RSV antigen, nucleic acid, pharmaceutical composition and application thereof

Abstract

The invention discloses an RSV antigen, nucleic acid, a pharmaceutical composition and application thereof. The RSV antigen has stronger immunogenicity than RSV in the prior art, can obtain higher neutralizing antibody titer, and has higher protective efficacy. And the RSV antigen provided by the invention can maintain more prefusion configurations, can stimulate generation of more neutralizing antibodies aiming at the prefusion configurations and generation of less neutralizing antibodies aiming at the postfusion configurations, and has relatively high safety. The RSV antigen, nucleic acid and pharmaceutical composition provided by the invention have good application prospects in preparation of drugs for relieving, preventing and / or treating diseases caused by RSV.

Description

[0001] This application claims priority to Chinese patent application No. 2024101736872, filed on February 6, 2024. This application incorporates the entirety of the aforementioned Chinese patent application. Technical Field

[0002] The present invention belongs to the field of vaccines, and specifically relates to an RSV antigen, nucleic acid, a pharmaceutical composition and applications thereof. Background Art

[0003] Respiratory syncytial virus (RSV) can cause respiratory tract infections in children, adolescents, and the elderly. Children, in particular, may develop severe infections and serious consequences such as respiratory sequelae. RSV belongs to the genus Pneumovirus of the family Pneumoviridae and has only one serotype. Its genome is 15.2 kb long and encodes 10 proteins. After infection, it mainly causes lower respiratory tract infections such as bronchiolitis and pneumonia in infants under 6 months old, and upper respiratory tract infections such as rhinitis and colds in older children and adults. There are three types of membrane surface proteins: fusion glycoprotein (F), attachment glycoprotein (G), and small hydrophobic protein (SH).

[0004] The RSV infection process primarily relies on the action of the F protein. During infection, the G protein attaches the virus particles to the cell surface through interaction with host cell attachment factors. The RSV F protein drives the fusion of the virus and host cell membranes by changing from a prefusion conformation to a postfusion conformation, thus initiating the entire viral infection cycle. Natural RSV infection induces neutralizing antibodies that are primarily directed against the RSV F protein, and therefore, most current RSV vaccine designs are designed specifically for the RSV F protein. Three other viral protein-based vaccine candidates that utilize non-F viral antigens are in clinical development.

[0005] RSV is the most common viral pathogen causing acute lower respiratory tract infections (ALRTI) in children under 5 years of age worldwide and is the leading cause of hospitalization for viral respiratory infections in infants and young children. Data show that RSV infection accounts for 28% of all ALRTI cases, and RSV-related deaths in hospitalized children account for 13%-22% of ALRTI-related deaths (Lancet. 2017, 390:946). Available RSV vaccines are summarized in Table 1.

[0006] Table 1 Available vaccines

[0007]

[0008]

[0009] However, the current protective efficacy of RSV antigen vaccines is relatively low, especially the ability to stimulate the production of neutralizing antibodies is not strong; in addition, too many postfusion configurations of RSV antigens will also bring about greater immune side effects. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to overcome the defects of the existing RSV antigen vaccines in that the protective efficacy is low, especially the ability to stimulate the production of neutralizing antibodies is not strong, and the excessive postfusion configuration of RSV antigens also brings about large immune side effects. A RSV antigen, nucleic acid, pharmaceutical composition and application thereof are provided. The RSV antigen provided by the present invention has stronger immunogenicity than the existing RSV, can achieve higher neutralizing antibody titers, and has higher protective efficacy.

[0011] The present invention solves the above technical problems through the following technical solutions.

[0012] The first aspect of the present invention provides an RSV antigen, characterized in that the RSV antigen satisfies any of the following conditions:

[0013] (I) The RSV antigen differs from SEQ ID NO: 1 by having S155C, S290C, S190F, S215P, S46G, E92D, D486C, D489C, A102C, and S362C mutations, and positions 103-144 of SEQ ID NO: 1 are substituted with a linker having an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or an amino acid sequence as shown in GGS, and the RSV antigen further comprises one or more of the following: a back mutation P215S, an insert having an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 140, and / or a replacement having an amino acid sequence as shown in SEQ ID NO: 7;

[0014] (II) The RSV antigen differs from SEQ ID NO:80 in that it has S155C, S290C, S190F, S46G, E92D, D486C, D489C, A102C and S362C mutations, and positions 103-144 of SEQ ID NO:80 are replaced by a linker with an amino acid sequence as shown in SEQ ID NO:2. The RSV antigen also includes an insert with an amino acid sequence as shown in SEQ ID NO:6.

[0015] In some embodiments of the present invention, the RSV antigen further satisfies one or more of the following conditions:

[0016] (1) In the RSV antigen described in (I) or (II), the insertion site of the insert fragment is between positions 516 and 517 of SEQ ID NO: 1 or SEQ ID NO: 80;

[0017] (2) (I) In the RSV antigen, the replacement fragment replaces positions 525 to 574 of SEQ ID NO: 1;

[0018] (3) (I) The RSV antigen further comprises a mutation L513I compared to SEQ ID NO: 1;

[0019] (4) (I) In the RSV antigen, the insertion site of the inserted fragment is between positions 513 and 514, between positions 519 and 520, or between positions 522 and 523 of SEQ ID NO: 1.

[0020] In some embodiments of the present invention, the RSV antigen is further post-translationally modified, and the post-translational modification is preferably glycosylation modification;

[0021] In some embodiments of the present invention, (I) the RSV antigen further comprises the back mutations C486D and / or C489D;

[0022] In some embodiments of the present invention, the RSV antigen (II) further comprises the reversion mutations (a) G46S; (b) D92E; (c) F190S; (d) C155S and C290S; (e) C486D and C489D; or (f) C102A and C362S;

[0023] In some preferred embodiments of the present invention, the glycosylation modification is an N-linked glycosylation modification, and / or the glycosylation modification occurs on the inserted fragment.

[0024] In some specific embodiments of the present invention, the amino acid sequence of the RSV antigen comprises the amino acid sequence shown in any one of SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:35-40, SEQ ID NO:81-89, SEQ ID NO:120, SEQ ID NO:122, SEQID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155 or SEQ ID NO:157.

[0025] The second aspect of the present invention provides an isolated nucleic acid comprising a nucleotide sequence encoding the RSV antigen as described in the first aspect.

[0026] In some embodiments of the present invention, the nucleic acid is DNA.

[0027] In some optional embodiments of the present invention, the DNA comprises a sequence encoding one or more of the following elements: a 5'-cap structure, a 5'UTR, a 3'UTR, a 3'-Poly (A) and a protein tag.

[0028] In some embodiments of the present invention, the nucleic acid is mRNA, preferably codon-optimized mRNA.

[0029] In some specific embodiments of the present invention, the mRNA has a sequence as shown in any one of SEQ ID NO: 59, 64-70, 72-79, 92-118, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 142, 144, 146, 148, 150, 152, 154, 156 or 158; and / or, the mRNA further comprises one or more of the following elements: a 5'-cap structure, a 5'UTR, a 3'UTR and a 3'-Poly (A).

[0030] The third aspect of the present invention provides a recombinant expression vector, which comprises the nucleic acid described in the second aspect.

[0031] In some embodiments of the present invention, the promoter of the recombinant expression vector is a T7 promoter.

[0032] The fourth aspect of the present invention provides a transformant, which comprises the nucleic acid as described in the second aspect or the recombinant expression vector as described in the third aspect.

[0033] The fifth aspect of the present invention provides a method for preparing RSV antigens, which comprises culturing the transformant as described in the fourth aspect under conditions suitable for expression of the RSV antigens.

[0034] The sixth aspect of the present invention provides a method for preparing a nucleic acid encoding an RSV antigen, the method comprising in vitro transcription of the recombinant expression vector described in the third aspect.

[0035] The seventh aspect of the present invention provides a composition comprising (1) the nucleic acid as described in the second aspect or the recombinant expression vector as described in the third aspect; and (2) a delivery vector.

[0036] In some embodiments of the present invention, the delivery vehicle is a lipid nanoparticle.

