RSV F protein mutant and application thereof

Abstract

The invention relates to an RSV F protein mutant and application thereof, and belongs to the field of vaccines. On the basis of the amino acid sequence of wild RSV F protein, multiple aspects and multiple modes of modification are carried out, including disulfide bond mutation, proline mutation, cavity filling mutation, electrostatic mutation, p27 sequence modification and / or trimerization structural domain modification, and multiple RSV F protein mutants are designed. And then, through gene synthesis, expression and ELISA detection, the pre-conformation (pre-F) proportion of the RSV F protein mutants and the stability under high-temperature and low-temperature conditions are tested, and a plurality of RSV F protein mutants with relatively high pre-conformation proportion and stable structures are screened out. Finally, candidate antigen representatives are selected for immunogenicity detection, and it is proved that the candidate antigen representatives have good immunogenicity and can be used for developing RSV vaccines and preparing RSV specific antibodies or RSV detection reagents.

Description

Technical Field

[0001] The invention relates to RSV F protein mutants and applications thereof, belonging to the field of vaccines. Background Art

[0002] Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus belonging to the family Paramyxoviridae, with two subtypes, A and B. RSV is transmitted by direct contact, and infection can cause symptoms such as bronchitis, pneumonia, asthma, and respiratory failure. RSV is the leading pathogen causing respiratory tract infections in children worldwide, and can also cause severe respiratory and lung infections in infants, the elderly, and people with impaired immune function. In 2019 alone, there were approximately 3.6 million RSV-related hospitalizations for acute lower respiratory tract infections, approximately 26,300 RSV-related in-hospital deaths for acute lower respiratory tract infections, and approximately 11,010 RSV-related deaths in children aged 0 to 60 months worldwide. In China, RSV is the number one pathogen causing respiratory tract infections in children (accounting for 25.7%), and is also the second most common pathogen causing respiratory tract infections in the entire population (accounting for 16.8%). In 2019, RSV caused acute lower respiratory tract infections in 3.5 million children under the age of 5 in China, and 620,000 to 950,000 children under the age of 5 were hospitalized. There are currently two humanized monoclonal antibodies (Palivizumab and Nirsevimab) available overseas to help prevent RSV infection, but the protection provided by this passive immunity is short-lived and requires multiple injections. Monoclonal antibodies are also very expensive. In terms of preventive vaccines, three RSV vaccines have been approved for marketing overseas, including Arexvy from GSK, Abrysvo from Pfizer, and mRESVIA from Modena, but there is no RSV preventive vaccine available in China. Therefore, the development of safe, efficient, and affordable vaccines is of great significance for the prevention and control of RSV infection.

[0003] The RSV genome is 15.2kb long and encodes 11 proteins. Among them, attachment protein (G) and fusion protein (F) are the main glycoproteins on the surface of the virus membrane and are also the key to the virus invading the human body. Therefore, they are the main antigenic proteins in vaccine research. G protein helps virus particles recognize and attach to receptors on the surface of host cells. F protein can promote fusion between virus particles and host cell membranes, allowing the virus nucleocapsid to enter the host cell. During fusion with the host cell membrane, F protein changes from a metastable pre-fusion conformation (Pre-F) to a stable post-fusion conformation (Post-F). Compared with G protein, F protein is more conservative in RSV and is a good antigenic protein for vaccine development. The F protein precursor F0 contains 574 amino acids. The 27 amino acid short peptide (p27) between amino acid residues 110-136 will be hydrolyzed and released by furin protease to form two subunits, F1 (137-574aa) and F2 (26-109aa). The two subunits are connected by a disulfide bond, and the three F1+F2 subunits form a mature F protein trimer structure. The F1 subunit contains a fusion peptide (FP) responsible for inserting into the adjacent host cell membrane and heptad repeat A (HRA) and heptad repeat B (HRB). HRA and HRB are separated in Pre-F. During membrane fusion, HRB docks with the HRA coiled coil and forms a stable hexagonal helical structure, producing a stable Post-F. The C-terminus of the F1 subunit also contains a transmembrane region (525-550aa) and an intracellular region (551-574aa).

[0004] In recent years, scientists have used structural biology and other methods to analyze the process of F protein from Pre-F structure to Post-F, and discovered antigenic epitopes related to neutralizing activity. Among them, Site Ⅰ, Ⅱ, and Ⅳ co-exist in Pre-F and Post-F conformations, and can be recognized by antibodies with neutralizing activity, such as 131-2a (Site Ⅰ), Motavizumab (Site II) and 101F / mAb19 (Site IV) and other antibodies. At the same time, scientists have discovered neutralizing antibody epitopes unique to the Pre-F structure, including Site Ø, Site V, and an antigenic epitope spanning between two monomers (quaternary cleavage-dependent epitope). These epitopes can bind to potent neutralizing antibodies, such as D25 (Site Ø), hRSV90 (Site V) and AM14 (quaternary cleavage-dependent epitope). Therefore, the Pre-F structure can induce the production of stronger neutralizing antibodies with higher titers than the Post-F structure, and is the key to the development of RSV vaccines. At present, RSV vaccines based on the Pre-F structure have achieved high protection rates in clinical practice and have been successfully marketed abroad. The National Institute of Allergy and Infectious Diseases (NIAID) team designed a protein mutant named "DS-Cav1" to stabilize the Pre-F structure by forming disulfide bonds (S155C, S290C) and filling the structural cavity (S190F, V207L) through site mutations (Jason S. McLellan, et al., Science, 2013). The "847" protein mutant designed by Pfizer forms a disulfide bond (T103C, I148C) through site mutations, fills the structural cavity (S190I) and introduces electrostatic mutations (D486S) to produce a stable Pre-F structure (Ye Che, et al., Science Translational Medicine, 2023). In addition, the "SC-TM" protein mutant designed by Janssen Pharmaceuticals (Janssen) also obtained a relatively stable Pre-F structure through rigid amino acid mutation (S215P), cavity filling (N67I) and electrostatic mutation (E487Q) (Anders Krarup, et al., Nature communication, 2015). At present, no RSV vaccine based on the Pre-F structure has been successfully marketed in China. At the same time, the current strategy for constructing the Pre-F structure also has problems such as low protein expression and insufficient structural stability. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a variety of mutants of wild-type RSV F protein. By performing amino acid sequence mutation on the wild-type RSV F protein, the mutation includes substitution, deletion or addition of amino acids, and the mutant can improve the protein expression and stability of the Pre-F structure, which is helpful for the prevention, diagnosis and treatment of RSV infection.

[0006] In order to solve the above technical problems, the specific technical solutions of the present invention are as follows:

[0007] In a first aspect, the present invention provides a mutant of a wild-type RSV F protein, wherein the mutant has at least one of the following modifications relative to the wild-type RSV F protein:

[0008] (a) at least one engineered disulfide bond mutation;

[0009] (b) at least one cavity-filling mutation;

[0010] (c) at least one electrostatic mutation;

[0011] (d) at least one proline mutation;

[0012] (e) Modification of p27 sequence: deleting the sequence containing p27;

[0013] (f) Modification of the trimerization domain: deleting the transmembrane region and the intracellular region and adding an amino acid sequence containing the trimerization domain.

[0014] The following descriptions of amino acid sequence positions are all determined based on the amino acid sequence of the wild-type RSV F protein shown in SEQ ID NO.1.

[0015] The engineered disulfide bond mutation refers to the mutation of at least one pair of amino acid residues in the wild-type RSV F protein to a pair of cysteine ​​residues (Cys, C) to form a disulfide bond between the two, and the residues mutated to cysteine ​​are close in the RSV F protein pre-fusion configuration (pre F) but far away in the post-fusion configuration (Post F), so the formation of a disulfide bond between the introduced pair of cysteine ​​residues can stabilize the pre-fusion configuration (pre F) of the RSV F protein. In an optional embodiment, the at least one modification comprises that at least one pair of amino acid residues in the RSV F protein F1 subunit and / or F2 subunit is replaced by cysteine ​​(Cys, C) to form an engineered disulfide bond. In an optional embodiment, the engineered disulfide bond mutation comprises at least one pair of wild-type RSV F protein amino acid sequence positions 106+144, 486+487, 104+146, 104+147, 104+148, 105+145, 105+146, 105+147, 34+471, 171+191, 60+196, and 105+370. Preferably, the disulfide bond mutation comprises at least one pair of R106C+V144C, N104C+I148C, N105C+A147C, D486C+E487C, E60C+K196C, and S / N105C+M370C.

[0016] The target replacement amino acids for cavity filling include small aliphatic amino acids (such as Gly, Ala and Val) or small polar amino acids (such as Ser and Thr), which are replaced by large aliphatic amino acids (Ile, Leu and Met) or large aromatic amino acids (His, Phe, Tyr and Trp) with larger side chains to fill the structural cavity and stabilize the Pre-F conformation. In an optional embodiment, the cavity filling mutation is a mutation of the amino acid at position 190 of the wild-type RSV F protein amino acid sequence to isoleucine (Ile, I), valine (Val, V), leucine (Leu, L) and tryptophan (Trp, W). Preferably, the cavity filling mutation comprises a mutation of position 190S to any one of I, V, L, and W amino acids.

