RSV antigen, nucleic acid, expression element, pharmaceutical composition and application thereof

Abstract

The invention discloses an RSV antigen, nucleic acid, an expression element, a pharmaceutical composition and application thereof. The RSV antigen is characterized by comprising mutations A102C and S362C on the basis of SEQ ID NO: 1. The RSV antigen has stronger immunogenicity than RSV in the prior art, can obtain higher neutralizing antibody titer, and has higher protective efficacy.

Description

[0001] This application claims the priority of Chinese patent application CN2023114397970, filed on October 31, 2023. This application cites the full text of the above Chinese patent application. Technical Field

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

[0003] Respiratory syncytial virus (RSV) can cause respiratory tract infections in children, adolescents and the elderly. Especially for pediatric patients, severe infections and respiratory sequelae may occur. RSV belongs to the genus Pneumovirus of the family Pneumoviridae and has only one serotype. Its genome is 15.2kb 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) proteins.

[0004] The infection process of RSV mainly depends on the action of F protein. During the infection process, G protein attaches the virus particles to the cell surface by interacting with the host cell attachment factor. RSV F protein drives the fusion of the virus and host cell membrane by changing from prefusion conformation to postfusion conformation, thus starting the entire virus infection cycle. Neutralizing antibodies caused by natural RSV infection are mainly targeted at RSV F protein, so most of the current vaccine designs for RSV virus are designed for RSV F protein. Three other viral protein-based candidate vaccines using non-F viral antigens are under clinical development.

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

[0006] Table 1 Available vaccines

[0007]

[0008]

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

[0010] In order to solve the above technical problems, the present invention provides a technical solution: a RSV (respiratory syncytial virus) antigen, wherein the RSV antigen comprises mutations A102C and S362C based on SEQ ID NO:1.

[0011] In a more preferred embodiment of the present invention, the RSV antigen also includes one or more of S46G, E92D, S155C, S190F, S215P, S290C, D486C, D489C, A149C and Y458C. Preferably, the RSV antigen includes D486C, D489C, A149C and Y458C.

[0012] In a preferred embodiment of the present invention, the RSV antigen further comprises 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.

[0013] In a preferred embodiment of the present invention, the RSV antigen further comprises an insertion fragment, and the insertion fragment 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.

[0014] In a preferred embodiment of the present invention, the amino acid sequence of the RSV antigen comprises an amino acid sequence as shown in any one of SEQ ID NOs: 14-30.

[0015] In order to solve the above technical problems, another technical solution provided by the present invention is: an isolated nucleic acid, wherein the isolated nucleic acid comprises a nucleotide sequence encoding the RSV antigen as described in the present invention.

[0016] In a preferred embodiment of the present invention, the isolated nucleic acid is mRNA. Preferably, the mRNA comprises one or more of a promoter region, 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.

[0017] In a specific embodiment of the present invention, the nucleotide sequence encoding the RSV antigen comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 37-61.

[0018] In order to solve the above technical problems, another technical solution provided by the present invention is: an expression element, which comprises a starting plasmid and the isolated nucleic acid as described in the present invention.

[0019] To solve the above technical problems, another technical solution provided by the present invention is: a method for preparing the isolated nucleic acid as described in the present invention, wherein the preparation method is to perform in vitro transcription on the expression element as described in the present invention. The in vitro transcription is conventional in vitro transcription in the art.

[0020] To solve the above technical problems, another technical solution provided by the present invention is: a pharmaceutical composition, which comprises the isolated nucleic acid or expression element as described in the present invention, and LNP (Lipid Nanoparticle).

[0021] In a preferred embodiment of the present invention, the LNP is SM102 (CAS2089251-47-6) or a composition consisting of a cationic lipid RL151, DSPC (distearoylphosphatidylcholine), cholesterol and a pegylated lipid. Preferably, the ratio of the cationic lipid RL151, DSPC, cholesterol and the pegylated lipid is 50:10:38.5:1.5.

[0022] In a preferred embodiment of the present invention, the nitrogen-to-phosphorus ratio in the lipid nanoparticles may be (2-30):1, and the nitrogen-to-phosphorus ratio refers to the ratio of the molar number of ionizable nitrogen atoms in one or more ionizable lipid compounds to the molar number of phosphate groups in RNA, and in the present application, it refers to the ratio of the molar number of ionizable nitrogen atoms in the ionizable lipid nanoparticles to the molar number of phosphate groups in mRNA in the pharmaceutical composition. Preferably, the nitrogen-to-phosphorus ratio is (2-20):1, preferably (3-20):1, for example (3-16):1.

[0023] In a preferred embodiment of the present invention, the pharmaceutical composition is a vaccine preparation, and preferably, the pharmaceutical composition comprises an adjuvant.

[0024] To solve the above technical problems, another technical solution provided by the present invention is: use of the RSV antigen described in the present invention, the isolated nucleic acid described in the present invention, the expression element described in the present invention or the pharmaceutical composition described in the present invention in the preparation of a drug for preventing RSV infection.

[0025] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0026] The reagents and raw materials used in the present invention are commercially available.

[0027] The positive and progressive effects of the present invention are:

[0028] The RSV antigen provided by the present invention has stronger immunogenicity than the RSV in the prior art, can obtain a higher neutralizing antibody titer, and has a higher protective efficacy. In addition, the RSV antigen provided by the present invention can maintain more prefusion configurations, can stimulate the production of more neutralizing antibodies against the prefusion configuration, and produce less neutralizing antibodies against the postfusion configuration, and has higher safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 WB results of in vitro transfection designed for different antigens; negative indicates negative control.

[0030] Figure 2 Flow cytometric results of in vitro transfection designed for different antigens.

