RSV pre-f mutant, production method therefor and use thereof

Abstract

The present application relates to an RSV pre-Fusion protein (pre-F) mutant, a preparation therefor and a use thereof. For the mutant, a α3-helix region of an F1 polypeptide thereof has a cysteine substituent α3-1, a β3-strand region has a cysteine substituent β3-1 and a cysteine substituent β3-2, and a β4 sheet has a cysteine substituent β4-1, wherein the cysteine substituent β3-1 and the cysteine substituent β4-1 form a disulfide bond, and the cysteine substituent α3-1 and the cysteine substituent β3-2 form a disulfide bond.

Description

RSV pre-f mutants, methods of production and uses thereof Related Applications This application claims priority to the Chinese patent application No. 2024106423573, filed on May 22, 2024, entitled “RSV pre-F mutants, methods of production and uses thereof”, the contents of which are hereby incorporated by reference in their entirety. TECHNICAL FIELD The present application belongs to the field of biotechnology, and relates to a RSV pre-F mutant, methods of production and uses thereof. BACKGROUND Human respiratory syncytial virus (RSV) is a common respiratory virus belonging to the single-stranded RNA virus under the virus family, and it is one of the main pathogens causing respiratory tract infections in infants and young children worldwide. The most common epidemic season of RSV is autumn and winter, especially the most frequent transmission among young children. RSV infection can cause respiratory symptoms, including cold symptoms, sore throat, cough, sneezing, nasal congestion and fever. In infants and people with weak immune systems, RSV can cause severe lower respiratory tract infections, such as bronchitis and pneumonia. In the past few decades, many RSV vaccine candidates have been tested in clinical trials, including attenuated virus vaccines, subunit vaccines and genetically engineered vaccines, etc. However, due to the complex characteristics of RSV virus and immunological responses, the development of these vaccines has faced some challenges, including the inability to obtain a long-lasting protective immune response, and the possible insufficient effectiveness for high-risk groups such as children and the elderly. Despite this, scientists and pharmaceutical companies are still working hard to try to find more effective RSV vaccines. Some research teams are focusing on understanding the immunological characteristics of RSV virus and how to stimulate a long-lasting and comprehensive immune response. At the same time, some new technology platforms and strategies are also being tried in vaccine development, such as microparticulate vaccines, nanoparticle vaccines and the design of structure-specific antigens, etc. F protein belongs to type I glycoprotein. Inactive precursor F0 is synthesized in the cell, in the process of maturation, F0 is cleaved by furin to form F2 (aa: 1-109), P27 (aa: 110-136) and F1 (aa: 137-574), F2 and F1 form heterodimer with disulfide bond, i.e. monomer of F protein, three monomers assemble to form a trimer. F protein adopts metastable pre-fusion protein (pre-F) expressed on the surface of viral envelope, when contacting with host cell membrane, the secondary structure components a2, a3 and b3, b4 and a4 at the N-terminal of F1 protein rearrange together with the following a5 to form a larger a helix structure, this series of processes leads to the transformation of F protein from high-energy level metastable pre-F structure to stable post-fusion protein (post-F) structure, compared with post-F, studies have shown that most of the high-efficiency neutralizing antibodies target epitopes only in pre-F, antigen epitopes and pre-F trimer top antigen epitope V has been proved to have extremely strong efficacy. Therefore, pre-F as a target antigen has become the priority of RSV vaccine. The current direction of RSV vaccine research is to find a more stable conformation of RSV pre-F mutant. Based on the principle of protein structure and stability, DS-Cav1 mutant is designed by cavity filling mutation (S190F and V207L) and disulfide bond replacement (S155C and S290C), which fixes the unstable N-terminal of F1 protein to obtain stable and antigenic pre-F protein (McLellan et al., Science, 2013). The iterative mutant based on structural design also shows stable pre-fusion conformation (PCT / US2014 / 026714 family) by the National Institutes of Health (NIH). The design strategy of RSV F protein mutant (CN 108738312 A) disclosed by Pfizer is also similar, in addition to S155C and S290C, S55C and L188C, T103C and I148C, and L142C and N371C are also replaced by disulfide bond. The cavity filling includes the substitution of I at position 190 and H at position 54, etc. Mutations such as D486S and E487Q are added, and different combination mutants are also performed. SC-DM designed by Janssen (Anders Krarup et al., Nature Communication, 2015) is to delete and replace the furin cleavage site and P27 with GS linker and introduce mutations N67I and S215P to maintain protein stability, and finally use T4 phage fiber protein trimer domain to make protein secretory expression and form trimer; and Pre-F-GCN4t of GSK (Normand Blais et al., Journal of Virology, 2017) replaces the transmembrane domain and C-terminal domain of F0 protein with the engineered GCN4 trimerization domain of Saccharomyces cerevisiae; the furin cleavage site and P27 sequence are removed, and a lysine residue is reserved at this position between F2 and fusion peptide (FP); mutations L112Q, Q471G and L482K are added to increase the uniformity and stability of the protein, and it is proved to be able to bind Epitope-specific monoclonal antibody D25. As can be seen from the above description, based on the structure of RSV F protein, mutations at multiple sites are derived based on different design strategies, which can prevent the conformational change of RSV F protein to post-F protein, so as to stabilize the pre-F protein of RSV in the pre-fusion conformation. However, in order to find a more stable conformation of RSV pre-F protein, the current research is still insufficient, and the expression amount and stability of pre-F protein still have room for improvement. Therefore, new exploration and attempts are still in progress. In 2013, the epitope was found by antibody D25 and named site [McLellan, Jason S., et al. "Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody." Science 340.6136 (2013): 1113-1117.], to solve the instability problem in the absence of D25 antibody, site of further modification to obtain the engineered stable RSV F protein pre-F antigen [McLellan, Jason S., et al. "Structure-based design of a fusion glycoprotein vaccine for respiratory syncytial virus." Science 342.6158 (2013): 592-598.], mainly involving disulfide bond mutations: DS (S155C, S290C), cavity filling: Cav1 (S190F, V207L) and mutation combination DS-Cav1, also including other mutants derived therefrom (PCT / US2014 / 026714 family). In 2015, Janssen Pharmaceutical published research evidence on the construction of stable pre-F protein mutants, introducing a short chain linker between F1 and F2, and performing amino acid mutation modification after the variant binds to the fibrous domain to obtain SC-DM and SC-TM [Krarup, Anders, et al. "A highly stable prefusion RSV F vaccine derived from structural analysis of the fusion mechanism." Nature communications 6.1 (2015): 8143.], involving amino acid mutations: SC-DM (N67I, S215P), SC-TM (N67I, S215P, E487Q) (US_11338031_B2 family). GSK developed DS-Cavl further (US8563002) by focusing on the protection and constraint of the furin cleavage site and the trimerization of the C-terminal of the protein. Given its good immunogenicity and the ability to induce neutralizing antibodies, GSK announced in 2022 that its RSV vaccine had positive results in phase III clinical trials for adults over 60 years old, and on May 03, 2023, it obtained marketing authorization (trade name: Arexvy), which is also the first vaccine against the RSV virus. Pfizer's stable pre-F protein mutants based on structural design (PCT / IB2016 / 057502 family) mainly involve mutation strategies including disulfide bond mutations and cavity filling, and the disulfide bond mutation sites involved include: S55C / L188C, S155C / S290C, T103C / I148C, L142C / N371C, and other cavity filling and electrostatic mutation sites that enhance the stability of the pre-F protein. Due to the good immunogenicity and excellent vaccine efficacy of the mutant, Pfizer announced in 2022 that its bivalent RSV vaccine had positive results in phase III clinical trials, and obtained marketing authorization in May 2023 (trade name: Abrysvo), becoming the second vaccine against the RSV virus after Arexvy. Currently, the main problem with stable RSV pre-F protein is low expression and instability during long-term storage. To solve this problem, structural design of RSV F protein is needed to find more stable mutation strategies. SUMMARY Based on this, one or more embodiments of the present application provide a RSV pre-F mutant and a production method and use thereof. One or more embodiments of the present application provide a mutant of RSV pre-F, wherein the F1 polypeptide of the mutant satisfies the conditions shown in (1) or (2) below: (1) the β3 sheet region has a cysteine substituent β3-1, the β4 sheet region has a cysteine substituent β4-1, and the cysteine substituent β3-1 and the cysteine substituent β4-1 form a disulfide bond; (2) the α3 helix region has a cysteine substituent α3-1, the β3 sheet region has a cysteine substituent β3-1 and a cysteine substituent β3-2, the β4 sheet has a cysteine substituent β4-1, the cysteine substituent β3-1 and the cysteine substituent β4-1 form a disulfide bond, and the cysteine substituent α3-1 and the cysteine substituent β3-2 form a disulfide bond; The mutant has one of the following combinations of cysteine mutations: Combination 1 is S180C and S186C; and, Combination 2 is K176C and S190C. In some embodiments of the application, the species origin of the mutant of RSV pre-F is human or bovine. In some embodiments of the application, the F1 polypeptide of the mutant of RSV pre-F has an amino acid sequence as set forth in any one of SEQ ID NO. 4, SEQ ID NO. 71 and SEQ ID NO. 72, or has at least 80% homology with the amino acid sequence as set forth in any one of SEQ ID NO. 4, SEQ ID NO. 71 and SEQ ID NO. 72. Optionally, the F1 polypeptide satisfies one or more of the following conditions: (1) does not contain a transmembrane domain, and (2) does not contain an intracellular domain; Further optionally, the F1 polypeptide has one or more of the following mutations: I379V and M447V. In some embodiments of the application, the mutant has one of the following combinations of cysteine mutations: Combination 3 is S180C, S186C, A170C, A177C; Combination 4 is S180C, S186C, E163C, L181C; Combination 5 is S180C, S186C, A170C, V179C; Combination 6 is S180C, S186C, L171C, A177C; Combination 7 is K176C, S190C, A170C, A177C; Combination 8 is K176C, S190C, E163C, L181C; Combination 9 is K176C, S190C, A170C, V179C; and, Combination 10 is K176C, S190C, L171C, A177C. In some embodiments of the application, the mutant further has one or more of the following mutation sites: S55C, S155C, V207L and S290C. In some embodiments of the application, the mutant does not contain a furin cleavage site fragment. In some embodiments of the application, the mutant does not contain a pep27 polypeptide, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected by a flexible short peptide; Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS. In some embodiments of the present application, the F2 polypeptide of the mutant satisfies one or more of the following conditions: 1) the C-terminus does not contain NN, and 2) has the following mutation: P102A; In some embodiments of the present application, the C-terminus of the mutant is linked to a tag fragment; optionally, the tag fragment comprises a poly-histidine; further optionally, the poly-histidine comprises an 8His fragment. In some embodiments of the present application, the mutant further comprises one or more of a structural polypeptide and a functional polypeptide; the structural polypeptide causes monomers of the mutant to form a multimer, and the functional polypeptide enhances the biological activity of the mutant; Optionally, the structural polypeptide comprises one or more of an aggregation motif, a GCN4 leucine zipper, and a nanoparticle conjugation motif. Optionally, the functional polypeptide comprises an antigen-specific binding motif. Optionally, one or more of the structural polypeptide and the functional polypeptide. In some embodiments of the present application, the sequence of the mutant is as shown in any one of SEQ ID NO. 20, SEQ ID NO. 25 to SEQ ID NO. 70. One or more embodiments of the present application provide a nucleic acid molecule encoding the mutant. One or more embodiments of the present application provide a vector comprising the nucleic acid molecule. One or more embodiments of the present application provide an engineered cell expressing the mutant, or comprising the nucleic acid molecule or the vector. One or more embodiments of the present application provide a method of producing the mutant, comprising the steps of: culturing the engineered cell, and isolating the mutant from the resulting culture supernatant. One or more embodiments of the present application provide an immunological composition comprising the mutant, or the nucleic acid molecule, and an immunological adjuvant. Optionally, the immunological adjuvant comprises one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, and an oil emulsion adjuvant. One or more embodiments of this application provide the application of the mutant described herein in the preparation of a respiratory syncytial virus antibody detection kit. One or more embodiments of this application provide a respiratory syncytial virus antibody detection kit, which includes the mutant described above. Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 shows the expression plasmid used to construct the RSV pre-F protein; Figure 2 shows a schematic diagram of the RSV F0 precursor polypeptide structure (A, B, bovine). Figure 3 is a schematic diagram of the amino acid sequence of the stabilized mutant monomer RSV F protein; Figure 4 is a schematic diagram of the polymerized structure of the stabilized mutant RSV F protein; Figure 5 shows a schematic diagram of the positions of α3, β3, and β4 (partial and overall); Figure 6 shows the distance between Cα at positions β3 / β4. amino acid residue pairs; Figure 7 shows the distance between Cα at the α3 / β3 position. amino acid residue pairs; Figure 8 shows the expression levels of the pre-F mutants formed at positions β3 and β4; Figure 9 shows the thermal stability of the mutant pre-F formed at positions β3 and β4; Figure 10 shows the expression levels of the mutant pre-F formed at the α3 and β3 positions; Figure 11 shows the thermal stability of the mutant pre-F formed at the α3 and β3 positions; Figure 12 shows the expression level of the pre-F mutant, which forms a disulfide bond at the α3 / β3 and β3 / β4 positions; Figure 13 shows the thermal stability of the pre-F mutant with disulfide bonds formed at the α3 / β3 and β3 / β4 positions; Figure 14 shows the SDS-PAGE identification of RSV pre-F mutant monomers; Figure 15 is RSV pre-F mutant single trimer SDS-PAGE identification; Figure 16 is RSV pre-F mutant monomer HPLC identification; Figure 17 is RSV pre-F mutant trimer HPLC identification; Figure 18 is RSV pre-F mutant trimer antigenicity identification; Figure 19 is RSV pre-F mutant trimer binding antibody level; Figure 20 is RSV pre-F mutant trimer neutralizing antibody level. DETAILED DESCRIPTION The present application will be further described in conjunction with the drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to explain the present application and not intended to limit the scope of the present application, the purpose of providing these embodiments and examples is to make the understanding of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein, those skilled in the art can make various modifications or changes without departing from the spirit of the present application, and the equivalent forms thus fall within the scope of the present application. In addition, in the following description, a large number of specific details are given in order to provide a more complete understanding of the present application, it should be understood that the present application can be implemented without one or more of these details. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the specification of the present application herein is only for the purpose of describing the embodiments and examples and is not intended to limit the present application. Terminology Unless otherwise indicated or contradictory, the terms or phrases used herein have the following meanings: The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or a combination of all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and"). In the present application, "multiple", "various", "multiple times", "multiple", etc. are used without specific limitation, which means more than two or equal to two in quantity. For example, "one or more" means one or more than two. As used herein, "combinations thereof", "any combination thereof", "any combination thereof", etc. include all suitable combinations of any two or more listed items. In the present application, "suitable", "suitable", "any suitable way", etc. are described as being able to implement the technical solutions of the present application, solve the technical problems of the present application, and achieve the expected technical effects of the present application. In the present application, "preferably", "better", "better", "preferably" are only used to describe the implementation mode or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application. In the present application, "further", "further", "especially" and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the protection scope of the present application. In the present application, "further", "further", "especially" and the like are used to describe the purpose, indicating the difference in content, but should not be understood as a limitation on the protection scope of the present application. In the present application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified, and there is no contradictory relationship or mutual restriction, each "optional" is independent. In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity. In the present application, among the technical features described in an open manner, both the closed technical solution consisting of the listed features and the open technical solution containing the listed features are included. In the present application, with respect to a numerical interval (i.e. a numerical range), if no specific description is provided, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no specific description is provided, when a numerical interval only points to integers within the numerical interval, including the two end point integers of the numerical range and every integer between the two end points, in this document, it is equivalent to directly listing every integer, for example, t is an integer selected from 1 to 10, which means that t is any one integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. In the present application, the temperature parameters, if not specifically limited, allow both constant temperature treatment and variation within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. It is allowed to fluctuate within the range of, for example, ±5°C, ±4°C, ±3°C, ±2°C, ±1°C. In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage. All the documents mentioned in the present application are cited in the present application as references, as if each document is cited as a reference individually. Unless and to the extent that the cited documents conflict with the purpose and / or technical solution of the present application, the cited documents are cited in the present application in their entirety and for all purposes. When the present application refers to the cited documents, the definitions of the relevant technical features, terms, nouns, phrases and the like in the cited documents are also cited. When the present application refers to the cited documents, the examples and preferred modes of the cited relevant technical features can also be incorporated into the present application as references, but are limited to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is given priority or is modified adaptively according to the description in the present application. RSV: Respiratory Syncytial Virus is a single-stranded RNA virus under the family of Pneumoviridae, a common and contagious virus that mainly affects the respiratory tract. It is a virus of the family Pneumoviridae, genus Pneumovirus. The RSV genome is about 15Kb in length, containing 10 genes, encoding 11 proteins, of which the surface exhibits F protein, G protein and SH protein. RSV usually causes mild cold-like symptoms in most people, and infants and the elderly or people with heart, lung or immune system damage are more likely to have severe RSV symptoms, which can cause pneumonia, bronchitis, middle ear infection. F protein: F protein (Fusion protein) is a surface protein of Respiratory Syncytial Virus (RSV) that helps the virus to infect human cells. It has two states, pre-fusion and post-fusion, and the F protein is irreversibly allosteric from the metastable pre-fusion conformation to the more stable post-fusion conformation during the process of virus invading host. In the natural state, F protein is translated to form F0 precursor with a length of 574 amino acids, and in the process of F0 maturation, it is cut by two included furin cleavage sites to form F1 polypeptide, F2 polypeptide and p27 composed of 27 amino acids. Further, F1 polypeptide and F2 polypeptide form a trimer, and the final mature F protein exists in the form of a trimer. Pre-fusion F protein: RSV F protein in the conformation before the fusion is triggered. In this conformation, the F protein is in a metastable state and can be irreversibly allosteric to a more stable post-fusion conformation by external environment or other reasons. In this conformation, the F protein has epitopes different from the post-fusion conformation, such as Φ epitope V epitope. Whether it is in this pre-fusion conformation can be determined by specific monoclonal antibodies (such as D25, AM22, 5C4, ADI-15568, etc.) corresponding to these epitopes. Post-fusion F protein: RSV F protein in the conformation after being irreversibly allosteric after being triggered by external environment or other reasons. F protein in this conformation does not have Φ epitope V epitope, so it cannot be recognized by specific monoclonal antibodies (such as D25, AM22, 5C4, ADI-15568, etc.) corresponding to the epitopes. F1 polypeptide: A polypeptide fragment formed by the amino acids at positions 137-574 of F0 after the translation and modification of RSV F0 precursor. It has an extracellular domain (about 137-524 of F0), a transmembrane domain (about 524-550 of F0) and an intracellular domain (about 551-574 of F0). The F1 polypeptide used herein refers to the natural state F1 polypeptide or the amino acid sequence corresponding to the natural F1 polypeptide or part of the natural F1 polypeptide after mutation or modification. F2 polypeptide: a polypeptide fragment formed by the amino acids at positions 26-109 of F0 formed by post-translational modification and enzymatic cleavage of RSV F0 precursor. As used herein, F1 polypeptide refers to F1 polypeptide in natural state or amino acid sequence corresponding to natural F1 polypeptide or part of natural F1 polypeptide formed by mutation or modification. DS-Cav1: refers to the amino acid sequence having the same mutations as the RSV F protein described in Mclellan et al., Structure-Based Design of a Fusion Glycoprotein Vaccine for Respiratory Syncytial Virus, Science. 2013 November 1; 342(6158): 592-598. Epitope: Epitope refers to specific regions on a protein that specifically interact with antibodies or lymphocyte receptors. Epitopes are usually related to antigen recognition, such as the interaction between antibodies and antigens. These epitopes can be linear (along a part of the protein sequence) or three-dimensional (in the folded structure of the protein). There are Φ, I, II, III, IV, V, etc. epitopes in F protein. Disulfide bond replacement: Disulfide bond replacement refers to replacing one or more pairs of amino acids at appropriate positions of RSV F protein with cysteine, so that disulfide bonds are formed between the replaced cysteines, thereby enhancing the stability of the F protein in the pre-fusion state. AM22: AM22 refers to the antibody described in WO 2011 / 043643 Al. It can specifically bind to the Φ epitope of RSV F protein. AM14: AM14 refers to the antibody described in WO 2008 / 147196 A2. It can specifically bind to RSV F protein in a trimeric state. ADI-15568: ADI-15568 refers to the antibody described in US2022 / 0144922 Al. It can specifically bind to the V epitope of RSV F protein. In a first aspect of the embodiments of the present application, a mutant of RSV pre-F is provided, wherein the F1 polypeptide of the mutant satisfies the following (1) or (2): (1) the β3 sheet region has a cysteine substituent β3-1, and the β4 sheet region has a cysteine substituent β4-1, and the cysteine substituent β3-1 and the cysteine substituent β4-1 form a disulfide bond; (2) the a3 helix region has a cysteine substitution a3-1, the b3 sheet region has a cysteine substitution b3-1 and a cysteine substitution b3-2, the b4 sheet has a cysteine substitution b4-1, the cysteine substitution b3-1 and the cysteine substitution b4-1 form a disulfide bond, the cysteine substitution a3-1 and the cysteine substitution b3-2 form a disulfide bond; The mutant has one of the following combinations of cysteine mutations: Combination 1 is S180C and S186C; and, Combination 2 is K176C and S190C. The present application finds a method for generating stable pre-fusion conformation of RSV F protein. The method involves introducing cysteine substitutions between b3 / b4 to form a disulfide bond, or introducing cysteine substitutions between b3 / b4 and a3 / b3 to form a double disulfide bond, which can maintain the F protein of RSV in pre-fusion conformation. The combination of mutations found by the present application, and the mutant produced thereby, are not involved in the prior art, which is a new idea for generating stabilized RSV pre-F mutants. The mutant with disulfide bond introduced between b3 / b4 and disulfide bond introduced at a3 / b3 position can bind with specific monoclonal antibodies recognizing the site, has good stability and immunogenicity, can induce neutralizing antibodies capable of neutralizing the virus, and has great potential for developing RSV vaccines. mutation, substitution, amino acid substitution: substitution of one amino acid with another amino acid or deletion of an amino acid in an antigen. For example, substitution of an amino acid in an antigen with an amino acid from a homologous protein. homologous protein: a protein having similar structure and function, for example, a protein from two or more species or viral strains having similar structure and function in the two or more species or viral strains. For example, RSV F protein from RSV A is a homologous protein to RSV F protein from bovine RSV. Homologous proteins share similar protein folding characteristics and can be considered structural homologs. Homologous proteins often share a high degree of sequence conservation, for example at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence conservation, and a high degree of sequence identity, for example at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. There are several subtypes of RSV, including human subtype A, human subtype B, and bovine subtype. Within RSV subtypes, there are individual strains of each subtype. In some embodiments, the F1 polypeptide of the RSV pre-F has an amino acid sequence as set forth in any one of SEQ ID NO. 4, SEQ ID NO. 72, and SEQ ID NO. 73, or shares at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology with the amino acid sequence as set forth in any one of SEQ ID NO. 4, SEQ ID NO. 71, and SEQ ID NO. 72; Optionally, the F1 polypeptide satisfies one or more of the following conditions: (1) does not contain a transmembrane domain, and (2) does not contain an intracellular domain; Further optionally, the F1 polypeptide has one or more of the following mutations: I379V and M447V. In some embodiments, the mutant has one of the following combinations of cysteine mutations: Combination 3 is S180C, S186C, A170C, A177C; Combination 4 is S180C, S186C, E163C, L181C; Combination 5 is S180C, S186C, A170C, V179C; Combination 6 is S180C, S186C, L171C, A177C; Combination 7 is K176C, S190C, A170C, A177C; Combination 8 is K176C, S190C, E163C, L181C; Combination 9 is K176C, S190C, A170C, V179C; and, Combination 10 is K176C, S190C, L171C, A177C. In some embodiments, the mutant further has one or more of the following mutation sites: S55C, S155C, V207L, and S290C. In some embodiments, the mutant does not contain a furin cleavage site fragment. In some embodiments, the mutant does not contain a pep27 polypeptide, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected by a flexible short peptide; Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS. In some embodiments, the F2 polypeptide of the mutant satisfies one or more of the following conditions: 1) the C-terminus does not contain NN, and, 2) has a mutation of P102A; In some embodiments, the C-terminus of the mutant is linked to a tag fragment; optionally, the tag fragment comprises a poly-histidine; further optionally, the poly-histidine comprises an 8His fragment. In some embodiments, the mutant further comprises one or more of a structural polypeptide and a functional polypeptide; the structural polypeptide causes monomers of the mutant to form a multimer, and the functional polypeptide enhances a biological activity of the mutant; Optionally, the structural polypeptide comprises one or more of an aggregation motif, a GCN4 leucine zipper, and a nanoparticle conjugation motif. Optionally, the functional polypeptide comprises an antigen-specific binding motif. Optionally, one or more of the structural polypeptide and the functional polypeptide. In some embodiments, the sequence of the mutant is as set forth in any one of SEQ ID NO. 20, SEQ ID NO. 25 to SEQ ID NO. 70, or has at least 80% (at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homology to these sequences. The mutant provided by the embodiments of the present application has a stable conformation, and can induce an immune response of a subject as an antigen / immunogen to produce an antibody. A subject is an animal. An animal is a living multicellular vertebrate or invertebrate organism, and the class includes, for example, mammals. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as non-human primates. Thus, administration to a subject can include administration to a human subject. Non-limiting examples of veterinary subjects include domestic animals (e.g., cats and dogs), farm animals (e.g., cows, horses, pigs, sheep, and goats), and laboratory animals (e.g., mice, rabbits, rats, hamsters, guinea pigs, and non-human primates). Antibody: A polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes or fragments thereof which specifically binds and recognizes an analyte (e.g., an antigen or immunogen), such as a RSV F protein or an antigenic fragment thereof. The immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes. The term "antibody" as used herein includes antibody fragments, such as produced by the modification of whole antibodies and those produced by recombinant DNA methods. Antibodies exist, for example, in the form of intact immunoglobulins and in a variety of well-characterized antibody fragments. For example, Fab, Fv and single-chain Fv (SCFv) that bind to RSV F protein will be RSV F protein specific binding agents. This includes intact immunoglobulins and variants and portions thereof well known in the art, such as Fab' fragments, F(ab')2 fragments, single-chain Fv proteins ("scFv"), and disulfide-linked Fv proteins ("dsFv"). The scFv protein is a fusion protein in which the light chain variable region of an immunoglobulin is linked with the heavy chain variable region of an immunoglobulin by a linker, while in the dsFv, the chains have been mutated to introduce disulfide linkages to stabilize the association of the chains. The term also includes genetically engineered forms, such as chimeric antibodies (e.g., a humanized murine antibody), heteroconjugate antibodies (e.g., bispecific antibodies). See also Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997. Antibody fragments are defined as follows: (1) Fab, a fragment of an antibody molecule that contains the entire light chain and a portion of one heavy chain that is produced, for example, by digestion with the enzyme papain to yield an intact light chain and one heavy chain that is one portion of the original one; (2) Fab', a fragment of an antibody molecule that is obtained, for example, by treating whole antibody with the enzyme pepsin, followed by reduction to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, a fragment of an antibody that is obtained, for example, by treating whole antibody with the enzyme pepsin without subsequent reduction; (4) F(ab')2, a dimer of two Fab' fragments that are held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing the complete variable region of a light chain and the variable region of a heavy chain expressed as two chains; and (6) single chain antibody ("SCA"), a genetically engineered molecule containing the variable region of a light chain and the variable region of a heavy chain linked together by a short peptide linker expressed as a genetically fused single chain molecule. Generally, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains that interconnect via disulfide bonds. There are two types of light chain, lambda (l) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activities of an antibody molecule: IgM, IgD, IgG, IgA and IgE. The disclosed antibodies can be class switched. Each heavy and light chain contains a constant region and a variable region, (the regions are also referred to as "domains"). In several embodiments, the heavy and light chain variable domains combine to specifically bind an antigen. In other embodiments, only the heavy chain variable domain is required. For example, naturally occurring camelid antibodies consisting of only heavy chains are functional and stable in the absence of light chains (see, e.g., Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol., 3:733-736, 1996). The light and heavy chain variable domains contain "framework" regions interrupted by three hypervariable regions (also referred to as "complementarity determining regions" or "CDRs") (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serve to position and align the CDRs in three-dimensional space. The CDRs are primarily responsible for binding to the epitope of an antigen. The boundaries of the amino acid sequences of given CDRs can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. ("Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991; the "Kabat" numbering scheme), Al-Lazikani et al. (JMB 273, 927-948, 1997; the "Chothia" numbering scheme), and Lefranc et al. ("IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev. Comp. Immunol., 27:55-77, 2003; the "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from N- to C-terminal), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found. Light chain CDRs are sometimes referred to as CDRL1, CDRL2, and CDRL3. Heavy chain CDRs are sometimes referred to as CDRH1, CDRH2, and CDRH3. Antigen: A compound, composition, or substance that can stimulate the production of an antibody or T cell response in an animal, including compositions injected or absorbed into an animal. Antigens react with products of specific humoral or cellular immunity, including those induced by heterologous antigens, such as the disclosed mutants of the RSV pre-fusion F protein. Examples of antigens include, but are not limited to, polypeptides, peptides, lipids, polysaccharides, combinations thereof (e.g., glycopeptides), and nucleic acids containing antigenic determinants, such as those recognized by immune cells. In some examples, an antigen includes a peptide derived from a pathogen of interest, such as RSV. In particular examples, an antigen is derived from RSV, such as an antigen including a modified RSV F protein stabilized in a pre-fusion conformation. An "epitope" or "antigenic determinant" refers to a region of an antigen to which a B and / or T cell response. Immunogen: A protein or portion thereof capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection with a pathogen. Administration of an immunogen can result in protective immunity and / or active immunity against a pathogen of interest. As provided by the examples herein, PreF mutants. Immune response: A reaction of cells of the immune system, such as B cells, T cells, or monocytes, to a stimulus. In one embodiment, the reaction is specific for a particular antigen ("antigen-specific reaction"). In one embodiment, the immune response is a T cell response, such as a CD4+ response or a CD8+ response. In another embodiment, the reaction is a B cell response and results in the production of specific antibodies. One of ordinary skill in the art will recognize that individual substitutions, deletions or additions to a sequence, which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence, are conserva- tive variations wherein each modified amino acid residue is one that is chemically similar to the replaced amino acid residue is a conservative substitution, where a small percentage means less than 20%, 15, 10, 5%, etc. Conservative amino acid substitutions are provided in the art as well-known. The following six groups each contain amino acids that are conservative substitutions for one another: Amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and, 6) Phenylalanine (F), Tyrosine (Y),

