CN117586359A - Respiratory Syncytial Virus (RSV) polypeptides having immunogenicity - Google Patents

Respiratory Syncytial Virus (RSV) polypeptides having immunogenicity Download PDF

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Publication number
CN117586359A
CN117586359A CN202410079902.2A CN202410079902A CN117586359A CN 117586359 A CN117586359 A CN 117586359A CN 202410079902 A CN202410079902 A CN 202410079902A CN 117586359 A CN117586359 A CN 117586359A
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rsv
protein
polypeptide
recombinant
domain
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胡颖嵩
邓家荔
张元杰
任朵朵
李菡
杨克俭
洪坤学
刘勇
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Jiangsu Ruike Biotechnology Co ltd
Wuhan Ruike Biotechnology Co ltd
Abzymo Biosciences Co ltd
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Jiangsu Ruike Biotechnology Co ltd
Wuhan Ruike Biotechnology Co ltd
Abzymo Biosciences Co ltd
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Abstract

The invention provides an immunogenic Respiratory Syncytial Virus (RSV) polypeptide, and relates to the technical field of biological medicines. The antigen and the vaccine prepared by the antigen have high expression quantity, good stability and strong immunogenicity.

Description

Respiratory Syncytial Virus (RSV) polypeptides having immunogenicity
Technical Field
The application belongs to the technical field of biological medicine, and particularly relates to an immunogenic Respiratory Syncytial Virus (RSV) polypeptide, a preparation method thereof and application thereof in preparing vaccine for preventing RSV infection.
Background
Respiratory viral infections are one of the most important public health burdens worldwide, resulting in millions of hospitalizations worldwide each year. Respiratory syncytial virus (Respiratory Syncytial Virus, RSV) is the leading cause of acute lower respiratory tract infection in children under the age of 2 and adults over the age of 65. Although the respiratory tract disease caused by RSV infection is severely compromised, no effective therapeutic and prophylactic vaccine specific to RSV is currently available. The major direction of development of RSV vaccines worldwide into clinical stages has focused on live attenuated vaccines, recombinant subunit vaccines and viral vector vaccines. RSV vaccine development is mainly directed to 3 target populations, including infants, pregnant women, and the elderly (no less than 60 years). Infants older than 6 months, either achieved passive immune protection mainly by mAbs, or achieved passive protection by placental transfer of antibodies after vaccination with RSV vaccine by pregnant women; infants older than 6 months of age can be protected by active immunization with a live attenuated RSV vaccine; the development of RSV vaccines for pregnant women and the elderly is based on subunit vaccines.
The choice of mainstream vaccine antigens is mainly focused on F proteins, especially F proteins in pre-fusion conformation (PreF). The F protein in the pre-fusion conformation may expose more potential epitopes with neutralizing activity than the F protein in the post-fusion conformation, such as epitope Φ and epitope V only present in PreF. Antibodies generated against the epitope phi and the epitope V have excellent neutralizing activity, and after being combined with F protein in a pre-fusion conformation, the antibodies can block virus membrane fusion mediated by F protein conformational change. Despite extensive research on the conformational stability of RSV F protein prior to fusion, the safety and efficacy of its use as a vaccine remains a significant challenge.
Accordingly, it is an urgent need for a vaccine comprising the same to provide an RSV antigen having high safety and enhanced immunogenicity and improved stability.
Disclosure of Invention
To solve the above technical problems, the present invention provides a Respiratory Syncytial Virus (RSV) polypeptide having immunogenicity.
In one aspect, the invention discloses an immunogenic Respiratory Syncytial Virus (RSV) polypeptide comprising a recombinant ectodomain of an RSV F protein, amino acid residues 26-513 corresponding to the wild-type RSV F protein sequence depicted in SEQ ID NO. 1, comprising an intermolecular cysteine substitution mutation compared to the wild-type RSV F protein, wherein the intermolecular cysteine substitution mutation is selected from 1 pair or 2 pairs of K75C+E218C, S C+N460C and A149C+Y458C, preferably the intermolecular cysteine substitution mutation K75C+E218C.
The invention discloses an immunogenic Respiratory Syncytial Virus (RSV) polypeptide comprising a recombinant ectodomain of the RSV F protein, corresponding to amino acid residues 26-513 of the wild-type RSV F protein sequence shown in SEQ ID NO:1, wherein the recombinant ectodomain may preferably comprise 3 types of cysteine substitution mutations, which result in the formation of disulfide bonds between the introduced cysteine residues, which disulfide bonds serve to stabilize the conformation or oligomeric state of the protein, such as the pre-fusion conformation, wherein at least one type is an intermolecular cysteine substitution mutation selected from 1 or 2 pairs of K75C+E218C, S C+N460C and A149C+Y458C, preferably K75C+E218C. Another class is the class N cysteine substitution mutations, the site of which is between residues 1-300 of the amino acid sequence of the wild-type RSV F protein, including 1 pair or 2 cysteine substitutions; the third class is the class C cysteine substitution mutation, which is located between residues 320-500 of the amino acid sequence of the wild-type RSV F protein, including 1 pair or 2 cysteine substitutions.
The N-type cysteine substitution mutation is one or two pairs of I148C+Y286C, S C+L188C, H C+I291C and G151C+I 288C.
The C-type cysteine substitution mutation in the technical scheme of the invention is selected from K399C+S485C, S443C+S466C, T323C+I475C, I C+P480C or F387C+I492C.
The polypeptide of the present invention, wherein the recombinant ectodomain comprises one of the following combinations of cysteine substitution mutations:
A. k75c+e218C, I148 7c+y286C and f387c+i492C;
B、K75C+E218C、I148C+Y286C、T323C+I475C；
C、K75C+E218C、I148C+Y286C、S433C+S466C；
D、K75C+E218C、I148C+Y286C、K399C+S485C；
E、K75C+E218C、G151C+I288C、K399C+S485C；
F、K75C+E218C、H159C+I291C、K399C+S485C。
the polypeptide of the present invention, wherein the recombinant ectodomain further comprises a cavity-filling mutation, wherein the cavity-filling mutation site is selected from one, two, three or four of positions 67, 495, 296 and 190 of the amino acid sequence of wild-type RSV F protein.
