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

[0001] This application is a divisional application of the patent application with the application number 202510136318.0 filed on February 7, 2025. Technical Field

[0002] The present invention relates to RSV F protein mutants and their applications, belonging to the field of vaccines. Background Art

[0003] Respiratory syncytial virus (RSV) is a single-stranded negative-sense RNA virus belonging to the Paramyxoviridae family, with two subtypes, A and B. RSV is transmitted by direct contact. After infection, it can cause symptoms such as bronchitis, pneumonia, asthma, and respiratory failure. RSV is the leading pathogen causing respiratory tract infections in children globally and can also cause severe respiratory tract and lung infections in infants, the elderly, and immunocompromised populations. In 2019 alone, there were approximately 3.6 million hospitalizations for RSV-related acute lower respiratory tract infections globally, approximately 26,300 in-hospital deaths due to RSV-related acute lower respiratory tract infections, and approximately 11,010 deaths in children aged 0 to 60 months related to RSV. In China, RSV is the top pathogen causing respiratory tract infections in children (accounting for 25.7%) and the second leading pathogen causing respiratory tract infections in the entire population (accounting for 16.8%). In 2019, RSV caused 3.5 million acute lower respiratory tract infections in children under 5 years old in China and 620,000 to 950,000 hospitalizations for children under 5 years old. Currently, there are two commercially available humanized monoclonal antibodies (Palivizumab and Nirsevimab) abroad for helping to prevent RSV infection, but the protection formed by this passive immunity has a short duration, requires multiple injections, and the price of monoclonal antibodies is very expensive. Regarding prophylactic vaccines, three RSV vaccines have been approved for marketing abroad, including Arexvy from GSK, Abrysvo from Pfizer, and mRESVIA from Modena, but there is no commercially available RSV prophylactic vaccine in China. Therefore, developing safe, efficient, and affordable vaccines is of great significance for preventing and controlling RSV infection.

[0004] The RSV genome is 15.2 kb in length and encodes 11 proteins. Among them, the attachment protein (G) and the fusion protein (F) are the main glycoproteins on the viral membrane surface and are also the key to the virus invading the human body. Therefore, they are the main antigenic proteins in vaccine research. The G protein helps the virus particles recognize and attach to the receptors on the surface of host cells. The F protein can promote the fusion between the virus particles and the host cell membrane, enabling the viral nucleocapsid to enter the host cell. During the fusion with the host cell membrane, the F protein changes from the metastable pre-fusion conformation (Pre-F) to the stable post-fusion conformation (Post-F). Compared with the G protein, the F protein is more conserved in RSV and is a good antigenic protein for vaccine development. The F protein precursor F0 contains 574 amino acids, and a 27-amino acid short peptide (p27) between amino acid residues 110-136 is hydrolyzed and released by furin protease, forming two subunits, F1 (137-574aa) and F2 (26-109aa). The two subunits are connected by disulfide bonds, and three F1+F2 subunits form the mature F protein trimer structure. The F1 subunit contains a fusion peptide (FP) responsible for inserting into the adjacent host cell membrane, as well as heptad repeat A (HRA) and heptad repeat B (HRB). HRA and HRB are separated in Pre-F. During membrane fusion, HRB docks with HRA to form a stable hexagonal helical structure, generating a stable Post-F. The C-terminus of the F1 subunit also contains a transmembrane region (525-550aa) and an intracellular region (551-574aa).

[0005] In recent years, scientists have used structural biology and other means to analyze the process of the F protein from the Pre-F structure to the Post-F structure, and have discovered antigenic epitopes related to neutralizing activity. Among them, Site Ⅰ, Ⅱ, and Ⅳ coexist in both the Pre-F and Post-F conformations and can be recognized by antibodies with neutralizing activity, such as antibodies like 131-2a (Site Ⅰ), Motavizumab (Site II), and 101F / mAb19 (Site IV). At the same time, scientists have discovered neutralizing antibody epitopes unique to the Pre-F structure, including Site Ø, Site V, and the antigenic epitope across 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 higher titers of potent neutralizing antibodies compared to the Post-F structure, which is the key to developing RSV vaccines. Currently, foreign RSV vaccines based on the Pre-F structure have achieved high protection rates in clinical trials and have been successfully marketed. The team at the National Institute of Allergy and Infectious Diseases (NIAID) in the United States designed a protein mutant called "DS-Cav1" that forms disulfide bonds (S155C, S290C) through site-directed mutagenesis and fills structural cavities (S190F, V207L) to stabilize the Pre-F structure (Jason S. McLellan, et al., Science, 2013). The "847" protein mutant designed by Pfizer forms disulfide bonds (T103C, I148C), fills structural cavities (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 also obtained a relatively stable Pre-F structure through rigid amino acid mutations (S215P), cavity filling (N67I), and electrostatic mutations (E487Q) (Anders Krarup, et al., Nature communication, 2015). Currently, no RSV vaccine based on the Pre-F structure has been successfully marketed in China. At the same time, the current strategies for constructing the Pre-F structure also have problems such as low protein expression levels and insufficient structural stability. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the present invention provides various mutants of wild-type RSV F protein. By mutating the amino acid sequence of wild-type RSV F protein, the mutations include substitution, deletion or addition of amino acids, and the mutant can improve the protein expression level and stability of the Pre-F structure, which is helpful for the prevention, diagnosis and treatment of RSV infection.

[0007] To solve the above technical problems, the specific technical solutions of the present invention are as follows:

[0008] In the first aspect, the present invention provides a mutant of wild-type RSV F protein, and the mutant has at least one of the following modifications relative to the wild-type RSV F protein:

[0009] (a) At least one engineered disulfide bond mutation;

[0010] (b) At least one cavity filling mutation;

[0011] (c) At least one electrostatic mutation;

[0012] (d) At least one proline mutation;

[0013] (e) Modification of the p27 sequence: deleting the sequence containing p27;

[0014] (f) Modification of the trimerization domain: deleting the transmembrane region and the intracellular region and adding an amino acid sequence containing the trimerization domain.

[0015] 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.

[0016] Engineering-modified disulfide bond mutations refer to the mutation of at least one pair of amino acid residues in the wild-type RSV F protein into a pair of cysteine residues (Cys, C) to form a disulfide bond between them. The residues mutated to cysteine are close in the pre-fusion conformation (pre F) of the RSV F protein but far apart in the post-fusion conformation (Post F). Therefore, the formation of a disulfide bond between the introduced pair of cysteine residues can stabilize the pre-fusion conformation (pre F) of the RSV F protein. In an alternative embodiment, the at least one modification comprises the substitution of at least one pair of amino acid residues in the F1 subunit and / or F2 subunit of the RSV F protein with cysteine (Cys, C) to form an engineered disulfide bond. In an alternative embodiment, the engineered disulfide bond mutation comprises at least one pair among 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 in the amino acid sequence of the wild-type RSV F protein. Preferably, the disulfide bond mutation comprises at least one pair among R106C+V144C, N104C+I148C, N105C+A147C, D486C+E487C, E60C+K196C, and S / N105C+M370C.