[0031] Figure 3 These are the WB results of in vitro transfection of each antigen after different disulfide bond combinations; negative represents the negative control.

[0032] Figure 4 These are the flow cytometry results of in vitro transfection of various antigens after different disulfide bond combinations.

[0033] Figure 5 The WB results of in vitro transfection of the designed antigen are shown in Figure 2; negative indicates the negative control.

[0034] Figure 6 Flow cytometric results of in vitro transfection of designed antigens for elongation.

[0035] Figure 7 To detect the integrity of mRNA and mRNA-LNP in mouse immune samples.

[0036] Figure 8 The results are flow cytometry analysis of in vitro transfection of mouse immune samples.

[0037] Fig. 9The results are for the total antibody level detection in the serum of antigen-immunized mice.

[0038] Fig.10 The single factor levels of antigen-specific CD8+T cell immune responses in the spleen of Balb / c mice induced by different antigens.

[0039] Fig.11 The multi-factor levels of antigen-specific CD8+T cell immune responses in the spleen of Balb / c mice induced by different antigens.

[0040] Fig.12 The single factor levels of antigen-specific CD4+T cell immune responses in the spleen of Balb / c mice induced by different antigens.

[0041] Fig.13 The multi-factor levels of spleen antigen-specific CD4+T cell immune response in Balb / c mice induced by different antigens.

[0042] Fig.14 Elispot analysis of antigen-specific IFN-γ and IL-2 in the spleen of Balb / c mice induced by different antigens.

[0043] Fig.15 The structure of mRNA. DETAILED DESCRIPTION

[0044] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0045] Example 1 Experimental method

[0046] 1. In vitro transcription and mRNA transfection

[0047] 1. PCR to obtain linearized target fragment

[0048] According to the needs of PCR reaction, add 8.7 μL of ddHO into a 1.5 mL centrifuge tube. 2 O, 0.5 μL template DNA (pcDNA3.1(+), 3'UTR, 5'UTR, polyA were modified according to the expression routine of RSV virus, source: GenScript synthesis), 0.4 μL forward and 0.4 μL reverse primers (source: GenScript synthesis) and 10 μL PrimerSTAR MAX polymerase (source: TAKARA, catalog number: R450A), mix well.

[0049] As shown in Table 2:

[0050] Table 2 PCR system

[0051]

[0052] The final PCR reaction conditions are shown in Table 3 below:

[0053] Table 3 PCR conditions

[0054]

[0055]

[0056] After the PCR reaction is completed, gel electrophoresis is performed to verify that if the nucleic acid bands are uniform, the next IVT reaction can be performed. If not, it needs to be repeated.

[0057] 2. In vitro transcription (IVT)

[0058] A small amount of IVT testing was performed according to the following ratio in Table 4 (20 μL system):

[0059] Table 4 IVT system

[0060] <![CDATA[ddH 2 The]]> 11μL 10x HYB (Source: ApexBio, Catalog No.: K1083) 2μL Cap1 (B8176, 100 mM) (Source: ApexBio, Catalog No.: B8176) 0.4μL GTP (100 mM) (Source: Zhaowei, Product No.: R2331) 0.4μL ATP (100 mM) (Source: Zhaowei, Product No.: R1331) 0.4μL CTP (100mM) (Source: Zhaowei, Product No.: R3331) 0.4μL Pseudo-U (100mM) (Source: Zhaowei, Product No.: R5331) 0.4μL T7 mix (Source: ApexBio, Product No.: K1083) 2μL PCR products 3μL Total 20μL

[0061] After the preparation is completed, the entire reaction system is placed in a 37°C water bath and incubated for 2 hours. After the reaction is completed, the DNA template is digested. At this time, 1 μL DNase I (source: Vazymy, item number: EN401-01) (35 μL for 1mL IVT reaction) is added and digested at 37°C for 15 minutes before IVT recovery. The recovery steps are as follows:

[0062] I. Add 80 μL ddHO to 20 μL of digestion product 2 O, make up to 100 μL.

[0063] II. Add 350 μL Solution D and 250 μL anhydrous ethanol (source: Shanghai Test, catalog number: 801769722), mix thoroughly, transfer to a nucleic acid purification column (source: Solarbio, catalog number: N1012), and centrifuge at 10,000 g for 1 min.

[0064] 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.

[0065] IV. Add 70 μL of sodium citrate (source: Sigma, catalog number: C8532) to the purification column, place at room temperature for 1 minute, centrifuge at 10,000 g for 2 minutes, and collect the flow-through, which is the target RNA.

[0066] Prepare 1% agarose gel according to the above ratio and perform RNA electrophoresis at 160V for 20min. After the electrophoresis, observe it on a gel imaging system (source: Tianneng, item number: Tanon 4600SF). If there is a single band, the concentration can be determined.

[0067] 3. Cell Transfection

[0068] After 1 μg of mRNA sample was incubated with 3 μL of lipo2000 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% CO 2 for 18-20 h, and then the cells were collected.

[0069] 2. In vitro flow cytometry

[0070] 1. Wash the transfected cells three times with 1xPBS (Source: Bioengineering, Catalog No.: B548117-0500), and then add 500uL 1xPBS for resuspending.

[0071] 2. Take 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, item number: YB-1.5s), and mark them as Unstained group, T1 group, and T2 group respectively.