In some embodiments, the preferred cavity filling mutation is N67F, in other embodiments, the preferred cavity filling mutation is n67f+s190v+v296f+v495L.
The polypeptide in the technical scheme of the invention, wherein the recombinant extracellular domain further comprises mutation P102A+I379V+M V, S46G or G184N for increasing the expression level.
The recombinant ectodomain described in the present invention comprises an epitope that is present only in the pre-fusion conformation, said epitope comprising residues 62-69 and 196-209 of the wild-type RSV F protein sequence depicted in SEQ ID NO. 1.
The polypeptide in the technical scheme of the invention, wherein the recombinant ectodomain comprises a recombinant F2 domain at least comprising amino acid residues 26-105 of RSV F protein and a recombinant F1 domain at least comprising amino acid residues 145-513 of RSV F protein.
The polypeptide in the technical scheme of the invention, wherein the recombinant F2 domain is directly connected with the recombinant F1 domain or is connected with the recombinant F1 domain through a linker.
In some embodiments, wherein the recombinant F2 domain is linked to the recombinant F1 domain by a linker means that the C-terminus of the F2 domain and the N-terminus of the F1 domain are linked by a linker, e.g., residues 106-144 of the recombinant extracellular domain of the F protein are replaced by a linker sequence selected from GGPGGS, GAPEPGE or GGSGGSG.
In some embodiments, wherein the C-terminus of the polypeptide comprises a multimerization domain.
In some embodiments, wherein the multimerization domain is located at the C-terminus of the recombinant ectodomain, directly linked to the C-terminus of the recombinant ectodomain or linked via a linker sequence.
In some embodiments, optionally, the linker sequence is a GGGSSGS, GSGSG or G and S composed 4-10 amino acid sequence.
In some embodiments, wherein the multimerization domain is a trimerization domain, the trimerization domain promotes the formation of a trimer of 3F 1/F2 heterodimers.
In some embodiments, wherein the multimerization domain is a domain that can achieve more aggregation, e.g., ferritin that can self-assemble to form a twenty-four mer.
In some embodiments, the multimerization domain is preferably a foldon domain or a ferritin domain.
In the present application, "ferritin" refers to noctuid ferritin or helicobacter pylori ferritin. In the present application, H.pylori ferritin has a single-stranded structure. In the present application, the ferritin may be a mutant of spodoptera exigua or helicobacter pylori ferritin. In this application, "mutant" generally refers to a sequence that differs from a reference sequence by containing one or more differences (mutations). The difference may be a substitution, deletion or insertion of one or more amino acids. Illustratively, the ferritin amino acid sequence used herein is ESQVRQQFSKDIEKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS.
In some embodiments, wherein the recombinant ectodomain comprises the F2 domain of RSV F protein or a functionally active fragment thereof and the F1 domain or a functionally active fragment thereof.
In some embodiments, the F2 domain comprises a RSV F protein polypeptide corresponding to amino acids 26-105 of SEQ ID NO. 1, and the F1 domain comprises a RSV F protein polypeptide corresponding to amino acids 137-516 of SEQ ID NO. 1.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising at least amino acid residues 26-105 of the RSV F protein and a recombinant F1 domain comprising at least amino acid residues 145-513 of the RSV F protein.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising amino acid residues 26-105 of the RSV F protein and a recombinant F1 domain comprising amino acid residues 145-513 of the RSV F protein.
In some embodiments, wherein the recombinant F2 domain and the recombinant F1 domain are linked by a linker sequence.
In some embodiments, wherein the recombinant F2 domain and the recombinant F1 domain are linked by a GGGSSGS, GSGSG, GGPG or G to S composed of a 4-10 amino acid residue linker sequence.
In some embodiments, wherein the G and S constitute a 4-10 amino acid residue linker sequence, optionally GGSGGSG.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising at least amino acid residues 26-103 of the RSV F protein and a recombinant F1 domain comprising at least amino acid residues 146-513 of the RSV F protein.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising amino acid residues 26-103 of the RSV F protein and a recombinant F1 domain comprising amino acid residues 145-513 of the RSV F protein.
In some embodiments, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising amino acid residues 26-103 of the RSV F protein and a recombinant F1 domain comprising amino acid residues 147-513 of the RSV F protein.
In some embodiments, wherein the recombinant F2 domain and recombinant F1 domain are linked by a linker sequence of 5-8 amino acid residues consisting of glycine G, proline P, and one or more amino acids selected from serine S, alanine a, glutamic acid E, and lysine K.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, and serine S.
In some embodiments, the linker sequence is a 6 amino acid residue length linker sequence consisting of glycine G, proline P, and serine S, wherein the linker sequence is GGPGGS.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, serine S, and alanine a.
In some embodiments, the linker sequence is a 5 amino acid residue length linker sequence consisting of glycine G, proline P, serine S, and alanine a, wherein the linker sequence is GAPGS.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, alanine a, and glutamic acid E.
In some embodiments, the linker sequence is a 7 amino acid residue length linker sequence consisting of glycine G, proline P, alanine a, and glutamic acid E, wherein the linker sequence is gappge.
In some embodiments, wherein the linker sequence is a 5-8 amino acid residue linker sequence consisting of glycine G, proline P, alanine a, glutamic acid E, and lysine K.
In some embodiments, the linker sequence is a 7 amino acid residue length linker sequence consisting of glycine G, proline P, alanine a, glutamic acid E, and lysine K, wherein the linker sequence is gkppapge.
In some embodiments, wherein the cavity filling mutation is selected from one or more of N67F, S190V, V296F, and V495L. In some embodiments, wherein the cavity filling is abrupt to N67F. In some embodiments, wherein the cavity filling mutation is preferably a combination of N67F, S190V, V296F and V495L.
In some embodiments, wherein the substitution that increases expression is selected from p102a+i379v+m V, S46, G, L, M, I, 221M and/or E218A. In some embodiments, wherein the electrostatic mutation is selected from G184N, while in other embodiments, G184N also has an effect of increasing expression.
