RSV antigen, nucleic acid, recombinant expression vector, pharmaceutical composition and use thereof

Abstract

An RSV antigen, a nucleic acid, a recombinant expression vector, a pharmaceutical composition and the use thereof. The RSV antigen has stronger immunogenicity than that of the RSV in the prior art, can obtain a relatively high neutralizing antibody titer, and has higher protective efficacy. The provided RSV antigen can maintain more prefusion configurations, can stimulate the generation of more neutralizing antibodies against the prefusion configurations and less neutralizing antibodies against postfusion configurations, and has better safety. Furthermore, the provided RSV antigen, nucleic acid, recombinant expression vector and pharmaceutical composition have good application prospects with regard to the preparation of a drug for alleviating, preventing and / or treating a disease caused by the RSV.

Description

RSV antigen, nucleic acid, recombinant expression vector, pharmaceutical composition and application thereof

[0001] This application claims the benefit of priority to Chinese Patent Application No. 2023114397970, filed on October 31, 2023, the benefit of priority to Chinese Patent Application No. 2024100528143, filed on January 12, 2024, the benefit of priority to Chinese Patent Application No. 2024101736872, filed on February 6, 2024, and the benefit of priority to Chinese Patent Application No. 2024113315075, filed on September 23, 2024. The entire contents of the aforementioned Chinese patent applications are incorporated herein by reference. Technical Field

[0002] The present invention belongs to the field of vaccines, and specifically relates to an RSV antigen, nucleic acid, recombinant expression vector, pharmaceutical composition and application 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 (ALRTIs) in children under five 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 hospitalization deaths 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] 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.

[0008] Summary of the Invention

[0009] The technical problem to be solved by the present invention is to overcome the defects of low protective efficacy of vaccines with RSV antigens in the prior art, especially weak ability to stimulate the production of neutralizing antibodies, and the excessive postfusion configuration of RSV antigens that also brings about large immune side effects. A RSV antigen, nucleic acid, recombinant expression vector, pharmaceutical composition and application thereof are provided. The RSV antigen provided by the present invention has stronger immunogenicity than the RSV in the prior art, can achieve higher neutralizing antibody titers, and has higher protective efficacy.

[0010] A first aspect of the present invention provides an RSV antigen comprising mutations A102C and S362C based on SEQ ID NO: 1 or SEQ ID NO: 80.

[0011] In some embodiments of the present invention, the RSV antigen further comprises one or more of S46G, E92D, S155C, S190F, S215P, S290C, D486C, D489C, A149C and Y458C.

[0012] In some preferred embodiments of the present invention, the RSV antigens include D486C, D489C, A149C and Y458C.

[0013] In some embodiments of the present invention, the RSV antigen further comprises a linker.

[0014] In some preferred embodiments of the present invention, the amino acid sequence of the linker is any one of SEQ ID NOs: 2-5 and GGS, and / or the linker replaces positions 103-144 of SEQ ID NO: 1.

[0015] In some embodiments of the present invention, the RSV antigen further comprises an insertion fragment, wherein the insertion fragment is inserted between positions 516 and 517 of SEQ ID NO:1.

[0016] In some preferred embodiments of the present invention, the amino acid sequence of the inserted fragment is shown as SEQ ID NO: 6 or SEQ ID NO: 7.

[0017] In some embodiments of the present invention, the RSV antigen satisfies any of the following:

[0018] (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;

[0019] (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.

[0020] In some preferred embodiments of the present invention, the RSV antigen further satisfies one or more of the following conditions:

[0021] (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;

[0022] (2) (I) In the RSV antigen, the replacement fragment replaces positions 525 to 574 of SEQ ID NO: 1;

[0023] (3) (I) The RSV antigen further comprises a mutation L513I compared to SEQ ID NO: 1;

[0024] (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.

[0025] In some embodiments of the present invention, the RSV antigen is further post-translationally modified.

[0026] In some embodiments of the present invention, the RSV antigen (I) further comprises the back mutations C486D and / or C489D.

[0027] In some embodiments of the present invention, the RSV antigen (II) further comprises reversion mutations (a) G46S; (b) D92E; (c) F190S; (d) C155S and C290S; (e) C486D and C489D; or, (f) C102A and C362S.

[0028] In some preferred embodiments of the present invention, the post-translational modification is glycosylation modification.

[0029] In some more 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.

[0030] In some specific embodiments of the present invention, the amino acid sequence of the RSV antigen comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34-40, 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, SEQ ID 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, SEQ ID NO: 158. The amino acid sequence shown in any one of NO:166-176.

[0031] 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.

[0032] In some embodiments of the present invention, the isolated nucleic acid is mRNA.

[0033] In some preferred embodiments of the present invention, the mRNA is codon-optimized mRNA.

[0034] In some more preferred embodiments of the present invention, the mRNA comprises one or more of a promoter region, a 5'-cap structure, a 5'UTR, a protein tag, and a 3'UTR-polyA.

[0035] In some specific embodiments of the present invention, the promoter is a T7 promoter, and / or the protein tag is HA-HIBIT.

[0036] In some specific embodiments of the present invention, the nucleotide sequence encoding the RSV antigen includes a nucleotide sequence as shown in any one of SEQ ID NO:42, 43, 47, 51, 55, 59, 64-79, 92-118, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 142, 144, 146, 148, 150, 152, 154, 156, 158 or 182-200.

[0037] In some embodiments of the present invention, the nucleic acid is DNA.

[0038] In some preferred 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.

[0039] The third aspect of the present invention provides a recombinant expression vector comprising a starting plasmid and the isolated nucleic acid as described in the second aspect.

[0040] The fourth aspect of the present invention provides a transformant, which comprises the isolated nucleic acid according to the second aspect or the recombinant expression vector according to the third aspect.

[0041] The fifth aspect of the present invention provides a method for preparing the isolated nucleic acid as described in the second aspect, wherein the preparation method comprises in vitro transcription of the recombinant expression vector as described in the third aspect.

[0042] The sixth 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.

[0043] The seventh aspect of the present invention provides a pharmaceutical composition comprising (1) the nucleic acid as described in the second aspect, and (2) a delivery vector.

[0044] In some embodiments of the present invention, the delivery vehicle comprises LNP;

[0045] In some preferred embodiments of the present invention, the LNP is a composition consisting of (A) SM102, cationic lipid RL151, LQ104-54, LQ104-56, LQ104-E16b-2 or compound 6, and (B) cholesterol, (C) DSPC and (D) PEGylated lipid;

[0046] In some more preferred embodiments of the present invention, the molar ratio of cationic lipid RL151, cholesterol, DSPC and PEGylated lipid is 50:38.5:10:1.5.

[0047] In some specific embodiments of the present invention, the ratio of cationic lipid RL151, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; or, 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; or, the molar ratio of LQ104-54, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; or, the molar ratio of LQ104-56, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5.

[0048] In some embodiments of the present invention, the pharmaceutical composition is a vaccine formulation.

[0049] In some optional embodiments of the present invention, the pharmaceutical composition comprises a pharmaceutically acceptable carrier and / or excipient.

[0050] In some embodiments of the present invention, the pharmaceutical composition includes an adjuvant.

[0051] The eighth 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, and the pharmaceutical composition as described in the seventh aspect.

[0052] The ninth aspect of the present invention provides an application of one or more of the RSV antigen as described in the first aspect, the isolated 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 pharmaceutical composition as described in the seventh aspect in the preparation of a drug for alleviating, preventing and / or treating diseases caused by RSV. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG1 shows the in vitro flow cytometry results of RSV F antigens with different designs.

[0054] FIG2 is an in vitro flow cytometric assay of RSV F antigen with P215S reversion mutation.

[0055] FIG3 shows in vitro flow cytometric detection of RSV F antigens with different lengthening designs and sequence optimizations.

[0056] FIG4 shows in vitro flow cytometric detection of RSV F antigen with different codon optimization strategies.

[0057] FIG5 shows the effect of the intermolecular disulfide bond (C486 and C489, subsequently named: ΔF111) on the membrane surface expression and conformation of RSV F antigen.

[0058] FIG6 shows the effect of the intermolecular disulfide bond (C486 and C489, subsequently named: ΔF111) on the membrane surface expression and conformation of RSV F antigen.

[0059] FIG7 shows the in vitro flow cytometry detection (mRNA-Lipofectamine 2000) after the secondary structure of RNA molecules was optimized.

[0060] Figure 8 shows the in vitro flow cytometry detection of RNA molecule secondary structure after optimization (mRNA-RL151).

[0061] Figure 9 shows the total antibody titer in the serum of the 5 μg group two weeks after the second immunization.

[0062] FIG10 shows the neutralizing antibody titer of BALB / c mouse immune sera.

[0063] FIG11 shows the single factor levels of antigen-specific CD4+ T cell immune responses in the spleen of BALB / c mice induced by different antigens.