[0017] Electrostatic mutation is the use of amino acid mutations to reduce ionic repulsion between residues close to each other in the protein folding structure. In an optional embodiment, the electrostatic mutation comprises a mutation of amino acid 486, 487 or 489 in the wild-type RSV F protein amino acid sequence to asparagine (Asn, N), glutamine (Gln, Q), threonine (Thr, T) or proline (Pro, P). Preferably, the electrostatic mutation comprises a mutation of position 486D to N or Q, a mutation of position 487E to T, and a mutation of position 489D to P or Q.

[0018] Proline mutation is also called rigid amino acid mutation, and its purpose is to utilize the unique ring structure and geometric conformation restriction of proline to stabilize the correct folding conformation of the protein in the corner region or specific key position of the polypeptide main chain, thereby reducing the possibility of misfolding or unfolding and improving the structural stability of the protein. In an optional embodiment, the proline mutation comprises positions 210, 211, 212, 213, 214, 215, 216, 217, 218, 490, 210+212, 211+213, 212+214, 214+216, 216+218, 213+214+216 and 211+212+214 amino acid mutations to P and / or G. Preferably, the proline mutation comprises Q210P, S211P, C212P, S213P, I214P, S215P, N216P, I217P, E218P, Q210P+C212P, S211P+S213P, C212P+I214P, I214P+N216P, S215P+A490P, N216P+E218P, S213G+I214P+N216G or S211G+C212P+I214G.

[0019] p27 sequence modification refers to deleting the p27 sequence based on wild-type RSV F, with the purpose of deleting the furin cleavage site and connecting the F1 and F2 subunits with a covalent bond or a connecting peptide to stabilize the Pre-F conformation. Furin generally cleaves after the RXK / RR of the protein (where R represents arginine, K represents lysine, and X represents any amino acid). Combined with the sequence of the wild-type RSV F protein (SEQ ID NO: 1), it can be seen that the furin cleavage site is located after the 109th and 136th amino acid residues of RSV F, so the p27 sequence refers to the 110th to 136th amino acid residue fragment of the RSV F protein (aa 110–136). In the present invention, p27 sequence modification refers to deleting a fragment including p27 on the basis of RSV F protein, and the deleted fragment may be slightly longer than p27. For example, in an optional embodiment, the p27 sequence modification refers to deleting a fragment between amino acids 103 to 145, 104 to 148, or 105 to 147 on the basis of the amino acid sequence of wild-type RSV F protein or replacing it with a connecting peptide after deletion (the aforementioned "between" does not include the end value of the given data range), and the connecting peptide amino acid sequence may be a common form such as GS, GSGS, GGGS, etc. Preferably, the p27 sequence modification is selected from: N104-I148, N104-GS-I148, S / N105-A147, S / N105-GS-A147, or T103-GS-G145, indicating that the amino acid residues corresponding to the end value of the data range are retained and the fragments therebetween are deleted, or the deleted fragments are further replaced with GS.

[0020] The modification of the trimerization domain refers to deleting the sequence containing the transmembrane region and the intracellular region on the basis of the wild-type RSV F, and adding the amino acid sequence containing the trimerization domain, the purpose of which is to promote the F protein to form a trimer conformation and maintain its natural conformation. The transmembrane region and the intracellular region refer to the transmembrane region (aa 525-550) and the intracellular region (aa 551-574) located at the C-terminus of the wild-type RSV F protein; the trimerization domain can select a heterologous trimerization domain such as the Foldon domain, or a cysteine ​​zipper trimerization domain obtained by modifying the RSV F intracellular region fragment, etc. (1) The Foldon trimerization domain refers to the residues at the C-terminus of the phage T4 fiber protein. In the present application, the Foldon domain can be the 27 residues at the C-terminus of the phage T4 fiber protein or a mutant thereof. In an optional embodiment, the amino acid sequence of the Foldon trimerization domain is GYIPEAPRDGQAYVRKDGEWVLLSTFL; as an optional embodiment, the deletion of the transmembrane region and the intracellular region and the addition of an amino acid sequence comprising a trimerization domain refers to deleting aa 514~574 (covering the transmembrane region and the intracellular region) at the C-terminus of the wild-type RSV F protein, and adding a linker sequence (such as SAIG) and the aforementioned Foldon trimerization domain. (2) The cysteine ​​zipper trimerization domain refers to a sequence comprising four pairs of cysteine ​​mutations, which may be an ABCD-tag or an ABCH-tag. In an optional embodiment, the amino acid sequence of the ABCD-tag trimerization domain is CCHNVNACCSTTNICCTTTNICCTT, and the amino acid sequence of the ABCH-tag trimerization domain is CCHNVNACCSTTNICCTTIIICCIV; as an optional embodiment, the deletion of the transmembrane region and the intracellular region and the addition of an amino acid sequence comprising a trimerization domain refers to the deletion of aa 512 to 574 (covering the transmembrane region and the intracellular region) at the C-terminus of the wild-type RSV F protein and the addition of the aforementioned ABCD-tag or ABCH-tag

[0021] In an optional embodiment, the wild-type RSV F protein mutants of the present invention further include a linker sequence (such as GG, GS, SAIG) and an 8*His tag sequence (HHHHHHHH). The sequences are not required for the function of the RSV F protein, such as inducing an immune response. Those skilled in the art will recognize such sequences and understand, where appropriate, that the RSV F protein mutants disclosed in the present invention may or may not include such sequences.

[0022] As an optional embodiment, the present invention provides three groups of RSV F mutants:

[0023] The first group of RSV F mutants all had electrostatic mutations E487T, D486N, or D486Q;

[0024] The second group of RSV F mutants all have the disulfide bond mutations D486C+E487C;

[0025] The third group of RSV F mutants all have disulfide bond mutations selected from R106C+V144C, S / N105C+A147C, S / N105C+A146C, N104C+I148C, or N104C+S146C;

[0026] The three groups of RSV F mutants are described below:

[0027] (A) The first group of RSV F mutants (all with E487T, D486N or D486Q):

[0028] The present invention provides a RSV F protein mutant, which has multiple modifications relative to the wild-type RSV F protein to stabilize it in a pre-fusion conformation, wherein the modifications include:

[0029] (a) Proline mutation: S215P;

[0030] (b) Cavity-filling mutation: selected from S190V, S190L or S190I (preferably S190V or S190L, more preferably S190L);

[0031] (c) electrostatic mutation: selected from E487T, D486N or D486Q (E487T is preferred);

[0032] The position numbers are based on the sequence shown in SEQ ID NO: 1.

[0033] Preferably, the modification does not comprise an engineered disulfide bond mutation.

[0034] Preferably, the modifications present in the RSV F protein mutant include: S215P, S190L, E487T.

[0035] Optionally, the modification further comprises p27 sequence modification, i.e., deleting the p27 sequence. Optionally, the p27 sequence modification adopts one of the following methods: (1) deleting the p27-containing fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) deleting the p27-containing fragment and introducing a linker sequence: connecting the sequences before and after the deleted fragment using a linker (e.g., GG, GS, GSGS, GGGS, etc.). Preferably, the p27 sequence modification is selected from one of the following: T103-GS-G145, N104-I148, N104-GS-I148, S / N105-A147, S / N105-GS-A147, wherein T103-GS-G145, N104-GS-I148 or S / N105-A147 are further preferred, and S / N105-A147 is further preferred.

[0036] Optionally, the modification further comprises a trimerization domain modification: deleting the transmembrane region and the intracellular region and adding a sequence comprising a trimerization domain. Preferably, the trimerization domain is selected from Foldon, ABCD-tag or ABCH-tag, and their sequences are shown in SEQ ID NO: 2, 3, 4, respectively, wherein Foldon is preferred.

[0037] Preferably, the modification comprises or consists of any of the following modifications:

[0038] Group (1): S215P, S190L, E487T, T103-GS-G145, Foldon;

[0039] Group (2): S215P, S190L, E487T, N104-GS-I148, Foldon;

[0040] Group (3): S215P, S190L, E487T, S / N105-A147, Foldon;

[0041] Group (4): S215P, S190L, E487T, S / N105-GS-A147, Foldon.

[0042] Among them, group (3) is preferred.

[0043] In the above groups, "Foldon" means deleting the transmembrane and intracellular regions of RSV F and adding a sequence containing the Foldon trimerization domain.

[0044] Preferably, the modified RSV F protein mutants of group (1), group (2), group (3) or group (4) respectively comprise or have an amino acid sequence that is more than 99% (preferably 100%) identical to SEQ ID NO: 51, 53, 54 or 55.

[0045] (ii) the second group of RSV F mutants (all with D486C+E487C disulfide bond mutations);

[0046] The present invention also provides a RSV F protein mutant, which has multiple modifications relative to the wild-type RSV F protein to stabilize it in a pre-fusion conformation, wherein the modifications include:

[0047] (a) Engineered disulfide bond mutation: D486C+E487C;

[0048] (b) Proline mutation: selected from S215P, Q210P, S211P or S211P+S213P;

[0049] (c) Cavity-filling mutation: selected from S190V or S190L;

[0050] The position numbers are based on the sequence shown in SEQ ID NO: 1.

