Pre-fusion RSV F protein mutant and application thereof

Abstract

The invention discloses a pre-fusion RSV F protein mutant and application thereof. The invention provides a new RSV F protein mutation site, it is found for the first time that 157C and 183C substitution and / or 170C and 177C substitution in the respiratory syncytial virus F protein has an important effect on stabilizing conformation before fusion, S155C and S290C substitution can be not used, the immunogenicity of the respiratory syncytial virus F protein before fusion is improved, the neutralizing antibody titer induced by the respiratory syncytial virus F protein is greatly improved, and the immunogenicity of the respiratory syncytial virus F protein before fusion is improved. Meanwhile, the high expression level of the antigen in mammalian cells is achieved, and the method is used for preparing RSV diagnostic reagents, antibodies and various vaccines with the pre-fusion F protein as the antigen.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. CN202311762067.4, filed on December 20, 2023, the entire text of which is incorporated herein by reference. Technical Field

[0003] The present invention relates to the fields of molecular biology and immunology, and in particular to a pre-fusion RSV F protein mutant and application thereof. Background Art

[0004] Respiratory syncytial virus (RSV) belongs to the genus Orthopneumovirus of the family Pneumoviridae and is one of the main pathogens causing lower respiratory tract infections in infants, young children and the elderly. As early as the 1960s, the United States conducted research on inactivated HRSV vaccine (FI-RSV), but an enhanced respiratory disease (ERD) event occurred, which affected the development of HRSV vaccine (Kim HW, Canchola JG, Brandt CD, Pyles G, Chanock RM, Jensen K et al (1969) Respiratory syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am J Epidemiol 89(4):422–434).

[0005] RSV is an enveloped single-stranded negative-sense RNA virus, divided into two subtypes, A and B, with a total genome length of about 15,200 bp. HRSV virus encodes 11 proteins, including 3 non-structural proteins (NS1, NS2 and M2-2) and 8 structural proteins. Among the 8 structural proteins, 3 proteins are located on the surface membrane of the virus: small hydrophobic protein (SH), attachment protein G (G) and fusion protein (F) glycoprotein; 5 proteins are located inside the virus: nucleoprotein (N), phosphoprotein (P), matrix protein M (M), M2-1 protein expressed by M2 gene and RNA-dependent RNA polymerase L (Large polymerase protein, L) protein.

[0006] Among them, F protein and G protein are crucial to the infectivity and pathogenesis of the virus. G protein targets ciliated cells in the airways and mediates the adhesion of the virus to host cells. F protein initiates the entry of the virus by fusing the virus with the cell membrane, and causes cell fusion in the infected cells in the late stage of infection, resulting in the production of characteristic syncytia. The G and F proteins of HRSV carry antigenic determinants that can cause the host to produce neutralizing antibodies. Because the F protein plays an important role in the virus invading host cells and is relatively conserved in the two subtypes of HRSV, it is the preferred target for vaccine development. However, the F protein presents two different conformations before and after fusion. One is the lollipop-shaped pre-fusion conformation pre F, which exists on the virus surface before virus-cell interaction, and the other is the crutch-shaped post-fusion conformation post F, which is formed after the fusion of the virus and cell membrane. The antigenic epitopes of the F protein also vary with different conformations. Pre F has 6 antigenic epitopes (site Ⅰ, Ⅱ, Ⅲ, Ⅳ, Ⅴ and Ø), but post F has only 4 antigenic epitopes (site Ⅰ, Ⅱ, Ⅲ and Ⅳ). The Ø epitope with the highest ability to induce neutralizing antibodies only exists in the pre-fusion conformation (Li, H., Cao, L., Zhang, Y. et al. Human respiratory syncytial virus F protein expressed in Pichia pastoris or Escherichia coli induces protective immunity without inducing enhanced respiratory disease in mice. Arch Virol 165, 1057–1067 (2020). https: / / doi.org / 10.1007 / s00705-020-04578-7 ).

[0007] The F glycoprotein of human RSV is first translated from mRNA into a single 574 amino acid polypeptide precursor (referred to as "F0" or "F0 precursor"), which contains a signal peptide sequence (amino acid 1-25) at the N-terminus. After translation, the signal peptide is removed by a signal peptidase in the endoplasmic reticulum. The remainder of the F0 precursor (i.e., residue 26-574) can be further cut by a cell protease (especially furin) at two polybasic sites (positions 109 / 110 and 136 / 137), removing the intervening sequence (amino acid 110-136) of 27 amino acids named pep27 and generating two connected fragments named F1 (C-terminal portion; amino acid 137-574) and F2 (N-terminal portion; amino acid 26-109). F1 contains a hydrophobic fusion peptide and two heptad repeat regions (HRA and HRB) at its N-terminus. HRA is close to the fusion peptide, and HRB is close to the TM domain. F1 and F2 fragments are linked together by two disulfide bonds. The uncleaved F0 protein or F1-F2 heterodimer without a signal peptide sequence can form RSV F protomers. Three such protomers assemble to form the final RSV F protein complex, which is a trimer of three protomers.

[0008] In 2013, Jason S. McLellan et al. stabilized the pre F protein (DS-Cav1) by engineering the disulfide bond mutations S155C and S290C ("DS"), the cavity-filling mutations S190F and V207L ​​("Cav1"), and the attached C-terminal heterologous trimerization domain to the wild-type F protein amino acid sequence. The immunogenicity of the pre F protein was increased by about 70 times compared with the post F protein (McLellan JS, Chen M, Joyce MG, et al. Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus [published correction appears inScience. 2013 Nov 22;342(6161):931]. Science. 2013;342(6158):592-598. doi:10.1126 / science.1243283), and then further optimized it for four rounds. It was found that DS2-Cav1, which was designed by adding mutations and modifications such as 149C and 458C substitutions to the non-natural inter-protomer disulfide bonds between the cysteines introduced on the basis of DS-Cav1, induced neutralizing activity about four times higher than that of DS-Cav1 after two immunizations in RSV-naive CB1F6 / J mice (Joyce MG, Zhang B, Ou L, et al. Iterative structure-basedimprovement of a fusion-glycoprotein vaccine against RSV. Nat Struct MolBiol. 2016;23(9):811-820. doi:10.1038 / nsmb.3267). In 2023, the United States approved a vaccine based on DS-Cav1 (RSVPreF3) and AS01 E Adjuvanted RSV vaccine (https: / / www.fda.gov / vaccines-blood-biologics / arexvy), Moderna's mRNA vaccine (mRNA-1345) based on NIH's DS2-Cav1 has also entered clinical trials (NCT05127434). However, how to further improve the immunogenicity of antigens and improve the expression of antigens is an important research direction for the industrialization of recombinant RSV vaccines. Summary of the invention

[0009] The purpose of the present invention is to provide a pre-fusion RSV F protein mutant and application thereof.

