Respiratory syncytial virus prefusion F protein mutant and its application

Abstract

The present invention relates to the technical field of biological products, and in particular to a respiratory syncytial virus prefusion F protein mutant and its application. The RSV prefusion F protein mutant provided by the present invention can effectively stabilize the F protein in a prefusion conformation, has a high expression level, and has high stability, and has a high binding ability with key epitope antibodies. The results of in vivo immunogenicity studies in animals show that the mutant can induce animals to produce high-titer RSV neutralizing antibodies, and can be used to prepare RSV preventive and therapeutic drugs and diagnostic reagents.

Description

Technical Field

[0001] The invention relates to the technical field of biological products, and in particular to a respiratory syncytial virus pre-fusion F protein mutant and an application thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is a single-stranded, negative-sense, enveloped RNA virus that is the most common cause of acute lower respiratory tract infection in young children. RSV infection causes increased mucus and inflammation, which in turn leads to airway narrowing, posing a serious threat to children's health. Currently, treatment for RSV infection is largely supportive, such as bronchodilators, epinephrine, corticosteroids, and hypertonic saline.

[0003] In the 1960s, researchers tried to develop an inactivated human respiratory syncytial virus (HRSV) vaccine, but unfortunately, this study led to a phenomenon called "enhanced respiratory disease" (ERD) in vaccinated children when they were naturally exposed to HRSV. Vaccinated people showed more severe respiratory symptoms than unvaccinated people, some even required hospitalization, and a few died. This incident dealt a major blow to the development of HRSV vaccines, forcing scientists to re-examine the complexity of vaccine safety and immune response, especially the abnormal immune response that may be caused by vaccination in children. Since then, researchers have turned to exploring a variety of different vaccine platforms and technologies, such as live attenuated vaccines, recombinant subunit vaccines, DNA vaccines, and virus-like particle (VLP) vaccines, to avoid side effects similar to ERD (Zhang Lujing, Zheng Zizheng, Xia Ningshao (2018). Research progress on the mechanism of enhanced occurrence of human respiratory syncytial virus disease." Journal of Virology 34(03): 406-414.). In recent years, the development of HRSV vaccines has made some progress.

[0004] RSV is divided into two subtypes, A and B, with only one serotype. The genome is about 15,200 bp long and encodes 11 proteins, including 3 non-structural proteins (NS1, NS2 and M2-2) and 8 structural proteins. These 8 structural proteins are divided into three types of proteins located on the surface of the virus: small hydrophobic proteins, adhesion protein G and fusion protein F (referred to as F protein); and five proteins located inside the virus: nucleocapsid protein, phosphoprotein, matrix protein M, M2-1 protein expressed by the M2 gene, and RNA-dependent RNA polymerase L (Beugeling, M., J. De Zee, HJ Woerdenbag, HW Frijlink, JCWilschut and WLJ Hinrichs (2019). "Respiratory syncytial virus subunitvaccines based on the viral envelope glycoproteins intended for pregnant women and the elderly." Expert Rev Vaccines 18(9): 935-950.).

[0005] The F protein plays a key role in the infectivity and disease progression of HRSV. It can initiate the process of virus invasion of cells by fusing the virus with the host cell membrane and triggering cell-to-cell fusion in the late stage of infection to form a unique syncytial structure. In order to activate this process, the RSV F protein precursor F0 must be cleaved by the host's furin protease at two specific sites. This step removes the p27 fragment and produces a complex composed of the N-terminal small subunit F2 and the C-terminal large subunit F1. The F2 subunit is connected to the F1 subunit through a disulfide bond to form a dimer, which is then assembled into a functional trimer structure, which is the form in which the F protein performs its fusion function. During the process of virus fusion with the cell membrane, the conformation of the F protein changes from an unstable pre-fusion conformation (pre-F) to a more stable post-fusion trimer conformation (post-F). This change is crucial for the successful invasion of the virus into the cell.

[0006] The F protein contains key antigenic epitopes that stimulate the host to produce neutralizing antibodies. Its conservative structural characteristics may partially explain why HRSV has only a single serotype. It is worth noting that in HRSV-infected individuals, the neutralizing antibody titers induced by the F protein are significantly higher than those induced by the G protein and SH protein. In view of this, the F protein is regarded as the core target for HRSV vaccine development (Jiang Mingyue, Xu Yunshao, Xu Songtao, Feng Luzhao (2023). "Progress in Clinical Research on Human Respiratory Syncytial Virus Vaccine." Chinese Journal of Preventive Medicine 57(1): 70-77.). Pre-F-specific antibodies determine the magnitude of RSV neutralizing activity in human serum. Therefore, in the development of HRSV vaccines, the use of structure-based antigen design strategies is conducive to more effectively inducing high-affinity neutralizing antibodies against the pre-F protein, thereby improving the protective efficacy of the vaccine.

