RSV F protein mutants and their applications

Abstract

This invention relates to RSV F protein mutants and their applications, belonging to the field of vaccines. Based on the amino acid sequence of wild-type RSV F protein, this invention has carried out modifications in multiple aspects and methods, including disulfide bond mutations, proline mutations, cavity-filling mutations, electrostatic mutations, p27 sequence modifications, and / or trimerization domain modifications, designing a variety of RSV F protein mutants. Then, through gene synthesis, expression, and ELISA detection, this invention tested the pre-fusion conformation (pre-F) ratio and stability under high and low temperature conditions of the RSV F protein mutants, screening out several RSV F protein mutants with a high pre-fusion conformation ratio and stable structure. Finally, candidate antigens were selected for immunogenicity testing, demonstrating good immunogenicity, and indicating that they can be used to develop RSV vaccines, prepare RSV-specific antibodies, or RSV detection reagents.

Description

[0001] This application is a divisional application of patent application No. 202510136318.0, filed on February 7, 2025. Technical Field

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

[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 through direct contact and infection can cause symptoms such as bronchitis, pneumonia, asthma, and respiratory failure. RSV is the leading cause of respiratory infections in children worldwide and can also cause serious respiratory and lung infections in infants, the elderly, and immunocompromised individuals. Currently, two humanized monoclonal antibodies (Palivizumab and Nirsevimab) are available internationally to help prevent RSV infection; however, the protection provided by this passive immunity is short-lived, requires multiple injections, and the monoclonal antibodies are very expensive. Regarding prophylactic vaccines, three RSV vaccines have been approved for marketing internationally: GSK's Arexvy, Pfizer's Abrysvo, and Modena's mRESVIA. However, there are currently no RSV prophylactic vaccines available in China. Therefore, developing safe, effective, and affordable vaccines is of great significance for the prevention and control of RSV infection.

[0004] The RSV genome is 15.2 kb long and encodes 11 proteins. Among them, the attachment protein (G) and fusion protein (F) are the main glycoproteins on the viral membrane surface and are crucial for viral invasion of the human body, thus being the primary antigenic proteins in vaccine research. The G protein helps viral particles recognize and attach to receptors on the surface of host cells. The F protein promotes fusion between the viral particle and the host cell membrane, allowing the viral nucleocapsid to enter the host cell. During fusion with the host cell membrane, the F protein changes from a metastable pre-fusion conformation (Pre-F) to a stable post-fusion conformation (Post-F). Compared to 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. A 27-amino acid short peptide (p27) between amino acid residues 110-136 is released by furin protease hydrolysis, forming two subunits: F1 (137-574 aa) and F2 (26-109 aa). The two subunits are linked by disulfide bonds, and the three F1+F2 subunits form a mature F protein trimer structure. The F1 subunit contains a fusion peptide (FP) responsible for insertion into the adjacent host cell membrane, as well as heptacap repeat regions HRA (HRA) and HRB (HRB). HRA and HRB are separated during pre-F; during membrane fusion, HRB docks with HRA, coils into a helical shape, and forms a stable hexagonal helical structure, resulting in a stable post-F. The C-terminus of the F1 subunit also contains a transmembrane region (525-550 aa) and an intracellular region (551-574 aa).

[0005] In recent years, scientists have used structural biology and other techniques to elucidate the process of F protein transformation from Pre-F to Post-F and have discovered antigenic epitopes associated with neutralizing activity. Site I, II, and IV coexist in both the Pre-F and Post-F conformations and can be recognized by neutralizing antibodies, such as 131-2a (Site I), Motavizumab (Site II), and 101F / mAb19 (Site IV). Simultaneously, scientists have discovered neutralizing antibody epitopes specific to the Pre-F structure, including Site Ø, Site V, and a quadrary cleavage-dependent epitope spanning two monomers. 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 higher titers of potent neutralizing antibodies compared to the Post-F structure, making it crucial for RSV vaccine development. Currently, RSV vaccines based on the Pre-F structure have achieved high protection rates in clinical trials and have been successfully marketed abroad. The National Institute of Allergy and Infectious Diseases (NIAID) team designed a protein mutant called "DS-Cav1," which stabilizes the Pre-F structure through site mutations to form disulfide bonds (S155C, S290C) and fill structural cavities (S190F, V207L) (Jason S. McLellan, et al., Science, 2013). Pfizer's "847" protein mutant generates a stable Pre-F structure through site mutations to form disulfide bonds (T103C, I148C), fill structural cavities (S190I), and introduce electrostatic mutations (D486S) (Ye Che, et al., Science Translational Medicine, 2023). Furthermore, Janssen Pharmaceuticals' "SC-TM" protein mutant also achieved a relatively stable Pre-F structure through rigid amino acid mutation (S215P), cavity filling (N67I), and electrostatic mutation (E487Q) (Anders Krarup, et al., Nature communications, 2015). Currently, no RSV vaccine based on the Pre-F structure has been successfully marketed in China. At the same time, current strategies for constructing the Pre-F structure also suffer from problems such as low protein expression levels and insufficient structural stability. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides several mutants of wild-type RSV F protein. By mutating the amino acid sequence of wild-type RSV F protein, including substitution, deletion, or addition of amino acids, these mutants can enhance the 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-mentioned technical problems, the specific technical solution of the present invention is as follows:

[0008] In a first aspect, the present invention provides a mutant of the wild-type RSV F protein, wherein 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 abrupt change;

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

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

[0014] (f) Trimerization domain modification: delete the transmembrane region and intracellular region and add an amino acid sequence containing the trimerization domain.