[0037] In some preferred embodiments of the present invention, the lipid nanoparticles are composed of (A) cationic lipid RL151, LQ104-E16b-2 or compound 6, and (B) cholesterol, (C) DSPC and (D) DMG-PEG2000;

[0038]

[0039] In some specific embodiments of the present invention, the molar ratio of cationic lipid RL151, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; the molar ratio of LQ104-E16b-2, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; or, the molar ratio of compound 6, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5.

[0040] The eighth aspect of the present invention provides a pharmaceutical composition, which comprises the composition as described in the seventh aspect, and optionally a pharmaceutically acceptable carrier and / or excipient.

[0041] The ninth aspect of the present invention provides a vaccine comprising the RSV antigen as described in the first aspect, the nucleic acid as described in the second aspect, one or more of the composition as described in the seventh aspect and the pharmaceutical composition as described in the eighth aspect, and an adjuvant.

[0042] The tenth aspect of the present invention provides a kit or a medicine box, which comprises one or more of the RSV antigen as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant expression vector as described in the third aspect, the transformant as described in the fourth aspect, the composition as described in the seventh aspect, the pharmaceutical composition as described in the eighth aspect, and the vaccine as described in the ninth aspect.

[0043] The eleventh aspect of the present invention provides the use of one or more of the RSV antigen as described in the first aspect, the nucleic acid as described in the second aspect, the recombinant expression vector as described in the third aspect, the transformant as described in the fourth aspect, and the composition as described in the seventh aspect in the preparation of a drug for alleviating, preventing and / or treating diseases caused by RSV.

[0044] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0045] The reagents and raw materials used in the present invention are commercially available.

[0046] The positive progress of the present invention is that the RSV antigen provided by the present invention has stronger immunogenicity than the existing RSV, can achieve higher neutralizing antibody titers, and has higher protective efficacy. In addition, the RSV antigen provided by the present invention can maintain more prefusion configurations, can stimulate the production of more neutralizing antibodies against the prefusion configuration, and reduce the production of neutralizing antibodies against the postfusion configuration, thus having higher safety. The RSV antigen, nucleic acid, and pharmaceutical composition provided by the present invention have good application prospects in the preparation of drugs for alleviating, preventing, and / or treating diseases caused by RSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 These are the in vitro flow cytometry results of RSV F antigens with different designs.

[0048] Figure 2 In vitro flow cytometric detection of RSV F antigen with P215S reversion mutation.

[0049] Figure 3 In vitro flow cytometric detection of RSV F antigen with different lengthening designs and sequence optimization.

[0050] Figure 4 In vitro flow cytometric analysis of RSV F antigen with different codon optimization strategies.

[0051] Figure 5 The effect of the intermolecular disulfide bond (C486 and C489, subsequently named: ΔF111) on the membrane surface expression and conformation of RSV F antigen.

[0052] Figure 6The effect of the intermolecular disulfide bond (C486 and C489, subsequently named: ΔF111) on the membrane surface expression and conformation of RSV F antigen.

[0053] Figure 7 In vitro flow cytometry detection (mRNA-Lipofectamine 2000) was performed after optimization of RNA molecule secondary structure.

[0054] Figure 8 In vitro flow cytometry detection after optimization of RNA molecule secondary structure (mRNA-RL151).

[0055] Figure 9 This is the total antibody titer in the serum of the 5μg group two weeks after the second immunization.

[0056] Figure 10 is the neutralizing antibody titer of BALB / c mouse immune serum.

[0057] Figure 11 To induce antigen-specific CD4 in spleen of BALB / c mice with different antigens + T cell immune response single factor level.

[0058] Figure 12 To induce antigen-specific CD4 in spleen of BALB / c mice with different antigens + Multifactor levels of T cell immune response.

[0059] Figure 13 To induce antigen-specific CD8 in spleen of BALB / c mice with different antigens + T cell immune response single factor level.

[0060] Figure 14 To induce antigen-specific CD8 in spleen of BALB / c mice with different antigens + Multifactor levels of T cell immune response.

[0061] Figure 15 Elispot analysis of antigen-specific IFN-γ and IL-2 in the spleen of BALB / c mice induced by different antigens.

[0062] Figure 16 In vitro flow cytometric detection of RSV F antigen with extended sequences of different glycosylation modifications.

[0063] Figure 17 The neutralizing antibody levels of RSV F antigen with extended sequences of different glycosylation modifications.

[0064] Figure 18 In vitro flow cytometric analysis of RSV F mutants with different 1×GCN4t insertion sites.

[0065] Figure 19In vitro flow cytometric analysis of RSV F mutants with different linker lengths.

[0066] Figure 20 The neutralizing antibody levels of RSV F antigen with different linker lengths.

[0067] Figure 21 In vitro flow cytometric detection of RSV B mutants.

[0068] Figure 22 is the neutralizing antibody level of RSV B candidate vaccine. DETAILED DESCRIPTION

[0069] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0070] Unless otherwise specified, the same sequence names with different upper / lower case letters in the present invention represent the same sequence, for example, "003m-P215S-1×GCN4t" and "003M-P215S-1×GCN4t" refer to the same sequence, and so on.

[0071] Example 1 Experimental method

[0072] 1. In vitro transcription and mRNA transfection

[0073] 1. PCR to obtain linearized target fragment

[0074] According to the requirements of the PCR reaction, add 8.7 μL of ddH2O, 0.5 μL of template DNA (source: GenScript synthesis), 0.4 μL of forward and 0.4 μL of reverse primers (source: GenScript synthesis), and 10 μL of PrimerSTARMAX polymerase (source: TAKARA, product number: R450A) into a 1.5 mL centrifuge tube and mix well.

[0075] As shown in Table 2 below:

[0076] Table 2 PCR system

[0077]

[0078]

[0079] The final PCR reaction conditions are shown in Table 3 below:

[0080] Table 3 PCR conditions

[0081]

[0082] After the PCR reaction is completed, gel electrophoresis is performed to verify that the nucleic acid bands are uniform. If the bands are uniform, the next IVT reaction can be carried out. If not, the reaction needs to be repeated.

[0083] 2. In vitro transcription (IVT)

[0084] A small amount of IVT testing was performed according to the following ratio in Table 4 (20 μL system):

[0085] Table 4 IVT system

[0086] <![CDATA[ddH2O]]> 11μL 10×HYB (Source: ApexBio, Product No.: K1083) 2μL Cap1 (B8176, 100 mM) (Source: ApexBio, Catalog No.: B8176) 0.4μL GTP (100 mM) (Source: Zhaowei, Product No.: R2331) 0.4μL ATP (100 mM) (Source: Zhaowei, Product No.: R1331) 0.4μL CTP (100 mM) (Source: Zhaowei, Product No.: R3331) 0.4μL Pseudo-U (100 mM) (Source: Zhaowei, Cat. No.: R5331) 0.4μL T7 mix (Source: ApexBio, Catalog No.: K1083) 2μL PCR products 3μL Total 20 μL

[0087] After the preparation is complete, the entire reaction system is placed in a 37°C water bath and incubated for 2 hours. After the reaction is complete, the DNA template is digested by adding 1 μL DNase I (source: Vazyme, product number: EN401-01) (35 μL for 1 mL IVT reaction) and digesting at 37°C for 15 minutes before IVT recovery. The recovery steps are as follows:

[0088] I. Add 80 μL of ddH2O to 20 μL of digestion product to make up to 100 μL.

[0089] II. Add 350 μL of Solution D and 250 μL of anhydrous ethanol (Source: Shanghai Test, Catalog No.: 801769722), mix thoroughly, transfer to a nucleic acid purification column (Source: Solarbio, Catalog No.: N1012), and centrifuge at 10,000 g for 1 min.

[0090] III. Add 500 μL of 70% ethanol and centrifuge at 10,000 g for 1 min. Repeat once and then centrifuge at 10,000 g for 2 min.

[0091] IV. Add 70 μL of sodium citrate (source: Sigma, catalog number: C8532) to the purification column. After standing at room temperature for 1 minute, centrifuge at 10,000 g for 2 minutes. Collect the flow-through, which is the target RNA.