[0182] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). Not all residue positions within a protein will tolerate an originally "conservative" substitution. For example, if an amino acid residue is essential for the function of the protein, even a conservative substitution can destroy the activity, e.g., the specific binding of an antibody to its target epitope can be destroyed by a conservative mutation in the target epitope. Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on an antigenic molecule that cause them to induce a specific immune response, e.g., an epitope is a region of an antigen that reacts with B and / or T cells. Antibodies bind to specific antigenic epitopes, e.g., epitopes of RSV F protein, e.g., the D25 or AM22 epitopes present on the prefusion conformation of RSV F protein. Epitopes can be formed by contiguous amino acids or noncontiguous amino acids brought into proximity by tertiary folding of a protein. Epitopes formed by contiguous amino acids are usually maintained in exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost upon treatment with denaturing solvents. Epitopes usually include at least 3 and more usually at least 5, about 9, or about 8-10 amino acids in a unique spatial conformation. Methods of determining the spatial conformation of an epitope include, e.g., x-ray crystallography and nuclear magnetic resonance. Epitopes can also include post-translational modifications of amino acids, e.g., N-linked glycosylation. A "target epitope" is a particular epitope on an antigen that specifically binds to an antibody of interest, e.g., a monoclonal antibody. In some examples, a target epitope includes amino acid residues that contact an antibody of interest such that the target epitope can be selected by determining the amino acid residues that contact the antibody of interest. Amino acids in peptides, polypeptides, or proteins are typically chemically bonded together via amide bonds (CONH). In addition, amino acids can be bonded together by other chemical bonds. For example, the linkage of amino acids or amino acid analogs can include CH2NH-, -CH2S-, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, -CH(OH)CH2-, and -CH2SO- (these and others are found in Spatola, in Chemistry and Biochemistry of Amino Acids, Peptides, and Proteins, B. Weinstein, ed., Marcel Dekker, New York, pp. 267 (1983); Spatola, A. F., Vega Data (March 1983), Vol. 1, No. 3, Peptide Backbone Modifications (general review); Morley, Trends Pharm Sci pp. 463-468, 1980; Hudson et al., Int J Pept Prot Res 14:177-185, 1979; Spatola et al., Life Sci 38:1243-1249, 1986; Harm J. Chem. Soc Perkin Trans. 1 307-314, 1982; Almquist et al., J. Med. Chem. 23:1392-1398, 1980; Jennings-White et al., Tetrahedron Lett 23:2533, 1982; Hollada V et al., Tetrahedron. Lett 24:4401-4404, 1983; and Hruby Life Sci 31 :189-199, 1982). Peptide Modifications: Peptides, such as the pre-fusion conformation stabilizing mutants of the embodiments of the application, can be modified, for example, to include amino acid substitutions compared to the native RSV protein sequence, or by various chemical techniques to produce derivatives having substantially the same activity and conformation as the unmodified peptide and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxy-terminal or side chain, can be provided in salt form with a pharmaceutically acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of the formula NR1R2, where R1and R2are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6-membered ring. Amino groups of the peptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically acceptable acid addition salt such as HC1, HBr, acetate, benzoate, toluenesulfonate, maleate, tartrate, and other organic salts, or can be modified to C1-C16 alkyl or dialkylamino groups or further converted to amides. The hydroxyl groups of the peptide side chains can be converted to C1-C16 alkoxy groups or C1-C16 esters using art-recognized techniques. The phenyl and phenol rings of the peptide side chains can be substituted with one or more halogen atoms such as F, CI, Br, or I or with C1-C16 alkyl groups, C1-C16 alkoxy groups, carboxylic acids and esters thereof or amides of these carboxylic acids. The methylene groups of the peptide side chains can be extended to homologous C2-C4 alkylene groups. The thiols can be protected with any of a variety of art-recognized protecting groups such as an acetamide group. In a second aspect, the present application provides a nucleic acid molecule encoding the mutant. Nucleic acid molecule, nucleic acid: a polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof) joined by phosphodiester bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof. Thus, the term includes nucleotide polymers in which the nucleotides and the linkages between them include synthetic non-naturally occurring analogs such as, and without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs), and the like. These polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "oligonucleotide" generally refers to a short polynucleotide, typically no greater than about 50 nucleotides. It is understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes the RNA sequence (i.e., A, U, G, C) wherein "U" is instead of "T." "Nucleotide" includes, but is not limited to, a monomer comprising a base linked to a sugar (e.g., a pyrimidine, purine, or synthetic analog thereof) or a monomer comprising a base linked to an amino acid (as in a peptide nucleic acid (PNA)). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide. "Encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes. Thus, for example, if a gene encodes a protein, then the DNA sequence of the gene is said to encode not only the mRNA, but also the protein. As used herein, the term "coding strand" refers to the nucleotide sequence of a polynucleotide that is identical to the mRNA sequence and is used as the template for transcription. The term "non-coding strand" refers to the nucleotide sequence of a polynucleotide that is complementary to the mRNA sequence and is not used as the template for transcription. Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences which are degenerate versions of each other and which encode the same amino acid sequence. Nucleotide sequences which encode proteins and RNA can include introns. In view of degenerate variants and conservative variants, the present application does not specifically limit nucleic acid molecules. Degenerate variants and conservative variants: Polynucleotides which encode the same or a substantially similar amino acid sequence due to the degeneracy of the genetic code. For example, polynucleotides which encode the disclosed antigens or antibodies which specifically bind the disclosed antigens. There are 20 naturally occurring amino acids, most of which are specified by more than one codon. Thus, a multitude of polynucleotide sequences will encode the same or a substantially similar amino acid sequence. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given polypeptide. For instance, the codons CGU, CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine. Thus, at every position in a protein coding sequence where an arginine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and are of a kind that do not alter the polypeptide encoded by the nucleic acid. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill in the art will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine) can be modified to yield a functionally identical molecule. Accordingly, each "silent variation" of a nucleic acid which encodes a polypeptide is implicit to each described sequence. In some examples, which are codon-optimized for expression in a cell of a mammal, is operably linked to a promoter. Expression control sequence: a nucleic acid sequence that regulates the expression of an operably linked heterologous nucleic acid sequence. An expression control sequence is operably linked to a nucleic acid sequence when the expression control sequence controls and regulates the transcription of the nucleic acid sequence, and, where appropriate, translation. Thus, an expression control sequence can include a suitable promoter, enhancer, transcription terminators, a start codon (ATG) in front of a protein-encoding gene, splicing signal at the intron sequence, a suitable restriction site for insertion of a heterologous nucleic acid sequence, a polyadenylation site, and / or any other sequence segment that reversibly modulates the transcription of a heterologous nucleic acid sequence. The term "control sequences" is intended to include, at a minimum, components whose presence is necessary for expression, and also includes other components that can favorably influence expression. An expression control sequence can include a promoter. A promoter is a minimal sequence sufficient to direct transcription. Also included are those promoter elements sufficient to allow promoter-independent gene expression with respect to cell type-specificity, tissue-specificity, controllability by external signals or agents; these elements can be located in the 5' or 3' region of the gene. Both constitutive and inducible promoters (see, e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987) are included. For example, when cloning in bacterial systems, inducible promoters such as the pl of bacteriophage lambda, plac, ptrp, ptac (a hybrid trp-lac promoter), and the like can be used. In one embodiment, when cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from the genome of mammalian viruses (e.g., retroviral long terminal repeat; adenovirus late promoter; vaccinia virus 7.5K promoter) can be used. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide transcription of the nucleic acid sequence. A polynucleotide can be inserted into an expression vector containing a promoter sequence that promotes transcription of the inserted genetic sequence by the host. The expression vector typically contains an origin of replication, a promoter, and specific nucleic acid sequences that allow transformed cells to be selected against untransformed cells. RSV F proteins from different RSV subgroups, as well as nucleic acid sequences encoding these proteins and methods for manipulating and inserting these nucleic acid sequences into vectors are disclosed herein and known in the art (see, e.g., Tan et al., PLOSone, 7: e51439, 2011; Sambrook et al., Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994)). In a third aspect of the embodiments herein, there is provided a vector comprising the nucleic acid molecule. In a fourth aspect of the embodiments herein, there is provided an engineered cell expressing the mutant, or comprising the nucleic acid molecule or the vector. Expression: The translation of a nucleic acid into a protein. The protein can be expressed and remain within the cell, become a component of the cell surface membrane, or be secreted into the extracellular matrix or culture medium. Engineered cell, host cell: A cell in which a vector can propagate and express its DNA. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It is understood that all progeny can not be identical to the parental cell since there can be mutations that occur during replication. However, such progeny are included when the term "host cell" is used. In a fifth aspect of the embodiments herein, there is provided a method of producing the mutant, comprising the steps of: Culturing the engineered cell, and isolating the mutant from the resulting culture supernatant. In a sixth aspect of the embodiments herein, there is provided an immunological composition comprising the mutant, or the nucleic acid molecule, and an immunological adjuvant. Immune adjuvants: mediators used to enhance antigenicity. Adjuvants include suspensions of antigen adsorbed onto a mineral (alum, aluminum hydroxide, or phosphate) or a water-in-oil emulsion, e.g., in which an antigen solution is emulsified in mineral oil (Freund's incomplete adjuvant), sometimes including killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibit degradation of the antigen and / or cause influx of macrophages). Immune stimulatory oligonucleotides (e.g., those including a CpG motif) can also be used as adjuvants. Adjuvants include biological molecules ("biological adjuvants"), e.g., costimulatory molecules. Exemplary adjuvants include IL-2, RANTES, GM-CSF, TNF-alpha, IFN-gamma, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 4-1BBL, and toll-like receptor (TLR) agonists, e.g., TLR-9 agonists. Adjuvants are well known to those of ordinary skill in the art (see, e.g., Singh (ed.) Vaccine Adjuvants and Delivery Systems. Wiley-Interscience, 2007). Adjuvants can be used in combination with the disclosed PreF antigens. Alternatively, the immune adjuvant in the embodiments of the present application is one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, an oil-in-water emulsion adjuvant. For example, Alum, CpG, Alum+CpG, MF59, AS04, AS01E, etc. MF59 is a water-in-oil emulsion adjuvant, which is mainly composed of three parts: oil phase, emulsifier and auxiliary agent; the oil phase is a mixture of micro-particleized short-chain triglycerides suitable for human injection; the emulsifier is a surfactant that can mix the oil phase and water uniformly; the auxiliary agent mainly includes moisturizers and buffers such as glycerol, sorbitol, ATP, etc. AS04 adjuvant is a mixture of AS03 adjuvant and MPL adjuvant. AS03 adjuvant is a mixture of liposomes, TWEEN 80, SORBITAN three surfactants. AS01E is a nanoscale liposome solution prepared from DOPC, Chol, MPL and QS-21. The main component of MPL adjuvant is lipopolysaccharide. Immunological composition (immunogenic composition): A composition that includes an antigen that induces an immune response, e.g., a measurable CTL response against a virus expressing the antigen or a measurable B cell response (e.g., antibody production) against the antigen. Thus, an immunogenic composition includes one or more antigens (e.g., polypeptide antigens) or antigenic epitopes. An immunogenic composition can also include one or more additional components capable of inducing or enhancing an immune response, e.g., excipients, carriers, and / or adjuvants. In certain instances, an immunogenic composition is administered to induce an immune response that protects a subject from a symptom or condition induced by a pathogen. In some instances, a symptom or disease caused by a pathogen is prevented (or reduced or ameliorated) by inhibiting replication of the pathogen after the subject is exposed to the pathogen (e.g., RSV). In one example, an "immunogenic composition" includes a recombinant RSV F protein stabilized in a prefusion conformation that induces a measurable CTL response against a virus expressing the RSV F protein or induces a measurable B cell response (e.g., antibody production) against the RSV F protein. It further refers to an isolated nucleic acid encoding an antigen, e.g., a nucleic acid that can be used to express an antigen (and thus to induce an immune response against such a polypeptide). For in vitro use, an immunogenic composition can include an antigen or a nucleic acid encoding an antigen. For in vivo use, an immunogenic composition will typically include a protein, immunogenic peptide, or nucleic acid in a pharmaceutically acceptable carrier and / or other agents. Any particular peptide, e.g., a disclosed RSV F protein stabilized in a prefusion conformation or a nucleic acid encoding a disclosed RSV F protein stabilized in a prefusion conformation, can be readily tested for its ability to induce a CTL or B cell response by well-recognized assays. An immunogenic composition can include an adjuvant well known to those of skill in the art. Immunological reaction conditions: Include conditions that refer to the binding of an antibody raised against a particular epitope to that epitope and to the extent of its binding being detectably greater than that of substantially all other epitopes and / or substantially excluding binding to substantially all other epitopes. Immunological reaction conditions depend on the form of the antibody binding reaction and are typically those used in immunoassay protocols or those encountered in vivo. The immunological reaction conditions used in the methods are "physiological conditions," which include reference to the typical conditions (e.g., temperature, osmotic pressure, pH) inside a living mammal or mammalian cell. While it is recognized that some organs are subjected to extreme conditions, the intra-organismal and intracellular environment is generally about pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the principal solvent, and exists at temperatures above 0°C and below 50°C. The osmotic pressure is in a range that supports cell viability and proliferation. In a seventh aspect of the embodiments herein, there is provided use of the mutant in the preparation of a respiratory syncytial virus antibody detection kit. In the embodiments of the present application, the respiratory syncytial virus antibody detection kit can be used for detecting the corresponding antibody. The definition of the antibody refers to the first aspect, which can be a neutralizing antibody or a binding antibody. In the eighth aspect of the embodiments of the present application, a respiratory syncytial virus antibody detection kit is provided, which comprises the mutant. The definition of the respiratory syncytial virus antibody detection kit refers to the seventh aspect. In the ninth aspect of the embodiments of the present application, the embodiments of the present application provide a method for preventing and treating lower respiratory tract infection caused by respiratory syncytial virus, which comprises the following steps: administering a therapeutically effective amount of the immunological composition of the sixth aspect to a subject. Administration: introducing a composition into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intravenous, the composition is administered by introducing the composition into the subject's vein. Effective amount: the amount of an agent, such as a PreF antigen or a nucleic acid encoding a PreF antigen, or other agent, sufficient to produce a desired response, such as an immune response to RSV F protein, or to reduce or eliminate signs or symptoms of a condition or disease, such as RSV infection. For example, this can be the amount required to inhibit viral replication or measurably alter the outward symptoms of viral infection. Generally, such an amount will be sufficient to measurably inhibit viral (e.g., RSV) replication or infectivity. When administered to a subject, a dose will typically be used that will achieve a target tissue concentration (e.g., in respiratory tissue) that has been demonstrated to achieve in vitro inhibition of viral replication. In some examples, an "effective amount" is an amount that treats (including preventing) one or more symptoms and / or underlying cause of any condition or disease, such as treating RSV infection. In one example, an effective amount is a therapeutically effective amount. In one example, an effective amount is an amount that prevents development of one or more signs or symptoms of a particular disease or condition (e.g., one or more signs or symptoms associated with RSV infection). Prevent, prophylactic, therapeutic: For example, in a subject at risk of a disease (e.g., RSV infection), to inhibit the full development of the disease or condition. "Treatment" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term "amelioration" of a disease or pathological condition refers to any observable beneficial result. Beneficial or desired results can include, e.g., a decrease in the clinical symptoms of the disease in a susceptible subject, a decrease in severity of some or all clinical symptoms of the disease, slower progression of the disease, improved overall health status or well-being of the subject, or other parameters well known in the art that are specific to the particular disease. A "prophylactic" treatment is a treatment administered to a subject who does not yet exhibit signs of a disease or exhibits only early signs of a disease, for the purpose of decreasing the risk of developing the disease. The term "decrease" is a relative term, such that a drug decreases a response or condition if the response or condition is quantitatively lessened after administration of the drug, or if it is lessened relative to a reference drug. Similarly, the term "prevention" does not necessarily refer to a drug that completely eliminates a response or condition, but only that at least one characteristic of the response or condition is eliminated. Thus, an immunogenic composition that decreases or prevents an infection or response (e.g., a pathological response, e.g., a vaccine-enhanced viral disease) can but does not necessarily completely eliminate such infection or response, provided that the infection or response is measurably lessened, e.g., at least about 50%, e.g., at least about 70%, or about 80%, or even about 90% (i.e., down to 10% or less) than the infection or response in the absence of the drug, or relative to a reference drug. In a tenth aspect of the embodiments of the present application, a method for detecting or isolating RSV F binding antibodies in a sample is provided, comprising the steps of: contacting the mutant of the first aspect with RSV F binding antibodies in a sample to form an immunocomplex; and, detecting the immunocomplex, thereby detecting or isolating RSV F binding antibodies in a sample. The embodiments of the present application will be described in detail with reference to the following Examples. It is to be understood that these Examples are intended to illustrate the application and not to limit its scope. The experimental methods in the following Examples, for which specific conditions are not indicated, are preferably carried out according to the indications given in the present application, but also according to the protocols or general conditions known in the art, or according to the conditions suggested by the manufacturer, or according to the experimental methods known in the art. In the following specific examples, the measurement parameters of the raw material components, if not specified, can have slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by the instrument testing accuracy or operation accuracy are allowed. (1) RSV pre-F mutant vector construction According to the mutation strategy, the amino acid sequence of the RSV pre-F mutant is determined, and the nucleic acid sequence corresponding thereto is codon-optimized, and the optimized codon is beneficial for expression in Chinese hamster ovary cells (CHO cells). Finally, it is connected to the pcDNA3.1 vector through BamH I and Xho I enzyme digestion sites. The substitution, insertion and deletion of the vector are performed using the ClonExpress II Recombinant Cloning Kit (Novagen Biotechnology Co., Ltd.). The fragments at both ends of the mutation site are amplified by polymerase chain reaction (PCR) using the high-fidelity enzyme Phanta Max (Novagen Biotechnology Co., Ltd.), and the two fragments are fused by PCR after the amplification products are recovered. The fused fragments are homologously recombined into the pcDNA3.1 vector digested by BamH I and Xho I. The constructed point mutation vector is sequenced by Beijing Qikexin Biotechnology Co., Ltd. and confirmed to have no errors. Figure 1 is an expression plasmid for constructing the RSV pre-F mutant. The cloning vector is inoculated into 300 mL of LB (Amp+) medium, cultured at 37°C and 180 rpm for 16 h, and the plasmid is extracted using a large-scale plasmid extraction kit; finally stored in 1 mL of sterile TE Buffer buffer. For all commercial kits or reagents, follow the manufacturer's instructions. Figure 2 describes the schematic diagram of the amino acid structure of the pre-F polypeptide of the wild-type RSV virus F protein. The amino acid sequence shown in the figure can be derived from The A subtype or B subtype of the human respiratory syncytial virus F protein. The natural F protein forms an F0 precursor polypeptide after ribosome translation, and the full-length precursor polypeptide includes about 574 amino acids, including a signal peptide (1 aa-25 aa), an F2 polypeptide (26 aa-109 aa), a pep27 polypeptide (110 aa-136 aa), and an Fl polypeptide (137 aa-574 aa). Among them, the Fl polypeptide includes a transmembrane domain (514 aa-550 aa) and an intracellular domain (551 aa-574 aa). For the precursor polypeptide of the natural full-length human RSV A2 and human RSV B, the full-length pre-F polypeptide of the natural full-length bovine RSV B is 572 aa, and the Fl polypeptide corresponds to aa137-572 aa, containing the furin cleavage sites RAKR and KKRKRR. The signal peptide of RSV pre-F is cleaved during endoplasmic reticulum translocation, and the two furin cleavage sites (RARR and KKRKRR) contained in the polypeptide are processed by intracellular furin-like proteases to produce three peptide segments, F1 polypeptide, F2 polypeptide and Pep27 polypeptide, and the pep27 polypeptide is deleted during the maturation of F protein, and finally the F2 polypeptide at the N-terminus and the F1 polypeptide at the C-terminus are contained, and the F1 polypeptide is