A self-replicating mRNA sequence and a vaccine for preventing MenB and a preparation method thereof

Abstract

The present invention relates to the technical field of MenB vaccines, and specifically to a self-replicating mRNA sequence, a vaccine for preventing MenB, and a method for preparing the same. A self-replicating mRNA sequence comprises RNA encoding a region in an amino acid sequence, wherein the amino acid sequence comprises a single sequence shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, which is concatenated in any order to form a tandem sequence. The present application screens amino acid sequences for strains found in China's monitoring coverage, provides a vaccine for preventing MenB, and reduces the mortality rate caused by meningococcal B infection in China. The vaccine for preventing MenB provided by the present application has a low number of injection needles, a low dose, and a high expression effect, which can reduce the dosage and frequency of administration, while improving the expression efficiency of the vaccine, in order to achieve a better immune effect.

Description

Technical Field

[0001] The present invention relates to the technical field of MenB vaccines, and in particular to a self-replicating mRNA sequence and a vaccine for preventing MenB and a preparation method thereof. Background Art

[0002] Epidemic meningitis is caused by Neisseria meningitidis. Serotype B meningitis has been prevalent in my country and many other countries in recent years, with infants and children under five years old being at high risk. Current Serotype B meningococcal vaccines do not cover the strain prevalent in my country. Therefore, developing an effective vaccine to prevent Serotype B meningitis is crucial for its prevention and control.

[0003] Recombinant protein vaccine production is plagued by complex processes, high costs, and difficult and time-consuming purification, hindering public acceptance. Therefore, the development of an efficient and safe self-replicating mRNA molecule and a vaccine for MenB prevention is a major technical challenge currently in need of resolution. Summary of the Invention

[0004] This patented self-replicating mRNA vaccine targets four antigens of meningitis B. The vaccine, encapsulated in lipid nanoparticles, encodes a tandem sequence of four antigenic proteins. Cell and animal experiments have demonstrated that the designed self-replicating mRNA vaccine molecule can consistently and efficiently produce proteins targeting the four antigens of meningitis B, demonstrating its broad potential for application.

[0005] During the experiment, the inventors discovered that a self-replicating mRNA sequence is used to obtain a mRNA vaccine sequence for preventing MenB, which produces a specific immune response, has high immunogenicity, a long-lasting immune response, and a low vaccine cost.

[0006] The first aspect of the present invention provides a self-replicating mRNA sequence, comprising an RNA coding region in an amino acid sequence, wherein the amino acid sequence comprises a tandem sequence formed by connecting the single sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in any order.

[0007] For strains found in China's surveillance, the protein sequence of this application covers the ST-4821, ST-41 / 44, ST-32, ST-198, and ST-175 clone complexes in Neisseria meningitidis serogroup B bacteria. Amino acid sequences were screened for Chinese strains, and the four antigenic proteins fHbp, NHBA, NadA, and PorA were optimized and the original signal peptide sequence was deleted. The optimized protein amino acid sequence includes SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0008] In one embodiment, the amino acid sequence is optimized for fHbp, NHBA, NadA and PorA antigenic proteins and the original signal peptide sequence is deleted.

[0009] In one embodiment, the fHbp antigen protein molecule deletes the original 1-19 signal peptide sequence to form the amino acid sequence SEQ ID NO: 7.

[0010] In one embodiment, the NHBA antigen protein molecule deletes the original 1-17 signal peptide sequence as the amino acid sequence SEQ ID NO: 8.

[0011] In one embodiment, the NadA antigen protein molecule deletes the original 1-23 signal peptide sequence as the amino acid sequence SEQ ID NO: 9.

[0012] In one embodiment, the PorA antigen protein molecule deletes the original 1-18 signal peptide sequence to form the amino acid sequence SEQ ID NO: 10.

[0013] In one embodiment, a signal peptide sequence of one or more of the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 is added to the 5' end of the tandem sequence.

[0014] In one embodiment, the amino acid sequence is a tandem sequence formed by connecting SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in any order.

[0015] During their experiments, the inventors discovered that designing based on Chinese strain sequences ensures that the constructed tandem sequences more accurately match the characteristics of the MenB strains prevalent in China, thus laying the foundation for the effectiveness of the MenB vaccine. During their experiments, the inventors discovered that different permutation orders can affect the spatial structure and immunogenicity of the final protein. The multiple permutation possibilities provide more options for screening the most immunologically active amino acid sequences.

[0016] In one embodiment, the signal peptide sequence added to the 5' end of the tandem sequence includes a new coronavirus signal peptide, the sequence of which is SEQ ID NO: 1.

[0017] In one embodiment, the signal peptide sequence added to the 5' end of the tandem sequence includes a tPA signal peptide, the sequence of which is SEQ ID NO: 2.

[0018] In one embodiment, the signal peptide sequence added to the 5' end of the tandem sequence includes an fHbp signal peptide, the sequence of which is SEQ ID NO: 3.

[0019] In one embodiment, the signal peptide sequence added to the 5' end of the tandem sequence includes an NHBA signal peptide, the sequence of which is SEQ ID NO: 4.

[0020] In one embodiment, the signal peptide sequence added to the 5' end of the tandem sequence includes a NadA signal peptide, the sequence of which is SEQ ID NO: 5.

[0021] In one embodiment, the signal peptide sequence added to the 5' end of the tandem sequence includes a PorA signal peptide, the sequence of which is SEQ ID NO: 6.

[0022] In order to further optimize the tandem sequence, the inventors added a signal peptide sequence at the 5' end of the tandem sequence to guide the newly synthesized protein into a specific secretory pathway within the cell, ensuring that the protein can be correctly positioned and function, and ensuring the secretion of self-replicating enzymes and antigens; during the experiment, the inventors found that different signal peptides can have different guiding properties, which are suitable for different cellular environments and protein functional requirements.

[0023] In one embodiment, a signal peptide sequence shown in SEQ ID NO: 1 is added to the 5' end of the tandem sequence.

[0024] To further optimize the tandem sequence, a signal peptide sequence represented by SEQ ID NO: 1 was added to the 5' end of the tandem sequence. During the experiment, the inventors found that the signal peptide of SEQ ID NO: 1 performed best in directing the protein encoded by the tandem sequence into the secretory pathway, significantly improving protein expression efficiency and correct localization rate, thereby enhancing the immunogenicity of the vaccine.

[0025] In one embodiment, the individual sequences in the tandem sequence are connected using a flexible linker in the amino acid sequence SEQ ID NO: 11.

[0026] In one embodiment, the tandem sequences are connected using a flexible linker shown in SEQ ID NO: 11 between the individual sequences.

[0027] In one embodiment, the amino acid sequence is a tandem sequence formed by connecting SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in series in any order, and the signal peptide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 is added to the 5' end of the tandem sequence, and the individual sequences in the tandem sequence are connected using the flexible linker shown in SEQ ID NO: 11.

[0028] In one embodiment, the amino acid sequence is a tandem sequence formed by connecting SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in any order, and the signal peptide sequence shown in SEQ ID NO: 1 is added to the 5' end of the tandem sequence, and the individual sequences in the tandem sequence are connected using the flexible linker shown in SEQ ID NO: 11.

[0029] During the experiment, the inventors found that in order to ensure that the protein domains encoded by each individual sequence in the tandem sequence can function independently and avoid steric hindrance and interference between each other, limiting the tandem sequence to use a flexible linker as shown in SEQ ID NO: 11 between individual sequences can provide sufficient spatial freedom for each protein domain, allowing it to fold and move freely, thereby ensuring the correct conformation and functional integrity of the entire tandem protein.

[0030] In one embodiment, a MITD molecule sequence is added to the end of the tandem sequence, and the MITD molecule sequence includes the amino acid sequence SEQ ID NO: 12 or SEQ ID NO: 13.

[0031] In one embodiment, the preferred MITD sequence is SEQ ID NO: 12, and the MITD sequence designed by adding mutations to the SEQ ID NO: 12 is more preferably SEQ ID NO: 13.

[0032] In experiments, the inventors discovered that MITD is a special sequence that can be added to vaccine antigens. It can guide the antigens into specific areas of dendritic cells, making them easier to be processed by dendritic cells and presented to T cells, thereby initiating an immune response.

[0033] Regarding mutation design, the inventors discovered in experiments a wild-type sequence of the antigen fHbp, SEQ ID NO: 27, and a mutated sequence of the antigen fHbp, SEQ ID NO: 28.

[0034] Regarding mutation design, the inventors discovered in experiments a wild-type sequence of SEQ ID NO: 29 based on the antigen NHBA and a mutated sequence of SEQ ID NO: 30 based on the antigen NHBA.

[0035] Regarding mutation design, the inventors discovered in experiments a wild-type sequence of the antigen NadA, SEQ ID NO: 31, and a mutated sequence of the antigen NadA, SEQ ID NO: 32.

[0036] Regarding mutation design, the inventors discovered in experiments a wild-type sequence of the antigen PorA, SEQ ID NO: 33, and a mutated sequence of the antigen PorA, SEQ ID NO: 34.

[0037] In one embodiment, the self-replicating mRNA sequence includes target protein amino acids for molecular tandem design of fHbp, NHBA, NadA and PorA antigenic proteins.

[0038] In one embodiment, the self-replicating mRNA sequence comprises the RNA coding region of the amino acid sequence provided by the first set of experiments.

[0039] In one embodiment, molecules of four antigenic proteins, fHbp, NHBA, NadA, and PorA, are designed in tandem as self-replicating mRNA sequences.

[0040] In one embodiment, molecular tandem design is performed on the four antigenic proteins fHbp, NHBA, NadA, and PorA to obtain the new coronavirus SP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid sequence, and the target protein amino acid sequence is SEQ ID NO: 14.

[0041] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 14 is adjusted to optimize its GC content and secondary structure, and the optimized base sequence is SEQ ID NO: 15.

[0042] In one embodiment, molecular tandem design is performed on four antigenic proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid sequence of tPASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the amino acid sequence of the target protein is SEQ ID NO: 16.

