Pertussis mRNA vaccine

Abstract

The invention provides a pertussis mRNA (messenger Ribonucleic Acid) vaccine, the pertussis mRNA vaccine comprises one or more mRNA molecules, and antigens coded by the mRNA molecules comprise pertussis toxin and filamentous hemagglutinin; and pertussis adhesin. In some cases, the antigens encoded by the mRNA molecule also include pilin 2 and pilin 3. According to the invention, on the basis of gC180-mRNA and FHA456-mRNA vaccines, the protection effect of the pertussis mRNA vaccine is improved by compatibility of a three-component pertussis mRNA vaccine added with PRN-mRNA or compatibility of a five-component pertussis mRNA vaccine added with PRN and Fim < 2 > / 3.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a pertussis mRNA vaccine. Background Technology

[0002] Pertussis, an acute respiratory infectious disease caused by Bordetella pertussis, was once a leading cause of illness and death in infants and young children. Vaccination has significantly reduced the incidence and mortality of pertussis, but in recent years, the incidence has been rising and even experiencing outbreaks, a phenomenon known as the "re-emergence of pertussis." In China, the vaccination rate for three doses of DPT (diphtheria, pertussis, tetanus) vaccine remains above 99%, yet the incidence of pertussis is on the rise.

[0003] Analysis of the reasons for the resurgence of pertussis reveals that, in addition to changes in the pertussis gene and improved surveillance capabilities, another important factor is the weakening of vaccine-preventive immunity. There are two types of pertussis vaccines: whole-cell pertussis vaccine (wP), made from inactivated Bordetella pertussis, which has a high adverse reaction rate due to the presence of various impurity antigens; and acellular pertussis vaccine (aP), composed of purified detoxified pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN), and fimbriae type 2 and 3 (Fim2 / 3) antigens, which has a low adverse reaction rate. The aP vaccine primarily induces a Th2-type cellular immune response, mediating humoral immunity. High levels of antibodies can prevent clinical symptoms but cannot prevent Bp colonization or transmission in the nasopharynx, and resistance to pertussis infection is reduced after 5 years. wP (whole pox vaccine) can better mimic natural infection, inducing Th1 / Th17 cellular immune responses. It not only prevents disease but also engulfs and kills Bordetella pertussis, providing longer-lasting protection. Therefore, there is a need to explore novel pertussis vaccines with good safety profiles that can induce strong Th1 / Th17 cellular immune responses.

[0004] mRNA vaccines can simultaneously induce high levels of humoral and cellular immune responses and have been used to prevent various viral diseases, such as rabies and respiratory syncytial virus. Antibacterial mRNA vaccines have demonstrated effectiveness in preclinical studies. Developing a pertussis mRNA vaccine using an mRNA platform holds promise as a new strategy to address the resurgence of pertussis.

[0005] This invention, based on previous research, constructed mRNAs expressing truncated forms of the PT and FHA antigens. The truncated PT includes the 9K / 129G mutated S1 subunit (named gS1) and the N-terminal 180 amino acid residues of the 9K / 129G mutated S1 subunit (named gC180). The truncated FHA includes amino acid sequences 1655-2111 (named FHA456), 1545-1917 (named FHA373), and 1655-1917 (named FHA233). In a pertussis immunization challenge protection experiment, the survival rate of mice protected by the gC180 single-component pertussis mRNA vaccine group was 31.25%, higher than the 12.5% ​​survival rate of mice protected by the gS1 single-component pertussis mRNA vaccine group. GC180 was combined with FHA373, FHA456, and FHA233 respectively to form a two-component pertussis mRNA vaccine. In a pertussis challenge protection experiment, the survival rates of mice were 37.5%, 56.25%, and 50%, respectively. The two-component pertussis mRNA vaccine showed improved protective efficacy compared to the single-component mRNA vaccine, but the protective effect was still limited, and further research is needed to improve the protective effect. Summary of the Invention

[0006] In view of this, the present invention provides a pertussis mRNA vaccine, which is a three-component pertussis mRNA vaccine based on gC180-mRNA and FHA456-mRNA vaccines, with added PRN-mRNA, thereby improving the protective effect of the pertussis mRNA vaccine.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The present invention provides a pertussis mRNA vaccine, wherein the pertussis mRNA vaccine comprises one or more mRNA molecules, and the antigens encoded by the mRNA molecules include: pertussis toxin, filamentous hemagglutinin; and pertussis adhesin.