[0072] 3. Sample preparation for single staining group and LIVE&DEAD group: Since the RSV F protein conformation is divided into postfusion and prefusion conformations, and the prefusion conformation is further divided into monomer and trimer, this experiment used three antibodies for detection, namely 4D7 (source: Baiying Bio, batch number: 20220808M011), D25 (source: Baiying Bio, batch number: 20220808M010), and AM14 (source: Baiying Bio, batch number: 20220808M009). At the same time, referring to the literature knowledge, the above three antibodies were labeled with AF647 (source: thermo Fisher, product number: A20186) and AF488 fluorescent dyes (source: thermoFisher, product number: A20181), and named 4D7-AF647, D25-AF488, and AM14-AF488, respectively. The single-stained tubes were stained with 4D7-AF647 and D25-AF488 respectively. The LIVE&DEAD group was prepared by incubating in a 65°C metal bath (source: Hangzhou Ruicheng Instrument Co., Ltd., catalog number: DH300) for 30 minutes, and then immediately placed on ice. When loading the sample, SYTOX (source: thermo, catalog number: S34861) with a final concentration of 1 / 5000 was added for signal reading.

[0073] 4. Sample preparation for the experimental group: 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 h, centrifuge at 4°C, 500×g for 4 min, discard the supernatant, wash three times with 150uL 1xPBS, and resuspend in 75 μL 1xPBS.

[0074] 5. Before loading, add 75 μL of SYTOX with a final concentration of 1 / 5000 to the LIVE&DEAD group, T1 group, and T2 group. Incubate at room temperature for 5 min and perform flow cytometry detection on a flow cytometer (source: Cytek, catalog number: NL-CLC V16 B14 R8).

[0075] 3. Western Blot

[0076] 1. Preparation of cell lysis buffer: Add the transfected cell sample to the cell lysis buffer containing protease inhibitors (source: ApexBio, catalog number: K1012), incubate on ice for 10 minutes, centrifuge at 12000g for 10 minutes, and collect the supernatant.

[0077] 2. Preparation of electrophoresis samples: Divide the above lysed samples into two equal parts, add 4xLDS loading (source: invitrogen, catalog number: NP0008) containing 40mM DTT and 4x LDS loading without DTT respectively. The sample containing DTT needs to be boiled at 95℃ for 8min, and immediately placed on ice for cooling after boiling. The sample without DTT does not need any treatment.

[0078] 3. Add the prepared electrophoresis samples to the corresponding channels of the protein gel (source: Tanno, catalog number: 180-9115H), and use 1xMOPS Running buffer (source: Tanno, catalog number: BT8100-2002H) to perform protein electrophoresis in the corresponding electrophoresis tank (source: Tanno, catalog number: VE-180).

[0079] 4. Color development of target protein: 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 the end, use ECL luminescent color development solution (source: ApexBio, catalog number: K1200) for analysis in a gel imaging system.

[0080] 5. After the target protein is developed, use 1x stripping buffer to wash the PVDF membrane (source: Cytia, catalog number: 10600023) to remove the bound primary and secondary antibodies, then block it, incubate with appropriate concentrations of primary antibody (source: PROTEINTECH, catalog number: 60004-1-Ig) and secondary antibody (source: Abcam, catalog number: ab6728), and after completion, use ECL luminescent colorimetric solution (source: ApexBio, catalog number: K1200) for analysis in a gel imaging system.

[0081] 4. mRNA-LNP integrity detection

[0082] 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.

[0083] 2. Pipette 400ng of the above sample, add 1μL RNA loading and 1μL ddH 2 O, boil the sample at 70℃ for 8min and then immediately place the sample on ice. Centrifuge at short speed after cooling. Take an appropriate volume of sample and perform agarose gel electrophoresis on an electrophoresis instrument.

[0084] 5. Total antibody level detection

[0085] 1. Antigen coating: Dilute the target antigen (source: Shanghai Lanque Biotechnology, batch number: rq20221031) to 1 μg / mL, and draw 100 μL into a 96-well ELISA coated plate (source: Nest, catalog number: 514201), and incubate overnight in a 4°C refrigerator away from light. Block the plate with 1xPBST solution containing 3% bovine serum albumin for 2 hours at room temperature. Dilute the immune mouse serum 100 times as the initial concentration, and then make 4-fold serial dilutions in PBS buffer, with a total of 11 gradients. 1xPBST wash plate incubated with serially diluted serum at room temperature for 2 hours. To determine RSV F-specific antibody response, the plate 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 about 12 minutes, the color development reaction was terminated with 1 M sulfuric acid, and the absorbance was measured at a wavelength of 450 nm using a microplate reader (model: Synergy H1, purchased from BioTek).

[0086] 6. Neutralizing antibody level detection

[0087] 1. Novozyme detection method:

[0088] 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 and placed in a cell culture incubator (source: Thermo, catalog number: 3111) (37°C, 5% CO 2 ) Culture overnight and ensure that the cell confluence is about 90% on the next day before starting the experiment.

[0089] II. Gradient dilution: dilute the sample 20 times in the first well, and perform 3-fold gradient dilution, with a total of 8 dilutions (including the first well), single well or 2 replicate wells.

[0090] III. Virus dilution: Add an appropriate amount of virus according to the PFU value of the virus.

[0091] IV. Neutralization reaction: Add the diluted virus to the sample well and virus control well, and dilute it down to 4 dilutions in 2-fold gradient in the back-drip wells. Incubate at 37°C and 5% CO 2 Incubate in the incubator for approximately 1 hour.

[0092] V. Virus adsorption: Add the above viruses, serum neutralization products, and positive and back-tick well viruses to the cells prepared in advance, 50 μl per well, 2 replicates, at 37°C, 5% CO 2 After culturing in the incubator for about 2 hours, the medium was changed and 100 μl of culture medium was added to each well and the culture was continued for about 22 hours.

[0093] VI. Plate measurement: discard the supernatant, fix the cells, add fluorescently labeled detection antibodies, and read the plate using a CTL instrument.