[0007] According to the structural information, RSV Pre-F has six major antigenic sites (I, II, III, IV, V and Ø epitopes), of which Ø and V epitopes are specific to the prefusion structure, which lays the foundation for the structural design of RSV Pre-F. The corresponding specific neutralizing antibodies such as D25, AM22, 5C4, etc. are the key to determining the prefusion structure of RSV F. Based on this, the first Pre-F design was generated: DS-Cav1 (McLellan, JS, M. Chen, MG Joyce, M. Sastry, GB Stewart-Jones, Y. Yang, B. Zhang, L. Chen, S. Srivatsan, A. Zheng, T. Zhou, KWGraepel, A. Kumar, S. Moin, JC Boyington, GY Chuang, C. Soto, U. Baxa,AQ Bakker, H. Spits, T. Beaumont, Z. Zheng, N. Xia, SY Ko, JP Todd,S. Rao, BS Graham and PD Kwong (2013). "Structure-based design of afusion glycoprotein vaccine for respiratory syncytial virus." Science 342(6158): 592-598.). The recently developed F protein antigen can induce a strong neutralizing antibody response. Based on this, researchers have developed two recombinant protein vaccines (AREXVY and ABRYSVO) and one mRNA vaccine (mRESVIA). All three vaccines are currently on the market.Among them, ABRYSVO contains two subtypes of RSV Pre-F recombinant protein vaccine (code: pXCS847) (Che Ye, PR Dormitzer, AV Gribenko, LD Hanke, AK Prasad, Qiu Xiayang, ME Lu Peng, Song Xi, KA Swanson, S. Kodali, Xu Xin, KS Efferen, Cai Ping, KR Tompkins and LDP Nunez (2018). RSV F protein mutants: 128; Che, Y., AV Gribenko, X. Song, LD Hanke, KS Efferen, K. Tompkins, S. Kodali, L. Nunez, AK Prasad, LMPhelan, M. Ammirati, X. Yu, JA Lees, W. Chen, L. Martinez, V. Roopchand,S. Han, X. Qiu, JP DeVincenzo, KU Jansen, PR Dormitzer and KASwanson (2023). "Rational design of a highly immunogenic prefusion-stabilizedF glycoprotein antigen for a respiratory syncytial virus vaccine." Sci TranslMed 15(693): eade6422.) Its advantages are that it does not require adjuvants and has fewer adverse reactions. It is currently the best antigen design for Pre-F in the RSV vaccines that have been marketed. In order to improve the prevention and treatment efficacy of RSV, it is necessary to develop F protein antigens with better performance. Summary of the invention

[0008] The invention provides a respiratory syncytial virus pre-fusion F protein mutant and application thereof.

[0009] For RSV pre-fusion F protein (RSV pre-F), the content and stability of its trimer are important goals for improving its immunogenicity, among which the stability of the trimer is correlated with the level of induced neutralizing antibodies. Based on the above strategy, the present invention designs a new antigen for the respiratory syncytial virus (RSV) pre-fusion F protein so that it can be stable in the pre-fusion trimer conformation for a long time, and the key epitopes (Ø and V) maintain the correct spatial structure. The immunogenicity of the RSV pre-fusion F protein mutant obtained by antigen design is significantly enhanced, and it can induce a high level of neutralizing antibody titers, which can be used for RSV diagnosis, antibody therapy and the development of various vaccines for the pre-fusion F protein conformation.

[0010] Specifically, the present invention provides the technical solution described below.

[0011] In a first aspect, the present invention provides a respiratory syncytial virus pre-fusion F protein mutant, which has the following mutations compared to the wild-type respiratory syncytial virus F protein: the amino acid at position 100 mutates to P, the amino acid at position 101 mutates to A or G, the amino acid at position 102 mutates to C, and the amino acid at position 152 mutates to C.

[0012] Preferably, the mutant has the following mutations compared to the wild-type respiratory syncytial virus F protein: T at position 100 mutates to P, P at position 101 mutates to A or G, A at position 102 mutates to C, and V or I at position 152 mutates to C.

[0013] In some embodiments of the present invention, a protein with a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2 is used as an example of a wild-type respiratory syncytial virus F protein, and the mutant has the following mutations compared to the wild-type respiratory syncytial virus pre-fusion F protein: T100P, P101A or G, A102C, and V or I152C.

[0014] In the antigen design process for RSV pre-fusion F protein, the present invention screened a large number of mutants and verified their performance. It was found that the combination mutation of amino acid 100 to P, amino acid 101 to A or G, amino acid 102 to C, and amino acid 152 to C can significantly increase the expression of pre-fusion F protein and the stability of pre-fusion conformation. The pre-fusion F protein mutant containing the above four site combination mutations can effectively stabilize the F protein in the pre-fusion conformation, which can effectively improve the structural stability of RSV pre-fusion F protein. After being treated at 50°C and 60°C for 1 hour, the relative binding activity with specific antibodies against key epitopes remains relatively stable. The combination of the above mutation sites can not only maintain high binding force with antibodies against key epitopes (Ø and V), but also help to enhance the specific immunogenicity of RSV pre-fusion F protein and significantly improve the level of induced neutralizing antibodies.

[0015] Among the above mutants, the mutation of amino acid at position 100 to P and the mutation of amino acid at position 101 to A or G can shorten the distance between amino acid at position 102 and amino acid at position 152. On this basis, the mutation of amino acid at position 102 to C and the mutation of amino acid at position 152 to C can introduce intramolecular disulfide bonds, thereby enhancing the structural stability between the F1 subunit and the F2 subunit before fusion, thereby effectively improving the structural stability of RSV F protein before fusion.

[0016] Illustratively, the amino acid sequence of the mutant comprising the above mutation site is shown in SEQ ID NO.12, 13, 15, 16, 17, 19 or 20.

[0017] Preferably, the mutant further has one or more mutations selected from the following compared to the wild-type respiratory syncytial virus F protein: S46G, N or T67F or I, A or T74P, E92D, S190I, N227P, K465Q, D486S, E487Q.

[0018] Based on the combined mutations of amino acid position 100 to P, amino acid position 101 to A or G, amino acid position 102 to C, and amino acid position 152 to C, the mutants obtained by further combining one or more of the above-mentioned S46G, N or T 67F or I, A or T74P, E92D, S190I, N227P, K465Q, D486S, and E487Q mutations can still maintain the correct spatial structure at the key epitopes (Ø and V), and the stability and immunogenicity of the mutant pre-fusion conformation are significantly improved, and as an antigen, it can induce the production of higher levels of neutralizing antibody titers.

[0019] Preferably, the mutant further has the following mutation compared to the wild-type respiratory syncytial virus F protein: amino acids 106-137 are replaced by the QARGSGSGRS (SEQ ID NO. 21) linker.

[0020] In some embodiments of the present invention, the mutant contains the following mutations compared to the wild-type respiratory syncytial virus F protein: S46G, N or T67F, A or T74P, E92D, T100P, P101A, A102C, V or I152C, amino acids 106-137 are replaced by a QARGSGSGRS linker, S190I, N227P, K465Q and E487Q.