[0008] Preferably, the antigen encoded by the mRNA molecule further includes fimbriae 2 and fimbriae 3.

[0009] Preferably, the mRNA encoding the pertussis adhesin antigen gene comprises a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:1-SEQ ID NO:6; and / or, The mRNA encoding the fimbriae protein 2 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:7-SEQ ID NO:12; and / or, The mRNA encoding the fimbriae protein 3 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:13-SEQ ID NO:18.

[0010] Preferably, the DNA molecule encoding the pertussis adhesin antigen gene has the sequence shown in SEQ ID NO:19-SEQ ID NO:24; and / or, DNA molecules encoding the fimbriae protein 2 antigen gene, the sequences of which are shown in SEQ ID NO:25-SEQ ID NO:30; and / or, The DNA molecule encoding the fimbriae protein 3 antigen gene has the sequence shown in SEQ ID NO:31-SEQ ID NO:36.

[0011] Preferably, the mRNA molecules encoding the genes for pertussis toxin, filamentous hemagglutinin, pertussis adhesin, fimbriaein 2, and fimbriaein 3 antigens all include: (a) 5'-cap structure; (b) 5' non-translation area; (c) Kozak sequence; (d) The antigen gene sequence of Bordetella pertussis, which encodes the pertussis adhesin and fimbriae 2 and 3 antigens that can induce an immune response; (e) 3' untranslated region; (f) Polyadenylate tail elements with a total length of 100 nt or more.

[0012] Preferably, the vaccine also includes an antigen delivery vector for encapsulating the antigen of the pertussis mRNA vaccine.

[0013] Preferably, the antigen delivery carrier comprises ionizable lipids, neutral lipids, PEGylated lipids, and cholesterol.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides mRNAs for expressing PRN and Fim2 / 3, which, when combined with mRNAs expressing gC180 and FHA456 genes, form a three-component pertussis mRNA vaccine (including PT, FHA, and PRN) and a five-component pertussis mRNA vaccine (including PT, FHA, PRN, and Fim2 / 3), which can be used to prevent Bordetella pertussis infection. This invention optimizes the sequences encoding the PRN, Fim2, and Fim3 antigen genes. Sequence optimization includes mRNA structural stability, frequency of use of commonly used human codons, various cis-elements, and codon adaptability, etc. The DNA sequences are shown in SEQ ID NO:19-SEQ ID NO:24, SEQ ID NO:25-SEQ ID NO:30, and SEQ ID NO:31-SEQ ID NO:36.

[0015] Using sequence-optimized DNA as a template (linearized plasmid), transcription was performed to obtain mRNA molecules encoding the PRN, Fim2, and Fim3 antigen genes of Bordetella pertussis, with sequences shown in SEQ ID NO:1-SEQ ID NO:6, SEQ ID NO:7-SEQ ID NO:12, and SEQ ID NO:13-SEQ ID NO:18. The optimized nucleotide sequences result in a more stable mRNA structure and higher translation efficiency of the target protein in mammals and humans. This addresses technical problems in existing technologies, such as the instability of mRNA chemical properties, easy degradation, and insufficient protein expression, and can efficiently induce an immune response in the body.

[0016] (2) The immune response of the human body to mRNA is related to uridine. In this invention, all uracil (U) in the mRNA nucleic acid molecule is replaced with pseudouridine (ψ), which reduces the content of uracil in the mRNA molecule and reduces the recognition of mRNA by the immune system.