In some embodiments, the present invention contemplates intermolecular disulfide bonds, meaning disulfide bonds that link monomers, which can assist in locking the F protein in a pre-fusion conformation, such as k75c+e218C, V c+i407C or s150c+y458C, etc. In some embodiments, the invention contemplates disulfide mutations of the distal region of the immobilized membrane-class N disulfide, such as, for example, I164C+Y 286C, S C+L188C, H C+I291C or G151C+I288C. In some embodiments, the invention contemplates disulfide mutations in the proximal region of the immobilized membrane-class C disulfide, such as K399C+S485C, S443C+S466C, T C+I475C, I332C+P480C or F387C+I492C.
In some embodiments, wherein the mutation that increases expression comprises S46G.
In some embodiments, wherein the mutation that increases expression comprises G184N, S46G, L M, and I221M.
The polypeptides disclosed herein comprise signal peptide sequences required for expression or linker sequences linked to purification tags, which may be removed in the final product.
In some embodiments, the polypeptide further comprises a heterologous signal peptide at the time of construction.
In some embodiments, the heterologous signal peptide is an IgG signal peptide having an amino acid sequence of MGWSCIILFLVATATGVHS.
In some embodiments, the polypeptide also incorporates affinity tags such as strep Tag (amino acid sequence: WSHPQFEK), monoclone Flag-Tag (amino acid sequence: DYKDDDK) and His Tag (amino acid sequence: HHHHHHH) at the time of vector construction, which Tag sequences can be removed in the final product.
The invention also provides application of the polypeptide in preparing vaccine for preventing RSV infection.
The invention also provides a vaccine for preventing RSV infection, comprising a polypeptide of any of the above; the vaccine provides protection against infection by at least one of the RSV subtypes a and/or B.
The invention discloses a vaccine composition for preventing RSV infection, further comprising an immunoadjuvant comprising at least one of aluminum adjuvant, squalene, tocopherol, MPL, LPA, cpG, poly (I: C) and QS-21.
In some embodiments, the invention discloses a vaccine for preventing RSV infection comprising an adjuvant in the form of liposomes, the major components of the adjuvant in the form of liposomes comprising MPL, QS-21, DOPC and cholesterol. The dose of the adjuvant may be 0.5ml per dose, containing 50 μg MPL, 50 μg QS-21, 1mg DOPC, 0.25mg cholesterol, 4.835mg sodium chloride, 0.15mg anhydrous disodium hydrogen phosphate, 0.54mg potassium dihydrogen phosphate and water for injection, or may be one half, one quarter or less of the above dose, for example, the main components include 25 μg MPL, 25 μg QS-21, 0.5mg DOPC, 0.125mg cholesterol.
The invention also provides a method for preparing the polypeptide, which comprises the following steps:
s1, synthesizing a DNA sequence corresponding to the polypeptide, and cloning the DNA sequence into a plasmid vector;
s2, transfecting a host cell with a recombinant plasmid vector and expressing the host cell, wherein the host cell is a eukaryotic cell;
s3, purifying the culture product to obtain the polypeptide.
In some embodiments, the vaccines of the present invention generally further comprise a pharmaceutically acceptable carrier and/or excipient such as a buffer. Pharmaceutically acceptable carriers and excipients are well known and may be selected by those skilled in the art. Optionally, the pharmaceutically acceptable carrier or excipient further comprises at least one component that stabilizes solubility and/or stability. Examples of solubilizing/stabilizing agents include detergents, such as laurel sarcosine and/or tween. Other examples of solubilizing agents/stabilizers include arginine, sucrose, trehalose, and the like. Thus, one skilled in the art can select suitable excipients and carriers to produce a formulation suitable for administration to a subject by a selected use. Suitable excipients include, but are not limited to, glycerol, polyethylene glycol, KCl, calcium ions, magnesium ions, manganese ions, zinc ions, and other divalent cation related salts, and the like.
The adjuvant should generally be capable of enhancing a Th 1-biased immune response in a subject or population of subjects receiving vaccine administration and be safe and effective in the subject or population of subjects at the time of selection.
The invention also provides a method for preparing the vaccine for preventing RSV infection, which comprises the following steps:
s1, culturing a mammalian host cell to express the polypeptide;
s2, purifying the polypeptide expressed in the step (1);
s3, fully mixing the purified polypeptide with an immunoadjuvant according to a proportion.
In some embodiments, additional steps are included in gene synthesis, construction of expression vectors, lyophilization of purified proteins, and the like. The expressed fusion polypeptide may be recovered and purified from recombinant cell culture by any one or several of a number of methods well known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, filtration, ultrafiltration, centrifugation, anion/cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. Finally, high performance liquid chromatography HPLC can be employed in the final purification step.
Compared with the prior art, the invention has the following beneficial effects:
the invention obtains RSV polypeptide with high expression quantity and immunogenicity which has specific epitope to Pre-F through the design of stabilizing modification, multimerization (such as trimerization) and the like of wild RSV F protein, and ensures that the RSV polypeptide can cause effective neutralizing antibody reaction and binding antibody reaction to respiratory syncytial virus while maintaining stable Pre-fusion conformation.
Drawings
The following drawings are only for purposes of illustration and explanation of the present invention and are not intended to limit the scope of the invention. Wherein:
fig. 1 (a): collecting the outflow peak and chromatographic spectrum of DS-Cav 1A;
fig. 1 (B): collecting the outflow peak and chromatographic spectrum of DS-Cav 1B;
fig. 2 (a): electrophoresis results of DS-Cav 1A;
fig. 2 (B): electrophoresis results of DS-Cav 1B.
Detailed Description
The invention will be further illustrated by the following non-limiting examples, which are well known to those skilled in the art, that many modifications can be made to the invention without departing from the spirit thereof, and such modifications also fall within the scope of the invention. The following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention as embodiments are necessarily varied. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting, the scope of the present invention being defined in the appended claims.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Preferred methods and materials of the invention are described below, but any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. The following experimental methods are all methods described in conventional methods or product specifications unless otherwise specified, and the experimental materials used are readily available from commercial companies unless otherwise specified.