[0043] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 16 is adjusted to optimize its GC content and secondary structure, thereby obtaining an optimized base sequence of SEQ ID NO: 17.

[0044] In one embodiment, molecular tandem design is performed on the four antigenic proteins fHbp, NHBA, NadA, and PorA to obtain the fHbpSP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid sequence, and the target protein amino acid sequence is SEQ ID NO: 18.

[0045] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 18 is adjusted to optimize its GC content and secondary structure, thereby obtaining an optimized base sequence of SEQ ID NO: 19.

[0046] In one embodiment, molecular tandem design is performed on four antigenic proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid sequence of NHBASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence is SEQ ID NO: 20.

[0047] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 20 is adjusted to optimize its GC content and secondary structure, and the optimized base sequence is SEQ ID NO: 21.

[0048] In one embodiment, molecular tandem design is performed on the four antigenic proteins fHbp, NHBA, NadA, and PorA to obtain the target protein amino acid sequence NadASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA, and the target protein amino acid sequence is SEQ ID NO: 22.

[0049] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 22 is adjusted to optimize its GC content and secondary structure, and the optimized base sequence is SEQ ID NO: 23.

[0050] In one embodiment, molecular tandem design is performed on the four antigenic proteins fHbp, NHBA, NadA, and PorA to obtain the PorASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid sequence, and the target protein amino acid sequence is SEQ ID NO: 24.

[0051] In one embodiment, the codon sequence of the target protein amino acid sequence SEQ ID NO: 24 is adjusted to optimize its GC content and secondary structure, thereby obtaining an optimized base sequence of SEQ ID NO: 25.

[0052] In one embodiment, the sequence stop codon of the self-replicating mRNA comprises the amino acid sequence SEQ ID NO:26.

[0053] During their experiments, the inventors discovered that to achieve precise control of protein translation, they could terminate protein synthesis by adding a stop codon, thereby producing the desired protein variant or avoiding the production of unnecessary proteins. Specifically, the stop codon sequence was designed as SEQ ID NO: 26.

[0054] During their experiments, the inventors discovered that to improve gene expression, high-frequency synonymous codons should be selected based on the codon usage preference of the host cell. The stability of the mRNA sequence's secondary structure is closely related to the half-life of the mRNA sequence and protein expression, and enhancing the stability of the mRNA sequence's secondary structure is beneficial for the expression of the target protein. High guanine and cytosine (G and C) content can improve mRNA sequence stability and translation efficiency. Adjusting the GC content of the target gene's codon sequence can improve its expression level in the host cell. By adjusting the GC content of the target gene's codon sequence, the optimized amino acid sequences of SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQ ID NO:25 have GC contents of 58.85%, 58.34%, 58.58%, 58.10%, 58.88%, and 58.77%, respectively.

[0055] In one embodiment, the self-replicating mRNA sequence includes one or more sequences shown in SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 or SEQ ID NO: 25.

[0056] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 15.

[0057] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 17.

[0058] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 19.

[0059] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 21.

[0060] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 23.

[0061] In one embodiment, the self-replicating mRNA sequence is the sequence shown in SEQ ID NO: 25.

[0062] In one embodiment, the self-replicating mRNA sequence comprises the amino acid sequence shown in SEQ ID NO:17.

[0063] A second aspect of the present invention provides a vaccine for preventing MenB, comprising a self-replicating mRNA sequence.

[0064] A third aspect of the present invention provides a method for preparing a vaccine for preventing MenB, comprising the following steps:

[0065] Vector construction, plasmid preparation, mRNA stock solution preparation, mRNA stock solution encapsulation, to obtain the vaccine for preventing MenB.

[0066] In one embodiment, the method for preparing the vaccine for preventing MenB comprises the following steps:

[0067] S1. Vector construction: The self-replicating mRNA sequence was cloned into a self-replicating vector by SalI / XbaI double digestion to construct a self-replicating mRNA plasmid; the DNA sequence of the self-replicating mRNA plasmid was then introduced into Escherichia coli cells using a heat shock method to establish a seed bank;

[0068] S2. Plasmid preparation:

[0069] ① Fermentation culture: Take engineered bacteria seeds containing plasmids from the seed bank, inoculate them into antibiotic screening culture medium, and amplify the culture in a fermenter to replicate the plasmid in large quantities to obtain culture;

[0070] ② Crude extraction: The culture in the fermentation tank is centrifuged to obtain bacterial sludge, and the resuspension solution is added to the bacterial sludge in sequence for mixing, standing, removing floating impurities, filtering and ultrafiltration concentration to obtain crude plasmid DNA;

[0071] ③Purification: Purify crude plasmid DNA to obtain supercoiled plasmid;

[0072] ④ Linearization: Cut the supercoiled plasmid with restriction endonucleases and purify it by anion chromatography to obtain the linearized plasmid template;

[0073] S3. Preparation of mRNA stock solution:

[0074] ① Synthesize mRNA by mixing linearized plasmid DNA template, RNA polymerase, and NTPs to synthesize mRNA;

[0075] ② Purification: purify the synthesized mRNA to obtain mRNA stock solution;

[0076] S4. Encapsulation of mRNA stock solution:

[0077] ① Obtain nanoparticles by diluting the mRNA stock solution as the aqueous phase, mixing the aqueous phase with the organic phase and preparing nanoparticles using a microfluidic preparation instrument;

[0078] ② Solidification and purification: PBS buffer is added to the nanoparticles for solidification to obtain LNP particles, which are then purified and filtered to obtain the LNP-encapsulated mRNA pharmaceutical composition, namely the MenB vaccine.

[0079] In one embodiment, the method for preparing the vaccine for preventing MenB comprises the following steps:

[0080] S1. Construction of recombinant strains:

[0081] Ⅰ. Construction of recombinant plasmid:

[0082] ① According to the self-replicating mRNA sequences optimized in Examples 1, 2, 3, 4, 5, and 6 in the second group of experiments, after sequencing verification, the 4 antigen linker genes carrying SalI upstream and XbaI downstream and the self-replicating vector JJ-Sap-2 were double-digested with SalI+XbaI;

[0083] ② Recover the 4 antigen ligation genes and self-replicating vector after enzyme digestion, prepare the reaction system according to the molar molecular ratio of ligation gene: vector of 3-5:1, add T4 DNA ligase and reaction buffer, mix well, and ligate at room temperature for 30-60 minutes or at 16°C overnight to obtain the recombinant plasmid.

[0084] II. Obtaining recombinant strains:

[0085] ① Take 3-5 μL of the ligated product of the recombinant plasmid and add it to 50 μL of thawed competent cells DH5α, mix well and heat shock at 42°C for 30-60 seconds;

[0086] ② Add 500 μL of liquid LB medium to the heat-shocked E. coli cells, culture them in a constant temperature shaking incubator at 37°C and 200 rpm for about one hour, then spread them on solid LB medium and culture them in a constant temperature incubator at 37°C for 16-18 hours.

[0087] S2. Preparation of recombinant plasmid:

[0088] ① Select a well-growing single colony and add it to liquid synthetic culture medium. Culture it in a constant temperature shaking incubator at 37°C and 200 rpm for 4-6 hours. Take a small amount of bacterial liquid and use a plasmid extraction kit to extract the plasmid and perform SalI+XbaI enzyme digestion for identification.

[0089] ② The bacterial solution with the correct enzyme digestion results was cultured in a constant temperature shaking incubator at 37°C and 200 rpm for 14-16 hours, and then centrifuged at 4000 rpm for about 5-10 minutes to harvest the bacterial slurry;

[0090] ③ Lyse the collected bacterial sludge, add Lysis Buffer I, and thoroughly resuspend the bacteria using an oscillator. The volume ratio of bacterial sludge to Lysis Buffer I is 4-6:250. Once the bacteria are fully suspended, add Lysis Buffer II and gently invert to lyse the bacteria. Add Lysis Buffer III and immediately and gently invert to mix thoroughly to neutralize the mixture. Let it stand at room temperature for 5-10 minutes. The volume ratio of Lysis Buffer I: Lysis Buffer II: Lysis Buffer III is 5:5:7. Centrifuge the lysed mixture at 12,000 rpm for 5-10 minutes. Remove the supernatant and apply it to a DNA adsorption column. Centrifuge at 12,000 rpm for 1 minute to remove the waste liquid. Add the wash solution to the adsorption column and centrifuge at 12,000 rpm for 1 minute to remove the waste liquid. Add the eluent, let it stand at 37°C for 2-5 minutes, and then centrifuge at 12,000 rpm for 1 minute. Collect the eluate to obtain the circular plasmid DNA solution.

[0091] S3. Preparation of mRNA stock solution:

[0092] I. Plasmid linearization:

[0093] ① Use the BspQ1 enzyme digestion system and incubate at 50°C for 1-2 hours to digest the circular plasmid into a linearized plasmid. Take a portion of the digestion product for agarose gel electrophoresis verification;

[0094] ② Add 0.5 times the volume of magnetic beads to the remaining enzyme digestion product and mix thoroughly. Incubate at room temperature for 10-15 minutes, then place on a magnetic stand. After the solution is clear, remove the supernatant.

[0095] ③ Rinse the magnetic beads with freshly prepared 80% ethanol solution, remove the supernatant and dry the magnetic beads, add an appropriate volume of Nuclease-free H2O, pipette and mix well, then aspirate the supernatant to obtain the purified linear plasmid DNA solution and store it at -20℃.

[0096] II. In Vitro Transcription (IVT)

[0097] ① Mix the linear plasmid DNA template, NTPs, CleanCapAU, buffer and other substrates, then add T7 RNA polymerase. Mix well and place in a 37°C PCR instrument for in vitro transcription reaction. After 1-3 hours of reaction, add DNaseI to terminate the reaction.