[0017] (3) The mRNA molecules of the present invention are encapsulated in a lipid shell to form lipid nanoparticles, wherein the lipid nanoparticles include ionizable lipids, neutral lipids, PEGylated lipids and cholesterol. Attached Figure Description

[0018] Figure 1 The bar chart shows the levels of PT, FHA, PRN, and Fim antibodies in the serum of CD1 mice after immunizing them with the three-component and five-component pertussis mRNA vaccines provided in Example 4 of this invention, and the results were detected by ELISA. Figure 2 After CD1 mice were immunized with the three-component and five-component pertussis mRNA vaccines provided in Example 4 of this invention, the spleen immune cells of the mice secreted CD4+ IFN-γ and IL-4. + Bar chart of T cells; Figure 3 The bar chart shows the ratio of central memory T cells and effector memory T cells in the spleen of CD1 mice after immunizing them with the three-component and five-component pertussis mRNA vaccines provided in Example 4 of the present invention. Figure 4 The graphs show the number of Bordetella pertussis colonies in the lungs of BALB / c mice after immunizing them with the three-component and five-component pertussis mRNA vaccines provided in Example 6 of this invention. Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0020] Example 1 The sequences SEQ ID NO:19-SEQ ID NO:24, SEQ ID NO:25-SEQ ID NO:30, and SEQ ID NO:31-SEQ ID NO:36 were synthesized and constructed into polyadenylated tail plasmid templates containing UTR (untranslated region) and 120nt (120 adenosine nucleotides (A)). These were then transformed into TOP10 competent cells. The strain was fermented in shake flasks, and the transcription templates were then purified using an endotoxin-free plasmid large-scale extraction kit.

[0021] 1.1 Preparation of mRNA The linearization of the template plasmid is shown in Table 1 below.

[0022] Table 1

[0023] Incubate at 37°C for 4 hours.

[0024] 1.2 Purification of linearized plasmids The enzyme digestion products were purified using the Tiangen Biotech standard DNA purification and recovery kit. The products were purified according to the experimental instructions and dissolved in Endo-free H2O. The linearized plasmid concentration was determined using NanoDrop and stored at -20℃ for later use.

[0025] 1.3 In vitro transcription mRNA was prepared using the commercial mRNA IVT kit, HanHai New Enzyme-High-Yield T7 In Vitro Transcription Reagent (N1-Me-pUTP)-HBP001505. The obtained linearized plasmid was processed according to the kit instructions, as shown in Table 2 below. Table 2

[0026] The reaction was carried out at 37°C for 4 hours, and then DNase I was added to the reaction system. The reaction was carried out at 37°C for 15 minutes to digest the transcribed DNA template.

[0027] 1.4 Lithium Chloride Purification Lithium chloride purification can remove proteins and most free nucleotides. Add 20 μL of lithium chloride precipitation buffer (5M lithium chloride) to the transcription product from the previous step. Gently pipette to mix, incubate at -20°C, and centrifuge to obtain the RNA precipitate. Wash the RNA precipitate with 70% ice-point ethanol, centrifuge again to obtain the RNA precipitate, and repeat 2-3 times. Open the lid and dry for 5-10 min. After confirming complete drying, add 20-50 μL of RNase-free ddH2O to dissolve the RNA precipitate.

[0028] 1.5 with cap First, the purified uncapped mRNA was heated at 65℃ for 10 min to open its secondary structure, and then immediately placed in an ice bath. Then, capping was performed using the HanHai New Enzyme-Enzymatic Capping Reagent Cap1 Capping System-HBP001513, as shown in Table 3. Table 3

[0029] Reaction conditions: 37℃ for 60 min.

[0030] 1.6 Then purify with lithium chloride. mRNA can also be prepared using other methods, and is not limited to the methods and reagents mentioned above.