[0092] Prepare 1% agarose gel according to the above ratio and perform RNA electrophoresis at 160V for 20min. After the electrophoresis, observe the gel using a gel imaging system (source: Tianneng, catalog number: Tanon 4600SF). If there is a single band, the concentration can be determined.

[0093] 3. Cell Transfection

[0094] After 1 μg of mRNA sample was incubated with 3 μL of lipofectamine 2000 or RL151 at room temperature for 20 min, the mixed sample was added to 1e6 Expi293F (Thermo Fisher, catalog number: A39241) cells and cultured at 37°C, 5% CO2 for 18-20 h. The cells were then collected.

[0095] 2. In vitro flow cytometry

[0096] 1. Wash the transfected cells three times with 1×PBS (Source: Sangon, Cat. No.: B548117-0500), then resuspend in 500 μL 1×PBS.

[0097] 2. Pipette 3 equal portions of 100uL from the single-cell suspension of each sample and add them into 1.5mL EP tubes (source: Shanghai Yueyi Biological, product number: YB-1.5s), and mark them as Unstained group, T1 group, and T2 group respectively.

[0098] 3. Sample preparation for the single staining group and the LIVE&DEAD group: Since the RSV F protein conformation is divided into postfusion and prefusion conformations, and the prefusion conformation is further divided into monomers and trimers, this experiment used three antibodies for detection, namely 4D7 (source: Bio-Bio, batch number: 20220808M011), D25 (source: Bio-Bio, batch number: 20220808M010), and AM14 (source: Bio-Bio, batch number: 20220808M009). At the same time, based on the knowledge in the literature, the above three antibodies were labeled with AF647 (source: thermo Fisher, product number: A20186) and AF488 fluorescent dyes (source: thermoFisher, product number: A20181), and named 4D7-AF647, D25-AF488, and AM14-AF488, respectively. Single-stained tubes were stained with 4D7-AF647 and D25-AF488, respectively. The LIVE & DEAD group was prepared by incubating in a 65°C metal bath (Source: Hangzhou Ruicheng Instrument Co., Ltd., Catalog No. DH300) for 30 minutes, then immediately placed on ice. SYTOX (Source: Thermo, Catalog No. S34861) was added at a final concentration of 1 / 5000 for signal reading upon sample loading.

[0099] 4. Sample preparation for the experimental groups: Add 50 μL of AM14-AF488 and 4D7-AF647 to the T1 group in step 2, and add 50 μL of D25-AF488 and 4D7-AF647 to the T2 group. Incubate at 4°C for 1 hour, centrifuge at 4°C, 500 × g for 4 minutes, discard the supernatant, wash three times with 150 μL of 1× PBS, and resuspend in 75 μL of 1× PBS.

[0100] 5. Before loading, add 75 μL of SYTOX (final concentration 1 / 5000) to the LIVE&DEAD, T1, and T2 groups. Incubate at room temperature for 5 minutes and perform flow cytometric analysis on a flow cytometer (Source: Cytek, Catalog Number: NL-CLC V16 B14 R8).

[0101] 3. Total Antibody Level Detection

[0102] 1. Antigen Coating: Dilute the target prefusion conformation antigen (Source: Shanghai Bio-Tech Biotechnology Co., Ltd., Lot No. M-202309120335) and postfusion conformation antigen (Source: Shanghai Bio-Tech Biotechnology Co., Ltd., Lot No. M-202309160679) to 400 ng / mL and 300 ng / mL, respectively. Pipette 100 μL of the solution into a 96-well ELISA-coated plate (Source: Nest, Catalog No. 514201) and incubate overnight at 4°C in the dark. Block the plate with 1× PBST containing 3% bovine serum albumin for 2 hours at room temperature. Dilute the immune mouse serum 200-fold as the initial concentration, then perform a 4-fold serial dilution in PBS buffer for a total of 10 dilutions. Wash the plate with 1× PBST and incubate the serially diluted serum for 2 hours at room temperature. To determine RSV F-specific antibody responses, the plate was incubated with rabbit anti-mouse IgG HRP (source: abcam, catalog number: ab6728) at 25°C for 1 hour, followed by color development using the substrate tetramethylbenzidine (TMB) solution (source: Invitrogen, catalog number: 00-4201-56). After approximately 12 minutes, the color development reaction was terminated with 1M sulfuric acid, and the absorbance was measured at a wavelength of 450 nm using a microplate reader (model: Synergy H1, purchased from BioTek).

[0103] 4. Neutralizing Antibody Level Detection

[0104] 1. Guangzhou HUAWEI Testing Co., Ltd.

[0105] I. One day before the experiment, Hep-2 cells were seeded into a 96-well cell culture plate and allowed to grow to a confluent monolayer.

[0106] II. Take the inactivated mouse serum and make a 2-fold serial dilution in DMEM in a 96-well plate, making different dilutions according to 1:20, 1:40...1:5120;

[0107] III. Remove the frozen RSV virus solution from the -80°C freezer and dilute the RSV virus to the target concentration in serum-free DMEM. Then, add 50 μL of the virus solution to each well of the diluted serum and incubate at room temperature for 2 hours.

[0108] IV. Dilute the virus diluent used in the experiment at 1, 10, 100, and 1000 times, add 100 μL / well to a 96-well plate, and perform a virus regression experiment in 12 replicates for each dilution.

[0109] V. Remove cells from the incubator, digest and pellet, count, and adjust the cell density to 1×10 cells / mL using DMEM containing 4% FBS and 1% double-antibody. 5 cells / mL;

[0110] VI. After the neutralization and incubation, add 100 μL / well of the prepared cell suspension to a 96-well plate and incubate in a 37°C, 5% CO2 incubator for 5 days. During this time, CPE was continuously observed and recorded.

[0111] Judgment criteria: 1) Cell control (CC) should be free of lesions, and virus control (VC) should be lesions;

[0112] 2) The neutralization titer of the negative serum control (NC) should be <1:40;

[0113] 3) The neutralization titer of the positive serum control (PC) should be within the compliance range;

[0114] 4) In the viral regression test, the viral titer of 1g TCID50 / mL should be between 630 and 6310 TCID50 / mL;

[0115] If any of the above items are not met, the experiment will be invalid.

[0116] 2. Fluorescence method

[0117] II. One day before the experiment, A549 cells were seeded into a 96-well cell culture plate and kept aside until the cells grew confluent to a monolayer;

[0118] II. Inactivated mouse serum was diluted 4-fold in DMEM in a 96-well plate, with dilutions ranging from 1:25, 1:100, to 1:1638400.

[0119] III. Remove the frozen RSV-GFP virus solution from the -80°C freezer and dilute the RSV virus to the target concentration in serum-free DMEM. Then, add an equal amount of virus solution to each well of the diluted serum and incubate at room temperature for 1 hour.

[0120] IV. After the neutralization and incubation are completed, culture in a 3+1, 37°C, 5% CO2 incubator for 2 days

[0121] Vi detection: Green fluorescence signals were detected by a fully automated live cell imaging instrument.

[0122] Data analysis: Cells expressing GFP are RSV-GFP virus-infected cells. The NT50 of the serum to be tested is calculated by nonlinear fitting based on the percentage of positive cell area in each well and the serum dilution factor.

[0123] 5. Antigen-specific T cell response detection

[0124] Antigen-specific T cell responses in mouse spleens were measured by intracellular cytokine staining (ICS). Briefly, RSV F peptide pools or medium containing an equal amount of DMSO were added to 96-well plates as negative controls. Mouse spleen cells were resuspended in RIPM1640 complete medium and incubated at 37°C for 1 hour before adding protein transport inhibitors and incubating again for 5 hours. The cells were washed once with PBS and stained with Fixable Viability Stain 510 (Cat. No. 564406, purchased from BD Bioscience). After incubation for 10 minutes, the cells were washed and anti-mouse CD16 / CD32 (Cat. No. 553142, purchased from BD Bioscience) was added and incubated at 4°C for 10 minutes. A mixture of anti-mouse CD3-FITC, CD4-APC, and CD8-Percp-cy5.5 antibodies (Cat. Nos. 553061, 553051, and 551162, purchased from BD Bioscience) was added for staining against surface molecules. After a 30-minute incubation, cells were washed twice, fixed and permeabilized for 20 minutes, and then washed once. Cells were then stained with a cocktail of anti-cytokine antibodies against mouse IFN-γ-Pe-Cy7, IL-2-BV605, TNF-α-BV650, IL-4-BV711, and IL-5-PE (catalog numbers 557649, 563943, 563911, 564005, and 562049, respectively, purchased from BD Biosciences). After a 30-minute incubation, cells were washed twice and resuspended in 200 μL of PBS. Fluorescence signals were analyzed using a CYTEK Aurora / NL flow cytometer (model NL-CLC V16B14R8, purchased from Cytek Biosciences).