[0094] 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.

[0095] 2. Extraordinary detection method:

[0096] I. One day before the experiment, Hep-2 cells were seeded into a 96-well cell culture plate and kept aside until the cells grew and converged to a monolayer;

[0097] II. Take the inactivated mouse serum and dilute it 2-fold in DMEM in a 96-well plate, and dilute it at different multiples of 1:20, 1:40...1:5120;

[0098] III. Take out the frozen RSV virus solution from the -80℃ refrigerator, dilute the RSV virus to the target concentration with serum-free DMEM, then add 50μL of virus solution to each well of the diluted serum and incubate at room temperature for 2h;

[0099] IV. Dilute the virus diluent used in the experiment by 1, 10, 100, and 1000 times, add 100 μL / well to a 96-well plate, and perform 12 replicates for each dilution to perform a virus regression experiment;

[0100] V. Remove cells from the incubator, digest and suspend the cells, and adjust the cell density to 1×10 cells / mL using DMEM containing 4% FBS and 1% double antibody. 5 cells / mL;

[0101] VI. After the neutralization and incubation, add 100 μL / well of the prepared cell suspension to the 96-well sample plate and culture in a 37°C, 5% CO2 incubator for 5 days, during which the CPE situation is continuously observed and recorded;

[0102] Judgment criteria: 1) The cell control (CC) should be free of lesions, and the virus control (VC) should have lesions;

[0103] 2) The neutralization titer of the negative serum control (NC) should be <1:40;

[0104] 3) The neutralization titer of the positive serum control (PC) should be within the compliance range;

[0105] 4) In the virus regression test, the virus titer of lgTCID50 / ml should be between 630 and 6310TCID 50 / ml;

[0106] If any of the above items are not met, this experiment will be invalid.

[0107] 3. Zhongke Guobang testing method:

[0108] I. Sample preparation: Thaw samples at room temperature. Heat inactivate serum samples at 56°C for 30 min. Take out samples after 30 min and cool to room temperature.

[0109] II. Sample loading and dilution: 40-fold initial dilution, followed by 4-fold dilution, for 6 dilutions. Take a 96-well plate, use the second column as the cell control (CC), and the third column as the virus control (VC). Add the 20-fold diluted sample to wells B4-B11, use a multichannel pipette to gently mix the liquid in wells B4-B11, and then perform a 4-fold dilution until wells G4-G11. Take the positive reference and negative reference for loading and dilution.

[0110] III. Virus dilution: dilute the virus to 800-1200 FFU / well on ice and add to each well except the cell control.

[0111] IV. Place the above 96-well plate in a 37°C, 5% CO2 incubator and incubate for 1 hour.

[0112] V. Digest and count HEp-2 cells and adjust the number to 2.0 × 10 using complete culture medium. 5 pcs / mL, 100 μL / well was added into a 96-well plate.

[0113] 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.

[0114] 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.

[0115] VII. Antigen-specific T cell response detection

[0116] Antigen-specific T cell responses in mouse spleens were measured by intracellular factor 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 resuspended in RIPM1640 complete medium were added, incubated at 37°C for 1 hour, and protein transport inhibitors were added and incubated 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. Anti-mouse CD3-FITC, CD4-APC and CD8-Percp-cy5.5 antibody mixtures (Cat. Nos.: 553061, 553051, 551162, purchased from: BD Bioscience) against surface molecules were added for staining. After 30 minutes of incubation, the cells were washed twice, fixed and permeabilized for 20 minutes, and then washed once, and stained with a mixed solution of anti-cytokine anti-mouse IFN-γ-Pe-Cy7, IL-2-BV605, TNF-α-BV650, IL-4-BV711, and IL-5-PE (Cat. Nos. 557649, 563943, 563911, 564005, 562049, purchased from BD Bioscience). After 30 minutes of incubation, the cells were washed twice and resuspended in 200 μL PBS. The fluorescence signal was analyzed by CYTEK Aurora / NL flow cytometer (model: NL-CLC V16B14R8, purchased from CytekBiosciences).

[0117] 8. Antigen-specific T cell response detection

[0118] Day 1

[0119] 1. Activation of pre-coated plates

[0120] Dayu: Add 200 μL of Dayu Elispot-specific serum-free medium to each well and remove it after standing at room temperature for 5-10 minutes.

[0121] Mabtech: Wash the plate 4 times with sterile 1xPBS, add 200 μL / well of serum-free medium, and incubate at room temperature for at least 30 min.

[0122] 2. After adding cells to the wells, add stimulants, cover the plate, and place in a 37°C, 5% CO2 incubator for 23 hours. Avoid shaking during the incubation period.

[0123] Day 2:

[0124] Post-culture operation (sterile operation is no longer required)

[0125] 1. Lyse cells: Pour out the culture medium in the wells. Add pre-cooled deionized water, 200 μL / well, and place in a 4°C refrigerator for 10 minutes to lyse the cells hypotonicly.

[0126] 2. Prepare 1× washing buffer: dilute with deionized water (1:50) to make 1x washing buffer for later use. Store in a 4℃ refrigerator after use (make as much as you need).

[0127] 3. Wash the plate: Pour the liquid in the wells, 1x washing buffer, 250μL / well, wash 6 times, stay for 1min each time. Dry the plate on absorbent paper each time.

[0128] 4. Detection antibody incubation: Add the diluted biotin-labeled antibody working solution to each experimental well, 100 μL / well. Incubate at 37°C for 1 hour.

[0129] Dilution Buffer R (10×): Dilute with 1× PBS (1:9) to prepare 1× Dilution Buffer R working solution for later use.