[0031] Example 2 Ionizable lipids, neutral lipids, PEGylated lipids, and cholesterol were dissolved in anhydrous ethanol at a molar ratio of 50:2.5:10:37.5 and thoroughly mixed. A 50 mmol / mL citrate buffer (pH 4) was prepared using citric acid and trisodium citrate. The prepared mRNA was diluted with this citrate buffer to a concentration of approximately 170 ng / μL and stored at 4°C. The diluted mRNA and the prepared lipid mixture were then passed through a nanomedicine preparation system at flow rates of 9 mL / min and 3 mL / min to prepare mRNA-LNPs. The prepared mRNA-LNPs were added to a 30 kDa ultrafiltration tube, and an appropriate amount of DPBS was added. Ultrafiltration was performed to remove the ethanol, repeated several times until the anhydrous ethanol concentration was reduced to 1% of its original level. Finally, the mixture was filtered through a 0.22 μm filter for sterilization.

[0032] Add 10 μL of the prepared mRNA-LNPs to 1 mL of purified water, and analyze the particle size and PDI using a particle size analyzer. Detect the RNA concentration before and after mRNA-LNPs lysis, and calculate the mRNA encapsulation efficiency. The procedure is as follows: 1) Prepare 20% Triton X-100: Take 320 μL of Equalbit RNA BR Buffer, preheat at 37℃, add 80 μL of Triton X-100, incubate at 37℃ for 15 min, and then vortex to mix.

[0033] 2) mRNA-LNP disruption: Take 80 μL of Equalbit RNA BR Buffer, add 10 μL of 20% Triton X-100 and 10 μL of mRNA-LNP, mix well by pipetting, and incubate at 37°C for 10 min.

[0034] 3) Take 10 μL of mRNA-LNP and use the Equalbit RNA HS (High Sensitivity) Assay Kit to detect the mRNA concentration of mRNA-LNP before fragmentation.

[0035] 4) Take 10 μL of the broken mRNA-LNP and use the Equalbit RNA BR (Broad-Range) Assay Kit to detect the mRNA concentration after the mRNA-LNP is broken.

[0036] 5) The amount of mRNA coated is the amount of mRNA after mRNA-LNP fragmentation minus the amount of mRNA before mRNA-LNP fragmentation.

[0037] 6) Encapsulation efficiency = (encapsulated mRNA concentration / total mRNA concentration after fragmentation) × 100% result: The prepared mRNA-LNP was a pale blue, opalescent, transparent solution with an average particle size of approximately 80 nm, a PDI < 0.1, and good sample homogeneity. The encapsulation efficiency, as detected by the Qubit fluorescent dye method, was > 95%. Other conventional methods can also be used to prepare mRNA delivery formulations, and the methods and reagents are not limited to those described above.

[0038] Example 3: Vaccine Compatibility PT-mRNA-LNPs, FHA-mRNA-LNPs, PRN-mRNA-LNPs, Fim2-mRNA-LNPs, and Fim3-mRNA-LNPs were prepared. PT-mRNA-LNPs, FHA-mRNA-LNPs, and PRN-mRNA-LNPs were combined in equal proportions to form a three-component pertussis mRNA vaccine, with each dose containing 75 μg of mRNA. PT-mRNA-LNPs, FHA-mRNA-LNPs, PRN-mRNA-LNPs, Fim2-mRNA-LNPs, and Fim3-mRNA-LNPs were also combined in equal proportions to form a five-component pertussis mRNA vaccine, with each dose containing 100 μg of mRNA.