[0010] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a pre-fusion RSV F protein mutant with higher immunogenicity and higher expression level, wherein the mutant comprises at least one intra-protomer disulfide bond mutation and / or at least one inter-protomer disulfide bond mutation relative to the native RSV F protein.

[0011] Wherein, the intra-protomer disulfide bond mutation comprises the following (1) and / or (2): (1) 157C and 183C substitution; (2) Replaced by 170C and 177C.

[0012] In the present invention, the RSV F protein position corresponds to the amino acid sequence of the reference F0 polypeptide shown in SEQ ID NO:1.

[0013] Furthermore, the intra-protomer disulfide bond mutation further comprises any one or a combination of the following: (3) S55C and L188C substitutions; (4) L142C and N371C substitutions; (5) T103C and I148C substitutions; (6) L142C and N371C substitutions.

[0014] Furthermore, the inter-protomer disulfide bond mutation comprises any one or a combination of the following: 1) 96C and 279C substitutions (e.g. L96C and G279C substitutions); 2) 149C and 458C substitutions (e.g. A149C and Y458C substitutions); 3) 183GC and 428C substitution (for example: N183GC and N428C substitution); 4) 369C and 455C substitution; 5) 100C and 362C replacement; 6) 99C and 362C substitution; 7) 98C and S362C substitutions; 8) 100C and 361C replaced; 9) 99C and 361C substitution; 10) 98C and S361C substitutions.

[0015] Furthermore, the mutant further comprises a glycine-serine peptide linker (GS) at amino acids 104-144 or 104-136 of the RSV F protein. n Substitution, wherein 1≤n≤3.

[0016] Furthermore, the mutant also comprises that the 190th amino acid of the RSV F protein is replaced by F, I, Y, L, H or M, and the 207th amino acid is replaced by L, I or Y.

[0017] Furthermore, the mutant also comprises RSV F protein L373R substitution.

[0018] Furthermore, the mutant is selected from any one of the following I to XI: I. RSV F protein A170C and A177C are replaced, S55C and L188C are replaced, A149C and Y458C and the 190th amino acid is replaced by F, I, Y, L, H or M, and the 207th amino acid is replaced by L, I or Y; preferably, the amino acid sequence of the mutant is shown in SEQ ID NO: 5; II, comprising RSV F protein A170C and A177C substitutions, V157C and A183C substitutions, A149C and Y458C substitutions, and amino acid position 190 substitutions by F, I, Y, L, H or M, and amino acid position 207 substitutions by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 2; III, comprising RSV F protein A170C and A177C substitutions, V157C and A183C substitutions, L142C and N371C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 3; IV, comprising RSV F protein A170C and A177C substitutions, V157C and A183C substitutions, T103C and I148C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 4; V, comprising RSV F protein A170C and A177C substitutions, L96C and G279C substitutions, A149C and Y458C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 7; VI, comprising RSV F protein A170C and A177C substitutions, L96C and G279C substitutions, T103C and I148C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 8; VII. comprising RSV F protein V157C and A183C substitutions, S55C and L188C substitutions, and / or A149C and Y458C substitutions and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 9; VIII. comprising substitutions of V157C and A183C, A149C and Y458C in RSV F protein, and substitutions of amino acid 190 with F, I, Y, L, H or M, and substitutions of amino acid 207 with L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 10; IX, comprising RSV F protein V157C and A183C substitutions, L142C and N371C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 11; X, comprising RSV F protein V157C and A183C substitutions, T103C and I148C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 12; XI. Comprising RSV F protein L96C and G279C substitutions, A149C and Y458C substitutions, S55C and L188C substitutions, and the 190th amino acid is substituted by F, I, Y, L, H or M, and the 207th amino acid is substituted by L, I or Y. Preferably, the amino acid sequence of the mutant is shown in SEQ ID NO:6.

[0019] In a second aspect, the present invention provides a recombinant extracellular domain trimer of the mutant, wherein the three recombinant F2-F1 extracellular domain protomers each comprise a C-terminus connected to a trimerization domain.

[0020] Further, three recombinant F2-F1 ectodomain protomers each comprise RSV F positions 26-103 and 145-513, or RSV F positions 26-103 and 137-513, and are linked to a C-terminal trimerization domain.

[0021] The trimerization domain is a T4 Fibritin trimerization domain, and the amino acid sequence is preferably: GYIPEAPRDGQAYVRKDGEWVLLSTF.

[0022] Further, each of the three recombinant F2-F1 extracellular domain protomers comprises a C-terminus connected to a transmembrane domain (such as a RSV F protein transmembrane domain).

[0023] Further, each of the three recombinant F2-F1 ectodomain protomers comprises an amino acid substitution that increases the stability or expression of the RSV F ectodomain trimer.

[0024] Furthermore, the amino acid substitution that can increase the stability or expression of the RSV F extracellular domain trimer can be selected from at least one of the following: S46G, N67I, E92D, L95M, I221M, R429K, K465Q, S215P or I217P, etc.

[0025] Preferably, the amino acid substitutions that can increase the stability or expression of the RSV F extracellular domain trimer comprise L95M, I221M and R429K substitutions.

[0026] Preferably, the amino acid substitutions that can increase the stability or expression of the RSV F extracellular domain trimer comprise L95M, I221M, R429K and I217P substitutions.

[0027] Preferably, the amino acid substitutions that can increase the stability or expression of the RSV F extracellular domain trimer comprise S46G, K465Q, S215P and E92D substitutions.

[0028] Preferably, the amino acid substitutions that can increase the stability or expression of the RSV F extracellular domain trimer comprise S46G, K465Q, S215P, E92D and N67I substitutions.

[0029] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the mutant or the recombinant extracellular domain trimer.

[0030] In a fourth aspect, the present invention provides biological materials containing the nucleic acid molecule, wherein the biological materials include but are not limited to recombinant DNA, mRNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria or transgenic cell line.

[0031] In a fifth aspect, the present invention provides a virus-like particle comprising the mutant or the recombinant extracellular domain trimer.

[0032] In a sixth aspect, the present invention provides a composition comprising the mutant or the recombinant extracellular domain trimer or the nucleic acid molecule or the biological material or the virus-like particle, and a pharmaceutically acceptable carrier.