[0130] Biotinylated antibody: dilute (1:100) with Dilution buffer R (1x) to prepare the working solution.

[0131] 5. Wash the plate: Pour the liquid in the wells, 1x washing buffer, 250μL / well, wash 6 times, stay for 1min each time. Dry the plate on absorbent paper each time.

[0132] 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.

[0133] Streptavidin-HRP: dilute with Dilution buffer R (1x) (1:100) to prepare the working solution.

[0134] 7. Wash the plate: Pour the liquid in the wells, 1x washing buffer, 250μL / well, wash 6 times, stay for 1min each time. Dry the plate on absorbent paper each time.

[0135] 8. Color development: Add the prepared ACE color development solution to each experimental well, 100μL / well. Incubate at room temperature away from light for 5-30 minutes, and choose the end time of color development according to the spot formation. If the room temperature is lower than 20℃, it is recommended to develop the color in a 37℃ incubator and check every 5-10 minutes.

[0136] ACE colorimetric solution: Mix ACE dilution, ACE solution I (20x), ACE solution II (20x), and ACE solution III (200x) in a clean container at a ratio of 180:10:10:1 to prepare the working solution. See Table 5 below. The half-life of ACE colorimetric solution at room temperature is 30 minutes. Prepare it for use immediately.

[0137] Table 5 ACE system

[0138]

[0139] 9. Stop color development: Pour the liquid in the wells, wash the front and back of each experimental well and the base 3 times with deionized water to stop color development. Place the plate in a cool place at room temperature, wait for it to dry naturally, and then close the base.

[0140] 10. Count the spots on the ELISPOT plate and record various parameters of the spots for statistical analysis. If readings cannot be taken in time, seal and store the plates away from light and try to read the plates within one week.

[0141] Antigen design:

[0142] The wild-type RSV sequence (SEQ ID NO: 1) is as follows:

[0143] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPAANSRARRELPRFMNYTLNNTKNTNVTLSKKRKRRFLGFLLG VGSAIASGIAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYS IMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0144] The mutation sites involved in 003m and the extended version are shown in Table 6 below:

[0145] Table 6 003m

[0146]

[0147] 003m-1xGCN4t is an insertion of EDKIEEILSKIYHIENEIARIKKLIGEA (SEQ ID NO: 6) between positions 516 and 517 of SEQ ID NO: 14.

[0148] 003m-2xGCN4t is an insertion of EDKIEEILSKIYHIENEIARIKKLIGEAIYHIENEIARIKKLIGEA (SEQ ID NO: 7) between positions 516 and 517 of SEQ ID NO: 14.

[0149] The lengthened sequence is shown in Table 7 below:

[0150] Table 7 Lengthened sequence

[0151]

[0152] The mutation sites involved in 006m and its derivative variants are shown in Table 8 below:

[0153] Table 8 006m

[0154]

[0155] The mutation sites involved in 007m and its derivative variants are shown in Table 9 below:

[0156] Table 9 007m

[0157]

[0158] The mutation sites involved in 008m and its derivative variants are shown in Table 10 below:

[0159] Table 10 008m

[0160]

[0161] The mutation sites involved in 009m and its derivative variants are shown in Table 11 below:

[0162] Table 11 009m

[0163]

[0164]

[0165] The mutation sites involved in 010m and its derivative variants are shown in Table 12 below:

[0166] Table 12 010m

[0167]

[0168] The mutation sites involved in DS-Cav1 and its derivative variants are shown in Table 13 below

[0169] Table 13 DS-Cav1

[0170] name Known anthropogenic mutations Linker Sequence DS-Cav1 S155C, S290C, S190F, V207L No linker

[0171] DS-Cav1-1xGCNt is obtained by inserting EDKIEEILSKIYHIENEIARIKKLIGEA (SEQ ID NO: 6) between positions 516 and 517 of SEQ ID NO: 10.

[0172] DS-Cav1-2xGCNt is obtained by inserting EDKIEEILSKIYHIENEIARIKKLIGEAIYHIENEIARIKKLIGEA (SEQ ID NO: 7) between positions 516 and 517 of SEQ ID NO: 10.

[0173] The structures of all mRNAs in this application experiment are as follows Fig.15 shown.

[0174] The serial codes are explained in Table 14 below:

[0175] Table 14 Code Explanation

[0176] Type Mutation site _Cys D486C / D489C -ΔCys C102A / C362A -DS A149C / Y458C

[0177] Example 2 Antigen screening

[0178] This example uses the operations of “1. In vitro transcription and mRNA transfection”, “2. In vitro flow cytometry” and “3. Western Blot” in Example 1.

[0179] Each antigen expression gene was synthesized by GenScript.

[0180] The coding genes of each antigen are shown in SEQ ID NO:31-61, and the amino acid sequences of each antigen are shown in SEQ ID NO:8-30. (Among them, the coding nucleic acid sequence used by 003m is SEQ ID NO:37)

[0181] result:

[0182] Figure 1 : From the WB results of in vitro transfection, m5K6I, 006m-010m, and 003m all have relatively obvious expressions, and because the above mutants all contain linkers, from the results of reduced gel, they all show obvious single-chain forms. In the absence of linkers, the F1 and F2 domains are clearly distinguished.

[0183] From the perspective of the average 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.

[0184] Table 15 Fluorescence data

[0185]

[0186] Figure 2:There are three flow cytometry detection antibodies, namely 4D7 (labeled with Alexa Flour 647 to detect postfusion conformation), AM14 (labeled with Alexa Flour 488 to detect trimer prefusion conformation), and D25 (labeled with Alexa Flour 488 to detect prefusion conformation). From the results, the conformation of mA2F (i.e. wild type, WT) is basically equal to that of postfusion and prefusion, and the conformation of DS-Cav1 is mostly prefusion conformation, but some postfusion conformation exists. Compared with other mutants, the membrane surface conformation of 003m is basically prefusion conformation, and no obvious postfusion conformation exists. The membrane surface conformations of 006m-010m, m5K6I, mF111, and mSC-TM are also largely postfusion conformations.