[0039] Example 4: In vivo immunogenicity evaluation of three-component and five-component pertussis mRNA vaccines (1) Mouse immunization Mice were immunized with the three-component and five-component pertussis mRNA vaccines prepared above; empty LNPs were used as a negative control, and Sanofi Pasteur's hexavalent vaccine (vaxelis) was used as a positive control. Female SPF-grade CD1 mice (20-24g) were used in four groups. Each vaccine was administered at a 5-fold dilution to 15 mice (500 μL / mouse). Ten mice were used as the humoral immunity assay group, and five mice as the cellular immunity assay group. Three weeks after the first immunization, the five mice in the cellular immunity assay group were re-injected with the 5-fold dilution vaccine. Four weeks after the initial immunization, whole blood was collected from the humoral immunity assay group via enucleation, and spleens were collected from the cellular immunity assay group.

[0040] (2) Evaluation of humoral immune response The levels of PT, FHA, PRN, and Fim antibodies were detected by ELISA.

[0041] (3) Evaluation of cellular immune response Spleen samples were collected from the cellular immunoassay assay group for flow cytometry T-cell typing. The specific procedure is as follows: Single-cell preparation: Grind spleen tissue into a single-cell suspension; Lymphocyte separation: Spleen single-cell suspension is slowly added along the tube wall into a centrifuge tube containing lymphocyte separation solution, and density gradient centrifugation is performed; Cell collection: Collect cells from the white membrane layer; Cell washing: Wash twice with PBS; Cell division: After washing the cell pellet, resuspend it in 1 mL of PBS. Divide the obtained cells into two parts. Take one-third of the total volume of cells for Panel-1 detection, and the detection indicators are: CD3, CD4, CD44, CD62L. Take two-thirds of the total volume for Panel-2 detection, and the detection indicators are: CD3, CD4, IL-4, IFN-γ.

[0042] Two Panel Detection Methods Panel-1 testing method: 1. Cell collection and tube preparation: Collect cells and prepare blank tubes, single-stain tubes, and sample tubes; 2. Dyeing live and dead dyes: Add the prepared live and dead dyes and incubate at room temperature for 10 minutes; 3. Cell membrane staining: Wash cells with PBS, add cell membrane dye, and stain at room temperature for 15-30 min; 4. Cell washing: Wash twice with PBS; 5. On-machine testing and analysis: Based on the blank and single-color tube, establish the scheme and perform on-machine analysis.

[0043] Panel-2 testing method: 1. Cocktail blocking: Add blocking agent and incubate for 4 hours; 2. Cell collection and tube preparation: Collect cells and prepare blank tubes, single-stain tubes, and sample tubes; 3. Dyeing live and dead dyes: Add the prepared live and dead dyes and incubate at room temperature for 10 minutes; 4. Cell membrane staining: Wash cells with PBS, add cell membrane dye, and stain at room temperature for 15-30 min; 5. Cell fixation and permeabilization: Add fixative and fix at room temperature for 20-30 minutes; after fixation, perform permeabilization. 6. Intracellular staining: Add intracellular dye and stain at room temperature for 15-30 minutes; 7. On-machine testing and analysis: Based on the blank and single-color tube, establish a scheme and perform on-machine analysis.

[0044] result: (1) Evaluation of humoral immune response: such as Figure 1 As shown, both the three-component and five-component pertussis mRNA vaccine groups produced high levels of antibodies. The serum levels of PT IgG, FHA IgG, and Fim IgG antibodies in mice in the Sanofi Pasteur hexavalent vaccine group were significantly higher than those in the pertussis mRNA vaccine group, while there were no significant differences between the three-component and five-component pertussis mRNA vaccine groups. The serum PRN antibody level in mice in the pertussis mRNA vaccine group was significantly higher than that in the Sanofi Pasteur hexavalent vaccine group.

[0045] (2) Evaluation of cellular immune response: Inducing a cellular immune response against Bordetella pertussis and improving the durability of protection are important directions in the development of novel pertussis vaccines. The response of mouse spleen memory T cells was detected by flow cytometry, and the results are as follows: Figure 2The results showed that the proportions of central memory T cells (Tcm) and effector memory T cells (Tem) in mice immunized with the three-component and five-component pertussis mRNA vaccine groups were significantly different from those in the negative control group, while there was no significant difference between the Sanofi Pasteur hexavalent vaccine group and the negative control group. This suggests that the three-component and five-component pertussis mRNA vaccines may provide more durable immune protection.

