Recombinant mRNA (messenger ribonucleic acid), DNA (deoxyribonucleic acid) molecule, pharmaceutical composition, vaccine and preparation method

Abstract

The invention discloses a recombinant mRNA (messenger ribonucleic acid), a DNA (deoxyribonucleic acid) molecule, a pharmaceutical composition, a vaccine and a preparation method. The mRNA hepatitis B therapeutic vaccine provided by the invention can generate dual mechanisms of activating humoral immunity and T cell immunity so as to resist hepatitis B virus, and is a therapeutic mRNA vaccine which is prepared for the first time and can effectively induce a specific neutralizing antibody (protective antibody) aiming at HBV. By optimizing the nucleotide sequence, more mRNA can be translated under the condition of the same inoculum size, and a greater biological effect can be exerted.

Description

[0001] This application is a divisional application with application number CN202211159116.0, application date September 22, 2022, and invention name “A therapeutic mRNA vaccine for hepatitis B virus and its preparation method and application”. Technical Field

[0002] The present invention belongs to the technical field of mRNA vaccines, and specifically relates to a recombinant mRNA, a DNA molecule, a pharmaceutical composition, a vaccine and a preparation method. Background Art

[0003] Hepatitis B is a global public health problem. Current clinical treatment drugs include interferon and nucleoside (acid) analogs, which can effectively inhibit HBV transcription or replication, but cannot completely eliminate HBV. There are also problems such as low treatment response rate or low antigen negative conversion rate, making it difficult to cure hepatitis B.

[0004] Most chronic hepatitis B patients are caused by mother-to-child transmission or infection before the age of 1. The long-term latency of the virus leads to overexpression of immunosuppressive molecules, loss of co-stimulatory molecules or abnormal increase in Treg cell levels, which leads to exhaustion of the body's antiviral immunity and non-responsiveness to virus-specific immunity. Therefore, activating innate or adaptive immune responses and inducing T cell and B cell antiviral immunity is a new strategy for curing hepatitis B.

[0005] mRNA vaccine technology can be used as a fast and flexible technology platform. With the development and maturity of chemically modified mRNA and nanolipid carrier technology, the advantages of mRNA in the research and development of various viral vaccines have become increasingly prominent. Among them, the new crown mRNA vaccine has good prospects in the vaccine field due to its outstanding performance in effectiveness and safety. Due to the specificity of the immune response, the target antigen epitope sequence (including B cell epitopes and T cell epitopes) directly determines the type of immune response. Given that both B cell immunity and T cell immunity are indispensable in the clearance of HBV virus, selecting suitable immunogenic fragments as much as possible is a key step in the design of mRNA vaccines.

[0006] The document with application publication number CN114903987A discloses an mRNA drug for treating chronic hepatitis B virus infection, its preparation method and application, IL-21mRNA is prepared according to the IL-21 gene, and then the IL-21mRNA is mixed with lipids to prepare LNP-IL-21. It can effectively induce CD8T cell immunity and humoral immunity in vivo. It encodes IL-21 protein with the mouse gene IL-21, and uses 1-methyl pseudoUTP to replace UTP and cap structure to improve the translation efficiency of IL-21mRNA, but the IL-21 protein expression finally verified still has the defects of long transfection time and low expression efficiency.

[0007] There are a large number of cis-elements and special structures on the UTR of eukaryotic mRNA, which interact with a variety of specific RNA-binding proteins (RBPs) and then recruit other proteins to form complexes, helping cells to precisely regulate the translation of mRNA according to different signaling pathways. In order to maximize the translation efficiency and stability of mRNA vaccines, the present invention screens and optimizes the non-coding regions (5'UTR and 3'UTR) on the basis of optimizing the sequence SFORF, which can translate more mRNA under the same inoculation conditions and exert a greater biological effect. Summary of the invention

[0008] The purpose of the present invention is to provide a recombinant mRNA, a DNA molecule, a pharmaceutical composition, a vaccine and a preparation method.

[0009] In one aspect, the present invention discloses a recombinant mRNA encoding a protein having the same amino acid sequence as the large protein region of the hepatitis B virus envelope protein, wherein the amino acid sequence of the large protein region of the hepatitis B virus envelope protein is as follows:

[0010] As shown in SEQ ID NO:1.

[0011] In one aspect, the present invention discloses the use of recombinant mRNA in the preparation of a medicament for treating hepatitis B virus infection.

[0012] In one aspect, the present invention discloses a recombinant mRNA comprising a nucleic acid sequence as shown in SEQ ID NO:2.

[0013] In some embodiments, the recombinant mRNA further comprises a 5'-UTR sequence and / or a 3'-UTR sequence, and the UTR sequence comprises a HIF2 sequence, a GAPDH sequence, an ACTA1 sequence, a CKMM sequence, a MYLPF sequence, a TNNC2 sequence, and an RPS27A-201 sequence.

[0014] In some embodiments, the UTR sequence further preferably includes a HIF2 sequence and a GAPDH sequence.

[0015] In some embodiments, the recombinant mRNA is further modified by 3' tailing, and / or at least one 5' capping modification.

[0016] In one aspect, the present disclosure provides a DNA molecule encoding a recombinant mRNA of the present technology.

[0017] In one aspect, the present invention discloses a recombinant plasmid comprising a DNA molecule of the present technology.

[0018] In one aspect, the present invention discloses an expression vector comprising the recombinant mRNA of the present technology and a pharmaceutically acceptable carrier.