[0017] The target substitute 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). By replacing these amino acids with large aliphatic amino acids (Ile, Leu, and Met) or large aromatic amino acids (His, Phe, Tyr, and Trp) with larger side chains, the structural cavity is filled to stabilize the Pre-F conformation. In an alternative embodiment, the cavity filling mutation is the mutation of the 190th amino acid in the amino acid sequence of the wild-type RSV F protein to isoleucine (Ile, I), valine (Val, V), leucine (Leu, L), and tryptophan (Trp, W). Preferably, the cavity filling mutation comprises the mutation of position 190S to any one of I, V, L, and W.

[0018] Electrostatic mutations utilize amino acid mutations to reduce the ionic repulsion between residues close to each other in the protein folding structure. In an alternative embodiment, the electrostatic mutation comprises the mutation of the 486th, 487th, or 489th amino acid in the amino acid sequence of the wild-type RSV F protein to asparagine (Asn, N), glutamine (Gln, Q), threonine (Thr, T), or proline (Pro, P). Preferably, the electrostatic mutation comprises the mutation of position 486D to N or Q, the mutation of position 487E to T, and the mutation of position 489D to P or Q.

[0019] Proline mutations, also known as rigid amino acid mutations, aim to utilize the unique cyclic structure and geometric conformation constraints of proline to stabilize the correct folding conformation of proteins in the turn regions or specific key positions of the polypeptide backbone, thereby reducing the possibility of misfolding or unfolding and improving the structural stability of the protein. In an alternative embodiment, the proline mutations include mutations of amino acids at 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 to P and / or G. Preferably, the proline mutations include 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.

[0020] The modification of the p27 sequence refers to the deletion of the p27 sequence based on the wild-type RSV F, aiming to remove the furin cleavage site and connect the F1 and F2 subunits by covalent bonds or linker peptides to stabilize the Pre-F conformation. Furin generally cleaves behind R-X-K / R-R in a protein (where R represents arginine, K represents lysine, and X represents any amino acid). Combining 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 behind the 109th and 136th amino acid residues of RSV F. Therefore, the p27 sequence refers to the fragment of amino acid residues 110-136 of the RSV F protein (aa 110–136). In the present invention, the modification of the p27 sequence refers to the deletion of the fragment containing p27 on the basis of the RSV F protein, and the deleted fragment can be slightly longer than p27. For example, in an alternative embodiment, the modification of the p27 sequence refers to the deletion or replacement by a linker peptide of the fragment between the 103rd and 145th, 104th and 148th, or 105th and 147th amino acids on the basis of the amino acid sequence of the wild-type RSV F protein (the "between" does not include the end values of the given data range). The amino acid sequence of the linker peptide can be common forms such as GS, GSGS, GGGS, etc. Preferably, the modification of the p27 sequence 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 values of the data range are retained and the fragment between them is deleted, or further replaced by GS for the deleted fragment.

[0021] Trimerization domain modification refers to deleting the sequence containing the transmembrane region and intracellular region based on the wild-type RSV F, and adding an amino acid sequence containing a trimerization domain, with the aim of promoting the formation of a trimer conformation of the F protein and maintaining its native conformation. The transmembrane region and intracellular region refer to the transmembrane region (aa 525 - 550) and intracellular region (aa 551 - 574) located at the C-terminus of the wild-type RSV F protein; the trimerization domain can be a heterotrimeric trimerization domain such as the Foldon domain, or a cysteine zipper trimerization domain obtained by modifying a fragment of the RSV F intracellular region, etc. (1) The Foldon trimerization domain refers to the residues at the C-terminus of the bacteriophage T4 fibritin. In the present application, the Foldon domain can be the 27 residues at the C-terminus of the bacteriophage T4 fibritin or its mutants. In an alternative embodiment, the amino acid sequence of the Foldon trimerization domain is GYIPEAPRDGQAYVRKDGEWVLLSTFL; as an alternative embodiment, deleting the transmembrane region and intracellular region and adding an amino acid sequence containing a trimerization domain means deleting aa 514 - 574 (covering the transmembrane region and 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 containing 4 pairs of cysteine mutations, which can be ABCD-tag or ABCH-tag. In an alternative 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 alternative embodiment, deleting the transmembrane region and intracellular region and adding an amino acid sequence containing a trimerization domain means deleting aa 512 - 574 (covering the transmembrane region and intracellular region) at the C-terminus of the wild-type RSV F protein, and adding the aforementioned ABCD-tag or ABCH-tag

[0022] In an alternative embodiment, the wild-type RSV F protein mutant of the present invention further includes a linker sequence (such as GG, GS, SAIG) and an 8*His tag sequence (HHHHHHHH). Such sequences are not essential for the functions of the RSV F protein, such as inducing an immune response. Those skilled in the art will recognize such sequences and understand, when appropriate, that the RSV F protein mutants disclosed in the present invention may or may not contain such sequences.

[0023] As an alternative embodiment, the present invention provides a total of three groups of RSV F mutants:

[0024] The first group of RSV F mutants all have the electrostatic mutations E487T, D486N or D486Q;

[0025] The second group of RSV F mutants all have the disulfide bond mutation D486C+E487C;

[0026] 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;

[0027] The following separately describes the above three groups of RSV F mutants:

[0028] (I) The first group of RSV F mutants (all having E487T, D486N or D486Q):

[0029] The present invention provides an RSV F protein mutant, which has a variety of modifications relative to the wild-type RSV F protein to stabilize it in the prefusion conformation, and the modifications include:

[0030] (a) Proline mutation: S215P;

[0031] (b) Cavity filling mutation: selected from S190V, S190L or S190I (wherein S190V or S190L is preferred, and S190L is further preferred);

[0032] (c) Electrostatic mutation: selected from E487T, D486N or D486Q (wherein E487T is preferred);

[0033] Among them, the position numbers are based on the sequence shown in SEQ ID NO: 1.

[0034] Preferably, the modification does not include an engineered disulfide bond mutation.

[0035] Preferably, the modifications present in the RSV F protein mutant include: S215P, S190L, E487T.

[0036] Optionally, the modification further includes modification of the p27 sequence, i.e., deletion of the p27 sequence. Optionally, the p27 sequence modification is carried out in one of the following ways: (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: using a linker (such as GG, GS, GSGS, GGGS, etc.) to connect the sequences before and after the deleted fragment. 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, among which T103-GS-G145, N104-GS-I148 or S / N105-A147 is further preferred, and S / N105-A147 is even more preferred.

[0037] Optionally, the modification further includes modification of the trimerization domain: deleting the transmembrane region and the intracellular region and adding a sequence containing the 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, among which Foldon is preferred.

[0038] Preferably, the modification includes the modification listed in any one of the following groups, or consists of the modification listed in any one of the following groups:

[0039] Group (1): S215P, S190L, E487T, T103-GS-G145, Foldon;

[0040] Group (2): S215P, S190L, E487T, N104-GS-I148, Foldon;

[0041] Group (3): S215P, S190L, E487T, S / N105-A147, Foldon;

[0042] Group (4): S215P, S190L, E487T, S / N105-GS-A147, Foldon.

[0043] Among them, Group (3) is preferred.

[0044] In the above groups, "Foldon" refers to deleting the transmembrane region and the intracellular region of RSV F and adding a sequence containing the Foldon trimerization domain.