[0125] 6. Antigen-specific T cell response detection (Elispot)

[0126] Day 1

[0127] 1. Activation of Pre-coated Plates

[0128] Dayu: Add 200 μL of Dayu Elispot-specific serum-free medium to each well, let it stand at room temperature for 5-10 minutes, and then remove it.

[0129] Mabtech: Wash the plate four times with sterile 1× PBS, add 200 μL / well of serum-free medium, and incubate at room temperature for at least 30 min.

[0130] 2. After adding cells to the wells, add stimulants, cover the plate, and incubate in a 37°C, 5% CO2 incubator for 23 hours. Avoid shaking during incubation.

[0131] Day 2:

[0132] Post-culture operation (sterile operation is no longer required)

[0133] 1. Cell Lysis: Pour out the culture medium from the wells. Add 200 μL / well of pre-chilled deionized water and place in a 4°C refrigerator for 10 minutes to induce hypotonic cell lysis.

[0134] 2. Prepare 1× washing buffer: Dilute with deionized water (1:50) to make 1× washing buffer. Store in a 4°C refrigerator after use (make as much as you need).

[0135] 3. Wash the plate: Pour the liquid from the wells and add 1× washing buffer (250 μL / well). Wash six times, each time for 1 minute. Dry the plate on absorbent paper after each wash.

[0136] 4. Detection Antibody Incubation: Add the diluted biotinylated antibody working solution to each well at 100 μL / well. Incubate at 37°C for 1 hour.

[0137] Dilution Buffer R (10×): Dilute with 1× PBS (1:9) to prepare 1× Dilution Buffer R working solution for later use.

[0138] Biotinylated antibody: Dilute with Dilution buffer R (1×) (1:100) to prepare the working solution.

[0139] 5. Wash the plate: Pour the liquid from the wells and add 1× washing buffer (250 μL / well). Wash six times, each time for 1 minute. Dry the plate on absorbent paper after each wash.

[0140] 6. Enzyme-linked avidin incubation: Add the diluted enzyme-linked avidin working solution to each experimental well, 100 μL / well, and incubate at 37°C for 1 hour.

[0141] Streptavidin-HRP: Dilute with Dilution buffer R (1×) (1:100) to prepare the working solution.

[0142] 7. Wash the plate: Pour the liquid from the wells and add 1× washing buffer (250 μL / well). Wash six times, each time for 1 minute. Dry the plate on absorbent paper after each wash.

[0143] 8. Color development: Add freshly prepared ACE color development solution to each well (100 μL / well). Incubate at room temperature, away from light, for 5-30 minutes. Terminate color development based on spot formation. If the room temperature is below 20°C, it is recommended to incubate at 37°C, checking every 5-10 minutes.

[0144] ACE Colorimetric Solution: In a clean container, mix ACE solution dilution, ACE solution I (20×), ACE solution II (20×), and ACE solution III (200×) in a ratio of 180:10:10:1 to prepare the working solution. Refer to Table 5 below. The half-life of ACE colorimetric solution at room temperature is 30 minutes. Prepare and use immediately.

[0145] Table 5 ACE system

[0146]

[0147] 9. Stop color development: Pour out the liquid in the wells and wash the front and back of each well and the base three times with deionized water to stop color development. Place the plate in a cool place at room temperature and allow it to dry naturally before closing the base.

[0148] 10. Count the spots on the ELISPOT plate and record various spot parameters for statistical analysis. If reading cannot be done immediately, store the plate in a sealed container away from light and read the plate within one week.

[0149] 7. Preparation of FI-RSV Inactivated Vaccine

[0150] 1. Cell and Reagent Preparation:

[0151] I. Hep-2 cells: Prepare 10 T75 culture flasks and allow them to grow completely. Spread 30 10 cm culture dishes, with 1.2 × 10 cells per 10 cm dish. 7 When the density is >90%, proceed to the next step.

[0152] II.D10: 1×DMEM+10% FBS+1% PS

[0153] III.SF-DMEM: 1×DMEM + 1% PS

[0154] IV. 10% Formalin

[0155] V. 40 mg / mL Al(OH)3 adjuvant

[0156] 2. Experimental materials and equipment:

[0157] I. Constant temperature shaker

[0158] II. Hitachi Ultracentrifuge CP100NX

[0159] 3. Experimental steps:

[0160] I. When the Hep-2 cell density is >90%, wash the cells once with SF-DMEM.

[0161] II. Dilute RSV-A2 (Genbank: KT992094; diluted with SF-DMEM), inoculum MOI = 0.1

[0162] III. Add 3 mL of virus dilution to each 10 cm dish and incubate at 37°C, 5% CO2 for 1 hour.

[0163] IV. Discard the viral supernatant and wash the cells once with SF-DMEM.

[0164] V. Add 10 mL of SF-DMEM to each 10 cm dish.

[0165] VI. Incubate at 37°C, 5% CO2 for 3 days, cytopathic effect ~50% (Note: Inoculation started at 15:30 on August 10, 2024 - Amplification ended at 16:30 on August 13, 2024)

[0166] VII. Collect the virus solution: Collect the cell culture supernatant into a 50 mL centrifuge tube and centrifuge at 1000 × g, 4°C, for 10 min. Collect the supernatant.

[0167] 120uL of virus solution was retained for virus titration.

[0168] VIII. Add 10% neutral Formalin to the shake flask at a ratio of 1:400, mix well, seal and place on a shaker at 37°C, 50 rpm,

[0169] Inactivated for 3 days.

[0170] IX. After inactivation, collect the virus by ultracentrifugation at room temperature and resuspend in 10 mL of PBS.

[0171] X. Add Al(OH)3 adjuvant at a final concentration of 4 mg / mL and adsorb overnight at room temperature.

[0172] XI. Centrifuge at 1000×g for 10 minutes and resuspend the pellet in 3 mL of PBS (+1% PS).

[0173] Example 2 1×GCN4t, STM and 1×GCN4t-STM Transformation of Candidate Antigens

[0174] There are three types of flow cytometry detection antibodies, namely 4D7 (labeled with Alexa Flour 647, i.e. AF647, to detect the postfusion conformation), AM14 (labeled with Alexa Flour 488, i.e. AF488, to detect the trimer prefusion conformation), and D25 (labeled with Alexa Flour 488, i.e. AF488, to detect the prefusion conformation).

[0175] The wild-type RSV sequence (SEQ ID NO: 1) is shown below:

[0176] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLGVGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQ LLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSS VITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0177] Compared with the wild-type RSV sequence, the mutation sites involved in 003m are shown in Table 6.

[0178] Table 6 003m mutation site and linker sequence

[0179]

[0180] 007m-Cys, 008m-Cys, 009m-Cys and 010m-Cys are shortened linkers in the sequence based on 003m, as shown in Table 7.

[0181] Table 7 Mutation sites and linker sequences

[0182]

[0183] 1×GCN4t is a lengthened modification by inserting a specific sequence. 003m-1×GCN4t, 007m-Cys-1×GCN4t, 008m-Cys-1×GCN4t, 009m-Cys-1×GCN4t, and 010m-Cys-1×GCN4t have EDKIEEILSK IYHIENEIARIKKLIGEA (SEQ ID NO: 6) inserted between V516 and N517.

[0184] 003m-STM, 007m-Cys-STM, 008m-Cys-STM, 009m-Cys-STM, and 010m-Cys-STM were transformed into lengthened types by replacing all I525-N574 at the C-terminus with WPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCK (SEQ ID NO: 7).

