Ev71 virus mRNA vaccine and application thereof

Abstract

This invention relates to an EV71 virus mRNA vaccine and its application, wherein the EV71 virus mRNA vaccine contains an encoding EV71 virus. vp1 The mRNA molecule of the gene is encapsulated by lipid nanoparticles; the nucleic acid sequence of the mRNA molecule is shown in SEQ ID NO.1; the sequence encoding the amino acids of the mRNA molecule is shown in SEQ ID NO.2. The mRNA vaccine provided by this invention significantly enhances the production of anti-EV71 virus in mice. vp1 Neutralizing antibody titers and specific cellular immune responses.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine industry, specifically relating to an EV71 virus mRNA vaccine and its application. Background Technology

[0002] EV71 virus belongs to the Picornaviridae family and the Enterovirus genus. It is one of the important pathogens that cause hand-foot-mouth disease in infants and young children, and is especially closely related to severe hand-foot-mouth disease and neurological complications in children.

[0003] The EV71 viral capsid is composed of four structural proteins, among which vp1 The protein, located on the surface of the virus particle, contains several important neutralizing epitopes and is one of the key antigens that induce a protective immune response in the body. Although EV71 inactivated vaccines are already on the market and used clinically, there is still room for further optimization in terms of vaccine production cycle, duration of immunity, and response to viral mutations.

[0004] In recent years, the development of novel mRNA-based vaccines has shown promising application prospects in the field of infectious disease prevention and control due to their advantages such as high safety, short production cycle, and high antigen expression efficiency.

[0005] Efficient delivery of mRNA vaccines is crucial for their successful clinical translation. Lipid nanoparticle (LNP) formulations are the most successful, effective, and safe method for delivering mRNA.

[0006] Therefore, the key antigens of the EV71 virus vp1 The development of an EV71 virus mRNA vaccine by combining mRNA vaccine and LNP technology is of great significance for the prevention and control of hand-foot-mouth disease in infants and young children. Summary of the Invention

[0007] The purpose of this invention is to provide an EV71 virus mRNA vaccine for the prevention and control of EV71 virus infection; another purpose of this invention is to provide the application of this EV71 virus mRNA vaccine.

[0008] The technical solution adopted by this invention to solve its technical problem is: this EV71 virus mRNA vaccine contains an encoding EV71 virus. vp1 The mRNA molecule of the gene is encapsulated by lipid nanoparticles.

[0009] The nucleic acid sequence of the mRNA molecule in the above scheme is shown in SEQ ID NO.1; it includes the following elements from the 5' end to the 3' end: AflⅡ site, 5'UTR, Kozak sequence, SP sequence, BamHI site, vp1Gene, Myc tag, EcoRI site, 3'UTR, Poly(A) tail, NotI site.

[0010] The sequence of the amino acid encoding the mRNA molecule in the above scheme is shown in SEQ ID NO.2; including the following elements from the 5' end to the 3' end: SP sequence, VP1 protein, and Myc tag.

[0011] In the above scheme, the mRNA nucleotide sequence is obtained by in vitro transcription, specifically: synthesis vp1 It is inserted into the BamHI / EcoRI site and tandem with the T7 promoter, 5'UTR, Kozak, signal peptide, 3'UTR, and polyA. The T7 RNA polymerase then transcribes the mRNA molecule in vitro to obtain the mRNA molecule.

[0012] The mRNA molecule preparation method in the above scheme is as follows: 1) Synthesizing the EV71 virus vp1 Gene; 2) Construct the DNA template for the mRNA molecule; 3) Obtained by in vitro transcription using T7 RNA polymerase vp1 mRNA.

[0013] The lipid nanoparticles in the above scheme include: SM-102, DSPC (a type of phospholipid), cholesterol, and PEG-PEG2000 (a type of PEGylated lipid).

[0014] The lipid nanoparticles in the above scheme are prepared through the following steps: 1) Preparation of the lipid phase: SM-102, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol, with a molar ratio of 50:10:38.5:1.5. 2) Preparation of the aqueous phase: vp1 mRNA was dissolved in sodium citrate buffer; 3) The lipid and aqueous phases are mixed to form lipid nanoparticles LNP-mRNA that encapsulate mRNA. The volume ratio of the lipid phase to the aqueous phase is 1:3.

