and amino acid at position 234; or

[0070] (39) amino acid at position 93, amino acid at position 227, amino acid at position 193, and amino acid at position 234; or

[0071] (40) amino acid at position 93, amino acid at position 234, and amino acid at position 373; or

[0072] (41) amino acid 230, amino acid 238 and amino acid 373; or

[0073] (42) amino acid at position 93, amino acid at position 141, and amino acid at position 234; or

[0074] (43) amino acid at position 93, amino acid at position 141, amino acid at position 230, and amino acid at position 234; or

[0075] (44) amino acid 141, amino acid 230, and amino acid 238; or

[0076] (45) amino acid 227, amino acid 230 and amino acid 373; or

[0077] (46) amino acid at position 93, amino acid at position 227, and amino acid at position 234; or

[0078] (47) amino acid at position 93, amino acid at position 227, amino acid at position 230, and amino acid at position 234; or

[0079] (48) amino acid at position 93, amino acid at position 227, amino acid at position 234, and amino acid at position 373; or

[0080] (49) amino acid 227, amino acid 230, amino acid 238 and amino acid 373.

[0081] Furthermore, in some embodiments, the amino acid mutation sites may include the following:

[0082] (1) amino acid at position 92; or

[0083] (2) amino acid at position 96; or

[0084] (3) amino acid 227; or

[0085] (4) amino acid 230; or

[0086] (5) amino acid 238; or

[0087] (6) amino acid 227 and amino acid 230; or

[0088] (7) amino acid at position 92 and amino acid at position 227; or

[0089] (8) amino acid at position 92 and amino acid at position 230; or

[0090] (9) amino acid 227 and amino acid 238; or

[0091] (10) amino acid 230 and amino acid 238; or

[0092] (11) amino acid at position 93 and amino acid at position 234; or

[0093] (12) amino acid at position 92 and amino acid at position 234; or

[0094] (13) amino acid 193, amino acid 227 and amino acid 230; or

[0095] (14) amino acid 190, amino acid 193, amino acid 227, and amino acid 230; or

[0096] (15) amino acid at position 93, amino acid at position 230, and amino acid at position 234; or

[0097] (16) amino acid at position 93, amino acid at position 234, and amino acid at position 373; or

[0098] (17) amino acid 230, amino acid 238 and amino acid 373; or

[0099] (18) amino acid at position 93, amino acid at position 141, and amino acid at position 234; or

[0100] (19) amino acid at position 93, amino acid at position 141, amino acid at position 230, and amino acid at position 234; or

[0101] (20) amino acid 141, amino acid 230, and amino acid 238; or

[0102] (21) amino acid 227, amino acid 230, and amino acid 373; or

[0103] (22) amino acid at position 93, amino acid at position 227, and amino acid at position 234; or

[0104] (23) amino acid at position 93, amino acid at position 227, amino acid at position 234, and amino acid at position 373; or

[0105] (24) amino acid 190, amino acid 193 and amino acid 230; or

[0106] (25) amino acid 193 and amino acid 230; or

[0107] (26) amino acid at position 93, amino acid at position 193, amino acid at position 230, and amino acid at position 234; or

[0108] (27) amino acid at position 93, amino acid at position 190, amino acid at position 193, and amino acid at position 234; or

[0109] (28) amino acid at position 93, amino acid at position 227, amino acid at position 193, and amino acid at position 234; or

[0110] (29) amino acid 93, amino acid 227, amino acid 230, and amino acid 234; or

[0111] (30) amino acid 227, amino acid 230, amino acid 238 and amino acid 373.

[0112] In order to further improve the stability of the RSV F protein trimer before fusion conformation, in some embodiments, the amino acid mutation site further includes: at least one site selected from any site among amino acids 486 to 513.

[0113] The binding experiment of the pre-fusion conformation trimer-specific monoclonal antibody AM14 showed that the mutant provided in the embodiments of the present disclosure has strong binding activity with the AM14 monoclonal antibody, while the wild-type RSV F protein before artificial mutation has no binding activity with the AM14 monoclonal antibody. The binding experiment shows that the mutant provided in the embodiment of the present disclosure and the wild-type RSV F protein before artificial mutation have strong binding activity with palivizumab. The above experimental results show that the introduced mutation successfully stabilizes the RSV F protein in a pre-fusion conformational trimer state and folds well. In the embodiment of the present disclosure, an accelerated stability test was also carried out on the above-mentioned expressed F protein mutant. The results showed that after being placed in a 37°C environment for 4 weeks, there was no obvious change in the binding activity of AM14 monoclonal antibody, indicating that the mutant protein pre-fusion conformational trimer remained stable. Animal immunization experiments were also carried out in the embodiment of the present disclosure. The experimental results showed that compared with the wild-type RSV F protein before artificial mutation, the mutant provided in the embodiment of the present disclosure can induce the production of higher titers of neutralizing antibodies. Therefore, any combination of the technical solutions provided in the above-mentioned embodiments of the present disclosure can solve the problems in the prior art and achieve the same or similar technical effects.

[0114] In the embodiments of the present disclosure, the amino acids 486 to 513 are located in the tail domain of the trimer of the wild-type RSV F protein before fusion (e.g. Figure 1A The tail domain is defined in the present invention for the purpose of describing the structure of RSV F protein in an image manner. We define the amino acids 1 to 485 as the head domain, and the amino acids 486 to the C-terminus of the F protein as the tail domain. The approximate spatial positions of the head domain and the tail domain are shown in FIG. Figure 1A As shown. Based on the research results disclosed in the present invention, it can be considered that the mutation of any amino acid in the amino acid positions 486 to 513 of the tail domain of the RSV F protein pre-fusion conformation trimer will further stabilize the RSV F protein pre-fusion conformation trimer structure. Therefore, the amino acids 486 to 513 described in the present invention may have one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid position offsets, which are also considered to be included in the protection scope of the present invention.

[0115] Therefore, in some embodiments, the above-mentioned amino acid mutation site also includes: at least one selected from the 501st amino acid, the 502nd amino acid, the 505th amino acid, the 509th amino acid or the 512th amino acid.

[0116] Furthermore, in some other embodiments, the amino acid mutation site may also include the following situations:

[0117] (1) amino acid 509; or

[0118] (2) amino acid 505; or

[0119] (3) amino acid 502; or

[0120] (4) amino acid 501; or

[0121] (5) amino acid 505 and amino acid 509; or

[0122] (6) amino acid at position 502 and amino acid at position 509; or

[0123] (7) amino acid at position 501 and amino acid at position 509; or

[0124] (8) amino acid at position 502 and amino acid at position 505; or

[0125] (9) amino acid at position 501 and amino acid at position 505; or

[0126] (10) amino acid at position 502, amino acid at position 505, and amino acid at position 509; or

[0127] (11) amino acid at position 501, amino acid at position 505 and amino acid at position 509.

[0128] Furthermore, in some embodiments, the amino acid mutation site also includes the following situations:

[0129] (1) amino acid 509; or

[0130] (2) amino acid 505; or

[0131] (3) amino acid at position 505 and amino acid at position 509; or

[0132] (4) amino acid at position 502 and amino acid at position 509; or

[0133] (5) Amino acid at position 501 and amino acid at position S509.

[0134] RSV F protein has two subtypes, A and B, and there is a very high sequence conservation between the two subtypes, and its amino acid sequence homology reaches 90%. The amino acid sequence homology between different strains within subtype A and subtype B is higher. And the RSV F protein of various subtypes and strains thereof is composed of 574 amino acids. In view of the high conservation of amino acid sequences between different subtypes of RSV F protein, the mutation scheme provided in the present disclosure is applicable to any strain sequence of RSV F protein subtype A and subtype B or strain sequence that may appear in the future. In the embodiment of the present disclosure, the wild-type RSV F protein sequence can be from any known or future F protein sequence that may appear, including but not limited to the F protein sequence from any RSV strain of subtype A or subtype B, such as the F protein sequence of A2 strain of subtype A shown in SEQ ID NO.1-2, the F protein sequence of ON1 strain of subtype A shown in SEQ ID NO.3, and the F protein sequence of subtype B strain shown in SEQ ID NO.4-6. Because the sequence of wild-type RSV F protein is highly conservative or identical between RSV subtypes and strains, the wild-type RSV F protein sequence is allowed to contain slight differences, which may be substitutions, deletions or insertions between amino acid residues. Therefore, in some embodiments, the sequences of the wild-type RSV F protein may have more than 90% identity, for example, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and have the same or substantially the same biological function.

[0135] Based on the research results disclosed herein, in some embodiments, the wild-type RSV F protein can be in the following form:

[0136] (a) a F0 precursor protein comprising two protease cleavage sites; or

[0137] (b) a F0 precursor protein that is incapable of binding a protease; or

[0138] (c) a combination of an independent F1 polypeptide and an independent F2 polypeptide; or

[0139] (d) a combination of F2 polypeptide-linker-F1 polypeptide; or

[0140] (e) A combination of F2' polypeptide-connector-F1' polypeptide, wherein the F2' polypeptide has 1 to 10 fewer amino acid residues than the F2 polypeptide along the N-terminal direction, and the F1' polypeptide has 1 to 10 fewer amino acid residues than the F1 polypeptide along the C-terminal direction.