[0046] Detection of CD4 IFN-γ secreted by mice in the three-component and five-component pertussis mRNA vaccine groups + The proportion of T cells increased significantly, and CD4 cells secreting IL-4 were detected. + T cells showed no significant difference between the three-component pertussis mRNA vaccine, the five-component pertussis mRNA vaccine, and the Sanofi Pasteur hexavalent vaccine and the negative control group. This indicates that the three-component and five-component pertussis mRNA vaccines can induce a Th1-prone cellular immune response in mice. Figure 3 ).

[0047] Example 5: Potency testing of three-component and five-component pertussis mRNA vaccines 1) Mouse immunization The procedure for testing vaccine immunoprotection followed the MICA method in the Chinese Pharmacopoeia (Volume III, 2020 edition). The vaccines required for immunization included the pertussis reference vaccine, the three-component pertussis mRNA vaccine, the five-component pertussis mRNA vaccine, and the Sanofi Pasteur hexavalent vaccine. Mice were immunized with three dilutions of the pertussis reference vaccine and the test sample: 5-fold, 25-fold, and 125-fold dilutions, respectively. Each dilution was used to immunize 20 equal-sized (10-12g male and female) NIH mice, with each mouse receiving an intraperitoneal injection of 0.5 mL. Simultaneously, 55-60 mice were housed as controls.

[0048] 2) Attack Twenty-one days after immunization, each mouse was challenged intracerebrally with 0.03 mL of bacterial suspension (containing 8.0 × 10⁻⁶ bacteria) using a 0.25 mL syringe. 4 The LD50 of the challenge bacteria was determined in the control group, with five dilutions set for each group, each containing 8.0 × 10⁻⁶ bacteria. 4 8.0×10 3 8.0×10 2 8.0×10 1 In addition to the control group, 10 mice were challenged per dilution. The challenge bacteria was Bordetella pertussis strain CMCC58030 (18323).

[0049] 3) Result Calculation and Judgment The number of dead mice was observed and recorded over 14 days. LD50 values ​​were calculated using the Reed-Muench method. Vaccine efficacy was calculated using parallel-line determinations correlated with qualitative responses.

[0050] result: Fourteen days after the virus challenge, the number of surviving mice in each group was observed and shown in Table 4. The results were then entered into the statistical software Statis and calculated using the "Probit Analysis" method to obtain the vaccine potency and 95% confidence interval for each group, as shown in Table 5.

[0051] Table 4

[0052] Table 5

[0053] Example 6: Bordetella pertussis strain clearance experiment of three-component and five-component pertussis mRNA vaccines 1) Mouse immunization The three-component and five-component pertussis mRNA vaccines prepared above were administered intraperitoneally to mice. Empty LNPs were used as a negative control, and Sanofi Pasteur hexavalent vaccine was used as a positive control. Female SPF-grade BALB / c mice weighing 18-20g were used. Each vaccine was administered to 20 mice with a 5-fold diluted vaccine, via intraperitoneal injection of 500 μL per mouse.

[0054] 2) Attacking the poison Twenty-one days after immunization, mice from each group were sequentially placed into a mouse exposure chamber equipped with a nebulizer and treated with 10... 11 Atomize and expose to CFU / mL Bordetella pertussis CMCC58030(18323) bacterial suspension for 30 min.

[0055] 3) Colony count in the lungs of infected mice Lung tissue was collected from mice at 0, 3, 7, and 14 days post-exposure to the virus, with 5 mice in each group. A tissue homogenizer was placed on ice, and 1 mL of pre-cooled physiological saline was added to the homogenizer before adding the mouse lung tissue. The homogenate was then prepared. After appropriate dilution, 50 μL of the diluted solution was evenly spread onto sheep blood BG plates and incubated at 37°C for 5 days. The plates were then observed, and the colony forming units (CFU) were calculated.