[0019] In one aspect, the present invention discloses a use of the recombinant mRNA of the present technology in the preparation of a medicine for treating hepatitis B virus infection.

[0020] In some embodiments, the medicine for treating hepatitis B virus infection may contain a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be a carrier, a diluent, an adjuvant or a nucleotide sequence encoding an adjuvant, a solubilizing agent, an adhesive, a lubricant, a suspending agent, a transfection facilitator, etc. The transfection facilitator includes, but is not limited to, a surfactant such as an immunostimulating complex, Freunds incomplete adjuvant, an LPS analog (e.g., monophosphoryl ester A), a cell wall peptide, a quinone analog, squalene, hyaluronic acid, a lipid, a lipid, a calcium ion, a viral protein, a cation, a polycation (e.g., poly-L-glutamic acid (LGS)) or a nanoparticle or other known transfection facilitators.

[0021] In one aspect, the present invention discloses a therapeutic vaccine for chronic hepatitis B virus infection, which comprises lipid nanoparticles mRNA-LNP made of recombinant mRNA and lipids of the present technology.

[0022] In some embodiments, the lipid nanoparticle mRNA-LNP comprises mRNA and lipids, wherein the lipids comprise: (a) one or more of positively charged lipids and / or ionizable lipids; (b) neutral auxiliary lipids; (c) cholesterol; (d) PEG-modified lipids.

[0023] In some embodiments, the method for preparing a therapeutic vaccine includes the following steps: (1) synthesizing a DNA fragment encoding a natural domain peptide of a large protein region and cloning it into a plasmid as a template; (2) preparing a target mRNA by transcription; and (3) preparing the above-mentioned lipid nanoparticle mRNA-LNP.

[0024] In some embodiments, the target mRNA in step (2) is a purified mRNA molecule.

[0025] In some embodiments, the process of preparing lipid nanoparticles mRNA-LNP in step (3) includes: dissolving the target mRNA molecule in an aqueous phase composed of a citrate buffer, mixing it with a lipid component dissolved in an ethanol phase using impact jet or microfluidics methods, to prepare lipid nanoparticles loaded with mRNA.

[0026] In one aspect, the present invention discloses a method for inducing an immune response against hepatitis B virus in a subject, comprising administering the above-mentioned therapeutic vaccine to the subject.

[0027] In some embodiments, the subject's immune response includes production of protective antibodies against hepatitis B virus.

[0028] Compared with the prior art, the present invention adopts the above technical solution to have the following beneficial effects:

[0029] The mRNA hepatitis B therapeutic vaccine provided by the present invention can produce dual mechanisms of activating humoral immunity and T cell immunity to fight against hepatitis B virus. It is a therapeutic mRNA vaccine prepared for the first time that can effectively induce specific neutralizing antibodies (protective antibodies) against HBV.

[0030] The present invention can translate more mRNA under the condition of the same inoculation amount through the optimization of nucleic acid sequence, thereby exerting a greater biological effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram showing the strategy for constructing the DNA transcription template used in the present invention.

[0032] Figure 2 The results of the dual luciferase reporter gene experiment screening UTR in Example 1 of the present invention are shown.

[0033] Figure 3 The DNA linearization product obtained by enzyme digestion in Example 1 of the present invention is shown.

[0034] Figure 4 The size and degradation degree test results of the mRNA prepared in Example 1 of the present invention are shown.

[0035] Figure 5 It shows the S antigen level of the experimental mice in Example 2 of the present invention.

[0036] Figure 6 It shows the E antigen level of the experimental mice in Example 2 of the present invention.

[0037] Figure 7 It shows the S antibody level of the experimental mice in Example 2 of the present invention.

[0038] Figure 8 The figure shows the relationship between the S antigen level and the S antibody level of the experimental mice in Example 2 of the present invention.

[0039] Fig. 9 It shows the HBV DNA level of the experimental mice in Example 2 of the present invention.

[0040] Fig.10 The figure shows the ALT level of the experimental mice in Example 2 of the present invention.

[0041] Fig.11The figure shows the weight status of the experimental mice in Example 2 of the present invention.

[0042] Fig.12 The S antigen level of clinically cured mice in Example 2 of the present invention is shown.

[0043] Fig.13 It shows the HBV DNA level of clinically cured mice in Example 2 of the present invention.

[0044] Fig.14 The figure shows the immune activation of the experimental mice in Example 2 of the present invention.

[0045] Fig.15 The mRNA vaccine of the present invention shows the neutralizing activity of antibodies induced. DETAILED DESCRIPTION

[0046] Ⅰ. Summary

[0047] Hepatitis B virus (HBV) is the pathogen that causes hepatitis B (abbreviated as HBV). HBV is a partially double-stranded circular DNA molecule, and its genome contains four open reading frames. They are called S, C, P, and X regions respectively. The S region is divided into three segments: S gene, preS1 gene, and preS2 gene, which encode S protein, preS1 protein, and preS2 protein respectively. The three are collectively called large S protein. Due to the specificity of the immune response, the target antigen epitope sequence (including B cell epitopes and T cell epitopes) directly determines the type of immune response. Given that both B cell immunity and T cell immunity are indispensable in the clearance of HBV virus, selecting suitable immunogenic fragments as much as possible is a key step in the design of mRNA vaccines.