[0141] In some embodiments, the wild-type RSV F protein can be an original F0 precursor protein, i.e., comprising F1 polypeptide, F2 polypeptide, P27 peptide, optionally comprising a protein of a transmembrane domain and an intracellular domain. The F0 precursor protein contains a cleavage site of a protease, and the protease can be, for example, furin, trypsin, factor Xa, thrombin or cathepsin L, etc. Generally, the protease is furin. During the expression process, it is naturally cut by the protease in the expression system, thereby losing the P27 peptide, and the F1 polypeptide and the F2 polypeptide jointly form a mature form of the F protein. In other embodiments, the wild-type RSV F protein can be an F0 precursor protein that cannot bind to a protease, i.e., comprising F1 polypeptide, F2 polypeptide, P27 peptide, optionally comprising a protein of a transmembrane domain and an intracellular domain, but cannot bind to a protease, thereby retaining the P27 peptide. The protease can be, for example, furin, trypsin, factor Xa, thrombin or cathepsin L, etc. Generally, the protease is furin. The above-mentioned reason that can not bind to protease can be the deletion, substitution or insertion of the amino acid residue of the restriction site of the protease. In other embodiments, the wild-type RSV F protein can be a combination of an independent F1 polypeptide and an independent F2 polypeptide, which can selectively include a protein of a transmembrane domain and an intracellular domain. The F1 polypeptide and the F2 polypeptide spontaneously form a F2-F1 polypeptide heterodimer. When expressed in vitro, the F1 polypeptide and the F2 polypeptide need to be expressed respectively. In other embodiments, the wild-type RSV F protein can be a combination of F2 polypeptide-connector-F1 polypeptide, i.e., comprising F1 polypeptide, F2 polypeptide and connector (Linker-1), which can selectively include a protein of a transmembrane domain and an intracellular domain. In some other embodiments, the wild-type RSV F protein can be a combination of F2' peptide-connector-F1' peptide, wherein the F2' polypeptide has 1 to 10 fewer amino acid residues than the F2 polypeptide along the N-terminal direction, and the F1' polypeptide has 1 to 10 fewer amino acid residues than the F1 polypeptide along the C-terminal direction, that is, the longer sequence containing the P27 peptide in the F0 sequence is replaced with a connector. Studies have shown that replacing the P27 peptide with a short connector can increase the expression and trimer content of the protein (Ref: Krarup A. et al. Nat. Commun., 2015, 6: 8143). Therefore, in one embodiment, the 107-138 sequence (RARRELPRFMNYTLNNAKKTNVTLSKKRKRRF) containing the P27 peptide and the furin cleavage site is replaced with a QARGSGSGRS sequence.

[0142] In some embodiments, the wild-type RSV F protein before artificial mutation may include a sequence as shown in SEQ ID No. 7-34 in the sequence list, or a sequence having more than 90% identity thereto, for example, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and having the same or substantially the same biological function.

[0143] Based on the research results disclosed herein, in some embodiments, a combination of amino acid residues after the amino acid mutation site is substituted is also provided. The amino acid mutation site includes at least one of the following situations:

[0144] (1) amino acid 79 is substituted by C and amino acid 220 is substituted by C; or

[0145] (2) amino acid 82 is substituted by C and amino acid 224 is substituted by C; or

[0146] (3) amino acid 92 is substituted by C and amino acid 234 is substituted by C; or

[0147] (4) amino acid 93 is substituted by C and amino acid 234 is substituted by C; or

[0148] (5) the amino acid at position 82 is substituted by L; or

[0149] (6) the amino acid at position 92 is substituted by L, I, M, F, Y or W; or

[0150] (7) the amino acid at position 96 is substituted by M or Y; or

[0151] (8) the amino acid at position 141 is substituted by M, F, Y or W; or

[0152] (9) amino acid 190 is substituted by V; or

[0153] (10) the amino acid at position 193 is substituted by M, F, Y or W; or

[0154] (11) the amino acid at position 195 is substituted by M, F, Y or W; or

[0155] (12) the amino acid at position 199 is substituted by M, F, Y or W; or

[0156] (13) the amino acid at position 227 is substituted by A, V, L, I, M, S, T or Q; or

[0157] (14) amino acid 230 is substituted by F; or

[0158] (15) the amino acid at position 238 is substituted by L, I, M, F, Y or W; or

[0159] (16) The amino acid at position 373 is substituted by M, F or Y.

[0160] Furthermore, in some embodiments, the amino acid mutation sites include the following:

[0161] (1) the amino acid at position 92 is substituted by I, M, F, Y or W; or

[0162] (2) the amino acid at position 96 is substituted by Y; or

[0163] (3) the amino acid at position 227 is substituted by M, V or I; or

[0164] (4) amino acid 230 is substituted by F; or

[0165] (5) the amino acid at position 238 is substituted by L, I, M or W; or

[0166] (6) the amino acid at position 227 is substituted by M or V, and the amino acid at position 230 is substituted by F; or

[0167] (7) the amino acid at position 82 is substituted by L and the amino acid at position 227 is substituted by M; or

[0168] (8) the amino acid at position 92 is substituted by M or L, and the amino acid at position 227 is substituted by M or V; or

[0169] (9) the amino acid at position 92 is substituted by M or L, and the amino acid at position 230 is substituted by F; or

[0170] (10) the amino acid at position 96 is substituted by Y and the amino acid at position 227 is substituted by M or V; or

[0171] (11) the amino acid at position 96 is substituted by Y and the amino acid at position 230 is substituted by F; or

[0172] (12) the amino acid at position 96 is substituted by Y, and the amino acid at position 238 is substituted by L, I, M or W; or

[0173] (13) amino acid at position 89 is substituted by L and amino acid at position 230 is substituted by F; or

[0174] (14) the amino acid at position 227 is substituted by M and the amino acid at position 238 is substituted by L, I, M or W; or

[0175] (15) the amino acid at position 230 is substituted by F, and the amino acid at position 238 is substituted by L, I, M or W; or

[0176] (16) the amino acid at position 193 is substituted by M, and the amino acid at position 195 is substituted by M, F or Y; or

[0177] (17) the amino acid at position 190 is substituted by V, the amino acid at position 193 is substituted by M, and the amino acid at position 195 is substituted by M, F or Y; or

[0178] (18) the amino acid at position 193 is substituted by M, and the amino acid at position 199 is substituted by M or F; or

[0179] (19) the amino acid at position 141 is substituted by M or F, and the amino acid at position 373 is substituted by M, F or Y; or

[0180] (20) amino acid at position 82 is substituted by C and amino acid at position 224 is substituted by C; or

[0181] (21) the amino acid at position 92 is substituted by C, and the amino acid at position 238 is substituted by C; or

[0182] (22) amino acid 79 is substituted by C and amino acid 220 is substituted by C; or

[0183] (23) amino acid 93 is substituted by C and amino acid 234 is substituted by C; or

[0184] (24) amino acid at position 92 is substituted by C and amino acid at position 234 is substituted by C; or

[0185] (25) amino acid position 193 is substituted by M, amino acid position 227 is substituted by M or V, and amino acid position 230 is substituted by F; or

[0186] (26) the amino acid at position 190 is substituted by V, the amino acid at position 193 is substituted by M, and the amino acid at position 230 is substituted by F; or

[0187] (27) amino acid 193 is substituted by M and amino acid 230 is substituted by F; or

[0188] (28) the amino acid at position 190 is substituted by V, the amino acid at position 193 is substituted by M, the amino acid at position 227 is substituted by M or V, and the amino acid at position 230 is substituted by F; or

[0189] (29) amino acid position 93 is substituted by C, amino acid position 230 is substituted by F, and amino acid position 234 is substituted by C; or

[0190] (30) the amino acid at position 93 is substituted by C, the amino acid at position 193 is substituted by M, the amino acid at position 230 is substituted by F, and the amino acid at position 234 is substituted by C; or

[0191] (31) the amino acid at position 93 is substituted by C, the amino acid at position 190 is substituted by V, the amino acid at position 193 is substituted by M, and the amino acid at position 234 is substituted by C; or

[0192] (32) the amino acid at position 93 is substituted by C, the amino acid at position 227 is substituted by M or V, and the amino acid at position 234 is substituted by C; or

[0193] (33) the amino acid at position 93 is substituted by C, the amino acid at position 193 is substituted by M, the amino acid at position 227 is substituted by M or V, and the amino acid at position 234 is substituted by C; or

[0194] (34) amino acid position 93 is substituted by C, amino acid position 234 is substituted by C, and amino acid position 373 is substituted by M, F or Y; or

[0195] (35) the amino acid at position 230 is substituted by F, the amino acid at position 238 is substituted by L, I, M or W, and the amino acid at position 373 is substituted by M, F or Y; or

[0196] (36) the amino acid at position 93 is substituted by C, the amino acid at position 141 is substituted by M or F, and the amino acid at position 234 is substituted by C; or

[0197] (37) the amino acid at position 93 is substituted by C, the amino acid at position 141 is substituted by M or F, the amino acid at position 230 is substituted by F, and the amino acid at position 234 is substituted by C; or

[0198] (38) the amino acid at position 141 is substituted by M or F, the amino acid at position 230 is substituted by F, and the amino acid at position 238 is substituted by L, I, M or W; or

[0199] (39) the amino acid at position 227 is substituted by M or V, the amino acid at position 230 is substituted by F, and the amino acid at position 373 is substituted by M, F or Y; or

[0200] (40) the amino acid at position 93 is substituted by C, the amino acid at position 227 is substituted by M or V, the amino acid at position 230 is substituted by F, and the amino acid at position 234 is substituted by C; or

[0201] (41) the amino acid at position 93 is substituted by C, the amino acid at position 227 is substituted by M or V, the amino acid at position 234 is substituted by C, and the amino acid at position 373 is substituted by M, F or Y; or

[0202] (42) The amino acid at position 227 is substituted by M or V, the amino acid at position 230 is substituted by F, the amino acid at position 238 is substituted by L, I, M or W, and the amino acid at position 373 is substituted by M, F or Y.