[0056] result: The colonies on sheep blood agar plates in each experimental group showed morphology consistent with known Bordetella pertussis colonies. For example... Figure 4As shown, lung tissue homogenates were immediately cultured after challenge, and the number of Bordetella pertussis colonies in the lungs of all groups was consistent. Three days later, the colony count in the lung tissue homogenates decreased in all experimental groups. No Bordetella pertussis colonies were detected at day 7. At day 14, Bordetella pertussis colonies were still undetectable in the three-component and five-component Bordetella pertussis mRNA vaccine groups, while a small number of Bordetella pertussis colonies were detected in the lung tissue homogenate culture of the Sanofi Pasteur hexavalent vaccine group. The lung tissue homogenate culture of the negative control group immunized with empty LNPs consistently showed a large number of Bordetella pertussis colonies.

[0057] Example 7: Specific toxicity testing of three-component and five-component pertussis mRNA vaccines 1) Mouse immunization The tests were conducted according to the "Specific Toxicity Test Method for Pertussis Vaccine Original Solution" in the Chinese Pharmacopoeia (Volume III, 2025 Edition). Ten 14-16g NIH mice (equal sexes per group) were administered 500μL intraperitoneally per mouse. The vaccines administered included a three-component pertussis mRNA vaccine at a pre-packaged dose, a five-component pertussis mRNA vaccine at a pre-packaged dose, a Sanofi Pasteur hexavalent vaccine at a pre-packaged dose, and a toxicity reference.

[0058] 2) Mouse leukocytosis test Three days after injection, peripheral blood was collected from mice for white blood cell count. The results were processed using statistical methods.

[0059] 3) Mouse histamine sensitization test Four days after injection, each mouse was intraperitoneally injected with 0.5 mL of a solution (containing 4 mg of histamine dihydrochloride or 2 mg of histamine diphosphate), and the rectal temperature of the mice was measured 30 minutes later. The experimental results were processed using statistical methods.

[0060] result: The experimental results, processed using statistical software, showed that the leukocytosis toxicity activities in mice injected with the three-component pertussis mRNA vaccine, the five-component pertussis mRNA vaccine, and the Sanofi Pasteur hexavalent vaccine were 0.024 LPU / mL, 0.019 LPU / mL, and 0.065 LPU / mL, respectively, all below the 0.5 LPU / mL requirement stipulated in the Chinese Pharmacopoeia. The experimental results, processed using the "Parallel Line Assay" method in the Statis software, showed that the histamine sensitization toxicity activities in mice injected with the three-component pertussis mRNA vaccine, the five-component pertussis mRNA vaccine, and the Sanofi Pasteur hexavalent vaccine were 0.487 HSU / mL, 0.108 HSU / mL, and 0.220 HSU / mL, respectively, all below the 0.8 HSU / mL requirement stipulated in the Chinese Pharmacopoeia, and no animal deaths were observed.