[0051] Preferably, the modification does not include other electrostatic mutations (the disulfide bond mutation D486C+E487C can serve the purpose of electrostatic mutation to a certain extent, because the originally negatively charged D486 and E487 are replaced by uncharged C, thus avoiding charge repulsion between adjacent amino acids; therefore, when using D486C+E487C, no other electrostatic mutations need to be introduced).

[0052] Optionally, the modification further comprises p27 sequence modification, i.e., deleting the p27 sequence. Optionally, the p27 sequence modification adopts one of the following methods: (1) deleting the p27-containing fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) deleting the p27-containing fragment and introducing a linker sequence: connecting the sequences before and after the deleted fragment using a linker (e.g., GG, GS, GSGS, GGGS, etc.). Preferably, the p27 sequence modification is selected from one of the following: T103-GS-G145, N104-I148, N104-GS-I148, S / N105-A147, S / N105-GS-A147, wherein T103-GS-G145 or S / N105-A147 is preferred.

[0053] Optionally, the modification further comprises a trimerization domain modification: deleting the transmembrane region and the intracellular region and adding a sequence comprising a trimerization domain. Preferably, the trimerization domain is selected from Foldon, ABCD-tag or ABCH-tag, and their sequences are shown in SEQ ID NO: 2, 3, 4, respectively, wherein Foldon is preferred.

[0054] Preferably, the modification comprises or consists of any of the following groups:

[0055] Group (1): D486C+E487C, S215P, S190V, T103-GS-G145, Foldon;

[0056] Group (2): D486C+E487C, S215P, S190L, T103-GS-G145, Foldon;

[0057] Group (3): D486C+E487C, S215P, S190L, T103-GS-G145;

[0058] Group (4): D486C+E487C, S215P, S190L, S / N105-A147, Foldon;

[0059] Group (5): D486C+E487C, Q210P, S190L, S / N105-A147, Foldon;

[0060] Group (6): D486C+E487C, S211P, S190L, S / N105-A147, Foldon;

[0061] Group (7): D486C+E487C, S211P+S213P, S190L, S / N105-A147, Foldon.

[0062] Among them, group (2), group (4), group (5) or group (6) is preferred.

[0063] In the above groups, "Foldon" means deleting the transmembrane and intracellular regions of RSV F and adding a sequence containing the Foldon trimerization domain.

[0064] Preferably, the modified RSV F protein mutants of group (1), group (2), group (3), group (4), group (5), group (6) or group (7) respectively contain or are amino acid sequences that are more than 99% (preferably 100%) identical to SEQ ID NO: 46, 47, 50, 58, 64, 65, 73.

[0065] (iii) Group III RSV F mutants (all with R106C+V144C, S / N105C+A147C, S / N105C+A146C, N104C+I148C, or N104C+S146C disulfide bond mutations);

[0066] The present invention also provides a RSV F protein mutant, which has multiple modifications relative to the wild-type RSV F protein to stabilize it in a pre-fusion conformation, wherein the modifications include:

[0067] (a) Engineered disulfide bond mutations selected from R106C+V144C, N104C+S146C, N104C+I148C, S / N105C+A146C, S / N105C+A147C or S / N105C+M370C;

[0068] (b) Proline mutation: S215P;

[0069] (c) cavity-filling mutation: selected from S190I, S190L, S190V or S190W;

[0070] (d) an electrostatic mutation selected from D486N, D486Q, E487T, D489P or D489Q;

[0071] The position numbers are based on the sequence shown in SEQ ID NO: 1.

[0072] An optional scheme is: in the modification, the disulfide bond mutation is selected from R106C+V144C, the cavity filling mutation is selected from S190I, S190L, S190V or S190W (preferably S190L, S190V or S190W), and the electrostatic mutation is selected from D486N, D486Q, E487T, D489P or D489Q (preferably D486N, D486Q, E487T or D489P).

[0073] Another optional scheme is: in the modification, the disulfide bond mutation is selected from N104C+S146C, N104C+I148C, S / N105C+A146C or S / N105C+A147C, the cavity filling mutation is selected from S190I, S190L or S190V (S190I is preferred), and the electrostatic mutation is selected from D486N, D486Q, E487T (D486N is preferred).

[0074] Optionally, the modification may or may not include p27 sequence modification, wherein the p27 sequence modification refers to deleting the p27 sequence. Optionally, the p27 sequence modification adopts one of the following methods: (1) deleting the p27-containing fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) deleting the p27-containing fragment and introducing a linker sequence: connecting the sequences before and after the deleted fragment using a linker (e.g., GG, GS, GSGS, GGGS, etc.). Preferably, the p27 sequence modification is selected from one of the following: T103-GS-G145, N104-I148, N104-GS-I148, S / N105-A147, S / N105-GS-A147.

[0075] Optionally, the modification further comprises a trimerization domain modification: deleting the transmembrane region and the intracellular region and adding a sequence comprising a trimerization domain. Optionally, the trimerization domain is selected from Foldon, ABCD-tag or ABCH-tag, and their sequences are shown in SEQ ID NO: 2, 3, 4, respectively, wherein Foldon is preferred.

[0076] Preferably, the modification comprises or consists of any of the following groups:

[0077] (1) R106C+V144C, S215P, S190I, D486N, Foldon;

[0078] (2) R106C+V144C, S215P, S190V, D486N, Foldon;

[0079] (3) R106C+V144C, S215P, S190L, D486N, Foldon;

[0080] (4) R106C+V144C, S215P, S190W, D486N, Foldon;

[0081] (5) R106C+V144C, S215P, S190V, D486Q, Foldon;

[0082] (6) R106C+V144C, S215P, S190V, E487T, Foldon;

[0083] (7) R106C+V144C, S215P, S190L, D486Q, Foldon;

[0084] (8) R106C+V144C, S215P, S190L, E487T, Foldon;

[0085] (9) R106C+V144C, S215P, S190L, D489P, Foldon;

[0086] (10) R106C+V144C, S215P, S190W, D486Q, Foldon;

[0087] (11) R106C+V144C, S215P, S190W, E487T, Foldon;

[0088] (12) N104C+S146C, S215P, S190I, D486N, Foldon;

[0089] (13) N104C+I148C, S215P, S190I, D486N, Foldon;

[0090] (14) S / N105C+S146C, S215P, S190I, D486N, Foldon;

[0091] (15) S / N105C+A147C, S215P, S190I, D486N, Foldon;

[0092] (16) S / N105C+M370C, S190L, E487T, S / N105-A147, Foldon.

[0093] In the above groups, "Foldon" means deleting the transmembrane and intracellular regions of RSV F and adding a sequence containing the Foldon trimerization domain.

[0094] Further preferably, the RSV F protein mutant having any modification described in any one of groups (1) to (16) has the following amino acid sequence characteristics: comprising or being an amino acid sequence that is 99% or more (preferably 100%) identical to SEQ ID NO: 5, 6, 7, 8, 9, 10, 14, 15, 16, 19, 20, 24, 26, 28, 29 or 83.

[0095] As an optional embodiment, the present invention provides 79 specific RSV F mutants, the modifications present in which relative to the wild-type RSV F protein are specifically shown in Table 1, Table 3, Table 5 and Table 7.

[0096] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial comprises any one of the following:

[0097] (a) A nucleic acid molecule encoding the RSV F protein mutant.

[0098] (b) A recombinant vector containing the nucleic acid molecule described in (a). Preferably, the original plasmid of the recombinant vector is pOET1.1.

[0099] (c) a recombinant virus containing the nucleic acid molecule of (a) or the recombinant vector of (b), preferably, the recombinant virus comprises insect cell baculovirus, adenovirus, adeno-associated virus, vaccinia virus, herpes virus or retrovirus.

[0100] (d) a transformed cell containing (a) the nucleic acid molecule, (b) the recombinant vector or (c) the recombinant virus, wherein the host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells; preferably, the insect cell is Sf9 cell, Sf21 cell or High-Five cell.

[0101] In a third aspect, the present invention also provides a method for preparing the above RSV F protein mutant, the steps comprising: constructing a recombinant vector, extracting the recombinant vector, transfecting the recombinant vector into a host cell to express the protein, and purifying the expression product.

[0102] In a fourth aspect, the present invention also provides an immunogenic composition comprising the above-mentioned RSV F protein mutant, nucleic acid molecule, recombinant vector, recombinant virus or transformed cell.

[0103] In a fifth aspect, the present invention further provides the use of any of the above-mentioned RSV F protein mutants, nucleic acid molecules, recombinant vectors, recombinant viruses, transformed cells, and immunogenic compositions in any of the following aspects:

[0104] (a) Application of preparing RSV-specific antibodies.

[0105] (b) preparing a drug for preventing and / or treating RSV infection; preferably, the drug comprises a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.

[0106] (c) Preparation of diagnostic reagents for RSV.