[0015] The following descriptions of amino acid sequence positions are all based on the amino acid sequence of the wild-type RSV F protein shown in SEQ ID NO.1.

[0016] 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 into a pair of cysteine ​​residues (Cys, C) to form a disulfide bond between them. The mutated cysteine ​​residues are close together 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 optional embodiment, the at least one modification involves the substitution of at least one pair of amino acid residues in the F1 and / or F2 subunits of the RSV F protein with cysteine ​​(Cys, C) to form an engineered disulfide bond. In an optional embodiment, the engineered disulfide bond mutation comprises at least one pair of the following amino acid sequence positions in the wild-type RSV F protein: 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 the following: R106C+V144C, N104C+I148C, N105C+A147C, D486C+E487C, E60C+K196C, and S / N105C+M370C.

[0017] The target amino acids for cavity filling include small aliphatic amino acids (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr). These amino acids are replaced with 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 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 includes a mutation at position 190S to any one of the amino acids I, V, L, and W.

[0018] Electrostatic mutations utilize amino acid mutations to reduce ion repulsion between closely spaced residues in a protein's folded structure. In an optional embodiment, the electrostatic mutation comprises a mutation at amino acid positions 486, 487, or 489 of the wild-type RSV F protein sequence to asparagine (Asn, N), glutamine (Gln, Q), threonine (Thr, T), or proline (Pro, P). Preferably, the electrostatic mutation comprises a mutation at position 486D to N or Q, at position 487E to T, and at 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 confinement of proline to stabilize the correct folding conformation of the protein at the turn regions or specific critical positions of the polypeptide backbone, thereby reducing the possibility of misfolding or unfolding and improving the structural stability of the protein. In optional embodiments, the proline mutation includes mutating 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 mutation includes 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] p27 sequence modification refers to deleting the p27 sequence from the wild-type RSV F protein. The purpose is to remove the furin cleavage site and connect the F1 and F2 subunits using covalent bonds or linker peptides to stabilize the Pre-F conformation. Frin typically cleaves proteins after the RXK / RR sequence (where R represents arginine, K represents lysine, and X represents any amino acid). Based on the sequence of the wild-type RSV F protein (SEQ ID NO:1), the furin cleavage site is located after amino acid residues 109 and 136 of RSV F. Therefore, the p27 sequence refers to the segment of amino acid residues 110–136 of the RSV F protein (aa 110–136). In this invention, p27 sequence modification refers to deleting a fragment containing p27 from the RSV F protein. The deleted fragment can be slightly longer than p27. For example, in an optional embodiment, p27 sequence modification means that, based on the amino acid sequence of the wild-type RSV F protein, the fragment between amino acids 103-145, 104-148, or 105-147 is deleted or replaced by a linker peptide (the aforementioned "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 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 values ​​of the data range are retained and the fragments between them are deleted, or further, GS is used to replace the deleted fragment.

[0021] Trimerization domain modification refers to deleting sequences containing transmembrane and intracellular regions from wild-type RSV F protein and adding amino acid sequences containing trimerization domains. The aim is to promote the formation of a trimer conformation of the F protein and maintain its native conformation. The transmembrane and intracellular regions 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 heterologous trimerization domain, such as a Foldon domain, or a cysteine ​​zipper trimerization domain obtained by modifying the intracellular region fragment of RSV F. (1) The Foldon trimerization domain refers to the residue at the C-terminus of phage T4 fibrin. In this application, the Foldon domain can be 27 residues at the C-terminus of phage T4 fibrin 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 containing the trimerization domain means 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 containing four pairs of cysteine ​​mutations, which can be ABCD-tag or 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 the amino acid sequence containing the trimerization domain refers to deleting aa 512~574 (covering the transmembrane region and the intracellular region) at the C-terminus of the wild-type RSV F protein and adding the aforementioned ABCD-tag or ABCH-tag.

[0022] In optional embodiments, the wild-type RSVF protein mutant of the present invention further includes an adapter sequence (such as GG, GS, SAIG) and an 8*His tag sequence (HHHHHHHH). These sequences are not essential for the function of the RSVF protein, such as for inducing an immune response. Those skilled in the art will recognize such sequences and, where appropriate, understand that the RSVF protein mutant disclosed in this invention may or may not contain such sequences.

[0023] As an optional implementation, the present invention provides three groups of RSV F mutants:

[0024] The first group of RSV F mutants all have 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, which are selected from R106C+V144C, S / N105C+A147C, S / N105C+A146C, N104C+I148C or N104C+S146C.

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

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

[0029] This invention provides an RSV F protein mutant with various modifications relative to the wild-type RSV F protein to stabilize it in its pre-fusion conformation, said modifications including:

[0030] (a) Proline mutation: S215P;

[0031] (b) Cavity filling mutation: selected from S190V, S190L or S190I (preferably S190V or S190L, and more preferably S190L).

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

[0033] The position number is based on the sequence shown in SEQ ID NO: 1.

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

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

[0036] Optionally, the modification further includes p27 sequence modification, 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 fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) deleting the p27 fragment while introducing a linker sequence: using a linker (e.g., 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, wherein T103-GS-G145, N104-GS-I148 or S / N105-A147 are further preferred, and S / N105-A147 is even more preferred.