[0045] Preferably, the RSV F protein mutants having the modifications of group (1), group (2), group (3) or group (4) respectively contain or are amino acid sequences having more than 99% (preferably 100%) identity with SEQ ID NO: 51, 53, 54 or 55.

[0046] (ii) The second group of RSV F mutants (all having the D486C+E487C disulfide bond mutation);

[0047] The present invention also provides an RSV F protein mutant which has a variety of modifications relative to the wild-type RSV F protein to stabilize it in the prefusion conformation, and the modifications include:

[0048] (a) Engineered disulfide bond mutations: D486C+E487C;

[0049] (b) Proline mutations: selected from S215P, Q210P, S211P or S211P+S213P;

[0050] (c) Cavity filling mutations: selected from S190V or S190L;

[0051] Wherein the position numbers are based on the sequence shown in SEQ ID NO: 1.

[0052] Preferably, the modifications do 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, avoiding charge repulsion between adjacent amino acids; therefore, in the case of using D486C+E487C, other electrostatic mutations may not be introduced).

[0053] Optionally, the modifications further include p27 sequence modification, that is, deleting the p27 sequence. Optionally, the p27 sequence modification is carried out in one of the following ways: (1) deleting the fragment containing p27 without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) deleting the fragment containing p27 and introducing a linker sequence: using a linker (such as GG, GS, GSGS, GGGS, etc.) to connect the sequences before and after the deleted fragment. 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, and preferably T103-GS-G145 or S / N105-A147.

[0054] Optionally, the modification further includes trimerization domain modification: deleting the transmembrane region and intracellular region and adding a sequence containing 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, and Foldon is preferred among them.

[0055] Preferably, the modification includes any one group or consists of any one group of the following:

[0056] Group (1): D486C + E487C, S215P, S190V, T103-GS-G145, Foldon;

[0057] Group (2): D486C + E487C, S215P, S190L, T103-GS-G145, Foldon;

[0058] Group (3): D486C + E487C, S215P, S190L, T103-GS-G145;

[0059] Group (4): D486C + E487C, S215P, S190L, S / N105-A147, Foldon;

[0060] Group (5): D486C + E487C, Q210P, S190L, S / N105-A147, Foldon;

[0061] Group (6): D486C + E487C, S211P, S190L, S / N105-A147, Foldon;

[0062] Group (7): D486C + E487C, S211P + S213P, S190L, S / N105-A147, Foldon.

[0063] Among them, group (2), group (4), group (5) or group (6) is preferred.

[0064] In the above groups, "Foldon" means deleting the transmembrane region and intracellular region of RSV F and adding a sequence containing the Foldon trimerization domain.

[0065] Preferably, the RSV F protein mutants with the modifications described in group (1), group (2), group (3), group (4), group (5), group (6) or group (7) respectively contain or are amino acid sequences having more than 99% (preferably 100%) identity with SEQ ID NO: 46, 47, 50, 58, 64, 65, 73.

[0066] (iii) The third group of RSV F mutants (all having disulfide mutations of R106C+V144C, S / N105C+A147C, S / N105C+A146C, N104C+I148C or N104C+S146C);

[0067] The present invention also provides an RSV F protein mutant, which has a variety of modifications relative to the wild-type RSV F protein to stabilize it in the pre-fusion conformation, and the modifications include:

[0068] (a) Engineered disulfide mutations: selected from R106C+V144C, N104C+S146C, N104C+I148C, S / N105C+A146C, S / N105C+A147C or S / N105C+M370C;

[0069] (b) Proline mutation: S215P;

[0070] (c) Cavity filling mutations: selected from S190I, S190L, S190V or S190W;

[0071] (d) Electrostatic mutations: selected from D486N, D486Q, E487T, D489P or D489Q;

[0072] Among them, the position numbers are based on the sequence shown in SEQ ID NO: 1.

[0073] An optional scheme is: among the modifications, the disulfide mutation selects R106C+V144C, the cavity filling mutations are selected from S190I, S190L, S190V or S190W (wherein S190L, S190V or S190W are preferred), and the electrostatic mutations are selected from D486N, D486Q, E487T, D489P or D489Q (wherein D486N, D486Q, E487T or D489P are preferred).

[0074] Another optional scheme is: among the modifications, the disulfide mutations are selected from N104C+S146C, N104C+I148C, S / N105C+A146C or S / N105C+A147C, the cavity filling mutations are selected from S190I, S190L or S190V (wherein S190I is preferred), and the electrostatic mutations are selected from D486N, D486Q, E487T (wherein D486N is preferred).

[0075] Optionally, the modification may or may not include modification of the p27 sequence, and the p27 sequence modification refers to deletion of the p27 sequence. Optionally, the p27 sequence modification is 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 while introducing a linker sequence: using a linker (such as GG, GS, GSGS, GGGS, etc.) to connect the sequences before and after the deleted fragment. 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.

[0076] Optionally, the modification further includes modification of the trimerization domain: deleting the transmembrane region and the intracellular region and adding a sequence containing the 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, and Foldon is preferred.

[0077] Preferably, the modification includes any one group of the following or consists of any one group:

[0078] (1) R106C + V144C, S215P, S190I, D486N, Foldon;

[0079] (2) R106C + V144C, S215P, S190V, D486N, Foldon;

[0080] (3) R106C + V144C, S215P, S190L, D486N, Foldon;

[0081] (4) R106C + V144C, S215P, S190W, D486N, Foldon;

[0082] (5) R106C + V144C, S215P, S190V, D486Q, Foldon;

[0083] (6) R106C + V144C, S215P, S190V, E487T, Foldon;

[0084] (7) R106C + V144C, S215P, S190L, D486Q, Foldon;

[0085] (8) R106C + V144C, S215P, S190L, E487T, Foldon;

[0086] (9) R106C + V144C, S215P, S190L, D489P, Foldon;

[0087] (10) R106C + V144C, S215P, S190W, D486Q, Foldon;

[0088] (11) R106C + V144C, S215P, S190W, E487T, Foldon;

[0089] (12) N104C + S146C, S215P, S190I, D486N, Foldon;

[0090] (13) N104C + I148C, S215P, S190I, D486N, Foldon;

[0091] (14) S / N105C + S146C, S215P, S190I, D486N, Foldon;

[0092] (15) S / N105C + A147C, S215P, S190I, D486N, Foldon;

[0093] (16) S / N105C + M370C, S190L, E487T, S / N105 - A147, Foldon.

[0094] In each of the above groups, "Foldon" refers to deleting the transmembrane region and intracellular region of RSV F and adding a sequence containing the Foldon trimerization domain.

[0095] Further preferably, the RSV F protein mutants with any of the modifications described in groups (1) to (16) respectively have the following amino acid sequence characteristics: containing or being an amino acid sequence having more than 99% (preferably 100%) identity with SEQ ID NO: 5, 6, 7, 8, 9, 10, 14, 15, 16, 19, 20, 24, 26, 28, 29 or 83.

[0096] As an alternative embodiment, the present invention provides 79 specific RSV F mutants, and the modifications they have relative to the wild - type RSV F protein are specifically shown in Tables 1, 3, 5 and 7.