[0185] 003m-1×GCN4t-STM, 007m-Cys-1×GCN4t-STM, 008m-Cys-1×GCN4t-STM, 009m-Cys-1×G CN4t-STM, and 010m-Cys-1×GCN4t-STM were transformed into lengthened types by inserting EDKIEEILS KIYHIENEIARIKKLIGEA (SEQ ID NO: 6) between V516 and N517, and replacing I525-N574 with WPWYIWLGFIAGLIAIV MVTIMLCCMTSCCSCLKGCCSCGSCCK (SEQ ID NO: 7).

[0186] result:

[0187] Figure 1 : The 1×GCN4t and 1×GCN4t-STM mutation designs will lead to a significant decrease in the membrane surface expression of RSV F antigen, but the STM modification will not have a significant change in the protein expression level, and both can well maintain the trimeric prefusion conformation of the protein; the difference in linker length has no significant effect on the membrane surface expression level, and the RSV F antigen can maintain a good trimeric conformation.

[0188] Example 3 Effect of P215S reversion mutation on RSV F antigen membrane surface conformation and expression

[0189] Figure 2: For 003m, a P215S reversion mutation was made, and further 1×GCN4t and 2×GCN4t extension mutations were made. Flow cytometry results showed that the P215S reversion mutation, 1×GCN4t extension, and 2×GCN4t extension significantly reduced RSV F antigen surface expression, with the extension having a more significant effect (Table 8). However, none of these mutations altered the prefusion trimer conformation.

[0190] Table 8 Mean fluorescence intensity of different mutant designs of P215S by flow cytometry in vitro

[0191]

[0192] Example 4 Effects of different lengthening designs and sequence optimization on antigen expression and conformation

[0193] The S and L modifications are lengthened modifications that add the L513I mutation and insert specific sequences. 003M-P215S-S further inserts NEKINQISASIRKIDESISQI (SEQ ID NO: 8) between V516 and N517. 003M-P215S-L inserts NEKINQISASIRKIDESINEKINQISASIRKIDESISQI (SEQ ID NO: 9) between V516 and N517. 003M-P215S-S-3N and 003M-P215S-L-3N, based on 003M-P215S-S and 003M-P215S-L, modify some of the amino acid residues in the inserted sequences with N-linked glycosylation mutations.

[0194] Figure 3 Comparison of 003M-P215S-S with 003M-P215S-S-3N, and 003M-P215S-L with 003M-P215S-L-3N, respectively, showed that the increased glycosylation site modification significantly increased the membrane surface expression of RSV F antigen, which was much higher than the membrane surface expression of 1×GCN4t and 2×GCN4t (Table 9), and the trimer prefusion conformation of RSV F antigen did not change significantly.

[0195] Table 9 In vitro flow cytometry mean fluorescence intensity with different lengthening designs

[0196]

[0197] Example 5 Codon Optimization Strategy Screening

[0198] Figure 4: According to the results of in vitro flow cytometry and mean fluorescence intensity (Table 10), the expression of membrane surface antigens was significantly increased when the codon-optimized 003m-P215S-1×GCN4t was used for cell transfection.

[0199] Table 10 Mean fluorescence intensity of different codon optimized 003m-P215S-1×GCN4t in vitro flow cytometry

[0200]

[0201]

[0202] Example 6 Intermolecular disulfide bond screening

[0203] Figure 5 When transfected in vitro using Lipofectamine 2000, reverse mutation of the intermolecular disulfide bond between C486 and C489 (hereinafter referred to as ΔF111) significantly increased the membrane surface expression of RSV F antigen (Table 11), while also leading to the production of some postfusion conformational antigens. Furthermore, all extended designs showed a significant decrease in membrane surface expression.

[0204] Table 11 In vitro flow cytometry mean fluorescence intensity (Lipofectamine) of different mutants with intermolecular disulfide bond design

[0205]

[0206] Figure 6 In vitro transfection using mRNA-LNP (RL151, Catalog No.: W211-YB211202, purchased from Zhejiang Shenzhou Pharmaceutical; for the mRNA-LNP preparation method, see Example 8, except that LQ104-E16b-2 was replaced with RL151) resulted in a significant decrease in membrane surface protein expression compared to transfection using Lipofectamine 2000 (Table 12). However, all of the above antigen designs were able to maintain the trimer prefusion conformation well. After the ΔF111 backmutation, the membrane surface expression of the RSV F antigens of all designs, except 003m-P215S-ΔF111-1×GCN4t, was still significantly improved compared to the unmutated design.

[0207] Table 12 In vitro flow cytometry mean fluorescence intensity (LNP) of different mutants with intermolecular disulfide bond design

[0208]

[0209] Example 7 RNA secondary structure optimization

[0210] Figure 7 When using Lipofectamine 2000-mRNA, the effect of RNA secondary structure optimization on membrane surface expression was limited (Table 13), and some RSV F antigens still presented a postfusion conformation.

[0211] Table 13 In vitro flow cytometry mean fluorescence intensity (Lipofectamine) of different RNA secondary structure optimizations

[0212]

[0213] Figure 8 When mRNA-LNP (RL151) was used for in vitro transfection, optimization of RNA secondary structure had a significant effect on membrane surface expression, with an approximately 8-fold difference between the high-expression group and the low-expression group (Table 14). The antigen protein was able to maintain a good trimer prefusion conformation, but the membrane surface expression level was significantly lower than that when transfected using Lipofectamine 2000.

[0214] Table 14 In vitro flow cytometry mean fluorescence intensity (LNP) of different RNA secondary structure optimization

[0215]

[0216] Example 8 Preparation of lipid nanoparticles from BALB / c mouse immune samples

[0217] The target sequence mRNAs screened in the above examples (mDS-Cav1, 003m-1×GCN4t, 003m-1×GCN4t-STM, 003m-STM, 003m-P215S, 003m-P215S-ΔF111, 003m-P215S-S-3N, 003m-P215S-ΔF111-S-3N, 003m-P215S-1×GCN4t, 003m-P215S-ΔF111-1×GCN4t -opti3) were ionized (cationic) at low pH and coated into nanoparticles with two helper lipids: DSPC (distearylphosphatidylcholine, Catalog No. B90536, manufactured by Nippon Seika Co., Ltd.) and cholesterol (Catalog No. C00373, manufactured by Nippon Seika Co., Ltd.), along with LQ104-E16b-2 (referenced from CN117534584A) and a pegylated lipid (DMG-PEG2000, Catalog No. M-DMG-2000, purchased from JenKem). An aqueous solution of mRNA was prepared by mixing mRNA dissolved in ultrapure water with 100 mM (millimoles per liter, or mmol / L) citrate buffer at pH 4.0 in a 1:1 volume ratio. The four lipid components are adjusted in molar ratios (e.g., LQ104-E16b-2: cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5) and dissolved in 99.5% ethanol to form a lipid solution. The mRNA and lipid solution are mixed in a NanoAssemblr (Precision Nanosystems) microfluidic mixing system at a volumetric mixing ratio of HO:EtOH = 3:1 and a constant total flow rate of 12 mL / min. Lipid nanoparticles are prepared with a nitrogen-phosphorus ratio of ionizable lipid to mRNA of 3-15:1. This yields mRNA-containing lipid nanoparticles (mRNA-LNPs). The lipid nanoparticles are then dialyzed, concentrated, filtered, and stored to yield a vaccine containing the target antigen.

[0218] Example 9 Immunization of BALB / c mice

[0219] 1. Animal Vaccination and Serum Collection

[0220] BALB / c mice: For mouse vaccination, 5- to 7-week-old female BALB / c mice (purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized with the target antigen vaccine prepared in Example 8 (hereinafter referred to as "immunization"). A second dose was administered on day 21 after vaccination to boost the immune response. Each group consisted of 8 or 14 BALB / c mice (n = 8 / 14). The immunization regimen is shown in Table 15 below.

[0221] Table 15 BALB / c mouse immunization schedule

[0222]

[0223] Administer the test solution twice, three weeks apart. The dose for each dose is shown in the table above. Use a sterile insulin syringe of appropriate size to accurately draw out 400 μL of the test solution (slowly invert the syringe 5-10 times to mix thoroughly before drawing out the test solution). Inject 100 μL into a single intramuscular injection into the right lower limb of the animal.