connected to the F2 polypeptide by two disulfide bonds. The mature F protein forms a trimer and is anchored to the cell membrane by the transmembrane domain through the F1 polypeptide. Figure 3 describes the structural schematic diagram of the constructed stabilized RSV pre-F mutant monomer (SEQ ID NO. 11). Different from the wild-type RSV pre-F monomer: 1) the p27 peptide is replaced by GS, which is not affected by furin, and the mature monomer F protein will be composed of a single-chain polypeptide; 2) the transmembrane domain (TM, 514aa-550aa) and the intracellular domain (CT, 551aa-574aa) are deleted at the same time, and the final monomer protein is expressed in a secreted form; 3) and His-Tag is added at the C-terminus of the protein to facilitate purification. Unless otherwise specified, other monomer RSV F protein mutations involved in the embodiments of the present application are designed on this basis, but it can be understood that the design starting point polypeptide of the RSV pre-F mutant monomer is not limited to SEQ ID NO: 10 which meets the above conditions based on the design concept of the embodiments of the present application. Figure 4 describes the structural schematic diagram of the monomer contained in the constructed stabilized RSV pre-F trimer (SEQ ID NO. 12). Based on the stabilized RSV pre-F mutant monomer, the trimerization motif of phage T4 fiber protein (foldon) (T4 foldon) is fused to the C-terminus thereof, so that the finally formed mature F protein forms a trimer to simulate the natural trimer state of F protein, and a purification tag is added at this C-terminus. Unless otherwise specified, other trimer RSV pre-F protein mutations involved in the embodiments of the present application are designed on this basis. Other multimers based on the mutant construction, such as dimers based on antibody Fc fragments, or nanoparticles based on ferritin, can replace T4 foldon with the corresponding antibody Fc fragment, etc. in the structure shown in the figure legend. Figure 5 depicts a schematic diagram of the three-dimensional structure of the positions of the mutations that can stabilize the RSV pre-F mutant, and a local enlarged schematic diagram (identified in green) of the positions of the mutations, including the a3 helix, the b3 sheet, and the b4 sheet. The schematic diagram shows the a3 helix and the b3 sheet, and the b3 sheet and the b4 sheet are parallel and close in distance. The RSV pre-F conformation is metastable, and is triggered by the environment to form an irreversible rearrangement, thereby forming a more stable non-functional post-F conformation. Among them, the a2, a3, b3, b4, a4 and a5 in the sequence are conformationally formed into a long helical bundle, and form a long handle composed of 6 helices with the long helix formed at the C terminal in the trimer case, which is a typical feature of post-F. In the embodiments of the present application, we found that the introduction of cysteine substitution between the b3 sheet and the b4 sheet can fix the b3 sheet and the b4 sheet, prevent their conformational change, and enable the RSV F protein to maintain the pre-fusion conformation. When the b3 and b4 and the a3 and b3 are fixed by the action of the disulfide bond bridge, the RSV F protein can be in a more stable pre-fusion conformation. Figure 6 depicts the amino acids involved in the b3 / b4 position and the interatomic distance between the two amino acids prone to form disulfide bonds in the embodiments of the present application The amino acids forming disulfide bonds include: 181L and 185V 180S and 186S 179V and 187V 178V and 188L 177A and 189T 176K and 190S Figure 7 depicts the amino acids involved in the a3 / b3 position and the interatomic distance between the two amino acids prone to form disulfide bonds in the embodiments of the present application The amino acids forming disulfide bonds include: 163E and 181L 166K and 179V 167I and 179V 170A and 179V 170A and 177A 171L and 189T 171L and 177A 191 K and 175 N Table 1. Amino acids comprised in the a3 helix, b3 sheet and b4 sheet in the RSV F0 protein Table 2. Amino acid pairing prone to form disulfide bonds after cysteine mutation Table 3. RSV pre-F mutant sites Table 4, (2) Expression of RSV pre-F mutant proteins Mutant proteins were expressed by ExpiCHO TM Expression system (Thermofisher). Transient expression was performed exactly according to the manufacturer's standard protocol. Briefly, one day before transfection (day -1), ExpiCHO-S TM cultures were subcultured to a final density of 3 x 10 6 ~ 4 x 10 6 viable cells / mL. The next day (day 0), the viable cell density and percentage of viability were determined. The cell density should be about 7 x 10 6 ~ 10 x 10 6 viable cells / mL. The viability should be 95% ~ 99% before transfection can proceed. Freshly pre-warmed ExpiCHO TM Expression Medium was used to dilute the cells to a final density of 6 x 10 6 viable cells / mL. The flasks were gently swirled to mix the cells and the remaining cells were discarded. ExpiFectamine TM CHO / Plasmid DNA complex was prepared according to the reagent instruction using cold reagents (4°C). After gentle inversion to mix, the ExpiFectamine™ CHO / DNA complex was added to the flasks and the cells were incubated on a shaker (8% CO2, 37°C, 120 rpm). ExpiFectamine TM CHO Enhancer and ExpiCHO TM Supplement. The culture was terminated on day 8 after transfection according to the standard test protocol and samples were taken for testing. The determination of expression amount refers to "(7) Detection of the content of RSV pre-F mutant by double antibody sandwich method". Method for determining thermal stability: Thermal stability was evaluated by comparing the loss of pre-fusion conformation of mutant proteins before and after heat treatment. Briefly, the mutant proteins were heat-treated at 50°C for 3h. The pre-fusion conformation protein concentration before and after heat-treatment was detected by referring to "(7) Detection of RSV pre-F mutant content by double-antibody sandwich method". The pre-fusion conformation protein concentration after heat-treatment was divided by the pre-fusion conformation protein concentration before heat-treatment to calculate the remaining percentage, and the heat stability of the mutant was evaluated by this percentage. Table 5, mutant expression and heat stability Figure 8 describes the expression of mutant pre-F formed at the β3 and β4 positions. Among them, JW-31, JW-34, and JW-35 were significantly expressed in CHO cells, especially JW-31 and JW-35, whose expression levels were significantly higher than those of WT (SEQ ID NO. 11) and F0-GS (SEQ ID NO. 12). JW-32 and JW-33 were almost not expressed. It is confirmed that mutations at some β3 and β4 positions can produce stable pre-F proteins. Figure 9 describes the heat stability of mutant pre-F formed at the β3 and β4 positions. The detection results show that, compared with WT and F0-GS, the mutant with high pre-F protein expression also has good heat stability. WT and F0-GS without cysteine mutation are very unstable after 50°C treatment. Figure 10 describes the expression of mutant pre-F formed at the α3 and β3 positions. Among the mutations introduced at this position, except that JW-03 has a low expression, the rest have significantly improved expression compared with WT and F0-GS. It is confirmed that mutations at some α3 and β3 positions can also produce stable pre-F proteins. Figure 11 describes the heat stability of mutant pre-F formed at the α3 and β3 positions. Mutations at different positions of α3 and β3 have a significant impact on stabilizing pre-F. JW-04 and JW-06 show good heat stability. Figure 12 describes the expression of double-disulfide bond mutant pre-F formed at the α3 / β3 and β3 / β4 positions. Figure 13 describes the heat stability of double-disulfide bond mutant pre-F formed at the α3 / β3 and β3 / β4 positions. Compared with single-disulfide bond mutations, double-disulfide bond mutations significantly improve the heat stability of the mutations, so that the mutant can still retain >60% of pre-F protein after 3h treatment at 50°C. (3) Purification of recombinant mutant proteins The protein purification was performed in two steps using affinity chromatography and ion exchange chromatography. In summary, the expression product was harvested by centrifugation (8000 rpm, 20 min) to remove cells and cell debris from the culture supernatant. The culture supernatant was filtered through a 0.45 pm filter to remove impurities. The treated supernatant was applied to a Ni-Sepharose 6 FF column (cytiva) equilibrated with 25 mM Tris-HCl, 0.15 M NaCl (Buffer A; pH 8.0). The column was then further washed with 25 mM Tris-HCl (150 mM NaCl, pH 8.0) until A280 reached baseline level, and eluted with a linear gradient of elution buffer (Buffer B: 25 mM Tris-HCl, 150 mM NaCl, 500 mM imidazole, pH 8.0) from 0 to 100%, and the eluate was collected and the imidazole was removed by using equilibration buffer as displacement buffer. After the buffer exchange, the sample was diluted with PBS buffer to a conductivity of less than 4 ms / cm and the pH was adjusted to 6.0. The protein was loaded onto a Capto S Impres packing (Cytiva) equilibrated with 20 mM phosphate buffer pH 6.0, and after loading, the column was eluted with 20 mM phosphate buffer pH 6.0 until A280 reached baseline level, and eluted with a linear gradient of elution buffer (20 mM phosphate buffer, 1 M NaCl pH 6.0) from 0 to 100%, and the eluate was collected. The pH was adjusted to 7.4-8.0 with 0.2 M Na2HP04, and the eluate was further filtered through a 0.45 pm filter to remove insoluble particles. (4) SDS-PAGE identification of mutant proteins The purified recombinant protein was detected by SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis). The detection sample after purification was added to a sample buffer containing SDS and a reducing agent (such as dithiothreitol or mercaptoacetic acid), and then loaded after boiling in a water bath. After electrophoresis, the gel was stained for protein using the Coomassie blue staining method, and the gel image was obtained using a transparent scanner or a protein imaging system. Figure 14 describes the results of SDS-PAGE identification of RSV pre-F mutant monomers. Different mutants only have mutations in individual amino acids, and the molecular weight only changes slightly. The electrophoretic bands on SDS-PAGE show no difference, and the bands are larger than the theoretical molecular weight (50.55 kDa), which may be due to the band shift caused by glycosylation modification. Figure 15 depicts the results of RSV pre-F mutant trimer SDS-PAGE identification. Different mutants only mutate individual amino acids, and the molecular weight size only changes slightly, and the electrophoretic bands on SDS-PAGE show no difference, and the theoretical molecular weight (53.99 kDa) is larger, which may be due to the glycosylation modification causing the band to shift. (5) HPLC detection of the purity and uniformity of mutant proteins The purity of mutant proteins was analyzed by HPLC (High Performance Liquid Chromatography) using a Thermo U3000 high performance liquid chromatograph and a (Thermo) TSKgel UP-SW2000 molecular exclusion column (TOSOH) chromatographic column. The mobile phase was 20 mM PBS buffer at a flow rate of 1 mL / min, and the elution was detected at 280 nm. HPLC was used to detect the purity and uniformity of the target protein. Figure 16 depicts the purity and uniformity of mutant monomer JW038 detected by HPLC. The mutant monomer appears a specific chromatographic peak at 18.713 min. The target peak is single, and the mutant monomer shows good uniformity. Figure 17 depicts the purity and uniformity of mutant trimer JW038-T4 detected by HPLC. The mutant trimer appears a specific chromatographic peak at 15.958 min. The mutant trimer shows good uniformity. (6) Differential scanning calorimetry detection of the thermal stability of mutant proteins Differential scanning calorimetry (DSC) was performed using a DSC Q2000 (TA instrument) system. The temperature range of the device was 0°C and 400°C, and the thermal expansion rate was 10°C / min in a nitrogen flow of 8 mL / min. Measurements were made under a nitrogen atmosphere. Table 6, mutant Tm value detection According to Table 6, the Tm values of different mutant proteins detected by differential scanning calorimetry are all >60°C, indicating that the thermal stability of the mutant proteins is good, which also corresponds to the good stability of the mutant proteins at 50°C. (7) Double antibody sandwich method for detecting the content of RSV pre-F mutant The RSV