[0203] Furthermore, in some embodiments, the amino acid mutation sites may include the following:

[0204] (1) the amino acid at position 92 is substituted by I, M, F, Y or W; or

[0205] (2) the amino acid at position 96 is substituted by Y; or

[0206] (3) the amino acid at position 227 is substituted by M or V; or

[0207] (4) amino acid 230 is substituted by F; or

[0208] (5) the amino acid at position 238 is substituted by L, I, M or W; or

[0209] (6) the amino acid at position 227 is substituted by M or V, and the amino acid at position 230 is substituted by F; or

[0210] (7) the amino acid at position 92 is substituted by M, and the amino acid at position 227 is substituted by M or V; or

[0211] (8) the amino acid at position 92 is substituted by M, and the amino acid at position 230 is substituted by F; or

[0212] (9) the amino acid at position 227 is substituted by M or V, and the amino acid at position 238 is substituted by L or I; or

[0213] (10) the amino acid at position 230 is substituted by F, and the amino acid at position 238 is substituted by L or I; or

[0214] (11) the amino acid at position 96 is substituted by Y and the amino acid at position 227 is substituted by M or V; or

[0215] (12) the amino acid at position 96 is substituted by Y and the amino acid at position 230 is substituted by F; or

[0216] (13) the amino acid at position 96 is substituted by Y, and the amino acid at position 238 is substituted by L, I, M or W; or

[0217] (14) the amino acid at position 93 is substituted by C, and the amino acid at position 234 is substituted by C; or

[0218] (15) the amino acid at position 92 is substituted by C, and the amino acid at position 234 is substituted by C; or

[0219] (16) amino acid position 193 is substituted by M, amino acid position 227 is substituted by M or V, and amino acid position 230 is substituted by F; or

[0220] (17) the amino acid at position 190 is substituted by V, the amino acid at position 193 is substituted by M, the amino acid at position 227 is substituted by M or V, and the amino acid at position 230 is substituted by F; or

[0221] (18) amino acid position 93 is substituted by C, amino acid position 230 is substituted by F, and amino acid position 234 is substituted by C; or

[0222] (19) the amino acid at position 93 is substituted by C, the amino acid at position 234 is substituted by C, and the amino acid at position 373 is substituted by M or F; or

[0223] (20) the amino acid at position 230 is substituted by F, the amino acid at position 238 is substituted by L or I, and the amino acid at position 373 is substituted by M or F; or

[0224] (21) the amino acid at position 93 is substituted by C, the amino acid at position 141 is substituted by M or F, and the amino acid at position 234 is substituted by C; or

[0225] (22) the amino acid at position 93 is substituted by C, the amino acid at position 141 is substituted by M or F, the amino acid at position 230 is substituted by F, and the amino acid at position 234 is substituted by C; or

[0226] (23) the amino acid at position 141 is substituted by M or F, the amino acid at position 230 is substituted by F, and the amino acid at position 238 is substituted by L or I; or

[0227] (24) amino acid position 227 is substituted by M or V, amino acid position 230 is substituted by F, and amino acid position 373 is substituted by M or F; or

[0228] (25) the amino acid at position 93 is substituted by C, the amino acid at position 227 is substituted by M or V, and the amino acid at position 234 is substituted by C; or

[0229] (26) the amino acid at position 93 is substituted by C, the amino acid at position 227 is substituted by M or V, the amino acid at position 234 is substituted by C, and the amino acid at position 373 is substituted by M or F; or

[0230] (27) the amino acid at position 190 is substituted by V, the amino acid at position 193 is substituted by M, and the amino acid at position 230 is substituted by F; or

[0231] (28) the amino acid at position 193 is substituted by M, and the amino acid at position 230 is substituted by F; or

[0232] (29) the amino acid at position 93 is substituted by C, the amino acid at position 193 is substituted by M, the amino acid at position 230 is substituted by F, and the amino acid at position 234 is substituted by C; or

[0233] (30) amino acid position 93 is substituted by C, amino acid position 190 is substituted by V, amino acid position 193 is substituted by M and amino acid position 234 is substituted by C; or

[0234] (31) The amino acid at position 93 is substituted by C, or the amino acid at position 227 is substituted by M or V, the amino acid at position 193 is substituted by M, and the amino acid at position 234 is substituted by C.

[0235] (32) The amino acid at position 93 is substituted by C, the amino acid at position 227 is substituted by M or V, the amino acid at position 230 is substituted by F, and the amino acid at position 234 is substituted by C.

[0236] (33) The amino acid at position 227 is substituted by M or V, the amino acid at position 230 is substituted by F, the amino acid at position 238 is substituted by L or I, and the amino acid at position 373 is substituted by M or F.

[0237] In order to further improve the stability of the RSV F protein pre-fusion conformation trimer, in some embodiments, an amino acid mutation site in the tail domain is also introduced, and the amino acid mutation site also includes the following situations:

[0238] (1) the amino acid at position 501 is substituted by V, L, I, M, F, W, Y, P or R; or

[0239] (2) the amino acid at position 502 is substituted by V, L, I, M, F, W, Y, P or R; or

[0240] (3) the amino acid at position 505 is substituted by M or W; or

[0241] (4) the amino acid at position 509 is substituted by G, A, V, L, I, M, F, W, P, S, T, C, Y, Q, K, R or H; or

[0242] (5) The amino acid at position 512 is substituted by I, F, M, Y, R or K.

[0243] Furthermore, in some embodiments, the amino acid mutation site also includes the following situations:

[0244] (1) the amino acid at position 509 is substituted by F, M or L; or

[0245] (2) the amino acid at position 505 is substituted by W; or

[0246] (3) the amino acid at position 502 is substituted by Y; or

[0247] (4) the amino acid at position 501 is substituted by M; or

[0248] (5) the amino acid at position 505 is substituted by W and the amino acid at position 509 is substituted by F, L or M; or

[0249] (6) the amino acid at position 502 is substituted by Y and the amino acid at position 509 is substituted by F; or

[0250] (7) amino acid 501 is substituted by M and amino acid 509 is substituted by F; or

[0251] (8) the amino acid at position 502 is substituted by Y and the amino acid at position 505 is substituted by W; or

[0252] (9) the amino acid at position 501 is substituted by M and the amino acid at position 505 is substituted by W; or

[0253] (10) the amino acid at position 502 is substituted by Y, the amino acid at position 505 is substituted by W, and the amino acid at position 509 is substituted by F; or

[0254] (11) The amino acid at position 501 is substituted by M, the amino acid at position 505 is substituted by W, and the amino acid at position 509 is substituted by F.

[0255] Furthermore, in some embodiments, the amino acid mutation site also includes the following situations:

[0256] (1) the amino acid at position 509 is substituted by F, M or L; or

[0257] (2) the amino acid at position 505 is substituted by W; or

[0258] (3) the amino acid at position 505 is substituted by W, and the amino acid at position 509 is substituted by F or L; or

[0259] (4) the amino acid at position 502 is substituted by Y, and the amino acid at position 509 is substituted by F; or

[0260] (5) The amino acid at position 501 is substituted by M, and the amino acid at position 509 is substituted by F.

[0261] In some embodiments, specific examples of the mutants may include sequences selected from sequences shown in SEQ ID No. 35-166 in the sequence listing, or sequences having more than 90% identity thereto, for example, more than 91%, more than 92%, more than 93%, more than 94%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, and having the same or substantially the same biological function.

[0262] The C-terminus of the full-length RSV F protein includes a transmembrane region and an intracellular region. In some embodiments, the transmembrane region and / or intracellular region may be retained or removed. In order to facilitate secretory expression, in some embodiments, the transmembrane region and intracellular region at the C-terminus of the F protein are removed to obtain a soluble F protein. In some other embodiments of the present disclosure, the intracellular region and all or part of the transmembrane region may be removed, or a longer sequence including the transmembrane region and the intracellular region at the C-terminus may be removed. In some specific embodiments of the present disclosure, amino acids 514 to 574 are removed from the C-terminus.

[0263] In some embodiments, the mutant can be in a trimer form. The native structure of RSV F protein is a homotrimer. Therefore, preferably, in some embodiments, the trimer form can be a homotrimer form. When the transmembrane region and the intracellular region are retained or removed, the C-terminus of the F protein can be directly or indirectly connected to an exogenous trimerization domain to promote the formation of a trimer structure. Exogenous multimerization domains are known in the art, such as GCN4 leucine zipper (Ref: Sliepen K. et al. J. Biol. Chem., 2015, 290: 7436-7442.), collagen (Ref: McAlinden A. et al. J. Biol. Chem., 2003, 278: 42200-42207), T4 foldon (Ref: 1. McLellan J. S. et al. Science, 2013, 342: 592-598. 2. Joyce M Get al. Nat. Struct. Mol. Biol., 2016, 23: 811-820), etc. In some specific examples, the C-terminus of the F protein is connected to T4 foldon to promote its trimerization.

[0264] In some embodiments, the mutant may be modified in order to achieve certain functions without affecting the overall biological activity of the mutant, for example, immunogenicity. The modification may be at least one selected from glycosylation modification, phosphorylation modification, methylation modification, coenzyme modification or acylation modification.

[0265] When the exogenous multimerization domain is connected to the F protein, it can be directly connected to the C-terminus of the F protein, or it can be connected through a linker (Linker-2). The linker can be long or short. In some embodiments, it can be, for example, a repeating sequence of GS, a repeating sequence of GGS, a repeating sequence of SAIG, a repeating sequence of GSS, or a repeating sequence of GG. These linkers and other similar linkers are well known in the art and can be used in the mutants disclosed herein. In some specific examples, the F protein is connected to the exogenous multimerization domain through a SAIG ​​linker.