[0033] In a seventh aspect, the present invention provides an immunogenic composition comprising the mutant or the recombinant extracellular domain trimer or the nucleic acid molecule or the biological material or the virus-like particle or the composition.

[0034] In an eighth aspect, the present invention provides a method for preparing the mutant or the recombinant extracellular domain trimer, comprising the following steps: S1. expressing the nucleic acid molecule or a vector containing the nucleic acid molecule in a host cell (such as a CHO cell) to produce a target protein; and S2. Purify the target protein.

[0035] In a ninth aspect, the present invention provides use of the mutant or the recombinant extracellular domain trimer or the nucleic acid molecule or the biological material or the virus-like particle or the composition or the immunogenic composition in the preparation of an RSV vaccine.

[0036] In a tenth aspect, the present invention provides the use of the mutant or the recombinant extracellular domain trimer or the nucleic acid molecule or the biological material or the virus-like particle or the composition or the immunogenic composition in the preparation of a medicament for inducing an immune response against RSV F protein in a subject.

[0037] Furthermore, the immune response inhibits, prevents or treats RSV infection.

[0038] In an eleventh aspect, the present invention provides use of the mutant or the recombinant extracellular domain trimer or the nucleic acid molecule or the biological material or the virus-like particle or the composition or the immunogenic composition in the preparation of RSV diagnostic reagents.

[0039] The RSV F protein mutants of the invention can be used to induce an immune response against RSV (eg, RSV A, RSV B, or bovine RSV) in vertebrates (eg, mammals, such as humans and cattle).

[0040] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The present invention provides new mutation sites of RSV F protein, and for the first time finds that 157C and 183C substitutions and / or 170C and 177C substitutions in the respiratory syncytial virus F protein play an important role in stabilizing its pre-fusion conformation, and S155C and S290C substitutions may not be used to improve the immunogenicity of the respiratory syncytial virus pre-fusion F protein, thereby greatly improving the titer of the neutralizing antibodies induced by the protein, and achieving a high expression level of the antigen in mammalian cells, and is used for the preparation of RSV diagnostic reagents, antibodies and various types of vaccines using the pre-fusion F protein as an antigen. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 An overview of the design of RSV F protein mutants of the present invention.

[0042] Figure 2 Schematic diagram of the structural location of the key mutation sites of the RSV F protein of the present invention.

[0043] Figure 3 This is the detection of the expression level of the F protein mutant in the preferred embodiment of the present invention.

[0044] Figure 4 It is the binding ability of the F protein mutant and the key epitope antibody in the preferred embodiment of the present invention.

[0045] Figure 5 This is the expression confirmation of the mRNA vaccine based on the F protein mutant in the preferred embodiment of the present invention.

[0046] Figure 6 This is the neutralizing antibody detection after immunization with the mRNA vaccine based on the F protein mutant in the preferred embodiment of the present invention. In the figure, IT50 is the competitive antibody titer when the inhibition rate is 50%.

[0047] Figure 7 This is the detection of epitope competing antibodies in the serum after immunization with the mRNA vaccine in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0048] The present invention provides a respiratory syncytial virus prefusion F protein mutant with higher immunogenicity and higher expression, comprising at least one engineered intra-protomer disulfide bond mutation and at least one engineered inter-protomer disulfide bond. The intra-protomer disulfide bond mutation comprises any one or a combination of the following: (1) 157C and 183C substitutions (e.g. V157C and A183C substitutions); and / or, (2) 170C and 177C substitution (e.g. A170C and A177C substitution); and / or, Wherein the RSV F protein position corresponds to the amino acid sequence of the reference F0 polypeptide shown in SEQ ID NO:1.

[0049] Preferably, in the respiratory syncytial virus pre-fusion F protein mutant, compared with the natural F0 polypeptide sequence, the amino acids at positions 104-144 or 104-136 are replaced by a glycine-serine peptide linker.

[0050] Preferably, in the respiratory syncytial virus prefusion F protein mutant, the inter-protomer disulfide bond mutation comprises any one or a combination of the following: (1) 96C and 279C substitutions (e.g., L96C and G279C substitutions); and / or, (2) 149C and 458C substitutions (e.g., A149C and Y458C substitutions); and / or, (3) 183GC and 428C substitutions (e.g. N183GC and N428C substitutions); and / or, (4) 369C and 455C substitution; and / or, (5) 100C and 362C replacement; and / or, (6) 99C and 362C substitutions; and / or, (7) 98C and S362C substitutions; and / or, (8) 100C and 361C substitution; and / or, (9) 99C and 361C substitutions; and / or, (10)98C and S361C substitutions.

[0051] Preferably, in the respiratory syncytial virus pre-fusion F protein mutant, the disulfide bond mutation in the protomer may also include any one or a combination of the following: (1) S55C and L188C substitutions; and / or, (2) L142C and N371C substitutions; and / or, (3) T103C and I148C substitutions; and / or, (4) L142C and N371C substitutions.

[0052] Preferably, the respiratory syncytial virus pre-fusion F protein mutant further comprises amino acid 190 substituted by F, I, Y, L, H or M, and amino acid 207 substituted by L, I or Y.

[0053] Furthermore, the RSV F protein mutant sequence disclosed in the present invention also comprises a L373R substitution, and the L373R mutation is introduced as a compatible solvent-exposed residue to increase protein production.

[0054] In some embodiments, the RSV F protein mutants disclosed herein can be expressed in the form of recombinant trimers, and the F2-F1 extracellular domain protomer of the trimer can include a C-terminus connected to a trimerization domain (eg, a T4 Fibritin trimerization domain).

[0055] In some embodiments, the RSV F protein mutant extracellular region trimer disclosed herein comprises RSV F0 positions 26-103 and 145-513, or RSV F0 positions 26-103 and 137-513, and is connected to a C-terminal trimerization domain.

[0056] In some embodiments, the recombinant F2-F1 ectodomain protomer of the RSV F protein mutant ectodomain trimer disclosed herein may include a C-terminus linked to a transmembrane domain (eg, a RSV F transmembrane domain).

[0057] In some embodiments, the recombinant F2-F1 ectodomain protomer in the RSV F protein mutant ectodomain trimer disclosed herein may contain additional amino acid substitutions known to increase the stability or expression of the RSV F ectodomain trimer.

[0058] For example, in some embodiments, the recombinant F2-F1 extracellular domain protomer in the RSV F protein mutant extracellular domain trimer disclosed herein can contain at least one of the following amino acid substitutions: S46G, N67I, E92D, L95M, I221M, R429K, K465Q, S215P, or I217P.