[0021] In other embodiments of the present invention, the mutant contains the following mutations compared to the wild-type respiratory syncytial virus F protein: S46G, N or T67F, A or T74P, E92D, T100P, P101G, A102C, V or I152C, amino acids 106-137 are replaced by a QARGSGSGRS linker, S190I, K465Q and D486S.

[0022] In the present invention, the mutated amino acid position is given with reference to the sequence of the wild-type respiratory syncytial virus F protein shown in SEQ ID NO.1 or SEQ ID NO.2. In the present invention, the mutated amino acid position and the amino acid before mutation shown in each mutation site are preferably referenced to the wild-type respiratory syncytial virus F protein shown in SEQ ID NO.1 or SEQ ID NO.2, but it can be understood by those skilled in the art that the sequence of the wild-type respiratory syncytial virus F protein is not limited to the sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and the sequence of the F protein of other wild strains of RSV may have conservative mutations at certain sites with the sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and these conservatively mutated amino acids are similar to the original amino acids in physical and chemical properties, and the conservative mutations have no effect or little effect on the function and structure of the protein. Therefore, the F protein of other wild-type RSVs carrying these conservative mutations can also be used as a reference for the amino acid position of the mutant. In this case, the amino acid before the mutation of a certain mutation site is correspondingly changed to the amino acid at the corresponding site of the wild-type RSV F protein (for example, when the sequence shown in SEQ ID NO.1 is used as a reference, the 152nd amino acid is V, and when the sequence shown in SEQ ID NO.2 is used as a reference, the 152nd amino acid is correspondingly changed to I, and V152I is a conservative mutation and will not affect the function of the protein). Those skilled in the art can reasonably expect that these mutants also have the performance claimed in the present invention.

[0023] In some embodiments of the present invention, the amino acid sequence of the wild-type respiratory syncytial virus F protein is shown as SEQ ID NO.1 or 2.

[0024] Based on the above mutants, the present invention further designs a truncation of the F protein before fusion.

[0025] Preferably, the mutant is truncated to amino acid position 510, 511, 512, 513, 514 or 515 compared to the wild-type respiratory syncytial virus F protein.

[0026] In some embodiments of the present invention, the mutant is truncated to amino acid 513 of wild-type respiratory syncytial virus F protein.

[0027] Preferably, the mutant is linked to a trimerization domain at the truncated position. The trimerization domain is linked to the C-terminus of the truncated position via a linker, such as an amino acid linker, for example the sequence GG, GS or SAIG.

[0028] The present invention does not impose any special restrictions on the trimerization domain, and any domain that can trimerize the pre-fusion F protein can be selected, including but not limited to the T4 Fibritin trimerization domain. The amino acid sequence of the optional trimerization domain is as follows: GYIPEAPRDGQAYVRKDGEWVLLSTFLGGIEGR (SEQ ID NO. 22).

[0029] In some embodiments of the present invention, the amino acid sequence of the mutant is as shown in SEQ ID NO.16, 17, 19 or 20. In the above mutants, T100P and P101A or G mutations can shorten the distance between amino acid 102 and amino acid 152, and further A102C and V or I152C substitutions, and the addition of intramolecular disulfide bond mutations can enhance the structural stability between F1 subunit and F2 subunit before fusion, while maintaining or enhancing the binding activity of important sites such as the middle epitope, and improving the pre-fusion F protein-specific immunogenicity of the mutant antigen. In the performance verification experiment, the mutant with an amino acid sequence as shown in SEQ ID NO.16, 17, 19 or 20 showed a high expression level, and the content of trimer formation was significantly increased, with high binding force to key epitope antibodies, the ability to efficiently induce the production of neutralizing antibodies, and excellent storage stability and thermal stability, and its performance is comparable to or even better than that of the pXCS847 mutant, and is an ideal antigen for RSV diagnostic reagents and preventive and therapeutic drugs.

[0030] In a second aspect, the present invention provides a biomaterial, wherein the biomaterial comprises any one of the following (1) to (5):

[0031] (1) a nucleic acid molecule encoding the respiratory syncytial virus pre-fusion F protein mutant described above;

[0032] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0033] (3) a recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2);

[0034] (4) A transformed cell containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3);

[0035] (5) A recombinant virus containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

[0036] For the nucleic acid molecule encoding the mutant described in (1), if the amino acid sequence of the mutant is known, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding it according to the codon rule. Based on the degeneracy of codons, nucleic acid molecules encoding a mutant are not unique, but all fall within the scope of protection of the present invention. In addition to the coding sequence of the mutant, the above-mentioned nucleic acid molecule may also contain structural elements, and the structural elements include one or more of a 5'UTR sequence, a 3'UTR sequence, and a polyA tail sequence. The nucleic acid molecule may be DNA or RNA.

[0037] The expression cassette described in (2) above can be obtained by connecting the nucleic acid molecule to transcription and expression structural elements, and the transcription and expression structural elements include one or more of a promoter, a terminator, and a ribosome binding site.

[0038] The recombinant vector described in (3) above may be a plasmid vector, a viral vector, etc.

[0039] The transformed cell described in (4) above is a host cell transformed with the nucleic acid molecule described in (1) or the expression cassette described in (2) or the recombinant vector described in (3), and the host cell is selected from mammalian cells, insect cells, bacteria or fungi; wherein the mammalian cells include BHK cells, Chinese hamster ovary cells (CHO), HEK293 cells, tumor cell lines, etc. The bacteria include Escherichia coli, etc. The fungi include yeast, etc.

[0040] The recombinant virus described in (5) above includes adenovirus, adeno-associated virus, vaccinia virus, herpes virus or retrovirus vector, etc.

[0041] In a third aspect, the present invention provides any one of the following (1) to (3) uses of the respiratory syncytial virus pre-fusion F protein mutant or the biomaterial described above:

[0042] (1) Preparation of drugs for preventing and / or treating respiratory syncytial virus infection;

[0043] (2) Preparation of antibodies against respiratory syncytial virus;

[0044] (3) Preparation of diagnostic reagents for respiratory syncytial virus.

[0045] In the application described in (1) above, the drug includes a vaccine or a pharmaceutical preparation for preventing respiratory syncytial virus infection.