[0064] Figure 12 shows the multi-factor levels of antigen-specific CD4+ T cell immune responses in the spleen of BALB / c mice induced by different antigens.

[0065] FIG13 shows the single factor levels of antigen-specific CD8+ T cell immune responses in the spleen of BALB / c mice induced by different antigens.

[0066] Figure 14 shows the multi-factor levels of antigen-specific CD8+ T cell immune responses in the spleen of BALB / c mice induced by different antigens.

[0067] FIG15 is an Elispot analysis of antigen-specific IFN-γ and IL-2 in the spleen of BALB / c mice induced by different antigens.

[0068] Figure 16 shows the percentage change (%) of body weight in BALB / c mice after challenge with the virus.

[0069] Figure 17 shows the virus titer and virus copy number after infection in BALB / c mice.

[0070] FIG18 shows the specific IgG antibody levels induced by BALB / c candidate antigens in BALB / c mice using different delivery systems.

[0071] FIG. 19 shows the levels of neutralizing antibodies induced by BALB / c candidate antigens in BALB / c mice using different delivery systems.

[0072] FIG20 shows the single factor levels of antigen-specific CD4+ T cell immune responses in the spleen of BALB / c mice.

[0073] FIG. 21 shows the multi-factor levels of antigen-specific CD4+ T cell immune responses in the spleen of BALB / c mice.

[0074] FIG. 22 shows the single factor levels of antigen-specific CD8+ T cell immune responses in the spleen of BALB / c mice.

[0075] FIG. 23 shows the multi-factor levels of antigen-specific CD8+ T cell immune responses in the spleen of BALB / c mice.

[0076] FIG. 24 shows the spleen cell immune response (Elispot) of BALB / c mice.

[0077] Figure 25 shows the percentage change (%) of body weight of cotton rats after challenge with poison.

[0078] FIG. 26 shows the neutralizing antibody levels of candidate antigens in cotton rats.

[0079] Figure 27 shows the virus titers in lung and nasal tissues of cotton rats after infection.

[0080] Figure 28 shows the cytokines in the lung tissue of cotton rats after infection.

[0081] Figure 29 shows the lung tissue pathology of cotton rats after infection.

[0082] FIG30 shows in vitro flow cytometric detection of RSV F antigen with extended sequences having different glycosylation modifications.

[0083] FIG. 31 shows the neutralizing antibody levels of RSV F antigen against extended sequences with different glycosylation modifications.

[0084] FIG32 shows in vitro flow cytometric detection of RSV F mutants at different 1×GCN4t insertion sites.

[0085] FIG33 shows in vitro flow cytometry detection of RSV F mutants with different linker lengths.

[0086] FIG34 shows the neutralizing antibody levels of RSV F antigen with different linker lengths.

[0087] FIG35 is an in vitro flow cytometric assay of RSV B mutants.

[0088] FIG. 36 shows RSV B vaccine candidate neutralizing antibody levels.

[0089] Figure 37 shows the WB results of in vitro transfection with different antigen designs; negative indicates negative control.

[0090] FIG38 shows the flow cytometry results of in vitro transfection with different antigen designs.

[0091] Figure 39 shows the WB results of in vitro transfection of various antigens after different disulfide bond combinations; negative indicates negative control.

[0092] FIG40 shows the flow cytometry results of in vitro transfection of various antigens after different disulfide bond combinations.

[0093] FIG41 shows the WB results of in vitro transfection of the lengthened designed antigen; negative indicates the negative control.

[0094] FIG42 shows the flow cytometry results of in vitro transfection of the lengthened designed antigen.

[0095] Figure 43 shows the integrity detection of mRNA and mRNA-LNP in mouse immune samples.

[0096] FIG44 shows the flow cytometry results of in vitro transfection of mouse immune samples.

[0097] FIG45 shows the results of total antibody level detection in the serum of antigen-immunized mice.

[0098] Figure 46 shows the single factor levels of antigen-specific CD8+ T cell immune responses in the spleen of Balb / c mice induced by different antigens.

[0099] Figure 47 shows the multi-factor levels of antigen-specific CD8+ T cell immune responses in the spleen of Balb / c mice induced by different antigens.

[0100] Figure 48 shows the single factor levels of antigen-specific CD4+ T cell immune responses in the spleen of Balb / c mice induced by different antigens.

[0101] Figure 49 shows the multi-factor levels of antigen-specific CD4+ T cell immune responses in the spleen of Balb / c mice induced by different antigens.

[0102] FIG50 is an Elispot analysis of antigen-specific IFN-γ and IL-2 in the spleen of Balb / c mice induced by different antigens.

[0103] Figure 51 shows the structure of mRNA. DETAILED DESCRIPTION

[0104] 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.

[0105] 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.

[0106] Example 1 Experimental method

[0107] 1. In vitro transcription and mRNA transfection

[0108] 1. PCR to obtain linearized target fragment

[0109] According to the requirements of the PCR reaction, add 8.7 μL of ddH2O, 0.5 μL of template DNA (using pcDNA(+) as the backbone, with conventional modifications to the 3'UTR and 5'UTR of the open reading frame, source: GenScript synthesis), 0.4 μL of forward and 0.4 μL of reverse primers (source: GenScript synthesis), and 10 μL of PrimerSTAR MAX polymerase (source: TAKARA, cat. no.: R450A) into a 1.5 mL centrifuge tube and mix well.

[0110] As shown in Table 2 below:

[0111] Table 2 PCR system

[0112] The final PCR reaction conditions are shown in Table 3 below:

[0113] Table 3 PCR conditions

[0114] 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 performed. If not, the reaction needs to be repeated.

[0115] 2. In vitro transcription (IVT)

[0116] A small amount of IVT testing was performed according to the following ratio in Table 4 (20 μL system):

[0117] Table 4 IVT system

[0118] 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:

[0119] I. Add 80 μL of ddH2O to 20 μL of digestion product to make up to 100 μL.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] 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.

[0124] 3. Cell Transfection

[0125] 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.

[0126] 2. In vitro flow cytometry

[0127] 1. Wash the transfected cells three times with 1×PBS (Source: Sangon, Cat. No.: B548117-0500), then resuspend in 500 μL 1×PBS.

[0128] 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.

[0129] 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 reference knowledge, the above three antibodies were labeled with AF647 (source: Thermo Fisher, product number: A20186) and AF488 fluorescent dyes (source: Thermo Fisher, 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 using 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 Fisher, Catalog No. S34861) was added at a final concentration of 1 / 5000 for signal reading upon sample loading.

[0130] 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.

[0131] 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).

[0132] 3. Western Blot

[0133] 1. Preparation of cell lysate: Add the transfected cell sample to cell lysis buffer containing protease inhibitors (source: ApexBio, catalog number: K1012), incubate on ice for 10 minutes, centrifuge at 12,000 × g for 10 minutes, and collect the supernatant.

[0134] 2. Electrophoresis Sample Preparation: Divide the lysed sample into two equal portions and add 4×LDS loading solution containing 40 mM DTT (source: Invitrogen, cat. no. NP0008) and 4×LDS loading solution without DTT, respectively. The sample containing DTT needs to be boiled at 95°C for 8 min and immediately placed on ice for cooling. The sample without DTT does not require any treatment.

[0135] 3. Add the prepared electrophoresis samples to the corresponding channels of a protein gel (source: Tanno, catalog number: 180-9115H) and perform protein electrophoresis in the corresponding electrophoresis tank (source: Tanno, catalog number: VE-180) using 1xMOPS Running buffer (source: Tanno, catalog number: BT8100-2002H).

[0136] 4. Target protein color development: After electrophoresis, transfer the protein gel to a membrane using a semi-dry transfer electrophoresis instrument (source: BIO-RAD, catalog number: 1704150EDU). After transfer, incubate with the corresponding primary antibody (source: PROMEGA, catalog number: CS2006A01) and secondary antibody (source: Abcam, catalog number: ab6728). After completion, use ECL luminescence color development solution (source: ApexBio, catalog number: K1200) for analysis using a gel imaging system.

[0137] 5. After the target protein is developed, wash the PVDF membrane (source: Cytia, catalog number: 10600023) with 1× stripping buffer to remove the bound primary and secondary antibodies. Then, after blocking, incubate with appropriate concentrations of primary antibody (source: PROTEINTECH, catalog number: 60004-1-Ig) and secondary antibody (source: Abcam, catalog number: ab6728). After completion, use ECL luminescence colorimetric solution (source: ApexBio, catalog number: K1200) for analysis on a gel imaging system.

[0138] 4. mRNA-LNP integrity detection

[0139] 1. Pipette 10 μL of mRNA-LNP (200 ng / μL) sample into a new EP tube, add 0.4 μL of 10% triton X-100, boil the sample at 70°C for 5 min, and immediately place on ice.