[0107] Definition of terms:

[0108] In this application, the term "respiratory syncytial virus" or "RSV" refers to a single-stranded negative-sense RNA virus belonging to the genus Pneumovirus of the family Paramyxoviridae. The virus can cause lower respiratory tract disease (LRTD) symptoms in infants, the elderly and immunocompromised adults, including interstitial pneumonia and bronchiolitis.

[0109] In this application, the term "mutant" refers to a protein that has one or more changes in amino acid sequence or protein structure compared to the wild-type protein. These changes may include, but are not limited to, deletion, insertion, replacement, shortening and / or loss of one or more amino acids, and modification or cleavage of protein structure. In this application, "mutant" refers specifically to respiratory syncytial virus (RSV) F protein mutants.

[0110] In the present application, the term "disulfide bond mutation" refers to the replacement of an amino acid in the wild-type RSV F protein by cysteine, and a disulfide bond is formed between the sulfur atoms in the two cysteine ​​residues to stabilize the RSV Pre-F structure.

[0111] In the present application, the term "cavity filling mutation" refers to that the amino acid residues in the wild-type RSV F protein are replaced by the amino acids that are expected to fill the cavity of the mature F protein structure to stabilize the RSV Pre-F structure. These amino acids that fill the cavity of the structure are often amino acids with larger side chain groups, such as large aliphatic amino acids (Ile, Leu and Met) or large aromatic amino acids (His, Phe, Tyr and Trp).

[0112] In the present application, the term "electrostatic mutation" refers to the replacement of an amino acid residue in the wild-type RSV F protein with an expected amino acid to reduce the ionic repulsion between residues close to each other in the protein folding structure to stabilize the RSV Pre-F structure. The electrostatic mutation comprises a mutation of the amino acid at position 486, 487 or 489 of the wild-type RSV F protein amino acid sequence to asparagine (Asn), glutamine (Gln) or threonine (Thr).

[0113] In this application, the term "proline mutation" refers to the replacement of an amino acid residue in the wild-type RSV F protein with proline to stabilize the RSV Pre-F structure. The amino acid and carboxyl group in proline are linked by an amino group to form a rigid five-membered ring structure. This ring structure limits the rotational freedom of proline in the protein, allowing proline to form fixed folds and turns, thereby increasing the stability of the protein.

[0114] In the present application, the term "p27" refers to a 27 amino acid short peptide between amino acid residues 110 and 136 in the wild-type RSV F protein. The amino acid short peptide is hydrolyzed and released by furin protease to form two subunits, F1 (137-574aa) and F2 (26-109aa).

[0115] In the present application, the term "p27 sequence modification" refers to deleting a fragment including p27 on the basis of the wild-type RSV F protein, that is, the deleted fragment should cover p27 and may be slightly longer than p27 (extending about 1 to 15 amino acid residues upstream and / or downstream of p27). For example, the fragment between amino acid residues 104 to 144, 105 to 147, or 106 to 146 of the RSV F protein may be deleted (the aforementioned "between" does not include the end value of the numerical range, for example, the fragment between amino acid residues 104 to 144 refers to a fragment consisting of amino acids 105 to 143), and after deleting the aforementioned fragment, the front and rear fragments may be directly covalently linked or linked using a connecting peptide (e.g., GG, GS, GSGS, GGGS, etc.), wherein GS is preferred.

[0116] In the present application, the term "trimerization domain" refers to an amino acid sequence that forms a trimer at the C-terminus of the wild-type RSV F protein by introducing a heterologous amino acid sequence (e.g., Foldon) or a cysteine ​​zipper amino acid sequence (e.g., ABCD-tag, ABCH-tag). The trimer in the present application refers to a complex formed by three RSV F protein F1+F2 subunits.

[0117] In the present application, the term "ABCD-tag" refers to the addition of a cysteine ​​zipper (an amino acid sequence containing 4 pairs of cysteine ​​mutations) to the C-terminus of the wild-type RSV F protein with the transmembrane region and the intracellular region deleted, and the ABCD-tag amino acid sequence is CCHNVNACCSTTNICCTTTNICCTT.

[0118] In the present application, the term "ABCH-tag" refers to the addition of a cysteine ​​zipper (an amino acid sequence containing 4 pairs of cysteine ​​mutations) to the C-terminus of the wild-type RSV F protein with the transmembrane region and the intracellular region deleted, and the amino acid sequence of the ABCH-tag is CCHNVNACCSTTNICCTTTNICCTT.

[0119] In this application, the term "Mota" or "motavizumab" refers to the antibody described in the paper by Herren et al. (Herren Wu, et al., Journal of Molecular Biology, 2007).

[0120] In this application, the term "D25" refers to the antibody described in the paper by Jason S McLellan, et al., Science, 2013.

[0121] In this application, the term "hRSV90" refers to the antibody described in the paper by Jarrod et al. Nature Microbiology 2, 16271 (2017).

[0122] In this application, the term "AM14" refers to the antibody described in the paper by Morgan et al. (Morgan SA Gilman, et al., PLoS Pathog, 2015).

[0123] In the present application, the term "conformational change" generally refers to a change in the spatial structure of a protein molecule; for example, the conformational change may include a change in the chemical bonds in the protein molecule, a change in the folding mode of a polypeptide.

[0124] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0125] Beneficial effects of the present invention:

[0126] The RSV F protein mutants of the present invention can significantly improve the stability of the pre-fusion conformation (pre-F) and the binding activity with neutralizing antibodies compared to the wild-type RSV F protein, indicating that it has a high potential to become an effective component of RSV vaccine. Compared with the prior art (such as 847 protein), some RSV protein mutants provided by the present invention have a higher pre-F ratio or better stability under high or low temperature conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 : Map of the pOET1.1 baculovirus transfer plasmid used in Example 1 of the present invention.

[0128] Figure 2 : Binding of the cell supernatants of each mutant to different monoclonal antibodies (double antibody sandwich ELISA).

[0129] Figure 3 : Reducing and non-reducing gel electrophoresis analysis of each mutant after purification.

[0130] Figure 4 : Western Blot analysis of each mutant after purification.

[0131] Figure 5 : Results of binding antibody assay in mice after immunization with different doses of BR47 antigen protein.

[0132] Figure 6 : Results of binding antibody assay in mice after immunization with different antigenic proteins. DETAILED DESCRIPTION

[0133] In order to more accurately and clearly understand and grasp the purpose, technical solution and advantages of the present invention, the following will be based on the relevant drawings in the embodiments of the present invention. The embodiments of the present invention are deeply interpreted and described in detail. Please note that the embodiments described here only represent some application examples of the present invention and do not include all embodiments.

[0134] Example 1: Design and screening of RSV F mutants

[0135] RSV F mutant design and screening include the following steps:

[0136] (1) Design of mutants and gene synthesis: First, the sequence of wild-type RSV F is obtained: Based on the F protein sequence of type A RSV strains published in the NCBI database, the amino acid with the highest number of occurrences at each site (common amino acid) is calculated to construct the RSV F common amino acid sequence (as shown in SEQ ID NO.1), which represents the sequence of type A wild-type RSV F. Then, based on the amino acid sequence of the wild-type RSV F protein shown in SEQ ID NO: 1, a modification is performed to design a recombinant RSV F protein (mutant): Based on the existing data and combined with structural biology analysis, the present invention designs a variety of RSV F mutants (recombinant proteins), which respectively include one or more of the following modifications: disulfide bond mutation, proline mutation, cavity filling mutation, electrostatic mutation, p27 sequence modification, and introduction of a trimerization domain. Then, codon optimization is performed according to the host Sf9 insect cell to determine the nucleic acid sequence and perform full gene synthesis.

[0137] (2) Construction of recombinant plasmid and expression of antigen protein: The synthesized target gene was inserted into the baculovirus transfer vector pOET1.1 ( Figure 1 ) and use flashBAC TM The baculovirus expression system kit co-transfects the recombinant plasmid and the baculovirus genome into Sf9 insect cells. The specific operation of transfection is carried out according to the instructions of the kit. The cell supernatant of Sf9 cells is collected 7 days after transfection. At this time, the supernatant contains the P0 generation virus of the recombinant exogenous gene. The P0 generation virus is transferred into a new Sf9 cell well plate to obtain the P1 generation virus. After the P1 generation virus infects the cells for 6-7 days, the cell supernatant is collected for ELISA to detect the conformation and stability of each antigen.

[0138] (3) ELISA detection of antigen proteins: Use the ELISA method to detect the conformation and stability of each antigen protein.