Definition of terms
In the present application, the term "DS-Cav1" is a PreF design concept with reference to CN105473604B, including pre-F trimers stabilized by disulfide bonds between residues 155 and 290 ("DS"), cavity filling mutations S190F and V207L ("Cav 1"), and additional C-terminal fibritin (i.e., foldon) trimerization domains, specific sequences being given in the examples.
In the present application, the term "D25" refers to the antibody described in CN200880023301.9, the specific amino acid sequence of which is referred to in the specification as SEQ ID NO 1-8.
In the present application, the term "101F" refers to the antibodies described in US11261356, the nucleotide and amino acid sequences of the heavy and light chains of which are referred to in the specification by SEQ ID NOS 3-6.
In the present application, the term "AM14" refers to the antibody described in CN200880023301.9, and specific sequences refer to the amino acid sequence of AM14 in example 4 of the patent specification.
In the present application, the term "Mota" or "motavizumab" refers to antibodies described in WO2007002543A2, the specific sequences of which refer to the amino acid sequences SEQ ID NO:48 and SEQ ID NO:11 of the A4B4L1FR-S28R antibodies in the patent specification.
In this application, each mutant of the present invention was designed and prepared based on the amino acid sequence of the wild-type (abbreviated as WT) RSV F protein described in SEQ ID NO. 1. The F2 polypeptide comprises at least a part or all of amino acids 26-106 of SEQ ID NO. 1, e.g.26-100, 26-102, 26-104, 26-105 or 26-106, except for the mutations introduced as described. The F1 polypeptide comprises at least a part or all of amino acid residues 136 to 513 of SEQ ID NO. 1, for example 136 to 500, 140 to 500, 136 to 510, 136 to 512 or 136 to 513, except for the mutations introduced as described.
The F2 domain may be at position 100, position 101, position 102, position 103, position 104, position 105 or position 106 in the sequence of SEQ ID NO. 1. The C end of the F2 domain is directly connected with the N end of the F1 domain or is connected with the N end of the F1 domain through a Linker. The N-terminal of the F1 domain may be 136 th, 137 th, 138 th, 140 th, 141 th, 142 th, 143 th, 144 th, 145 th or 146 th bit of SEQ ID NO. 1. The C-terminus of the F1 domain may be position 499, 500, 502, 503, 504, 505, 506, 508, 510 or 513 of SEQ ID NO. 1. The C-terminal F1 domain can be linked to the trimerization domain or the multimerization domain directly or via a Linker.
The terms "multimerization domain" and "multimerization" refer to the formation of dimers, trimers, tetramers, pentamers, hexamers, and/or tetracosamers of a polypeptide or a domain of a polypeptide with other, more multimerization domains to form a polymer. Trimerization domains such as foldon, GCN4 or MTQ.
In the present application, the term "GCN4" refers to the transcriptional activator GCN4 of yeast, a protein that binds to DNA through a leucine zipper (bZIP) structure, and the amino acid sequence of GCN4 in the present application is IKRMKQIEDKIEEIESKQKKIENEIARIKKIK.
In the present application, the term "MTQ" is a trimerization module, in which the amino acid sequence of MTQ is IKEEIAKIKEEQAKIKEKIAEIEKRIAEIEKRIAGGCC.
Example 1 plasmid construction, transformation and preparation
The sequence of the immunogenic RSV polypeptides, including wild-type (WT), ferritin, F ecto-Ferritin group (ectodomain of F protein+ferritin) and DS-Cav1B group were designed as shown in Table 1 below, and DS-Cav1A group was additionally designed by disulfide bond mutations S155C and S290C, and cavity filling mutations S190F and V207L on the basis of the WT sequence, and further P102A, I379V and M447V amino acid substitution mutations were performed on the sequence of DS-Cav1A group to obtain DS-Cav1B group.
The amino acid sequence was subjected to codon optimisation in accordance with the host CHO cell to determine the nucleic acid sequence for gene synthesis.
TABLE 1 amino acid sequence design of constructs
The synthesized target gene is inserted into eukaryotic plasmid pCHO3.1 vector for transfection and expression. Each recombinant plasmid constructed was transformed into E.coli DH 5. Alpha. Competent cells, glycerol bacteria transformed into plasmids containing the target gene were transferred into 150mL LB liquid medium (Amp final concentration 100. Mu.g/mL), shake cultured overnight at 37℃and 2000rpm, plasmid extraction was performed using endotoxin-free plasmid extraction kit, the plasmid concentration of the extract was measured, and enzyme digestion was performed, and specific reaction systems are shown in Table 2.
TABLE 2 cleavage reaction System
The concentration of the amplified plasmids is measured, and the measured concentration of each plasmid can obtain higher amplification concentration of about 600-900 mug/ml, and the total amount is about 1.0 mug. The cleavage results were detected by 1% agarose gel electrophoresis, and clear bands were visible at the corresponding size positions. All plasmids were sequenced and, as identified, all plasmids were completely correct in the gene sequence of interest. Recombinant plasmid vectors carrying the gene of interest have been successfully transferred into host cells and amplified.
The amplified concentration of plasmids having intermolecular cysteine substitution mutation (also referred to as "intermolecular disulfide bond mutation") was examined after plasmid amplification, and the results are shown in the following table.
TABLE 3 plasmid concentration measurement results
The results showed that the higher plasmid amplification concentrations were mainly pCHO3.1-F46, pCHO3.1-F47, pCHO3.1-F48, pCHO3.1-F49 and pCHO3.1-F50, with the most preferred intermolecular disulfide mutation at plasmid concentration being the combination of K75C+E218C.
Example 2 protein expression
The resulting plasmid was introduced into CHO cells by transient transfection, and protein supernatant was collected after culture.