[0097] In a second aspect, the present invention provides biological materials, and the biological materials include any one of the following:

[0098] (a) A nucleic acid molecule encoding the RSV F protein mutant.

[0099] (b) A recombinant vector containing the nucleic acid molecule described in (a), preferably, the original plasmid of the recombinant vector is pOET1.1.

[0100] (c) A recombinant virus containing the nucleic acid molecule described in (a) or the recombinant vector described in (b), preferably, the recombinant virus includes baculovirus of insect cells, adenovirus, adeno-associated virus, vaccinia virus, herpes virus or retrovirus.

[0101] (d) A transformed cell containing the nucleic acid molecule described in (a), the recombinant vector described in (b) or the recombinant virus described in (c), and the host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells; preferably, the insect cells are Sf9 cells, Sf21 cells or High-Five cells.

[0102] In the third aspect, the present invention also provides a method for preparing the above RSV F protein mutant, and the steps include: construction of a recombinant vector, extraction of the recombinant vector, transfection of the recombinant vector into a host cell and expression of the protein, and purification of the expression product.

[0103] In the fourth aspect, the present invention also provides an immunogenic composition comprising the above RSV F protein mutant, nucleic acid molecule, recombinant vector, recombinant virus or transformed cell.

[0104] In the fifth aspect, the present invention also provides the application of any one of the above RSV F protein mutant, nucleic acid molecule, recombinant vector, recombinant virus, transformed cell, immunogenic composition in any of the following aspects:

[0105] (a) Application for preparing RSV-specific antibodies.

[0106] (b) Preparation of drugs for preventing and / or treating RSV infection; preferably, the drugs include recombinant protein vaccines, vector vaccines or nucleic acid vaccines.

[0107] (c) Preparation of diagnostic reagents for RSV.

[0108] Term definition:

[0109] In the present application, the term "respiratory syncytial virus" or "RSV" belongs to a single-stranded negative-strand RNA virus of the genus Pneumovirus in the family Paramyxoviridae. This virus can cause lower respiratory tract disease symptoms (Lower respiratory tract disease, LRTD) in infants, the elderly and immunocompromised adults, including interstitial pneumonia and bronchiolitis.

[0110] In the present 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, as well as modification or cleavage of the protein structure. In the present application, the "mutant" specifically refers to a respiratory syncytial virus (RSV) F protein mutant.

[0111] In the present application, the term "disulfide bond mutation" refers to the replacement of an amino acid in the wild-type RSV F protein with cysteine, and the formation of a disulfide bond between the sulfur atoms of two cysteine residues to stabilize the RSV Pre-F structure.

[0112] In the present application, the term "cavity filling mutation" refers to the replacement of an amino acid residue in the wild-type RSV F protein with an amino acid that is expected to fill the cavity of the mature F protein structure to stabilize the RSV Pre-F structure. These amino acids that fill the structural cavity 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).

[0113] 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 that are close to each other in the protein folding structure to stabilize the RSV Pre-F structure. The electrostatic mutation includes the mutation of the amino acid at position 486, 487, or 489 in the amino acid sequence of the wild-type RSV F protein to asparagine (Asn), glutamine (Gln), or threonine (Thr).

[0114] In the present 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 group and carboxyl group in proline are linked by an amino group to form a rigid five-membered ring structure. This ring structure restricts the rotational freedom of proline in the protein, enabling proline to form fixed folds and turns, thereby increasing the stability of the protein.

[0115] In the present application, the term "p27" refers to a 27-amino acid short peptide between amino acid residues 110 - 136 in the wild-type RSV F protein. This amino acid short peptide is cleaved and released by furin protease, and two subunits, F1 (137 - 574aa) and F2 (26 - 109aa), are formed.

[0116] In the present application, the term "p27 sequence modification" refers to deleting the fragment containing p27 on the basis of the wild-type RSV F protein, that is, the deleted fragment should cover p27 and can be slightly longer than p27 (extended by about 1 to 15 amino acid residues upstream and / or downstream of p27). For example, the fragment between amino acid residues 104-144, 105-147, or 106-146 of the RSV F protein can be deleted (the "between" here does not include the end values of the numerical range. For example, the fragment between amino acid residues 104-144 refers to the fragment composed of amino acids 105-143), and after deleting the aforementioned fragment, the front and rear fragments can be directly covalently linked or linked using a linker peptide (such as GG, GS, GSGS, GGGS, etc.), among which GS is preferred.

[0117] In the present application, the term "trimerization domain" refers to the 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 (such as Foldon) or a cysteine zipper amino acid sequence (such as ABCD-tag, ABCH-tag). The trimer in the present application refers to a complex formed by three RSV F protein F1+F2 subunits.

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

[0119] In the present application, the term "ABCH-tag" refers to adding a cysteine zipper (amino acid sequence with 4 pairs of cysteine mutations) to the C-terminus of the wild-type RSV F protein after deleting the transmembrane region and the intracellular region, and the ABCH-tag amino acid sequence is CCHNVNACCSTTNICCTTTNICCTT.

[0120] In the present application, the term "Mota" or "motavizumab" refers to the antibody described in the paper by Herren et al. (Herren Wu, etal., Journal of Molecular Biology, 2007).

[0121] In the present application, the term "D25" refers to the antibody described in the paper by Jason et al. (Jason S McLellan, et al., Science,2013).

[0122] In the present application, the term "hRSV90" refers to the antibody described in the paper by Jarrod et al., Nature Microbiology 2, 16271 (2017).

[0123] In the present application, the term "AM14" refers to the antibody described in the paper by Morgan et al. (Morgan S A Gilman, et al., PLoSPathog, 2015).

[0124] 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 chemical bonds in the protein molecule and a change in the folding pattern of the polypeptide.

[0125] In the present application, the term "comprising" generally means including the expressly specified features, but not excluding other elements.

[0126] Advantages of the present invention:

[0127] The RSV F protein mutant described in the present invention can significantly improve the stability of the pre-fusion conformation (pre-F) and the binding activity with neutralizing antibodies compared with the wild-type RSV F protein, indicating that it has extremely high potential to become an effective component of an RSV vaccine. Compared with the prior art (such as the 847 protein), some of the 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

[0128] Figure 1 : Map of the pOET1.1 baculovirus transfer plasmid used in Example 1 of the present invention.

[0129] Figure 2 : Binding of supernatant of each mutant cell to different monoclonal antibodies (double antibody sandwich ELISA).

[0130] Figure 3 : Reducing and non-reducing gel electrophoresis analysis diagrams of each purified mutant.

[0131] Figure 4 : Western Blot analysis diagrams of each purified mutant.

[0132] Figure 5 : Results of binding antibody determination in mice immunized with different doses of BR47 antigen protein.

[0133] Figure 6 : Results of binding antibody determination in mice immunized with different antigen proteins. Detailed Description of the Invention

[0134] To more accurately, clearly understand and master the purpose, technical solution and advantages of the present invention, the following will deeply interpret and describe in detail the embodiments of the present invention based on the relevant drawings in the embodiments of the present invention. Please note that the embodiments described here only represent some application examples of the present invention and do not cover all embodiments.