[0139] The specific operation of ELISA detection is as follows: 100 μl of Motavizumab (abbreviated as Mota, can bind both Pre-F and Post-F), AM14 (only binds Pre-F), D25 (only binds Pre-F) or hRSV90 (only binds Pre-F) and other antibodies are coated on a 96-well plate at 4°C overnight. The next day, wash with PBST three times and pat dry, then block with 5% skim milk prepared in PBST (200 μl / well, block at room temperature for 1 hour). The cell supernatant collected in (2) is divided into 3 parts, and high temperature treatment (treatment above 50°C for 1 hour), low temperature treatment (long-term storage at 4°C or repeated freezing and thawing) and no treatment are performed respectively. After blocking, the 96-well plate is washed with PBST three times and pat dry. 100 μl of untreated and various stress-treated cell supernatant samples are added to each well and incubated at room temperature for 1-2 hours. After washing with PBST three times, 100 μl of Rabbit His-tag Antibody is added and incubated at room temperature for 1 hour. After washing 3 times with PBST, add 100 μl anti-rabbit IgG antibody (HRP) and incubate at room temperature for 1 hour. After washing 5 times with PBST, pat dry and add 100 μl freshly prepared TMB colorimetric solution to each well. After 10-15 minutes of color development, add stop solution to stop color development and read the absorbance at wavelengths of 450nm / 570nm with an enzyme reader. The final absorbance reading is A450nm-A570nm.

[0140] The method for evaluating stability is as follows: According to the ELISA test results, 1) on the one hand, the Pre-F ratio obtained by the RSV F protein designed by the present invention (hereinafter referred to as the candidate antigen protein) compared with the positive control 847 protein (the antigen protein used in Abrysvo, the RSV vaccine already marketed in the United States by Pfizer, as shown in SEQ ID NO: 84 in this application) is calculated. If the ratio is greater than 1, it means that the pre-F conformation ratio (% Pre-F, i.e. the ratio of Pre-F to the sum of Pre-F + Post-F) obtained by the candidate antigen protein is better than that of the 847 protein. If the Pre-F ratio is not less than 0.8 (at least not less than 0.75) compared with 847, it means that the candidate antigen protein is better. 2) On the other hand, the stability of the candidate antigen protein under high temperature and low temperature pressure conditions is calculated, wherein Mota protein and AM14 protein are used as antibodies to bind to the candidate antigen protein, respectively, to evaluate the overall stability of the candidate antigen protein and the stability of the Pre-F part. Based on the results of each round of screening, the present invention defines that if the corresponding value of protein stability after pressure treatment is not less than 0.6 (at least not less than 0.55), it means that the protein stability is relatively good.

[0141] (4) Scheme adjustment and improvement: Based on the results of the previous ELISA test and the antigens with excellent performance, select the better mutation types for the next round of screening.

[0142] According to the above research ideas, four rounds of RSV F mutant design and screening were carried out, as described in the following Examples 2 to 5.

[0143] Example 2: First round of RSV F mutant design and screening

[0144] The first round of mutant design and ELISA detection was carried out according to the operating steps described in Example 1. The main purpose of this round of experiments is to try various methods of disulfide bond mutation, cavity filling mutation, and electrostatic mutation, and test their combined effects through orthogonal experiments.

[0145] The design strategy of RSV F mutants is as follows: (1) For disulfide bond mutation, 8 designs were conducted (R106C+V144C, N104C+S146C, N104C+A147C, N104C+I148C, S / N105C+G145C, S / N105C+S146C, S / N105C+A147C, Q34C+G471C); (2) For proline mutation, S215P was used or A490P was further added on its basis; (3) Cavity filling In terms of mutation, four designs were carried out (S190V, S190L, S190W, S190I); (4) In terms of electrostatic mutation, five designs were carried out (D486N, D486Q, E487T, D489P, D489Q); (5) In terms of p27 sequence, no p27 sequence modification was performed, or T103-GS-G145 was adopted (deleting the fragment between amino acids 103~145 and introducing an additional linker GS); (6) In terms of trimerization domain: Foldon was used.

[0146] Based on the above-mentioned various types of modification methods, when they were combined to construct RSV F mutants, (1) on the one hand, fixed disulfide bond mutations (R106C+V144C), proline mutations (including S215P), p27 sequence modification (no modification) and trimerization domain (Foldon) were selected, and orthogonal design and screening of cavity filling mutations (S190I, S190V, S190L, S190W) and electrostatic mutations (D486N, D486Q, E487T, D489P, D489Q) were performed (BR01, BR06~BR23); (2) on the other hand, fixed proline mutations (including S215P), cavity filling mutations (S190I), electrostatic mutations (D486N, D486Q, E487T, D489P, D489Q) were performed. 486N), p27 sequence modification (without modification) and trimerization domain (Foldon) selection, disulfide bond mutation (N104C+S146C, N104C+A147C, N104C+I148C, S / N105C+G145C, S / N105C+S146C, S / N105C+A147C, Q34C+G471C) changes and comparisons (BR24~BR30); (3) In addition, p27 sequence modification (T103-GS-G145) was performed on the basis of BR30 to obtain BR31, and the two were compared to analyze the impact of p27 sequence modification on the technical effect.

[0147] The specific RSV F mutants designed in this round are shown in Table 1. In this table, the left column is the name of the mutant, and the same row lists the transformation of the mutant compared to the wild-type RSV F and its corresponding sequence number. The corresponding ELISA test results of each mutant are shown in Table 2.

[0148] Table 1: First round RSV F mutant design

[0149]

[0150] Remark:

[0151] (1) “ / ” indicates that this project has not been modified (the same below).

[0152] (2) "p27 sequence modification" column: " / " indicates that the p27 sequence in the wild-type RSV F protein has not been modified; "T103-GS-G145" means that the fragment between amino acids 103 to 145 of the wild-type RSV F protein (excluding the end values, i.e., amino acids 103 and 145, i.e., only the fragment between amino acids 104 to 144 is deleted) is deleted and replaced with GS, where GS serves as a linker.

[0153] (3) Regarding S / N105C+G145C: “S / N105” means that the amino acid position 105 in the wild-type RSV F protein can be S or N (S is the most common, followed by N), and “S / N105C” means that the position mutated from the wild-type S / N to C.

[0154] Table 2: Results of the first round of ELISA testing of RSV F mutants

[0155]

[0156] Remark:

[0157] 1 The calculation formula for "Pre-F ratio" is: %Pre-F=OD AM14 / OD Mota , the same below;

[0158] 2 "Pre-F ratio compared to 847" means the ratio of the Pre-F protein of each mutant to the positive control 847. For example, the calculation formula of the Pre-F ratio of BR06 compared to 847 is: (% Pre-F BR06 ) / (%Pre-F 847 ), the same below;

[0159] 3 "Protein stability (Mota)" refers to the change in the OD value of the Mota antibody in the sample before and after stress treatment. The specific calculation formula is: (Mota OD 处理后 ) / (Mota OD 处理前 ), the same below;

[0160] 4 "Protein stability (AM14)" refers to the change in OD value of the AM14 antibody sample before and after stress treatment. The specific calculation formula is: (AM14 OD 处理后 ) / (AM14 OD 处理前 ), the same below.

[0161] According to the results of the first round of screening, all antigen proteins can be expressed normally and can be screened by ELISA method. The first round of screening is mainly to preliminarily explore the combination effect of each design strategy:

[0162] In terms of disulfide bond mutations, R106C+V144C (corresponding to BR01, BR06~BR23), N104C+S146C (corresponding to BR24), N104C+I148C (corresponding to BR26), S / N105C+S146C (corresponding to BR28) and S / N105C+A147C (corresponding to BR29) can help stabilize the Pre-F protein conformation, while N104C+A147C (corresponding to BR25), S / N105C+G145C (corresponding to BR27) and Q34C+G471C (corresponding to BR30, BR31) performed generally. Since N104C+I148C and S / N105C+A147C are new disulfide bonds that have not been reported and have excellent performance, they were selected for the next round of screening.

[0163] In terms of cavity filling, the proportion of Pre-F conformation after adding S190V and S190L mutations was generally higher than that of S190W mutation, so they were selected to enter the next round of screening.

[0164] Regarding electrostatic mutations, adding mutations such as D486N, D486Q, and E487T stabilized the Pre-F conformation better than other mutations, and they were selected for the next round of antigen screening.

[0165] Example 3: Second round of RSV F mutant design and screening

[0166] The main purpose of this round of experiments is to test the combined effect of the selected disulfide bond mutations, cavity filling mutations, and electrostatic mutations; at the same time, the pressure conditions were increased in this round of experiments (the high temperature was increased from 50°C to 60°C) to test the thermal stability of the constructed RSV F mutants, and BR26 and BR29, which performed well in the previous round of screening, were also included in this round of pressure condition testing.

[0167] The RSV F mutant design strategy is as follows: Based on the results of the previous example, in this round of RSV F mutant design, (1) in terms of disulfide bond mutation, N104C+I148C and S / N105C+A147C were selected for this round of screening, and D486C+E487C were introduced; (2) in terms of proline mutation, S215P was still used; (3) in terms of cavity filling mutation, S190V or S190L was selected; (4) in terms of electrostatic mutation, D486N, D486Q or E487T was selected; (5) in terms of p27 sequence modification, T103-GS-G145 was still used, or no p27 sequence modification was performed; (6) in terms of trimerization domain, Foldon was still used.

[0168] The specific RSV F mutants designed in this round are shown in Table 3, in which the left column is the name of the mutant, and the same row lists the transformation of the mutant compared to the wild-type RSV F and its corresponding sequence number. The corresponding ELISA test results of each mutant are shown in Table 4.