[0187] Example 3 Disulfide bond modification of antigen

[0188] This example uses "1. In vitro transcription and mRNA transfection", "2. In vitro flow cytometry" and "3. Western Blot" in Example 1.

[0189] result:

[0190] Figure 3 : From the WB results, after adding the disulfide bond pair D486C / D489C, 007m-010m was renamed 007m_Cys-010_Cys, and there was no obvious decrease in the expression level; after adding the disulfide bond pair A149C / Y458C to 003m and 007m-010m (renamed as 003m_DS and 007m_DS-010m_DS), the expression level of the protein was significantly reduced; and after removing the disulfide bond pair A102C / S362C from 003m and 007m_Cys-010_Cys (renamed as 003m_ΔCys and 007m_Cys_ΔCys-010_Cys_ΔCys), its expression level also decreased significantly.

[0191] Figure 4:From the flow results, 003m, 007m_Cys-010_Cys can maintain the prefusion conformation relatively stably, while 007m-010m, 007m_DS-010m_DS, 007m_Cys_ΔCys-010_Cys_ΔCys can not maintain the prefusion conformation stably, and although 003m-DS can maintain the prefusion conformation, its membrane surface expression has a significant decrease, and combined with WB results, the overall expression has also been significantly reduced. The cell surface fluorescence signal values ​​of flow are shown in Table 16 below, which can reflect the content of the target protein on the cell surface.

[0192] Since the above-mentioned different disulfide bond combinations failed to significantly increase the protein expression level, it can be seen that the design of 003m is also relatively reasonable.

[0193] Table 16 Fluorescence data

[0194]

[0195] Example 4 Lengthened design of antigen

[0196] This example uses “1. In vitro transcription and mRNA transfection”, “2. In vitro flow cytometry” and “3. Western Blot” in Example 1.

[0197] Figure 5 : For 003m and DS-Cav1, we made the extended mutations of 1xGCN4t and 2xGCN4t respectively (both inserted between the 516th and 517th positions of 003m and DS-Cav1). From the WB results, the extended mutation design had a more obvious decrease in the expression level in 003m.

[0198] From the average fluorescence intensity (Table 17), after the lengthening design, the antigen expression on the membrane surface showed a clear downward trend.

[0199] Table 17 Fluorescence data

[0200]

[0201] Figure 6 : 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.

[0202] 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.

[0203] Example 5 Preparation of lipid nanoparticles

[0204] The mRNA of the antigen sequence selected in the above example was ionized (cationic) at low pH, and coated into nanoparticles by two auxiliary lipids DSPC (distearylphosphatidylcholine, item number: B90536, manufacturer: Nippon Seika Co., Ltd.) and cholesterol (item number: C00373, manufacturer: Nippon Seika Co., Ltd.), as well as RL151 (item number: W211-YB211202, purchased from: Zhejiang Shenzhou Pharmaceutical Co., Ltd.), pegylated lipid (DMG-PEG2000, item number: M-DMG-2000, purchased from: JenKem). The mRNA aqueous solution was prepared by mixing the mRNA dissolved in ultrapure water with a 100 mM (millimoles per liter, or mmol / L) citrate buffer at a volume ratio of 1:1 at pH 4.0. The ratio of the four lipid components was 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 were mixed in a NanoAssemblr (manufacturer: Precision Nanosystems) microfluidic mixing system with H 2 O:EtOH=3:1 volume mixing ratio and a constant total flow rate of 12mL / min are mixed, and lipid nanoparticles are prepared according to the nitrogen-phosphorus ratio of ionizable lipids to mRNA of (3-15):1. Lipid nanoparticles containing mRNA (mRNA-LNP) are obtained. Lipid nanoparticles containing mRNA (mRNA-LNP) are obtained. The lipid nanoparticles are dialyzed, concentrated, filtered and stored to obtain a vaccine containing the target antigen.

[0205] Figure 7 : From the gel electrophoresis results, neither the mRNA purification product nor the LNP-coated mRNA underwent obvious degradation.

[0206] Figure 8 : According to 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 forms 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.

[0207] Example 6 First round of animal immunization

[0208] 1. Animal vaccination and serum collection

[0209] 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"), 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).

[0210] The immunization method is shown in Table 19 below:

[0211] Table 19 Immunization method

[0212]

[0213]

[0214] The administration was performed twice, with an interval of 3 weeks. The dosage for each administration is shown in the table above. Use a sterile insulin syringe of appropriate specifications to accurately draw 400 μl of the test sample (slowly invert the syringe 5-10 times to mix before drawing the test sample), and select the right lower limb of the animal for intramuscular injection, with a single injection of 50 μl.

[0215] The sera of the immunized mice were collected and inactivated at 56°C for 0.5 hours to detect RSV F protein-specific IgG and neutralizing antibodies.

[0216] 2. Antibody Level Detection

[0217] The total antibody level detection method used the antibody level detection method in the fourth part of Example 1, and the neutralizing antibody detection method used the "fifth, total antibody level detection" and "sixth, neutralizing antibody level detection" in Example 1. Results:

[0218] Fig. 9 : From the perspective of total antibody levels in serum, the serum produced by mA2F (i.e. wild type) immunized mice mainly targets the postfusion conformation. In the serum of DS-Cav1 immunized mice, the total amount of antibodies against the prefusion conformation was the highest, followed by m5K6I and Moderna-ΔCT, and then 003m. The total antibody level against the postfusion conformation produced by mice immunized with 003m was the lowest. The total antibody against the prefusion conformation produced by 003m-1xGCN4t was significantly higher than that of 003m and 003m-2xGCN4t, and the total antibody level against the postfusion conformation was also at a low level.