[0059] In some embodiments, the recombinant F2-F1 ectodomain protomer in the RSV F protein mutant ectodomain trimer disclosed herein may comprise L95M, I221M, and R429K substitutions to increase the stability and / or expression of the RSV F ectodomain trimer.

[0060] In some embodiments, the recombinant F2-F1 ectodomain protomer in the RSV F protein mutant ectodomain trimer disclosed herein may further comprise L95M, I221M, R429K, and I217P substitutions to increase the stability and / or expression of the RSV F ectodomain trimer.

[0061] In some embodiments, the recombinant F2-F1 ectodomain protomer in the RSV F protein mutant ectodomain trimer disclosed herein may further comprise S46G, K465Q, S215P, and E92D substitutions to increase the stability and / or expression of the RSV F ectodomain trimer.

[0062] In some embodiments, the recombinant F2-F1 ectodomain protomer in the RSV F protein mutant ectodomain trimer disclosed herein may further comprise S46G, K465Q, S215P, E92D, and N67I substitutions to increase the stability and / or expression of the RSV F ectodomain trimer.

[0063] Furthermore, in the respiratory syncytial virus prefusion F protein mutant, the amino acid mutation is selected from a combination of the following mutations: (1) A170C and A177C are substituted, S55C and L188C are substituted, A149C and Y458C, and amino acid position 190 is substituted by F, I, Y, L, H or M, and amino acid position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 5; (2) A170C and A177C are substituted, V157C and A183C are substituted, A149C and Y458C and the amino acid at position 190 are substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y, the amino acid sequence of an exemplary mutant is SEQ ID NO: 2; (3) A170C and A177C are substituted, V157C and A183C are substituted, L142C and N371C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y, the amino acid sequence of an exemplary mutant is SEQ ID NO: 3; (4) A170C and A177C are substituted, V157C and A183C are substituted, T103C and I148C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 4; (5) A170C and A177C are substituted, L96C and G279C are substituted, A149C and Y458C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 7; (6) A170C and A177C are substituted, L96C and G279C are substituted, T103C and I148C are substituted, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 8; (7) V157C and A183C are substituted, S55C and L188C are substituted, and / or A149C and Y458C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 9; (8) V157C and A183C are substituted, A149C and Y458C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 10; (9) V157C and A183C are substituted, L142C and N371C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 11; (10) V157C and A183C are substituted, T103C and I148C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 12; (11) L96C and G279C are substituted, A149C and Y458C are substituted, S55C and L188C are substituted, and the amino acid at position 190 is substituted by F, I, Y, L, H or M, and the amino acid at position 207 is substituted by L, I or Y. The amino acid sequence of an exemplary mutant is SEQ ID NO: 6.

[0064] An overview of the design of RSV F protein mutants of the present invention is given in Figure 1 .

[0065] The structural position of the key mutation sites of the RSV F protein of the present invention is shown in FIG. Figure 2 .

[0066] The amino acid sequences of RSV F0 and exemplary mutants TS1 to TS11 are shown in Tables 1 to 3: Table 1 Table 2 Table 3 The present invention discloses that RSV F protein mutants can be used to induce an immune response against RSV (eg, RSV A, RSV B, or bovine RSV) in vertebrates (eg, mammals, such as humans and cattle).

[0067] (I) The mutant of the present invention removes the natural protease cleavage site and the pep27 peptide The recombinant RSV F extracellular domain trimer comprises three recombinant protomers, which comprise modified forms of RSV F2 protein and RSV F1 extracellular domain. Different from the natural RSV F protomer, the recombinant F2-F1 extracellular domain protomer in the disclosed recombinant RSV F extracellular domain trimer is a "single-chain" protein, which comprises a single polypeptide chain comprising F2 polypeptide and F1 extracellular domain. In particular, the recombinant F2-F1 extracellular domain protomer in the disclosed recombinant RSV F extracellular domain trimer comprises a deletion of RSV F position 104-144 or 104-136 to remove the natural protease cleavage site and pep27 peptide, and a glycine-serine peptide linker between RSV F position 103 and 145 or RSV F position 103 and 137.

[0068] In some embodiments, the RSV F extracellular domain trimer can be membrane-anchored, for example, for embodiments in which the RSV F extracellular domain trimer is expressed on an attenuated virus vaccine or a virus-like particle. In such embodiments, the recombinant F2-F1 extracellular domain protomers in the trimer generally each include a C-terminus connected to a transmembrane domain. In some embodiments, one or more peptide linkers (such as a Gly-Ser linker, for example a 10 amino acid glycine-serine peptide linker) can be used to connect the recombinant F2-F1 extracellular domain protomers to a transmembrane domain.

[0069] The recombinant F2-F1 extracellular domain protomer connected by the membrane spaning domain can include any stable mutation (or combination thereof) provided by the present invention, as long as the recombinant RSV F extracellular domain trimer formed by the recombinant F2-F1 extracellular domain protomer connected to the membrane spaning domain retains the desired characteristics (such as RSV F pre-fusion conformation including antigenic sites). In some embodiments, the recombinant RSV F extracellular domain trimer can be connected to the F1 membrane spaning domain and the cytoplasmic tail, for example, the protomer can include the RSV F position 26-103 connected (by a GS joint) to the C-terminal (about position 574) of RSV F position 145 to F1; or include the RSV F position 26-103 connected (by a GS joint) to the C-terminal (about position 574) of RSV F position 137 to F1.

[0070] (II) The mutation sites of the present invention can be used for recombinant vaccines In some embodiments, the recombinant RSV F extracellular domain trimer can be a soluble protein complex, for example, used as a recombinant subunit vaccine. In some such embodiments, the recombinant F2-F1 extracellular domain protomer can include a C-terminal connected to a trimerization domain (e.g., a T4 Fibritin trimerization domain). The trimerization domain promotes trimerization and stabilization of the membrane proximal aspects of the recombinant RSV F extracellular domain trimer.

[0071] (III) The mutation site of the present invention can be used for attenuated live vaccine or virus-like particle vaccine In some embodiments, the recombinant RSV F extracellular domain trimer can be a membrane-anchored protein complex, such as with an attenuated virus or virus-like particle vaccine. Membrane anchoring can be accomplished, for example, by connecting the C-terminus of the recombinant F2-F1 extracellular domain protomer to a transmembrane domain and an optional cytoplasmic tail (e.g., RSV F transmembrane domain and cytoplasmic tail).