[0169] Table 3: Second round RSV F mutant design

[0170]

[0171] Table 4: Second round of ELISA test results of RSV F mutants

[0172]

[0173] In the second round of screening, we increased the high temperature treatment pressure (from 50°C to 60°C) in the hope of screening out better antigen designs. The results showed:

[0174] (I) After high-temperature treatment at 60°C, the protein stability of most mutants (AM14) decreased significantly, while the Pre-F conformation of RSV F mutants (BR44~BR46) with added disulfide bond mutations D486C+E487C did not change significantly, especially BR46 and BR47, so the disulfide bond mutations D486C+E487C were considered to be retained in subsequent screening.

[0175] (ii) Relatively speaking, the contribution of disulfide bond mutations N104C+I148C or S / N105C+A147C to the stability of RSV F mutants is not as good as D486C+E487C. However, considering that the extraction, purification and storage of RSV F mutants generally do not involve high temperatures above 50°C, N104C+I148C or S / N105C+A147C are also available disulfide bond mutations under non-extreme temperature conditions. (1) For N104C+I148C, by comparing BR26, BR32~BR37, it can be seen that when N104C+I148C is used in combination with S190I and D486N, the technical effect of this mutation combination is not much different from that of S / N105C+A147C and other cavity-filling mutations (such as S190L / V) and electrostatic mutations (D486Q / E487T), but the protein stability (AM14) is slightly higher. (2) Similarly, for S / N105C+A147C, by comparing BR29, BR38~BR43, it can be seen that when S / N105C+A147C is used in combination with S190I and D486N, the technical effect of this mutation combination is not much different from that of S / N105C+A147C and other cavity-filling mutations (such as S190L / V) and electrostatic mutations (D486Q / E487T), but the protein stability (AM14) is slightly higher.

[0176] (III) At the same time, the results of this round of screening showed that p27 sequence modification (BR46 and BR47) can significantly improve the stability of Pre-F compared with no modification (BR44 and BR45), so p27 sequence modification is considered in subsequent screening. Among cavity filling mutations, S190L and S190V can help stabilize the Pre-F conformation, and S190L, which has a slightly better effect after p27 sequence modification, is selected for the next round of screening. Among electrostatic mutations, D486N, D486Q and E487T all performed well, and E487T, which has a slightly better effect, was selected to enter the next round of antigen screening.

[0177] Example 4: Third round of RSV F mutant design and screening

[0178] The main purpose of this round of experiments is to expand the modification methods of p27 sequence and trimerization domain, and to test their combined effects with disulfide bond mutations, cavity filling mutations, and electrostatic mutations screened out based on the results of the previous round; at the same time, in this round of testing, we further increased the pressure conditions (high temperature increased from 60°C to 65°C, and low temperature storage changed from 4°C to repeated freeze-thaw treatment), hoping to screen out antigen designs that can better withstand extreme conditions, and also include BR47, which performed relatively well in the previous round of screening, into this round of pressure condition testing.

[0179] The RSV F mutant design strategy is as follows: Based on the results of the previous example, in this round of RSV F mutant design, (1) in terms of disulfide bond mutation, D486C+E487C was selected to enter this round of screening, or disulfide bond mutation was not introduced; (2) in terms of proline mutation, S215P was still used, or proline mutation was not introduced; (3) in terms of cavity filling mutation, S190L was selected; (4) in terms of electrostatic mutation, E487T was selected, or electrostatic mutation was not introduced; (5) in terms of p27 sequence modification, in addition to T103-GS-G145 tested in the first and second rounds, another four modifications were added (6) In terms of trimerization domain, in addition to the previously tested Foldon, two options were added: ABCD-tag (CCHNVNACCSTTNICCTTTNICCTT) and ABCH-tag (CCHNVNACCSTTNICCTTIIICCIV), or no trimerization domain was introduced.

[0180] The specific RSV F mutants designed in this round are shown in Table 5, in which the left column is the name of the mutant, and the same row lists the transformation of the mutant compared to the wild-type RSV F and its corresponding sequence number. The corresponding ELISA test results of each mutant are shown in Table 6.

[0181] Table 5: Third round RSV F mutant design

[0182]

[0183] Remark:

[0184] p27 sequence modification column, 1) "S / N105-A147" means deleting the fragment between amino acids 105 and 147 of the wild-type RSV F protein (excluding the end values, i.e., amino acids 105 and 147, i.e., only deleting the fragment between amino acids 106 and 146). Similarly, "N104-I148" means deleting the fragment between amino acids 104 and 148 (excluding the end values). 2) "T103-GS-G145" means deleting the fragment between amino acids 103 and 145 of the wild-type RSV F protein (excluding the end values) and replacing it with GS. Similarly, "N104-GS-I148" means deleting the fragment between amino acids 104 and 148 of the wild-type RSV F protein (excluding the end values) and replacing it with GS. "S / N105-GS-A147" means deleting the fragment between amino acids 105 and 147 of the wild-type RSV F protein (excluding the end values) and replacing it with GS.

[0185] Table 6: Results of the third round of ELISA detection of RSV F mutants

[0186]

[0187] The third round of screening results showed:

[0188] In terms of p27 sequence modification, different modification methods have a significant effect on the stability of Pre-F conformation: among the five modification methods, S / N105-A147 has the best effect. As shown in Table 6, when no disulfide bond mutation is added: different p27 sequence modification schemes (i.e., antigen designs BR49, BR52, BR53, BR54 and BR55) are compared, among which BR54 (p27 sequence modified to S / N105-A147) is better than other designs after high temperature pressure treatment, and there is no significant difference with other designs after low temperature pressure treatment; similarly, when disulfide bond mutation is added: different p27 sequence modification schemes (i.e., antigen designs BR47, BR56, BR57, BR58 and BR59) are compared, among which BR58 (p27 sequence modified to S / N105-A147) is better than other designs in high temperature pressure treatment, and there is no significant difference with other designs after low temperature pressure treatment. Therefore, S / N105-A147 was selected to enter the next round of screening.

[0189] In addition, it is worth mentioning that although BR50 (D486C+E487C, S215P, S190L, T103-GS-G145) did not undergo trimerization domain modification, it was slightly weaker than BR47 (D486C+E487C, S215P, S190L, T103-GS-G145, Foldon) in terms of pre-F ratio and structural stability, but still performed well. It is speculated that the D486C+E487C disulfide bond near the C-terminus of the RSV F protein helps the protein trimerization to a certain extent. Therefore, when using the D486C+E487C disulfide bond mutation, it is not necessary to modify the trimerization domain.

[0190] Example 5: Fourth round of RSV F mutant design and screening

[0191] The main purpose of this round of experiments is to expand the modification methods of proline mutation, and the secondary purpose is to make more attempts in disulfide bond mutation and test its combined effect with cavity filling mutation, electrostatic mutation and p27 sequence modification screened out based on the results of the previous round.

[0192] The design strategy of RSV F mutants is as follows: Based on the results of the previous example, in this round of RSV F mutant design, (1) in terms of disulfide bond mutation, D486C+E487C is still the main one. In addition, L171C+K191C, E60C+K196C, S / N105C+M370C are introduced alone or in combination with D486C+E487C. The purpose of introducing these new disulfide bonds is to replace proline mutations and obtain a more stable Pre-F conformation; (2) in terms of proline mutation, in addition to the previous S215P, multiple possibilities (Q210P, S211P, C212P, S213P, I214P, N216P, I217P, E218P, Q219P, etc.) are tested. 10P+C212P, S211P+S213P, C212P+I214P, I214P+N216P, N216P+E218P, S213G+I214P+N216G, S211G+C212P+I214G) were used to compare their effects; (3) For cavity filling mutation, S190L was still used or no cavity filling mutation was introduced; (4) For electrostatic mutation, E487T was still used or no electrostatic mutation was introduced; (5) For p27 sequence modification, S / N105-A147 was uniformly used; (6) For trimerization domain, Foldon was uniformly used.

[0193] The specific RSV F mutants designed in this round are shown in Table 7, in which the left column is the name of the mutant, and the same row lists the transformation of the mutant compared to the wild-type RSV F and its corresponding sequence number. The corresponding ELISA test results of each mutant are shown in Table 8.

[0194] Table 7: Fourth round RSV F mutant design

[0195]

[0196] Table 8: Results of ELISA test of RSV F mutants in the fourth round

[0197]

[0198] The fourth round of screening results showed:

[0199] In terms of proline mutations, Q210P, S211P and S211P+S213P can all significantly improve the conformational stability of Pre-F protein.

[0200] In terms of disulfide bond mutations, the comparison of ELISA test results of BR79, BR80, BR81, BR82 and BR83 shows that: (1) BR83 performed relatively well, showing that S / N105C+M370C can help stabilize the Pre-F conformation in the absence of rigid amino acid mutations (proline mutations). (2) In comparison, although the data of BR80 are better on the surface (for example, the Pre-F ratio is as high as 2.18 compared with 847 protein), in fact, the expression level of this mutant protein is extremely low (for example, after treatment at 65℃ for 1h, OD Mota =0.111,OD AM14 =0.136, it can be seen that the OD value of the detection antibody is very low, although %Pre-F=OD AM14 / OD Mota =1.22 and the "Pre-F ratio compared to 847" is as high as 2.18), so the disulfide bond pair E60C+K196C is not preferred. (3) In addition, the Pre-F ratios of BR79 and BR81 are lower than those of the 847 protein, so the disulfide bond pair L171C+K191C is also not preferred.