[0015] The aforementioned EV71 virus mRNA vaccine is used to prevent EV71 virus infection.

[0016] The above-mentioned method for using the EV71 virus mRNA vaccine to prevent EV71 virus infection involves immunizing mice via intramuscular injection to induce the body's response to... vp1 Specific cellular immunity and humoral immunity. Beneficial effects

[0017] 1. The mRNA vaccine provided by this invention significantly enhances the production of anti-EV71 virus in mice. vp1 Neutralizing antibody titers and specific cellular immune responses.

[0018] 2. The mRNA vaccine of this invention exhibits high-efficiency expression in cells. vp1 protein.

[0019] 3. The mRNA vaccine of this invention can increase the proportion of CD4⁺T and CD8⁺T cells in mice and induce higher levels of antibodies. vp1 Specific IgG antibodies were detected, with the IgG2a subtype at a higher level than the IgG1 subtype, exhibiting an immune response characteristic dominated by the Th1 type.

[0020] 4. The mRNA vaccine of this invention has good immunogenicity and safety.

[0021] 5. The EV71 virus mRNA vaccine of the present invention is suitable for the rapid development of hand-foot-mouth disease vaccines and has broad application prospects.

[0022] 6. The lipid nanoparticles in this invention have a uniform particle size distribution, stable structure, and good delivery capability.

[0023] 7. The mRNA vaccine of this invention is highly expressed in cells and induces significant cellular and humoral immunity in mice, providing a safe and efficient novel candidate vaccine for the prevention and control of EV71 virus infection. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of an mRNA molecule.

[0025] Figure 2 The graph shows the particle size and zeta potential of LNP-mRNA, where a is the LNP particle size distribution, b is the LNP particle size distribution, c is the LNP potential distribution, and d is the LNP potential distribution.

[0026] Figure 3 This is an electron micrograph of LNP-mRNA.

[0027] Figure 4 This represents the expression of LNP-mRNA in cells.

[0028] Figure 5 Flow cytometry analysis of spleen T cells from mice immunized with LNP-mRNA.

[0029] Figure 6 The percentage of spleen T cells in mice immunized with LNP-mRNA.

[0030] Figure 7 The serum antibody titer of mice immunized with LNP-mRNA was determined.

[0031] Figure 8 The percentage of serum antibody subtypes in mice immunized with LNP-mRNA. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings: Combination Figures 1-8 As shown, this mRNA vaccine contains a coding vp1 The mRNA molecule of the gene has the nucleotide sequence shown in SEQ ID NO.1. The amino acid sequence encoded by the mRNA molecule is shown in SEQ ID NO.2. An EV71 virus mRNA vaccine was obtained through gene synthesis, in vitro transcription, and lipid nanoparticle preparation (LNP-mRNA). After transfection of cells with LNP-mRNA, it was efficiently expressed in cells. vp1 Protein. LNP-mRNA immunization of mice induces the body to target protein. vp1 Significant cellular and humoral immunity.

[0033] This EV71 virus mRNA vaccine contains the code for the EV71 virus. vp1 The mRNA molecule of the gene encapsulates the EV71 virus. vp1 Lipid nanoparticles containing mRNA molecules. The mRNA molecule nucleic acid sequence is shown in SEQ ID NO.1, obtained by in vitro transcription; it includes the following elements from the 5' end to the 3' end: AflⅡ site, 5'UTR, Kozak sequence, SP sequence, BamHI site, vp1 Gene, Myc tag, EcoRI site, 3'UTR, Poly(A) tail, NotI site.

[0034] The amino acid sequence encoded by the mRNA molecule is shown in SEQ ID NO.2; it includes the following elements from the 5' end to the 3' end: SP sequence, VP1 protein, and Myc tag.