[0048] The present invention has determined through preliminary studies that large S protein (S protein, preS1 protein and preS2 protein) is used as the active ingredient of the vaccine to stimulate the body to produce neutralizing antibodies or protective antibodies.

[0049] The UTR sequence of mRNA also affects the stability and translation efficiency of mRNA, and is a key factor in regulating the final protein expression level. In order to maximize the translation efficiency and stability of mRNA vaccines, the present invention, on the basis of optimizing the full-length coding region (SFORF) of HBVS protein, also screens and optimizes the non-coding region (5'UTR and 3'UTR). Through the dual luciferase reporter gene experiment, the optimal UTR is screened out, and more mRNA can be translated under the conditions of the same inoculum amount, exerting a greater biological effect.

[0050] II. HBV vaccine

[0051] When designing a target antigen for HBV virus, the present invention first optimizes the amino acid sequence of the target gene. The main optimization strategy is to adjust the codon preference when expressed in the human body, adjust the usage frequency of common codons, and increase the G / C content of the sequence, so that the mRNA structure is more stable and the translation efficiency of the target protein in mammals and humans is higher. The specific amino acid sequence is as follows:

[0052]

[0053] On the basis of optimizing the sequence SFORF, the gene sequence was designed according to the characteristics of the target mRNA sequence, mainly including the optimized HBVS protein full-length coding region (SFORF), non-coding region (UTR) and polyA. The sequence contains 5'UTR, SFORF, 3'UTR, and polyA from 5' to 3'. The key functional elements on the DNA transcription template include T7 promoter (transcription promoter), 5'UTR, SFORF, 3'UTR and polyA tail.

[0054] III. Preparation method

[0055] The present invention first analyzes the antigenic epitopes of hepatitis B virus (HBV), determines that the HBV large S protein is the antigenic target, LNP is the delivery vector, and the immunization method is the mRNA vaccine dosage form of subcutaneous injection. At the same time, the mRNA encoding the viral antigen fragment is synthesized by UTR and codon optimization and in vitro transcription method, and finally achieves efficient expression of human cells; the entire mRNA vaccine has a short production cycle, simple process operation, low production cost, and long storage time. The mRNA vaccine designed by the present invention can induce a stronger neutralizing antibody effect under the condition of the same inoculation amount.

[0056] In some embodiments of the present invention, the preparation method mainly includes: synthesizing a DNA fragment encoding a natural domain peptide of the large S protein, cloning it into a plasmid as a template, and finally transcribing it to prepare a target mRNA molecule.

[0057] Among them, the process of synthesizing the DNA fragment encoding the natural domain peptide of the large S protein can be synthesized by itself or commissioned.

[0058] Among them, according to the requirements for plasmid resistance in the pharmacopoeia, the plasmid backbone of the DNA transcription template includes key elements such as kanamycin resistance, CMV eukaryotic expression elements and T7 promoter sequence.

[0059] Among them, the target mRNA molecule can be prepared according to the following reaction:

[0060] a. Three-step method: uncapped and untailed mRNA is obtained by in vitro transcription, a polyA structure is added to the tail of the mRNA under the conditions of RNA polymerase and ATP (a linker may or may not be inserted in the middle of the polyA structure), and a cap structure is added to the 5' end of the uncapped mRNA under the catalysis of a capping enzyme to obtain a capped and tailed mRNA molecule;

[0061] b. Two-step method: in vitro transcription to obtain tailed but uncapped mRNA; under the catalysis of capping enzyme, a cap structure is added to the 5' end of the uncapped mRNA to obtain a capped and tailed mRNA molecule;

[0062] c. One-step method: perform in vitro transcription and capping to obtain capped and tailed mRNA molecules.

[0063] In some specific embodiments of the present invention, the method for preparing the mRNA molecule further comprises a purification process of the mRNA: the mRNA is subjected to lithium chloride / ethanol precipitation, centrifugal column, chlorine extraction / ethanol precipitation or gel purification to obtain purified mRNA. The nucleic acid sequence of the mRNA is as follows:

[0064]

[0065] Example 1: Preparation of mRNA vaccine

[0066] Step 1: Dual luciferase reporter gene assay to screen UTR

[0067] Based on the results of the preliminary experiments and research experience, the UTRs of seven genes with high expression levels in multiple tissue proteins, including H1F2, GAPDH, RPS27A-201, and ACTA1, CKMM, MYLPF, and TNNC2, were selected, and the 3'UTR and 3'+5'UTR of these seven genes were cloned into the target vector Psicheck. 293T cells were prepared and plasmid transfection was completed.

[0068] The dual luciferase reporter assay kit from GenePharma was used.

[0069] Prepare passive lysis buffer 1×PLB: add 1 volume of 5×Passive Lysis Buffer (PLB) to 4 volumes of distilled water, mix well, and store at 4°C until use;

[0070] Preparation of LARII: Dissolve lyophilized Luciferase Assay Substrate in Luciferase Assay Buffer II and store at -20°C until use;

[0071] Preparation Reagents (prepared immediately before use): Take a certain amount of Add to the corresponding amount Buffer, mix to a final concentration of 1× and set aside.

[0072] (1) Pour off the culture medium in the 24-well plate and wash the cells twice with PBS. This process must be done carefully without touching the adherent cells.

[0073] (2) Add 1×PLB to the culture wells, passively lyse the cells in the culture plate at room temperature, and transfer the lysate to the test plate. The experiment was designed with 3 replicates.