[0037] Optionally, the modification further includes trimerization domain modification: 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, and 4, respectively, with Foldon being preferred.

[0038] Preferably, the modification includes, or consists of, any one of the following sets of modifications:

[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, the preferred group is (3).

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

[0045] Preferably, the RSV F protein mutants having the modifications described in group (1), group (2), group (3) or group (4) respectively contain or have an amino acid sequence that is 99% (preferably 100%) identical to SEQ ID NO: 51, 53, 54 or 55.

[0046] (ii) The second group of RSV F mutants (all with D486C+E487C disulfide bond mutations).

[0047] The present invention also provides an RSV F protein mutant having various modifications relative to the wild-type RSV F protein to stabilize it in its pre-fusion conformation, said modifications comprising:

[0048] (a) Disulfide bond mutation in engineering modification: D486C + E487C;

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

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

[0051] The position number is based on the sequence shown in SEQ ID NO: 1.

[0052] Preferably, the modification does not include other electrostatic mutations (the disulfide bond mutation D486C+E487C can achieve the purpose of electrostatic mutation to a certain extent, because the originally negatively charged D486 and E487 are replaced with uncharged C, avoiding charge repulsion between adjacent amino acids; therefore, when using D486C+E487C, no other electrostatic mutations need to be introduced).

[0053] Optionally, the modification further includes p27 sequence modification, 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 fragment without introducing a linker: directly covalently bonding the sequences before and after the deleted fragment; (2) deleting the p27 fragment while introducing a linker sequence: using a linker (e.g., 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, wherein T103-GS-G145 or S / N105-A147 is preferred.

[0054] Optionally, the modification further includes trimerization domain modification: 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, and 4, respectively, with Foldon being preferred.

[0055] Preferably, the modification includes any one or a combination 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, the preferred groups are (2), (4), (5) or (6).

[0064] In the above groups, "Foldon" refers to the deletion of the transmembrane and intracellular regions of RSV F and the addition of a sequence containing the Foldon trimerization domain.

[0065] Preferably, the RSV F protein mutants having the modifications described in group (1), group (2), group (3), group (4), group (5), group (6) or group (7) respectively contain or have an amino acid sequence that is 99% (preferably 100%) identical to SEQ ID NO: 46, 47, 50, 58, 64, 65, 73.

[0066] (iii) The third group of RSV F mutants (all have disulfide bond 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 having various modifications relative to the wild-type RSV F protein to stabilize it in its pre-fusion conformation, said modifications comprising:

[0068] (a) Disulfide bond mutations in engineering modification: 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 mutation: selected from S190I, S190L, S190V or S190W;

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

[0072] The position number is based on the sequence shown in SEQ ID NO: 1.

[0073] One possible approach is as follows: in the modification, the disulfide bond mutation is selected as R106C+V144C, the cavity filling mutation is selected as S190I, S190L, S190V or S190W (preferably S190L, S190V or S190W), and the electrostatic mutation is selected as D486N, D486Q, E487T, D489P or D489Q (preferably D486N, D486Q, E487T or D489P).

[0074] Another optional approach is that, 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 (preferably S190I), and the electrostatic mutation is selected from D486N, D486Q, E487T (preferably D486N).

[0075] Optionally, the modification may or may not include p27 sequence modification, where p27 sequence modification refers to deleting the p27 sequence. Optionally, the p27 sequence modification may take one of the following approaches: (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 (e.g., 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 trimerization domain modification: 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 its sequences are shown in SEQ ID NO: 2, 3, and 4, respectively, with Foldon being preferred.

[0077] Preferably, the modification includes any one or a combination of the following:

[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 the above groups, "Foldon" refers to the deletion of the transmembrane and intracellular regions of RSV F and the addition of a sequence containing the Foldon trimerization domain.

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

[0096] As an optional implementation, the present invention provides 79 specific RSV F mutants, the modifications of which relative to the wild-type RSV F protein are shown in Tables 1, 3, 5 and 7.

[0097] In a second aspect, the present invention provides biomaterials, said biomaterials comprising any one of the following:

[0098] (a) The 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 insect cell baculovirus, adenovirus, adeno-associated virus, vaccinia virus, herpesvirus 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), wherein the host cell of the transformed cell is selected from mammalian cells, bacteria, yeast, fungi, or insect cells; preferably, the insect cell is an Sf9 cell, an Sf21 cell, or a High-Five cell.

[0102] Thirdly, the present invention also provides a method for preparing the above-mentioned RSV F protein mutant, comprising the steps of: constructing a recombinant vector, extracting the recombinant vector, transfecting the recombinant vector into a host cell and expressing the protein, and purifying the expression product.

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

[0104] Fifthly, the present invention also 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:

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

[0106] (b) Prepare a drug for the prevention and / or treatment of RSV infection; preferably, the drug includes a recombinant protein vaccine, a vector vaccine or a nucleic acid vaccine.

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

[0108] Terminology definition:

[0109] 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. This virus can cause lower respiratory tract disease (LRTD) symptoms in infants, the elderly, and immunocompromised adults, including interstitial pneumonia and bronchiolitis.

[0110] In this application, the term "mutant" refers to a protein that has one or more changes in its amino acid sequence or protein structure compared to the wild-type protein. These changes may include, but are not limited to, the deletion, insertion, substitution, shortening, and / or loss of one or more amino acids, as well as modifications or cleavages of the protein structure. In this application, "mutant" specifically refers to a respiratory syncytial virus (RSV) F protein mutant.