[0035] The nucleotide sequence of the mRNA molecule is obtained by in vitro transcription, specifically: synthesis vp1 It is inserted into the BamHI / EcoRI site and tandem with the T7 promoter, 5'UTR, Kozak, signal peptide, 3'UTR, and polyA. The T7 RNA polymerase then transcribes the RNA in vitro to obtain mRNA molecules.

[0036] The method for preparing mRNA molecules is as follows: 1) Synthesizing the EV71 virus vp1 Gene; 2) Construct the DNA template for the mRNA molecule; 3) Obtained by in vitro transcription using T7 RNA polymerase vp1 mRNA.

[0037] The lipid nanoparticles of this invention comprise the following components: ionizable lipid material (SM-102), auxiliary phospholipid DSPC (distearate phosphatidylcholine), cholesterol, and polyethylene glycol-modified lipid DMG-PEG2000 (dimyristoyl glycerol-polyethylene glycol 2000). The lipid nanoparticles are prepared through the following steps: 1) Dissolve SM-102, DSPC, cholesterol, and DMG-PEG2000 in ethanol to prepare the lipid phase. The molar ratio of SM-102, DSPC, cholesterol, and DMG-PEG2000 is 50:10:38.5:1.5. 2) vp1 mRNA was dissolved in sodium citrate buffer to prepare an aqueous phase; 3) The lipid phase and aqueous phase are mixed to obtain lipid nanoparticles. The lipid phase and aqueous phase are mixed to form lipid nanoparticles LNP-mRNA that encapsulate mRNA. The volume ratio of the lipid phase to the aqueous phase is 1:3.

[0038] The lipid nanoparticles in this invention have a uniform particle size distribution, stable structure, and good delivery capability.

[0039] This invention is an encoding of the EV71 virus. vp1 The study explored the use of mRNA molecules of genes and mRNA vaccines encapsulated in lipid nanoparticles. RNA vaccines exhibit high cellular expression and induce significant cellular and humoral immunity in mice, providing a safe and highly effective novel candidate vaccine for the prevention and control of EV71 virus infection.

[0040] This mRNA vaccine is a lipid nanoparticle (LNP-mRNA) containing the mRNA molecule, encoding an amino acid sequence as shown in SEQ ID NO.2.

[0041] The aforementioned mRNA molecules and lipid nanoparticles are used to prepare a vaccine to prevent EV71 infection.

[0042] The above-mentioned mRNA vaccine was used in the prevention of EV71 virus infection by immunizing mice via intramuscular injection to induce specific cellular and humoral immunity against VP1.

[0043] Following transfection of cells with LNP-mRNA, VP1 protein is efficiently expressed intracellularly. Immunization of mice with LNP-mRNA induces significant cellular and humoral immunity against VP1. This invention provides a new option for developing safe and effective novel vaccines to control EV71 virus infection.

[0044] Example: Preparation of LNP-mRNA lipid nanoparticles Design the DNA coding sequence of mRNA molecules ( Figure 1 ), synthesized and constructed by Nanjing Qingke Biotechnology Co., Ltd.

[0045] mRNA was transcribed in vitro using the T7 RNA transcription kit. N1-methylpseuuridine was added for nucleotide modification during transcription, and a capping reaction was performed using the Cap1 capping kit. The mRNA was purified using the Tiangen Biotech RNA purification kit and analyzed by gel electrophoresis to confirm the mRNA molecule size.

[0046] The obtained mRNA molecules include the AflⅡ site, 5'UTR, Kozak sequence, SP sequence, BamHI site, vp1 sequence, Myc tag, EcoRI site, 3'UTR, Poly(A) tail, and NotⅠ site.

[0047] The mRNA molecular sequences are shown in the table below:

[0048] The amino acid sequence encoded by the mRNA molecule is shown in the table below:

[0049] Ionizable lipid material SM-102, auxiliary phospholipid DSPC (distearylphosphatidylcholine), cholesterol, and polyethylene glycol-modified lipid DMG-PEG2000 (dimyristicoglycerol-polyethylene glycol 2000) were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5 to prepare a lipid mixture solution.

[0050] Dilute the mRNA to a concentration of 170 μg / ml with sodium citrate buffer (20 mM, pH 4.0) and let stand for 20 min.