pre-F mutant was quantified by double antibody sandwich ELISA. Palivizumab, which can recognize pre-F, post-F and F proteins at the same time, was used as the coating antibody, and the specific recognition of RSV pre-F mutant was used as the detection antibody. The standard curve was drawn by using the standard sample of RSV pre-F mutant, and the content of the sample was calculated by the standard curve. The site is horseradish peroxidase labeled specific monoclonal antibody D25 as detection antibody (D25-HRP) detection. At the same time, the purified pre-F monomer protein and trimer protein are used as calibration standards, respectively, and the content of the sample is obtained by detecting the absorbance at OD450 and the dilution factor through the standard curve. Through this method, the expression of mutant pre-F protein under transient expression and the residual content of pre-F mutant protein in mutant thermal stability test can be detected. The sample detection process is as follows: monoclonal antibody is diluted to 1 μg / L with carbonate buffer, and coated enzyme-labeled plate (Corning 9018) is used. 100 μL per well, 37°C for 1 h, then 2-8°C overnight; discard the liquid in the 96-well plate, wash 3 times with 20 mM PBS, then add 200 μL blocking solution (2% bovine serum albumin, component V) per well, and block at room temperature for 60 min; aspirate the blocking solution in the wells, wash 3 times with 20 mM PBS-T solution, add the mutant fusion pre-conformational F protein to the first column of the 96-well enzyme-labeled plate after a series of 3-fold dilution, and the negative control is PBS, 37°C for 60 min, aspirate the blocking solution in the wells, wash 3 times with 20 mM PBS-T solution; take anti-HIS-HRP conjugate, dilute with enzyme conjugate diluent 1:2000, then add to the 96-well enzyme-labeled plate, 100 μL per well, 37°C for 10 min; aspirate the secondary antibody in the wells, wash 3 times with 20 mM PBS-T solution, add 100 μL TMB color developing liquid per well, add 50 μL stop solution to stop the reaction after 10 min, then measure A450 and absorbance with an enzyme-labeled instrument. Figure 8, Figure 10 and Figure 12 show the pre-F protein expression in the cell culture supernatant after transient transfection of the monomeric RSV pre-F mutant proteins in Expi-CHO cells. In this example, Palivizumab, which can bind without relying on the conformational change of the F antigen, was used as the capture antibody, and D25 antibody, which can specifically detect the epitope Φ, was used as the detection antibody labeled with horseradish peroxidase. The purified DS-Cav1 monomeric protein or trimer (consistent with the expression form of the target) was used as a reference to establish a standard curve, and the pre-F protein expression in the culture supernatant was calibrated and converted according to the standard curve. Through the above operation, the following results were obtained: it was shown that the conservation of the epitope Φ was significantly improved in the mutants having cysteine point mutations in α3 and β3 to form disulfide bonds, and the pre-F was highly expressed in Expi-CHO cells. In terms of thermal stability, the stability of the protein was evaluated by assessing the proportion of the protein remaining bound to the D25 monoclonal antibody after treatment at 50°C (1h, 2h, 3h), and the purified protein sample was diluted to 0.1 mg / mL with PBS (pH 7.4) after heat treatment, and was detected by double antibody sandwich ELSIFA method, as shown in Figure 9, Figure 11 and Figure 13. (8) Antigenicity detection of RSV pre-F mutants Each purified mutant protein was coated in a 96-well enzyme plate (Corning 9018) at a starting dilution concentration of 10 μg / mL using a carbonate buffer (pH 9.6) with 3-fold dilution, and then 100 μL / well. After 1h at 37°C, it was placed overnight at 2-8°C; the liquid in the 96-well plate was discarded, and it was washed 3 times with 20 mM PBS, and then 200 μL of blocking solution (2% bovine serum albumin, component V) was added to each well, and it was blocked at room temperature for 60 min; the blocking solution in the well was aspirated, and it was washed 3 times with 20 mM PBS-T solution, and then the anti-D25-HRP, AM22-HRP, AM14-HRP, ADI-15568-HRP, etc. conjugate was diluted 1:2000 with enzyme conjugate diluent, and then added to the 96-well enzyme plate, 100 μL per well, and incubated at 37°C for 10 min; the excess antibody in the well was aspirated, and it was washed 3 times with 20 mM PBS-T solution, 100 μL of TMB color developing solution was added to each well, and after 10 min, 50 μL of stop solution was added to stop the reaction, and then the A450 and A630 absorbance values were measured with an enzyme marker. Figure 18 describes the results of the antigenicity identification of each mutant. Figure 18, panel A and panel B show that the purified mutants can be recognized by Epitope specific monoclonal antibodies D25 and AM22 recognized, confirming that the mutant is able to maintain the pre-F conformation of RSV F protein. At the same time, the mutant can also be recognized by ADI-15568 monoclonal antibody of epitope V (Figure 18, panel D). However, the trimer-specific antibody (AM14) is able to recognize the mutant trimer but not the mutant monomer, and the mutations at different positions have a significant impact on the stability of the trimer. (9) Immunogenicity evaluation of RSV pre-F protein In order to evaluate the immunogenicity of different pre-fusion conformation RSV F, different mutant proteins were used to immunize mice. Female Balb / c mice, 14-16 g, were immunized with 5 μg vaccine antigen added with aluminum hydroxide as an adjuvant. Intramuscular injection was performed at weeks 0 and 4 (28 d). Two weeks after the booster immunization (42 d), serum was collected for determination of total IgG antibody titer and neutralizing antibody titer. The immunization groups are shown in the following table: Table 7 (10) Detection of antigen-specific antibody levels Total IgG antibody detection was performed by indirect ELISA method. Pre-F protein was pre-coated in a 96-well plate, and after blocking, the serum to be tested was added. The serum to be tested was diluted by a factor of 800, and then diluted by a factor of 3. After washing to remove unbound serum, HRP-labeled goat anti-mouse secondary antibody was added for incubation. After incubation, unbound secondary antibody was removed by washing. After washing, substrate was added for color development. The mouse serum titer was determined by detecting the absorbance at 450 nm and 630 nm. The dilution titer was calculated according to the serum dilution factor of the last well diluted to an absorbance greater than the CUT-OFF value. Different mutant trimer proteins were added with aluminum hydroxide adjuvant to immunize BalB / c mice. The pre-F mutant trimer was used as the coating antigen to detect the antigen-specific IgG antibody levels in the mouse serum. Figure 19 describes the RSV pre-F specific binding antibody levels induced by different mutants in BalB / c mice. Both the mutant monomer and the mutant trimer have good immunogenicity and induce a high level of humoral immune response in mice. Among them, JW-38-T4 and JW-48-T4 induced binding antibody levels are superior to mutant monomer (JW-36), F0 protein and post-F protein, with statistically significant differences. (11) Detection of neutralizing antibody levels The neutralizing antibody titer detection adopts a high-throughput microwell plate method. Specifically, the density of Hep-2 cells is adjusted, inoculated into a 96-well plate, and placed in a cell incubator (37°C, 5% CO2) for overnight culture to ensure that the cell confluence is about 90% the next day, and then the test can be started. Then, the serum is inactivated in a water bath at 56°C for 30 min to prepare inactivated serum. Subsequently, the first hole of the sample to be tested is diluted 100 times, and 3 times of gradient dilution is performed, a total of 8 dilution degrees (including the first hole), 2 duplicate holes; then, according to the PFU value of the virus, the appropriate amount of virus is added. The sample hole and the virus control hole are added with the diluted virus, and the back-dropping hole is sequentially diluted by 2 times of gradient for a total of 4 dilution degrees, and then incubated in a 37°C, 5% CO2 incubator for about 1 h. Then, the above virus and serum neutralization product and positive and back-dropping hole virus are added to the previously prepared cells, 100 μL of culture medium is added to each well, and the culture is continued for about 22 h. The supernatant is discarded, the cells are fixed, and then the fluorescently labeled detection antibody is added, and the CTL instrument is used to read the plate. The neutralizing antibody titer is set as the serum dilution fold that causes a 50% reduction in the infectious unit (NT50). FIG. 20 describes the neutralizing antibody levels induced by different mutants in mice. The results show that the serum of the mutant immunized mice can neutralize the RSV: A2 strain virus. The neutralizing antibodies induced by different mutants are different, but they are all better than F0 and post-F protein. The stabilized pre-F protein retains the key neutralizing epitope and induces higher neutralizing antibodies than post-F. At the same time, the level of neutralizing antibodies induced by F0 is also higher than that of post-F protein. The embodiments of the present application provide a mutation strategy for generating a stabilized RSV pre-F protein. By forming a disulfide bond between β3 and β4, or simultaneously forming a disulfide bond between α3 and β3 and between β3 and β4, the conformational change of the F protein is prevented. This mutation strategy is different from the current mainstream mutation strategy, which is an innovative mutation, enriching the structure-based antigen design for RSV F protein. The introduction of the disulfide bond generates a stable RSV F protein pre-F conformation with high expression, and the expression in CHO cell transient expression reaches 300 mg / L. Moreover, the mutant has very high thermal stability, and the pre-F protein can remain more than 85% after being treated at 50°C for 3 h. The mutant has the potential to be used as a vaccine. Site recognized by D25 antibody Antigen epitope, retains other antigen epitopes recognized by the currently identified antibodies specific to RSV F protein. Immunization with the mutant can induce higher neutralizing antibodies in animals. The mutant has great potential as a vaccine. Each of the technical features of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure. The above-described embodiments only express several implementation manners of the present application, facilitate understanding of the technical solutions of the present application in detail, but should not be understood as a limitation on the patent protection scope of the present application. It should be noted that, for those skilled in the art, without departing from the concept of the present application, a number of modifications and improvements can be made, which are all within the protection scope of the present application. In addition, it should be understood that, after reading the above description of the present application, those skilled in the art can make various modifications or improvements to the present application, and the equivalent forms also fall within the protection scope of the present application. It should also be understood that, based on the technical solutions provided by the present application, those skilled in the art can obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be subject to the content of the appended claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Mutants of RSV pre-F characterized in that, The F1 polypeptide of the mutant satisfies either (1) or (2) as follows: (1) the β3 sheet region has a cysteine substitution β3-1, and the β4 sheet region has a cysteine substitution β4-1, the cysteine substitution β3-1 and the cysteine substitution β4-1 form a disulfide bond; (2) the α3 helix region has a cysteine substitution α3-1, the β3 sheet region has a cysteine substitution β3-1 and a cysteine substitution β3-2, the β4 sheet has a cysteine substitution β4-1, the cysteine substitution β3-1 and the cysteine substitution β4-1 form a disulfide bond, the cysteine substitution α3-1 and the cysteine substitution β3-2 form a disulfide bond; The mutant has one of the following combinations of cysteine mutations: Combination 1 is S180C and S186C; and, Combination 2 is K176C and S190C.