[0072] (IV) The mutation sites of the present invention can be used to prepare protein nanoparticles In some embodiments, RSV F mutants are provided for protein nanoparticle preparation schemes, including one or more disclosed recombinant RSV F extracellular domains. Non-limiting examples of nanoparticles include ferritin nanoparticles, encapsulated protein nanoparticles, sulfur oxygenase reductase (SOR) nanoparticles and dioxathiapterin synthase nanoparticles, which are respectively composed of the assembly of monomer subunits including ferritin, encapsulated protein, SOR protein and dioxathiapterin synthase. The exemplary sequence of the recombinant RSV F extracellular domain connected to the nanoparticle subunit is provided below. In order to construct a protein nanoparticle including a recombinant RSV F extracellular domain, the RSV F extracellular domain can be connected to the subunit (such as ferritin, encapsulated protein, SOR protein or dioxathiapterin synthase protein) of the protein nanoparticle. The fusion protein self-assembles into nanoparticles under appropriate conditions. In some embodiments, the protein nanoparticle comprises two or more recombinant RSV F extracellular domains, wherein two or more recombinant RSV F extracellular domains are from at least two different RSV virus strains. For specific implementation methods, please refer to patent document CN112851766A.

[0073] (V) The mutation sites of the present invention can be used to prepare nucleic acid vaccines In some embodiments, nucleotide sequences encoding recombinant F2-F1 extracellular domain protomers of any disclosed recombinant RSV F extracellular domain trimer are provided. These nucleotide sequences include DNA, cDNA, and RNA sequences encoding recombinant F2-F1 extracellular domain protomers. Those skilled in the art can easily use the genetic code to construct a variety of functionally equivalent nucleic acids, such as nucleic acids with different sequences but encoding the same antibody sequence, or nucleic acids encoding conjugated proteins or fusion proteins including nucleic acid sequences.

[0074] (VI) The mutation sites of the present invention can be used to prepare viral vector vaccines The nucleic acid molecule of disclosed recombinant RSV F protein extracellular domain can be included in a viral vector, for example, for expressing recombinant RSV F protein extracellular domain to produce corresponding recombinant RSV F extracellular domain trimer in host cell, or for the immunization of object, as disclosed in the present invention. In some embodiments, the viral vector is administered to the object as a part of primary immunity-boosting immunization. Generally, such viral vectors include nucleic acid molecules encoding disclosed recombinant RSV F protein extracellular domain protomer, which is connected to the C-terminal transmembrane domain, such as recombinant RSV F protein extracellular domain protomer can be connected to RSV F transmembrane domain and cytoplasmic tail. In some embodiments, the viral vector is included in a vaccine, for example, for the primary immunity vaccine or booster vaccine of primary immunity-boosting immunization.

[0075] In several examples, the viral vector encoding the recombinant F2-F1 extracellular domain protomer to produce the recombinant RSV F extracellular domain trimer can be replication-competent. For example, the viral vector can have a mutation in the viral genome (e.g., insertion of nucleic acid encoding the recombinant F2-F1 extracellular domain protomer) that attenuates but does not completely block viral replication in the host cell.

[0076] In some embodiments, virus-like particles (VLPs) are provided, which include disclosed recombinant RSV F extracellular domain trimers. Typically, such VLPs include recombinant RSV F extracellular domain trimers anchored by C-terminal transmembrane domain membranes, such as recombinant F2-F1 extracellular domain protomers in trimers, each of which can be connected to RSV F transmembrane domains and cytoplasmic tails.

[0077] Also provided are immunogenic compositions comprising a disclosed immunogen (e.g., a recombinant RSV F ectodomain trimer, a nucleic acid molecule or vector encoding a recombinant F2-F1 ectodomain protomer of a disclosed recombinant RSV F ectodomain trimer, or a protein nanoparticle or virus-like particle comprising a disclosed recombinant RSV F ectodomain trimer) and a pharmaceutically acceptable carrier. Such compositions can be administered to a subject by various modes of administration known to those of ordinary skill in the art, such as intramuscular, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, or parenteral routes. In several embodiments, a pharmaceutical composition comprising one or more disclosed immunogens is an immunogenic composition.

[0078] The present invention further protects the nucleic acid encoding the above-mentioned respiratory syncytial virus prefusion F protein mutant with stable conformation and high expression level, wherein the nucleic acid sequence is a codon-optimized sequence, so that it conforms to the mammalian codon usage preference.

[0079] (VII) The mutation sites of the present invention can be derived based on the nucleic acid sequences of different wild-type encoded proteins. Natural RSV F protein from different RSV groups and the nucleotide sequence and method for encoding these proteins are known, such as NCBI, Uniprot, GISAID and other databases to obtain. Disclosed recombinant RSV F extracellular domain trimer can be derived from any type of RSV, such as subtype A (such as A1 or A2), subtype B (such as B1 or B2) or cattle RSV. RSVF protein from different RSV subtypes, and the nucleotide sequence encoding these proteins and the method for manipulating and inserting these nucleotide sequences into vectors are known. Exemplary natural RSV F protein sequences are shown in patent documentation CN109069611A.

[0080] In some embodiments, any RSV F ectodomain trimer including the recombinant F2-F1 ectodomain protomer disclosed herein can be derived from the amino acid sequence of a native RSV F of subtype A (e.g., A1 or A2), subtype B (e.g., B1 or B2), or bovine RSV (e.g., positions 26-103 and 145-513), or an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical thereto, which has been modified by one or more amino acid substitutions and / or insertions provided herein.

[0081] The sequences of some recombinant RSV F extracellular domains provided by the present invention include protease cleavage sites (e.g., thrombin sites), protein tags (e.g., His tags, Strep tags II, Avi tags, etc.), and signal peptide sequences that are obviously known to those of ordinary skill in the art and will not be included in isolated immunogens (including recombinant RSV F extracellular domain trimers for prophylactic / therapeutic purposes), see patent documents CN105473604A, CN108738312A, and CN109069611A.