[0046] Among them, vaccines include recombinant protein vaccines, nucleic acid vaccines, vector vaccines or virus-like particle vaccines.

[0047] The application described in (2) above includes immunizing animals with the respiratory syncytial virus pre-fusion F protein mutant or the biological material as an antigen to induce the production of anti-respiratory syncytial virus antibodies.

[0048] In the above (3), the respiratory syncytial virus pre-fusion F protein mutant can be used as a positive control reagent for preparing a diagnostic reagent.

[0049] In a fourth aspect, the present invention provides a pharmaceutical composition comprising the respiratory syncytial virus pre-fusion F protein mutant or the biological material described above.

[0050] The above-mentioned pharmaceutical composition includes a vaccine or a pharmaceutical preparation for resisting respiratory syncytial virus infection.

[0051] The pharmaceutical composition may also include pharmaceutically acceptable adjuvants and / or auxiliary materials. The pharmaceutical composition may be in the form of injection, nasal drops, aerosol or oral preparation.

[0052] In some embodiments of the present invention, the pharmaceutical composition is a vaccine.

[0053] The vaccine includes a recombinant protein vaccine, a nucleic acid vaccine, a vector vaccine or a virus-like particle vaccine.

[0054] Wherein, the nucleic acid vaccine includes mRNA vaccine and DNA vaccine. The vector vaccine includes recombinant virus vector vaccine.

[0055] The subjects of administration of the above-mentioned pharmaceutical composition are animals, including mammals, preferably humans, livestock or poultry.

[0056] In a fifth aspect, the present invention provides a diagnostic reagent for respiratory syncytial virus, wherein the reagent comprises the respiratory syncytial virus pre-fusion F protein mutant or the biological material described above.

[0057] In a seventh aspect, the present invention provides an immunogenic composition comprising the respiratory syncytial virus pre-fusion F protein mutant or the biological material described above.

[0058] In an eighth aspect, the present invention provides a method for preparing the respiratory syncytial virus pre-fusion F protein mutant described above, the method comprising: introducing a nucleic acid molecule encoding the respiratory syncytial virus pre-fusion F protein mutant into a host cell to obtain a transformed cell, culturing the transformed cell, and collecting the respiratory syncytial virus pre-fusion F protein mutant in the culture.

[0059] The beneficial effects of the present invention include at least: the RSV pre-fusion F protein mutant provided by the present invention maintains the correct spatial structure at the key epitopes (Ø and V), has a high binding force with the key epitope antibody, can effectively stabilize the F protein in the pre-fusion conformation, has a high expression level, and has high storage stability and thermal stability. The results of the in vivo immunogenicity study in animals show that the mutant can induce animals to produce high-titer RSV neutralizing antibodies and can be used to prepare RSV prevention and treatment drugs and diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0061] Figure 1 Schematic diagram of the location of the disulfide bond in the Pre-F protein structure in Example 1 of the present invention.

[0062] Figure 2 Comparison of the neutralizing antibody levels induced by the mutant mRNA vaccines in animals in Example 5 of the present invention.

[0063] Figure 3 and Figure 4 The binding ability of TS68 and TS71 mutants in Example 6 of the present invention with the key epitope antibody.

[0064] Figure 5 This is a comparison of the neutralizing antibody levels induced by the TS68 and TS71 mutants and pXCS847A in animals in Example 7 of the present invention.

[0065] Figure 6 and Figure 7 This is an evaluation of the storage stability of the TS68 and TS71 mutants in Example 8 of the present invention at 5°C and room temperature.

[0066] Figure 8 This is an evaluation of the thermal stability of the TS68 and TS71 mutants in Example 9 of the present invention.

[0067] Figure 3-Figure 8 In the table, TS68 represents the TS68 A2 mutant, and TS71 represents the TS71 A2 mutant. DETAILED DESCRIPTION

[0068] The present invention studies the spatial structure of the pre-fusion F protein of the respiratory syncytial virus, takes the amino acid sequence of the wild-type respiratory syncytial virus F protein (type A2 or type B1, whose sequence is shown in SEQ ID NO.1, 2, or other wild-type virus strains, such as the RSV strain disclosed in patent application CN108738312A, pages 48-49) as a template, and attempts to design amino acid mutations that can stabilize the pre-fusion conformation based on the principles of structural biology.

[0069] The present invention provides biological materials such as a respiratory syncytial virus pre-fusion F protein mutant, an immunogenic composition comprising the respiratory syncytial virus pre-fusion F protein mutant, a method for producing the respiratory syncytial virus pre-fusion F protein mutant, a composition comprising the respiratory syncytial virus pre-fusion F protein mutant, and a nucleic acid molecule encoding the respiratory syncytial virus pre-fusion F protein mutant.

[0070] The respiratory syncytial virus pre-fusion F protein (RSV pre-F) mutant provided by the present invention, compared with the wild-type RSV F protein (preferably the sequence is shown in SEQ ID NO.1 or 2), simultaneously includes the following mutations: the amino acid at position 100 mutates to P, the amino acid at position 101 mutates to A or G, the amino acid at position 102 mutates to C, and the amino acid at position 152 mutates to C.

[0071] In some embodiments of the present invention, the pre-fusion F protein mutant comprises the following mutations (1) to (4) compared to the wild-type RSV F protein (preferably the sequence is shown in SEQ ID NO. 1 or 2):

[0072] The tyrosine (T) at position 100 was mutated to proline (P);

[0073] (2) Proline (P) at position 101 mutates to alanine (A) or glycine (G);

[0074] Alanine (A) at position 102 was mutated to cysteine ​​(C);

[0075] (4) The valine (V) or isoleucine (I) at position 152 is mutated to cysteine ​​(C).

[0076] In the above mutant, cysteine ​​at position 102 forms a disulfide bond with cysteine ​​at position 152 to stabilize the spatial conformation of the protein.

[0077] In some embodiments of the present invention, the mutant has one or more mutations selected from the following compared to the wild-type respiratory syncytial virus F protein: S46G, N67F or I, A74P, E92D, S190I, N227P, K465Q, D486S, E487Q.