[0219] Judging from the results of the neutralization experiment (Table 20 and Table 21), the neutralizing antibody titer produced by 003m-1xGCN4t is also high.

[0220] Table 20 Serum neutralizing antibody titers in the second week of the second immunization

[0221]

[0222]

[0223] Table 21 Neutralizing antibody titers of each subtype in serum two weeks after second immunization

[0224]

[0225] 3. Cellular Immune Response in Balb / c Mice

[0226] The detection process refers to "VII. Antigen-specific T cell response detection" in Example 1.

[0227] The results of intracellular factor detection were basically consistent with those of serum titer detection.

[0228] The expression levels of IL-4 and IL-5 in each experimental group were low, suggesting no associated risks.

[0229] Fig.10 and Fig.11 : Judging from the results of CD8 single factor and multi-factor, the trend is basically consistent with the total anti-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.

[0230] Fig.12 and Fig.13 : Judging from the CD4 single factor and multifactor results, mA2F induced higher levels of IL-4 and IL-5, while the other groups were slightly lower. In the extended type, both IL-4 and IL-5 levels were lower, indicating that the VED (vaccine enhanced disease) phenomenon it may induce is weaker.

[0231] IV. Cellular Immune Response of Balb / c Mice (Elispot)

[0232] The detection process refers to "VIII. ELISPot" in Example 1.

[0233] Detection time: 95 days after the second vaccination

[0234] Immunoassay: 2.5μg

[0235] like Fig.14As shown: The results of Elispot and intracellular factor detection are basically consistent, and Moderna-ΔCT has the highest levels of IFN-γ and IL-2. In the elongated mutation of DS-Cav1, the results of the two mutants are not much different, while in the elongated form of 003m, the results of 1xGCN4t are slightly better than 2xGCN4t.

[0236] All sequences involved in this application are as follows

[0237] Nucleic acid sequence

[0238] mA2F encoding nucleic acid sequence (SEQ ID NO:31)

[0239]

[0240] m5K6I encoding nucleic acid sequence (SEQ ID NO:32)

[0241]

[0242] DS-Cav1 encoding nucleic acid sequence (SEQ ID NO: 33)

[0243]

[0244] DS-Cav1-1xGCN4t encoding nucleic acid sequence (SEQ ID NO: 34)

[0245]

[0246] DS-Cav1-2xGCN4t encoding nucleic acid sequence (SEQ ID NO: 35)

[0247]

[0248] Moderna-ΔCT encoding nucleic acid sequence (SEQ ID NO: 36)

[0249]

[0250] 003m encoding nucleic acid sequence (SEQ ID NO: 37)

[0251]

[0252] 003m-1xGCN4t encoding nucleic acid sequence (SEQ ID NO: 38)

[0253]

[0254] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 1) (SEQ ID NO: 39)

[0255]

[0256] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 2) (SEQ ID NO: 40)

[0257]

[0258] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 3) (SEQ ID NO: 41)

[0259]

[0260] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 4) (SEQ ID NO: 42)

[0261]

[0262] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 5) (SEQ ID NO: 43)

[0263]

[0264] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 6) (SEQ ID NO: 44)

[0265]

[0266] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 7) (SEQ ID NO: 45)

[0267]

[0268] 003m-1xGCN4t optimized coding nucleic acid sequence (optimize 8) (SEQ ID NO: 46)

[0269]

[0270] 003m-2xGCN4t encoding nucleic acid sequence (SEQ ID NO: 47)

[0271]

[0272] 003s encoding nucleic acid sequence (SEQ ID NO:48)

[0273]

[0274] 006m encoding nucleic acid sequence (SEQ ID NO: 49)

[0275]

[0276] 007m encoding nucleic acid sequence (SEQ ID NO: 50)

[0277]

[0278] 007m-cys encoding nucleic acid sequence (SEQ ID NO:51)

[0279]

[0280]

[0281] 008m encoding nucleic acid sequence (SEQ ID NO:53)

[0282]

[0283] 008m-cys encoding nucleic acid sequence (SEQ ID NO:54)

[0284]

[0285] 008m-Cys-Δcys encoding nucleic acid sequence (SEQ ID NO: 55)

[0286]

[0287] 009m encoding nucleic acid sequence (SEQ ID NO:56)

[0288]

[0289] 009m-cys encoding nucleic acid sequence (SEQ ID NO:57)

[0290]

[0291] 009m-Cys-Δcys encoding nucleic acid sequence (SEQ ID NO: 58)

[0292]

[0293] 010m encoding nucleic acid sequence (SEQ ID NO: 59)

[0294]

[0295] 010m-Cys encoding nucleic acid sequence (SEQ ID NO: 60)

[0296]

[0297] 010m-Cys-Δcys encoding nucleic acid sequence (SEQ ID NO: 61)

[0298]

[0299] Amino acid sequence

[0300] mA2F amino acid sequence (SEQ ID NO:8)

[0301] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0302] m5K6I amino acid sequence (SEQ ID NO:9)

[0303] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKRKRRFLGFLLG VGSAIASGVAVSKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKQLLPIVNKQSCSISNIETVIEFQQKNNRLEITREFSVNAGVTTPVSTYMLTNSELLSLINMPITNDQKKLMSNNVQIVRQQSYS IMSIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKTASNKNRG IIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFDYPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYKARSTPVTLSKDQLSGINNIAFSN