[0224] The serum of the immunized mice was collected and inactivated at 56° C. for 0.5 h to detect RSV F protein-specific IgG and neutralizing antibodies.

[0225] 2. Antibody Level Testing

[0226] 1. The total antibody level detection method used the "3. Total antibody level detection" described in Example 1, and the neutralizing antibody detection method used the "4. Neutralizing antibody level detection" described in Example 1.

[0227] Figure 9 : From the perspective of total antibody levels in serum, all antigen designs containing 1×GCN4t have low levels of antibodies against the postfusion conformation in serum. Among them, 003m-1×GCN4t and 003m-P215S-1×GCN4t have the highest ratio of antibodies against the prefusion conformation to antibodies against the postfusion conformation. Although the antigen design with the ΔF111 reversion mutation has a high expression level in vitro, the total antibody level produced in vivo is not high. At the same time, the antigen containing the ΔF111 reversion mutation has some postfusion conformation proteins in vitro, and the total antibody level in vivo also shows that it produces more antibodies against the postfusion conformation.

[0228] 2. Neutralizing Antibody Titer

[0229] Figure 10 : According to the neutralizing antibody test results, in the high-dose group, the neutralizing antibody titer produced by mice immunized with 003m-P215S-1×GCN4t was the highest, followed by 003m-P215S, 003m-P215S-S-3N, and 003m-P215S-ΔF111-S-3N.

[0230] 3. Cellular Immune Response in BALB / c Mice

[0231] Figure 11 and Figure 12 :From CD4 single factor and multi-factor levels and total antibody levels ( Figure 10), the 003m-P215S-1×GCN4t high-dose group induced the highest level of cellular immunity, and this result also had a similar trend to the ratio of antibodies against prefusion conformation and postfusion conformation in the total antibody level.

[0232] Figure 13 and Figure 14 :From the CD8 single factor and multi-factor levels and total antibody levels ( Figure 10 ), 003m-P215S induced the highest level of cellular immunity, but there was no significant difference among the doses.

[0233] IV. Cellular Immune Response in BALB / c Mice (Elispot)

[0234] Figure 15 : In the Elispot test results, the high-dose group of 003m-P215S-1×GCN4t was slightly better than 003m-P215S, and this result was basically consistent with the CD4 test results.

[0235] Example 10 Screening of extended sequences with different glycosylation modifications

[0236] According to literature reports, in addition to GCN4t's trimer-forming properties, the T4 foldon (fibritin) can also promote trimer formation. Therefore, an extended version of the fibritin was also designed. The extended 1×fibritin was modified by inserting SAIGGYIPEAPRDGQAYVRKDGEWVLLSTFLGGLVPR (SEQ ID NO: 140) between the amino acid sequences L513 and H514 of SEQ ID NO: 1. Furthermore, since both GCN4t and T4 have certain immunogenic properties, both GCN4t and fibritin were glycosylated to a certain extent to silence their immunogenicity. Glycosylation modifications were made to three sites on 1×GCN4t: 1N, 2N, and 3N, in order. 1,2N refers to glycosylation modifications at positions 1 and 2, and subsequent 1,3N, etc., refer to this description. Glycosylation modification was performed on 1×Fibritin. The modified 1×Fibritin-1N was characterized by inserting SAIGGYIPEAPNDTQAYVRKDGEWVLLSTFLGGLVPR (SEQ ID NO: 159) between the amino acid sequence L513 and H514 shown in SEQ ID NO: 1.

[0237] 1. In vitro flow cytometry

[0238] Figure 16In vitro flow cytometry showed that elongation of 1×Fibritin and glycosylation modifications of 1×Fibritin and 1×GCN4t significantly increased the expression of RSV F protein in monomeric or trimer prefusion conformations on the membrane surface (Table 16). Compared with other glycosylation combinations of 1×GCN4t, diglycosylation (003m-P215S-1×GCN4t-1,3N) and triglycosylation (003m-P215S-1×GCN4t-1,2,3N) significantly increased membrane surface protein expression. Therefore, 003m-P215S-1×Fibritin, 003m-P215S-1×Fibritin-1N, 003m-P215S-1×GCN4t-1,3N, 003m-P215S-1×GCN4t-1,2,3N, 003m-P215S-1×GCN4t and 003m-P215S were selected for animal immunization.

[0239] Table 16 In vitro flow cytometry mean fluorescence intensity of the extended sequences with different glycosylation modifications

[0240]

[0241]

[0242] 2. Animal Vaccination and Serum Collection

[0243] BALB / c mice: For mouse vaccination, lipid LQ104-E16b-2 was prepared according to CN117534584A.

[0244]

[0245] Five- to seven-week-old female BALB / c mice (purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized (hereinafter referred to as "immunization") with the target antigen-containing vaccine prepared by coating with the target antigen as described in Example 8. A second dose was administered 21 days after vaccination to enhance the immune response. Each group consisted of 6 or 12 BALB / c mice (n = 6 / 12). The immunization regimen is shown in Table 17 below.

[0246] Table 17 BALB / c mouse immunization schedule

[0247]

[0248] Administer the test solution twice, three weeks apart. The dose for each dose is shown in the table above. Use a sterile insulin syringe of appropriate size to accurately draw out 400 μL of the test solution (slowly invert the syringe 5-10 times to mix thoroughly before drawing out the test solution). Inject 100 μL into a single intramuscular injection into the right lower limb of the animal.

[0249] The serum of the immunized mice was collected, inactivated at 56°C for 0.5 h, and stored in a -80°C refrigerator for subsequent detection.

[0250] 3. Antibody Level Testing

[0251] The neutralizing antibody detection method used the “fluorescence method” described in “IV. Neutralizing Antibody Level Detection” in Example 1.

[0252] Figure 17 Neutralizing antibody results: ① Glycosylation in both 1×Fibritin and 1×GCN4t decreased neutralizing antibody titers. ② Compared to 1×GCN4t, the extended 1×Fibritin form induced higher neutralizing antibody titers. ③ In the extended 1×GCN4t form, the higher the degree of glycosylation, the lower the decrease in neutralizing antibody titers.

[0253] Example 11 Effect of 1×GCN4t Insertion Site and Linker Length on Antigen Immunogenicity

[0254] To investigate the universality of the insertion position of 1×GCN4t (SEQ ID NO: 6), extended versions of 1×GCN4t with different insertion sites were designed: insertions were made at intervals of three amino acids based on the insertion position of 003m-P215S-1×GCN4t. The results are shown in Table 18. To investigate the effect of linker length on RSV F mutants, RSV F mutants with different linker lengths were also designed. The results are shown in Table 19.

[0255] 1. In vitro flow cytometry

[0256] Figure 18 and Figure 19 In vitro flow cytometry results showed that different insertion positions of 1×GCN4t and different linker lengths had no significant effect on the monomeric prefusion conformation of RSV F protein on the membrane surface (Tables 18 and 19).

[0257] Table 18 Average fluorescence intensity of RSV F mutants at different 1×GCN4t insertion sites detected by flow cytometry in vitro

[0258]

[0259] Table 19 Average fluorescence intensity of RSV F mutants with different linker lengths detected by flow cytometry in vitro and in vivo

[0260]

[0261] 2. Animal Vaccination and Serum Collection

[0262] BALB / c mice: For mouse vaccination, refer to "II. Animal Vaccination and Serum Collection" in Example 10 to prepare a vaccine containing the target antigen as shown in Table 20. Five- to seven-week-old female BALB / c mice (purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized with the vaccine containing the target antigen (hereinafter referred to as "immunization"). A second dose was administered on day 21 after vaccination to enhance the immune response. Each group consisted of six BALB / c mice (n=6). The immunization method is shown in Table 20 below.

[0263] Table 20 1×GCN4t insertion site and linker length Animal immunization schedule

[0264]

[0265]

[0266] Administer the test solution twice, three weeks apart. The dose for each dose is shown in the table above. Use a sterile insulin syringe of appropriate size to accurately draw out 400 μL of the test solution (slowly invert the syringe 5-10 times to mix thoroughly before drawing out the test solution). Inject 100 μL into a single intramuscular injection into the right lower limb of the animal.