[0078] In some embodiments of the present invention, the mutant further has one or more mutations selected from the following compared to the wild-type respiratory syncytial virus F protein: N67F, N227P, E487Q.

[0079] In some embodiments of the present invention, the mutant has the following mutations compared to the wild-type respiratory syncytial virus F protein: TPA102PAC-V152C, N67F, N227P, E487Q.

[0080] In some embodiments of the present invention, the mutant has the following mutations compared to the wild-type respiratory syncytial virus F protein: TPA102PGC-V152C, N67I, S190I, D486S.

[0081] In some embodiments of the present invention, the mutant has the following mutations compared to the wild-type respiratory syncytial virus F protein:

[0082] (1) TPA102PAC-V152C, N67F, A74P, E92D, S46G, K465Q, E487Q;

[0083] (2) TPA102PAC-V152C, N67F, A74P, E92D, S46G, K465Q, S190I, E487Q, N227P;

[0084] (3) TPA102PGC-I152C, E66F, N67I, S190I, D486S, A74P, E92D, S46G, K465Q;

[0085] (4) TPA102PGC-I152C, N67F, S190I, D486S, A74P, E92D, S46G, K465Q.

[0086] In some embodiments of the present invention, the mutant further has one or more of the following disulfide bond mutations compared to the wild-type respiratory syncytial virus F protein:

[0087] (1) 157C and 183C substitution;

[0088] (2) 170C and 177C replacement;

[0089] (3) S55C and L188C substitutions;

[0090] (4) L142C and N371C substitutions;

[0091] (5) T103C and I148C substitutions;

[0092] (6) L142C and N371C substitutions;

[0093] (7) 96C and 279C substitutions;

[0094] (8) 149C and 458C substitutions;

[0095] (9) 183GC and 428C substitution;

[0096] (10) 369C and 455C substitution;

[0097] (11) 100C and 362C substitution;

[0098] (12) 99C and 362C substitutions;

[0099] (13) 98C and S362C substitutions;

[0100] (14) 100C and 361C replacement;

[0101] (15) 99C and 361C substitutions;

[0102] (16)98C and S361C substitutions.

[0103] In some embodiments of the present invention, the respiratory syncytial virus prefusion F protein mutant may also contain at least one of the following amino acid substitutions: S46G, N67I, E92D, L95M, I221M, R429K, K465Q, S215P, I217P.

[0104] In some embodiments of the present invention, the respiratory syncytial virus prefusion F protein mutant may also include an amino acid at position 190 replaced by F, I, Y, L, H or M, and an amino acid at position 207 replaced by L, I or Y.

[0105] In some embodiments of the present invention, the respiratory syncytial virus prefusion F protein mutant may further comprise a L373R substitution, wherein the L373R mutation is introduced as a compatible solvent-exposed residue to increase protein production.

[0106] In some embodiments of the present invention, the mutant further has the following mutation compared to the wild-type respiratory syncytial virus F protein: amino acids 106-137 are replaced by a QARGSGSGRS linker.

[0107] In some embodiments of the present invention, the mutant has the following mutations compared to the wild-type respiratory syncytial virus F protein: truncation to amino acid position 510, 511, 512, 513, 514 or 515, preferably truncation to amino acid position 513.

[0108] In some embodiments of the present invention, the truncated mutants described above may be expressed in the form of a recombinant trimer, and the F2-F1 extracellular domain protomer of the trimer may include a C-terminus connected to a trimerization domain (eg, a T4 Fibritin trimerization domain).

[0109] In some embodiments of the invention, the truncated mutants described above comprise RSV F0 positions 26-105 and 138-513, or RSV F0 positions 26-103 and 137-513, and are linked to a C-terminal trimerization domain.

[0110] In some embodiments of the present invention, the recombinant F2-F1 extracellular domain protomer of the extracellular domain trimer of the truncated mutants described above may comprise a C-terminus connected to a transmembrane domain (eg, RSV F transmembrane domain).

[0111] In some embodiments of the invention, the recombinant F2-F1 ectodomain protomer in the above ectodomain trimer may contain additional amino acid substitutions known to increase the stability or expression of the RSV F ectodomain trimer.

[0112] In some embodiments of the present invention, the recombinant F2-F1 extracellular domain protomer in the respiratory syncytial virus prefusion F protein mutant may also contain at least one of the following amino acid substitutions: S46G, N67I, E92D, L95M, I221M, R429K, K465Q, S215P or I217P.

[0113] In some embodiments of the present invention, the recombinant F2-F1 extracellular domain protomer in the respiratory syncytial virus prefusion F protein mutant may also contain L95M, I221M and R429K substitutions to increase the stability and / or expression of its extracellular domain trimer.

[0114] In some embodiments of the present invention, the recombinant F2-F1 ectodomain protomer in the respiratory syncytial virus prefusion F protein mutant may further comprise L95M, I221M, R429K and I217P substitutions to increase the stability and / or expression of the RSV F ectodomain trimer.

[0115] In some embodiments of the present invention, the recombinant F2-F1 ectodomain protomer in the ectodomain trimer of the respiratory syncytial virus prefusion F protein mutant may further comprise S46G, K465Q, S215P and E92D substitutions to increase the stability and / or expression of the RSV F ectodomain trimer.

[0116] In some embodiments of the present invention, the recombinant F2-F1 ectodomain protomer in the ectodomain trimer of the respiratory syncytial virus prefusion F protein mutant may further comprise S46G, K465Q, S215P, E92D and N67I substitutions to increase the stability and / or expression of the RSV F ectodomain trimer.