[0098] ② Add nuclease-free water and LiCl solution to the reaction solution. The volume ratio of the reaction solution to water and LiCl is 1:1.5:1.5. After inversion to mix, incubate at -20°C for 15-30 minutes. Centrifuge at 4°C and 12000g for 10-15 minutes to remove the waste liquid. Add 70% ethanol to wash impurities. After removing the supernatant, add nuclease-free water to dissolve the precipitate to obtain the mRNA stock solution, which is stored at -80°C.

[0099] S4. Encapsulation of mRNA Stock Solution

[0100] ① Thaw the mRNA stock solution in a cold water bath and dilute it with citrate buffer to a concentration of about 0.1-0.5 mg / ml, which is the mRNA working solution;

[0101] ② The self-replicating mRNA is encapsulated in LNP, and the molar concentration ratio of the cation to DSPC, CHO-HP and DMP-PEG2000 in the lipid phase is (30-60):(5-10):(30-60):(2-5), and the total molar concentration of the lipid phase is 15-20 mmol / L; the cation in the lipid phase includes DLin-MC3-DMA, JK-0042, JK-0043, and JK-0045.

[0102] ③ The volume ratio of the organic phase to the mRNA working solution is 1:(2-4), and nanoparticles LNP are prepared using a microfluidic preparation instrument;

[0103] ④ Add 5-10 times the volume of PBS buffer to dilute the harvested nanoparticles, use a 100KD ultrafiltration centrifuge tube to centrifuge and concentrate at 4°C and 1500g. When the volume is concentrated to approximately the volume of the initially obtained LNP, add 5-10 times the volume of PBS buffer to dilute, continue to concentrate to the expected concentration volume, and obtain the finished LNP product after filtering through a 0.2μm membrane to obtain the vaccine for preventing MenB.

[0104] The preventive MenB vaccine was stored at -20°C.

[0105] In one embodiment, the method for preparing the vaccine for preventing MenB comprises the following steps:

[0106] S1. Vector construction: The self-replicating mRNA sequence was cloned into a self-replicating vector by SalI / XbaI double digestion to construct a self-replicating mRNA plasmid; the DNA sequence of the self-replicating mRNA plasmid was then introduced into Escherichia coli cells using a heat shock method to establish a research and development seed bank;

[0107] S2. Plasmid preparation:

[0108] ① Take the engineered bacteria seeds from the R&D seed bank and add them to a liquid synthetic medium shaking incubator. Check that the seeds have an OD600 of 0.6-2.5. The expanded seeds are cultured in a fermenter for 38-45 hours. The fermentation temperature is controlled at 28-37°C, the speed is 100-1000 rpm, the dissolved oxygen level is linked to the stirring speed, the pH is controlled at 6.3-7.0, and the feed is linked to the pH. After the fermentation is completed, the supernatant is removed by centrifugation at 4000 rpm for about 5 minutes to harvest the bacterial sludge.

[0109] ② Mix the bacterial sludge with the resuspension S1 at a mass ratio of 1:5-15. Add S2 solution at a ratio of 1-3 times the volume of S1 solution, let it stand for 2-10 minutes, then add 1-5 times the volume of S3 solution and ammonium bicarbonate, and let it stand for 30-60 minutes. Remove floating impurities and filter through a filter. Concentrate the solution using an ultrafiltration system. The pH value of S1 solution is 7.5-8.5 and it is composed of 25mM-100mM glucose, 15mM-35mM Tris-HCl, and 5mM-20mM EDTA. The S2 solution is composed of a freshly prepared solution of 0.1M-0.3M NaOH and 0.5%-2% SDS. The S3 solution is composed of a mixture of 3MKAc and 2MHAc.

[0110] ③ Use molecular sieve chromatography column chromatography for chromatography, the chromatography linear flow rate is 1-4 cm / h, about 1 / 3CV after the start of sample loading, the target product will flow through, the UV curve rises, and the flow-through peak is collected. The buffer solution used for the molecular sieve chromatography column chromatography is composed of 1.8M-2.3M (NH4)2SO4, 0.2M-0.5MNaCl, 0.1MTris-HCl and 0.01MEDTA-2Na.

[0111] ④ Perform chromatography using an affinity chromatography column, equilibrated with buffer solution A, at a linear flow rate of 10-15 cm / h. After loading, wash impurities with 5%-15% buffer solution B for approximately 2-5 CV.

[0112] The buffer solution A consists of 1.8M-2.3M (NH4)2SO4, 0.1MTris-HCl, and 0.01MEDTA-2Na;

[0113] The buffer solution B is composed of 1.8M-2.3M (NH4)2SO4, 0.2M-0.5MNaCl, 0.1M Tris-HCl, and 0.01MEDTA-2Na; and 20%-50% buffer solution B is used for elution, and the elution peak is collected.

[0114] ⑤ Use an ultrafiltration system to change the solution, replace the salt solution with 1×TE buffer solution, and select a hollow fiber column with a diameter of 100kD-500kD.

[0115] ⑥ Mix the supercoiled plasmid DNA, linearized enzyme, and water, incubate at 45°C-55°C for 0.5-4 hours, purify using anion chromatography column chromatography and ultrafiltration to obtain a linearized plasmid DNA template, and store at -80°C; the chromatography linear flow rate is 10-15 cm / h, the chromatography solution A is composed of EDTA-2Na and Tris-HCl, the mass ratio of EDTA-2Na to Tris-HCl is 1.681:7.88, and the pH of the chromatography solution A is 7.5;

[0116] The chromatography solution B consists of EDTA-2Na and Tris-HCl, the mass ratio of EDTA-2Na to Tris-HCl is 1.681:7.88, and the pH of the chromatography solution B is 7.5.

[0117] S3. Preparation of mRNA stock solution:

[0118] ① Take the linearized plasmid DNA template, NTPs and buffer substrate, mix them, add polymerase, and react at 33℃-37℃ in the synthesis workstation for 1-3 hours;

[0119] ② Purify by affinity chromatography using OligodT as the chromatographic medium. Before purification, dilute the reaction solution 15-30 times with an equilibration solution at a linear flow rate of 6 cm / h-20 cm / h, and wash the impurities for 2-8 CV. The equilibration solution consists of NaCl, EDTA, and TrisHCl in a mass ratio of 9.35:0.067:0.32. After washing, collect the eluted peak with pure water.

[0120] ③ Purify using a TFF system with pure water as the replacement solution. Use a transmembrane pressure of 5-20 psi and a shear rate of 1000-4000 / sec. Diafiltration is performed 8-15 times, followed by reconcentration to the target concentration. The resulting sample is filtered through a 0.2 μm filter to obtain the mRNA stock solution, which is then stored at -80°C.

[0121] S4. Encapsulation of mRNA stock solution:

[0122] ① Thaw the mRNA stock solution in a cold water bath, dilute the mRNA stock solution with buffer salt to a concentration of 0.1mg / ml-0.5mg / ml, and mark it as RNA working solution.

[0123] The volume ratio of the organic phase to the aqueous phase RNA working solution is 1:3, and the nanoparticles are prepared by a microfluidic preparation instrument.

[0124] ② Add 1× PBS (4-20 times the volume of the harvested nanoparticles) to solidify the LNP particles. Purify the LNPs using a TFF ultrafiltration system, replacing the solution with 1× PBS. A transmembrane pressure of 10 psi and a shear rate of 2000 / sec are used. Dilution and concentration are repeated 4-8 times, followed by 4-8 cycles of diafiltration and reconcentration 2-3 times. The resulting sample is filtered through a 0.2 μm filter to obtain the mRNA-encapsulated LNP pharmaceutical composition, which is the MenB vaccine and stored at -20°C.

[0125] During the experiment, the inventors found that the MenB vaccine obtained by the preparation method of the above-mentioned vaccine can achieve a low injection number, low dose, and high expression effect. The possible reason is that saRNA can self-replicate in cells as a carrier, which can reduce the dosage and frequency of administration, while improving the expression efficiency of the vaccine, thereby achieving a better immune effect.

[0126] During the experiment, the inventors found that the use of the above-mentioned method for preparing a MenB preventive vaccine to obtain different formulations of the MenB preventive vaccine can improve the efficacy of the vaccine. In addition, efficacy experiments have shown that different formulations of the MenB preventive vaccine have different effects on efficacy. The vaccine after the improved process has the advantages of quickly triggering an immune response, high antibody levels, and providing long-term immune protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 These are the electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 15 and its recombinant plasmid.

[0128] Figure 2 These are the electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 17 and its recombinant plasmid.

[0129] Figure 3 These are the electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 19 and its recombinant plasmid.

[0130] Figure 4 These are the electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 21 and its recombinant plasmid.

[0131] Figure 5 These are the electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 23 and its recombinant plasmid.

[0132] Figure 6 These are the electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 25 and its recombinant plasmid.

[0133] Figure 7 The protein band detection results obtained by immunoblotting for the sequences provided in Examples 7, 8, 9, 10, 11 and 12.

[0134] Figure 8 This is the result of fHbp binding antibody detection.

[0135] Figure 9 This is the result of NHBA binding antibody detection.

[0136] Figure 10 This is a diagram showing the results of NadA binding antibody detection.

[0137] Figure 11 This is the result of porA binding antibody detection.

[0138] Figure 12 This is the FACs test result on D34 days after the injection of MenB vaccine.

[0139] Figure 13 This is the FACs test result 70 days after the injection of MenB vaccine.

[0140] Figure 14 This is the ELISPOT test result on day 34 after the injection of the MenB vaccine.

[0141] Figure 15 This is the ELISPOT test result 70 days after the injection of MenB vaccine.

[0142] Figure 16 This is the result of MenB (PorA) binding antibody titer test.

[0143] Figure 17 This is the result of MenB (NadA) binding antibody titer test.

[0144] Figure 18 This is the result of MenB (NHBA) binding antibody titer test.

[0145] Figure 19 This is the result of MenB (fHbp) binding antibody titer test.

[0146] Figure 20 This is the result of CD4+IFNg+T cell typing test.

[0147] Figure 21 This is the result of CD4+CD69+T cell typing test.