2. The mutant of RSV pre-F according to claim 1, characterized in that, The mutant of the RSV pre-F is of human or bovine origin.

3. Mutant of RSV pre-F according to claim 1 or 2, characterized in that, The F1 polypeptide of the RSV pre-F has an amino acid sequence as shown in any one of SEQ ID NO. 4, SEQ ID NO. 71 and SEQ ID NO. 72, or has at least about 80% homology with the amino acid sequence as shown in any one of SEQ ID NO. 4, SEQ ID NO. 71 and SEQ ID NO. 72; Optionally, the F1 polypeptide satisfies one or more of the following conditions: (1) does not contain a transmembrane domain, and (2) does not contain an intracellular domain; Further optionally, the F1 polypeptide has one or more of the following mutations: I379V and M447V.

4. The mutant of the RSV pre-F according to any one of claims 1 to 3, wherein The mutant has one of the following combinations of cysteine mutations: Combination 3 is S180C, S186C, A170C, A177C; Combination 4 is S180C, S186C, E163C, L181C; Combination 5 is S180C, S186C, A170C, V179C; Combination 6 is S180C, S186C, L171C, A177C; Combination 7 is K176C, S190C, A170C, A177C; Combination 8 is K176C, S190C, E163C, L181C; Combination 9 is K176C, S190C, A170C, V179C; and, Combination 10 is K176C, S190C, L171C, A177C.