ExpiCHO-S cells (Thermo) were cultured in EmCD CHO-S203 (Eminance/L20301) medium supplemented with 6mM L-glutamine (sigma/G5146), 96 well plates (supplemented with 10% FBS) were plated at 0.25E5 cells/well density one day in advance, 37℃at 8% CO 2 Is subjected to stationary culture in an incubator. The day of transfection, 96-well plates plated with cells were replaced with fresh L-glutamine-containing EmCD CHO-S203 medium. A transfection incubation system (volume 50% of the transfection volume) was prepared with L-glutamine containing EmCD CHO-S203 medium with 1. Mu.g/ml DNA and transfection reagent PEI (Polysci)The ratio of the ends/24765-1) to the DNA was 3, PEI and the DNA were mixed, allowed to stand and incubated for 5min, then added to a 96-well plate with cells spread thereon, and the culture was incubated at 37℃with 8% CO 2 Is subjected to stationary culture in an incubator. After 24h of transfection, the temperature was reduced to 32℃for incubation, and a 5% by volume Feed supplement of Advanced Feed1 (sigma/24368-1L) was added. Culture was stopped at day 7 post-transfection to harvest supernatant.
Example 3 evaluation experiments on increased protein expression
And (3) carrying out molecular sieve chromatography by taking a DS-Cav1A protein supernatant sample stored at 4 ℃ and a DS-Cav1B protein supernatant sample as two groups of parallel samples to be tested. The height-diameter ratio of the molecular sieve chromatographic column is 40:1, and the filler is Superdex200; the mobile phase is 2.5% mercaptoethanol, 20mmol/L Tris-HCl with pH of 8.0; the flow rate is 50cm/hr; the sample loading amount is less than 5% (v/v) of the bed volume, and the flow-out peaks are collected step by step (chromatographic patterns are shown in figures 1 (A) and 1 (B), and electrophoresis results are shown in figures 2 (A) and 2 (B)).
The applicant has unexpectedly found that, in contrast, the three natural substitutions of P102A, I379V and M447V of DS-Cav1B can greatly increase the expression level of the protein. DS-Cav1 used in the control experiments of the following examples refers to optimized DS-Cav1B.
EXAMPLE 4F protein Individual mutagenesis protocol design
Each mutant of the present invention was designed and prepared based on the amino acid sequence of the wild-type (WT) RSV F protein described in SEQ ID NO. 1. This example illustrates the design of various F protein single mutants provided in the form of trimers, including the foldon domain and introduced amino acid mutations, such as cavity filling mutations. These F protein single mutants contained an IgG signal peptide (MGWSCIILFLVATATGVHS), two separate polypeptide chains. One of the polypeptide chains, the F2 polypeptide, comprises amino acids 26-106 of SEQ ID NO. 1, except for the mutations introduced as described. Another polypeptide chain comprises the F1 polypeptide (residues 136-513) linked to the N-terminus of the foldon domain, and their commonly introduced modifications include the substitution of del_P27 with ARGSG, R106Q and F137S in addition to the introduced mutations as described. Further included are purification tags His-Tag (HHHHHH), affinity Tag Monoclone Flag-Tag (DYKDDDK) and linker sequences (e.g., GSGSG). The signal peptide of SEQ ID NO. 1 (residues 1-25) and pep27 (residues 110-136) are cleaved from the F0 precursor during the expression process.
The amino acid sequences of the individual mutants of the RSV F protein and the control proteins were designed as follows, and the amino acid sequences were determined by codon optimization in accordance with the host CHO cells and were subjected to gene synthesis.
Table 4 mutants containing only cavity filling mutations
In addition, F protein single mutant only containing the linker sequence shown in the following table is designed, and is constructed by substituting residues 104-144 in the template amino acid sequence by the linker sequence, and the amino acid sequence is subjected to codon optimization according to a host CHO cell to determine a nucleic acid sequence and to perform gene synthesis.
Template amino acid sequence:
MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIATVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLSAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPRGSHHHHHHSAWSHPQFEK。
TABLE 5 design of linker sequences
Plasmid transformation, preparation and enzyme digestion identification were performed according to the method of example 1, and the concentration was measured after plasmid amplification, so that each plasmid could obtain a higher amplification concentration. All plasmids were sequenced and the sequence of the gene of interest was identified to be completely correct. Recombinant plasmid vectors carrying the gene of interest have been successfully transferred into host cells and amplified.
EXAMPLE 5 screening of intermolecular disulfide mutation schemes
The resulting plasmid was introduced into CHO cells by transient transfection, and protein supernatant was collected after culture. The coated antibody D25 or coated antibody motavizumab (abbreviated as mota) was diluted with PBS to 5 ng/. Mu.L, 200. Mu.L was added to each well of a 96-well plate, and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to an adaptation dilution (D25-1:8000, mota-1:16000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST. 100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read.
TABLE 6 binding of mutants designed with different intermolecular disulfide mutations to D25 and Mota
Selection of different intermolecular disulfide bond designs preferentially refers to the D25 antibody OD values, more preferably F49, F54, F56, while referring to the mota antibody OD values, F50, F51, F54, F56, F49 are also preferred, further in combination with the amplification concentration measurements of plasmids with intermolecular disulfide bond mutations in the plasmid construction, preferred mutation designs include F49, F54, F50 and F56, most preferred intermolecular disulfide bond mutation scheme is k75c+e218C.
EXAMPLE 6 selection of class N cysteine substitution mutation schemes
Regarding the selection of N-type cysteine substitution mutation scheme, the following single mutant plasmids were introduced into CHO cells by transient transfection, and after culturing, the protein supernatant was collected and allowed to stand at 4℃for 7 days for detection.
The coated antibody D25 or coated antibody motavizumab (abbreviated as mota) was diluted with PBS to 5 ng/. Mu.L, 200. Mu.L was added to each well of a 96-well plate, and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to an adaptation dilution (D25-1:8000, mota-1:16000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST. 100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read, and the sample concentration was calculated according to the regression analysis equation. The PreF conformational ratio of the protein after 7 days of standing at 4 ℃ was calculated and expressed as the ratio of D25 to Mota active concentration.