[0266] In order to achieve certain specific purposes or functions, in some embodiments, the mutant may also include a functional tag. It can be added at a suitable position. For example, the functional tag may be selected from one or more of a signal peptide, a tag or an immune enhancing peptide. The function of the above-mentioned signal peptide may be to be more conducive to the expression of the protein; the above-mentioned tag may be, for example, a Flag tag, an enhanced green fluorescent protein (eGFP), a glutathione sulfhydryl transferase (GST), etc., and its function may be used for detection, purification, separation, etc. The above-mentioned functional sequences may be used in any combination. In some embodiments, in order to facilitate the purification of the target protein, the constructed F protein mutant may contain a His tag (formed by connecting multiple His), a streptomycin tag II, an AviTag tag, and may also contain a TEV protease cleavage site for the removal of an additional sequence. The additional sequence is well known to those skilled in the art, and its addition or not does not affect the biological function of the F protein. In other embodiments, after the F protein is connected to the exogenous multimerization domain, it is connected to the His tag (HHHHHHHH) through the GSGSGS connecting arm to facilitate the purification of the target protein. In some other embodiments, in addition to the His tag, a streptomycin tag II, an AviTag tag, and a TEV protease cleavage site are also connected. The mutant sequences disclosed in the present disclosure do not contain these additional tag sequences.

[0267] In the present disclosure, the mutant of the wild-type RSV F protein can be prepared by conventional means in the prior art according to the content recorded in the present disclosure, for example, the nucleotide sequence encoding the mutant is loaded into a suitable vector and expressed and purified in a corresponding expression system. Li Qiming et al. disclose several new expression vectors for respiratory syncytial virus in Chinese patents with publication numbers CN111166881A, CN106986942A and CN106986943A. The expression of RSV F protein mutants can use suitable host cells in the prior art, for example, Escherichia coli, yeast cells, insect cells or mammalian cells. Examples of Escherichia coli expression systems, for example, GI698, ER2566, BL21 (DE3), XA90, DH (5a), B834 (DE3), BLR (DE3). Examples of yeast expression systems, for example, Saccharomyces cerevisiae, Pichia pastoris, Hansenula. Examples of insect cells include, for example, Sf9 cells, Sf21 cells, Tn5 cells, Schneider S2 cells, and High Five cells. Examples of mammalian cells include, for example, CHO cells, human embryonic kidney cells, NIH-3T3 cells, 293-T cells, Vero cells, and HeLa cells. The obtained protein can be purified by any suitable method, for example, salting out, precipitation, dialysis or ultrafiltration, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0268] Another aspect of the present invention is to provide a virus-like particle (VLPs), wherein the virus-like particle comprises the mutant described in the present disclosure. The virus-like particle can be prepared by any suitable method in the prior art. For example, VLPs can be expressed using a specific insect or mammalian cell expression system.

[0269] Another aspect of the present invention is to provide an isolated nucleic acid molecule encoding the mutant described in the present disclosure. In some embodiments, the isolated nucleic acid molecule is a DNA molecule. Generally, the codons encoding the amino acids of the mutant described in the embodiments of the present disclosure can be optimized to optimize its expression in the host cell. In other embodiments, the isolated nucleic acid molecule is an RNA molecule, and the RNA molecule includes an mRNA molecule.

[0270] Another aspect of the present invention is to provide a vector, wherein the vector comprises the sequence of the nucleic acid molecule described in the present disclosure or the mutant described in the present disclosure. In some embodiments, the vector is a protein expression vector. In other embodiments, the vector is a gene delivery vector. For example, a viral vector or a non-viral vector. In other embodiments, the viral vector includes a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpes simplex virus vector or a Sendai virus vector. The vector may contain regulatory sequences such as promoters and terminators, as well as marker sequences such as drug resistance genes and reporter genes. In addition, the above-mentioned vector may also include a sequence encoding a suicide gene. In other embodiments, the vector is a non-viral vector. In other embodiments, the non-viral vector includes, for example, a liposome, a cationic polymer vector, a lipid nanoparticle vector (LNP) or a multifunctional envelope nanocarrier.

[0271] Another aspect of the present invention provides a host cell, wherein the host cell comprises the nucleic acid molecule described in the present disclosure or the vector described in the present invention.

[0272] Another aspect of the present invention provides a protein conjugate, wherein the protein conjugate comprises the mutant described in the present invention.

[0273] Another aspect of the present invention is to provide a pharmaceutical composition, which contains the mutant described in the present invention, the isolated nucleic acid molecule described in the present invention, the vector described in the present invention, the host cell described in the present invention or the protein conjugate described in the present invention, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be any pharmaceutically acceptable additive, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, amino acids and their combinations, stabilizers, surfactants, preservatives, isotonic agents, etc. In some embodiments, the pharmaceutical composition can be an immunogenic composition. In some embodiments, the pharmaceutical composition is a vaccine. The vaccine contains the mutant described in the present invention, the isolated nucleic acid molecule described in the present invention, the vector described in the present invention, the host cell described in the present invention or the protein conjugate described in the present invention. The vaccine is a preventive or therapeutic vaccine. In some embodiments, the pharmaceutical composition can contain one or more different mutants described in the present invention, isolated nucleic acid molecules described in the present invention, vectors described in the present invention, host cells described in the present invention or protein conjugates described in the present invention, for example, two or three. In some embodiments, the vaccine is a protein vaccine, a nucleic acid vaccine or a vector vaccine.

[0274] For protein vaccines, in some embodiments, the vaccine may further include an immunomodulator, such as an adjuvant. Any suitable adjuvant may be selected, such as aluminum hydroxide, aluminum phosphate, MF59 or CpG, etc. In some embodiments, the immunomodulator also includes an immunopotentiator, such as a cytokine, a chemokine PAMP, a TLR-ligand, an immunostimulatory sequence, a DNA containing CpG, dsRNA, a recognition receptor ligand of an endocytic mode, LPS, a saponin from a quillaja tree, tucaresol, etc.

[0275] Another aspect of the present invention is to provide use of the mutant described in the present invention, the isolated nucleic acid molecule described in the present invention, the vector described in the present invention, the host cell described in the present invention, the protein conjugate described in the present invention or the pharmaceutical composition described in the present invention in the preparation of a product for preventing or treating RSV infection.

[0276] In some embodiments, a method for making a subject produce an immune response to RSV is also provided. The method includes administering to the subject an effective dose of a mutant described in the present invention, an isolated nucleic acid molecule described in the present invention, a carrier described in the present invention, a host cell described in the present invention, a protein conjugate described in the present invention, or a pharmaceutical composition described in the present invention. In some embodiments, the immune response can produce one or more neutralizing antibodies in the subject. In some embodiments, the immune response can trigger cellular immunity in the subject. In some embodiments, the immune response is to reduce or suppress the replication of RSV in the subject. In some embodiments, the administration can be one or more administrations. In some embodiments, the method of administration can be intramuscular injection, intraperitoneal injection, subcutaneous injection, mucosal immunization, etc.

[0277] The mutant described in the present invention, the isolated nucleic acid molecule described in the present invention, the vector described in the present invention, the host cell described in the present invention, the protein conjugate described in the present invention or the pharmaceutical composition described in the present invention can be administered simultaneously or in combination with other drugs including immunogenic compositions.

[0278] Another aspect of the present invention is to provide the use of the mutant described in the present invention, the isolated nucleic acid molecule described in the present invention, the vector described in the present invention, the host cell described in the present invention or the protein conjugate described in the present invention in the preparation of antibodies against the pre-fusion conformation of RSV F protein.

[0279] Another aspect of the present invention is to provide the use of the mutant described in the present invention, the isolated nucleic acid molecule described in the present invention, the vector described in the present invention, the host cell described in the present invention or the protein conjugate described in the present invention in detecting whether RSV antibodies are present in a sample. For example, the presence of RSV infection in a subject can be diagnosed by detecting whether RSV antibodies are present in a sample derived from the subject.

[0280] Another aspect of the present invention is to provide a method for preventing or treating RSV infection, wherein an effective dose of the pharmaceutical composition of the present invention is administered to a patient.

[0281] Beneficial effects of the present disclosure:

[0282] In the disclosed embodiment, based on the structural analysis of the RSV F protein before and after fusion conformation, other conformational change regions coupled with the F protein RR1 from before fusion to after fusion conformational change are obtained, and by introducing stability mutations, conformational motion is prevented from occurring, and / or mutations are directly introduced in the interaction region involved in RR1 to prevent its conformational transition, and the F protein is stabilized to the pre-fusion conformational trimer state. In addition, stability mutations are introduced in the tail domain of the F protein to further enhance the stability of the RSV F protein pre-fusion conformational trimer structure. The binding experiment of the pre-fusion conformational trimer-specific monoclonal antibody AM14 shows that the mutant provided in the disclosed embodiment can have strong binding activity with the AM14 monoclonal antibody, while the wild-type RSV F protein before the artificial mutation has no binding activity with the AM14 monoclonal antibody. The palivizumab binding experiment shows that the mutant provided in the disclosed embodiment and the wild-type RSV F protein before the artificial mutation have strong binding activity with palivizumab. The above experimental results show that the introduced mutations successfully stabilize the RSV F protein in the pre-fusion conformational trimer state and fold well.

[0283] In the disclosed embodiments, an accelerated stability test was also conducted on the F protein mutant expressed above, and the results showed that after being placed at 37°C for 4 weeks, there was no significant change in the binding activity of AM14 monoclonal antibody, indicating that the trimer conformation of the mutant protein before fusion remained stable.

[0284] Animal immunization experiments were also conducted in the embodiments of the present disclosure, and the experimental results showed that compared with the wild-type RSV F protein before artificial mutation, the mutants provided by the present disclosure can induce the production of higher titers of neutralizing antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0285] Figure 1A This is the structure diagram of the trimer of RSV F protein before fusion. Figure 1B The figure is a structure diagram of the trimer of the RSV F protein after fusion, wherein the head domain and the tail domain defined in the present invention for the purpose of image description are marked in the figure, and the RR1 region (located in the head domain) and the RR2 region (located in the C-terminus of the head domain and the tail domain) where a significant conformational change occurs during the transformation of the F protein from the pre-fusion conformation to the post-fusion conformation are marked in the figure, Figure 1A and Figure 1B They are drawn based on the coordinate files with PDB codes 4jhw and 3rrr respectively;

[0286] Figure 2The structure overlay diagram of the pre-fusion conformation (light color) and post-fusion conformation (dark color) of RSV F protein monomer. In the process of F protein changing from pre-fusion conformation to post-fusion conformation, the head domain RR1 undergoes a large conformational change, and α1 and α5 also undergo obvious conformational movement accordingly. The enlarged diagram on the right shows the conformational changes of α1 and α5 before and after fusion. The pre-fusion conformation and post-fusion conformation of F protein monomer are drawn according to the coordinate files with PDB codes 4jhw and 3rrr, respectively.