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

[0112] In this application, the term "cavity-filling mutation" refers to the replacement of amino acid residues in the wild-type RSV F protein with amino acids intended to fill the structural cavities of the mature F protein to stabilize the RSV Pre-F structure. These cavity-filling amino acids are often amino acids with large 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 this application, the term "electrostatic mutation" refers to the substitution of amino acid residues in wild-type RSV F protein with a desired amino acid, which reduces ionic repulsion between closely spaced residues in the protein fold structure, thereby stabilizing the RSV Pre-F structure. The electrostatic mutation includes a mutation at amino acid position 486, 487, or 489 of the wild-type RSV F protein sequence to asparagine (Asn), glutamine (Gln), or threonine (Thr).

[0114] In this application, the term "proline mutation" refers to the substitution of amino acid residues in 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 restricts the rotational freedom of proline within the protein, allowing proline to form fixed folds and turns, thus increasing protein stability.

[0115] In this 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. This amino acid short peptide is hydrolyzed by furin protease and released to form two subunits, F1 (137-574 aa) and F2 (26-109 aa).

[0116] In this application, the term "p27 sequence modification" refers to deleting a segment containing p27 from the wild-type RSV F protein. That is, the deleted segment should cover p27 and may be slightly longer than p27 (extending by about 1 to 15 amino acid residues upstream and / or downstream of p27). For example, the segment 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 of the numerical range; for example, the segment between amino acid residues 104 to 144 refers to the segment composed of amino acids 105 to 143). After deleting the aforementioned segment, the preceding and following segments can be directly covalently linked or linked using a linker peptide (e.g., GG, GS, GSGS, GGGS, etc.), with GS being preferred.

[0117] In this application, the term "trimerization domain" refers to an amino acid sequence that forms a trimer at the C-terminus of a 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). In this application, a trimer refers to a complex formed by the three RSV F protein F1+F2 subunits.

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

[0119] In this application, the term "ABCH-tag" refers to the addition of a cysteine ​​zipper (containing an amino acid sequence with 4 cysteine ​​mutations) to the C-terminus of a wild-type RSV F protein with its transmembrane and intracellular regions deleted. The amino acid sequence of the ABCH-tag is CCHNVNACCSTTNICCTTTNICCTT.

[0120] In this application, the terms “Mota” or “motavizumab” refer to the antibody described in the paper by Herren et al. (Herren Wu, et al., Journal of Molecular Biology, 2007).

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

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

[0124] In this 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 changes in the chemical bonds in a protein molecule or changes in the folding pattern of a polypeptide.

[0125] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0126] The beneficial effects of this invention are:

[0127] The RSV F protein mutants described in this invention significantly enhance 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 they have extremely high potential to become an effective component of RSV vaccines. Compared to existing technologies (such as the 847 protein), some RSV protein mutants provided by this invention have a higher pre-F ratio or better stability under high or low temperature conditions. Attached Figure Description

[0128] Figure 1 The pOET1.1 baculovirus transfer plasmid map used in Example 1 of this invention.

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

[0130] Figure 3 : Gel electrophoresis analysis of the reducing and non-reducing properties of each mutant after purification.

[0131] Figure 4 Western blotting analysis of each mutant after purification.

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

[0133] Figure 6 Results of antibody binding assays in mice immunized with different antigen proteins. Detailed Implementation

[0134] To more accurately and clearly understand and grasp the objectives, technical solutions, and advantages of this invention, the following will provide an in-depth interpretation and detailed description of the embodiments of this invention based on the relevant accompanying drawings. Please note that the embodiments described herein represent only some application examples of this invention and do not encompass all embodiments.

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

[0136] RSVF mutant design and screening includes the following steps:

[0137] (1) Design and gene synthesis of mutants: First, the sequence of wild-type RSV F was obtained: Based on the publicly available F protein sequence of type A RSV strains in the NCBI database, the amino acid with the most occurrences at each site (common amino acid) was calculated, and the common amino acid sequence of RSV F was constructed (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 wild-type RSV F protein shown in SEQ ID NO: 1, modifications were made to design recombinant RSV F protein (mutants): Based on existing data and combined with structural biology analysis, this invention designed a variety of RSV F mutants (recombinant proteins), which respectively contain 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, the nucleic acid sequence was determined by codon optimization according to the host Sf9 insect cell and the whole gene was synthesized.

[0138] (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 In ), and using flashBAC TM The baculovirus expression system kit co-transfects recombinant plasmids and baculovirus genomes into Sf9 insect cells. Transfection procedures are performed according to the kit instructions. Seven days after transfection, the cell supernatant is collected, containing P0 generation virus with the recombinant exogenous gene. The P0 generation virus is then transfected into new Sf9 cell wells to obtain P1 generation virus. Six to seven days after P1 generation virus infection, the cell supernatant is collected for ELISA to detect the conformation and stability of various antigens.

[0139] (3) ELISA detection of antigen proteins: The conformation and stability of each antigen protein are detected by ELISA.