[0061] gene sequence PRN amino acid sequence: DWNNQSIVKTGERQHGIHIQGSDPGGVRTASGTTIKVSGRQAQGILLENPAAELQFRNGSVTSSGQLSDDGIRRFLGTVTVKAGKLVADHATLANVGDTWDDDGIALYVAGEQAQASIADSTLQGAGGVQIERGANVTVQRSAIVDGGLHIGALQSLQPEDLPPSRVVLRDTNVTAVPASGAPAAVSVLGASELTLDGGHITGGRAAGVAAMQGAVVHLQRATIRRGDAPAGGAVPGGAVPGGAVPGGFGPGGFGPVLDGWYGVDVSGSSVELAQSIVEAPELGAAIRVGRGARVTVSGGSLSAPHGNVIETGGARRFAPQAAPLSITLQAGAHAQGKALLYRVLPEPVKLTLTGGADAQGDIVATELPSIPGTSIGPLDVALASQARWTGATRAVDSLSIDNATWVMTDNSNVGALRLASDGSVDFQQPAEAGRFKVLTVNTLAGSGLFRMNVFADLGLSDKLVVMQDASGQHRLWVRNSGSEPASANTLLLVQTPLGSAATFTLANKDGKVDIGTYRYRLAANGNGQWSLVGAKAPPAPKPAPQPGPQPPQPPQPQPEAPAPQPPAGRELSAAANAAVNTGGVGLASTLWYAESNAL Fim2 amino acid sequence MQIPFQRALRLCLRAALAAIASAAHADDGTIVITGTITDTTCVIEDPSGPNHTKVVQLPKISKNALKANGDQAGRTPFIIKLKDCPSSLGNGVKAYFEPGPTTDYSTGDLRAYKMVYATNPQTQLSNITAATEAQGVQVRISNLNDSKITMGANEATQQAAGFDPEVQTGGTSRTVTMRYLASYVKKNGDVEASAITTYVGFSVVYP Fim3 amino acid sequence ALANDGTIVITGSISDQTCVIEEPSTLNHIKVVQLPKISKNALRNDGDTAGATPFDIKLKECPQALGALKLYFEPGITTNYDTGDLIAYKQTYNASGNGNLSTVSSATKAKGVEFRLANLNGQHIRMGTDKTTQAAQTFTGKVTNGSKSYTLRYLASYVKKPKEDVDAAQITSYVGFSVVYP Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0062] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A pertussis mRNA vaccine, characterized in that, The pertussis mRNA vaccine comprises one or more mRNA molecules, wherein the antigens encoded by the mRNA molecules include: whooping cough toxin, Filamentous hemagglutinin; and, Pertussis adhesin.

2. The pertussis mRNA vaccine according to claim 1, characterized in that, The antigens encoded by the mRNA molecules also include fimbriae 2 and fimbriae 3.

3. The pertussis mRNA vaccine according to claim 2, characterized in that, The mRNA encoding the pertussis adhesin antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:1-SEQ ID NO:6; and / or, The mRNA encoding the fimbriae protein 2 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:7-SEQ ID NO:12; and / or, The mRNA encoding the fimbriae protein 3 antigen gene contains a nucleotide sequence having at least 85%, at least 90%, at least 95%, or 100% identity with the nucleotide sequences shown in SEQ ID NO:13-SEQ ID NO:

18.

4. The pertussis mRNA vaccine according to claim 2, characterized in that, A DNA molecule encoding the pertussis adhesin antigen gene, the sequence of which is shown in SEQ ID NO:19-SEQ ID NO:24; and / or, DNA molecules encoding the fimbriae protein 2 antigen gene, the sequences of which are shown in SEQ ID NO:25-SEQ ID NO:30; and / or, The DNA molecule encoding the fimbriae protein 3 antigen gene has the sequence shown in SEQ ID NO:31-SEQ ID NO:

36.

5. The pertussis mRNA vaccine according to claim 2, characterized in that, The mRNA molecules encoding the genes for pertussis toxin, filamentous hemagglutinin, pertussis adhesin, fimbriaein 2, and fimbriaein 3 antigens all include: (a) 5'-cap structure; (b) 5' non-translation area; (c) Kozak sequence; (d) The antigen gene sequence of Bordetella pertussis, which encodes the pertussis adhesin and fimbriae 2 and 3 antigens that can induce an immune response; (e) 3' untranslated region; (f) Polyadenylate tail elements with a total length of 100 nt or more.

6. The pertussis mRNA vaccine according to claim 1 or 2, characterized in that, It also includes an antigen delivery vector for encapsulating the antigen of the pertussis mRNA vaccine.

7. The pertussis mRNA vaccine according to claim 6, characterized in that, The antigen delivery carrier includes ionizable lipids, neutral lipids, PEGylated lipids, and cholesterol.

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