[0140] 2. Pipette 400 ng of the above sample, add 1 μL RNA loading and 1 μL ddH2O, boil the sample at 70℃ for 8 min, and immediately place the sample on ice. After cooling, centrifuge at a short speed. Pipette an appropriate volume of sample and perform agarose gel electrophoresis on an electrophoresis instrument.

[0141] 5. Total Antibody Level Detection

[0142] 1. Antigen coating:

[0143] (1) Dilute the target antigen (source: Shanghai Lanque Biotechnology, batch number: rq20221031) to 1 μg / mL and pipette 100 μL of the aliquot into a 96-well ELISA coated plate (source: Nest, catalog number: 514201). Incubate in a 4°C refrigerator in the dark overnight. Block the plate with 1×PBST solution containing 3% bovine serum albumin for 2 hours at room temperature. Dilute the immune mouse serum 100-fold as the initial concentration, and then perform a 4-fold serial dilution in PBS buffer for a total of 11 dilution steps.

[0144] (2) The target prefusion conformational antigen (source: Shanghai Bio-Tech Co., Ltd., batch number: M-202309120335) and postfusion conformational antigen (source: Shanghai Bio-Tech Co., Ltd., batch number: M-202309160679) were diluted to 400 ng / mL and 300 ng / mL, respectively, and 100 μL was added to a 96-well ELISA coated plate (source: Nest, catalog number: 514201). Incubate in a 4°C refrigerator in the dark overnight. Block the plate with 1×PBST solution containing 3% bovine serum albumin for 2 hours at room temperature. The immune mouse serum was diluted 200 times as the initial concentration, and then serially diluted 4-fold in PBS buffer for a total of 10 dilution gradients.

[0145] The plate was washed with 1×PBST and incubated with serially diluted serum at room temperature for 2 hours. To determine RSV F-specific antibody response, the plate obtained after step (1) or (2) was incubated with rabbit anti-mouse IgG HRP (source: abcam, catalog number: ab6728) at 25°C for 1 hour, and then the substrate tetramethylbenzidine (TMB) solution (source: Invitrogen, catalog number: 00-4201-56) was used for color development. 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).

[0146] 6. Neutralizing Antibody Level Detection

[0147] 1. Novozyme detection method

[0148] I. Cell Preparation: Hep-2 cells (source: ATCC, catalog number: CCL-23) were seeded into 96-well plates (source: Nest, catalog number: 701001) at an adjusted density. Cultured overnight in a cell culture incubator (source: Thermo, catalog number: 3111) (37°C, 5% CO2). The experiment was initiated upon reaching approximately 90% confluence the following day.

[0149] II. Serial dilution: Dilute the sample 20-fold in the first well, and perform a 3-fold serial dilution, with a total of 8 dilution levels (including the first well), in single wells or in duplicate.

[0150] III. Virus dilution: Add an appropriate amount of virus based on the PFU value of the virus.

[0151] IV. Neutralization reaction: Add diluted virus to sample wells and virus control wells respectively, and dilute the virus in the back-drip wells in a 2-fold gradient down to a total of 4 dilutions. Neutralize in a 37°C, 5% CO2 incubator for about 1 hour.

[0152] V. Virus adsorption: Add the above viruses, serum neutralization products, and positive and back-tick well viruses to the prepared cells, 50 μl per well, in duplicate, and culture in a 37°C, 5% CO2 incubator for about 2 hours. After that, change the medium and add 100 μl of culture medium to each well and continue to culture for about 22 hours.

[0153] VI. Plate Assay: Discard the supernatant, fix the cells, add fluorescently labeled detection antibodies, and read the plate using a CTL instrument.

[0154] Judgment criteria: By comparing the number of viruses with infectious activity with that of the RSV control group, the ID50 (the dilution multiple of the antibody when 50% of the RSV virus is inhibited) is calculated to indicate the neutralizing activity of the antibody against RSV virus.

[0155] 2. Methods of Guangzhou HUAWEI Testing Co., Ltd.

[0156] 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.

[0157] 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;

[0158] 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.

[0159] 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.

[0160] 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;

[0161] 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.

[0162] Judgment criteria: 1) Cell control (CC) should be free of lesions, and virus control (VC) should be lesions;

[0163] 2) The neutralization titer of the negative serum control (NC) should be <1:40;

[0164] 3) The neutralization titer of the positive serum control (PC) should be within the compliance range;

[0165] 4) In the viral regression test, the viral titer of 1g TCID50 / ml should be between 630 and 6310 TCID50 / ml;

[0166] If any of the above items are not met, the experiment will be invalid.

[0167] 3. Zhongke Guobang testing method

[0168] I. Sample preparation: Thaw samples at room temperature. Heat inactivate serum samples at 56°C for 30 min, remove from the heat and cool to room temperature after 30 min.

[0169] II. Sample Addition and Dilution: Perform a 40-fold initial dilution, followed by 4-fold dilutions for a total of 6 dilutions. In a 96-well plate, use column 2 as the cell control (CC) and column 3 as the virus control (VC). Add the 20-fold diluted sample to wells B4-B11. Gently mix the sample in wells B4-B11 using a multichannel pipette, then perform 4-fold dilutions until the sample is diluted in wells G4-G11. Separately, add the positive and negative reference samples for sample addition and dilution.

[0170] III. Virus dilution: Dilute the virus to 800-1200 FFU / well on ice and add it to all wells except the cell control.

[0171] IV. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 1 hour.

[0172] V. Digest and count HEp-2 cells and adjust the number to 2.0 × 10 using complete culture medium. 5 100 μL / well was added to a 96-well plate.

[0173] VI. Culture the cells in a 37°C, 5% CO2 incubator. After 24-28 hours, count the GFP fluorescent spots using a cell imaging multifunctional microplate detection system and record the results.

[0174] Judgment criteria: The fluorescent spot values ​​read by the instrument are input into the data analysis template, and the 50% neutralizing antibody inhibition rate (ND50) of the sample is calculated using the Reed-Muench method.

[0175] 4. Fluorescence

[0176] 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;

[0177] 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.

[0178] 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.

[0179] IV. After the neutralization and incubation, culture in a 37°C, 5% CO2 incubator for 2 days

[0180] Vi detection: Green fluorescence signals were detected by a fully automated live cell imaging instrument.

[0181] 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.

[0182] 7. Antigen-specific T cell response detection

[0183] 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).

[0184] 8. Antigen-specific T cell response assay (Elispot)

[0185] Day 1

[0186] 1. Activation of Pre-coated Plates

[0187] 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.

[0188] 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.

[0189] 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.

[0190] Day 2:

[0191] Post-culture operation (sterile operation is no longer required)

[0192] 1. Cell Lysis: Pour 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.

[0193] 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).

[0194] 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.

[0195] 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.

[0196] Dilution Buffer R (10×): Dilute with 1× PBS (1:9) to prepare 1× Dilution Buffer R working solution for later use.

[0197] Biotinylated antibody: Dilute with Dilution buffer R (1×) (1:100) to prepare the working solution.

[0198] 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.

[0199] 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.

[0200] Streptavidin-HRP: Dilute with Dilution buffer R (1×) (1:100) to prepare the working solution.

[0201] 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.

[0202] 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.

[0203] ACE Colorimetric Solution: In a clean container, mix ACE 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.

[0204] Table 5 ACE system

[0205] 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.

[0206] 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.

[0207] IX. Preparation of FI-RSV Inactivated Vaccine

[0208] 1. Cell and Reagent Preparation:

[0209] 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.

[0210] II.D10: 1×DMEM+10% FBS+1% PS

[0211] III.SF-DMEM: 1×DMEM + 1% PS

[0212] IV. 10% Formalin

[0213] V.40mg / mL Al(OH)3 adjuvant

[0214] 2. Experimental materials and equipment:

[0215] I. Constant temperature shaker

[0216] II. Hitachi Ultracentrifuge CP100NX

[0217] 3. Experimental steps:

[0218] I. When the Hep-2 cell density is >90%, wash the cells once with SF-DMEM.

[0219] II. Dilute RSV-A2 (Genbank: KT992094; diluted with SF-DMEM), inoculation amount MOI = 0.1.

[0220] III. Add 3 mL of virus dilution to each 10 cm dish and incubate at 37°C, 5% CO2 for 1 h.

[0221] IV. Discard the viral supernatant and wash the cells once with SF-DMEM.

[0222] V. Add 10 mL of SF-DMEM to each 10 cm dish.

[0223] 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)

[0224] VII. Collect the virus solution: Collect the cell culture supernatant into a 50 mL centrifuge tube and centrifuge at 1000 × g at 4°C for 10 min. Collect the supernatant and reserve 120 μL of the virus solution for virus titration.

[0225] VIII. Add 10% neutral Formalin at a ratio of 1:400 to the shake flask, mix thoroughly, seal, and place on a shaker at 37°C, 50 rpm, and inactivate for 3 days.

[0226] IX. After inactivation, collect the virus by ultracentrifugation at room temperature and resuspend in 10 mL of PBS.