[0148] Figure 22 This is the result of CD8+IFNg+T cell typing test.

[0149] Figure 23 This is the result of CD8+CD69+T cell typing test.

[0150] Figure 24 The ELISPOT test results are shown on day 35 after injection.

[0151] Figure 25 The ELISPOT test results are shown on day 70 after injection.

[0152] Figure 26The results of serum bactericidal titer test of SEQ ID NO: 17 split-preparation mixture.

[0153] Figure 27 The results of serum bactericidal titer test of SEQ ID NO: 15 split-preparation mixture.

[0154] Figure 16 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with PorA antigen protein for serum binding antibody detection.

[0155] Figure 16 In b, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with PorA antigen protein for serum binding antibody detection results when the dosage is 5 μg.

[0156] Figure 16 In c, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with PorA antigen protein for serum binding antibody detection results.

[0157] Figure 17 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with NadA antigen protein for serum binding antibody detection results.

[0158] Figure 17 In b, the serum binding antibody test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture and the split-preparation mixture provided in Example 16 were combined with NadA antigen protein when the dosage was 5 μg.

[0159] Figure 17In c, the serum binding antibody test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture and the split-preparation mixture provided in Example 16 were combined with NadA antigen protein when the dosage was 15 μg.

[0160] Figure 18 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with NHBA antigen protein for serum binding antibody detection results.

[0161] Figure 18 In b, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with NHBA antigen protein for serum binding antibody detection results when the dosage is 5 μg.

[0162] Figure 18 In c, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with NHBA antigen protein for serum binding antibody detection results.

[0163] Figure 19 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with fHbp antigen protein for serum binding antibody detection results.

[0164] Figure 19 b is the result of serum binding antibody detection when the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture and the split-preparation mixture provided in Example 16 are used at a dosage of 5 μg in combination with fHbp antigen protein.

[0165] Figure 19 In c, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 15 μg in combination with fHbp antigen protein for serum binding antibody detection results.

[0166] Figure 24 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 are injected into mice at D35 days at a dosage of 1 μg.

[0167] Figure 24 Figure b shows the ELISPOT test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice on day 35 at a dosage of 5 μg.

[0168] Figure 24 Figure c is the ELISPOT test result of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice on D35 days at a dosage of 5 μg.

[0169] Figure 25 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15-split-preparation mixture provided in Example 14 are injected into mice at D70 days at a dosage of 1 μg.

[0170] Figure 25Figure b shows the ELISPOT test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice at a dosage of 5 μg on day 70.

[0171] Figure 25 Figure c is the ELISPOT test result of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice at a dosage of 5 μg on day 70.

[0172] Beneficial effects

[0173] 1. This application screens amino acid sequences for strains found in China's surveillance coverage, provides a preventive MenB vaccine, and reduces the mortality rate caused by meningococcal B infection in China.

[0174] 2. In this application, the MITD molecular sequence is added to the end of the tandem sequence for amino acids that are prone to mutation, which can guide the antigen into specific areas of dendritic cells, making the antigen more easily processed by dendritic cells and presented to T cells, thereby initiating an immune response.

[0175] 3. In this application, a signal peptide sequence is added to the 5' end of the tandem sequence to guide the newly synthesized protein into a specific secretory pathway in the cell, ensuring that the protein can be correctly positioned and function, and ensuring the secretion of self-replicating enzymes and antigens; during the experiment, the inventors found that different signal peptides can have different guiding properties, which are suitable for different cellular environments and protein functional requirements.

[0176] 4. In order to achieve precise control of protein translation, the present application can terminate the synthesis of optimized proteins by adding stop codons, thereby producing the desired protein variants or avoiding the production of unnecessary proteins.

[0177] 5. The tandem sequence provided in this application can ensure that the protein domains encoded by each individual sequence in the tandem sequence can function independently, avoiding steric hindrance and interference between each other. Limiting the tandem sequence to use a flexible linker connection as shown in SEQ ID NO: 11 between individual sequences can provide sufficient spatial freedom for each protein domain, enabling it to fold and move freely, thereby ensuring the correct conformation and functional integrity of the entire tandem protein.

[0178] 6. This application uses saRNA as a carrier that can self-replicate in cells, so that the anti-MenB vaccine can have a low injection number, low dose, and high expression effect, which can reduce the dosage and frequency of administration, while improving the expression efficiency of the vaccine, in order to achieve better immune effects. DETAILED DESCRIPTION

[0179] In order to better understand the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that the embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0180] Unless otherwise specified, the technical means adopted in this embodiment are conventional technical means in this field.

[0181] The first set of experiments

[0182] The first set of experiments screened amino acid sequences.

[0183] For strains found in China's surveillance, the protein sequence of this application covers the ST-4821, ST-41 / 44, ST-32, ST-198, and ST-175 clone complexes in Neisseria meningitidis serogroup B bacteria. Amino acid sequences were screened for Chinese strains, and the four antigenic proteins fHbp, NHBA, NadA, and PorA were optimized and the original signal peptide sequence was deleted. The amino acid sequence is SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0184] The fHbp antigen protein molecule deletes the original 1-19 signal peptide sequence to form the amino acid sequence SEQ ID NO: 7.

[0185] The NHBA antigen protein molecule deletes the original 1-17 signal peptide sequence as the amino acid sequence SEQ ID NO: 8.

[0186] The NadA antigen protein molecule deletes the original 1-23 signal peptide sequence to form the amino acid sequence SEQ ID NO: 9.

[0187] The PorA antigen protein molecule deletes the original 1-18 signal peptide sequence to form the amino acid sequence SEQ ID NO: 10.

[0188] The second set of experiments

[0189] The second set of experiments provided self-replicating mRNA sequences, and molecular tandem design was performed on four antigenic proteins, fHbp, NHBA, NadA, and PorA, as self-replicating mRNA sequences.

[0190] The second group of experiments includes Example 1, Example 2, Example 3, Example 4, Example 5, and Example 6.

[0191] The self-replicating mRNA sequence provided in Example 1 is SEQ ID NO: 15.

[0192] The self-replicating mRNA sequence provided in Example 2 is SEQ ID NO: 17.

[0193] The self-replicating mRNA sequence provided in Example 3 is SEQ ID NO: 19.

[0194] The self-replicating mRNA sequence provided in Example 4 is SEQ ID NO: 21.

[0195] The self-replicating mRNA sequence provided in Example 5 is SEQ ID NO: 23.

[0196] The self-replicating mRNA sequence provided in Example 6 is SEQ ID NO: 25.

[0197] The self-replicating mRNA sequence includes the RNA of the coding region in the amino acid sequence provided by the first group of experiments, and the amino acid sequence is formed by connecting the single sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in series in any order to form a tandem sequence.

[0198] The signal peptide sequence added to the 5' end of the tandem sequence is a novel coronavirus signal peptide, and the sequence is SEQ ID NO: 1.

[0199] The tandem sequences were connected using a flexible linker shown in SEQ ID NO: 11 between the individual sequences.

[0200] A MITD molecular sequence with a mutation design is added to the end of the tandem sequence, and the MITD molecular sequence is the amino acid sequence SEQ ID NO: 13.

[0201] Example 1

[0202] Based on the self-replicating mRNA sequence provided by the second set of experiments, this example performs molecular tandem design on the four antigenic proteins fHbp, NHBA, NadA, and PorA to obtain the novel coronavirus SP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acids, the amino acid sequence of which is SEQ ID NO: 14.

[0203] In this example, the codon sequence of the target gene SEQ ID NO: 14 was adjusted to optimize its GC content and secondary structure to obtain the optimized base sequence SEQ ID NO: 15, thereby obtaining a self-replicating mRNA sequence.

[0204] Example 2

[0205] Based on the self-replicating mRNA sequence provided by the second set of experiments, this example performed molecular tandem design for four antigenic proteins, fHbp, NHBA, NadA, and PorA, to obtain the tPASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid sequence, the target protein amino acid sequence of which is SEQ ID NO: 16.

[0206] In this example, the codon sequence of the target gene SEQ ID NO: 16 was adjusted to optimize its GC content and secondary structure to obtain the optimized base sequence SEQ ID NO: 17, thereby obtaining a self-replicating mRNA sequence.

[0207] Example 3

[0208] Based on the self-replicating mRNA sequence provided by the second set of experiments, this example performs molecular tandem design on four antigenic proteins, fHbp, NHBA, NadA, and PorA, to obtain the fHbpSP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA target protein amino acid sequence, the target protein amino acid sequence of which is SEQ ID NO: 18.

[0209] In this example, the codon sequence of the target gene SEQ ID NO: 18 was adjusted to optimize its GC content and secondary structure to obtain the optimized base sequence SEQ ID NO: 19, thereby obtaining a self-replicating mRNA sequence.

[0210] Example 4

[0211] Based on the self-replicating mRNA sequence provided by the second set of experiments, this example performs molecular tandem design on four antigenic proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid sequence of NHBASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA. The amino acid sequence of the target protein is SEQ ID NO: 20.

[0212] In this example, the codon sequence of the target gene SEQ ID NO: 20 was adjusted to optimize its GC content and secondary structure to obtain the optimized base sequence SEQ ID NO: 21, thereby obtaining a self-replicating mRNA sequence.

[0213] Example 5

[0214] Based on the self-replicating mRNA sequence provided by the second set of experiments, this example performs molecular tandem design on four antigenic proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid sequence of NadASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA. The amino acid sequence of the target protein is SEQ ID NO: 22.

[0215] In this example, the codon sequence of the target gene SEQ ID NO: 22 was adjusted to optimize its GC content and secondary structure to obtain the optimized base sequence SEQ ID NO: 23, thereby obtaining a self-replicating mRNA sequence.

[0216] Example 6

[0217] Based on the self-replicating mRNA sequence provided by the second set of experiments, this example performs molecular tandem design on four antigenic proteins, fHbp, NHBA, NadA, and PorA, to obtain the target protein amino acid sequence of PorASP-fHbp-Linker-NHBA-Linker-NadA-Linker-PorA. The amino acid sequence of the target protein is SEQ ID NO: 24.