[0140] The specific steps for ELISA detection are as follows: Coat 100 μl of antibodies such as Motavizumab (abbreviated as Mota, which can bind to both Pre-F and Post-F), AM14 (binds only to Pre-F), D25 (binds only to Pre-F), or hRSV90 (binds only to Pre-F) into a 96-well plate at 4°C overnight. The next day, wash the plate three times with PBST and pat dry. Then, block the plate with 5% skim milk prepared with PBST (200 μl / well, room temperature for 1 hour). Divide the cell supernatant collected in (2) into three equal portions and treat them at high temperature (above 50°C for 1 hour), low temperature (stored at 4°C for a long time or repeatedly frozen and thawed), and no treatment, respectively. After blocking, wash the 96-well plate three times with PBST and pat dry. Add 100 μl of untreated and cell supernatant samples treated under various pressure conditions to each well and incubate at room temperature for 1-2 hours. After washing three times with PBST, add 100 μl of Rabbit His-tag Antibody and incubate at room temperature for 1 hour. After washing three times with PBST, add 100 μl of anti-rabbit IgG antibody (HRP) and incubate at room temperature for 1 hour. After washing five times with PBST, blot dry and add 100 μl of freshly prepared TMB chromogenic solution to each well. After 10-15 minutes of development, add stop solution to terminate the development and read the absorbance at wavelengths of 450 nm and 570 nm using a microplate reader. The final absorbance readings are A450 nm - A570 nm.

[0141] The stability assessment method is as follows: Based on the ELISA test results, 1) On the one hand, the Pre-F ratio of the RSV F protein (hereinafter referred to as the candidate antigen protein) designed in this invention compared with the positive control 847 protein (the antigen protein used in Pfizer's RSV vaccine Abrysvo, which is already marketed in the United States, shown in SEQ ID NO: 84 in this application) is calculated. If the ratio is greater than 1, it indicates that the pre-F conformation ratio (%Pre-F, i.e., the proportion of Pre-F to the sum of Pre-F and 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 indicates 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 to evaluate the overall stability of the candidate antigen protein and the stability of the Pre-F portion. Based on the results of each round of screening, this invention defines a protein stability value of not less than 0.6 (at least not less than 0.55) after stress treatment as indicating excellent protein stability.

[0142] (4) Scheme adjustment and improvement: Based on the ELISA test results of the previous step, select the better mutation type for the next round of screening based on the antigen with excellent performance.

[0143] Following the research approach described above, four rounds of RSV F mutant design and screening were carried out, as detailed in Examples 2-5 below.

[0144] Example 2: First round of RSV F mutant design and screening

[0145] Following the operating procedures described in Example 1, the first round of mutant design and ELISA detection was conducted. The main purpose of this round of experiments was to explore various methods of mutation, including disulfide bond mutation, cavity-filling mutation, and electrostatic mutation, and to test their combined effects through orthogonal experiments.

[0146] The design strategy for RSV F mutants is as follows: (1) Regarding disulfide bond mutations, eight designs were carried out (R106C+V144C, N104C+S146C, N104C+A147C, N104C+I148C, S / N105C+G145C, S / N105C+S146C, S / N105C+A147C, Q34C+G471C); (2) Regarding proline mutations, S215P was used or A490P was added to it; (3) Cavity filling Regarding mutations, four designs were proposed (S190V, S190L, S190W, S190I); (4) Regarding electrostatic mutations, five designs were proposed (D486N, D486Q, E487T, D489P, D489Q); (5) Regarding the p27 sequence, no p27 sequence modification was performed, or T103-GS-G145 was adopted (deleting the segment between amino acids 103 and 145 and introducing an additional linker GS); (6) Regarding the trimerization domain: Foldon was adopted.

[0147] Based on the above-mentioned various modification methods, when using them in combination to construct RSV F mutants, (1) on the one hand, the selection of disulfide bond mutation (R106C+V144C), proline mutation (including S215P), p27 sequence modification (without modification) and trimerization domain (Foldon) is fixed, and orthogonal design and screening (BR01, BR06~BR23) of cavity-filling mutation (S190I, S190V, S190L, S190W) and electrostatic mutation (D486N, D486Q, E487T, D489P, D489Q) are carried out; (2) on the other hand, the proline mutation (including S215P), cavity-filling mutation (S190I), and electrostatic mutation (D486N, D486Q) are fixed. (486N), p27 sequence modification (without modification) and selection of trimerization domain (Foldon) were carried out to make 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, p27 sequence modification (T103-GS-G145) was carried out 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.

[0148] 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 modifications made to the mutant compared to wild-type RSV F and their corresponding sequence numbers. The corresponding ELISA test results for each mutant are shown in Table 2.

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

[0150]

[0151] Remark:

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

[0153] (2) In the column “p27 sequence modification”: “ / ” indicates that the p27 sequence in the wild-type RSV F protein has not been modified; “T103-GS-G145” indicates that the segment between amino acids 103 and 145 of the wild-type RSV F protein has been deleted (excluding the terminal values, i.e., amino acids 103 and 145, i.e., only the segment between amino acids 104 and 144 has been deleted) and replaced with GS, where GS is a linker.

[0154] (3) Regarding S / N105C+G145C: "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" means that the site is mutated from S / N in the wild type to C.

[0155] Table 2: ELISA results of the first round of RSV F mutant detection

[0156]

[0157] Remark:

[0158] 1 The formula for calculating the "Pre-F ratio" is: %Pre-F = OD AM14 / OD Mota The same applies below;

[0159] 2 "Pre-F ratio compared to 847" represents the ratio of the Pre-F protein proportion of each mutant to the positive control 847. For example, the formula for calculating the Pre-F ratio of BR06 compared to 847 is: (%Pre-F) BR06 ) / (%Pre-F 847 (The same applies below);

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

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

[0162] Based on the results of the first round of screening, all antigen proteins were expressed normally and could be screened using the ELISA method. The first round of screening was mainly to explore the combined effects of various design strategies:

[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) all helped 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) showed only moderate performance. Since N104C+I148C and S / N105C+A147C are novel, unreported disulfide bonds with excellent performance, they were selected for the next round of screening.