[0227] X. Add Al(OH)3 adjuvant at a final concentration of 4 mg / mL and adsorb overnight at room temperature.

[0228] XI. Centrifuge at 1000×g for 10 minutes and resuspend the pellet in 3 mL of PBS (+1% PS).

[0229] Antigen design:

[0230] The wild-type RSV sequence (SEQ ID NO: 1) is as follows:

[0231] The mutation sites involved in 003m and the extended version are shown in Table 6 below:

[0232] Table 6 003m mutation site and linker sequence

[0233] 003m-1xGCN4t is an insertion of EDKIEEILSKIYHIENEIA RIKKLIGEA (SEQ ID NO: 6) between positions 516 and 517 of SEQ ID NO: 14.

[0234] 003m-2xGCN4t is an insertion of EDKIEEILSKIYHIENEIA RIKKLIGEAIYHIENEIARIKKLIGEA (SEQ ID NO: 160) between positions 516 and 517 of SEQ ID NO: 14.

[0235] The lengthened sequence is shown in Table 7 below:

[0236] Table 7 Lengthened sequence

[0237] The mutation sites involved in 006m and its derivative variants are shown in Table 8 below:

[0238] Table 8 006m mutation site and linker sequence

[0239] The mutation sites involved in 007m and its derivative variants are shown in Table 9 below:

[0240] Table 9 007m mutation site and linker sequence

[0241] The mutation sites involved in 008m and its derivative variants are shown in Table 10 below:

[0242] Table 10 008m mutation site and linker sequence

[0243] The mutation sites involved in 009m and its derivative variants are shown in Table 11 below:

[0244] Table 11 009m mutation site and linker sequence

[0245] The mutation sites involved in 010m and its derivative variants are shown in Table 12 below:

[0246] Table 12 010m mutation site and linker sequence

[0247] The mutation sites involved in DS-Cav1 and its derivative variants are shown in Table 13 below

[0248] Table 13 DS-Cav1 mutation sites and linker sequences

[0249] DS-Cav1-1xGCNt is obtained by inserting EDKIEEILSKIYHIENEIARIKKLIGEA (SEQ ID NO: 6) between positions 516 and 517 of SEQ ID NO: 10.

[0250] DS-Cav1-2xGCNt is obtained by inserting EDKIEEILSKIYHIENEIARIKKLIGEAIYHIENEIARIKKLIGEA (SEQ ID NO: 160) between positions 516 and 517 of SEQ ID NO: 10.

[0251] The structures of all mRNAs used in the experiments of this application are shown in FIG51 .

[0252] The serial numbers are explained in Table 14 below:

[0253] Table 14 Code Explanation

[0254] Example 2 Antigen screening

[0255] This example uses the procedures of “1. In vitro transcription and mRNA transfection”, “2. In vitro flow cytometry” and “3. Western Blot” in Example 1.

[0256] Each antigen expression gene was synthesized by GenScript.

[0257] The coding genes of each antigen are shown in SEQ ID NO: 42, 43, 47, 51, 55, 71, 182-200, and the amino acid sequences of each antigen are shown in SEQ ID NO: 10, 11, 16, 20, 24, 34, 161-169, 12, 170-176,

[0258] (The encoding nucleic acid sequence used in 003m is SEQ ID NO: 42)

[0259] result:

[0260] Figure 37: From the WB results of in vitro transfection, m5K6I, 006m-010m, and 003m all have relatively obvious expression. And because the above mutants all contain the presence of linker, the results of reducing gel show that they all appear in a clear single-chain form. In the absence of linker, the F1 and F2 domains are clearly distinguished.

[0261] Judging from the mean fluorescence intensity (Table 15), the expression levels of m5K6I and 006m-010m antigens on the membrane surface were also high, and the expression level of 003m on the membrane surface was second.

[0262] Table 15 Fluorescence data

[0263] Figure 38: Three flow cytometry antibodies were used: 4D7 (labeled with Alexa Flour 647, detecting the postfusion conformation), AM14 (labeled with Alexa Flour 488, detecting the trimer prefusion conformation), and D25 (labeled with Alexa Flour 488, detecting the prefusion conformation). The results show that mA2F (wild type, WT) has a roughly equal proportion of postfusion and prefusion conformations. DS-Cav1 is mostly prefusion, but some postfusion conformations are present. Compared to the other mutants, 003m's membrane surface conformation is primarily prefusion, with no obvious postfusion conformation. 006m-010m, m5K6I, mF111, and mSC-TM also exhibit a significant proportion of postfusion conformations on the membrane surface.

[0264] Example 3 Disulfide bond modification of antigens

[0265] This example uses "1. In vitro transcription and mRNA transfection", "2. In vitro flow cytometry" and "3. Western Blot" in Example 1.

[0266] result:

[0267] Figure 39: From the WB results, after the addition of the D486C / D489C disulfide bond pair, 007m-010m was renamed 007m_Cys-010_Cys, and there was no obvious decrease in the expression level; however, after the addition of the A149C / Y458C disulfide bond pair to 003m and 007m-010m (renamed 003m_DS and 007m_DS-010m_DS), the expression level of the protein was significantly reduced; and after the removal of the A102C / S362C disulfide bond pair from 003m and 007m_Cys-010_Cys (renamed 003m_ΔCys and 007m_Cys_ΔCys-010_Cys_ΔCys), its expression level also decreased significantly.

[0268] Figure 40: Flow cytometry results show that 003m and 007m_Cys-010_Cys can maintain a relatively stable prefusion conformation, while 007m-010m, 007m_DS-010m_DS, and 007m_Cys_ΔCys-010_Cys_ΔCys cannot. While 003m-DS can maintain a prefusion conformation, its surface expression level decreases significantly. Combined with Western blotting results, overall expression levels also decrease significantly. Flow cytometry cell surface fluorescence signal values, shown in Table 16 below, reflect the level of target protein on the cell surface.

[0269] Since the above-mentioned different disulfide bond combinations failed to significantly increase the expression level of the protein, it can be seen that the design of 003m is also relatively reasonable.

[0270] Table 16 Fluorescence data

[0271] Example 4 Lengthened design of antigen

[0272] This example uses "1. In vitro transcription and mRNA transfection", "2. In vitro flow cytometry" and "3. Western Blot" in Example 1.

[0273] Figure 41: Lengthened mutations of 1×GCN4t and 2×GCN4t were performed on 003m and DS-Cav1, respectively (1×GCN4t and 2×GCN4t were inserted between positions 516 and 517 in 003m and DS-Cav1, respectively). Western blot results show that the lengthened mutation design resulted in a significant decrease in expression in 003m.

[0274] Judging from the mean fluorescence intensity (Table 17), the amount of antigen expression on the membrane surface showed a clear downward trend after the lengthening design.

[0275] Table 17 Fluorescence data

[0276] Figure 42: From the flow cytometry results, after the extension of 003m, except for 2xGCN4t, the other mutations can maintain the prefusion conformation well in in vitro expression.

[0277] Conclusion: Based on the above comparison, not all antigens can achieve good results (including maintenance of conformation and expression level) by lengthening with GCN4t. Generally speaking, 003m is more suitable for GCN4t lengthening design.

[0278] Example 5 Preparation of lipid nanoparticles

[0279] The mRNAs of the antigen sequences selected in the above examples were ionized (cationic) at low pH and coated into nanoparticles with two helper lipids: DSPC (distearoylphosphatidylcholine, Catalog No. B90536, manufactured by Nippon Seika Co., Ltd.) and cholesterol (Catalog No. C00373, manufactured by Nippon Seika Co., Ltd.), as well as RL151 (Catalog No. W211-YB211202, purchased from Zhejiang Shenzhou Pharmaceutical Co., Ltd.) and a pegylated lipid (DMG-PEG2000, Catalog No. M-DMG-2000, purchased from JenKem). An aqueous solution of the mRNA was prepared by mixing the mRNA dissolved in ultrapure water with 100 mM (millimoles per liter, or mmol / L) citrate buffer at pH 4.0 at a volume ratio of 1:1. The ratio of the four lipid components is adjusted (molar ratio, for example, cationic lipid RL151: 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 (manufacturer: Precision Nanosystems) microfluidic mixing system at a volume mixing ratio of H2O: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. Lipid nanoparticles containing mRNA (mRNA-LNPs) are obtained. Lipid nanoparticles containing mRNA (mRNA-LNPs) are obtained. The lipid nanoparticles are dialyzed, concentrated, filtered, and stored to obtain a vaccine containing the target antigen.

[0280] Figure 43: Gel electrophoresis results show that neither the purified mRNA nor the LNP-coated mRNA has undergone significant degradation.

[0281] Figure 44: From the flow cytometry results of mRNA-LNP expression in vitro for 20 hours, the membrane surface expression level of 003m-1xGCN4t was significantly lower than that of 003m, and also much lower than that of m5K6I and Moderna-ΔCT; the membrane surface expression levels of the 1xGCN4t extended form of 003m and DS-Cav1 were slightly lower than those of 2xGCN4t; all extended forms of 003m and DS-Cav1 could maintain the trimer prefusion conformation well; the membrane surface expression levels of 10m-Cys and 003m were basically the same.