[0051] The liposomes and mRNA were rapidly mixed at a volume ratio of 1:3. The liposome-mRNA mixture was collected and allowed to stand at room temperature for 30 min. The mixture was then diluted 10-40 times with RNase-free PBS, centrifuged and concentrated using an ultrafiltration tube to remove ethanol. The volume was brought to 1 mL and sterilized by 0.22 μm filtration.

[0052] After encapsulating liposome LNP-mRNA, 30 μL of LNP-mRNA and LNP solution were taken and added to 1 mL of PBS, mixed well, and used for particle size and potential testing.

[0053] Particle size determination: Take 50 μL of diluted LNP-mRNA and LNP solution, add them to the micro sample cell, and use a particle size analyzer at room temperature to determine the particle size and polydispersity index.

[0054] Zeta potential assay: Take 50 μL of diluted LNP-mRNA and LNP solution, add them to the Malvern Zeta potential sample cell, and measure the Zeta potential using a Zeta potential analyzer at room temperature.

[0055] Transmission electron microscopy observation: Dilute LNP-mRNA with PBS to an appropriate concentration, take 10 μL of sample solution, drop it onto a membrane-covered TEM copper grid, let it stand for 10 min, remove excess liposome solution with filter paper, add 2% phosphotungstic acid negative staining solution, stain at room temperature for 10 min, let it stand and dry to prepare TEM sample, and observe and photograph the sample using transmission electron microscopy.

[0056] Cells were cultured to 80% confluence, transfected with LNP-mRNA for 24 hours, and transfected cells were collected, lysed on ice, and protein samples were obtained. SDS-PAGE electrophoresis was performed, and the proteins were transferred to a PVDF membrane. Hybridization was performed using antibodies against Myc, EV71, and β-Actin, respectively. The cells were incubated with HRP-labeled secondary antibody, and the target proteins were detected using a Western blot chemiluminescence imaging system.

[0057] Cells were transfected with LNP-mRNA for 24 hours. The samples were then observed and photographed under a fluorescence microscope after fixation, permeabilization, blocking, antibody incubation, and mounting.

[0058] Validation example: Immunogenicity evaluation of mRNA vaccines Thirty 6-8 week old BALB / c mice were randomly divided into 5 groups (n=6 per group): PBS control group, mRNA group, LNP-mRNA group, VP1 protein group, and plasmid group. Mice were immunized intramuscularly with booster immunizations every 14 days, for a total of 3 immunizations. The immunization doses were: 100 μL / mouse for the PBS control group; 10 μg / mouse for the mRNA, LNP-mRNA, and plasmid groups; and 50 μg / mouse for the VP1 protein group. Blood samples were collected on days 0, 10, 24, and 35 after the first immunization and stored at -20 ℃. Splenic cells were isolated and collected after immunization.

[0059] Seven days after the third immunization, the spleen of the mice was dissected, the outer mucosa was removed, the spleen was washed with PBS, the spleen was minced, and the filtrate was filtered through a 100μm cell filter. The filtrate was collected and centrifuged at 4 ℃ and 1000 rpm for 5 min. The supernatant was discarded, and the filtrate was resuspended in BoyoFC flow cytometry buffer. The supernatant was then discarded after centrifugation. 6-8 mL of ACK lysis buffer was added at a ratio of 1:3-5, and the lysis was allowed to stand for 3-5 min. An equal volume of BoyoFC buffer was added to terminate the lysis, and the supernatant was discarded after centrifugation. 2 mL of BoyoFC buffer was added for resuspending, and the lysis was 10-fold diluted before counting. The concentration was adjusted to 5-10 × 10⁻⁶. 6 Take 50 μL (approximately 2.5-5 × 10⁻⁶ cells / mL).5 (1 cell), 4 ℃ for later use.

[0060] Flow cytometry assay: Take 50 μL of cell suspension, add 2.5 μL of FITC mouse CD4 and 2.5 μL of FITC mouse CD8 antibody, and incubate on ice in the dark for 30-60 min; add 0.5 mL of BoyoFC cell staining buffer, centrifuge at 1000 rpm for 5 min, and then perform flow cytometry analysis.