[0218] In this example, the codon sequence of the target gene SEQ ID NO: 24 was adjusted to optimize its GC content and secondary structure to obtain the optimized base sequence SEQ ID NO: 25, thereby obtaining a self-replicating mRNA sequence.

[0219] The second set of experiments provided a self-replicating mRNA sequence that was optimized using SEQ ID NO: 26 as the stop codon.

[0220] The self-replicating mRNA sequence includes the amino acid sequence shown in SEQ ID NO: 17.

[0221] The third group of experiments

[0222] The third group of experiments provides a preventive MenB vaccine and its preparation method.

[0223] The preventive MenB vaccine comprises a self-replicating mRNA sequence provided by the second set of experiments.

[0224] The third group of experiments includes Example 7, Example 8, Example 9, Example 10, Example 11, and Example 12.

[0225] The self-replicating mRNA sequence used in the vaccine for preventing MenB provided in Example 7 is SEQ ID NO: 15.

[0226] The self-replicating mRNA sequence used in the MenB preventive vaccine provided in Example 8 is SEQ ID NO: 17.

[0227] The self-replicating mRNA sequence used in the MenB preventive vaccine provided in Example 9 is SEQ ID NO: 19.

[0228] The self-replicating mRNA sequence used in the vaccine for preventing MenB provided in Example 10 is SEQ ID NO: 21.

[0229] The self-replicating mRNA sequence used in the MenB preventive vaccine provided in Example 11 is SEQ ID NO: 23.

[0230] The self-replicating mRNA sequence used in the MenB preventive vaccine provided in Example 12 is SEQ ID NO: 25.

[0231] The preparation methods of the vaccines for preventing MenB provided in Examples 7, 8, 9, 10, 11 and 12 are prepared according to the following steps.

[0232] The method for preparing the vaccine for preventing MenB comprises the following steps:

[0233] S1. Construction of recombinant strains:

[0234] Ⅰ. Construction of recombinant plasmid:

[0235] ① According to the self-replicating mRNA sequences optimized in Examples 1, 2, 3, 4, 5, and 6 in the second group of experiments, after sequencing verification, the 4 antigen linker genes carrying SalI upstream and XbaI downstream and the self-replicating vector JJ-Sap-2 were double-digested with SalI+XbaI;

[0236] ② Recover the 4 antigen-linked genes and self-replicating vector after enzyme digestion, prepare a reaction system according to the molar molecular ratio of the linked gene to the vector of 5:1, add T4 DNA ligase and reaction buffer, mix well, and connect at room temperature for 30 minutes to obtain the recombinant plasmid.

[0237] II. Obtaining recombinant strains:

[0238] ① Take 5 μL of the ligated product of the recombinant plasmid and add it to 50 μL of thawed competent cells DH5α, mix well and heat shock at 42°C for 60 seconds;

[0239] ② Add 500 μL of liquid LB medium to the heat-shocked E. coli cells, culture them in a constant temperature shaking incubator at 37°C and 200 rpm for about one hour, then spread them on solid LB medium and culture them in a constant temperature incubator at 37°C for 16 hours.

[0240] S2. Preparation of recombinant plasmid:

[0241] ① Select a well-growing single colony and add it to liquid synthetic culture medium. Culture it in a constant temperature shaking incubator at 37°C and 200 rpm for 4 hours. Take a small amount of bacterial liquid and use a plasmid extraction kit to extract the plasmid and perform SalI+XbaI enzyme digestion for identification.

[0242] ② The bacterial solution with the correct enzyme digestion results was cultured in a constant temperature shaking incubator at 37°C and 200 rpm for 14 hours, and the bacterial slurry was harvested by centrifugation at 4000 rpm for about 10 minutes;

[0243] ③ Lyse the collected bacterial sludge, add Lysis Buffer I, and thoroughly resuspend the bacteria using an oscillator. The volume ratio of bacterial sludge to Lysis Buffer I is 4:250. Once the bacteria are fully suspended, add Lysis Buffer II and gently invert to lyse the bacteria. Add Lysis Buffer III and immediately and gently invert to fully neutralize the mixture. Let it stand at room temperature for 5 minutes. The volume ratio of Lysis Buffer I, Lysis Buffer II, and Lysis Buffer III is 5:5:7. Centrifuge the lysed mixture at 12,000 rpm for 5 minutes. The supernatant is added to a DNA adsorption column and centrifuged at 12,000 rpm for 1 minute to remove the waste liquid. Add the wash solution to the adsorption column and centrifuge at 12,000 rpm for 1 minute to remove the waste liquid. Add the eluent, let it stand at 37°C for 5 minutes, and then centrifuge at 12,000 rpm for 1 minute to collect the eluate to obtain the circular plasmid DNA solution.

[0244] S3. Preparation of mRNA stock solution:

[0245] I. Plasmid linearization:

[0246] ① Use the BspQ1 enzyme digestion system and incubate at 50°C for 1 hour to cut the circular plasmid into a linearized plasmid. Take 20 μg of the digestion product for agarose gel electrophoresis verification;

[0247] ② Add 1.0 times the volume of the digested product to the reaction system, then add 0.5 times the volume of magnetic beads and mix thoroughly. Incubate at room temperature for 10 minutes, then place on a magnetic stand. After the solution is clear, remove the supernatant.

[0248] ③ Rinse the magnetic beads with freshly prepared 80% ethanol solution, remove the supernatant and dry the magnetic beads, add 0.1 times the reaction system Nuclease-free H2O, pipette and mix well, then aspirate the supernatant to obtain the purified linear plasmid DNA solution and store it at -20℃.

[0249] The enzyme cleavage product is XbaI enzyme, and the manufacturer brand of the enzyme is Yisheng Biotechnology (Shanghai) Co., Ltd.

[0250] II. In Vitro Transcription (IVT)

[0251] ① Mix the linearized plasmid DNA template, NTPs, CleanCapAU, and Tris-HCl buffer as substrates, then add T7 RNA polymerase. Mix thoroughly and place in a 37°C PCR instrument for in vitro transcription. After 3 hours of reaction, add DNase I to terminate the reaction. The ratio of the DNA template mass to the DNase I volume is 1 g: 3 μL.

[0252] ② Add nuclease-free water and LiCl solution to the reaction solution. The volume ratio of the reaction solution to water and LiCl is 1:1.5:1.5. After inversion to mix, incubate at -20°C for 15 minutes. Centrifuge at 4°C and 12,000g for 10 minutes to remove the waste liquid. Add 70% ethanol to wash impurities. After removing the supernatant, add nuclease-free water to dissolve the precipitate to obtain the mRNA stock solution, which is stored at -80°C.

[0253] The concentration of the DNA template is 0.05-0.1 μg / μL.

[0254] The amount of NTPs used is 0.1 times the total volume.

[0255] The amount of CleanCapAU used is 0.1 times the total volume.

[0256] The amount of Tris-HCl buffer used is 0.1 times the total volume.

[0257] The amount of T7 RNA polymerase used is 0.5 times the total volume.

[0258] The DNA template, NTPs, CleanCapAU, Tris-HCl buffer, T7 RNA polymerase, and DNaseI are all commercially available products.

[0259] The DNA template can be purchased from Nanjing Novozymes Biotech Co., Ltd.

[0260] The NTPs can be purchased from Nanjing Novozymes Biotech Co., Ltd.

[0261] The manufacturer brand of the CleanCapAU can be Jiangsu Shenji Biotechnology Co., Ltd.

[0262] The Tris-HCl buffer may be purchased from Thermo Fisher Scientific.

[0263] The T7 RNA polymerase can be purchased from Thermo Fisher Scientific.

[0264] The DNaseI can be purchased from Thermo Fisher Scientific.

[0265] S4. Encapsulation of mRNA Stock Solution

[0266] ① Thaw the mRNA stock solution in a cold water bath and dilute it with citrate buffer to a concentration of about 0.5 mg / ml, which is the mRNA working solution; the pH value of the citrate buffer is 5.0.

[0267] ② Encapsulating the self-replicating mRNA in LNPs to obtain an organic phase (lipid phase), wherein the molar concentration ratio of the cation to DSPC, CHO-HP, and DMP-PEG2000 in the lipid phase is 30:5:30:2, and the total molar concentration of the lipid phase is 15 mmol / L;

[0268] ③ The volume ratio of the organic phase to the mRNA working solution is 1:2, and nanoparticles LNP are prepared using a microfluidic preparation instrument;

[0269] ④ Add 5 times the volume of PBS buffer to dilute the harvested nanoparticles, use a 100KD ultrafiltration centrifuge tube to centrifuge and concentrate at 4°C and 1500g. When the volume is concentrated to approximately the volume of the initially obtained LNP, add 5 times the volume of PBS buffer to dilute, and continue to concentrate to 1-0.5 times the volume before dilution. After filtering through a 0.2μm membrane, the LNP product is obtained to obtain the vaccine for preventing MenB.

[0270] The cationic model in the lipid phase can be selected from any one of DLin-MC3-DMA, JK-0042, JK-0043 and JK-0045; the cationic model in the lipid phase used in this experiment is JK-0042.

[0271] The preventive MenB vaccine was stored at -20°C.

[0272] The pH value of the PBS buffer solution is 7.5.

[0273] The DLin-MC3-DMA was purchased from Xiamen Sinobond Biotechnology Co., Ltd.

[0274] The JK-0042 is sourced from Ningbo Junjian Biotechnology Co., Ltd.

[0275] The JK-0043 is sourced from Ningbo Junjian Biotechnology Co., Ltd.

[0276] The JK-0045 is sourced from Ningbo Junjian Biotechnology Co., Ltd.

[0277] The fourth set of experiments

[0278] The fourth group of experiments includes Example 13, Example 14, Example 15, and Example 16.