[0117] As examples of some specific embodiments, the respiratory syncytial virus pre-fusion F protein mutant comprises a combination of mutations shown in any one of the following (1) to (14) compared to the wild-type respiratory syncytial virus F protein:

[0118] (1) I148C-V243C, S190I and D486S; the amino acid sequence of an exemplary mutant (TS14) is shown in SEQ ID NO.5;

[0119] (2) P101C-V152C, S190I and D486S; the amino acid sequence of an exemplary mutant (TS18) is shown in SEQ ID NO.6;

[0120] (3) N67F, N227P and E487Q; the amino acid sequence of an exemplary mutant (TS21) is shown in SEQ ID NO.7;

[0121] (4) A147C, M370GC, N67I, S215P and E487Q; the amino acid sequence of an exemplary mutant (TS25) is shown in SEQ ID NO.8;

[0122] (5) A147C, M370GC, N67F, S190I, D486S and E487Q; the amino acid sequence of an exemplary mutant (TS26) is shown in SEQ ID NO.9;

[0123] (6) A147C, M370GC, N67F, N227P, S190I, D486S and E487Q; the amino acid sequence of an exemplary mutant (TS27) is shown in SEQ ID NO. 10;

[0124] (7) TPA102PGC, I148C, N67F, N227P and E487Q; the amino acid sequence of an exemplary mutant (TS49) is shown in SEQ ID NO.11;

[0125] (8) TPA102PAC, V152C, N67F, S215P and E487Q; the amino acid sequence of an exemplary mutant (TS50) is shown in SEQ ID NO.12;

[0126] (9) TPA102PAC, V152C, N67F, N227P and E487Q; the amino acid sequence of an exemplary mutant (TS51) is shown in SEQ ID NO. 13;

[0127] (10) TPA102PGC, I148C, E66F, N67I, S190I and D486S; the amino acid sequence of an exemplary mutant (TS53) is shown in SEQ ID NO. 14;

[0128] (11) TPA102PGC, V152C, E66F, N67I, S190I and D486S; the amino acid sequence of an exemplary mutant (TS54) is shown in SEQ ID NO.15;

[0129] (12) TPA102PAC, V or I152C, S46G, N or T67F, A or T74P, E92D, S190I, N227P, K465Q and E487Q; the amino acid sequence of an exemplary mutant (TS68 A2 or TS68 B1) is shown in SEQ ID NO. 16 or 17;

[0130] (13) I148C-V243C, S46G, N67F, A74P, E92D, S190I, K465Q and D486S; the amino acid sequence of an exemplary mutant (TS69) is shown in SEQ ID NO. 18;

[0131] (14) TPA102PGC, V or I152C, S46G, N or T67F, A or T74P, E92D, S190I, D486S and K465Q; the amino acid sequence of an exemplary mutant (TS71 A2 or TS71 B1) is shown in SEQ ID NO. 19 or 20.

[0132] The mutants provided by the present invention can be used to induce an immune response against RSV (eg, RSV A or RSV B) in vertebrates (eg, mammals, such as humans and cattle).

[0133] In the present invention, the term "AM14" refers to the antibody described in WO2008 / 147196A2, which has a heavy chain variable domain and a light chain variable domain comprising an amino acid sequence, and is also disclosed in patent document CN108738312A. The antibody can be used to characterize whether a pre-fusion trimer conformation is formed.

[0134] The term "D25" refers to an antibody described in WO2008 / 147196A2, which has a heavy chain variable domain and a light chain variable domain comprising an amino acid sequence, and is also disclosed in patent document CN108738312A. The antibody can be used to characterize the presence of the Ø epitope conformation.

[0135] The term "Motavizumab" is a monoclonal antibody of Motavizumab, which is a derivative of Palivizumab. A variety of Palivizumab mutant antibodies were obtained by replacing amino acids in the CDR regions of the heavy and light chains of the monoclonal antibody of Palivizumab, among which Motavizumab's ability to bind to F protein is 70 times that of Palivizumab, and its activity in neutralizing HRSV is 20 times higher than that of Palivizumab. Motavizumab monoclonal antibody can be used to characterize the presence of II epitope conformation.

[0136] The term "RSV fusion (F) protein" is a RSV envelope glycoprotein that promotes fusion of viral and cellular membranes. In nature, the RSV F protein is initially synthesized as a single polypeptide precursor of approximately 574 amino acids in length, designated 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 proteolytically processed by cellular proteases at two conserved consensus furin cleavage sequences, approximately at positions 109 and 136 of F0; for example, RARR109 and RKRR136, to produce two disulfide-linked fragments F1 and F2. The smaller of these fragments, F2, is derived from the N-terminal portion of the F0 precursor and includes approximately residues 26-109 of F0. The larger of these fragments, F1, includes the C-terminal portion of the F0 precursor (approximately residues 137-574), including the extracellular / luminal region (approximately residues 137-529), the transmembrane domain (approximately residues 530-550), and the C-terminal cytoplasmic tail (approximately residues 551-574). The extracellular portion of the RSV F protein is the RSV F ectodomain, which includes the F2 protein and the F1 ectodomain.

[0137] The term "prefusion conformation" refers to the structural configuration adopted by the RSV F protein or mutant, which can be specifically bound by the following antibodies: (1) antibody D25 when the RSV F protein or mutant is in monomeric or trimer form, or (2) antibody AM14 when the RSV F protein mutant is in trimer form.

[0138] The term "RSV F protein mutant before fusion" refers to a mutant based on wild-type RSV F protein, wherein the mutant exhibits a mutation introduced therein relative to the amino acid sequence of the corresponding wild-type RSV F protein and has immunogenicity for wild-type RSV F protein or for a virus comprising wild-type F protein. In certain embodiments, RSV F protein mutants have some beneficial features before fusion, such as enhanced immunogenicity properties compared to the corresponding wild-type F protein or improved stability of the pre-fusion configuration of mutants or pre-fusion trimer configurations of mutants. In other embodiments, RSV F protein mutants exhibit one or more mutations introduced as described herein before fusion and combine pre-fusion specific antibodies selected from antibody D25 or antibody AM14.

[0139] The amino acid sequences of some RSV F proteins and RSV pre-fusion F protein mutants involved in the present invention are shown in Table 1, wherein pXCS847A and SC-TM are currently known RSV pre-fusion F protein mutants, used as controls.

[0140] Table 1

[0141]

[0142] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0143] In the following examples, the position of the RSV pre-fusion F protein corresponds to the amino acid sequence of the reference F0 polypeptide shown in SEQ ID NO. 1 or 2.