[0135] Example 1: Design and screening of RSV F mutants

[0136] The design and screening of RSV F mutants include the following steps:

[0137] (1) Design of mutants and gene synthesis: First, obtain the sequence of wild-type RSV F: According to the F protein sequence of the publicly available A-type RSV strain in the NCBI database, calculate the amino acid (common amino acid) that appears most frequently at each site, and construct the RSV F common amino acid sequence (as shown in SEQ ID NO.1), which represents the sequence of the A-type wild strain RSV F. Then, based on the amino acid sequence of the wild-type RSV F protein shown in SEQ ID NO: 1, it is modified to design a recombinant RSV F protein (mutant): According to existing data and combined with structural biology analysis, the present invention designs a variety of RSV F mutants (recombinant proteins), each of which contains one or more of the following modifications: disulfide bond mutation, proline mutation, cavity filling mutation, electrostatic mutation, p27 sequence modification, introduction of a trimerization domain. Then, codon optimization is carried out according to the host Sf9 insect cells to determine the nucleic acid sequence and full gene synthesis is carried out.

[0138] (2) Construction of recombinant plasmids and expression of antigen proteins: Insert the synthesized target gene into the baculovirus transfer vector pOET1.1 ( Figure 1 ) and use the flashBAC TM baculovirus expression system kit to co-transfect the recombinant plasmid and the baculovirus genome into Sf9 insect cells. The specific operation of transfection is carried out according to the kit instructions. The cell supernatant is collected 7 days after transfection of Sf9 cells. At this time, the supernatant contains the P0 generation virus of the recombinant foreign gene. Transfer the P0 generation virus into a new Sf9 cell well plate to obtain the P1 generation virus. The cell supernatant is collected 6 - 7 days after the P1 generation virus infects the cells for ELISA detection of the conformation and stability of each antigen.

[0139] (3) ELISA detection of antigen proteins: Use the ELISA method to detect the conformation and stability of each antigen protein.

[0140] The specific operation of ELISA detection is as follows: Coat 100 μl of antibodies such as Motavizumab (abbreviated as Mota, which can bind both Pre-F and Post-F), AM14 (which only binds to Pre-F), D25 (which only binds to Pre-F), or hRSV90 (which only binds to Pre-F) overnight at 4°C in a 96-well plate. The next day, wash it 3 times with PBST and then pat it dry, and then block it with 5% skim milk prepared with PBST (200 μl / well, block at room temperature for 1 hour). Divide the cell supernatant collected in (2) into 3 equal parts, and perform high-temperature treatment (treat at 50°C or above for 1 h), low-temperature treatment (store at 4°C for a long time or freeze-thaw repeatedly), and no treatment respectively. After washing the blocked 96-well plate 3 times with PBST and patting it dry, add 100 μl of the cell supernatant samples without treatment and treated under various stress conditions to each well, and incubate at room temperature for 1-2 hours. After washing 3 times with PBST, add 100 μl of Rabbit His-tag Antibody and incubate at room temperature for 1 hour. After washing 3 times with PBST, add 100 μl of anti-rabbit IgG antibody (HRP) and incubate at room temperature for 1 hour. After washing 5 times with PBST and patting it dry, add 100 μl of freshly prepared TMB chromogenic solution to each well. After chromogenic reaction for 10-15 minutes, add the termination solution to terminate the chromogenic reaction, and read the absorbance value at wavelengths of 450 nm / 570 nm with an enzyme-linked immunosorbent assay reader. The final absorbance value reading is A450nm - A570nm.

[0141] The method for stability evaluation is as follows: According to the ELISA detection results, 1) On the one hand, calculate the proportion of Pre-F obtained by the RSV F protein (abbreviated as the candidate antigen protein) designed in the present invention compared to the positive control 847 protein (the antigen protein used in the RSV vaccine Abrysvo already on the market in the United States by Pfizer, SEQ ID NO: 84 shown in this application). If this proportion is greater than 1, it means that the proportion of the pre-F conformation obtained by the candidate antigen protein (%Pre-F, that is, the proportion of Pre-F compared to the sum of Pre-F + Post-F) is more excellent than that of the 847 protein. If the Pre-F proportion is not lower than 0.8 (at least not lower than 0.75) compared to 847, it indicates that the candidate antigen protein is better. 2) On the other hand, calculate the stability of the candidate antigen protein under high-temperature and low-temperature stress conditions, where the 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. Combining the screening results of each round, the present invention defines that if the corresponding value of the protein stability after stress condition treatment is not lower than 0.6 (at least not lower than 0.55), it means that the protein stability is relatively excellent.

[0142] (4) Scheme adjustment and improvement: According to the ELISA detection results in the previous step, based on the excellent antigens, select better mutation types for the next round of screening.

[0143] According to the above research ideas, four rounds of RSV F mutant design and screening were carried out, as specifically described in Examples 2 to 5 below.

[0144] Example 2: The First Round of RSV F Mutant Design and Screening

[0145] According to the operation steps described in Example 1, the first round of mutant design and ELISA detection were carried out. The main purpose of this round of experiment was to make various attempts on the methods of disulfide bond mutation, cavity filling mutation, and electrostatic mutation, and to test their combined effects through orthogonal experiments.

[0146] The RSV F mutant design strategy is as follows: (1) In terms of disulfide bond mutation, 8 designs were carried out (R106C + V144C, N104C + S146C, N104C + A147C, N104C + I148C, S / N105C + G145C, S / N105C + S146C, S / N105C + A147C, Q34C + G471C); (2) In terms of proline mutation, S215P was used or A490P was further added on this basis; (3) In terms of cavity filling mutation, 4 designs were carried out (S190V, S190L, S190W, S190I); (4) In terms of electrostatic mutation, 5 designs were carried out (D486N, D486Q, E487T, D489P, D489Q); (5) In terms of the p27 sequence, no p27 sequence modification was carried out, or T103-GS-G145 was adopted (delete the fragment between amino acids 103 and 145 and additionally introduce the linker GS); (6) In terms of the trimerization domain: Foldon was adopted.

[0147] Based on the above various types of modification methods, when combining them to construct RSV F mutants, (1) on the one hand, fix the disulfide bond mutations (R106C + V144C), proline mutations (including S215P), p27 sequence modification (without modification), and the selection of the trimerization domain (Foldon), and conduct orthogonal design and screening of cavity filling mutations (S190I, S190V, S190L, S190W) and electrostatic mutations (D486N, D486Q, E487T, D489P, D489Q) (BR01, BR06 - BR23); (2) on the other hand, fix the proline mutations (including S215P), cavity filling mutations (S190I), electrostatic mutations (D486N), p27 sequence modification (without modification), and the selection of the trimerization domain (Foldon), and conduct changes and comparisons of disulfide bond mutations (N104C + S146C, N104C + A147C, N104C + I148C, S / N105C + G145C, S / N105C + S146C, S / N105C + A147C, Q34C + G471C) (BR24 - BR30); (3) in addition, based on BR30, conduct p27 sequence modification (T103 - GS - G145) to obtain BR31, and compare the two to analyze the influence of p27 sequence modification on the technical effect.

[0148] The specific RSV F mutants designed in this round are shown in Table 1. In this table, the left column lists the names of the mutants, and the same row lists the modifications made to this mutant compared to the wild - type RSV F and their corresponding sequence numbers. The corresponding ELISA test results of each mutant are shown in Table 2.