[0279] In Example 13, the four antigenic proteins fHbp, NHBA, NadA, and PorA of sequence 1 of SEQ ID NO: 15 in the self-replicating mRNA sequence were designed separately, and the stock solutions were mixed before encapsulation to prepare the formulation, which was defined as SEQ ID NO: 15-split-stock solution mixture.

[0280] In Example 14, the four antigenic proteins fHbp, NHBA, NadA, and PorA of sequence 1 of SEQ ID NO: 15 in the self-replicating mRNA sequence were designed separately, mixed after encapsulation, and then purified and packaged as a preparation, which was defined as a SEQ ID NO: 15-split-preparation mixture.

[0281] In Example 15, the four antigenic proteins fHbp, NHBA, NadA, and PorA of sequence 1 of SEQ ID NO: 17 in the self-replicating mRNA sequence were designed separately, and the stock solutions were mixed before encapsulation to prepare the formulation, which was defined as SEQ ID NO: 17-split-stock solution mixture.

[0282] In Example 16, the four antigenic proteins fHbp, NHBA, NadA, and PorA of sequence 1 of SEQ ID NO: 17 in the self-replicating mRNA sequence were designed separately, mixed after encapsulation, and then purified and packaged as a preparation, which was defined as a SEQ ID NO: 17-split-preparation mixture.

[0283] The encapsulation method used in Example 13, Example 14, Example 15 and Example 16 includes the following steps:

[0284] S1. Vector construction: The self-replicating mRNA sequence was cloned into a self-replicating vector by SalI / XbaI double digestion to construct a self-replicating mRNA plasmid; the DNA sequence of the self-replicating mRNA plasmid was then introduced into Escherichia coli cells using a heat shock method to establish a research and development seed bank;

[0285] S2. Plasmid preparation:

[0286] ① Take engineered bacteria seeds from the R&D seed bank and add them to a liquid synthetic medium shaking incubator. Check that the seed OD600 reaches 0.6-2.5. The expanded seeds are cultured in a fermenter for 40 hours. The fermentation temperature is controlled at 30°C, the speed is 500 rpm, the dissolved oxygen content is linked to the stirring speed, the pH is controlled at 6.3-7.0, and the feed is linked to the pH. After the fermentation is completed, the supernatant is removed by centrifugation at 4000 rpm for about 5 minutes to harvest the bacterial sludge.

[0287] ② Mix the bacterial sludge with the resuspension S1 at a mass ratio of 1:10. Add S2 solution at a ratio twice that of S1 solution, let it stand for 5 minutes, then add 3 times S3 solution and ammonium bicarbonate, and let it stand for 60 minutes. Remove floating impurities and filter through a filter. Concentrate the solution using an ultrafiltration system. The pH value of S1 solution is 7.5-8.5 and it is composed of 50mM glucose, 30mM Tris-HCl, and 15mM EDTA. The S2 solution is composed of a fresh solution of 0.2M NaOH and 1% SDS. The S3 solution is composed of a mixture of 3M KAc and 2M HAc.

[0288] ③ Use molecular sieve chromatography column chromatography for chromatography, the chromatography linear flow rate is 3 cm / h, about 1 / 3CV after the start of sample loading, the target product will flow through, the UV curve rises, and the flow-through peak is collected. The buffer solution used for the molecular sieve chromatography column chromatography is composed of 2.0M (NH4)2SO4, 0.5MNaCl, 0.1MTris-HCl and 0.01MEDTA-2Na.

[0289] ④ Perform chromatography using an affinity chromatography column, equilibrated with buffer solution A, at a linear flow rate of 10 cm / h. After loading, wash impurities with 15% buffer solution B for approximately 5 CV.

[0290] The buffer solution A consists of 2.3M (NH4)2SO4, 0.1M Tris-HCl, and 0.01M EDTA-2Na;

[0291] The buffer solution B consists of 2.3M (NH4)2SO4, 0.2MNaCl, 0.1M Tris-HCl, and 0.01MEDTA-2Na; 30% buffer solution B is used for elution, and the elution peak is collected.

[0292] ⑤ Use an ultrafiltration system to change the solution, replace the salt solution with 1×TE buffer solution, and select 300kD3 for the hollow fiber column.

[0293] ⑥ The supercoiled plasmid DNA, linearized enzyme, and water were mixed and incubated at 50°C for 2 hours. The linearized plasmid DNA template was purified using anion chromatography column chromatography and ultrafiltration, and stored at -80°C; the chromatography linear flow rate was 10-15 cm / h, the chromatography solution A consisted of EDTA-2Na and Tris-HCl, the mass ratio of EDTA-2Na to Tris-HCl was 1.681:7.88, and the pH of the chromatography solution A was 7.5;

[0294] The chromatography solution B consists of EDTA-2Na and Tris-HCl, the mass ratio of EDTA-2Na to Tris-HCl is 1.681:7.88, and the pH of the chromatography solution B is 7.5.

[0295] S3. Preparation of mRNA stock solution:

[0296] ① Take the linearized plasmid DNA template, NTPs and Tris-HCl buffer as substrates, mix them, add T7 RNA polymerase, and react at 33°C in the synthesis workstation for 3 hours;

[0297] ② Purify by affinity chromatography using OligodT as the chromatographic medium. Dilute the reaction solution 20-fold with an equilibration solution prior to purification. The linear flow rate for purification is 10 cm / h, and the impurities are washed for 5 CV. The equilibration solution consists of NaCl, EDTA, and TrisHCl in a mass ratio of 9.35:0.067:0.32. After washing, the eluted peak is collected using pure water.

[0298] ③ Purify using a TFF system with pure water as the replacement solution. Using a transmembrane pressure of 10 psi and a shear rate of 3000 / sec, perform diafiltration 10-fold and reconcentrate to the target concentration. Filter the resulting sample through a 0.2 μm filter to obtain the mRNA stock solution, which is then stored at -80°C.

[0299] The concentration of the DNA template is 0.05-0.1 μg / μL.

[0300] The amount of NTPs used is 0.1 times the total volume.

[0301] The amount of Tris-HCl buffer used is 0.1 times the total volume.

[0302] The amount of T7 RNA polymerase used is 0.5 times the total volume.

[0303] The DNA template, NTPs, Tris-HCl buffer, and T7 RNA polymerase are all commercially available products.

[0304] The DNA template can be purchased from Nanjing Novozymes Biotech Co., Ltd.

[0305] The NTPs can be purchased from Nanjing Novozymes Biotech Co., Ltd.

[0306] The Tris-HCl buffer may be purchased from Thermo Fisher Scientific.

[0307] The T7 RNA polymerase can be purchased from Thermo Fisher Scientific.

[0308] S4. Encapsulation of mRNA stock solution:

[0309] ① Thaw the mRNA stock solution in a cold water bath and dilute it with citrate buffer to a concentration of about 0.5 mg / ml, which is the mRNA working solution; the pH value of the citrate buffer is 5.0.

[0310] ② The self-replicating mRNA was encapsulated in LNP to obtain an organic phase (lipid phase), wherein the molar concentration ratio of the cation to DSPC, CHO-HP, and DMP-PEG2000 in the lipid phase was 30:5:30:2, and the total molar concentration of the lipid phase was 15 mmol / L.

[0311] ③ The volume ratio of the organic phase to the mRNA working solution was 1:3, and nanoparticles LNP were prepared using a microfluidic preparation instrument;

[0312] ④ Add 10 times the volume of the harvested nanoparticles in 1× PBS to solidify the LNP particles. Purify the LNPs using a TFF ultrafiltration system with 1× PBS as the replacement solution. The system is operated at a transmembrane pressure of 10 psi and a shear rate of 2000 / sec. The mixture undergoes a six-fold dilution and concentration cycle, followed by six cycles of diafiltration and reconcentration three times. The resulting sample is filtered through 0.2 μm to obtain the LNP-encapsulated mRNA pharmaceutical composition, which is then stored at -20°C.

[0313] The cationic model in the lipid phase can be selected from any one of DLin-MC3-DMA, JK-0042, JK-0043 and JK-0045; the cationic model in the lipid phase used in this experiment is JK-0042.

[0314] Performance testing:

[0315] 1. Electrophoresis test:

[0316] The self-replicating mRNA sequences optimized in Examples 1, 2, 3, 4, 5, and 6 in the second group of experiments and the recombinant plasmids obtained according to the preparation method provided in the third group of experiments were subjected to gel electrophoresis testing to obtain plasmid identification maps.

[0317] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 15 and its recombinant plasmid are shown in Figure 1 .

[0318] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 17 and its recombinant plasmid are shown in Figure 2 .

[0319] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 19 and its recombinant plasmid are shown in Figure 3 .

[0320] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 21 and its recombinant plasmid are shown in Figure 4 .

[0321] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 23 and its recombinant plasmid are shown in Figure 5 .

[0322] The electrophoresis test results obtained for the self-replicating mRNA sequence SEQ ID NO: 25 and its recombinant plasmid are shown in Figure 6 .

[0323] 2. Cell experiments:

[0324] (1) Cell protein sample collection:

[0325] ① Culture HEK293 cells on the cell well plate until the confluence of each well reaches 80%-90%;

[0326] ② Take two sterile EP tubes, add 2-5 μg mRNA sample to one tube, dilute to 150 μL with Opti-MEM (serum-free medium), and add 3-5 μL transfection reagent Lipofectamine™ MessengerMAX™ Reagent to the other tube, and also make up to 150 μL with Opti-MEM. Mix the liquids in the two tubes and pipette to mix thoroughly. Let it stand for 5 minutes. After standing, add the solution dropwise to the well plate and incubate in a CO2 incubator for 24 hours.

[0327] ③ Take out the cell well plate, observe the cell status under a microscope, then place it on ice, collect the culture medium in the well, add 1% PMSF according to volume, mix well, and place it on ice;

[0328] ④ After the culture medium is aspirated, the cell well plate is washed once with 1× PBS after ice bath, and 200 μL of RIPA lysis buffer is added to each well. 1% PMSF is added to the lysis buffer, and the cells are lysed on ice for 5-10 minutes. The lysate is collected and vortexed;

[0329] ⑤ Centrifuge the collected culture medium and lysate at 4°C, 17000g for 15-30min, collect the supernatant to obtain the cell protein sample, and store it at -20°C.