[0282] Example 6 First round of animal immunization

[0283] 1. Animal Vaccination and Serum Collection

[0284] Balb / c mice: For mouse vaccination, 5- to 7-week-old female Balb / c mice (purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.) were intramuscularly immunized with the target antigen vaccine prepared in Example 5 (hereinafter referred to as "immunization"). A second dose was administered on day 21 after vaccination to boost the immune response. Six BALB / c mice were administered in each group (n=6).

[0285] The immunization method is shown in Table 18 below:

[0286] Table 18 Immunization methods

[0287] 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 solution). Inject 50 μl into a single intramuscular injection into the right lower limb of the animal.

[0288] 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.

[0289] 2. Antibody Level Testing

[0290] The total antibody level detection method used the method in "V. Total Antibody Level Detection" of Example 1, specifically the method described in 1.(1) for antigen coating. The neutralizing antibody detection method used the method in "VI. Neutralizing Antibody Level Detection" of Example 1. Results:

[0291] Figure 45: In terms of total serum antibody levels, sera produced by mice immunized with mA2F (i.e., wild-type) primarily target the postfusion conformation. In sera from mice immunized with DS-Cav1, antibodies against the prefusion conformation were highest, followed by m5K6I and Moderna-ΔCT, and then 003m. Mice immunized with 003m produced the lowest total antibody levels against the postfusion conformation. 003m-1×GCN4t produced significantly higher total antibodies against the prefusion conformation than 003m and 003m-2×GCN4t, while total antibodies against the postfusion conformation were also lower.

[0292] Judging from the neutralization experiment results (Tables 19 and 20), the neutralizing antibody titer produced by 003m-1xGCN4t is also high.

[0293] Table 19 Serum neutralizing antibody titers two weeks after the second immunization

[0294] Table 20 Neutralizing antibody titers of various subtypes in serum two weeks after the second immunization

[0295] 3. Cellular Immune Response in Balb / c Mice

[0296] The detection process refers to "VII. Antigen-specific T cell response detection" in Example 1.

[0297] The results of intracellular factor detection were basically consistent with those of serum titer detection.

[0298] The expression levels of IL-4 and IL-5 in each experimental group were low, suggesting no associated risk.

[0299] Figure 46 and Figure 47: Judging from the CD8 single factor and multifactor results, the trend is basically consistent with the total antibody level. The cellular immunity level induced by 003m is lower than that of m5K6I, DS-Cav1, and Moderna-ΔCT, and the cellular immunity level induced by 003m-1xGCN4t is higher than that of 003m-2xGCN4t.

[0300] Figures 48 and 49: Based on the CD4 single-factor and multi-factor results, mA2F induced higher levels of IL-4 and IL-5, while the levels in the other groups were slightly lower. In the extended-type group, both IL-4 and IL-5 levels were lower, indicating that the VED (vaccine enhanced disease) phenomenon it may induce is weaker.

[0301] IV. Cellular Immune Response in Balb / c Mice (Elispot)

[0302] The detection process refers to "VIII. Antigen-specific T cell response detection (ELISPot)" in Example 1.

[0303] Detection time: 95 days after the second vaccination

[0304] Immunoassay: 2.5 μg

[0305] As shown in Figure 50, Elispot and intracellular factor assay results were generally consistent, with Moderna-ΔCT producing the highest levels of IFN-γ and IL-2. In the DS-Cav1 elongated mutant, the results were similar between the two mutants, while in the 003m elongated mutant, 1×GCN4t produced slightly better results than 2×GCN4t.

[0306] In Examples 7-18, a second round of screening and improvement was performed on RSV and its applications:

[0307] Example 7 Effect of P215S reversion mutation on RSV F antigen membrane surface conformation and expression

[0308] 003m-STM, 007m-Cys-STM, 008m-Cys-STM, 009m-Cys-STM, 010m-Cys-STM, etc., wherein the STM modification contained therein is to replace all I525-N574 with WPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCK (SEQ ID NO: 7).

[0309] Figure 1: The 1×GCN4t and 1×GCN4t-STM mutation designs will lead to a significant decrease in the expression of RSV F antigen on the membrane surface, but the STM modification will not have a significant change in the expression level of the protein, 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, and the RSV F antigen can maintain a good trimeric conformation.

[0310] Figure 2: Targeting 003m, a P215S reversion mutation was made, and further 1×GCN4t and 2×GCN4t extension mutations were made. Flow cytometry results show that the P215S reversion mutation, 1×GCN4t extension, and 2×GCN4t extension significantly reduced RSV F antigen surface expression, with extension having a more pronounced effect (Table 21). However, none of these mutations altered the prefusion trimer conformation.

[0311] Table 21 Mean fluorescence intensity of different mutant designs of P215S by flow cytometry in vitro

[0312] Example 8 Effects of Different Lengthening Designs and Sequence Optimization on Antigen Expression and Conformation

[0313] 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.

[0314] Figure 3: Comparison of 003M-P215S-S and 003M-P215S-S-3N, 003M-P215S-L and 003M-P215S-L-3N, respectively. 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 22), and the trimer prefusion conformation of RSV F antigen did not change significantly.

[0315] Table 22 In vitro flow cytometry mean fluorescence intensity with different lengthening designs

[0316] Example 9 Codon Optimization Strategy Screening

[0317] Figure 4: Based on the in vitro flow cytometry results and mean fluorescence intensity (Table 23), the expression of membrane surface antigens was significantly increased when the codon-optimized 003m-P215S-1×GCN4t was used for cell transfection.

[0318] Table 23 Mean fluorescence intensity of different codon optimized 003m-P215S-1×GCN4t in vitro flow cytometry

[0319] Example 10 Intermolecular disulfide bond screening

[0320] 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 24), while also leading to the production of some postfusion conformational antigens. Furthermore, all extended designs showed a significant decrease in membrane surface expression.

[0321] Table 24 In vitro flow cytometry mean fluorescence intensity (Lipofectamine) of different mutants with intermolecular disulfide bond design

[0322] Figure 6: When in vitro transfection was performed using mRNA-LNP (RL151, Catalog No.: W211-YB211202, purchased from Zhejiang Shenzhou Pharmaceutical; the mRNA-LNP preparation method can be found in Example 8, except that LQ104-E16b-2 was replaced with RL151), the membrane surface protein expression was significantly reduced compared to transfection using Lipofectamine 2000 (Table 25). However, the above antigen designs were able to maintain the trimer prefusion conformation well. After the ΔF111 back mutation, the membrane surface expression of the RSV F antigen of the remaining designs, except for 003m-P215S-ΔF111-1×GCN4t, was still significantly improved compared to the unmutated design.

[0323] Table 25 Intramolecular disulfide bond design in different mutants in vitro flow mean fluorescence intensity (LNP)

[0324] Example 11 RNA secondary structure optimization

[0325] Figure 7: When using Lipofectamine 2000-mRNA, RNA secondary structure optimization had limited effect on membrane surface expression (Table 26), and some RSV F antigens still exhibited a postfusion conformation.

[0326] Table 26 In vitro flow cytometry mean fluorescence intensity (Lipofectamine) of different RNA secondary structure optimizations

[0327] Figure 8 shows that when mRNA-LNP (RL151) was used for in vitro transfection, the optimization of RNA secondary structure had a significant effect on the membrane surface expression level. The difference between the high expression group and the low expression group was approximately 8 times (Table 27). 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.

[0328] Table 27 In vitro flow cytometry mean fluorescence intensity (LNP) of different RNA secondary structure optimization

[0329] Example 12 Preparation of lipid nanoparticles from BALB / c mouse immune samples

[0330] The target sequence mRNAs screened in Examples 7-11 (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×GCN 4t-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 at 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.

[0331] Example 13 Immunization of BALB / c mice

[0332] 1. Animal Vaccination and Serum Collection

[0333] 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 schedule is shown in Table 28 below.

[0334] Table 28 BALB / c mouse immunization schedule

[0335] 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.

[0336] 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.

[0337] 2. Antibody Level Testing

[0338] 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.

[0339] 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.

[0340] 2. Neutralizing Antibody Titer

[0341] Figure 10: From 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.

[0342] 3. Cellular Immune Response in BALB / c Mice

[0343] Figures 11 and 12: Judging from the CD4 single factor and multifactor levels and the total antibody level (Figure 10), the 003m-P215S-1×GCN4t high-dose group induced the highest level of cellular immunity. This result also has a similar trend to the ratio of prefusion conformation antibodies to postfusion conformation antibodies in the total antibody level.

[0344] Figures 13 and 14: From the perspective of CD8 single factor and multifactor 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.