[0164] Regarding cavity filling, the proportion of Pre-F conformations was generally higher after adding S190V and S190L mutations than that of S190W mutations, and they were selected for the next round of screening.

[0165] Regarding electrostatic mutations, the addition of mutations such as D486N, D486Q, and E487T is more stable in the Pre-F conformation than other mutations, and they are selected for the next round of antigen screening.

[0166] Example 3: Second round of RSV F mutant design and screening

[0167] The main purpose of this round of experiments is to test the combined effect of the selected disulfide bond mutation, cavity filling mutation, and electrostatic mutation. At the same time, the pressure conditions were increased (the high temperature was increased from 50℃ to 60℃) in this round of experiments to test the thermal stability of the constructed RSV F mutant. BR26 and BR29, which performed well in the previous round of screening, were also included in this round of pressure condition testing.

[0168] The RSV F mutant design strategy is as follows: Based on the results of the previous embodiment, in this round of RSV F mutant design, (1) for disulfide bond mutation, N104C+I148C and S / N105C+A147C are selected for this round of screening, and D486C+E487C is introduced; (2) for proline mutation, S215P is still used; (3) for cavity filling mutation, S190V or S190L is selected; (4) for electrostatic mutation, D486N, D486Q or E487T is selected; (5) for p27 sequence modification, T103-GS-G145 is still used, or no p27 sequence modification is performed; (6) for 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 is the name of the mutant, and the same row lists the modifications made to the mutant compared to wild-type RSV F and their corresponding sequence numbers. The corresponding ELISA test results for each mutant are shown in Table 4.

[0170] Table 3: Second Round RSV F Mutant Design

[0171]

[0172] Table 4: ELISA results of the second round of RSV F mutant detection

[0173]

[0174] In the second round of screening, we increased the high-temperature treatment pressure (from 50℃ to 60℃) in hopes of finding a superior antigen design. The results showed:

[0175] (a) After treatment at 60℃, the protein stability (AM14) of most mutants decreased significantly, while the Pre-F conformation of RSV F mutants (BR44~BR46) with added D486C+E487C disulfide bond mutation did not change significantly, especially BR46 and BR47. Therefore, the disulfide bond mutation D486C+E487C was considered to be retained in subsequent screening.

[0176] (ii) Relatively speaking, the disulfide bond mutations N104C+I148C or S / N105C+A147C contribute less to the stability of RSV F mutants than D486C+E487C. However, considering that the extraction, purification and preservation of RSV F mutants generally do not involve high temperatures above 50°C, N104C+I148C or S / N105C+A147C are also usable disulfide bond mutation methods under non-extreme temperature conditions. (1) Regarding N104C+I148C, compared with 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, compared with 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 combined with other cavity-filling mutations (such as S190L / V) and electrostatic mutations (D486Q / E487T), but the protein stability (AM14) is slightly higher.

[0177] (III) Meanwhile, the results of this round of screening showed that p27 sequence modification (BR46 and BR47) significantly improved Pre-F stability compared to no modification (BR44 and BR45). Therefore, p27 sequence modification was considered for all subsequent screenings. In cavity-filling mutations, both S190L and S190V helped stabilize the Pre-F conformation; S190L, which showed slightly better results after p27 sequence modification, was selected for the next round of screening. In electrostatic mutations, D486N, D486Q, and E487T all performed well; E487T, which showed slightly better results, was selected for the next round of antigen screening.

[0178] Example 4: Third round of RSV F mutant design and screening

[0179] The main purpose of this round of experiments is to expand the modification methods of the p27 sequence and trimerization domain, and to test the combined effect with disulfide bond mutations, cavity-filling mutations, and electrostatic mutations selected 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℃ to 65℃, and low temperature storage changed from 4℃ to repeated freeze-thaw treatment) in the hope of screening antigen designs that can better tolerate extreme conditions. BR47, which performed well in the previous round of screening, was also included in this round of pressure condition testing.

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

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

[0182] Table 5: Third Round RSV F Mutant Design

[0183]

[0184] Remark:

[0185] The p27 sequence modification column indicates the following: 1) "S / N105-A147" means the deletion of amino acids 105-147 of the wild-type RSV F protein (excluding the terminal values, i.e., amino acids 105 and 147, i.e., only amino acid 106-146 is deleted). Similarly, "N104-I148" means the deletion of amino acids 104-148 of the wild-type RSV F protein (excluding the terminal values); 2) "T103-GS-G145" means the deletion of amino acids 103-145 of the wild-type RSV F protein (excluding the terminal values) and its replacement with GS. Similarly, "N104-GS-I148" means the deletion of amino acids 104-148 of the wild-type RSV F protein (excluding the terminal values) and its replacement with GS, and "S / N105-GS-A147" means the deletion of amino acids 105-147 of the wild-type RSV F protein (excluding the terminal values) and its replacement with GS.