[0074] (3) Turn on the Tecan M1000 microplate reader, preheat, and select the dual luciferase detection system.

[0075] (4) Add LARII reagent to each well, select 1-2 seconds delay, 5-10 readings, and detect firefly luciferase activity on a microplate reader.

[0076] (5) Take out the test plate and add Reagent, select 1-2 seconds delay, 5-10 readings, and detect Renilla luciferase activity on a microplate reader.

[0077] (6) Results statistics and analysis Figure 2 As shown, according to the results of the dual luciferase reporter gene experiment, HIF2-5'UTR and GAPDH-5'UTR have the highest translation efficiency and stability, so HIF2-5'UTR and GAPDH-5'UTR are selected as the UTR sequences of the mRNA vaccine, as shown below:

[0078]

[0079] Step 2: Extract plasmid from E. coli

[0080] The genus-specific common antigenic determinant cluster "a" is located at the amino acid sequence of positions 101-172. According to the analysis of the amino acid sequence of the large surface protein, the large surface protein can completely retain the antigenic epitope, so the hepatitis B large surface protein gene was selected as the template for encoding mRNA.

[0081] According to the screened UTR sequence and the nucleotide sequence of the hepatitis B surface protein shown in the database after preliminary research, the T7 promoter sequence (Taatacgactcactatagg) is added before the 5'UTR sequence, and the polyA tail, 3'UTR, and Basl restriction site (GTCTGGAGACC) are added in sequence after the CDS region to synthesize the corresponding DNA fragment, and the DNA fragment is cloned into the expression plasmid to obtain a recombinant plasmid, and the recombinant plasmid is transferred into the host cell to obtain a recombinant cell. The plasmid is extracted from the recombinant cell after propagation.

[0082] The plasmid was extracted using the TIANGEN endotoxin-free plasmid extraction kit. The product components of the kit included balance solution BL, solution P1, solution P2, solution P4, rinse solution PW, elution buffer TB, RNase A, filter CS1, adsorption column CP6, and collection tube.

[0083] (1) Column equilibration step: Add 2.5 ml of equilibration solution BL to the adsorption column CP6 (the adsorption column is placed in a 50 ml collection tube), centrifuge at 8000 rpm (-8228 × g), pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0084] (2) Take the overnight culture solution and add it to a centrifuge tube. Centrifuge it at 8000 rpm at room temperature.

[0085] (3) Aspirate the supernatant.

[0086] (4) Add solution P1 (P1 solution in TIANGEN Endotoxin-free Plasmid Extraction Kit) (RNase A has been added) to the centrifuge tube containing the bacterial cell pellet to thoroughly suspend the bacterial cell pellet.

[0087] (5) Add solution P2 (P2 solution in TIANGEN Endotoxin-free Plasmid Extraction Kit) to the centrifuge tube, immediately and gently invert it upside down 6-8 times to fully lyse the bacteria, and leave it at room temperature for 5 minutes.

[0088] (6) Add solution P4 (P4 solution in TIANGEN Endotoxin-free Plasmid Extraction Kit) to the centrifuge tube, and immediately and gently turn it upside down 6-8 times to mix thoroughly until white dispersed flocculent precipitates appear in the solution, and then leave it at room temperature for about 10 minutes. Centrifuge at 8000rpm (-8228×g) to make the white precipitate fall to the bottom of the tube, pour all the solution into filter CS1, filter, and collect the filtrate in the collection tube.

[0089] (7) Add 0.3 times the volume of isopropanol to the filtrate, mix well, and transfer to the adsorption column CP6 (the adsorption column is placed in the collection tube).

[0090] (8) Centrifuge at 8000 rpm (-8228 × g) at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube.

[0091] (9) Add rinse solution PW (with anhydrous ethanol added) to the adsorption column CP6, centrifuge at 8000 rpm (-8228 × g) for 2 min, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0092] (10) Repeat step (9).

[0093] (11) Add anhydrous ethanol to the adsorption column CP6, centrifuge at 8000 rpm (-8228 × g) at room temperature for 2 min, and discard the waste liquid.

[0094] (12) Place the adsorption column CP6 back into the collection tube and centrifuge at 8000 rpm (-8228 × g).

[0095] (13) Place the adsorption column CP6 in a clean collection tube, add 1-2 ml of elution buffer TB (preheated elution buffer TB) to the middle part of the adsorption membrane, leave it at room temperature for 5 minutes, and then centrifuge it at 8000 rpm at room temperature; transfer all the centrifuge liquid into a clean 1.5 ml centrifuge tube and store it at -20°C.

[0096] Step 3: Linearize the constructed plasmid

[0097] The enzyme digestion reaction system is as follows:

[0098] Plasmid DNA: 1 μg;

[0099] 10x enzyme buffer: 5 μl;

[0100] Bsal enzyme (purchased from New England Biolab) 10 Unit

[0101] Make up to 50 μL with enzyme-free ddH2O;

[0102] The above enzyme digestion system was placed at 37°C for 15 minutes.

[0103] Step 4: Purify the linearized DNA

[0104] Purify DNA according to the instructions of TIANGEN DNA purification kit.

[0105] (1) Column equilibration step: Add equilibration solution BL to adsorption column CB2 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm (-13400×g), pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0106] (2) Estimate the volume of the PCR reaction solution or enzyme digestion reaction solution, add an equal volume of solution PC (TIANGEN DNA Purification Kit) into it, and mix thoroughly.