[0144] Example 1 Design and screening of truncated pre F protein mutants (1)

[0145] Based on the proof-of-concept work that has been carried out in the early stage, it was found that non-natural disulfide bonds are one of the important factors affecting the expression of RSV preF protein. Therefore, in the first round of design, by studying the spatial conformation of the pre-fusion F protein of the respiratory syncytial virus, using the amino acid sequence of the wild-type respiratory syncytial virus F protein (SEQ ID NO.1 or SEQ ID NO.2) as a template, we tried to introduce non-natural disulfide bond mutations based on the principles of structural biology, so as to obtain amino acid mutations with higher expression and stable pre-fusion conformation.

[0146] To achieve this goal, the present invention attempts a new approach: by using T100P and P101A or G mutations to shorten the distance between amino acids 102 and 152, and further performing A102C, V or I152C substitutions ( Figure 1 ), intramolecular disulfide bond mutations are added to enhance the structural stability between the pre-fusion F1 subunit and the F2 subunit, while maintaining or enhancing the binding activity of important sites such as the middle epitope, thereby improving the pre-fusion F protein-specific immunogenicity of the mutant antigen.

[0147] The mutation sites mentioned above include:

[0148] (1) Tyrosine (T) at position 100 mutated to proline (P);

[0149] (2) Proline (P) at position 101 mutates to alanine (A) or glycine (G);

[0150] (3) Alanine (A) at position 102 mutated to cysteine ​​(C);

[0151] (4) The valine (V) or isoleucine (I) at position 152 is mutated to cysteine ​​(C).

[0152] In addition, in order to further increase the stability of the pre-fusion F protein, the above mutation design was combined with S46G, N67F, A74P, V76I, E92D, R106Q, F137S, V152C, S190I, I379V, M447V, K465Q, D486S, etc., and further, a soluble T4 Fibritin trimerization domain was added at position 513 of the transmembrane region of the C-terminus of the pre-fusion F protein to increase the stability of the trimer structure of the F protein. The mutants designed according to the above objectives are shown in Table 2.

[0153] Table 2 Design of truncated pre F protein mutants

[0154]

[0155] By standard molecular biological techniques, the nucleic acid sequences of the truncated pre F protein mutants described in Table 2 were inserted into the pcDNA3.4 expression vector sequence and then the whole plasmid was synthesized. Then, the mutant plasmid was transfected into 10 mL of CHO cells using PEI. After continuous culture for 5 days, the supernatant culture fluid was collected and the expression of each mutant protein in the culture supernatant was detected by ELISA method using epitope II specific monoclonal antibody Motavizumab as capture antibody and epitope Ø specific antibody D25 as detection antibody. The OD value (OD 450 -OD 630 ) indicates that the pre-fusion conformation activity of the mutant is higher, and the results are shown in Table 3. The results show that the expression levels of TS50, TS51, and TS54 are comparable to those of the wild type, and the expression levels of TS14, TS18, TS26, and TS53 are comparable to those of pXCS847A and SC-TM.

[0156] Table 3 Expression of truncated pre F protein mutants

[0157]

[0158] Example 2 Design and screening of truncated pre F protein mutants (2)

[0159] In order to further improve the expression level and stability of the mutants, new combinations of mutation sites were screened based on the mutants TS14, TS51 and TS54 obtained in Example 1, and the stability of the mutants was further evaluated. The mutants designed according to the above objectives are shown in Table 4.

[0160] Table 4 Design of truncated pre F protein mutations

[0161]

[0162] The plasmid synthesis, transfection and fermentation culture of the truncated pre F protein mutants in this example were the same as in Example 1, and the expression level of each mutant in the cell supernatant was detected using the Human respiratory syncytial virus (RSV) (A2) Prefusion glycoprotein F / RSV-preF ELISA kit of Sino Biological, and the results are shown in Table 5. The results showed that the expression levels of mutants TS68 A2 and TS71 A2 were better than those of wild-type pre F and mutants pXCS847A and SC-TM.

[0163] Table 5 Expression of truncated pre F protein mutants

[0164]

[0165] Example 3 Purification of recombinant protein antigen of truncated pre F protein mutant

[0166] To further evaluate the properties of the antigen, affinity chromatography was used to purify the target protein. The storage solution in the chromatography column was rinsed with 3 to 5 column volumes of deionized water. The chromatography column was equilibrated with 5 column volumes of equilibration solution (100 mM Tris-HCl, 150 mM NaCl, 1mM EDTA, pH 8.0). The sample was diluted 2 times with the equilibration solution and loaded at a pump speed of 1 mL / min. After loading, the column was rinsed with equilibration solution for 10 column volumes, followed by elution with 5 to 10 column volumes of eluent (equilibration solution containing 2.5 mM desthiobiotin), and the eluent was collected. Finally, the eluent was concentrated and replaced with a 30KD ultrafiltration tube, which was the target protein.

[0167] Example 4 Preparation of RSV Pre-F protein mutant mRNA vaccine

[0168] GenScript Biotech Inc. was commissioned to synthesize the DNA template sequences of TS68 A2, TS69, and TS71 A2 mutants, and mRNAs were synthesized using the above mutant templates, which were named RVD68, RVD69, and RVD71, respectively. Lipids were dissolved in anhydrous ethanol at a molar ratio of 50:10:38.5:1.5, including cationic lipids, distearoylphosphatidylcholine (DSPC), cholesterol, and DMG-PEG2000 (the cationic lipid was SM102, and all four lipids were purchased from Sinobank), as the alcohol phase; mRNA was dissolved in 50mM citric acid buffer (pH 4.0) as the aqueous phase; the alcohol phase and the aqueous phase were then mixed and encapsulated using a microfluidic system at a ratio of 1:3 (volume ratio), and the preparation was diluted with PBS buffer and concentrated using a 30KD ultrafiltration tube for liquid exchange. After adjusting the mRNA concentration to 200μg / mL, an equal volume of PBS solution with a sucrose concentration of 20% was added to make the mRNA concentration to 100μg / mL and the sucrose concentration to 10%, and the mixture was filtered using a 0.22μm filter membrane to obtain the mRNA vaccine preparation, which was stored at 4°C for later use.