[0345] IV. Cellular Immune Response in BALB / c Mice (Elispot)

[0346] 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.

[0347] Example 14 BALB / c mice challenge

[0348] 1. Animal Weight Changes

[0349] Figure 16: Results show that the 5μg vaccine effectively alleviated RSV A2-induced weight loss, with the 003m-P215S-1×GCN4t group showing the best effect. However, the 003m-STM (1μg) group experienced more severe weight loss than the placebo group (Figure 16), suggesting a risk of VED.

[0350] 2. Viral titer and copy number

[0351] Figure 17: The results showed that the vaccine groups were able to significantly reduce the viral titers and copy numbers in the lung and nasal tissues. The viral titers in the lungs and nasal cavity of animals in each vaccine immunization group decreased by more than 2 orders of magnitude, indicating that each candidate antigen has good protective properties.

[0352] (Note: The lower limit of viral titer detection is: lung tissue: 417 PFU / g tissue, nasal tissue: 1630 PFU / g tissue; the lower limit of viral copy number detection is: 1 Log10 copies / μg RNA)

[0353] Example 15 Immunogenicity of different delivery systems for candidate antigens

[0354] 1. Animal Vaccination and Serum Collection

[0355] BALB / c mice: For mouse vaccination, a vaccine containing the target antigen was prepared by referring to the mRNA-LNP preparation methods described in Examples 12 and 17. Five- to seven-week-old female BALB / c mice (purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized (hereinafter referred to as "immunization"), and a second dose was administered on day 21 after vaccination to boost the immune response. Each group consisted of eight BALB / c mice (n=8). The immunization schedule is shown in Table 29.

[0356] Preparation of LQ104-54:

[0357] Step 1: Preparation of compound 54-1

[0358] To a reaction flask, 5-bromovaleric acid (10 g, 55 mmol, 1.1 eq), DCC (20.6 g, 100 mmol, 2 eq), DMAP (610 mg, 5 mmol, 0.1 eq), and DCM (300 mL) were added, followed by 9-heptadecanol (12.8 g, 50 mmol, 1 eq). The mixture was stirred at room temperature for 12 h. The reaction mixture was filtered, concentrated, and purified by column chromatography to yield 14 g of a colorless oil. The product rf value was 0.6 on TLC (PE:EA = 20:1).

[0359] Step 2: Preparation of compound 16-3

[0360] To a reaction flask, E16b-2 (5 g, 11.5 mmol, 0.8 eq, cited from CN117534584A), N,N'-bis(2-hydroxyethyl)ethylenediamine (2.2 g, 14.4 mmol, 1 eq), K2CO3 (3 g, 21.6 mmol, 1.5 eq), KI (2.4 g, 14.4 mmol, 1 eq), and acetonitrile (80 mL) were added and stirred at 70°C for 16 h. The reaction mixture was filtered, concentrated, and purified by column chromatography to yield 4 g of a colorless oil. TLC (DCM:MeOH = 10:1) revealed an rf value of 0.5.

[0361] Step 3: Preparation of LQ104-54

[0362] To a reaction flask were added 16-3 (1.6 g, 3.2 mmol, 1.1 eq), 54-1 (1.63 g, 3.9 mmol, 1.2 eq), KCO (1 g, 6.4 mmol, 2 eq), KI (0.6 g, 3.2 mmol, 1 eq), and acetonitrile (40 mL). The mixture was heated to 70°C and stirred for 16 h. The reaction mixture was filtered, concentrated, and purified by column chromatography to yield 700 mg of a colorless oil. TLC (DCM:MeOH = 10:1) revealed an rf value of 0.4.

[0363] 1H NMR(600MHz,Chloroform-d)δ4.86(p,J=6.3Hz,2H),3.63(q,J=5.0Hz,4H),2.70–2.55(m,12H),2.30(dt,J=15 .2,7.3Hz,4H),1.62(dt,J=18.1,7.7Hz,4H),1.55–1.47(m,12H),1.30–1.22(m,50H),0.87(t,J=6.9Hz,12H).

[0364] MS(ES+)m / z): 839(M),840(M+H).

[0365] Preparation of LQ104-56:

[0366] Step 1: Preparation of compound 56-1

[0367] To a reaction flask, 8-bromooctanoic acid (12.2 g, 55 mmol, 1.1 eq), DCC (20.6 g, 100 mmol, 2 eq), DMAP (610 mg, 5 mmol, 0.1 eq), and DCM (300 mL) were added, followed by 9-heptadecanol (12.8 g, 50 mmol, 1 eq). The mixture was stirred at room temperature for 12 h. The reaction mixture was filtered, concentrated, and purified by column chromatography to yield 15.5 g of a colorless oil. The product rf value was 0.6 on TLC (PE:EA = 20:1).

[0368] Step 2: Preparation of LQ104-56

[0369] To a reaction flask were added 16-3 (1.6 g, 3.2 mmol, 1.1 eq), 20-1 (1.8 g, 3.9 mmol, 1.2 eq), K2CO3 (1 g, 6.4 mmol, 2 eq), KI (0.6 g, 3.2 mmol, 1 eq), and acetonitrile (40 mL). The mixture was heated to 70°C and stirred for 16 h. The reaction solution was filtered, concentrated, and purified by column chromatography to afford 1.2 g of a colorless oil. TLC (DCM:MeOH = 10:1) revealed an rf value of 0.4.

[0370] 1H NMR(400MHz,Chloroform-d)δ4.85(p,J=6.2Hz,2H),3.64–3.57(m,4H),2.67–2.57(m,8H),2.52(t,J=7.9Hz,4H) ,2.27(td,J=7.5,4.7Hz,4H),1.68–1.57(m,4H),1.49(t,J=6.2Hz,12H),1.33–1.22(m,56H),0.90–0.84(m,12H).

[0371] MS(ES+)m / z): 882(M),883(M+H).

[0372] Table 29 BALB / c mouse immunization schedule

[0373] 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.

[0374] 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.

[0375] 2. Antibody Level Testing

[0376] 1. Total Antibody Level

[0377] The detection method used was the same as that described in "V. Total Antibody Level Detection" in Example 1, specifically antigen coating was performed using the method described in 1.(2). The neutralizing antibody detection method used was the same as that described in "VI. Neutralizing Antibody Level Detection" in Example 1.

[0378] Figure 18: ① Compared to the 1μg group, the 5μg group induced higher total antibody levels, showing an overall dose-dependent pattern. ② After immunization with different liposomes, 003m-P215S-1xGCN4t produced lower levels of antibodies targeting the postfusion conformation, and 003m-P215S-1xGCN4t induced significantly lower levels of antibodies targeting the postfusion conformation than 003m-P215S. ③ When using the same liposomes, 003m-P215S produced higher total antibody titers than 003m-P215S-1xGCN4t. ④ The majority of antibodies produced by Post-F were directed against the postfusion conformation. ⑤ Compared to the other three liposomes, RL151 induced higher levels of antibodies targeting the prefusion conformation in 003m-P215S and 003m-P215S-1xGCN4t.

[0379] In general, both 003m-P215S and 003m-P215S-1xGCN4t can induce the production of more antibodies against the prefusion conformation, and the ratio of antibodies against the prefusion conformation to the postfusion conformation induced by 003m-P215S-1xGCN4t is better than that of 003m-P215S.

[0380] 2. Neutralizing Antibody Level Detection

[0381] Figure 19: Consistent with total antibody levels: ① Post-F induced lower neutralizing antibody titers. ② Compared to the 1μg group, the 5μg group induced higher neutralizing antibody titers. ③ Compared to 003m-P215S, 003m-P215S-1xGCN4t induced higher neutralizing antibody titers in BALB / c cells. ④ Both the RL151 and iPLX delivery systems induced higher neutralizing antibody titers.

[0382] 3. Spleen Cell Immune Response of BALB / c Mice

[0383] Figures 20 and 21 (CD4): ① 003m-P215S, 003m-P215S-1xGCN4t, and Post-F all induced strong cellular immunity, with the 5μg group slightly higher than the 1μg group. ② Compared to E16b2, RL151, LQ104-56, and LQ104-54 all induced lower levels of Th2-related factors, suggesting that immunization with E16b2 carries a risk of VED. ③ Even with RL151 delivery, Post-F still carries a risk of VED.

[0384] Figures 22 and 23 (CD8): ① Cytokine production was linearly correlated with dose, with the 5μg group exhibiting slightly higher levels than the 1μg group. ② RL151 delivery induced significantly higher levels of cellular immunity than LQ104-56 and LQ104-54. ③ 003m-P215S-1xGCN4t induced higher levels of cellular immunity than 003m-P215S, demonstrating that 003m-P215S-1xGCN4t is superior to 003m-P215S in BALB / c mice.