[0107] (3) Add the solution obtained in the previous step to an adsorption column CB2 (the adsorption column is placed in a collection tube), place at room temperature for 2 minutes, centrifuge at 12000 rpm (-13,400 × g) for 1 minute, pour out the waste liquid in the collection tube, and place the adsorption column in the collection tube.

[0108] (4) Add rinse solution PW (TIANGEN DNA purification kit) to the adsorption column CB2 (please check whether anhydrous ethanol has been added before use), centrifuge at 12000 rpm (-13400×g), pour out the waste liquid in the collection tube, and place the adsorption column CB2 in the collection tube.

[0109] (5) Repeat step (4).

[0110] (6) Place the adsorption column CB2 back into the collection tube, centrifuge at 12000 rpm (-13400 × g) for 2 minutes, and remove the rinse solution. Place the adsorption column CB2 at room temperature until it is dry.

[0111] (7) Place the adsorption column CB2 in a clean centrifuge tube, add an appropriate amount of elution buffer EB (TIANGEN DNA purification kit) to the middle of the adsorption membrane, and leave it at room temperature for 2 minutes. Centrifuge at 12000 rpm (-13,400 × g) for 2 minutes to collect the DNA solution.

[0112] Step 5: DNA gel examination

[0113] After the purification reaction is completed, 1 μl of the system before and after the enzyme digestion reaction is taken for DNA agarose gel electrophoresis (1.5% agarose gel, 5 V / min, 40 min). The electrophoresis results are compared to show whether the recombinant plasmid is completely digested.

[0114] Qualification criteria: a single band appears in electrophoresis detection; compared with the supercoiled plasmid before enzyme digestion, the band is located above the supercoiled plasmid; the size meets the expected requirements.

[0115] Test results: Figure 3 As shown, there is a single band; the size is as expected and the band is located above the supercoiled plasmid.

[0116] Step 6. In vitro synthesis of mRNA

[0117] Use NEB's S1404 series m7G(5')ppp(5')G RNA Cap Structure Analog with the E2040 series. There are two options, one is to add the cap during the synthesis of mRNA, and the other is to add the cap after the synthesis. To add the cap during the synthesis:

[0118] Proceed according to the following synthesis system (reaction reagents are added in order):

[0119] Reaction volume, 20 μl (placed in a 1.5 RNase-free tube, the reaction volume of a single tube, multiple tubes reacted at one time):

[0120] enzyme-free ddH2O (4-X) μl;

[0121] ATP (100 mM) 2 μl 10 mM final;

[0122] UTP (100mM) 2 μl 10mMfinal;

[0123] CTP (100 mM) 2 μl 10 mM final;

[0124] GTP (20 mM) 2 μl 2 mMfinal;

[0125] CapAnalog (40mM) 4 μl 8mMfinal;

[0126] Template DNA X μl 1 μg;

[0127] 10X reaction buffer 2 μl;

[0128] T7 RNA Polymerase Mix 2 μl;

[0129] The total reaction volume was 20 μl;

[0130] The procedure for the in vitro RNA synthesis was 37° C. for 2 hours.

[0131] Output: 1 μg DNA can produce 40 μg-50 μg mRNA in 2 hours under the above conditions, of which 80% of the mRNA will have an m7G cap.

[0132] Step 7: DNaseI digestion to remove DNA template

[0133] Add 2 μl of DNaseI to each centrifuge tube after in vitro synthesis of mRNA.

[0134] Mix by inversion and centrifuge at 1000 rpm.

[0135] The mixture was placed back in a thermostatic reactor (37°C) for reaction.

[0136] After the reaction, the reaction solution was combined into RNase-free 1.5 ml centrifuge tubes to detect the residual DNA fragments. The results of three measurements were a ng, b ng, c ng per d μg mRNA (to be measured).

[0137] Step 8. Purify mRNA

[0138] NEB RNACleanupKit, T2040 series kits for purifying mRNA.

[0139] (1) Add 100 μl RNACleanup Binding Buffer to 50 μl of mRNA to be purified.

[0140] (2) Add 150 μl of 95% ethanol to the mixture and mix thoroughly.

[0141] (3) Place the adsorption column in the collection tube, add the mixed solution into the adsorption column, then centrifuge and discard the filtrate.

[0142] (4) Place the adsorption column in a collection tube, add 500 μL RNACleanup Wash Buffer, centrifuge, and discard the filtrate.

[0143] (5) Repeat step (4).

[0144] (6) Transfer the adsorption column to a new RNase-free 1.5 ml centrifuge tube.

[0145] (7) Add a specified volume of enzyme-free water to the center of the adsorption column, centrifuge, elute the mRNA, and store the purified mRNA at -70°C.

[0146] Step 9: RNA gel examination

[0147] The purified mRNA was diluted 10 times and the integrity of the fragments was tested by RNA Screen Tapeassay and agarose gel electrophoresis. Figure 4 Indicates the size of synthesized mRNA and the degree of degradation.

[0148] The test results showed that the bands were of the same size and the fragments were complete.