[0169] Example 5 Evaluation of the immunogenicity of mRNA vaccines

[0170] 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, with 5 mice in each group. The mutant mRNA vaccine prepared in Example 4 was injected into the quadriceps femoris of the thigh on one side at 10 μg / mouse, and the negative control group was PBS; a second booster immunization was performed 3 weeks after the initial immunization, for a total of two immunizations. Orbital blood was collected 14 days after the booster immunization, and the serum was separated for neutralizing antibody detection. The results showed that among the mRNA vaccines, the mRNA vaccine RVD71 prepared with the sequence of the mutant TS71 induced the highest level of neutralizing antibodies in mice ( Figure 2 ).

[0171] Example 6 Detection of epitope binding ability of truncated pre F protein mutants

[0172] According to the above results, the mutants TS68 A2 and TS71 A2 with high expression levels were selected as the objects for protein vaccine evaluation. The epitope activity of the purified TS68 A2 and TS71 A2 mutant antigens was detected. Monoclonal antibody D25 targeting epitope Ø and monoclonal antibody AM14 targeting epitope V were used as primary antibodies to detect the binding with each mutant. The purified mutants were diluted to 100 μg / well, diluted 3 times in a gradient, and the antigens were coated in a 96-well plate using coating buffer. The primary antibody and HRP anti-human IgG1 Fc Antibody were added in sequence for incubation and TMB color development was performed. The absorbance at 450 nm was detected by an enzyme reader. The data were counted and analyzed, and pXCS847A was used as a positive control.

[0173] The results are as follows Figure 3 and Figure 4 As shown, mutants TS68 A2 and TS71 A2 have strong binding activity with each monoclonal antibody, among which TS71 A2 has a significantly better binding activity with AM14 than the positive control pXCS847A, indicating that the trimer content formed by mutant TS71 A2 is significantly higher than that of pXCS847A.

[0174] Example 7 Evaluation of the immunogenicity of RSV Pre-F protein mutant protein vaccines

[0175] The experimental animals were BALB / c female mice purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd., 6-8 weeks old, and raised in the Experimental Animal Center of Beijing Minhai Biotechnology Co., Ltd. The mice were randomly divided into groups, with 10 mice in each group. The mutant antigen purified in Example 3 was injected into the quadriceps femoris of the thigh at 10 μg / mouse and 0.25 μg / mouse. The negative control group was PBS, and the positive control group was pXCS847A antigen; a secondary booster immunization was performed 3 weeks after the initial immunization, for a total of two immunizations. Blood was collected from the orbit 14 days after the booster immunization, and the serum was separated for analysis.

[0176] The separated serum was incubated at 56°C for 30 minutes to inactivate complement; the serum was diluted in a gradient: 4 times in the first well, 2 times in a gradient, and 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, the fluorescently labeled antibody was added to the 96-well plate, incubated for 1 hour, and fixed; after washing, the fluorescence of each well was observed under a fluorescent inverted microscope to determine the neutralizing antibody titer, and the results are shown in Figure 5 The results showed that the RSV neutralizing antibody titers induced by mutants TS68 A2 and TS71 A2 in mice were higher than those induced by pXCS847A. The neutralizing antibody level induced by 10 μg of mutant TS71 A2 was twice that of 10 μg of mutant pXCS847A.

[0177] Example 8 Storage stability evaluation of mutant antigens

[0178] After the TS68 A2 and TS71 A2 mutant antigens were placed in a 5°C environment and a room temperature environment for 0, 4, 7, 14 and 28 days, the Motavizumab antibody was coated on a high-adsorption 96-well plate. The Motavizumab antibody can capture RSV antigens, and then D25-HRP / AM14-HRP was added. Horseradish peroxidase (HRP) catalyzed TMB substrate for color development, and the absorbance (OD) was tested at 450 / 630nm on an ELISA reader. The OD value is proportional to the sample concentration. The four-parameter fitting regression model was used to draw the curve and calculate the antigen content of the sample. The results are shown in Figure 6 and Figure 7 As shown, the TS68 A2 and TS71 A2 mutants remained stable after 28 days without significant changes.

[0179] Example 9 Evaluation of thermal stability of mutant antigens

[0180] After the TS68 A2 and TS71 A2 mutant antigens were treated at 50°C and 60°C for 1 hour, the monoclonal antibodies D25 and AM14 were used to detect the binding of the antibodies to the mutant antigens. The method was the same as in Example 2, and the ratio of the OD values ​​before and after heat treatment was used as the relative binding activity of the antibodies. Figure 8 As shown, both TS68 A2 and TS71 A2 mutant antigens have good thermal stability, among which TS71 A2 mutant antigen has better thermal stability.

[0181] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A respiratory syncytial virus prefusion F protein mutant, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.16 or 19.

2. Biomaterial, characterized in that The biological material includes any one of the following (1) to (5): (1) A nucleic acid molecule encoding the respiratory syncytial virus pre-fusion F protein mutant according to claim 1; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (2); (4) A transformed cell containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3); (5) A recombinant virus containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

3. The respiratory syncytial virus pre-fusion F protein mutant according to claim 1 or the biomaterial according to claim 2 for use in any one of the following (1) to (3): (1) Preparation of drugs for preventing and / or treating respiratory syncytial virus infection; (2) Preparation of antibodies against respiratory syncytial virus; (3) Preparation of diagnostic reagents for respiratory syncytial virus.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the respiratory syncytial virus pre-fusion F protein mutant according to claim 1 or the biological material according to claim 2.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition is a vaccine; The vaccine includes a recombinant protein vaccine, a nucleic acid vaccine, a vector vaccine or a virus-like particle vaccine.

6. A diagnostic reagent for respiratory syncytial virus, characterized in that: The reagent comprises the respiratory syncytial virus pre-fusion F protein mutant according to claim 1 or the biological material according to claim 2.

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