[0330] The protein bands of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 sequences provided in Examples 7, 8, 9, 10, 11 and 12 and the positive and blank control samples were detected by immunoblotting. The test results are shown in FIG. Figure 7 .

[0331] Test methods include:

[0332] ① Add 4×LDS sample buffer and 1×MDTT (10×) according to the concentration of the cell protein sample, add deionized water to make up to 60μl system, incubate at 90℃ for 5-10min, centrifuge and mix well before loading;

[0333] ② Electrophoresis: Run the stacking gel at 80V constant voltage for 20-30 minutes, use a polyacrylamide separation gel with a concentration of 6%, and run the separation gel at 150V constant voltage for 35 minutes. Stop the electrophoresis when the dye reaches the bottom of the separation gel.

[0334] ③Electroporation: Activate the PVDF membrane in activation solution; remove the gel, cut off the excess gel, and create a sandwich structure in 1× transfer buffer: sponge-filter paper-gel-PVDF membrane-filter paper-sponge. Ensure that there are no bubbles in the sandwich structure. Add an appropriate amount of 1× transfer buffer to the transfer tank, place it in an ice box, and perform electrotransfer at 200mA constant current for 100 minutes.

[0335] ④ Blocking: Use 1×TPBT to prepare 5% BSA as blocking solution, place the transferred PVDF membrane in it, and incubate on a shaker at room temperature for 60 minutes; rinse with 1×TBST buffer three times, each time for 10 minutes;

[0336] ⑤ Primary antibody incubation: Dilute the primary antibody in 1× antibody diluent according to the instructions, incubate at room temperature with shaking for 1 hour, incubate at 4°C overnight, and return to room temperature with shaking for 1 hour the next day; wash three times with 1× TBST, 5 minutes each time;

[0337] ⑥ Secondary antibody incubation: Dilute the secondary antibody in 1× antibody diluent according to the instructions, incubate at 37°C in a dark shaking incubator for 1 hour, then incubate at room temperature in a shaking incubator for 20 minutes; wash three times with 1× TBST, 5 minutes each time;

[0338] ⑦ Development: Mix developer A and developer B in a 1:1 ratio, add them dropwise onto the membrane, and place it in a chemiluminescence imaging system for imaging.

[0339] Figure 7 The marker is a protein molecular weight standard containing specific molecular weight markers, such as 245kD and 180kD, which are used to indicate the molecular weight of the protein in the sample. Lane 1 is labeled blank and is a blank control sample. In theory, there should be no target protein-specific bands. It is used to detect the background signal of the experimental system and eliminate interfering factors such as nonspecific binding. Lane 2 is labeled positive and is a positive control sample. It contains samples known to express the target protein and will show characteristic target protein bands. It is used to verify the effectiveness of the experimental system and ensure that the experimental process can correctly detect the target protein. Lanes 3-8 are detection bands for samples containing SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, and SEQ ID NO: 25 sequences, respectively. By comparing with the marker, the molecular weight of the target protein in the sample can be determined. By comparing with the positive control, the expression of the target protein in these samples can be evaluated, including whether the target protein is successfully expressed and the relative expression level. Information such as the position and grayscale of the bands can provide a basis for analyzing the characteristics of the target protein in each sample.

[0340] 4. Animal experiments and serum binding antibody detection:

[0341] 4.1 Animal Experiments

[0342] ① Grouping principle: Balb / c mice were randomly divided into groups, with 5 mice in each group; each group was injected with the mRNA sequence of the preventive MenB vaccine, and the day of grouping was Day 0.

[0343] ② Animal administration: Different control groups were set up, and the prepared self-replicating mRNA-LNP samples were used for administration at a dose of 15 μg / animal. The empty LNP group served as the control group and was administered once and twice on Day 0 and Day 21, respectively.

[0344] ③ Recording of experimental indicators: Observe the animals daily and record their clinical symptoms. Clinical symptoms include, but are not limited to, 0: Normal, 1: Decreased activity, 2: Restless, 3: Trembling, 4: Spinning or backwards, 5: Piloeresis, 6: Abnormal breathing, 7: Arched back, 8: Significant decrease in body temperature, 9: Hair loss, 10: Eye abnormalities, 11: Abdominal swelling, and 12: Other.

[0345] ④ Sampling: Blood was collected from the retroorbital space on Day 0, Day 13, Day 20, Day 29, Day 34, Day 50, Day 72, and Day 90.

[0346] ⑤ Termination of the experiment: After all drug administration and observation are completed, the experiment will be terminated after 1-2 months of observation.

[0347] 4.2 Serum binding antibody detection:

[0348] The self-replicating mRNA sequence provided in Example 1 is SEQ ID NO: 15, the self-replicating mRNA sequence provided in Example 2 is SEQ ID NO: 17, the self-replicating mRNA sequence provided in Example 3 is SEQ ID NO: 19, the self-replicating mRNA sequence provided in Example 4 is SEQ ID NO: 21, the self-replicating mRNA sequence provided in Example 5 is SEQ ID NO: 23, and the self-replicating mRNA sequence provided in Example 6 is SEQ ID NO: 25, respectively, combined with fHbp, NHBA, NadA or PorA antigen protein for serum binding antibody detection, and the test results are shown in FIG. Figures 8-11 . Figure 8 This is the result of fHbp binding antibody detection; Figure 9 This is the result of NHBA binding antibody detection; Figure 10 This is the result of NadA binding antibody detection; Figure 11 This is the result of porA binding antibody detection.

[0349] 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1 were respectively taken; 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2 were respectively taken; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 15-split-stock solution mixture provided in Example 13 were respectively taken; 1 μg, 5 μg, and 15 μg of the split-preparation mixture provided in Example 14 were respectively taken; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 17-split-stock solution mixture provided in Example 15 were respectively taken; and 1 μg, 5 μg, and 15 μg of the split-preparation mixture provided in Example 16 were respectively combined with fHbp, NHBA, NadA, or PorA antigen proteins for serum binding antibody detection. The test results are shown in FIG. Figure 16-19 .

[0350] Detection method:

[0351] ① Dilute MenB (fHbp), MenB (NHBA), MenB (NadA), or MenB (PorA) antigen to 1 μg / mL in ELISA coating buffer. Add the diluted antigen to the wells of the ELISA plate at 100 μL / well and coat at 4°C overnight (14-18 hours).

[0352] ② The next day, the wells were washed three times with PBST for 5 minutes each time, and 200 μL of 1% BSA was added to each well for blocking after patting dry. The wells were incubated at 37°C for 1 hour, and then washed three times with PBST for 5 minutes each time, and the wells were patted dry.

[0353] ③ Add mouse serum of the corresponding dilution ratio, 100 μL / well, and perform one replicate for each sample. Set up negative / positive controls at the same time. Incubate at 37°C for 1 hour, rinse with PBST three times, 5 minutes each time, and pat dry.

[0354] ④ Add anti-mouse IgG heavy chain antibody (HRP labeled) at 100 μL / well, incubate at 37°C for 1 hour, rinse with PBST three times for 5 minutes each time, and pat dry;

[0355] ⑤ Add TMB colorimetric solution (100 μL / well) for color development. Incubate at room temperature in the dark for 10-15 minutes. Terminate the reaction by adding stop solution (50 μL / well). Measure the absorbance at 450 nm using a microplate reader. Calculate the titer of each bound antibody in the sample based on the OD value.

[0356] Figure 16 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with PorA antigen protein for serum binding antibody detection.

[0357] Figure 16 In b, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with PorA antigen protein for serum binding antibody detection results when the dosage is 5 μg.

[0358] Figure 16 In c, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with PorA antigen protein for serum binding antibody detection results.

[0359] pass Figure 16 It can be seen that the binding antibody titers of each sample group were generally low 14 days and 21 days after administration; as time went on, the antibody titers of some sample groups increased significantly 35 days, 49 days, and 70 days after administration, reflecting that vaccines with different sequences, doses, and formulations have different abilities and time courses to induce the production of MenB (porA) binding antibodies, which can be used to evaluate the immune effect of vaccines and optimize vaccine design.

[0360] Figure 17 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with NadA antigen protein for serum binding antibody detection results.

[0361] Figure 17 In b, the serum binding antibody test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture and the split-preparation mixture provided in Example 16 were combined with NadA antigen protein when the dosage was 5 μg.

[0362] Figure 17 In c, the serum binding antibody test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture and the split-preparation mixture provided in Example 16 were combined with NadA antigen protein when the dosage was 15 μg.

[0363] pass Figure 17 It can be seen that at 14 and 21 days after administration, the binding antibody titers of most sample groups were at low levels, indicating that the body's immune response to the vaccine had not yet fully initiated. 35 days after administration, the antibody titers of some sample groups began to rise significantly, indicating that the body's immune response was gradually strengthening. At 49 and 70 days after administration, the antibody titers of several sample groups reached high levels, indicating that the ability and time course of MenB (NadA) binding antibodies induced by vaccines with different sequences, doses, and formulations vary.

[0364] Figure 18 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with NHBA antigen protein for serum binding antibody detection results.

[0365] Figure 18In b, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with NHBA antigen protein for serum binding antibody detection results when the dosage is 5 μg.

[0366] Figure 18 In c, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used in combination with NHBA antigen protein for serum binding antibody detection results.

[0367] pass Figure 18 It can be seen that 14 days and 21 days after administration, the binding antibody titers of each sample group were generally low, indicating that the body's immune response was weak at this time; 35 days after administration, the antibody titers of some sample groups began to rise significantly; 49 days and 70 days after administration, the antibody titers of multiple sample groups reached a high level, indicating that there are differences in the ability and time process of vaccines with different sequences, doses and formulations to induce the production of MenB (NHBA) binding antibodies.