[0267] The serum of the immunized mice was collected, inactivated at 56°C for 0.5 h, and stored in a -80°C refrigerator for subsequent detection.

[0268] 3. Antibody Level Testing

[0269] The neutralizing antibody detection method used the “fluorescence method” described in “IV. Neutralizing Antibody Level Detection” in Example 1.

[0270] Figure 20 Neutralizing antibody results: ① The insertion position of 1×GCN4t has a certain impact on neutralizing antibody titers. The later the insertion position, the lower the neutralizing antibody titer, but all are higher than 003m-1×GCN4t and DS-Cav1. ② RSV F mutants with different linker lengths have a certain impact on neutralizing antibody titers, but there is no significant difference with 003m-P215S-1×GCN4t. In summary, different insertion sites and linker lengths of 1×GCN4t can all induce high neutralizing antibody titers, that is, the selection of 1×GCN4t insertion position and linker length is universal.

[0271] Example 12: Modification of candidate B-type antigens

[0272] There are three types of flow cytometry detection antibodies, namely 4D7 (labeled with Alexa Flour 647, i.e. AF647, to detect the postfusion conformation), AM14 (labeled with Alexa Flour 488, i.e. AF488, to detect the trimer prefusion conformation), and D25 (labeled with Alexa Flour 488, i.e. AF488, to detect the prefusion conformation).

[0273] The amino acid sequence of wild-type RSV B (SEQ ID NO: 80) is shown below:

[0274] MELLIHRSSAIFLTLAINALYLTSSQNITEEFYQSTCSAVSRGYLSALRTGWYTSVITIELSNIKETKCNGTDTKVKLIKQELDKYKNAVTELQLLMQNTPAVNNRARREAPQYMNYTINTTKNLNVSISKKRKRRFLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKNALQLTNKAVVSLSNGVSVLTSKVLDLKNYINNQLLPIVNQQSCRISNIETVIEFQQKNSRLLEITREFSVNAGVTTPLSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMSIIKEEVLAYVVQLPIYGVIDTPCWKLHTSPLCTTNIKEGSNICLTRTDRGWYCDNAGSVSFFPQADTCKVQSNRVFCDTMNSLTLPSEVSLCNTDIFNSKYDCKIMTSKTDISSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKNLYVKGEPIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIMITAIIIVIIVVLLSLIAIGLLLYCKAKNTPVTLSKDQLSGINNIAFSK

[0275] Modification based on RSV B:

[0276] 003m is compared with the amino acid sequence of wild-type RSV B (SEQ ID NO: 80), and the mutation sites and linker sequences involved are shown in Table 6 in Example 2.

[0277] 1×GCN4t is a lengthened modification in which EDKIEEILSKIYHIENEIARIKKLIGEA (SEQ ID NO: 6) is inserted between V516 and N517. At the same time, G46S, D92E, F190S, C155S / C290S, C486D / C489D, C102A / C362S back mutations, linker replacement, and dGCN4t modification were also performed: RSV B-003m-P215S-1×GCN4t-G46S, RSVB-003m-P215S-1×GCN4t-D92E, RSV B-003m-P215S-1×GCN4t-F190S, RSV B-003m-P215S-1×GCN4t-C155S-C290S, RSV B-003m-P215S-1×GCN4t-C486D-C489D, RSV B-003m-P215S-1×GCN4t-C102A-C362S, RSV B-003m-P215S-1×GCN4t-ori_linker and RSV B-003m-P215S, and each mutant sequence optimized three different nucleic acid sequences (i.e., RQ1-RQ3), and the mutant amino acid sequences are shown in SEQ ID NO:82-89.

[0278] The above modifications can be combined. For example, a P215S reversion mutation was performed on RSV B-003m, and on this basis, a 1×GCN4t elongation mutation was further performed. The amino acid sequence of RSV B-003m-P215S-1×GCN4t is shown in SEQ ID NO: 81.

[0279] Figure 21 : In vitro flow cytometry results showed that single-point mutations or mutations of disulfide bond pairs would result in the presence of some postfusion conformation F protein on the membrane surface. At the same time, without the use of a linker, the expression of the membrane surface protein would be seriously affected. After removing 1×GCN4t, although the expression of the membrane surface protein was increased, it also led to an increase in the content of the postfusion conformation F protein. At the same time, RSV B-003m-P215S-1×GCN4t-RQ1 showed a certain degree of improvement in expression compared to the other two sequence optimizations. Therefore, RSV B-003m-P215S-1×GCN4t can still maintain the monomer and trimer prefusion conformations well in RSV B type. Among the three sequence optimizations, RQ1 is the best. (Table 21)

[0280] Table 21 Average fluorescence intensity of RSV F mutants detected by flow cytometry in vitro

[0281]

[0282]

[0283] Example 13 Immunization and Neutralization of BALB / c Mice with Candidate Type B Antigens

[0284] 1. Animal Vaccination and Serum Collection

[0285] BALB / c mice: For mouse vaccination, target antigen vaccines were prepared by coating target sequence mRNA (RSV B-003m-P215S-1×GCN4t-RQ1 and RSV B-003m-P215S-RQ1) with lipid compound 6.

[0286] Preparation of lipid compound 6:

[0287] Step 1: Preparation of 6-1

[0288] Reaction formula:

[0289]

[0290] The material ratio is shown in Table 22:

[0291] Table 22 Material ratios for preparation 6-1

[0292] Material Name Molecular weight Feed ratio Feeding amount mmol 1,2-Epoxydodecane 184 1eq 5g 27 6-Bromohexanoic acid 209 1.1eq 5.85g 30 Ferric chloride 162 0.025 eq 220mg 1.35 Pyridine 79 0.0125eq 53mg 0.68

[0293] Operation process:

[0294] To a 250 mL reaction flask, add 6-bromohexanoic acid, 1,2-epoxydodecane, ferric chloride, and pyridine (see Table 22). Stir and react at room temperature for 16 hours. TLC (PE:EA = 4:1) indicated completion of the reaction (product rf value 0.5). Purification by column chromatography yielded 6.8 g of a colorless oil.

[0295] Step 2: Preparation of 6-2

[0296] Reaction formula:

[0297]

[0298] The material ratio is shown in Table 23:

[0299] Table 23 Material ratios for preparation 6-2

[0300] Material Name Molecular weight Feed ratio Feeding amount mmol 6-1 379.38 1eq 6.6g 17.5 bitter 144 1.1 eq 2.8g 19.2 EDCI 192 2 eq 6.7g 35 DMAP 122 0.2 eq 420mg 3.5 dichloromethane - - 80mL -

[0301] Operation process:

[0302] To the reaction flask, 6-1, octanoic acid, EDCI, DMAP, and dichloromethane were added and stirred at room temperature for 16 hours. TLC (PE:EA = 20:1) indicated completion of the reaction (product rf value 0.6). The reaction mixture was washed twice with 100 mL of water. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to yield 6.5 g of a colorless oil.

[0303] Step 3: Preparation of compound 6

[0304] Reaction formula:

[0305]

[0306] The material ratio is shown in Table 24:

[0307] Table 24 Material ratios for preparing compound 6

[0308] Material Name Molecular weight Feed ratio Feeding amount mmol 6-2 505.58 2.2eq 4.3g 8.55 Ethanolamine 61 1 eq 230mg 3.9 <![CDATA[K2CO3]]> 138 2 eq 1.1g 7.8 KI 166 2 eq 1.3g 7.8 Acetonitrile - - 40mL -

[0309] Operation process:

[0310] 6-2, ethanolamine, KCO, KI, and acetonitrile were added to the reaction flask and heated to 65°C with stirring for 16 hours. TLC (DCM:MeOH = 10:1) indicated completion of the reaction (product rf value 0.5). The reaction mixture was filtered, concentrated, and purified by column chromatography to yield 2.8 g of a colorless oil.