[0082] the term: Unless otherwise noted, technical terms are used according to conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes X, Jones & Bartlett Publishers, 2009; Meyers et al. (eds.), The Encyclopedia of Cell Biology and Molecular Medicine, Wiley-VCH, 16 volumes, 2008; and other similar references. As described in the present invention, unless the context otherwise indicates, the singular forms "one" and "the" refer to the singular form as well as the plural form. For example, the term "antigen" includes a single or multiple antigens and can be considered equivalent to the phrase "at least one antigen". As used in the present invention, the term "comprising" means "including". Therefore, "comprising an antigen" means "including an antigen" without excluding other elements. It should also be understood that, unless otherwise stated, any and all base sizes or amino acid sizes given for nucleic acids or polypeptides, and all molecular weights or molecular mass values ​​are approximate and are provided for descriptive purposes. Although many methods and materials similar or equivalent to those described in the present invention can be used, particularly suitable methods and materials are described below. In the event of a conflict, the present specification (including term explanations) shall prevail. In addition, materials, methods and examples are illustrative and non-restrictive only. In order to facilitate the review of various embodiments, the following term explanations are provided: Mutant: refers to a mutant obtained by at least 2 or more mutations in the sequence compared to the amino acid sequence of the natural RSV F protein, and retains the antigenic site Ø, including but not limited to mutants that have been truncated or deleted in the intracellular region, transmembrane region or extracellular region.

[0083] D25: A neutralizing monoclonal antibody that specifically binds to an antigenic site present in the prefusion conformation of the RSV F protein. The D25 protein and nucleic acid sequence are known, for example, the heavy chain and light chain amino acid sequences of the D25 antibody are disclosed in patent document CN101778866A, all of which are incorporated into the present invention; see also Kwakkenbos et al., Nat. Med., 16: 123-128, 2009.

[0084] Engineered intra-protomer disulfide bond mutations: mutant forms of disulfide bonds within the single chain of the RSV F protein protomer itself.

[0085] Engineered inter-protomer disulfide bond mutation: A mutant form of the disulfide bonds formed between the single chains of the RSV F protein protomer.

[0086] RSV fusion (F) protein: RSV envelope glycoprotein that promotes fusion of viral and cell membranes. In nature, RSV F protein is initially synthesized as a single polypeptide precursor of approximately 574 amino acids in length, named F0. F0 includes an N-terminal signal peptide that directs localization to the endoplasmic reticulum, where the signal peptide (approximately the first 25 residues of F0) is proteolytically cleaved. The remaining F0 residues oligomerize to form trimers and are bound at two conserved consensus furin cleavage sequences (approximately F0 positions 109 and 136; e.g., RARR 109 and RKRR 136 RSV F protein is proteolytically processed by cell protease to produce two disulfide-linked fragments F1 and F2. In these fragments, smaller F2 derives from the N-terminal portion of F0 precursor, and comprises the residue 26-109 of about F0. In these fragments, larger F1 comprises the C-terminal portion (about residue 137-574) of F0 precursor, comprises extracellular / cavity area (about residue 137-529), transmembrane domain (about residue 530-550) and the cytoplasmic tail (about residue 551-574) of C-terminus. The extracellular part of RSV F protein is RSV F extracellular domain, which comprises F2 protein and F1 extracellular domain.

[0087] In the prefusion conformation, the RSV F protein contains an antigenic site at its membrane-distal apex called “antigenic site Ø” that includes RSV F residues 62-69 and 196-209, and also includes the epitopes of the 5C4, D25, and AM22 monoclonal antibodies (see McLellan et al., Science, 340(6136): p. 1113-7, 2013, McLellan et al., Science, 342: 592-598, 2013, and the structural coordinates provided as PDB Accession No. 4JHW, each of which is incorporated herein by reference in its entirety).

[0088] The "DS2" described in the present invention is a second-generation RSV F protein pre-fusion structural mutant developed by NIH, and the mutation sites include: S155C and S290C substitutions, S190F and V207L ​​substitutions, and A149C and Y458C substitutions. See patent documents CN109069611A, CN115103682A and journal articles (Joyce MG, Zhang B, Ou L, et al. Nat Struct Mol Biol . 2016;23(9):811-820.) Based on the above mutation site of the respiratory syncytial virus F protein, the results of the in vivo immunogenicity study in animals showed that high titers of virus neutralizing antibodies and key epitope antibodies were produced in the animal serum. Compared with the NIH second-generation vaccine, it has stronger immunogenicity (with the potential to improve the effectiveness of the vaccine) and expression level (reducing industrial production costs). It can be used for the preparation of RSV diagnostic reagents, antibodies and various types of vaccines with pre-fusion F protein as antigen.

[0089] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0090] Example 1 Expression detection of recombinant RSV F mutants The mutant RSV F mutant protein gene was cloned into a vector and then transfected into CHO cells. Seven days after transfection, the cell supernatant was collected and centrifuged. The supernatant was purified by a nickel column, the protein concentration in the sample was determined, and the relative expression of F protein in each group of samples was calculated. The target protein was confirmed by ELISA. After each sequence was constructed into the vector pEE12.4 vector (purchased from Lonza), the plasmid was transiently transfected into CHO cells for 7 days, and then the test was performed. The experimental results are shown in the figure. Figure 3As shown, the expression levels of TS1, TS2 and DS2 were 3.0 μg / ml, 14 μg / ml and 3.6 μg / ml, respectively. It can be seen that the expression level of TS1 is equivalent to that of DS2, and the expression level of TS2 is 3.6 times that of DS2. It can be seen that the expression level of the soluble F protein mutant in CHO cells is better than that of DS2.

[0091] Example 2 Evaluation of the binding ability of RSV F mutants with key epitope antibodies The ELISA method was used to analyze the binding ability of the purified antigen with different antibodies. In brief, a 96-well plate was coated with Palivizumab monoclonal antibody at a concentration of 100 μL / well and 1 μg / mL, and incubated overnight at 4°C. The plate was washed once with PBST solution using a plate washer and blocked with 2% BSA at 37°C for 1 hour. After washing five times with PBST solution, the antigen was diluted 10 times in series and added to the wells at 100 μL / well. After incubation at room temperature for 2 hours, the plate was washed 5 times with PBST solution and incubated with HRP-labeled D25 antibody (20 μg / mL) for 1 hour. The plate was washed 5 times with PBST solution and incubated with TMB substrate (100 μL / well) for 15 minutes. The color development was terminated with 2M H2SO4 (50 μL), and the plate was read with an enzyme reader at a wavelength of 450 nm. The experimental results are shown in the figure. Figure 4 As shown, the ability of purified TS1 and TS2 to bind D25 antibody was comparable to that of DS2.