[0287] Figure 3 The SDS-PAGE electrophoresis diagram of exemplary mutants FHM-24 and FHM-25 with mutations introduced into the head domain in the embodiments of the present disclosure;

[0288] Figure 4 The results of the AM14 monoclonal antibody binding experiment of exemplary mutants FHM-24 and FHM-25 with mutations introduced into the head domain in the embodiments of the present disclosure, as well as wild-type RSV F protein 1 before artificial mutation and wild-type RSV F protein 2 before artificial mutation;

[0289] Figure 5 The results of the Palivizumab binding experiment of the exemplary mutants FHM-24 and FHM-25 with mutations introduced into the head domain in the embodiments of the present disclosure, as well as the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation;

[0290] Figure 6 The SDS-PAGE electrophoresis diagram of the FHCS-1, FHCS-2, FHCS-3, FHCS-4, FHCS-5, FHCM-1, FHCM-2, FHCM-3 and FHCM-4 mutant proteins constructed in the embodiments of the present disclosure after purification;

[0291] Fig. 7A A comparison chart of the AM14 monoclonal antibody binding test results of the FHCS-1, FHCS-2, FHCS-3, FHCS-4, FHCS-5, FHCM-1 and FHCM-2 mutants and the FHM-24 mutant, as well as the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation constructed in the embodiments of the present disclosure; Figure 7B A comparison chart of the AM14 monoclonal antibody binding test results of the FHCM-3 and FHCM-4 mutants and the FHM-25 mutant constructed in the embodiments of the present disclosure, as well as the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation;

[0292] Fig. 8AA comparison chart of the results of palivizumab binding experiments of the FHCS-1, FHCS-2, FHCS-3, FHCS-4, FHCS-5, FHCM-1 and FHCM-2 mutants and the FHM-24 mutant, as well as the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation constructed in the embodiments of the present disclosure; Figure 8B A comparison chart of the results of palivizumab binding experiments of the FHCM-3 and FHCM-4 mutants and the FHM-25 mutant constructed in the embodiments of the present disclosure, as well as the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation;

[0293] Fig. 9 : This is a comparison chart of the RSV A2 strain live virus neutralizing antibody detection results of the FHCS-1, FHCS-2, FHCS-3 and FHCS-4 mutants constructed in the embodiments of the present disclosure, the FHM-24 mutant, and the wild-type RSV F protein 2 before artificial mutation.

[0294] Sequence Listing

[0295] Exemplary sequences provided by the present disclosure are shown in Tables 1-3.

[0296] Table 1. Wild-type RSV F protein amino acid sequence

[0297]

[0298]

[0299]

[0300] Table 2. Amino acid sequence of wild-type RSV F protein before artificial mutation

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309] Table 3 Amino acid sequences of exemplary wild-type RSV F protein mutants (after artificial mutation)

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351] DETAILED DESCRIPTION

[0352] The present disclosure discloses mutants of RSV F protein, and those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present disclosure, and relevant personnel can obviously modify or appropriately change and combine the contents described herein without departing from the content, spirit and scope of the present disclosure to implement and apply the disclosed technology.

[0353] In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Definitions of common terms in molecular biology can be found in Lewin's GENES XII, Jocelyn E. Krebs / Elliott S. Goldstein / Stephen T. Kilpatrick, published by Jones & Bartlett, 2018.

[0354] Unless otherwise expressly indicated, throughout the specification and claims, the term "comprising" or variations thereof such as "including" or "comprising" and the like will be understood to include the elements or components stated without excluding other elements or other components. The terms "a", "an", and "the" include plural referents. The term "plurality" refers to two or more. The terms "such as", "for example", and the like are intended to refer to exemplary embodiments and are not intended to limit the scope of the present disclosure.

[0355] In order to enable those skilled in the art to better understand the technical solution of the present disclosure, the present disclosure is further described in detail below in conjunction with specific embodiments.

[0356] I. Definitions

[0357] The term "wild type", which may also be referred to as "native" or "natural", refers to a protein without the addition of non-natural mutations. In some embodiments, exemplary examples of wild-type RSV F proteins are sequences such as those shown in SEQ ID No. 1-6.

[0358] The term "mutation" refers to a change in a sequence (such as a nucleotide or amino acid sequence) relative to the corresponding sequence in a natural, wild-type, standard, or reference form (i.e., a non-mutated sequence). Amino acid mutations generally include substitutions, deletions, or insertions of amino acid residues. Mutations can be artificial or naturally occurring.

[0359] The terms "peptide", "polypeptide" and "protein" can be used interchangeably and generally refer to peptides and proteins of amino acids covalently linked by peptide bonds. The term "protein" encompasses purified natural products, or products that can be produced in part or in whole using recombinant or synthetic techniques. The terms "peptide" and "protein" can refer to aggregates of proteins, such as dimers or other polymers, fusion proteins, protein variants or derivatives thereof. The term also includes modifications of proteins, for example, proteins modified by glycosylation, acetylation, phosphorylation, pegylation, ubiquitination, etc. Proteins may include amino acids that are not encoded by nucleic acid codons. Proteins may have amino acid sequences that are long enough to produce higher levels of tertiary and / or quaternary structures.

[0360] The term "protease cleavage site" refers to a specific amino acid motif within a protein or fusion protein amino acid. Within the amino acid sequence of a protein or fusion protein, it is cleaved by a specific protease that recognizes the amino acid motif. In some embodiments, examples of specific proteases include furin, trypsin, factor Xa, thrombin, or cathepsin L, etc.

[0361] The term "F0 precursor protein" refers to the RSV F protein first translated into a single-chain inactive precursor protein F0 consisting of 574 amino acids in vivo. The signal peptide consisting of amino acids 1 to 25 at the N-terminus of the F0 protein is removed by a signal peptidase. In addition, there are two furin protease cleavage sites at amino acids 109 / 110 and 136 / 137 of F0. By enzymatic cleavage by protease, the amino acid polypeptide at positions 110 to 136 (called p27) is removed to generate F1 consisting of amino acids 137 to 574 and F2 consisting of amino acids 26 to 109.

[0362] The terms "head domain" and "tail domain" are defined in the present invention for the purpose of describing the structure of RSV F protein. We define amino acids 1 to 485 as the head domain, and amino acids 486 to the C-terminus of the F protein as the tail domain. The approximate spatial positions of the head domain and the tail domain are as follows: Figure 1A shown.

[0363] The term "virus-like particles (VLPs)" or "pseudoviruses" refers to a multiprotein structure composed of the structural proteins of the corresponding natural virus, but lacking all or part of the viral genome, especially the replication and infection components of the viral genome, and therefore not replicable and infective. The multiprotein structure highly mimics its corresponding natural virus particles in morphology and size, and can be formed spontaneously after recombinant expression of the structural proteins of the virus.

[0364] The term "protein delivery vector" refers to a protein that can act as a carrier of an epitope peptide, that is, it can insert an epitope peptide at a specific position (e.g., inside the protein, at the N-terminus or C-terminus) so that the epitope peptide can be presented, thereby enabling the epitope peptide to be recognized by antibodies or the immune system.

[0365] The term "immunogenicity" refers to the ability of a substance to elicit, trigger, stimulate or induce an immune response in an animal to a specific antigen, with or without an adjuvant.

[0366] The term "conjugate" generally refers to any substance formed by joining two or more separate parts together. For example, a conjugate may include a substance formed by joining one polypeptide to another or more polypeptides. When one part of the conjugate is a protein, the conjugate is a protein conjugate.

[0367] II.RSV Mutation design of the F protein head domain to stabilize the F protein to a trimer before fusion state

[0368] Existing literature reports that most of the highly active neutralizing antibodies against RSV F protein discovered so far target the pre-fusion conformation (Ref: Battles MB, McLellan JSNat. Rev. Microbiol., 2019, 17: 233-245; Gilman M. SA et al. PLoS Pathog., 2015, 11: e1005035; Harshbarger W., et al. MAbs, 2021, 13: 1955812.), and the neutralizing antibody titer induced by the F protein trimer in the pre-fusion conformation is much higher than that of the post-fusion conformation trimer (Ref: Battles MB, McLellan JSNat. Rev. Microbiol., 2019, 17: 233-245; Krarup A.etal.Nat.Commun.,2015,6:8143), therefore, the F protein trimer in the prefusion conformation is the preferred antigen for developing recombinant subunit vaccines. However, the F protein trimer in the prefusion conformation is a metastable state and can easily be transformed from the prefusion conformation (such as Figure 1A ) into a post-fusion conformation (as shown Figure 1B Stabilizing the F protein to the pre-fusion conformational trimer state through modification of key sites is one of the key challenges that need to be addressed in the development of recombinant subunit vaccines.