[0201] After completing four rounds of screening, mutants with better overall performance (BR47, BR54, BR58, BR64, BR65, and BR73) were selected as representatives and then tested with other neutralizing antibodies by ELISA to further confirm the conformational stability of the designed antigen and its potential to induce multiple neutralizing antibodies. Figure 2 As shown, multiple mutants can bind to different strong neutralizing antibodies, and their Pre-F conformations are relatively stable.

[0202] Example 6: Mass Expression and Purification of Antigen Protein

[0203] The recombinant baculovirus expressing the antigen protein was used to infect 1L of HighFive cells in the logarithmic growth phase (the viable cell density was about 2×10 6 cells / ml, viability greater than 95%). The infected cells were placed at 27°C and cultured at 120 rpm for 3-4 days. When the cell diameter increased significantly and the viable cell density and cell viability decreased significantly, the cell supernatant was collected by centrifugation at 4000 rpm for 20 minutes. The collected supernatant was filtered through a 0.22 μm filter membrane and then purified by a nickel column. The specific steps of purification are:

[0204] Equilibration: Equilibrate a nickel column (UniNTA-80Ni) with binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Wash the nickel column with 10 column volumes of binding buffer.

[0205] Loading: Slowly add the filtered sample to the equilibrated nickel column and flow through the sample 3 times to allow the target protein to fully bind to the nickel column.

[0206] Wash: Use 10-20 column volumes of wash buffer (50 mM NaH2PO4, 300 mM NaCl, 20-50 mM imidazole, pH 8.0) to wash away non-specifically bound proteins.

[0207] Elution: Elute the bound target protein using elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250-500 mM imidazole, pH 8.0). Collect the eluate and divide it into 1 mL fractions.

[0208] The collected eluate was placed in a SnakeSkin dialysis bag (Thermo Scientific™), and the salt ions in the eluate were replaced by PBS dialysis solution. After dialysis, the solution in the dialysis bag was collected and stored in a -80°C refrigerator. At the same time, a small amount of sample was taken for reducing and non-reducing electrophoresis, as well as Western blot analysis (primary antibody: D25 antibody, secondary antibody: Goat Anti-Human IgG-Fc Secondary Antibody HRP).

[0209] The results are as follows Figure 3 and Figure 4 As shown in the figure, the molecular weight of monomers and trimers of each antigen protein expressed by the insect cell-baculovirus expression system is in line with expectations, and Western blot detection using specific antibody D25 confirms that the protein band is RSV F protein. The purity of each mutant antigen is in line with expectations and can be used for subsequent experiments.

[0210] Example 7: Animal Experiment

[0211] Female Balb / C mice aged 6-8 weeks were randomly divided into groups, with 5 mice in each group. Different doses of antigen protein purified by insect cell expression or antigen protein plus aluminum adjuvant were injected intramuscularly on days 0 and 21, 50 μl in each leg, for a total of 100 μl. The negative control group was immunized with 100 μl of saline. Blood was collected from mice on days 14 and 35, and serum was separated for binding antibody detection as follows:

[0212] 100 μl of purified antigen protein was coated into a 96-well plate. The next day, the plate was washed with PBST for 3 times and then patted dry. The plate was then blocked with 5% skim milk prepared with PBST (200 μl / well, blocked for 1 hour at room temperature). Wash with PBST for 3 times and patted dry. Serum samples were serially diluted 2-fold using PBST solution containing 1% skim milk. 1:100 was used as the first detection point. 100 μl was added to each well and incubated for 1 hour at room temperature. Wash with PBST for 3 times and then 100 μl Goat Anti-Mouse IgG-HRP antibody was added. The plate was incubated for 1 hour at room temperature. Wash with PBST for 5 times and then patted dry. 100 μl of freshly prepared TMB colorimetric solution was added to each well. After 10-15 minutes of color development, the stop solution was added and the absorbance at a wavelength of 450 nm was read using an ELISA reader. The cut-off value was 2.1 times the absorbance of the negative control. The maximum dilution factor when the sample absorbance value was ≥ the cut-off value was the binding antibody titer of the sample.

[0213] The results are as follows Figure 5 and Figure 6 As shown: In the dose and adjuvant exploration experiment ( Figure 5 ), immunization with different doses of antigen protein BR47 stock solution or vaccine combined with aluminum adjuvant can induce mice to produce high titer binding antibodies, showing a high level of immunogenicity. In the experiment comparing different antigen designs ( Figure 6 ), multiple antigen proteins obtained through in vitro screening can induce mice to produce high-titer binding antibodies, proving that the established in vitro screening method can be used for the screening of RSV F antigen proteins, and the screened antigens have the potential to develop excellent RSV vaccines.

[0214] The in-depth analysis and detailed description of the embodiments of the present invention are not intended to limit the scope of application of the present invention, but are only used as representatives to illustrate the process, results and application of the present invention. According to the embodiments of the invention described this time, all other implementation methods that can be derived or obtained by equivalent substitution by specific technicians in the same field without creative efforts are included in the scope of protection of the present invention.

Claims

1. A RSV F protein mutant having multiple modifications relative to the wild-type RSV F protein to stabilize it in a pre-fusion conformation, the modifications comprising: (a) Proline mutation: S215P; (b) Cavity-filling mutation: selected from S190V, S190L or S190I; (c) electrostatic mutation: selected from E487T, D486N or D486Q; in, Position numbering is based on the sequence shown in SEQ ID NO:

1.

2. The RSV F protein mutant of claim 1, characterized in that The modifications do not comprise engineered disulfide bond mutations.

3. The RSV F protein mutant according to any one of claims 1 to 2, characterized in that The cavity filling mutation is selected from S190V or S190L.

4. The RSV F protein mutant of claim 3, characterized in that The cavity filling mutation: S190L was selected.

5. The RSV F protein mutant according to any one of claims 1 to 2, characterized in that The electrostatic mutation: E487T was selected.

6. The RSV F protein mutant according to any one of claims 1 to 2, characterized in that The modifications include: S215P, S190L, E487T.

7. The RSV F protein mutant according to any one of claims 1 to 2, characterized in that The modification further comprises p27 sequence modification, namely, deletion of the p27 sequence.

8. The RSV F protein mutant of claim 7, characterized in that The p27 sequence modification adopts one of the following methods: (1) Deleting the p27-containing fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) Delete the fragment containing p27 and introduce a linker sequence: Use a linker to connect the sequences before and after the deleted fragment.

9. The RSV F protein mutant of claim 8, characterized in that The p27 sequence modification is selected from one of the following: T103-GS-G145, N104-I148, N104-GS-I148, S / N105-A147, S / N105-GS-A147.

10. The RSV F protein mutant of claim 9, characterized in that The p27 sequence modification is selected from one of the following: T103-GS-G145, N104-GS-I148, S / N105-A147.

11. The RSV F protein mutant of claim 10, characterized in that The p27 sequence modification selected S / N105-A147.

12. The RSV F protein mutant according to any one of claims 1 to 2, characterized in that The modification also includes the modification of the trimerization domain: deleting the transmembrane region and the intracellular region and adding a sequence containing the trimerization domain.

13. The RSV F protein mutant of claim 12, characterized in that The trimerization domain is selected from Foldon, ABCD-tag or ABCH-tag, which have the sequences shown in SEQ ID NO: 2, 3, 4, respectively.

14. The RSV F protein mutant according to any one of claims 1 to 2, characterized in that The modification comprises or consists of any of the following: Group (1): S215P, S190L, E487T, T103-GS-G145, Foldon; Group (2): S215P, S190L, E487T, N104-GS-I148, Foldon; Group (3): S215P, S190L, E487T, S / N105-A147, Foldon; Group (4): S215P, S190L, E487T, S / N105-GS-A147, Foldon.

15. The RSV F protein mutant of claim 14, characterized in that The modification comprises or consists of group (3).

16. The RSV F protein mutant of claim 14, characterized in that The amino acid sequence of the RSV F protein mutant comprises or is a sequence that is more than 99% identical to SEQ ID NO:51, 53, 54 or 55.

17. The RSV F protein mutant of claim 16, characterized in that The amino acid sequence of the RSV F protein mutant comprises or is the sequence shown in SEQ ID NO:51, 53, 54 or 55.

18. A RSV F protein mutant having multiple modifications relative to the wild-type RSV F protein to stabilize it in a prefusion conformation, the modifications comprising: (a) Engineered disulfide bond mutation: D486C+E487C; (b) Proline mutation: selected from S215P, Q210P, S211P or S211P+S213P; (c) Cavity-filling mutation: selected from S190V or S190L; in, Position numbering is based on the sequence shown in SEQ ID NO:

1.

19. The RSV F protein mutant of claim 18, characterized in that The modifications did not include other electrostatic mutations.