[0092] Example 3 Preparation of mRNA vaccine based on F mutant According to the method described in the literature published by Andrew J Bett et al. (PMID: 32128257), RNA transcription-related elements were constructed into an in vitro transcription plasmid containing an F protein mutant, and the plasmid was linearized with the restriction endonuclease BspQ1 after large-scale extraction. Transcription was performed using a T7 in vitro transcription kit (manufacturer: Nanjing Novi Biotechnology Co., Ltd.; catalog number: DD4202) to obtain capped mRNA. The transcription template was digested with DNaseI. The mRNA was purified by precipitation using lithium chloride (manufacturer: Invitrogen; catalog number: AM9480).

[0093] The purified mRNA was dissolved in an acidic sodium citrate buffer for later use. Preparation of lipid nanoparticles LNP: The cationic lipids: DSPC: cholesterol: DMG-PEG2000 purchased from Sinobond Biotechnology Co., Ltd. were dissolved and mixed in ethanol at a molar ratio of 50:10:38.5:1.5. The mRNA and lipid mixed solution were encapsulated at a flow rate ratio of 3:1. After encapsulation, dialyzed in a PBS solution to obtain the mRNA vaccine.

[0094] Example 4 Detection of cell expression of mRNA vaccine After mRNA transfection, 293 cells (0.5×10 6 24 hours after the cells were added to a V-bottom 96-well plate (0.5×10 6 Cells were suspended in staining buffer (FBS) with D25-Alexa Fluor 488 or isotype control antibody (5 μg / mL each) and incubated on ice for 15 minutes in the dark. Cells were pelleted and washed twice with staining buffer (FBS) (300 μL / well), resuspended in staining buffer (FBS) (200 μL / well), analyzed by flow cytometry, and data were processed using Flow Jo software. The mRNA vaccine was constructed with the sequences of TS1 and TS2 mutants. After transfection into cells, the D25 antibody (see McLellan et al., Science, 340(6136): p. 1113-7, 2013, McLellan et al., Science, 342: 592-598, 2013 and the sequence disclosed in PDB accession number 4JHW) was synthesized. The heavy chain and light chain amino acid sequences of the D25 antibody are also disclosed in patent document CN101778866A) was detected by flow cytometry. It can be seen that the F protein can be successfully expressed. The experimental results are as follows: Figure 5 shown.

[0095] Example 5 In vivo immunization of animals The experimental animals were BALB / c female mice, 6-8 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in the Experimental Animal Center of Beijing Minhai Biotechnology Co., Ltd. The mice were randomly divided into groups, 5 in each group. The mRNA vaccine prepared in Example 3 was injected into the quadriceps femoris of the thigh at 10 μg / mouse, and the control group was PBS; a secondary booster immunization was performed 3 weeks after the initial immunization, for a total of two immunizations. Blood was collected from the orbits 14 days after the booster immunization, and serum was separated for analysis.

[0096] Example 6 Detection of RSV neutralizing antibody titer The serum separated in Example 5 was incubated at 56°C for 30 minutes to inactivate complement; Gradient dilution of serum: 10-fold in the first well, 4-fold gradient dilution for a total of 10 gradients; An equal volume of RSV-A2 virus (MOI = 0.1) was added, mixed, and incubated at 37°C for 1 hour; The serum-virus mixture was transferred to a 96-well plate with a monolayer of Hpe2 cells at a volume of 100 μL / well and incubated at 37°C; After 72 hours, fluorescently labeled antibodies were added to 96-well plates, incubated for 1 hour, and fixed; After washing, the fluorescence was observed under an inverted fluorescence microscope to determine the neutralizing antibody titer.

[0097] After mice were immunized with the F protein mutant mRNA vaccine, the neutralizing antibodies were detected. The results showed that the neutralizing antibodies induced by the TS1 and TS2 mRNA vaccines were higher than those induced by the DS2 mRNA vaccine, which were 1.2 times and 2.7 times higher than those induced by the DS2 mRNA vaccine, respectively. Figure 6 Among them, the DS2 mRNA vaccine is self-made based on patent CN115103682A.

[0098] Example 7 Detection of D25 Competitive Antibody Levels in Serum In order to further illustrate that the F protein mutant of the present invention has good immunogenicity, we further compared the level of D25 competitive antibodies in the post-immunization serum. The Pre-F antigen was coated on a high-adsorption 96-well plate at a total amount of 100 ng / well, incubated at 4°C overnight, and then blocked with 1% BSA; the RSV antibody serum was serially diluted and added at 100 μL / well. After incubation at room temperature for 1 hour, HRP-labeled D25 antibody was added and incubated at room temperature for 1 hour, and then TMB substrate was added for color development. The absorbance (OD) was tested at 450nm / 630nm on an enzyme reader. The OD value is inversely proportional to the sample 0 epitope concentration. The results show that the competitive antibodies induced by TS1 and TS2 mRNA vaccines are higher than those induced by DS2 mRNA vaccines. The experimental results are as follows Figure 7 shown.

[0099] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A pre-fusion RSV F protein mutant, characterized in that: The mutant comprises at least one intra-protomer disulfide bond mutation, and / or at least one inter-protomer disulfide bond mutation relative to the native RSV F protein; Wherein, the intra-protomer disulfide bond mutation comprises the following (1) and / or (2): (1) 157C and 183C substitution; (2) 170C and 177C replacement; Among them, the RSV F protein position corresponds to the amino acid sequence of the reference F0 polypeptide shown in SEQ ID NO:

1.

2. The mutant according to claim 1, characterized in that The intra-protomer disulfide bond mutations further include any one or a combination of the following: (3) S55C and L188C substitutions; (4) L142C and N371C substitutions; (5) T103C and I148C substitutions; (6) L142C and N371C substitutions.

3. The mutant according to claim 1, characterized in that The inter-protomer disulfide bond mutation comprises any one or a combination of the following: 1) 96C and 279C substitutions; 2) 149C and 458C substitution; 3) 183GC and 428C substitution; 4) 369C and 455C substitution; 5) 100C and 362C replacement; 6) 99C and 362C substitution; 7) 98C and S362C substitutions; 8) 100C and 361C replaced; 9) 99C and 361C substitution; 10) 98C and S361C substitutions.

4. The mutant according to claim 1, characterized in that The mutant further comprises amino acids 104-144 or 104-136 of RSV F protein replaced by a glycine-serine peptide linker.

5. The mutant according to claim 1, characterized in that The mutant also comprises that the 190th amino acid of RSV F protein is replaced by F, I, Y, L, H or M, and the 207th amino acid is replaced by L, I or Y.

6. The mutant according to claim 1, characterized in that The mutant also comprises the RSV F protein L373R substitution.