[0369] The spatial structure of the trimer of RSV F protein before and after fusion has been obtained by X-ray crystal structure analysis experiment (Ref:McLellan JS, et al. Science, 2013, 340: 1113-1117.McLellan JS, et al. J. Virol., 2011, 85: 7788-7796.), and submitted to the protein structure database (protein data bank, PDB). According to PDB codes 4jhw and 3rrr, the spatial structure coordinates of the trimer of the before and after fusion conformation can be downloaded from the PDB database. Using commonly used protein structure comparison software, such as Pymol, Chimera, etc., the pre-fusion and post-fusion conformations are superimposed, such as Figure 2 As shown. Figure 2The results of the structural comparison show that from the pre-fusion conformation to the post-fusion conformation, the RR1 region of the head domain, as well as the RR2 region located at the C-terminus of the head domain and the tail domain have undergone significant conformational changes. At the same time, α1 and α5 of the head domain have also undergone obvious conformational movements accordingly. Therefore, the conformational changes of α1 and α5 are coupled with the conformational changes of RR1. Based on the above structural analysis, by introducing stability mutations in the α1 or α5 region to prevent the conformational changes of α1 and α5, the occurrence of RR1 conformational changes can be prevented through coupling regulation, and the F protein can be stabilized in the pre-fusion conformation state. In addition, stability mutations can also be directly introduced in the interaction region involved in RR1 to prevent the transition from the pre-fusion conformation to the post-fusion conformation. To this end, the present disclosure introduces mutations in the α1 helix (amino acids 73 to 96) or the α5 helix (amino acids 216 to 240) or directly in the interaction region involved in RR1, or introduces any combination of these mutations, and stabilizes the conformation of α1, α5 or RR1 by enhancing the interaction between the residues, thereby stabilizing the F protein in the pre-fusion conformational trimer state. Some exemplary single-site mutation designs are shown in Table 4. These single-site mutations can be combined arbitrarily and can have similar or stronger stabilizing effects. Some exemplary mutation combinations are shown in Table 5.

[0370] Table 4. Example single-site mutation designs for the head domain of RSV F protein

[0371]

[0372]

[0373] Table 5. Example site mutation combination design of RSV F protein head domain

[0374]

[0375]

[0376] III. Embodiment

[0377] Example 1 Plasmid construction of mutants designed by introducing artificial mutations into the head domain (Table 4 and Table 5) Recombinant expression and effect verification

[0378] The natural RSV F protein is a homotrimer. In order to facilitate the secretory expression of the protein, the amino acids 514 to 574 containing the transmembrane region and the intracellular region are removed from the full-length sequence of F0. This region plays an important role in the formation of the F protein trimer. In order to enable the truncated sequence to assemble to form a trimer, the C-terminus of the truncated sequence is connected to the exogenous trimerization domain (the T4 Foldon trimerization motif is used in this embodiment) through a short connector (the sequence is SAIG). Studies have shown that removing the sequence of the p27 part in F0 contributes to the formation of trimers (Krarup A. et al. Nat. Commun., 2015, 6: 8143.). In this embodiment, the sequence 107 to 138 containing p27 and furin cleavage sites (RARRELPRFMNYTLNNAKKTNVTLSKKRKRRF) is replaced with the linker QARGSGSGRS sequence. In addition, the present invention replaces the signal peptide "MELLILKANAITTILTAVTFCFASG" composed of amino acids 1 to 25 at the N-terminus of the sequence with a signal peptide sequence "MDMRVPAQLLGLLLLWLRGARC" suitable for the recombinant expression system HEK293 to promote the secretory expression of the recombinant protein. On this basis, the corresponding RSV F protein mutants were constructed according to the head domain site mutations designed in Tables 4 and 5. The constructed mutant amino acid sequences (excluding signal peptides, T4 foldon and connectors) are shown in SEQ ID No.35-157.

[0379] According to the amino acid sequence of SEQ ID No.35-157, a signal peptide, T4 foldon trimerization motif, connector, and an external tag sequence that facilitates purification are added to the recombinant expression system HEK293, and codon optimization is performed according to the codon preference of HEK293 to form the corresponding coding gene sequence. The coding gene sequence is constructed by whole gene synthesis or point mutation, and the Kozak sequence "GCCACC" is added to the front end of the sequence and then connected to the recombinant expression vector pcDNA3.1 to form a recombinant expression plasmid. Expi293 is used. TMThe transient expression system (Thermo Fisher) recombinantly expresses the RSV F protein mutant before the fusion of the construction, and the cell secretion supernatant is harvested on the 5th day after transfection. As a control, based on the above method, we also recombinantly expressed the wild-type RSV F protein before artificial mutation according to SEQ ID No.12 (called "wild-type RSV F protein 1 before artificial mutation"). In addition, we also purchased RSV A2 strain F protein (Catalog Number: 11049-V08B) from Sino Biological Shenzhou Company as another control (called "wild-type RSV F protein 2 before artificial mutation"), and its amino acid sequence is shown in SEQ ID No.15.

[0380] In order to verify whether the introduction of the above mutations can stabilize the RSV F protein to the pre-fusion conformational trimer state, the present example uses the pre-fusion conformational trimer to specifically bind to the monoclonal antibody AM14, and the binding activity of the above-constructed mutants to the AM14 antibody is detected by the ELISA experimental method, and compared with the AM14 monoclonal antibody binding activity of the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation. Published literature shows that the AM14 antibody binds to the epitope region at the interface between two adjacent monomers in the pre-fusion conformation of the F protein trimer (Ref: Gilman MSA et al. PLoS Pathog., 2015, 11: e1005035; Harshbarger W., et al. MAbs, 2021, 13: 1955812.). When the F protein trimer changes from the pre-fusion conformation to the post-fusion conformation, the epitope is destroyed. Therefore, the AM14 antibody cannot bind to the post-fusion conformation of the F protein. This antibody is often used for specific recognition and detection of the pre-fusion conformation trimer structure of the F protein (Ref: Gilman M. S A et al. PLoS Pathog., 2015, 11: e1005035; Harshbarger W., et al.MAbs, 2021, 13: 1955812.), and there are literature reports that the binding activity of AM14 monoclonal antibody is positively correlated with the neutralizing antibody titer produced by immunized animals (Ref: Che Y. et al. Sci. Transl. Med., 2023, 15: eade6422.). At the same time, in order to verify whether the constructed mutant can be expressed, this example uses a His tag antibody to detect its expression.

[0381] The ELISA detection process of transfection supernatant is as follows:

[0382] (1) Detection of AM14 monoclonal antibody binding activity

[0383] AM14 monoclonal antibody (Costan Bio) was diluted to 1 μg / ml using coating solution (Na2CO3-NaHCO3 solution, pH 9.6) and then coated on a 96-well ELISA plate, 100 μl / well, and coated at 4°C for 8-12 hours; after washing the plate with PBST solution, blocking solution (20% calf serum, 0.2% yeast extract, dissolved in PBST) was added, 100 μl / well, and blocked at 37°C for 2 hours; after washing the plate with PBST solution, 10-fold diluted cell secretion supernatant after centrifugation of the recombinant expression mutant (containing wild-type RSV F protein 1 before artificial mutation) and 2 μg / ml wild-type RSV F protein 2 before artificial mutation were added, 100 μl / well, and incubated at 37°C for 1 hour; after washing the plate with PBST solution, diluted horseradish peroxidase-labeled RSV F protein rabbit monoclonal antibody (1:1000, Sino Biological), 100 μl / well, incubated at 37°C for 1 h; after washing with PBST solution, color development solution A and B were added successively, color development was carried out at room temperature for 5 min, and stop solution C was added to terminate the color development reaction; the optical density (OD) value was read at 450nm and 630nm wavelength.

[0384] (2) Detection of His tag antibody binding activity

[0385] The cell secretion supernatant after centrifugation of the recombinant mutant (containing the wild-type RSV F protein 1 before artificial mutation) was diluted 10 times using the coating solution and coated on a 96-well ELISA plate. F protein 2 was used as the control well, 100 μl / well, coated at 4°C for 8-12 h; after washing with PBST solution, blocking solution was added, 100 μl / well, and blocked at 37°C for 2 h; after washing with PBST solution, diluted His tag antibody (1:2000 dilution, Sino Biological) was added, 100 μl / well, and incubated at 37°C for 1 h; after washing with PBST solution, diluted horseradish peroxidase-labeled goat anti-mouse secondary antibody (1:10000, Zhongshan Jinqiao) was added, 100 μl / well, and incubated at 37°C for 1 h; after washing with PBST solution, color development solution A and B were added successively, color development was performed at room temperature for 5 min, and stop solution C was added to stop the color development reaction; OD values ​​were read at wavelengths of 450 nm and 630 nm.

[0386] The results of ELISA tests are shown in Tables 6, 7, and 8. The results show that the His tag antibody test of most mutants has a high OD value, indicating that most of the constructed mutants can be effectively expressed. Table 6 shows that the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation have no AM14 monoclonal antibody binding activity, while Tables 7 and 8 show that a variety of head domain single-site mutants and a variety of head domain multi-site combination mutants can bind to AM14 monoclonal antibody, including FHS-1, FHS-4, FHS-5, FHS-6, FHS-11, FHS-12, FHS-14, FHS-26, FHS-45, FHS-46, FHS-47, FHS-48, FHS-49, FHS-50, FHS-65 , FHS-66, FHS-67, FHS-68, FHS-69, FHS-76, FHS-78, FHS-84, FHS-87, FHS-88, FHM-1, FHM-2, FHM-5, FHM-9, FHM-12, FHM-13, FHM-15, FHM-16, FHM-18, FHM-19, FHM-20, FHM-22, FHM-23, FHM-24 and FHM-25 mutants. The OD values ​​of these single-site mutants or multi-site combination mutants detected by AM14 monoclonal antibody were significantly higher than those of wild-type RSV F protein 1 and wild-type RSV F protein 2 before artificial mutation, indicating that the introduction of the above mutations successfully stabilized the RSV F protein to the pre-fusion conformational trimer state.