20. The RSV F protein mutant according to any one of claims 18 to 19, characterized in that The modification further comprises p27 sequence modification, namely, deletion of the p27 sequence.

21. The RSV F protein mutant of claim 20, characterized in that The p27 sequence modification adopts one of the following methods: (1) Deleting the p27-containing fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) Delete the fragment containing p27 and introduce a linker sequence: Use a linker to connect the sequences before and after the deleted fragment.

22. The RSV F protein mutant of claim 21, characterized in that The p27 sequence modification is selected from one of the following: T103-GS-G145, N104-I148, N104-GS-I148, S / N105-A147, S / N105-GS-A147.

23. The RSV F protein mutant of claim 22, characterized in that The p27 sequence modification is selected from one of the following: T103-GS-G145, S / N105-A147.

24. The RSV F protein mutant according to any one of claims 18 to 19, characterized in that The modification also includes the modification of the trimerization domain: deleting the transmembrane region and the intracellular region and adding a sequence containing the trimerization domain.

25. The RSV F protein mutant of claim 24, characterized in that The trimerization domain is selected from Foldon, ABCD-tag or ABCH-tag, which have the sequences shown in SEQ ID NO: 2, 3, 4, respectively.

26. The RSV F protein mutant according to any one of claims 18 to 19, characterized in that The modification comprises or consists of any of the following: Group (1): D486C+E487C, S215P, S190V, T103-GS-G145, Foldon; Group (2): D486C+E487C, S215P, S190L, T103-GS-G145, Foldon; Group (3): D486C+E487C, S215P, S190L, T103-GS-G145; Group (4): D486C+E487C, S215P, S190L, S / N105-A147, Foldon; Group (5): D486C+E487C, Q210P, S190L, S / N105-A147, Foldon; Group (6): D486C+E487C, S211P, S190L, S / N105-A147, Foldon; Group (7): D486C+E487C, S211P+S213P, S190L, S / N105-A147, Foldon.

27. The RSV F protein mutant of claim 26, characterized in that The modification is selected from: group (2), group (4), group (5) or group (6).

28. The RSV F protein mutant of claim 26, characterized in that The amino acid sequence of the RSV F protein mutant comprises or is a sequence that is more than 99% identical to SEQ ID NO: 46, 47, 50, 58, 64, 65 or 73.

29. The RSV F protein mutant of claim 28, characterized in that The amino acid sequence of the RSV F protein mutant comprises or is the sequence shown in SEQ ID NO: 46, 47, 50, 58, 64, 65 or 73.

30. A RSV F protein mutant having multiple modifications relative to a wild-type RSV F protein to stabilize it in a prefusion conformation, the modifications comprising: (a) Engineered disulfide bond mutations selected from R106C+V144C, N104C+S146C, N104C+I148C, S / N105C+A146C, S / N105C+A147C or S / N105C+M370C; (b) Proline mutation: S215P; (c) cavity-filling mutation: selected from S190I, S190L, S190V or S190W; (d) an electrostatic mutation selected from D486N, D486Q, E487T, D489P or D489Q; in, Position numbering is based on the sequence shown in SEQ ID NO:

1.

31. The RSV F protein mutant of claim 30, characterized in that: The disulfide bond mutation selected is R106C+V144C; the proline mutation selected is S215P; the cavity filling mutation selected is S190I, S190L, S190V or S190W; the electrostatic mutation selected is D486N, D486Q, E487T, D489P or D489Q.

32. The RSV F protein mutant of claim 31, characterized in that The electrostatic mutation is selected from D486N, D486Q, E487T or D489P.

33. The RSV F protein mutant of claim 30, characterized in that: The disulfide bond mutation is selected from N104C+S146C, N104C+I148C, S / N105C+A146C or S / N105C+A147C; the proline mutation is selected from S215P; the cavity filling mutation is selected from S190I, S190L or S190V; the electrostatic mutation is selected from D486N, D486Q or E487T.

34. The RSV F protein mutant of claim 33, characterized in that The cavity-filling mutation was S190I, and the electrostatic mutation was D486N.

35. The RSV F protein mutant of any one of claims 30-34, characterized in that The modification may or may not include p27 sequence modification, wherein the p27 sequence modification refers to deletion of the p27 sequence.

36. The RSV F protein mutant of claim 35, characterized in that The p27 sequence modification adopts one of the following methods: (1) Deleting the p27-containing fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) Delete the fragment containing p27 and introduce a linker sequence: Use a linker to connect the sequences before and after the deleted fragment.

37. The RSV F protein mutant of claim 36, characterized in that The p27 sequence modification is selected from one of the following: T103-GS-G145, N104-I148, N104-GS-I148, S / N105-A147, S / N105-GS-A147.

38. The RSV F protein mutant of any one of claims 30-34, characterized in that The modification also includes the modification of the trimerization domain: deleting the transmembrane region and the intracellular region and adding a sequence containing the trimerization domain.

39. The RSV F protein mutant of claim 38, characterized in that The trimerization domain is selected from Foldon, ABCD-tag or ABCH-tag, which have the sequences shown in SEQ ID NO: 2, 3, 4, respectively.

40. The RSV F protein mutant of claim 30, characterized in that The modification comprises or consists of any of the following: (1) R106C+V144C, S215P, S190I, D486N, Foldon; (2) R106C+V144C, S215P, S190V, D486N, Foldon; (3) R106C+V144C, S215P, S190L, D486N, Foldon; (4) R106C+V144C, S215P, S190W, D486N, Foldon; (5) R106C+V144C, S215P, S190V, D486Q, Foldon; (6) R106C+V144C, S215P, S190V, E487T, Foldon; (7) R106C+V144C, S215P, S190L, D486Q, Foldon; (8) R106C+V144C, S215P, S190L, E487T, Foldon; (9) R106C+V144C, S215P, S190L, D489P, Foldon; (10) R106C+V144C, S215P, S190W, D486Q, Foldon; (11) R106C+V144C, S215P, S190W, E487T, Foldon; (12) N104C+S146C, S215P, S190I, D486N, Foldon; (13) N104C+I148C, S215P, S190I, D486N, Foldon; (14) S / N105C+S146C, S215P, S190I, D486N, Foldon; (15) S / N105C+A147C, S215P, S190I, D486N, Foldon; (16) S / N105C+M370C, S190L, E487T, S / N105-A147, Foldon.

41. The RSV F protein mutant of claim 40, characterized in that The amino acid sequence of the RSV F protein mutant comprises or is a sequence that is more than 99% identical to SEQ ID NO: 5, 6, 7, 8, 9, 10, 14, 15, 16, 19, 20, 24, 26, 28, 29 or 83.

42. The RSV F protein mutant of claim 41, characterized in that The amino acid sequence of the RSV F protein mutant comprises or is the sequence shown in SEQ ID NO: 5, 6, 7, 8, 9, 10, 14, 15, 16, 19, 20, 24, 26, 28, 29 or 83.

43. A nucleic acid molecule encoding the RSV F protein mutant of any one of claims 1-42. A recombinant vector comprising the nucleic acid molecule of claim 43 .

45. The recombinant vector according to claim 44, characterized in that The original plasmid of the recombinant vector is pOET1.

1.

46. ​​A recombinant virus comprising the nucleic acid molecule of claim 43 or the recombinant vector of any one of claims 44-45.

47. The recombinant virus according to claim 46, characterized in that The recombinant virus is selected from insect cell baculovirus, adenovirus, adeno-associated virus, vaccinia virus, herpes virus or retrovirus.

48. A transformed cell comprising one of the following: (1) The nucleic acid molecule according to claim 43; (2) The recombinant vector according to any one of claims 44 to 45; (3) The recombinant virus according to any one of claims 46 to 47.

49. The transformed cell according to claim 48, characterized in that The host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells.

50. The transformed cell according to claim 49, characterized in that The insect cells are Sf9 cells, Sf21 cells or High-Five cells.

51. An immunogenic composition comprising one of the following: (1) The RSV F protein mutant according to any one of claims 1 to 42; (2) The nucleic acid molecule according to claim 43; (3) The recombinant vector according to any one of claims 44 to 45; (4) The recombinant virus according to any one of claims 46 to 47; (5) The transformed cell according to any one of claims 48 to 50.

52. A method for preparing a RSV F protein mutant according to any one of claims 1 to 42, comprising the following steps: Construction of recombinant vectors, extraction of recombinant vectors, transfection of recombinant vectors into host cells to express proteins and purification of expression products.

53. Application of any of the following (1) to (6): (1) The RSV F protein mutant according to any one of claims 1 to 42; (2) The nucleic acid molecule according to claim 43; (3) The recombinant vector according to any one of claims 44 to 45; (4) The recombinant virus according to any one of claims 46 to 47; (5) The transformed cell according to any one of claims 48 to 50; (6) The immunogenic composition of claim 51; The application is selected from any one of the following (a) to (c): (a) preparing RSV-specific antibodies; (b) preparing a medicament for preventing and / or treating RSV infection; (c) Preparation of diagnostic reagents for RSV.

54. The use according to claim 53, characterized in that The medicine is a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine for preventing and / or treating RSV.

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