TABLE 7 PreF conformational occupancy of proteins with N class cysteine substitutions after 7 days of placement
The results show that F27, F25, F26 and F30 protein supernatants were able to detect higher PreF conformational ratios after 7 days at 4 ℃, indicating that they have N-class cysteine substitution mutations that are more favorable for maintaining the pre-F protein fusion conformation in a general vaccine storage environment, and therefore the preferred N-class cysteine substitution mutations are v151c+i288C, H159c+i291C, I c+y286C and s55c+v188C.
EXAMPLE 7 screening of class C cysteine substitution mutation schemes
Regarding the selection of the C-type cysteine substitution mutation scheme, the following single mutant plasmids were introduced into CHO cells by transient transfection, and protein supernatants were collected for detection after culturing.
The coated antibody, motavizumab (abbreviated as mota), was diluted with PBS to 5 ng/. Mu.L, and 200. Mu.L was added to each well of a 96-well plate and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to adaptation dilution (mota-1:16000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST. 100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read.
Table 8 ability of proteins with class C cysteine substitutions to bind to mota antibodies
The results show that F39, F42, F44, F41 and F45 are more capable of binding to Mota antibodies (a site II specific antibody that binds to both pre-and post-fusion F) than F39, F42, F44 and F45, so the preferred C-class cysteine substitution mutation schemes are l321c+i475C, I332 6c+s485C, F387c+i492C, I332 6c+p480C and k399c+s485C.
Example 8 combinatorial screening of intermolecular disulfide mutations and conformational thermal stability evaluation
With reference to the design scheme of intermolecular disulfide bonds, further designing polypeptides introducing combinatorial mutations in RSV F ecto (ecto), the combinatorial screening of intermolecular disulfide bond mutations is focused on.
Preparing a large amount of expressed candidate protein, purifying by using a nickel column affinity chromatography and molecular sieve chromatography two-step purification method, diluting the protein concentration to a detection concentration, and identifying the Pre-fusion conformational stability of the purified protein by using Pre-F Pre-fusion conformational specificity monoclonal antibody D25 at 60 ℃ and 70 ℃ for 1 h.
Design of individual mutations of the binding proteins the effect of the intermolecular disulfide bond (inter disulfide bond) mutation k75c+e218C on the conformational thermal stability of the F protein prior to fusion was first examined further. Conformational heat stability reference values were characterized using the sum of the D25 concentration ratios of different batches of protein after one hour of heat treatment to 4 ℃. The conformational thermal stability reference values in the following table are mainly the sum of the two batches, three times D25 concentration ratios, including the 0512 batch of protein at 60 ℃ for one hour and the 4 ℃ D25 concentration ratio at 0512 batch at 70 ℃ for one hour and the 4 ℃ D25 concentration ratio at 0509 batch at 70 ℃ for one hour, the results are shown in the table below.
TABLE 9 combinatorial screening of inter disulfide mutations and conformational thermal stability evaluation
In terms of PreF conformational thermostability, the pre-fusion conformation of the k75c+e218C mutant design with other combinations of F proteins is relatively most stable in the inter disulfide design compared to the F protein without the intermolecular disulfide design, while the N-type cysteine substitution mutation used in combination with the intermolecular k75c+e387 286C, G151 6c mutant design, preferably I148 c+y52387c+i288C, the C-type cysteine substitution mutation, preferably F387c+i492C, has a substantial beneficial contribution to the stability of the protein if the PreF conformational thermostability is very poor in the absence of the intermolecular disulfide design.
EXAMPLE 9 Secondary screening of disulfide mutation combination schemes
The amino acid sequences of the respective RSV F protein combinatorial mutants and the control proteins were designed as follows, and the amino acid sequences were determined by codon optimization of host CHO cells and gene synthesis was performed.
The resulting plasmid was introduced into CHO cells by transient transfection, and protein supernatant was collected after culture. The coated antibody D25 or AM14 was diluted with PBS to 5 ng/. Mu.L, 200. Mu.L was added to each well of a 96-well plate and incubated overnight at 4 ℃. PBST was washed 1 time. ELISA assay plates were blocked with 250. Mu.L of 2% BSA/PBS, blocked for 1 hour at room temperature and washed 1 time with PBST. 200. Mu.L of protein supernatant was added to each well and incubated for 1h at room temperature. Wash 3 times with PBST. HRP-labeled antibody was diluted to adaptation dilution (D25-1:8000) with 0.2% BSA/PBS, 200. Mu.L per well was added and incubated for 1 hour at room temperature. Wash 4 times with PBST. 100. Mu.L of substrate solution (TMB two-component solution A: B=1:1 mixture) was added, reacted at room temperature for 15-20min, 100. Mu.L of stop solution was added, and OD value was read, and the concentration of the sample after 1h at 60℃was calculated according to the regression analysis equation.
TABLE 10 evaluation of Pre-fusion conformation of different combination mutants
Note that: "/" means not measured or not detected
The results show that each designed combined mutant can be combined with the D25 antibody or the AM14 antibody, so that the proteins have different degrees of pre-fusion conformations, wherein the sum of the concentration of the two antibodies is larger than that of DS-Cav1 after the sample is placed at 60 ℃ for 1h, compared with a positive control DS-Cav1, the intermolecular cysteine substitution mutation K75C+E218C has a very prominent effect of protecting the pre-fusion conformations, and the combination of the K75C+E218C and N-type cysteine substitution mutation S55C+L168C or I148 C+Y7C, and the C-type cysteine substitution mutation S443C+S466C, K C+S485C or F387C+I492C is very beneficial to the acquisition of the pre-fusion conformations.
Example 10 immunoassay evaluation of bound antibody detection
Female BALB/C mice of 6-8 weeks of age are selected for immunization, randomly grouped, 6 mice in each group are subjected to vaccine double-dot immunization by intramuscular injection on days 0 and 21, and 50 mu L of each of the left leg and the right leg, wherein immunogens comprise candidate antigens F179 and F188, DS-Cav1, pXCS847 (SEQ ID NO: 11) and a positive control group of PreF-1345 (SEQ ID NO: 12); PBS negative control group. The antigen dose was 12. Mu.g/mouse, and the AS01 adjuvant dose was 1/20HD per mouse.