[0368] Figure 19 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 1 μg in combination with fHbp antigen protein for serum binding antibody detection results.

[0369] Figure 19 b is the result of serum binding antibody detection when the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture and the split-preparation mixture provided in Example 16 are used at a dosage of 5 μg in combination with fHbp antigen protein.

[0370] Figure 19In c, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 17 provided in Example 15-split-stock solution mixture, and the split-preparation mixture provided in Example 16 are used at a dosage of 15 μg in combination with fHbp antigen protein for serum binding antibody detection results.

[0371] pass Figure 19 It can be seen that 14 days and 21 days after administration, the binding antibody titers of each sample group were generally low, indicating that the body's immune response was weak at this time; 35 days after administration, the antibody titers of some sample groups began to increase significantly; 49 days and 70 days after administration, the antibody titers of multiple sample groups reached a high level, indicating that there are differences in the ability and time process of vaccines with different sequences, doses and formulations to induce the production of MenB (fHbp) binding antibodies.

[0372] 4.3. Immune cell typing test (FACs):

[0373] The spleens of mice injected with the MenB prevention vaccines provided in Examples 7, 8, 9, 10, 11, and 12 were examined for T cell activation; after polypeptide stimulation, spleen cells were stained with IFN-γ / CD69 dye and T cell activation was analyzed using the FACs method. The test results at 34 and 70 days after injection were recorded. Figure 12-13 .

[0374] Figure 12 This is the FACs test result on D34 days after the injection of MenB vaccine.

[0375] Figure 13 This is the FACs test result 70 days after the injection of MenB vaccine.

[0376] 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 15-split-stock solution mixture provided in Example 13 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the split-preparation mixture provided in Example 14 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 17-split-stock solution mixture provided in Example 15 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the split-preparation mixture provided in Example 16 were injected into mice respectively, and the results of immune cell typing test after injection were recorded. The test results are shown in FIG. Figure 20-23 .

[0377] Figure 20 It is the result of CD4+IFNg+T cell typing test; Figure 21 This is the result of CD4+CD69+T cell typing test; Figure 22 CD8+IFNg+T cell typing test results; Figure 23 This is the result of CD8+CD69+T cell typing test.

[0378] pass Figure 20-23 It can be seen that after administration, the different vaccine samples were able to induce changes in activation and function-related indicators of CD4⁺ and CD8⁺T cell subsets to a certain extent. The frequencies of CD4⁺IFNγ⁺T, CD4⁺CD69⁺T, CD8⁺IFNγ⁺T, and CD8⁺CD69⁺T cells increased across the different sample groups, indicating that the vaccine can activate the body's T cell immune response, prompting T cells to secrete cytokines or enter an early activation state, participating in immune defense. Vaccine samples containing different doses of SEQ ID NO.15 and SEQ ID NO.17, as well as different doses of split-stock solution mixtures and split-formulation mixtures, showed differences in their ability to induce T cell immune responses and the time course of their induction. As the number of days after administration increased, from 35 to 70 days, the T cell immune response indicators induced by most sample groups showed an increasing trend, reflecting that the body's cellular immune response to the vaccine is a dynamic process that gradually increases over a period of time after administration.

[0379] 4.4. IFN-γ cytokine secretion level detection (ELISOPT):

[0380] The ELISPOT method was used to detect IFN-γ secretion in spleen cells of mice injected with the MenB prevention vaccines provided in Examples 7, 8, 9, 10, 11, and 12. Mouse cell suspensions were prepared by taking spleen cells, and the polypeptide was added for stimulation to cause the spleen cells to secrete IFN-γ. Avidin-enzyme complex was added to bind to biotin, and a colorimetric substrate was added to form visible spots. The ELISOPT test results were recorded 34 days and 70 days after injection. Figure 14-15 .

[0381] Figure 14 This is the ELISPOT test result on day 34 after the injection of the MenB vaccine.

[0382] Figure 15 This is the ELISPOT test result 70 days after the injection of MenB vaccine.

[0383] 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 15-split-stock solution mixture provided in Example 13 were injected into mice respectively; 1 μg, 5 μg, and 15 μg of the SEQ ID NO: 15 split-preparation mixture provided in Example 14 were injected into mice respectively, and the ELISOPT test results after injection were recorded. Figure 24-25 .

[0384] Figure 24 ELISPOT test results on day 35 of injection; Figure 25 The ELISPOT test results are shown on day 70 after injection.

[0385] pass Figure 7 The results of Western Blotting for detecting protein bands showed that the six sequences of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23 and SEQ ID NO: 25 provided in Examples 7, 8, 9, 10, 11 and 12 all had target bands at corresponding positions, indicating that the above six sequences can all express the target proteins normally in the cells. Figures 8-11 The combined antibody results showed that the antibody titers of SEQ ID NO: 15 group and SEQ ID NO: 17 group were the highest. Figure 12-13 The results of FACs test after the injection of MenB vaccine showed that there was no significant difference in immune cell typing among the groups; Figure 14-15 The ELISPOT results after injection of the MenB vaccine showed slightly higher levels in the SEQ ID NO: 17 and SEQ ID NO: 19 groups. The test results presented by intracellular protein expression, in vivo binding antibody data, FACs assay data, and ELISPOT results indicate that the self-replicating mRNA sequence designed in this application can induce an immune response against meningococci in vivo, with the SEQ ID NO: 17 group showing the best experimental results.

[0386] Figure 24In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 are injected into mice at D35 days at a dosage of 1 μg.

[0387] Figure 24 Figure b shows the ELISPOT test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice on day 35 at a dosage of 5 μg.

[0388] Figure 24 Figure c is the ELISPOT test result of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice on D35 days at a dosage of 5 μg.

[0389] Figure 25 In a, the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15-split-preparation mixture provided in Example 14 are injected into mice at D70 days at a dosage of 1 μg.

[0390] Figure 25 Figure b shows the ELISPOT test results of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice at a dosage of 5 μg on day 70.

[0391] Figure 25Figure c is the ELISPOT test result of the self-replicating mRNA sequence SEQ ID NO: 15 provided in Example 1, the self-replicating mRNA sequence SEQ ID NO: 17 provided in Example 2, the SEQ ID NO: 15-split-stock solution mixture provided in Example 13, and the SEQ ID NO: 15 split-preparation mixture provided in Example 14 when injected into mice at a dosage of 5 μg on day 70.

[0392] pass Figure 24 and Figure 25 It can be seen that on D35 and D70, a certain number of cells secreting cytokines (reflected by SFU values) were detected in different sample groups under the stimulation of multiple antigens (fHbp, NHBA, NadA, PorA), indicating that vaccine samples of different sequences, doses and formulations can activate the body's immune cells to a certain extent, prompting them to secrete cytokines and initiate immune responses.

[0393] 4.5 Serum bactericidal titer detection:

[0394] The revived strain was cultured in a 37°C 5% CO2 environment for 16-24 hours. Approximately 20-40 colonies were subcultured and incubated in a 37°C 5% CO2 environment for 4 hours to prepare a bacterial suspension and adjust to the appropriate concentration. The serum to be tested was inactivated and diluted in a 96-well U-bottom microtiter plate with bactericidal buffer. SEQ ID NO: 15 split-preparation mixture and SEQ ID NO: 17 split-preparation mixture were added to the diluted serum as complement and bacterial suspension, respectively, and the corresponding complement SEQ ID NO: 15 split-preparation mixture or SEQ ID NO: 17 split-preparation mixture and bacterial suspension were added to the complement control and serum control wells. The titration plate was sealed and incubated at 37°C for 60 minutes. 10 μL was taken from each well and inoculated onto a Columbia blood agar plate using the tilt method. The plate was incubated overnight at 37°C 5% CO2 and the colonies were counted. The bactericidal titer of the sample to be tested is based on the live complement well, and the highest dilution factor at which the number of colonies is less than 50% of the live complement well; the test results of the SEQ ID NO: 17 split-preparation mixture are shown in Figure 26 ; SEQ ID NO: 15 split-preparation mixture test results are shown in Figure 27 .

[0395] from Figure 26 It can be seen that the serum bactericidal titer corresponding to the SEQ ID NO: 17 split-preparation mixture is 1:32, which means that when the serum is diluted to 1:32, it can still effectively kill a certain number of bacteria. Above this dilution, the bactericidal ability decreases and the number of colonies on the plate increases significantly.

[0396] from Figure 27It can be seen that the serum bactericidal titer corresponding to the SEQ ID NO: 15 split-preparation mixture is 1:256, which means that when the serum is diluted to 1:256, it can still effectively kill a certain number of bacteria. Above this dilution, the bactericidal ability weakens and the number of colonies on the plate increases.

Claims

1. A self-replicating mRNA sequence, characterized in that An mRNA sequence encoding an amino acid sequence, wherein the amino acid sequence comprises a tandem sequence formed by connecting the individual sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10 in any order; A signal peptide sequence of one or more of the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6 is added to the 5' end of the tandem sequence; The individual sequences in the tandem sequence are connected using the flexible linker in the amino acid sequence SEQ ID NO: 11; A MITD molecular sequence is added to the end of the tandem sequence, wherein the MITD molecular sequence includes the amino acid sequence SEQ ID NO: 13; The self-replicating mRNA sequence includes target protein amino acids for molecular tandem design of fHbp, NHBA, NadA and PorA antigen proteins.

2. The self-replicating mRNA sequence according to claim 1, wherein The sequence of the self-replicating mRNA includes an mRNA sequence encoding the corresponding amino acid sequence SEQ ID NO:

17.

3. A vaccine for preventing MenB, characterized in that The method comprises the self-replicating mRNA sequence according to any one of claims 1 to 2.

4. A method for preparing a vaccine for preventing MenB according to claim 3, characterized in that: The following steps are involved: Vector construction, plasmid preparation, mRNA stock solution preparation, mRNA stock solution encapsulation, to obtain the vaccine for preventing MenB.

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