[0149] Step 10: LNP preparation

[0150] One volume of the novel cationic lipid DLin-MC3-DMA (4-(N,N-dimethylamino)butyric acid (6Z,9Z,28Z,31Z)-heptatriacontac-6,9,28,31-tetraen-19-yl lipid) (Shanghai Avite Pharmaceutical Technology Co., Ltd.), DSPC (distearylphosphatidylcholine) (Shanghai Avite Pharmaceutical Technology Co., Ltd.), cholesterol, DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000) (Shanghai Avite Pharmaceutical Technology Co., Ltd.) was mixed in ethanol at a molar ratio of 50:10:38.5:1.5, and three volumes of mRNA (the mass ratio of mRNA to lipid was 1:25, and the nitrogen-phosphorus ratio was 6), and the buffer system was citrate buffer (PH4.0, 10mM). The lipid and mRNA were injected into a microfluidic mixing device (ZX-LS-32, FluidicLab) at a mixing rate of 1.5 ml / min. The resulting mixture was dialyzed against phosphate buffered saline (PBS, pH 7.4, 10 mM) for 16 hours to remove ethanol.

[0151] Example 2. Evaluation of the therapeutic effect of mRNA vaccines

[0152] Construction of mouse model

[0153] C57BL / 6 mice (6-8 weeks, male) were injected with 1x1010vg (viral genome) AAV-HBV1.3 (purchased from Beijing Wujiahe Molecular Medicine Research Institute Co., Ltd.) via the tail vein. Stable HBV tolerance and chronic HBV infection models were established 4 weeks after infection. Five mice in each group were randomly divided into 4 groups: vaccine alone group, antiviral drug alone group, antiviral drug + vaccine combination group, and normal saline group.

[0154] On the 1st and 14th days, each mouse in the vaccine alone group and the antiviral drug + vaccine combination group was given a hepatitis B virus mRNA vaccine by subcutaneous injection, intramuscular injection, or tail vein injection. The antiviral monotherapy group and the antiviral drug + vaccine group were regularly given new antiviral drugs, and the saline group was injected with saline at the same time. Blood was collected from the eye sockets once a week to obtain serum until the end of the experiment. The titers of HBVDNA, S antigen, E antigen, ALT, and anti-S protein-specific antibodies in the serum were tested.

[0155] S antigen, E antigen, and S antibody are detected using the Antu kit

[0156] 1. Preparation before the experiment

[0157] (1) Place the test kit (Antu test kit) at room temperature (18-25 degrees) for equilibrium.

[0158] (2) Adjust the thermostat or water bath to the reaction temperature and wait until the temperature stabilizes before use.

[0159] (3) Prepare washing solution: dissolve 1 package of solid washing solution in 500 ml of purified water.

[0160] (4) 60 minutes in advance, mix the luminescent substrate A and B solutions in equal proportions to the required volume for this test.

[0161] 2. Experimental Procedure

[0162] (1) Take out a certain number of coated wells, number them, and add 50 μl of calibrator and sample to each well.

[0163] (2) Add 50 μl of enzyme conjugate to each well.

[0164] (3) Oscillate on a micro-oscillator for 60 seconds to mix the liquid in the wells evenly.

[0165] (4) Apply the plate sticker and incubate at 37°C for 60 minutes.

[0166] (5) Washing the plate: ① Hand washing: shake off the liquid in the wells, then add 350 μl of washing solution per well, leave for 5 seconds and shake off, repeat 5 times, and finally pat dry on absorbent paper. ② Machine washing: inject 350 μl of washing solution into each well, wash the plate 6 times, and finally pat dry on absorbent paper.

[0167] (6) Add 50 μl of the mixed luminescent substrate to each well, shake for 10 seconds, and react at room temperature (18°C-25°C) in the dark for 10 minutes.

[0168] (7) The luminescence intensity is detected by a chemiluminescence detector.

[0169] The results of the test for HBsAg, HBeAg, and anti-HBs titers in mouse serum can be found in Figure 5 , 6, 7. The relationship between HBsAg and anti-HBs titers is shown in Figure 8 As shown.

[0170] The results showed that in the mRNA combined vaccine group, HBsAg decreased by about 3 log 10 , HBeAg decreased by more than 1 Log 10 In the mRNA combined vaccine group, the decreases in HBsAg and HBeAg were much higher than those in the antiviral drug monotherapy group, and anti-HBs was significantly higher than that in the antiviral drug monotherapy group. In particular, in the mRNA combined vaccine group, the HBsAg and HBV DNA of two mice decreased below the detection line, and high titers of anti-HBs were produced, achieving clinical cure of hepatitis B and s antigen serological conversion.

[0171] DNA detection using Shengxiang Hepatitis B Virus Nucleic Acid Assay Kit (PCR-fluorescent probe method)

[0172] 1. Reaction system configuration:

[0173] (1) Take out the sample release agent (Shengxiang Company, used in conjunction with the hepatitis B virus nucleic acid assay kit), wait for it to equilibrate to room temperature and mix thoroughly, then pipette 5 μl of the sample release agent into a 96-well PCR plate.

[0174] (2) Add 5 μl of the sample to be tested to each well, pipette up and down 3-5 times to mix thoroughly, and let stand at room temperature for 10 minutes to fully lyse.

[0175] (3) Each reaction requires 38 μl of PCR reaction solution, 2 μl of enzyme mixture, and 0.2 μl of internal standard. Pipette each solution according to the number of samples to be measured, mix thoroughly to form a PCR mixture, centrifuge briefly, and set aside.

[0176] (4) Add 40 μL of PCR mixture to each well, attach a qPCR-specific pressure-sensitive sealing film, tap to mix thoroughly and remove bubbles, and centrifuge briefly before PCR amplification.