AS01 adjuvant is liposome adjuvant, and comprises MPL, QS-21, DOPC and cholesterol AS main components, wherein 1HD AS01 adjuvant is 0.5ml, and contains 50 μg MPL, 50 μg QS-21, 1mg DOPC, 0.25mg cholesterol, 4.835mg sodium chloride, 0.15mg anhydrous disodium hydrogen phosphate, 0.54mg potassium dihydrogen phosphate and water for injection. Serum was collected on day 20 and day 35, respectively, and the collected serum was stored at-20 ℃ for subsequent detection of bound and neutralizing antibodies.
Detection of bound antibodies:
(1) Diluting a serum sample to be detected by using a sample diluent;
(2) The positive control serum is diluted 1:10000 times by using a sample diluent, and the negative control serum is diluted 1:200 times by using a sample diluent;
(3) Sample adding: serial diluted serum sample to be tested, negative control serum, positive control serum and blank control solution (sample diluent is taken as blank control) are added into a 100 mul/Kong Jiazhi pre-F ELISA plate, 1 hole is added into the serum sample to be tested, and 2 holes are added into the control solution; igG-UNLB standard yeast ELISA strip at 100 μl/well was added to the sample dilution;
(4) Incubation: the ELISA plate is placed at 37 ℃ for 30 minutes, and is washed by washing liquid for 5 times in an automatic plate washing machine;
(5) Adding enzyme: placing an enzyme-labeled secondary antibody (HRP coat Anti-Mouse IgG (H+L), ABclonal) in a 100 μl/Kong Jiazhi enzyme-labeled plate, incubating at 37deg.C for 30 min, and washing the plate with washing liquid for 5 times;
(6) Color development: mixing the color development A solution and the color development B solution (flying organisms) in equal volume, and incubating in a 100 μl/Kong Jiazhi ELISA plate at room temperature in a dark place for 15 minutes;
(7) And (3) terminating: stop the reaction by adding a stop solution at 50. Mu.l/well;
(8) Reading a plate: the absorbance at a wavelength of 450nm (reference wavelength of 620 nm) was measured by a microplate reader, and the results are shown in the following table.
TABLE 11 Pre-F specific IgG content (five weeks serum)
After boosting, pre-F IgG GMC (geometric mean concentration) was detected in the 5 week serum of each group of mice to a different extent than at 3 weeks. The preF IgG GMC (geometric mean concentration) of F179 is highest, above the preF design concept of the positive control DS-Cav1 (cf. CN 105473604B), including preF trimers stabilized by disulfide bonds between residues 155 and 290 ("DS"), cavity filling mutations S190F and V207L ("Cav 1") and additional C-terminal fibritin (i.e. foldon) trimerization domains.
EXAMPLE 11 immunoassay evaluation of neutralizing antibody detection
The neutralizing antibody can specifically identify an antigen neutralizing site on the surface of a virus, prevent the virus from invading cell reproduction, protect the organism from being damaged, and has an index for evaluating the immune effect of real antiviral effect. The state of the middle department was commissioned to detect neutralizing antibodies by fluorescence spot reduction neutralization assay (FRNT method) using RSV A2 strain. The method comprises the following specific steps:
(1) Sample preparation: thawing the serum stored at-20deg.C at room temperature, heat-inactivating at 56 deg.C for 30min, taking out and cooling to room temperature, and mixing 6 parts of serum of each group thoroughly to obtain each group of mixed serum samples.
(2) Sample addition and dilution: initial dilution 40-fold followed by 4-fold dilutions for 6 dilutions. 96 well plates were used, column 2 as Cell Control (CC), column 3 as Virus Control (VC). The diluted 20 times sample is added into the B4-B11 hole, the liquid in the B4-B11 hole is gently and repeatedly mixed evenly by using a multi-channel pipette, and then 4 times ratio dilution is carried out until the mixture is in the G4-G11 hole. And taking the positive reference and the negative reference for sample adding and dilution.
(3) Virus dilution: the virus was diluted to 800-1200 FFU/well on ice box and wells were added except for the cell control.
(4) The 96-well plate was placed at 37℃in 5% CO 2 Incubate in incubator for 1h.
(5) HEp-2 cells were digested and counted and adjusted to 2.0X10 using complete medium 5 mu.L/well was added to a 96-well plate at 100. Mu.L/mL.
(6) Placing at 37deg.C and 5% CO 2 After incubation in incubator for 24-28 hours, GFP fluorescence spot counts were performed using a cell imaging multifunctional microplate detection system and the results recorded.
(7) 50% neutralizing antibody inhibition (ND) 50 ) Is calculated by (1): the instrument-read fluorescence spot values were input into a data analysis template, and the 50% neutralizing antibody inhibition (ND) was calculated for the samples using the Reed-Muench method 50 ）。
In addition, yun Ling metering was also entrusted for auxiliary detection, mainly by using RSV long strain and using luciferase reporter gene by CPENT method and chemiluminescent method. The results of the detection are shown in the following table.
Table 12 neutralizing antibody detection results
From the above experiments, it can be seen that the antigen and vaccine composition provided by the invention can induce the generation of neutralizing antibodies with high titer against RSV virus, and the neutralizing effect on RSV is superior to that of each positive control group, which also suggests that the vaccine will provide better protective effect in virus invasion, and has important significance for the development of RSV vaccine candidates.
Example 12 detection of neutralizing antibodies in multimerization Domain modified samples
Yun Ling, the RSV long strain is detected by CPENT method and luciferase reporter gene and chemiluminescence method. Sample dilution: the first well was serially diluted 3-fold at 30-fold initial dilution, diluted 6 gradients, and duplicate wells were set for each dilution. Virus neutralization with sample: quantifying recombinant virus solution, adding and mixing with diluted sample half-amount RSV recombinant virus solution, and heating at 37deg.C and 5% CO 2 The cells were neutralized in a cell incubator for 1h. 6 cell control wells, 6 virus control wells, were set per plate. Cell culture: a concentration of Hep-2 cell suspension was added to 96-well cell plates containing sample and virus neutralizer at 37℃with 5% CO 2 Culturing in a cell culture box for 24 hours. Cell lysis: adding a luciferase detection reagent into the cell culture plate, and reacting for 2min at room temperature in a dark place. And (3) detection: the liquid in the reaction well is repeatedly blown and sucked by a multi-channel liquid-transferer, so that cells are fully cracked, 150 mu l of liquid is sucked out of each well and added into a corresponding 96-well chemiluminescent detection plate, and the chemiluminescent detection plate is placed into a chemiluminescent detector to read the luminescent value. And calculating the neutralization inhibition rate.