[0385] IV. BALB / c mouse spleen cell immune response (Elispot)

[0386] Figure 24: Elispot assay results are consistent with those of intracellular factor assays: ① The level of cellular immunity induced by RL151 delivery was significantly higher than that induced by LQ104-56 and LQ104-54, but slightly lower than that induced by E16b2. ② Compared to 003m-P215S, 003m-P215S-1xGCN4t induced higher levels of cellular immunity.

[0387] Example 16 Cotton rat challenge

[0388] 1. Animal Vaccination and Serum Collection

[0389] Cotton rat vaccination: For cotton rat vaccination, 6- to 8-week-old female cotton rats (purchased from Sibefor (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized with the target antigen vaccine prepared using the mRNA-LNP (RL151) preparation method described in Example 17 (hereinafter referred to as "immunization"). A second dose was administered 21 days after vaccination to enhance the immune response. In the Placeb group, the specific composition of Tris-Ac (pH 7.5) was: 20 mM Tris, 1.42 mM glacial acetic acid, and 2.89 mM sodium acetate. The number of cotton rats in each group was 3 / 4 (n=3 / 4). The immunization method is shown in Table 30 below.

[0390] Table 30 Cotton rat immunization schedule

[0391] 2. Weight Changes

[0392] Figure 25: Compared with the placebo group, all groups were able to alleviate the weight loss caused by RSV A2 virus (Genbank: KT992094) infection, among which 003m-P215S-1×GCN4t (25 μg) was more effective.

[0393] Neutralizing Antibodies

[0394] Figure 26: Compared with the inactivated vaccine, 003m-P215S-1xGCN4t induced higher neutralizing antibody titers at 5 μg, and had neutralizing effects on both RSV A2 and RSV B strains, but did not induce neutralizing antibody titers higher than the low-dose group at 25 μg.

[0395] IV. Virus Titer

[0396] Figure 27: ① Compared to the placebo group, the inactivated virus group (FI-RSV) significantly reduced lung viral titers, but the virus was still detectable in nasal tissue. ② 003m-P215S1×GCN4t significantly reduced viral titers in both lung and nasal tissues, with the virus titers in the animals' lungs and nasal cavities decreasing by more than two orders of magnitude, demonstrating the antigen's protective properties.

[0397] 5. Lung Tissue Cytokines

[0398] Figure 28: Cytokine detection results showed that the expression of Th2-related factors (IL-4, IL-2, IL-3) in the inactivated virus group (FI-RSV) was significantly higher than that in the 003m-P215S-1xGCN4t group and the placebo group, while in the 003m-P215S-1xGCN4t group it was basically the same as that in the placebo group, indicating that the inactivated virus group (FI-RSV) had a higher risk of VED.

[0399] 6. Lung Tissue Pathology

[0400] Figure 29: Results show that RSV A2 infection in mice causes pathological inflammation in the alveolar cavity, peribronchiolar, perivascular, and alveolar interstitial space. 003m-P215S-1xGCN4t significantly reduced lung inflammation in a dose-dependent manner. In contrast, the inactivated virus (FI-RSV) group developed more severe lung inflammation, indicating that FI-RSV can induce more severe VED.

[0401] Example 17 Screening of extended sequences with different glycosylation modifications

[0402] 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.

[0403] 1. In vitro flow cytometry

[0404] Figure 30 shows in vitro flow cytometry results showing that elongation of 1×Fibritin and glycosylation modifications of 1×Fibritin and 1×GCN4t significantly increased the expression of RSV F protein in the monomeric or trimer prefusion conformation on the membrane surface (Table 31). Compared to 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.

[0405] Table 31 In vitro flow cytometry mean fluorescence intensity of the extended sequences with different glycosylation modifications

[0406] 2. Animal Vaccination and Serum Collection

[0407] BALB / c mice: For mouse vaccination, lipid LQ104-E16b-2 was prepared according to CN117534584A.

[0408] Five- to seven-week-old female BALB / c mice (purchased from Sibeifu (Suzhou) Biotechnology Co., Ltd.) were intramuscularly immunized with the target antigen-containing vaccine prepared by coating with the target antigen as described in Example 8 (hereinafter referred to as "immunization"). A second dose was administered on day 21 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 32 below.

[0409] Table 32 Immunization schedule for BALB / c mice

[0410] 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.

[0411] 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.

[0412] 3. Antibody Level Testing

[0413] The neutralizing antibody detection method used the “fluorescence method” described in “VI. Neutralizing Antibody Level Detection” in Example 1.

[0414] Figure 31: 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.

[0415] Example 18 Effect of 1×GCN4t Insertion Site and Linker Length on Antigen Immunogenicity

[0416] 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.

[0417] 1. In vitro flow cytometry

[0418] Figures 32 and 33: In vitro flow cytometry results show that different insertion positions of 1×GCN4t and different linker lengths have no significant effect on the RSV F protein in the monomeric prefusion conformation on the membrane surface (Tables 33 and 34).

[0419] Table 33 Mean fluorescence intensity of RSV F mutants at different 1×GCN4t insertion sites detected by flow cytometry in vitro

[0420] Table 34 Average fluorescence intensity of RSV F mutants with different linker lengths detected by flow cytometry in vitro

[0421] 2. Animal Vaccination and Serum Collection

[0422] BALB / c mice: For mouse vaccination, refer to "II. Animal Vaccination and Serum Collection" in Example 17 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 target antigen vaccine (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 35 below.

[0423] Table 35 1×GCN4t insertion site and linker length Animal immunization schedule

[0424] 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.

[0425] 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.

[0426] 3. Antibody Level Testing

[0427] The neutralizing antibody detection method used the “fluorescence method” described in “VI. Neutralizing Antibody Level Detection” in Example 1.

[0428] Figure 34: Neutralizing antibody results: ① The insertion position of 1×GCN4t has a certain effect 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 effect on neutralizing antibody titers, but there is no significant difference with 003m-P215S-1×GCN4t. In general, different insertion sites and linker lengths of 1×GCN4t can all induce higher neutralizing antibody titers, that is, the selection of 1×GCN4t insertion position and linker length is universal.

[0429] Example 19: Modification of candidate B antigens

[0430] 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).

[0431] The amino acid sequence of wild-type RSV B (SEQ ID NO: 80) is shown below:

[0432] Modification based on RSV B:

[0433] 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 1.

[0434] 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, RSV B-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.

[0435] 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.

[0436] Figure 35: In vitro flow cytometry results show that single-point mutations or disulfide bond pair mutations will result in the presence of some postfusion conformation F protein on the membrane surface. At the same time, when no linker is used, the expression of the membrane surface protein will be severely affected. While the removal of 1×GCN4t increases the expression of the membrane surface protein, it also leads to an increase in the content of the postfusion conformation F protein. At the same time, RSV B-003m-P215S-1×GCN4t-RQ1 shows a certain degree of improvement in expression compared to the other two sequence optimizations. Therefore, RSV B-003m-P215S-1×GCN4t is still able to maintain the monomer and trimer prefusion conformations in RSV B type. Among the three sequence optimizations, RQ1 is the best (Table 36).

[0437] Table 36 Mean fluorescence intensity of RSV F mutants in vitro flow cytometry

[0438] Example 20 Immunization and Neutralization of BALB / c Mice with Candidate Type B Antigens

[0439] 1. Animal Vaccination and Serum Collection

[0440] 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.

[0441] Preparation of lipid compound 6:

[0442] Step 1: Preparation of 6-1

[0443] Reaction formula:

[0444] The material ratio is shown in Table 37:

[0445] Table 37 Material ratios for preparation 6-1

[0446] Operation process:

[0447] 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.

[0448] Step 2: Preparation of 6-2

[0449] Reaction formula:

[0450] The material ratio is shown in Table 38:

[0451] Table 38 Material ratios for preparation 6-2

[0452] Operation process:

[0453] 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.

[0454] Step 3: Preparation of compound 6

[0455] Reaction formula:

[0456] The material ratio is shown in Table 39:

[0457] Table 39 Material ratios for preparing compound 6

[0458] Operation process:

[0459] 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.

[0460] 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).

[0461] MS (ES+) m / z): 910.7 (M+H) + .

[0462] A control FI-RSV inactivated vaccine was prepared with reference to "IX. 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 12. Female BALB / c mice aged 5 to 7 weeks (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 as follows.

[0463] Table 40 Animal Immunization Schedule for Type B RSV F Mutants

[0464] 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.

[0465] 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.

[0466] 2. Antibody Level Testing

[0467] The neutralizing antibody detection method used the “fluorescence method” described in “VI. Neutralizing Antibody Level Detection” in Example 1.

[0468] Figure 36: 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 1×GCN4t produced higher neutralizing antibody titers, while at a 5 μg immunization dose, the RSV B design with 1×GCN4t produced higher neutralizing antibody titers. ③ Compared to the A strain, both RSV B antigen designs produced higher neutralizing antibody titers against the B strain.

[0469] In general, both mutant forms of RSV B can induce higher neutralizing antibody titers against type B strains.