[0177] 2. PCR reaction:

[0178] (1) Place the PCR plate in the reaction tank and set the negative control, positive control, standard and unknown samples in the software. The concentration of the standard is: Standard A: 4X10 7 IU / ml, Standard B: 4X10 6 IU / ml, Standard C: 4X10 6 IU / ml, Standard D: 4X10 5 IU / mL.

[0179] (2) Select the FAM channel to detect HBV-DNA, the VIC channel to detect the internal standard, and the ROX channel as the reference fluorescence.

[0180] (3) Cycle parameter setting:

[0181]

[0182] 3. Data processing:

[0183] The analysis software was used to fit the standard curve by measuring the Ct value of the standard, and the HBV-DNA concentration of the unknown sample was calculated using the standard curve.

[0184] 4.Quality Control:

[0185] a) Negative control: no Ct value is displayed; but the internal standard test is positive (Ct value ≤ 40);

[0186] b) Positive control: the detection concentration value is between 1.26X105-1.26X106IU / mi;

[0187] c) Quantitative standards: All tested positive, and the correlation coefficient of the standard curve was r≥0.98.

[0188] The results of HBV DNA test in mouse serum can be found in Fig. 9 Show.

[0189] The results showed that HBV DNA in the mRNA combined vaccine group decreased by about 6 log 10 HBVDNA in the antiviral drug monotherapy group decreased by about 5 log 10 The ability of mRNA combined vaccine to reduce HBV DNA is better than that of single antiviral drug.

[0190] ALT detection using Elabscience alanine aminotransferase (ALT) activity fluorescence kit

[0191] 1. Reagent Preparation

[0192] (1) Before testing, all reagents must be equilibrated to room temperature.

[0193] (2) Preparation of enzyme reagent working solution: Dissolve each enzyme reagent powder in 1.2 ml of buffer and place on ice for later use.

[0194] (3) Preparation of 1 mmol / L pyruvate standard stock solution: Take 10 μL of 100 mmol / L pyruvate standard and add it to 990 μL of buffer to prepare a 1 mmol / L pyruvate standard stock solution.

[0195] (4) Preparation of 50 μmol / L pyruvate standard solution: dilute 1 mmol / L pyruvate standard stock solution 20 times at a volume ratio of 1:19 to obtain 50 μmol / L standard solution. Prepare as needed and place on ice until use.

[0196] (5) Preparation of reaction working solution: Mix the buffer solution: probe: enzyme reagent working solution: substrate in a volume ratio of 56:4:20:20 and store in a dark place.

[0197] 2. Operation steps

[0198] (1) Standard wells: Add 20 μl of standard samples of different concentrations to the corresponding standard wells of the ELISA plate; Assay wells: Add 20 μl of the sample to be tested to the corresponding assay wells of the ELISA plate.

[0199] (2) Add 100 μl of the reaction working solution to each well in step (1).

[0200] (3) Vibrate the plate on the microplate reader for 5 seconds, let it stand at room temperature for 3 minutes, set the excitation wavelength of the fluorescence microplate reader to 535 nm and the emission wavelength to 587 nm, measure the fluorescence value of each well, and record it as F1. Then let it stand at room temperature in the dark for 60 minutes, and measure the fluorescence value of each well under the same wavelength conditions, and record it as F2. Then ΔF = F2-F1 (Note: There is no need to calculate the difference of the standard wells. The fluorescence value measured after 60 minutes of reaction can be directly used as the standard curve).

[0201] The ALT level in mouse serum Fig.10 The weight of mice was Fig.11 shown.

[0202] The results showed that there was no significant change in ALT levels and mouse weight, indicating that the mRNA vaccine had no obvious toxicity.

[0203] In summary, this embodiment verifies the following effects:

[0204] The mRNA vaccine of the present invention can effectively reduce HBV DNA, S antigen and E antigen in serum.

[0205] It has good immunogenicity and induced the production of specific antibodies against the S protein. Mice in the control group injected with normal saline could not produce specific antibodies against the S protein.

[0206] The mRNA candidate vaccine had no effect on ALT levels and had no effect on mouse weight, indicating that the mRNA vaccine had no obvious liver toxicity and good safety. Among them, two mice in the antiviral drug + mRNA vaccine combination group achieved clinical cure of hepatitis B. They were: D85 mouse surface antigen cleared, HBV DNA cleared, and high-titer antibodies were produced, reaching 896.69; D40 mouse surface antigen cleared, HBV DNA cleared, S antigen serological conversion occurred, and antibodies reached 194.175.

[0207] The HBsAg and HBV DNA levels of mice that achieved complete cure were as follows Fig.12 , 13, HBsAg and HBV DNA decreased below the detection line.

[0208] Example 3. Verification of the immune activation effect of mRNA vaccines on T cells and B cells

[0209] In this example, the immune activation effect of hepatitis B mRNA vaccine on T cells and B cells was investigated through experiments.

[0210] The mice in Example 2 were used and sacrificed after 15 weeks of administration, and the liver, spleen and peripheral blood were collected. The liver was subjected to mass spectrometry flow cytometry analysis of liver in situ immunity, and the spleen and peripheral blood were subjected to flow cytometry analysis of peripheral immunity.