[0149] Table 1: Design of RSV F mutants in the first round

[0150]

[0151] Remarks:

[0152] (1) " / " indicates that this item has not been modified (the same below).

[0153] (2) In the column of "p27 sequence modification": " / " indicates that the p27 sequence in the wild - type RSV F protein has not been modified; "T103 - GS - G145" indicates deleting the fragment between amino acids 103 - 145 in the wild - type RSV F protein (excluding the end values, i.e., amino acids 103 and 145, that is: only deleting the fragment of amino acids 104 - 144) and replacing it with GS, where GS serves as a linker.

[0154] (3) Regarding S / N105C+G145C: Among them, "S / N105" means that the 105th amino acid site in the wild-type RSV F protein can be S or N (S is the most common, followed by N), and "S / N105C" indicates that this site mutates from the wild-type S / N to C.

[0155] Table 2: ELISA test results of the first-round RSV F mutants

[0156]

[0157] Remarks:

[0158] 1 The calculation formula for "Pre-F ratio" is: %Pre-F = OD AM14 / OD Mota , and the same hereinafter;

[0159] 2 "Pre-F ratio compared with 847" represents the ratio of the Pre-F protein ratio of each mutant to the positive control 847. For example, the calculation formula for the Pre-F ratio of BR06 compared with 847 is: (%Pre-F BR06 ) / (%Pre-F 847 ), and the same hereinafter;

[0160] 3 "Protein stability (Mota)" represents the change in the OD value of the Mota antibody in the sample before and after treatment under stress conditions. The specific calculation formula is: (Mota OD 处理后 ) / (Mota OD 处理前 ), and the same hereinafter;

[0161] 4 "Protein stability (AM14)" represents the change in the OD value of the AM14 antibody in the sample before and after treatment under stress conditions. The specific calculation formula is: (AM14 OD 处理后 ) / (AM14 OD 处理前 ), and the same hereinafter.

[0162] According to the results of the first-round screening, each antigen protein can be normally expressed and the antigen protein can be screened by the ELISA method. The first-round screening is mainly to preliminarily explore the combined effects of each design strategy:

[0163] Regarding 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 all help stabilize the conformation of the Pre-F protein, while N104C + A147C (corresponding to BR25), S / N105C + G145C (corresponding to BR27), and Q34C + G471C (corresponding to BR30, BR31) show mediocre performance. Since N104C + I148C and S / N105C + A147C are unreported novel disulfide bonds and exhibit excellent performance, they are selected for the next round of screening.

[0164] Regarding cavity filling, the proportion of the Pre-F conformation is generally higher after adding S190V and S190L mutations than that after the S190W mutation. They are selected for the next round of screening.

[0165] Regarding electrostatic mutations, adding mutations such as D486N, D486Q, and E487T can stabilize the Pre-F conformation more effectively than other mutations. They are selected for the next round of antigen screening.

[0166] Example 3: Design and Screening of the Second Round of RSV F Mutants

[0167] The main purpose of this round of experiment is to test the combined effects of the selected disulfide bond mutations, cavity filling mutations, and electrostatic mutations. At the same time, the pressure conditions are increased in this round of experiment (the high temperature is increased from 50°C to 60°C) to test the thermal stability of the constructed RSV F mutants. Meanwhile, BR26 and BR29, which showed good performance in the previous round of screening, are also included in the pressure condition test of this round.

[0168] The RSV F mutant design strategy is as follows: According to the results of the previous example, in the design of the RSV F mutants in this round, (1) regarding disulfide bond mutations, N104C + I148C and S / N105C + A147C are selected for this round of screening, and in addition, D486C + E487C is introduced; (2) regarding proline mutations, S215P is still used; (3) regarding cavity filling mutations, S190V or S190L is selected; (4) regarding electrostatic mutations, D486N, D486Q, or E487T is selected; (5) regarding the p27 sequence modification, T103 - GS - G145 is still adopted, or the p27 sequence is not modified; (6) regarding the trimerization domain, Foldon is still used.

[0169] The specific RSV F mutants designed in this round are shown in Table 3. In this table, the left column lists the names of the mutants, and the modifications made to the mutant compared to the wild-type RSV F and their corresponding sequence numbers are listed in the same row. The corresponding ELISA test results of each mutant are shown in Table 4.

[0170] Table 3: Design of RSV F mutants in the second round

[0171]

[0172] Table 4: ELISA test results of RSV F mutants in the second round

[0173]

[0174] In the second-round screening, we increased the high-temperature treatment pressure (from 50 °C to 60 °C) in the hope of screening for a more excellent antigen design. The results showed that:

[0175] (I) After heat treatment at 60 °C, the protein stability (AM14) of most mutants decreased significantly, while the pre-F conformation of the RSV F mutants with the D486C+E487C disulfide bond mutation (BR44~BR46) did not change significantly. In particular, BR46 and BR47 showed outstanding performance. Therefore, the disulfide bond mutation D486C+E487C was considered for retention in the subsequent screening.

[0176] (II) Relatively speaking, the contribution of the disulfide bond mutations N104C+I148C or S / N105C+A147C to the stability of the RSV F mutants is less than that of D486C+E487C. However, considering that the extraction, purification, and preservation processes of the RSV F mutants generally do not involve high temperatures above 50 °C, N104C+I148C or S / N105C+A147C are also available disulfide bond mutation methods under non-extreme temperature conditions. (1) For N104C+I148C, comparing BR26, BR32~BR37, it can be seen that when N104C+I148C is used in combination with S190I and D486N, the technical effects of this mutation combination are not much different from those of S / N105C+A147C and other cavity-filling mutations (such as S190L / V) and electrostatic mutations (D486Q / E487T) types, but the protein stability (AM14) is slightly higher. (2) Similarly, for S / N105C+A147C, comparing BR29, BR38~BR43, it can be seen that when S / N105C+A147C is used in combination with S190I and D486N, the technical effects of this mutation combination are not much different from those of S / N105C+A147C and other cavity-filling mutations (such as S190L / V) and electrostatic mutations (D486Q / E487T) types, but the protein stability (AM14) is slightly higher.

[0177] (3) Meanwhile, the results of this round of screening showed that the modification of the p27 sequence (BR46 and BR47) could significantly improve the stability of Pre-F compared with no modification (BR44 and BR45). Therefore, the modification of the p27 sequence was considered in all subsequent screenings. Among the cavity filling mutations, both S190L and S190V could help stabilize the Pre-F conformation. S190L, which showed slightly better results after the modification of the p27 sequence, was selected for the next round of screening. Among the electrostatic mutations, D486N, D486Q, and E487T all showed good performance. E487T, which showed slightly better results, was selected for the next round of antigen screening.

[0178] Example 4: Design and Screening of the Third Round of RSV F Mutants

[0179] The main purpose of this round of experiment was to expand the modification methods of the p27 sequence and the trimerization domain, and to test their combined effects with the disulfide bond mutations, cavity filling mutations, and electrostatic mutations screened based on the previous round of results. Meanwhile, in this round of testing, we further increased the pressure conditions (the high temperature was increased from 60 °C to 65 °C, and the low temperature storage at 4 °C was changed to repeated freeze-thaw treatment), hoping to screen out antigen designs that were more tolerant to extreme conditions. At the same time, BR47, which showed outstanding performance in the previous round of screening, was also included in the pressure condition test of this round.