Inhibition ratio = [1- (mean of luminous intensity of sample group-mean of cell control CC)/(mean of luminous intensity of virus control VC-mean of cell control CC) ] ×100%.
The ED50 is calculated according to the neutralization inhibition rate result and the Reed-Muench method. The results of the detection are shown in the following table.
TABLE 13 neutralizing antibody detection results
In the antigen candidate of the experimental sample, F179 and F196 are connected with trimerization foldon at the C end of preF protein, and F206 (SEQ ID NO: 13) and F210 (SEQ ID NO: 14) are connected with multimerization domain ferritin at the C end, which proves that the multimerization domain modified RSV F protein can also induce the generation of neutralizing antibodies with high titer against RSV virus, and the neutralizing effect on RSV is superior to that of wild-type antigen and DS-Cav1, preF-1345 and PXCS847 positive control group, which also suggests that the vaccine designed by the invention will provide better protective effect in virus invasion and has important significance for the development of RSV vaccine candidate.
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims (18)

1. An immunogenic Respiratory Syncytial Virus (RSV) polypeptide comprising an RSV F protein recombinant ectodomain, corresponding to amino acid residues 26-513 of a wild-type RSV F protein sequence set forth in SEQ ID No. 1, comprising an intermolecular cysteine substitution mutation compared to the wild-type RSV F protein, wherein the intermolecular cysteine substitution is selected from 1 pair or 2 pairs of k75c+e218C, S c+n460C and a149 c+y458C.
2. The polypeptide of claim 1, further comprising N-type cysteine substitution mutations compared to the recombinant ectodomain of the wild-type RSV F protein, the N-type cysteine substitution being at a site between residues 1-300 of the amino acid sequence of the wild-type RSV F protein.
3. The polypeptide of claim 1, further comprising a class C cysteine substitution mutation compared to the recombinant ectodomain of the wild-type RSV F protein, the class C cysteine substitution being at a site between residues 320-500 of the amino acid sequence of the wild-type RSV F protein.
4. An immunogenic Respiratory Syncytial Virus (RSV) polypeptide comprising an RSV F protein recombinant ectodomain corresponding to amino acid residues 26-513 of the wild-type RSV F protein sequence set forth in SEQ ID No. 1, wherein the recombinant ectodomain comprises the intermolecular cysteine substitution mutation k75c+e218C.
5. The polypeptide of claim 4, further comprising an N-class cysteine substitution mutation compared to the wild-type RSV F protein recombinant ectodomain selected from one or two pairs of I148C + Y286C, S C + L188C, H159C + I291C and G151C + I288C; also included are class C cysteine substitution mutations, which refer to K399C + S485C, S443C + S466C, T C + I475C, I C + P480C or F387C + I492C.
6. The polypeptide of claim 5, wherein the recombinant ectodomain comprises a combination of cysteine substitution mutations:
A. an intermolecular cysteine substitution mutation selected from k75c+e218C;
B. a class N cysteine substitution mutation selected from I148c+y286C, G151c+i288C or H159 c+i291C;
C. a class C cysteine substitution mutation selected from F387c+i492C, K399c+s485C or T323 c+i475C.
7. The polypeptide of claim 5, wherein the recombinant ectodomain comprises one of the following combinations of cysteine substitution mutations:
A、K75C+E218C、I148C+Y286C、F387C+I492C；
B、K75C+E218C、I148C+Y286C、K399C+S485C；
C、K75C+E218C、I148C+Y286C、S443C+S466C；
D、K75C+E218C、I148C+Y286C、T323C+I475C；
E、K75C+E218C、G151C+I288C、K399C+S485C；
F、K75C+E218C、H159C+I291C、K399C+S485C。
8. The polypeptide of claim 7, wherein the recombinant ectodomain further comprises a cavity filling mutation, the site of the cavity filling mutation selected from positions 67, 495, 296, or 190 of the amino acid sequence of the wild-type RSV F protein.
9. The polypeptide of claim 8, wherein the recombinant ectodomain further comprises an expression-enhancing mutation p102a+i379v+m V, S46G or G184N.
10. The polypeptide of any one of claims 1-9, wherein the recombinant ectodomain comprises a recombinant F2 domain comprising at least amino acid residues 26-105 of an RSV F protein and a recombinant F1 domain comprising at least amino acid residues 145-513 of an RSV F protein.
11. The polypeptide of claim 10, wherein the recombinant F2 domain is linked to the recombinant F1 domain directly or through a linker.
12. The polypeptide of claim 11, wherein linking the recombinant F2 domain to the recombinant F1 domain by linker means that residues 106-144 of the recombinant extracellular domain of the F protein are replaced by linker selected from GGPGGS, GAPEPGE or GGSGGSG.
13. The polypeptide of any one of claims 1-9, wherein the C-terminus of the polypeptide comprises a trimerization domain or a multimerization domain.
14. The polypeptide of claim 13, wherein the trimerization domain is a foldon, GCN4, or MTQ domain and the multimerization domain is a ferritin domain.
15. Use of the polypeptide of any one of claims 1-9, 11, 12 or 14 in the preparation of a vaccine for preventing RSV infection.
16. A vaccine for preventing RSV infection comprising the polypeptide of any one of claims 1-14; the vaccine provides protection against infection by at least one of the RSV subtypes a and/or B.
17. The vaccine of claim 16, comprising an adjuvant in the form of liposomes that prevents RSV infection.
18. The vaccine for preventing RSV infection according to claim 17, wherein said adjuvant in liposome form comprises MPL, QS-21 and cholesterol components.
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