[0387] Table 6 Binding activity of wild-type RSV F protein 1 before artificial mutation and wild-type RSV F protein 2 before artificial mutation with AM14 monoclonal antibody and His tag antibody

[0388]

[0389] Table 7 Binding activity of exemplary mutants with single-site mutations introduced into the head domain of RSV F protein to AM14 monoclonal antibody and His tag antibody

[0390]

[0391] Table 8 Binding activity of exemplary mutants with multiple combined mutations introduced into the head domain of RSV F protein to AM14 monoclonal antibody and His tag antibody

[0392] Mutant number AM14 monoclonal antibody (OD value) His tag antibody (OD value) FHM-1 2.288 0.489 FHM-2 2.228 1.585 FHM-3 0.070 0.359 FHM-4 0.028 0.183 FHM-5 1.964 0.982 FHM-6 0.005 0.098 FHM-7 0.007 0.196 FHM-8 0.016 0.465 FHM-9 2.121 1.446 FHM-10 0.010 0.136 FHM-11 0.006 0.139 FHM-12 2.128 1.708 FHM-13 1.070 0.443 FHM-14 0.008 0.458 FHM-15 1.505 1.720 FHM-16 0.260 1.280 FHM-17 0.006 0.654 FHM-18 1.797 0.870 FHM-19 0.367 0.228 FHM-20 0.607 0.586 FHM-21 0.007 0.159 FHM-22 1.978 1.765 FHM-23 1.887 1.620 FHM-24 1.610 0.932 FHM-25 2.419 1.728

[0393] Example 2 Exemplary mutants FHM-24 and FHM- Purification, identification and biological activity evaluation of 25

[0394] The constructed RSV F protein head domain was introduced into the exemplary mutants FHM-24 and FHM-25 (corresponding amino acid sequences SEQ ID No.156 and SEQ ID No.157) and recombinant expression plasmids were cloned and expressed by Expi293 TM The expression system (Thermo Fisher) was used for transient expression, and the secretion supernatant was harvested on the 5th day after transfection. The transfection supernatant was purified by affinity chromatography using a nickel column (HisTrap FFcrude pre-packed column). The sample was loaded after equilibration of the chromatography column with an equilibrium buffer (20mM phosphate-150mM sodium chloride, pH7.4), and the target protein was collected after elution with 20% and 50% elution buffer (20mM phosphate-150mM sodium chloride-500mM imidazole, pH7.4). The purified protein was analyzed by 12% SDS-PAGE electrophoresis, and the results are shown in the figure. Figure 3 As shown, the molecular weight of the target protein monomer band is about 50-70 kD, which is basically consistent with the theoretical value, indicating that the FHM-24 and FHM-25 mutants can be expressed correctly.

[0395] After obtaining the purified samples of FHM-24 and FHM-25 mutants, the binding activity of the above two mutants with AM14 monoclonal antibody was detected by antibody binding experiment using the pre-fusion conformation trimer-specific monoclonal antibody AM14 to verify whether it was successfully stabilized to the pre-fusion conformation trimer state. At the same time, palivizumab was used to further verify the folding of FHM-24 and FHM-25 mutants. Palivizumab can specifically bind to the II epitope of RSV F protein. Both the pre-fusion and post-fusion F proteins contain the same conformation of the II epitope. Therefore, palivizumab can bind to both the pre-fusion conformation F protein and the post-fusion conformation F protein. In this experiment, wild-type RSV F protein 1 before artificial mutation and wild-type RSV F protein 2 before artificial mutation were used as controls. The specific experimental operation process is as follows: the purified FHM-24 and FHM-25 mutants, as well as the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation were used as controls. F protein 2 was diluted 2-fold using coating solution starting from 80 μg / ml (corresponding primary antibody was AM14 monoclonal antibody) and 10 μg / ml (corresponding primary antibody was palivizumab), 100 μl / well was coated on the ELISA plate, 3 replicates were made for each dilution, coated at 4°C for 8-12h, and blank wells were used as negative controls; after washing the plate with PBST solution, blocking solution was added, and the plate was blocked at 37°C for 2h; after washing the plate with PBST solution, diluted AM14 monoclonal antibody (0.1 μg / ml) was added or Palivizumab (2ug / ml), 100μl / well, incubate at 37℃ for 1h; after washing with PBST solution, add diluted horseradish peroxidase-labeled goat anti-human secondary antibody (1:10000), 100μl / well, incubate at 37℃ for 1h; after washing with PBST solution, add colorimetric solution A and B successively, color at room temperature for 5min, add stop solution C to stop the colorimetric reaction; read the OD value at 450nm and 630nm wavelength, and compare the OD values ​​of duplicate wells with different concentrations 450 / 630nm The mean of the values ​​was taken and the standard deviation was calculated. The protein concentration was used as the horizontal axis and the OD values ​​corresponding to each concentration were 450 / 630nm The mean is the vertical axis, and the line graph is drawn using Origin software.

[0396] Antibody binding assay results Figure 4 and Figure 5 As shown. AM14 monoclonal antibody binding experiment results ( Figure 4 ) showed that wild-type RSV F protein 1 and wild-type RSV F protein 2 before artificial mutation had no AM14 monoclonal antibody binding activity, while the exemplary mutants FHM-24 and FHM-25 with mutations introduced into the head domain showed AM14 monoclonal antibody binding activity. This shows that by introducing stabilizing mutations into the head domain, the RSV F protein can be stabilized in the pre-fusion conformational trimer state, preventing the occurrence of the transition from pre-fusion to post-fusion conformation. Palivizumab binding experiment results ( Figure 5 ) showed that the FHM-24 mutant, the FHM-25 mutant, the wild-type RSV F protein 1 before artificial mutation, and the wild-type RSV F protein 2 before artificial mutation all had palivizumab binding activity, indicating that these four proteins can be correctly folded.

[0397] Example 3: Based on the introduction of mutations in the head domain of RSV F protein, mutations in the tail domain are added. The aim is to further enhance the stability of the RSV F protein prefusion conformation trimer, thereby further improving its immunogenicity.

[0398] The stability of the trimer structure of the RSV F protein before fusion has an important influence on its immunogenicity as an antigen. Based on the head domain mutations introduced in the above embodiments, this embodiment also introduces mutations in the tail domain of the F protein, and enhances the stability of the trimer structure of the F protein by enhancing the interaction between the three helical bundles in the RR2 region, thereby enhancing the binding activity of the AM14 monoclonal antibody, thereby further improving its immunogenicity. It has been reported that the binding activity of the AM14 monoclonal antibody is positively correlated with the neutralizing antibody titer produced by the immunized animals (Ref: Che Y. et al. Sci. Transl. Med., 2023, 15: eade6422.). To this end, this embodiment introduces a single site mutation or a combination of multiple site mutations in the RR2 region of the RSV F protein tail domain, and further enhances the stability of the trimer structure of the F protein before fusion by enhancing the interaction between the three helical bundles in the RR2 region. This embodiment takes the mutants FHM-24 (head domain introduces 227N→M and 230L→F mutations) and FHM-25 (head domain introduces 227N→M, 230L→F mutations, 190S→V mutations and 193L→M mutations) introduced into the head domain as examples, and further enhances the stability of the RSV F protein trimer by introducing stability mutations into the tail domain. The single-site mutation designs of some exemplary tail domains are shown in Table 9. The single-site mutations of these tail domains can be combined arbitrarily, and can have similar or stronger stabilizing effects, and some exemplary mutation combinations are shown in Tables 10 and 11.

[0399] Table 9 Exemplary single-site mutation designs introduced into the tail domain based on the FHM-24 mutant to further enhance the stability of the RSV F protein prefusion conformation trimer

[0400] Mutant number Mutations introduced into the head domain Single-site mutation introduced into the tail domain FHCS-1 227N→M,230L→F 509S→M FHCS-2 227N→M,230L→F 509S→F FHCS-3 227N→M,230L→F 509S→L FHCS-4 227N→M,230L→F 505F→W FHCS-5 227N→M,230L→F 502S→Y

[0401] Table 10 Based on the FHM-24 mutant, exemplary site mutation combinations introduced into the tail domain are designed to further enhance the stability of the RSV F protein prefusion conformation trimer

[0402] Mutant number Mutations introduced into the head domain Combinations of site mutations introduced into the tail domain FHCM-1 227N→M,230L→F 505F→W, 509S→L FHCM-2 227N→M,230L→F 501Q→M, 509S→F

[0403] Table 11 Based on the FHM-25 mutant, exemplary site mutation combinations introduced into the tail domain are designed to further enhance the stability of the RSV F protein prefusion conformation trimer

[0404]

[0405] Example 4 Plasmid construction, recombinant expression, purification and biological activity verification of the mutant designed in Example 3

[0406] In order to verify whether the mutation introduced by the tail domain can further enhance the stability of the RSV F protein prefusion conformation trimer, this example uses the F protein prefusion conformation trimer specific binding monoclonal antibody AM14 to detect the antibody binding activity of the mutant designed in Example 3, and compares it with the AM14 antibody binding activity of the FHM-24 or FHM-25 mutants before the introduction of the tail domain mutation, the wild-type RSV F protein 1 before the artificial mutation, and the wild-type RSV F protein 2 before the artificial mutation, to evaluate the enhancing effect of the mutation introduced by the tail domain on the stability of the RSV F protein prefusion conformation trimer.

[0407] First, the amino acid sequences corresponding to the RSV F protein mutants designed in Example 3 are SEQ ID No. 158-166. According to the corresponding amino acid sequences, a signal peptide, T4foldon trimerization motif, connector, and an external tag sequence that facilitates purification are added to the recombinant expression system HEK293, and codon optimization is performed according to the codon preference of HEK293 to form the corresponding coding gene sequence. The plasmid construction and recombinant expression operation process described in Example 1 is used to construct a recombinant expression plasmid, and Expi293 is used to generate the recombinant expression plasmid. TM The mutants were recombinantly expressed using a transient expression system, and the cell secretion supernatant was harvested on the 5th day after transfection.