[0470] The sequences involved in the present invention are shown in Table 41 and in the following sequences:

[0471] Nucleic acid sequence

[0472] mA2F encoding nucleic acid sequence (SEQ ID NO: 177)

[0473] m5K6I encoding nucleic acid sequence (SEQ ID NO: 178)

[0474] DS-Cav1-1xGCN4t encoding nucleic acid sequence (SEQ ID NO: 179)

[0475] DS-Cav1-2xGCN4t encoding nucleic acid sequence (SEQ ID NO: 180)

[0476] Moderna-ΔCT encoding nucleic acid sequence (SEQ ID NO: 181)

[0477] 003m-1xGCN4t optimized encoding nucleic acid sequence (optimize 1) (SEQ ID NO: 182)

[0478] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 2) (SEQ ID NO: 183)

[0479] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 3) (SEQ ID NO: 184)

[0480] 003m-1xGCN4t optimized encoding nucleic acid sequence (optimize 4) (SEQ ID NO: 185)

[0481] 003m-1xGCN4t optimized encoding nucleic acid sequence (optimize 5) (SEQ ID NO: 186)

[0482] 003m-1xGCN4t optimized encoding nucleic acid sequence (optimize 6) (SEQ ID NO: 187)

[0483] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 7) (SEQ ID NO: 188)

[0484] 003m-1xGCN4t optimized encoding nucleic acid sequence (optimize 8) (SEQ ID NO: 189)

[0485] 003m-2xGCN4t encoding nucleic acid sequence (SEQ ID NO: 190)

[0486] 003s encoding nucleic acid sequence (SEQ ID NO: 191)

[0487] 006m encoding nucleic acid sequence (SEQ ID NO: 192)

[0488] 007m encoding nucleic acid sequence (SEQ ID NO: 193)

[0489] 007m-Cys-Δcys encoding nucleic acid sequence (SEQ ID NO: 194)

[0490] 008m encoding nucleic acid sequence (SEQ ID NO: 195)

[0491] 008m-Cys-Δcys encoding nucleic acid sequence (SEQ ID NO: 196)

[0492] 009m encoding nucleic acid sequence (SEQ ID NO: 197)

[0493] 009m-Cys-Δcys encoding nucleic acid sequence (SEQ ID NO: 198)

[0494] 010m encoding nucleic acid sequence (SEQ ID NO: 199)

[0495] 010m-Cys-Δcys encoding nucleic acid sequence (SEQ ID NO: 200)

[0496] Amino acid sequence

[0497] mA2F amino acid sequence (SEQ ID NO: 161)

[0498] m5K6I amino acid sequence (SEQ ID NO: 162)

[0499] DS-Cav1-1xGCN4t amino acid sequence (SEQ ID NO: 163)

[0500] DS-Cav1-2xGCN4t amino acid sequence (SEQ ID NO: 164)

[0501] Moderna-ΔCT amino acid sequence (SEQ ID NO: 165)

[0502] 003m-2xGCN4t amino acid sequence (SEQ ID NO: 166)

[0503] 003s amino acid sequence (SEQ ID NO: 167)

[0504] 006m amino acid sequence (SEQ ID NO: 168)

[0505] 007m amino acid sequence (SEQ ID NO: 169)

[0506] 007m-Cys-Δcys amino acid sequence (SEQ ID NO: 170)

[0507] 008m amino acid sequence (SEQ ID NO: 171)

[0508] 008m-Cys-Δcys amino acid sequence (SEQ ID NO: 172)

[0509] 009m amino acid sequence (SEQ ID NO: 173)

[0510] 009m-Cys-Δcys amino acid sequence (SEQ ID NO: 174)

[0511] 010m amino acid sequence (SEQ ID NO: 175)

[0512] 010m-Cys-Δcys amino acid sequence (SEQ ID NO: 176)

[0513] Table 41 Sequence information

[0514] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A RSV antigen, characterized in that The RSV antigen comprises mutations A102C and S362C based on SEQ ID NO: 1 or SEQ ID NO:

80.

2. The RSV antigen according to claim 1, characterized in that The RSV antigen further comprises one or more of S46G, E92D, S155C, S190F, S215P, S290C, D486C, D489C, A149C and Y458C; preferably, the RSV antigen comprises D486C, D489C, A149C and Y458C; And / or, the RSV antigen further includes a linker; preferably, the amino acid sequence of the linker is any one of SEQ ID NO:2-5 and GGS, and / or, the linker replaces positions 103-144 of SEQ ID NO:

1.

3. The RSV antigen according to claim 1 or 2, characterized in that The RSV antigen also includes an insertion fragment, which is inserted between positions 516 and 517 of SEQ ID NO:1; preferably, the amino acid sequence of the insertion fragment is shown in SEQ ID NO:6 or SEQ ID NO:

7.

4. The RSV antigen according to claim 1, characterized in that RSV antigen meets any of the following requirements: (I) The RSV antigen has the following differences compared to SEQ ID NO: 1: S155C, S290C, S190F, S215P, S46G, E92D, D486C, D489C, A102C and S362C mutations, and the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or the amino acid sequence of GGS linker replaces the 103-144th position of SEQ ID NO: 1, and the RSV antigen further includes one or more of the following: back mutation P215S, an insertion fragment of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8 or SEQ ID NO: 140, and / or a replacement fragment of the amino acid sequence of 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, and the RSV antigen also includes an insert with an amino acid sequence as shown in SEQ ID NO:

6.

5. The RSV antigen according to claim 4, characterized in that The RSV antigen also 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.

6. The RSV antigen according to claim 1, 4 or 5, characterized in that The RSV antigen is also post-translationally modified, and the post-translation modification is preferably glycosylation modification; 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.

7. The RSV antigen according to any one of claims 1 to 6, characterized in that The amino acid sequence of the RSV antigen comprises SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 34-40, 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, SEQ ID 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, SEQ ID NO:

158. The amino acid sequence shown in any one of NO:166-176.

8. An isolated nucleic acid, characterized in that The isolated nucleic acid comprises a nucleotide sequence encoding the RSV antigen of any one of claims 1-7.

9. The isolated nucleic acid of claim 8, wherein The isolated nucleic acid is mRNA, preferably codon-optimized mRNA; Preferably, the mRNA comprises one or more of a promoter region, a 5'-cap structure, a 5'UTR, a protein tag and a 3'UTR-polyA; More preferably, the promoter is a T7 promoter, and / or the protein tag is HA-HIBIT; and / or the nucleotide sequence encoding the RSV antigen includes a nucleotide sequence as shown in any one of SEQ ID NO:42, 43, 47, 51, 55, 59, 64-79, 92-118, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 142, 144, 146, 148, 150, 152, 154, 156, 158 or 182-200.

10. The isolated nucleic acid of claim 8, wherein The nucleic acid is DNA; preferably, 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.

11. A recombinant expression vector, characterized in that: The recombinant expression vector comprises a starting plasmid and the isolated nucleic acid according to any one of claims 8 to 10.

12. A transformant, characterized in that: The transformant comprises the isolated nucleic acid according to any one of claims 8 to 10 or the recombinant expression vector according to claim 11.

13. A method for preparing an isolated nucleic acid according to any one of claims 8 to 10, characterized in that: The preparation method comprises in vitro transcription of the recombinant expression vector as claimed in claim 11.

14. A method for preparing RSV antigen, characterized in that: The method comprises culturing the transformant according to claim 12 under conditions suitable for expression of the RSV antigen.

15. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises (1) the nucleic acid of claim 8 or 9, and (2) a delivery vector; Preferably, the delivery vehicle comprises LNP; More preferably, the LNP is a composition consisting of (A) SM102, cationic lipid RL151, LQ104-54, LQ104-56, LQ104-E16b-2 or compound 6, and (B) cholesterol, (C) DSPC and (D) pegylated lipid; Preferably, the molar ratio of the cationic lipid RL151, cholesterol, DSPC and pegylated lipid, such as DMG-PEG2000, is 50:38.5:10:1.5; or, 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; or, the molar ratio of LQ104-54, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; or, the molar ratio of LQ104-56, cholesterol, DSPC and DMG-PEG2000 is 50:38.5:10:1.5; Further preferably, the pharmaceutical composition is a vaccine preparation; and / or, the pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier and / or excipient; preferably, the pharmaceutical composition comprises an adjuvant.

16. A test kit or a medicine box, characterized in that: The test kit or drug kit comprises one or more of the RSV antigen according to any one of claims 1-7, the nucleic acid according to any one of claims 8-10, the recombinant expression vector according to claim 11, the transformant according to claim 12 and the pharmaceutical composition according to claim 15.

17. Use of one or more of the RSV antigen as described in any one of claims 1-7, the isolated nucleic acid as described in claims 8-10, the recombinant expression vector as described in claim 11, the transformant as described in claim 12 and the pharmaceutical composition as described in claim 15 in the preparation of a medicament for alleviating, preventing and / or treating diseases caused by RSV.

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