[0304] DS-Cav1 Amino Acid Sequence (SEQ ID NO:10) MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0305] DS-Cav1-1xGCN4t Amino Acid Sequence (SEQ ID NO:11)

[0306] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVEDKIEEILSKIYHIENEIARIKKLIGEANAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0307] Amino acid sequence of DS-Cav1-2xGCN4t (SEQ ID NO: 12)

[0308] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATNNRARRELPRFMNYTLNNAKKTNVTLSKKRKRRFLGFLLGVGSAIASGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVEDKIEEILSKIYHIENEIARIKKLIGEAIYHIENEIARIKKLIGEANAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0309] Moderna - ΔCT Amino Acid Sequence (SEQ ID NO:13)

[0310] MELLILKANAITTILTAVTFCFASGQNITEEFYQSTCSAVSKGYLSALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQSTPATGSGSAICSGVAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPILNKQSCSISNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSNNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSRTLPSEVNLCNVDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYCVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLSLIAVGLLLYC

[0311] 003m amino acid sequence (SEQ ID NO:14)

[0312] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0313] 003m-1xGCN4t Amino Acid Sequence (SEQ ID NO:15)

[0314] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNV EDKIEEILSKIYHIENEIARIKKLIGEA NAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0315] 003m-2xGCN4t Amino Acid Sequence (SEQ ID NO:16)

[0316] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNV EDKIEEILSKIYHIENEIARIKKLIGEAIYHIENEIARIKKLIGEA NAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0317] 003s Amino acid sequence (SEQ ID NO:17)

[0318] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELL

[0319] 006m Amino acid sequence (SEQ ID NO:18)

[0320] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0321] 007m Amino acid sequence (SEQ ID NO:19)

[0322] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0323] 007m-cys amino acid sequence (SEQ ID NO:20)

[0324] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYNAVTDLQLLMQSTPCGGSGGSGSAIASGIAVCKVLHLEGEVNKIKSA LLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPL YGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGGTKCTASNKNRGIIKTFSNGC DYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFDYPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILALLIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0325] 007m-Cys-Δcys Amino Acid Sequence (SEQ ID NO:21) MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPAGGSGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0326] 008m Amino Acid Sequence (SEQ ID NO:22)

[0327] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0328] 008m-cys amino acid sequence (SEQ ID NO:23)

[0329] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0330] 008 m-Cys-Δcys Amino Acid Sequence (SEQ ID NO:24)

[0331] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPAGGSGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0332] 009m amino acid sequence (SEQ ID NO:25)

[0333] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGSGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0334] 009m-cys amino acid sequence (SEQ ID NO:26)

[0335] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGSGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0336] 009 m-Cys-Δcys Amino Acid Sequence (SEQ ID NO: 27)

[0337] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPAGSGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0338] 010 m Amino acid sequence (SEQ ID NO:28)

[0339] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0340] 010 m-Cys Amino Acid Sequence (SEQ ID NO:29)

[0341] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPCGGSGSAIASGIAVCKVLHLEGEVNKIKSALLSTNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLYGVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQCNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0342] 010 m-Cys-Δcys Amino Acid Sequence (SEQ ID NO:30)

[0343] MELPILKTNAITTILAAVTLCFASSQNITEEFYQSTCSAVSKGYLGALRTGWYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTDLQLLMQSTPAGGSGSAIAASGIAVCKVLHLEGEVNKIKSALL STNKAVVSLSNGVSVLTFKVLDLKNYIDKQLLPIVNKQSCSIPNIETVIEFQQKNNRLLEITREFSVNAGVTTPVSTYMLTNSELLSLINDMPITNDQKKLMSSNVQIVRQQSYSIMCIIKEEVLAYVVQLPLY GVIDTPCWKLHTSPLCTTNTKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPSEVNLCNIDIFNPKYDCKIMTSKTDVSSSVITSLGAIVSCYGKTKCTASNKNRGIIKTFSNGCD YVSNKGVDTVSVGNTLYYVNKQEGKSLYVKGEPIINFYDPLVFPSCEFCASISQVNEKINQSLAFIRKSDELLHNVNAGKSTTNIMITTIIIVIIVILLALIAVGLLLYCKARSTPVTLSKDQLSGINNIAFSN

[0344] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited 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.

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 The amino acid sequence of the RSV antigen comprises the amino acid sequence shown in any one of SEQ ID NOs: 14-30.

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

6. The isolated nucleic acid according to claim 5, characterized in that The isolated nucleic acid is mRNA; preferably, the mRNA comprises one or more of a promoter region, 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; further preferably, the nucleotide sequence encoding the RSV antigen comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 37-61.

7. An expression element, characterized in that The expression element comprises a starting plasmid and the isolated nucleic acid according to claim 5 or 6.

8. A method for preparing an isolated nucleic acid according to claim 6, characterized in that: The preparation method comprises in vitro transcription of the expression element as claimed in claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the nucleic acid of claim 5 or 6 or the expression element of claim 7, and LNP; Preferably, the LNP is SM102 or a composition consisting of cationic lipid RL151, DSPC, cholesterol and PEGylated lipid; preferably, the ratio of cationic lipid RL151, DSPC, cholesterol and PEGylated lipid is 50:10:38.5:1.5; More preferably, the pharmaceutical composition is a vaccine preparation; preferably, the pharmaceutical composition comprises an adjuvant.

10. Use of the RSV antigen according to any one of claims 1 to 4, the isolated nucleic acid according to claim 5 or 6, the expression element according to claim 7 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for preventing RSV infection.

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