[0408] Then, all mutants designed in Example 3 were purified using the purification method described in Example 1 to obtain purified mutant proteins. The purified proteins were subjected to 12% SDS-PAGE electrophoresis analysis. The results were as follows: Figure 6 The molecular weight of the target protein monomer band is about 50-70 kD, indicating that the mutant can be expressed correctly.

[0409] Finally, the purified mutant samples were tested for binding activity of AM14 monoclonal antibody by the ELISA experiment described in Example 2 through the specific binding of RSV F protein pre-fusion conformation trimer, and compared with the AM14 monoclonal antibody binding activity of FHM-24 or FHM-25 mutants before the introduction of mutations in the tail domain, wild-type RSV F protein 1 before artificial mutation, and wild-type RSV F protein 2 before artificial mutation, to evaluate the enhancing effect of the mutation introduced by the tail domain on the stability of the RSV F protein pre-fusion head trimer. At the same time, the folding of the constructed mutant was further verified using Palivizumab.

[0410] The results of AM14 monoclonal antibody binding experiment are as follows Fig. 7A , Figure 7B As shown, the results showed that the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation had no AM14 monoclonal antibody binding activity, while the F protein mutants after the introduction of mutations in the head domain and tail domain were significantly improved in binding activity compared with the mutants without the introduction of tail domain mutations (FHM-24, FHM-25) and the wild-type RSV F protein 1 before artificial mutation and the wild-type RSV F protein 2 before artificial mutation. The results showed that the mutations introduced in the tail domain can further enhance the stability of the trimer structure of the RSV F protein before fusion conformation. The results of the Palifera antibody binding experiment are shown in Fig. 8A , Figure 8B As shown, the results showed that the wild-type RSV F protein 1 before artificial mutation, the wild-type RSV F protein 2 before artificial mutation, the F protein mutant without introduction of tail domain mutation, and the mutant with introduction of stability mutation in the tail domain all had strong palivizumab binding activity, indicating that these proteins can be folded correctly.

[0411] Example 5 Exemplary RSV constructed Study on the Accelerated Stability Test of F Protein Mutants

[0412] The constructed RSV F protein mutant is used as an antigen to develop a vaccine, and its stability is an important characteristic that needs to be paid attention to. In this example, the constructed exemplary RSV F protein mutants FHCS-1, FHCS-2 and FHCS-4 were subjected to thermal acceleration stability test. The mutant samples obtained after expression and purification were placed at 37°C for 2 weeks and 4 weeks, respectively. At the same time, the same samples were placed at -80°C for 2 weeks and 4 weeks as controls. The stability samples were diluted 2 times in series starting from 10 μg / ml (the corresponding primary antibody was AM14 monoclonal antibody) and 2.5 μg / ml (the corresponding primary antibody was Palivuzumab) concentrations using the coating solution, 100 μl / well was coated on an ELISA plate, and 2 duplicate wells were made for each dilution, and then the ELISA method described in Example 2 was used to detect the binding activity of AM14 monoclonal antibody and Palivuzumab in the sample.

[0413] The results of the heat accelerated stability test are shown in Tables 12, 13, 14, and 15. The results show that after the RSV F protein mutant constructed by the present invention was placed in a 37°C environment for 4 weeks, the binding activity of AM14 monoclonal antibody and palivizumab did not change significantly compared with the sample placed in a -80°C environment, indicating that the pre-fusion conformation trimer structure of the constructed RSV F protein mutant remained stable after being placed in a 37°C environment for 4 weeks.

[0414]

[0415]

[0416]

[0417]

[0418] Example 6 Exemplary RSV constructed Evaluation of the animal immune effect of F protein mutants

[0419] In order to verify whether the immune effect of the RSV F protein mutant constructed by the present invention is significantly enhanced, this example carried out an animal immune effect evaluation study on the mutants FHCS-1, FHCS-2, FHCS-3 and FHCS-4, and compared the immune effects of the wild-type RSV F protein before artificial mutation and the mutant protein FHM-24 with only the head domain mutation introduced.

[0420] The specific experimental method is: after the purified exemplary F protein mutant and the wild-type RSV F protein 2 before artificial mutation are mixed with aluminum hydroxide adjuvant respectively, the poly I-cell solution is added to mix and prepare the vaccine. The prepared vaccine is used to immunize BALB / c mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., SPF grade, female, 6-8 weeks old), 10 mice in each group. Muscle immunization was performed at week 0 and week 2, the antigen amount was 0.5 μg / mouse, the immunization volume was 0.1 ml / mouse, and blood was collected one week after the last immunization to separate the serum. The neutralizing antibody level of the immune serum was detected by the RSV A2 live virus neutralization experiment, as follows: the serum was serially diluted 2-fold using the maintenance solution (DMEM culture medium containing 2% fetal bovine serum and 1% penicillin and streptomycin), and the diluted RSV A2 virus was added, 100 TCID 50 / well, and each serum was made into duplicate wells; at the same time, virus control wells without serum and cell control wells without serum and virus were set up, and incubated at 4°C for 2h; 4 The number of cells in each well was added to Hep2 cell suspension and cultured in a 37°C, 5% CO2 incubator for 3 days; the neutralization result was determined by immunofluorescence method, and the serum dilution multiple lower than the mean value of the fluorescent spots in the 50% virus control wells was taken as the neutralizing antibody titer of the serum.

[0421] The results are as follows Fig. 9 As shown, compared with the wild-type RSV F protein before artificial mutation, the neutralizing antibody titer was improved after the head domain mutation was introduced, and the neutralizing antibody geometric mean titer (GMT) was increased from 147 to 239. Further, on the basis of introducing the head domain mutation, the neutralizing antibody titer was further significantly improved after the tail domain mutation was introduced at the same time, and the GMT increased to 588-1261, which was 4-8.58 times that of the wild-type RSV F protein before artificial mutation. The results show that the present invention stabilizes the RSV F protein to the pre-fusion conformation trimer structure state by introducing the head domain and tail domain mutations, significantly improving the immunogenicity of the protein, and providing a candidate antigen with significant technical advantages and immunological activity advantages for the development of recombinant RSV vaccines.

[0422] The above is only a preferred embodiment of the present disclosure. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present disclosure. These improvements and modifications should also be regarded as the scope of protection of the present disclosure.

Claims

1. A mutant of wild-type RSV F protein, wherein the mutant is an artificial mutant, characterized in that: The amino acid sequence of the mutant is as shown in SEQ ID No.156, 158-166.

2. A trimer of a mutant of the wild-type RSV F protein as claimed in claim 1, characterized in that The trimer is a homotrimer.

3. The trimer according to claim 2, characterized in that The C-terminus of the mutant is directly or indirectly linked to the trimerization domain.

4. The trimer according to claim 3, characterized in that The trimerization domain is GCN4 leucine zipper, collagen or T4 foldon.

5. The trimer according to claim 3, characterized in that The C-terminus of the mutant is connected to the trimerization domain via a linker.

6. The trimer according to claim 5, characterized in that The connector is a repeating sequence of GS, a repeating sequence of GGS, a repeating sequence of SAIG, a repeating sequence of GSS or a repeating sequence of GG.

7. A fusion protein, characterized in that The fusion protein consists of the mutant according to claim 1 or the trimer according to claim 2, and a signal peptide or a tag; The tag is a Flag tag, enhanced green fluorescent protein, glutathione S-transferase, a His tag, a streptomycin tag II or an AviTag tag.

8. A virus-like particle, characterized in that The virus-like particle comprises the mutant according to claim 1 , the trimer according to any one of claims 2 to 6 , or the fusion protein according to claim 7 .

9. An isolated nucleic acid molecule, characterized in that The isolated nucleic acid molecule encodes the mutant according to claim 1, the trimer according to any one of claims 2 to 6, or the fusion protein according to claim 7.

10. A carrier, characterized in that The vector comprises the sequence of the nucleic acid molecule according to claim 9.

11. A carrier, characterized in that The vector comprises the mutant according to claim 1 , the trimer according to any one of claims 2 to 6 , or the fusion protein according to claim 7 .

12. The carrier according to claim 10, characterized in that The vector is a protein expression vector or a gene delivery vector.

13. The carrier according to claim 11, characterized in that The vector is a protein delivery vector.

14. The carrier according to claim 12, characterized in that The gene delivery vector is a viral vector or a non-viral vector; The viral vector includes a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a poxvirus vector, a herpes simplex virus vector or a Sendai virus vector; the non-viral vector includes a liposome, a cationic polymer vector, a lipid nanoparticle vector or a multifunctional envelope nanocarrier.

15. A host cell, characterized in that The host cell comprises the nucleic acid molecule according to claim 9 or the vector according to any one of claims 10 to 14.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the mutant according to claim 1, the trimer according to any one of claims 2 to 6, the fusion protein according to claim 7, the isolated nucleic acid molecule according to claim 9, the vector according to any one of claims 10 to 14, the host cell according to claim 15, and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition according to claim 16, characterized in that The pharmaceutical composition is a vaccine.

18. The pharmaceutical composition according to claim 17, characterized in that The vaccine is a protein vaccine, a nucleic acid vaccine or a vector vaccine.

19. Use of the mutant as described in claim 1, the trimer as described in any one of claims 2 to 6, the fusion protein as described in claim 7, the virus-like particle as described in claim 8, the isolated nucleic acid molecule as described in claim 9, the vector as described in any one of claims 10 to 14, the host cell as described in claim 15, or the pharmaceutical composition as described in any one of claims 16 to 18 in the preparation of a product for preventing or treating RSV infection.

20. Use of the mutant according to claim 1, the trimer according to any one of claims 2 to 6, the fusion protein according to claim 7, the isolated nucleic acid molecule according to claim 9, the vector according to any one of claims 10 to 14, and the host cell according to claim 15 in preparing antibodies against the mutant, wherein the antibody is a polyclonal antibody.

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