[0186] Table 6: ELISA results of the third round of RSV F mutant detection

[0187]

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

[0189] Regarding p27 sequence modification, different modification methods significantly affected the stability of the Pre-F conformation: among the five modification methods, S / N105-A147 showed the best performance. As shown in Table 6, without adding disulfide bond mutations: comparing different p27 sequence modification schemes (i.e., antigen designs BR49, BR52, BR53, BR54, and BR55), BR54 (p27 sequence modified to S / N105-A147) outperformed other designs after high-temperature pressure treatment, but showed no significant difference after low-temperature pressure treatment. Similarly, with the addition of disulfide bond mutations: comparing different p27 sequence modification schemes (i.e., antigen designs BR47, BR56, BR57, BR58, and BR59), BR58 (p27 sequence modified to S / N105-A147) outperformed other designs in high-temperature pressure treatment, but showed no significant difference after low-temperature pressure treatment. Therefore, S / N105-A147 was selected for the next round of screening.

[0190] Furthermore, it's worth noting that although BR50 (D486C+E487C, S215P, S190L, T103-GS-G145) did not undergo trimerization domain modification, it still performed better than BR47 (D486C+E487C, S215P, S190L, T103-GS-G145, Foldon) in terms of pre-F ratio and structural stability. This is presumably because the D486C+E487C disulfide bond near the C-terminus of the RSV F protein helps with protein trimerization to some extent. Therefore, trimerization domain modification is not necessary when using the D486C+E487C disulfide bond mutation.

[0191] Example 5: Fourth round of RSV F mutant design and screening

[0192] The primary objective of this round of experiments is to expand the methods of proline mutation modification, with a secondary objective of making more attempts at disulfide bond mutation and testing its combined effect with cavity-filling mutation, electrostatic mutation, and p27 sequence modification selected based on the results of the previous round.

[0193] The RSV F mutant design strategy is as follows: Based on the results of the previous embodiment, 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, and S / N105C+M370C are introduced alone or in combination with D486C+E487C. The purpose of introducing these new disulfide bonds is to replace proline mutation and obtain a more stable Pre-F conformation; (2) in terms of proline mutation, in addition to the previous S215P, a variety of possibilities (Q210P, S211P, C212P, S213P, I214P, N216P, I217P, E218P, Q2) were tested. 10P+C212P, S211P+S213P, C212P+I214P, I214P+N216P, N216P+E218P, S213G+I214P+N216G, S211G+C212P+I214G), to compare their effects; (3) In terms of cavity filling mutation, S190L is still used or cavity filling mutation is not introduced; (4) In terms of electrostatic mutation, E487T is still used or electrostatic mutation is not introduced; (5) In terms of p27 sequence modification, S / N105-A147 is uniformly used; (6) In terms of 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 is the name of the mutant, and the same row lists the modifications made to the mutant compared to wild-type RSV F and their corresponding sequence numbers. The corresponding ELISA test results for each mutant are shown in Table 8.

[0195] Table 7: Fourth Round RSV F Mutant Design

[0196]

[0197] Table 8: ELISA results of the fourth round of RSV F mutant detection

[0198]

[0199] The results of the fourth round of screening show:

[0200] Regarding proline mutations, Q210P, S211P, and S211P+S213P all significantly improved the conformational stability of the Pre-F protein.

[0201] Regarding disulfide bond mutations, a comparison of the ELISA results of BR79, BR80, BR81, BR82, and BR83 reveals the following: (1) BR83 performs relatively well, indicating that S / N105C+M370C can help stabilize the Pre-F conformation in the absence of rigid amino acid mutations (proline mutations). (2) In contrast, although BR80 appears to have better data (e.g., a higher Pre-F ratio of 2.18 compared to 847 protein), the actual expression level of this mutant protein is extremely low (e.g., after treatment at 65℃ for 1 h, OD...). Mota =0.111, OD AM14 =0.136, indicating that the antibody OD value is very low, even though %Pre-F=OD AM14 / OD Mota =1.22 and “Pre-F ratio compared to 847” is as high as 2.18), so the E60C+K196C disulfide bond is not preferred. (3) In addition, the Pre-F ratio of BR79 and BR81 is lower than that of 847 protein, so the L171C+K191C disulfide bond is also not preferred.

[0202] After four rounds of screening, mutants with superior overall performance (BR47, BR54, BR58, BR64, BR65, BR73) were selected as representatives. These were then subjected to ELISA testing with other neutralizing antibodies to further confirm the conformational stability of the designed antigen and its potential to induce multiple neutralizing antibodies. Results are as follows: Figure 2 As shown, multiple mutants can bind to different strong neutralizing antibodies, and their Pre-F conformation is relatively stable.

[0203] Example 6: Large-scale expression and purification of antigen protein

[0204] Recombinant baculovirus expressing the antigen protein was used to infect 1 L of HighFive cells in logarithmic growth phase (viable cell density approximately 2 × 10⁶ cells / year) at a ratio of 1:100. 6 Cells / ml (viability greater than 95%). Inoculated cells were cultured at 27°C with shaking at 120 rpm for 3-4 days. When the cell diameter significantly increased and the viable cell density and viability significantly decreased, 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 using a nickel column. The specific purification steps are as follows:

[0205] Equilibration: Equilibrate the nickel column (UniNTA-80Ni) using 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.

[0206] Sample loading: Slowly add the filtered sample into the equilibrated nickel column, allowing the sample to flow through the column three times to ensure that the target protein is fully bound to the nickel column.