[0211] The specific operation is as follows: single-cell lymphocytes are isolated from liver and spleen tissues by percoll density gradient centrifugation, and PBMCs are isolated from fresh blood by Ficoll. After counting, the cells are diluted with culture medium and added to a 24-well plate. Appropriate amounts of S protein pepmix stimulators and blockers are added to the culture plate and incubated for 12 hours; the 24-well plate is incubated at 37°C CO 2 After 12 hours, the cells were recovered and further analyzed for CD4 and CD8 cells using mass spectrometry and flow cytometry extracellular and intracellular staining experiments.

[0212] The results of mass spectrometry and flow cytometry are as follows Fig.14 As shown, in situ immunity of the liver showed that the effect memory CD8+T and CD4+T cells in the antiviral drug + vaccine group were significantly more than those in the other three groups, and the difference was statistically significant; the immune activation indicators IL-2 and TNFα increased. Peripheral immunity of the spleen and peripheral blood showed that the memory CD8+T and CD4+T cells in the antiviral drug + vaccine group were significantly more than those in the other three groups, and the difference was statistically significant, and the immunosuppression indicator PD-1 decreased. The cellular immunity of in situ immunity and peripheral immunity in the antiviral drug + vaccine group was activated, breaking the state of immune tolerance. The combined use of hepatitis B mRNA vaccine and antiviral drugs has an immune activation effect.

[0213] Example 4. Verification of antibody titer of mRNA vaccine

[0214] In this embodiment, the antibody titer of the hepatitis B mRNA vaccine was investigated by experiment. The specific operation was as follows:

[0215] Vaccination: C57 mice aged 6-8 weeks were vaccinated with hepatitis B virus mRNA vaccine (subcutaneous injection, intramuscular injection or tail vein injection) at 0 days and 14 days respectively; peripheral blood and serum were collected from mice every week.

[0216] Prepare the virus produced by HepAD38 cells. Cultivate HepAD38 cells in a 10 cm dish with F12 medium containing tetracycline for 3-4 generations, then switch to medium without tetracycline, collect the cell supernatant, and store the supernatant at -80°C. Measure the HBV DNA concentration in the supernatant, and select cells with a HBV DNA titer higher than 1×10 5 / mL supernatant for virus concentration. Mix the supernatant to be concentrated with 25% PEG8000 to a final concentration of 10%. Centrifuge the mixture at 2000g, 4°C for 30 minutes, discard the supernatant, resuspend the precipitate with PMM solution, which is the concentrated virus, and store at -80°C.

[0217] HepG2-NTCP cells were plated in a 48-well plate with medium containing G418. Three hours after plating, the supernatant was discarded, an appropriate amount of PMM solution was added, and the cells were incubated at 37°C overnight. The serum was heat-inactivated, and 75 μL of serum-medium mixture was mixed with 75 μL of virus-PEG8000-medium mixture storage solution in equal volumes. At the same time, DMEM and virus solution were mixed in equal volumes as a negative control. Incubate at 37°C for 1-2 hours. The above virus-antibody serum-medium mixture was transferred to the 48-well plate HepG2-NTCP cells, and the plate was shaken at room temperature for 2-5 hours with an interval of 10 seconds. After the shaking was completed, the plate was placed in a 37°C incubator overnight. After 24 hours, the medium was discarded, the cells were washed 3 times with serum-free DMEM, and PMM was added to continue culturing. On Day 3, 5, and 7, the cell supernatant was collected, and PMM was added to continue culturing to detect HBVDNA and HBsAg.

[0218] The experimental results are as follows Fig.15 As shown, the HBV DNA in the supernatant of mice using mRNA vaccine was significantly lower than that of the control group, indicating that antibodies with neutralizing activity were present in the serum of mice using mRNA vaccine. The results of the neutralization experiment show that mRNA vaccine can induce the production of neutralizing antibodies and has good neutralizing activity and immunogenicity.

[0219] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A recombinant mRNA, characterized in that It comprises the nucleic acid sequence shown in SEQ ID NO: 2, and the recombinant mRNA further comprises a 5'-UTR sequence and a 3'-UTR sequence, and the UTR sequence comprises a HIF2 sequence and a GAPDH sequence.

2. The recombinant mRNA according to claim 1, characterized in that The recombinant mRNA is further modified by 3' tailing, and / or at least one 5' capping modification.

3. A DNA molecule encoding the recombinant mRNA according to claim 1 or 2. A recombinant plasmid comprising the DNA molecule according to claim 3. An expression vector comprising the recombinant mRNA according to claim 1 or 2.

6. A pharmaceutical composition comprising the recombinant mRNA according to claim 1 or 2 and a pharmaceutically acceptable carrier.

7. Use of the recombinant mRNA as claimed in claim 1 or 2 in the preparation of a medicine for treating hepatitis B virus infection.

8. A therapeutic vaccine for chronic hepatitis B virus infection, comprising lipid nanoparticles mRNA-LNP made from the recombinant mRNA according to claim 1 or 2 and lipids.

9. The method for preparing a therapeutic vaccine according to claim 8, characterized in that: The following steps are involved: (1) Synthesize a DNA fragment encoding a natural domain peptide of a large protein region and clone it into a plasmid as a template; (2) Transcribe and prepare the target mRNA; (3) Prepare lipid nanoparticles mRNA-LNP.

10. The preparation method according to claim 9, characterized in that: The method also includes step (4) preparing the lipid nanoparticle mRNA-LNP into a frozen preparation or a freeze-dried preparation.

Images