7. The mutant according to claim 1, characterized in that The mutant is selected from any one of the following I to XI: I. RSV F protein A170C and A177C are replaced, S55C and L188C are replaced, A149C and Y458C and the 190th amino acid is replaced by F, I, Y, L, H or M, and the 207th amino acid is replaced by L, I or Y; preferably, the amino acid sequence of the mutant is shown in SEQ ID NO: 5; II, comprising RSV F protein A170C and A177C substitutions, V157C and A183C substitutions, A149C and Y458C substitutions, and amino acid position 190 substitutions by F, I, Y, L, H or M, and amino acid position 207 substitutions by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 2; III, comprising RSV F protein A170C and A177C substitutions, V157C and A183C substitutions, L142C and N371C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 3; IV, comprising RSV F protein A170C and A177C substitutions, V157C and A183C substitutions, T103C and I148C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 4; V, comprising RSV F protein A170C and A177C substitutions, L96C and G279C substitutions, A149C and Y458C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 7; VI, comprising RSV F protein A170C and A177C substitutions, L96C and G279C substitutions, T103C and I148C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 8; VII. comprising RSV F protein V157C and A183C substitutions, S55C and L188C substitutions, and / or A149C and Y458C substitutions and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 9; VIII. comprising substitutions of V157C and A183C, A149C and Y458C in RSV F protein, and substitutions of amino acid 190 with F, I, Y, L, H or M, and substitutions of amino acid 207 with L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 10; IX, comprising RSV F protein V157C and A183C substitutions, L142C and N371C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 11; X, comprising RSV F protein V157C and A183C substitutions, T103C and I148C substitutions, and amino acid 190 is substituted by F, I, Y, L, H or M, and amino acid 207 is substituted by L, I or Y; preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO: 12; XI. Comprising RSV F protein L96C and G279C substitutions, A149C and Y458C substitutions, S55C and L188C substitutions, and the 190th amino acid is substituted by F, I, Y, L, H or M, and the 207th amino acid is substituted by L, I or Y. Preferably, the amino acid sequence of the mutant is shown in SEQ ID NO:

6.

8. The recombinant extracellular domain trimer of the mutant according to any one of claims 1 to 7, characterized in that: The three recombinant F2-F1 ectodomain protomers each comprise a C-terminus linked to a trimerization domain.

9. The recombinant extracellular domain trimer according to claim 8, characterized in that The three recombinant F2-F1 ectodomain protomers each comprise RSV F positions 26-103 and 145-513, or RSV F positions 26-103 and 137-513, and are linked to a C-terminal trimerization domain.

10. The recombinant extracellular domain trimer according to claim 8, characterized in that The trimerization domain is a T4Fibritin trimerization domain.

11. The recombinant extracellular domain trimer according to claim 8, characterized in that The three recombinant F2-F1 ectodomain protomers each comprise a C-terminus linked to a transmembrane domain.

12. The recombinant extracellular domain trimer according to claim 11, characterized in that The transmembrane domain is the RSV F protein transmembrane domain.

13. The recombinant extracellular domain trimer according to claim 8, characterized in that Each of the three recombinant F2-F1 ectodomain protomers contained an amino acid substitution that increased the stability or expression of the RSV F ectodomain trimer.

14. The recombinant extracellular domain trimer according to claim 13, characterized in that The amino acid substitution that increases the stability or expression of the RSV F extracellular domain trimer is selected from at least one of the following: S46G, N67I, E92D, L95M, I221M, R429K, K465Q, S215P or I217P.

15. The recombinant extracellular domain trimer according to claim 14, characterized in that The amino acid substitutions that can increase the stability or expression of RSV F extracellular domain trimers include L95M, I221M and R429K substitutions.

16. The recombinant extracellular domain trimer according to claim 14, characterized in that The amino acid substitutions that can increase the stability or expression of RSV F extracellular domain trimers include L95M, I221M, R429K and I217P substitutions.

17. The recombinant extracellular domain trimer according to claim 14, characterized in that The amino acid substitutions that can increase the stability or expression of RSV F extracellular domain trimers include S46G, K465Q, S215P and E92D substitutions.

18. The recombinant extracellular domain trimer according to claim 14, characterized in that The amino acid substitutions that can increase the stability or expression of RSV F extracellular domain trimers include S46G, K465Q, S215P, E92D and N67I substitutions.

19. An isolated nucleic acid molecule, characterized in that It encodes the mutant according to any one of claims 1 to 7 or the recombinant extracellular domain trimer according to any one of claims 8 to 18.

20. A biological material containing the nucleic acid molecule according to claim 19, characterized in that: The biological material is recombinant DNA, mRNA, expression cassette, transposon, plasmid vector, virus vector, engineering bacteria or transgenic cell line.

21. A virus-like particle, characterized in that It comprises the mutant according to any one of claims 1 to 7 or the recombinant extracellular domain trimer according to any one of claims 8 to 18.

22. A composition, characterized in that It comprises the mutant according to any one of claims 1 to 7, the recombinant extracellular domain trimer according to any one of claims 8 to 18, the nucleic acid molecule according to claim 19, the biological material according to claim 20, or the virus-like particle according to claim 21, and a pharmaceutically acceptable carrier.

23. An immunogenic composition, characterized in that Comprising the mutant according to any one of claims 1 to 7, or the recombinant extracellular domain trimer according to any one of claims 8 to 18, or the nucleic acid molecule according to claim 19, or the biological material according to claim 20, or the virus-like particle according to claim 21, or the composition according to claim 22.

24. Use of the mutant according to any one of claims 1 to 7, the recombinant extracellular domain trimer according to any one of claims 8 to 18, the nucleic acid molecule according to claim 19, the biological material according to claim 20, the virus-like particle according to claim 21, the composition according to claim 22, or the immunogenic composition according to claim 23 in the preparation of RSV vaccines.

25. Use of the mutant according to any one of claims 1 to 7, the recombinant extracellular domain trimer according to any one of claims 8 to 18, the nucleic acid molecule according to claim 19, the biological material according to claim 20, the virus-like particle according to claim 21, the composition according to claim 22, or the immunogenic composition according to claim 23 in the preparation of a medicament for inducing an immune response against RSV F protein in a subject.

26. The use according to claim 25, characterized in that The immune response suppresses, prevents or treats RSV infection.

27. Use of the mutant according to any one of claims 1 to 7, the recombinant extracellular domain trimer according to any one of claims 8 to 18, the nucleic acid molecule according to claim 19, the biological material according to claim 20, the virus-like particle according to claim 21, the composition according to claim 22, or the immunogenic composition according to claim 23 in the preparation of RSV diagnostic reagents.

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