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

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

[0209] The collected eluent was placed into a SnakeSkin dialysis bag (Thermo Scientific™), and the salt ions in the eluent were replaced with PBS dialysis buffer. After dialysis, the solution in the dialysis bag was collected, aliquoted, and stored at -80°C. Simultaneously, 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).

[0210] The results are as follows Figure 3 and Figure 4 As shown, the molecular weights of the monomers and trimers of the various antigenic proteins expressed using the insect cell-baculovirus expression system were as expected. Western blot analysis using the specific antibody D25 confirmed that the protein bands were RSVF proteins. The purity of each mutant antigen was as expected and can be used for subsequent experiments.

[0211] Example 7: Animal Experiment

[0212] Female Balb / C mice aged 6-8 weeks were randomly divided into groups of 5 mice each. On days 0 and 21, mice were immunized intramuscularly with different doses of either a vaccine prepared from purified antigen protein expressed in insect cells or a mixture of antigen protein and aluminum adjuvant, 50 μl in each leg, for a total of 100 μl. A negative control group received 100 μl of saline. Blood was collected from mice on days 14 and 35, and serum was separated for antibody detection, as follows:

[0213] 100 μl of purified antigen protein was coated onto a 96-well plate. The plate was washed three times with PBST the next day and then blotted dry. It was then blocked with 5% skim milk prepared with PBST (200 μl / well, 1 hour at room temperature). After washing three times with PBST, the plate was blotted dry. Serum samples were serially diluted 2-fold with PBST solution containing 1% skim milk, with a 1:100 detection threshold. 100 μl of this solution was added to each well and incubated at room temperature for 1 hour. After washing three times with PBST, 100 μl of Goat Anti-Mouse IgG-HRP antibody was added and incubated at room temperature for 1 hour. After washing five times with PBST, the plate was blotted dry, and 100 μl of freshly prepared TMB chromogenic solution was added to each well. After 10-15 minutes of development, stop solution was added, and the absorbance was read at 450 nm using a microplate reader. The cut-off value was defined as 2.1 times the absorbance of the negative control. The maximum dilution factor with an absorbance ≥ the cut-off value was used to determine the antibody titer of the sample.

[0214] The results are as follows Figure 5 and Figure 6 As shown: In the dosage and adjuvant exploration experiments ( Figure 5 Immunization with different doses of the BR47 antigen protein stock solution or vaccines combined with aluminum adjuvants induced high titers of binding antibodies in mice, demonstrating high levels of immunogenicity. In experiments comparing different antigen designs ( Figure 6 Multiple antigen proteins obtained through in vitro screening can induce mice to produce high-titer binding antibodies, demonstrating that the established in vitro screening method can be used to screen RSV F antigen proteins, and that the screened antigens have the potential to develop excellent RSV vaccines.

[0215] The detailed analysis and description of the embodiments of this invention are not intended to limit the scope of protection of this invention, but merely to illustrate the process, results, and application of this invention by using selected embodiments as examples. Based on the embodiments described herein, all other implementations that can be derived by those skilled in the art without inventive effort or through equivalent substitution are included within the scope of protection of this invention.

Claims

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

2. A nucleic acid molecule encoding the RSV F protein mutant of claim 1.

3. A recombinant vector comprising the nucleic acid molecule of claim 2.

4. The recombinant vector according to claim 3, characterized in that, The original plasmid of the recombinant vector is pOET1.

1.

5. A recombinant virus comprising the nucleic acid molecule of claim 2 or the recombinant vector of any one of claims 3-4.

6. The recombinant virus according to claim 5, characterized in that, The recombinant virus is selected from insect cell baculovirus, adenovirus, adeno-associated virus, vaccinia virus, herpesvirus, or retrovirus.

7. Transformed cells, which include one of the following: (1) The nucleic acid molecule according to claim 2; (2) The recombinant vector according to any one of claims 3-4; (3) The recombinant virus according to any one of claims 5-6.

8. The transformed cell according to claim 7, characterized in that, The host cells of the transformed cells are selected from mammalian cells, bacteria, fungi, or insect cells.

9. The transformed cell according to claim 8, characterized in that, The insect cells are Sf9 cells, Sf21 cells, or High-Five cells.

10. An immunogenic composition comprising one of the following: (1) The RSV F protein mutant of claim 1; (2) The nucleic acid molecule according to claim 2; (3) The recombinant vector according to any one of claims 3-4; (4) The recombinant virus according to any one of claims 5-6; (5) The transformed cells according to any one of claims 7-9.

11. The method for preparing the RSV F protein mutant according to claim 1, comprising the following steps: Construction of recombinant vectors, extraction of recombinant vectors, transfection of recombinant vectors into host cells and expression of proteins, and purification of expression products.

12. Application of any one of (1) to (6) below: (1) The RSV F protein mutant of claim 1; (2) The nucleic acid molecule according to claim 2; (3) The recombinant vector according to any one of claims 3-4; (4) The recombinant virus according to any one of claims 5-6; (5) The transformed cells according to any one of claims 7-9; (6) The immunogenic composition according to claim 10; The application is selected from any one of the following (a) to (b): (a) To prepare medicines for the prevention and / or treatment of RSV infection; (b) Preparation of diagnostic reagents for RSV.

13. The application according to claim 12, characterized in that, The drug is a recombinant protein vaccine, vector vaccine, or nucleic acid vaccine used for the prevention and / or treatment of RSV.

Images