[0180] The RSV F mutant design strategy was as follows: According to the results of the previous example, in the design of RSV F mutants in this round, (1) for disulfide bond mutations, D486C + E487C was selected for this round of screening, or no disulfide bond mutations were introduced; (2) for proline mutations, S215P was still used, or no proline mutations were introduced; (3) for cavity filling mutations, S190L was selected; (4) for electrostatic mutations, E487T was selected, or no electrostatic mutations were introduced; (5) for the modification of the p27 sequence, in addition to T103-GS-G145 tested in the first and second rounds, four other modification methods (N104-I148, N104-GS-I148, S / N105-A147, and S / N105-GS-A147) were added; (6) for the trimerization domain, in addition to Foldon tested previously, two other options, ABCD-tag (CCHNVNACCSTTNICCTTTNICCTT) and ABCH-tag (CCHNVNACCSTTNICCTTIIICCIV), were added, or no trimerization domain was introduced.

[0181] The specific RSV F mutants designed in this round are shown in Table 5. In this table, the left column lists the names of the mutants, and the modifications made to this mutant compared with the wild-type RSV F and their corresponding sequence numbers are listed in the same row. The corresponding ELISA test results of each mutant are shown in Table 6.

[0182] Table 5: Design of the Third Round of RSV F Mutants

[0183]

[0184] Remarks:

[0185] Regarding the modification of the p27 sequence: 1) "S / N105-A147" means deleting the fragment between amino acids 105 and 147 of the wild-type RSV F protein (excluding the terminal values, i.e., amino acids 105 and 147, that is, only deleting the fragment between amino acids 106 and 146). Similarly, "N104-I148" means deleting the fragment between amino acids 104 and 148 of it (excluding the terminal values); 2) "T103-GS-G145" means deleting the fragment between amino acids 103 and 145 of the wild-type RSV F protein (excluding the terminal 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 terminal values) and replacing it with GS, and "S / N105-GS-A147" means deleting the fragment between amino acids 105 and 147 of the wild-type RSV F protein (excluding the terminal values) and replacing it with GS.

[0186] Table 6: ELISA Detection Results of the Third Round of RSV F Mutants

[0187]

[0188] The results of the third round of screening show that:

[0189] Regarding the modification of the p27 sequence, different modification methods have a significant impact on the conformational stability of Pre-F: Among the five modification methods, S / N105-A147 shows the best effect. As shown in Table 6, when no disulfide bond mutations are added: Comparing different p27 sequence modification schemes (i.e., antigen designs BR49, BR52, BR53, BR54, and BR55), among them, BR54 (p27 sequence modified to S / N105-A147) is superior to other designs after high-temperature pressure treatment, and there is no significant difference from other designs after low-temperature pressure treatment; Similarly, when disulfide bond mutations are added: Comparing different p27 sequence modification schemes (i.e., antigen designs BR47, BR56, BR57, BR58, and BR59), among them, BR58 (p27 sequence modified to S / N105-A147) is superior to other designs during high-temperature pressure treatment, and there is no significant difference from other designs after low-temperature pressure treatment. Therefore, S / N105-A147 is selected for the next round of screening.

[0190] In addition, it is worth mentioning that although BR50 (D486C+E487C, S215P, S190L, T103-GS-G145) has not been modified in the trimerization domain, it still performs well in terms of the pre-F ratio and structural stability, although it is slightly weaker than BR47 (D486C+E487C, S215P, S190L, T103-GS-G145, Foldon). It is speculated that the reason is that the D486C+E487C disulfide bond near the C-terminus of the RSV F protein helps the protein trimerize to a certain extent. Therefore, when the D486C+E487C disulfide bond mutation is adopted, the modification of the trimerization domain is not necessary.

[0191] Example 5: Design and Screening of the Fourth Round of RSV F Mutants

[0192] The main purpose of this round of experiments is to expand the modification methods of proline mutations. The secondary purpose is to make more attempts in disulfide bond mutations and test their combined effects with cavity filling mutations, electrostatic mutations, and p27 sequence modifications screened based on the previous round of results.

[0193] The RSV F mutant design strategy is as follows: According to the results of the previous example, in the design of RSV F mutants in this round, (1) for disulfide bond mutations, 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 expect to replace proline mutations and obtain a more stable Pre-F conformation; (2) for proline mutations, various possibilities (Q210P, S211P, C212P, S213P, I214P, N216P, I217P, E218P, Q210P+C212P, S211P+S213P, C212P+I214P, I214P+N216P, N216P+E218P, S213G+I214P+N216G, S211G+C212P+I214G) are tested in addition to the previous S215P to compare their effects; (3) for cavity filling mutations, S190L is still used or no cavity filling mutation is introduced; (4) for electrostatic mutations, E487T is still used or no electrostatic mutation is introduced; (5) for p27 sequence modification, S / N105-A147 is uniformly used; (6) for the trimerization domain, Foldon is uniformly used.

[0194] The specific RSV F mutants designed in this round are shown in Table 7. In this table, the left column lists the names of the mutants, and the modifications made to the mutant compared to the wild-type RSV F and their corresponding sequence numbers are listed in the same row. The corresponding ELISA test results of each mutant are shown in Table 8.

[0195] Table 7: Design of the Fourth Round of RSV F Mutants

[0196]

[0197] Table 8: ELISA Detection Results of the Fourth Round of RSV F Mutants

[0198]

[0199] The results of the fourth round of screening showed that:

[0200] In terms of proline mutations, Q210P, S211P, and S211P + S213P could all significantly improve the conformational stability of the Pre-F protein.

[0201] In terms of disulfide bond mutations, by comparing the ELISA detection results of BR79, BR80, BR81, BR82, and BR83, it can be seen that: (1) BR83 performed relatively well, indicating that S / N105C + M370C could help stabilize the Pre-F conformation in the absence of rigid amino acid mutations (proline mutations). (2) In contrast, although the various data of BR80 seemed good on the surface (for example, the Pre-F ratio was as high as 2.18 compared with the 847 protein), in fact, the protein expression level of this mutant was extremely low (for example, after treatment at 65°C for 1 h, OD Mota = 0.111, OD AM14 = 0.136, showing that the OD values of the detection antibodies were very low. Although the calculated %Pre-F = OD AM14 / OD Mota = 1.22 and the "Pre-F ratio compared with 847" was as high as 2.18), the disulfide bond pair E60C + K196C was not preferred. (3) In addition, the Pre-F ratios of BR79 and BR81 compared with the 847 protein were relatively low, so the disulfide bond pair L171C + K191C was not preferred either.

[0202] After completing the four rounds of screening, mutants with relatively excellent overall performance (BR47, BR54, BR58, BR64, BR65, BR73) were selected as representatives, and then ELISA detection was performed with other neutralizing antibodies to further confirm the conformational stability of the designed antigen and its potential to induce various neutralizing antibodies. The results are as Figure 2 shown. Multiple mutants could bind to different strong neutralizing antibodies, and their Pre-F conformations were relatively stable.