[0311] 1 H NMR(600MHz,Chloroform-d)δ5.10–5.05(m,2H),4.22(dd,J=11.8,3.3Hz,2H),4.01(dd,J=11.8,6.8Hz,2H),3.61(d,J= 5.1Hz,2H),2.73–2.49(m,6H),2.30(td,J=7.5,5.1Hz,8H),1.65–1.50(m,16H),1.33–1.23(m,52H),0.91–0.85(m,12H).

[0312] MS (ES+) m / z): 910.7 (M+H) + .

[0313] A control FI-RSV inactivated vaccine was prepared with reference to "VII. Preparation of FI-RSV inactivated vaccine" in Example 1. A vaccine containing the target antigen was prepared by coating the target antigen listed in Table 25 with compound 6: cholesterol: DSPC: DMG-PEG2000 = 50:38.5:10:1.5 with reference to Example 8. 5 to 7 week old female BALB / c mice (purchased from: Sibeifu (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized (hereinafter referred to as "immunization") using the prepared target antigen-containing vaccine, and a second dose was given on the 21st day after vaccination to enhance the immune response. The number of BALB / c mice in each group was 6 (n=6). The immunization method is shown in Table 25 below.

[0314] Table 25B RSV F mutant animal immunization schedule

[0315]

[0316] Administer the test solution twice, three weeks apart. The dose for each dose is shown in the table above. Use a sterile insulin syringe of appropriate size to accurately draw out 400 μL of the test solution (slowly invert the syringe 5-10 times to mix thoroughly before drawing out the test solution). Inject 100 μL into a single intramuscular injection into the right lower limb of the animal.

[0317] The serum of the immunized mice was collected and inactivated at 56°C for 0.5 hours, and then stored in a -80°C refrigerator for subsequent detection.

[0318] 2. Antibody Level Testing

[0319] The neutralizing antibody detection method used the “fluorescence method” described in “IV. Neutralizing Antibody Level Detection” in Example 1.

[0320] Figure 22 Neutralizing antibody results: ① Both mutant designs induced higher neutralizing antibody titers compared to FI-RSV. ② At a 1 μg immunization dose, the mutant without the 1×GCN4t mutant produced higher neutralizing antibody titers, while at a 5 μg immunization dose, the RSV B design with the 1×GCN4t mutant produced higher neutralizing antibody titers. ③ Compared to the RSV A strain, both RSV B antigen designs produced higher neutralizing antibody titers against the RSV B strain.

[0321] In summary, both mutant forms of RSV B can induce higher neutralizing antibody titers against type B strains.

[0322] Table 26 Sequence information

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370] The sequences involved in the present invention are shown in Table 26.

Claims

1. A RSV antigen, characterized in that RSV antigen meets any of the following requirements: (I) The RSV antigen differs from SEQ ID NO: 1 by having S155C, S290C, S190F, S215P, S46G, E92D, D486C, D489C, A102C, and S362C mutations, and positions 103-144 of SEQ ID NO: 1 are substituted with a linker having an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, or an amino acid sequence as shown in GGS, and the RSV antigen further comprises one or more of the following: a back mutation P215S, an insert having an amino acid sequence as shown in SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 140, and / or a replacement having an amino acid sequence as shown in SEQ ID NO: 7; (II) The RSV antigen differs from SEQ ID NO:80 in that it has S155C, S290C, S190F, S46G, E92D, D486C, D489C, A102C and S362C mutations, and positions 103-144 of SEQ ID NO:80 are replaced by a linker with an amino acid sequence as shown in SEQ ID NO:

2. The RSV antigen also includes an insert with an amino acid sequence as shown in SEQ ID NO:

6.

2. The RSV antigen according to claim 1, wherein The RSV antigen further satisfies one or more of the following conditions: (1) In the RSV antigen described in (I) or (II), the insertion site of the insert fragment is between positions 516 and 517 of SEQ ID NO: 1 or SEQ ID NO: 80; (2) (I) In the RSV antigen, the replacement fragment replaces positions 525 to 574 of SEQ ID NO: 1; (3) (I) The RSV antigen further comprises a mutation L513I compared to SEQ ID NO: 1; (4) (I) In the RSV antigen, the insertion site of the inserted fragment is between positions 513 and 514, between positions 519 and 520, or between positions 522 and 523 of SEQ ID NO:

1.

3. The RSV antigen according to claim 1 or 2, wherein The RSV antigen is further post-translationally modified, and the post-translational modification is preferably glycosylation; and / or, (I) the RSV antigen further comprises the back mutations C486D and / or C489D; and / or, (II) the RSV antigen further comprises the reversion mutations (a) G46S; (b) D92E; (c) F190S; (d) C155S and C290S; (e) C486D and C489D; or, (f) C102A and C362S; Preferably, the glycosylation modification is an N-linked glycosylation modification, and / or the glycosylation modification occurs on the inserted fragment.

4. The RSV antigen according to any one of claims 1 to 3, wherein The amino acid sequence of the RSV antigen comprises the amino acid sequence shown in any one of SEQ ID NO:28, SEQ ID NO:33, SEQ ID NO:35-40, SEQ ID NO:81-89, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155 or SEQ ID NO:

157.

5. An isolated nucleic acid, characterized in that The isolated nucleic acid comprises a nucleotide sequence encoding the RSV antigen of any one of claims 1-4.

6. The nucleic acid according to claim 5, wherein The nucleic acid is DNA; Optionally, the DNA comprises a sequence encoding one or more of the following elements: a 5'-cap structure, a 5'UTR, a 3'UTR, a 3'-Poly (A) and a protein tag.

7. The nucleic acid according to claim 5, wherein The nucleic acid is mRNA, preferably codon-optimized mRNA; Preferably, the mRNA has a sequence as shown in any one of SEQ ID NO: 59, 64-70, 72-79, 92-118, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 142, 144, 146, 148, 150, 152, 154, 156 or 158; and / or, the mRNA further comprises one or more of the following elements: a 5'-cap structure, a 5'UTR, a 3'UTR and a 3'-Poly (A).

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to any one of claims 5 to 7; Preferably, the promoter of the recombinant expression vector is a T7 promoter.

9. A transformant, characterized in that The transformant comprises the nucleic acid according to any one of claims 5 to 7 or the recombinant expression vector according to claim 8.

10. A method for preparing RSV antigen, characterized in that: The method comprises culturing the transformant according to claim 9 under conditions suitable for expression of the RSV antigen.

11. A method for preparing a nucleic acid encoding an RSV antigen, characterized in that: The method comprises performing in vitro transcription on the recombinant expression vector according to claim 8.

12. A composition, characterized in that The composition comprises (1) the nucleic acid according to any one of claims 5 to 7 or the recombinant expression vector according to claim 8; and, (2) a delivery vector; Preferably, the delivery vehicle is a lipid nanoparticle; More preferably, the lipid nanoparticles are composed of (A) cationic lipid RL151, LQ104-E16b-2 or compound 6, and (B) cholesterol, (C) DSPC and (D) DMG-PEG2000; Preferably, the molar ratio of cationic lipid RL151, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; the molar ratio of LQ104-E16b-2, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; or the molar ratio of compound 6, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.

5.

13. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the composition according to claim 12, and optionally a pharmaceutically acceptable carrier and / or excipient.

14. A vaccine, characterized in that The vaccine comprises the RSV antigen according to any one of claims 1 to 4, the nucleic acid according to any one of claims 5 to 7, one or more of the composition according to claim 12 and the pharmaceutical composition according to claim 13, and an adjuvant.

15. A test kit or a medicine box, characterized in that: The test kit or medicine box comprises one or more of the RSV antigen according to any one of claims 1 to 4, the nucleic acid according to any one of claims 5 to 7, the recombinant expression vector according to claim 8, the transformant according to claim 9, the composition according to claim 12, the pharmaceutical composition according to claim 13, and the vaccine according to claim 14.

16. Use of one or more of the RSV antigen according to any one of claims 1 to 4, the nucleic acid according to any one of claims 5 to 7, the recombinant expression vector according to claim 8, the transformant according to claim 9, and the composition according to claim 12 in the preparation of a medicament for alleviating, preventing and / or treating a disease caused by RSV.

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