[0061] The levels of VP1-specific IgG antibodies and their subtypes IgG1 and IgG2a in the serum of immunized mice were detected by indirect ELISA.

[0062] Mouse serum was collected on days 0, 14, 28, and 35. VP1 protein was diluted to 2 μg / mL with carbonate buffer (pH 9.6), and 100 μL was added to each well. The plate was incubated overnight at 4°C. Each well was washed with 200 μL PBST for 5 min each time, for a total of 3 times. Each well was blocked with 200 μL BSA (3-5%) for 1 h. Mouse serum was serially diluted with PBS (1:200 to 1:3200), and 100 μL was added to each well. The plate was incubated at 37°C for 1 h, and washed 3 times with PBST. Each well was then incubated with 100 μL HRP-labeled goat anti-mouse IgG / IgG1 / IgG2a (1:5000) at room temperature for 1 h. After washing with PBS, 100 μL TMB chromogenic solution was added to each well, and the plate was incubated in the dark for 15-30 min. 100 μL of stop solution was added, and the absorbance at 450 nm and 650 nm was measured using a microplate reader. The OD value was calibrated as OD0. 450 -OD 650 .

[0063] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An EV71 virus mRNA vaccine, characterized in that: The EV71 virus mRNA vaccine comprises an mRNA molecule encoding an EV71 virus vp1 gene, the mRNA molecule being encapsulated by a lipid nanoparticle.

2. The EV71 virus mRNA vaccine according to claim 1, characterized in that: The nucleic acid sequence of the mRNA molecule is shown as SEQ ID NO. 1; comprising the following elements from 5' end to 3' end: Afl II site, 5' UTR, Kozak sequence, SP sequence, BamH I site, vp1 gene, Myc tag, EcoR I site, 3' UTR, Poly(A) tail, Not I site.

3. The EV71 virus mRNA vaccine according to claim 2, characterized in that: The sequence of the amino acids encoded by the mRNA molecule is shown in SEQ ID NO.2; it includes the following elements from the 5' end to the 3' end: SP sequence, VP1 protein, and Myc tag.

4. The EV71 virus mRNA vaccine according to claim 3, characterized in that: The mRNA nucleotide sequence was obtained by in vitro transcription, specifically: synthesis vp1 It is inserted into the BamHI / EcoRI site and tandem with the T7 promoter, 5'UTR, Kozak, signal peptide, 3'UTR, and polyA. The T7 RNA polymerase then transcribes the RNA in vitro to obtain mRNA molecules.

5. The EV71 virus mRNA vaccine according to claim 4, characterized in that: The method for preparing the mRNA molecule is as follows: 1) Synthesizing the EV71 virus vp1 Gene; 2) Construct the DNA template for the mRNA molecule; 3) Obtained by in vitro transcription using T7 RNA polymerase vp1 mRNA.

6. The EV71 virus mRNA vaccine according to claim 5, characterized in that: The lipid nanoparticles comprise SM-102, DSPC (a type of phospholipid), cholesterol, and PEG-PEG2000 (a type of PEGylated lipid).

7. The EV71 virus mRNA vaccine according to claim 6, characterized in that: The lipid nanoparticles were prepared by the following steps: 1) Preparation of the lipid phase: SM-102, DSPC, cholesterol, and DMG-PEG2000 were dissolved in ethanol, with a molar ratio of 50:10:38.5:1.

5. 2) Preparation of the aqueous phase: vp1 mRNA was dissolved in sodium citrate buffer; 3) The lipid and aqueous phases are mixed to form lipid nanoparticles LNP-mRNA that encapsulate mRNA. The volume ratio of the lipid phase to the aqueous phase is 1:

3.

8. The application of the EV71 virus mRNA vaccine according to claim 7, characterized in that: Used to prevent EV71 virus infection.

9. The application of the EV71 virus mRNA vaccine according to claim 8, characterized in that: When the EV71 virus mRNA vaccine is used to prevent EV71 virus infection, mice are immunized by intramuscular injection to induce the body's response to the virus. vp1 Specific cellular immunity and humoral immunity.

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