[0203] Example 6: Large-Scale Expression and Purification of Antigen Proteins

[0204] Infect 1 L of HighFive cells in the logarithmic growth phase (viable cell density is about 2×10 6 cells / ml and viability is greater than 95%) with the recombinant baculovirus expressing the antigen protein at a ratio of 1:100. Incubate the inoculated cells at 27°C with shaking at 120 rpm for 3 - 4 days. When the cell diameter significantly increases and the viable cell density and cell viability significantly decrease, centrifuge at 4000 rpm for 20 minutes to collect the cell supernatant. Filter the collected supernatant through a 0.22 μm filter membrane and then perform nickel column purification. The specific purification steps are as follows:

[0205] Equilibration: Equilibrate the nickel column (UniNTA - 80Ni) with the binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). Wash the nickel column with 10 column volumes of the binding buffer.

[0206] Loading: Slowly add the filtered sample to the equilibrated nickel column and allow the flow - through sample to pass through 3 times to enable the target protein to fully bind to the nickel column.

[0207] Washing away impurities: Wash away non - specifically bound proteins using 10 - 20 column volumes of the washing buffer (50 mM NaH2PO4, 300 mM NaCl, 20 - 50 mM imidazole, pH 8.0).

[0208] Elution: Elute the bound target protein using the elution buffer (50 mM NaH2PO4, 300 mM NaCl, 250 - 500 mM imidazole, pH 8.0). Collect the eluate and collect it in 1 mL fractions.

[0209] Load the collected eluate into a SnakeSkin dialysis bag (Thermo Scientific™) and replace the salt ions in the eluate with PBS dialysis buffer. After dialysis is completed, collect the solution in the dialysis bag, aliquot it, and store it in a - 80°C refrigerator. At the same time, take a small amount of the sample 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).

[0210] The results are as Figure 3 and Figure 4 shown. The molecular weights of the monomer and trimer of each antigen protein expressed using the insect cell - baculovirus expression system are as expected. Western blot detection using the specific antibody D25 confirms that the protein band is the RSVF protein. The purity of each mutant antigen is as expected and can be used for subsequent experiments.

[0211] Example 7: Animal Experiment

[0212] Female Balb / C mice at 6 - 8 weeks of age were randomly divided into groups of 5. On days 0 and 21 respectively, different doses of the antigen protein stock solution expressed and purified by insect cells or the vaccine prepared by mixing the antigen protein with aluminum adjuvant were immunized by intramuscular injection, 50 μl in each of the left and right legs, for a total of 100 μl. The negative control group was immunized with 100 μl of normal saline. Mouse blood was collected on days 14 and 35 respectively, and serum was separated for detection of binding antibodies. The method is as follows:

[0213] 100 μl of the purified antigen protein was coated onto a 96 - well plate. After washing 3 times with PBST the next day and patting dry, it was blocked with 5% skim milk prepared with PBST (200 μl / well, blocked at room temperature for 1 hour). After washing 3 times with PBST and patting dry. Serum samples were serially diluted 2 - fold with PBST solution containing 1% skim milk, starting from a detection point of 1:100, and 100 μl was added to each well, incubated at room temperature for 1 hour. After washing 3 times with PBST, 100 μl of Goat Anti - Mouse IgG - HRP antibody was added and incubated at room temperature for 1 hour. After washing 5 times with PBST and patting dry, 100 μl of freshly prepared TMB chromogenic solution was added to each well. After chromogenesis for 10 - 15 minutes, the stop solution was added, and the absorbance value at a wavelength of 450 nm was read with an enzyme - linked immunosorbent assay (ELISA) reader. Taking 2.1 times the absorbance value of the negative control as the Cut - off value, the maximum dilution factor at which the sample absorbance value ≥ Cut - off value is the binding antibody titer of the sample.

[0214] The results are as Figure 5 and Figure 6 shown: In the dose and adjuvant exploration experiment ( Figure 5 ), immunization with different doses of the antigen protein BR47 stock solution or the vaccine combined with aluminum adjuvant could 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 by in vitro screening could 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.

[0215] The in - depth analysis and detailed description of the embodiments of the present invention are not used to limit the scope of application for which the present invention should enjoy protection, but only to illustrate the process, results and applications of the present invention with the selected embodiments as representatives. According to the embodiments of the invention described herein, for those of ordinary skill in the same field, all other embodiments that can be deduced or obtained by equivalent substitution without creative efforts shall be included within the protection scope 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) 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.

2. The RSV F protein mutant of claim 1, 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.

3. The RSV F protein mutant of claim 2, characterized in that The electrostatic mutation is selected from D486N, D486Q, E487T or D489P.

4. The RSV F protein mutant of claim 1, 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.

5. The RSV F protein mutant of claim 4, characterized in that The cavity-filling mutation was S190I, and the electrostatic mutation was D486N.

6. The RSV F protein mutant according to any one of claims 1 to 5, 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.

7. The RSV F protein mutant of claim 6, 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.

8. The RSV F protein mutant of claim 7, 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.

9. The RSV F protein mutant according to any one of claims 1 to 5, 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.

10. The RSV F protein mutant of claim 9, characterized in that The trimerization domain is selected from Foldon, which has the sequence shown in SEQ ID NO:

2.

11. The RSV F protein mutant of claim 10, 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.

12. The RSV F protein mutant of claim 11, 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.

13. A nucleic acid molecule encoding the RSV F protein mutant of any one of claims 1-12. A recombinant vector comprising the nucleic acid molecule according to claim 13 .

15. The recombinant vector according to claim 14, characterized in that The original plasmid of the recombinant vector is pOET1.

1.

16. A recombinant virus comprising the nucleic acid molecule of claim 13 or the recombinant vector of any one of claims 14-15.

17. The recombinant virus according to claim 16, characterized in that The recombinant virus is selected from insect cell baculovirus, adenovirus, adeno-associated virus, vaccinia virus, herpes virus or retrovirus.

18. A transformed cell comprising one of the following: (1) The nucleic acid molecule according to claim 13; (2) The recombinant vector according to any one of claims 14 to 15; (3) The recombinant virus according to any one of claims 16 to 17.

19. The transformed cell according to claim 18, characterized in that The host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi or insect cells.

20. The transformed cell according to claim 19, characterized in that The insect cells are Sf9 cells, Sf21 cells or High-Five cells.

21. An immunogenic composition comprising one of the following: (1) The RSV F protein mutant according to any one of claims 1 to 12; (2) The nucleic acid molecule according to claim 13; (3) The recombinant vector according to any one of claims 14 to 15; (4) The recombinant virus according to any one of claims 16 to 17; (5) The transformed cell according to any one of claims 18 to 20.

22. A method for preparing a RSV F protein mutant according to any one of claims 1 to 12, 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.

23. Application of any of the following (1) to (6): (1) The RSV F protein mutant according to any one of claims 1 to 12; (2) The nucleic acid molecule according to claim 13; (3) The recombinant vector according to any one of claims 14 to 15; (4) The recombinant virus according to any one of claims 16 to 17; (5) The transformed cell according to any one of claims 18 to 20; (6) The immunogenic composition of claim 21; 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.

24. The use according to claim 23, 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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