5. The mutant of RSV pre-F according to any one of claims 1 to 4, characterized in that, The mutant further has one or more of the following mutation sites: S55C, S155C, V207L and S290C.

6. The mutant of RSV pre-F according to any one of claims 1 to 5, characterized in that, The mutant does not contain a furin cleavage site fragment.

7. The mutant of RSV pre-F according to any one of claims 1 to 6, characterized in that, The mutant does not contain a pep27 polypeptide, the C-terminus of the F2 polypeptide and the N-terminus of the F1 polypeptide are directly connected by an amide bond or indirectly connected by a flexible short peptide; Optionally, the flexible short peptide is GS, G, S, GS, SG, SS, GG, PG, GGG, GGS, SSS, GSG, SGS, GPG, GSGS, GGGS, GPGS, GGGG, GSGG, GGSG, SGGG, GSSG, SGSG, GSSG, GGPGG, or GGGGS.

8. The mutant of RSV pre-F according to any one of claims 1 to 7, characterized in that, The F2 polypeptide of the mutant satisfies one or more of the following conditions: 1) the C-terminus does not contain NN, and 2) has a mutation of P102A.

9. The mutant of RSV pre-F according to any one of claims 1 to 8, characterized in that, The C-terminus of the mutant is linked to a tag fragment; optionally, the tag fragment comprises a poly-histidine; further optionally, the poly-histidine comprises an 8His fragment.

10. The mutant of RSV pre-F according to any one of claims 1 to 9, characterized in that, The mutant further comprises one or more of a structural polypeptide and a functional polypeptide; the structural polypeptide causes monomers of the mutant to form a multimer, and the functional polypeptide enhances the biological activity of the mutant; Optionally, the structural polypeptide comprises one or more of an aggregation motif, a GCN4 leucine zipper, and a nanoparticle conjugation fragment; Optionally, the functional polypeptide comprises an antigen-specific binding fragment; Optionally, one or more of the structural polypeptide and the functional polypeptide.

11. The mutant of RSV pre-F according to any one of claims 1 to 10, characterized in that, The sequence of the mutant is as set forth in any one of SEQ ID NO. 20, SEQ ID NO. 25 to SEQ ID NO.

70.

12. A nucleic acid molecule encoding the mutant of any one of claims 1 to 11.

13. A vector comprising the nucleic acid molecule of claim 12.

14. An engineered cell expressing the mutant of any one of claims 1 to 11, or comprising the nucleic acid molecule of claim 12 or the vector of claim 13.

15. A method of producing the mutant of any one of claims 1 to 11, comprising the steps of: culturing the engineered cell of claim 14, and isolating the mutant from the resulting culture supernatant.

16. An immunological composition comprising the mutant of any one of claims 1 to 11, or the nucleic acid molecule of claim 12, and an immunological adjuvant; Optionally, the immunological adjuvant comprises one or more of an aluminum salt adjuvant, a surfactant, a polynucleotide, a lipopolysaccharide, a liposome, and an oil emulsion adjuvant.

17. Use of the mutant of any one of claims 1 to 11 in the manufacture of a respiratory syncytial virus antibody detection kit.

18. A respiratory syncytial virus antibody detection kit comprising the mutant of any one of claims 1 to 11.

19. A method of preventing or treating lower respiratory tract infection caused by respiratory syncytial virus, comprising the step of administering to a subject a therapeutically effective amount of the immunological composition of claim 16.

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