A rabies virus mRNA vaccine and preparation method thereof

Abstract

The present invention discloses a rabies virus mRNA vaccine and a preparation method thereof. The rabies virus nucleic acid vaccine provided by the present invention comprises RVG mRNA with optimized sequence and lipid nanoparticles containing novel cationic lipids, so that sufficient protection effect can be achieved by using a single immunization with a relatively low dose, and is superior to existing rabies vaccine technologies in terms of safety and effectiveness.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a rabies virus mRNA vaccine and a preparation method thereof. Background Art

[0002] Rabies is a zoonotic infectious disease that infects the nervous system and is caused by the rabies virus (RABV). It is mainly transmitted to humans by infected animals through bites and scratches. The virus enters the peripheral nerves through the bite site and is transported to the neurons of the central nervous system, causing functional disorders of the nervous system, manifested as clinical symptoms such as hydrophobia, wind fear, dyspnea, spasms and convulsions. The mortality rate of RAV infection is extremely high, posing a serious threat to global public health security. With the large-scale vaccination of rabies vaccines and the improvement of public awareness of post-exposure prevention, China has made significant progress in the prevention and control of rabies, and the number of rabies-related deaths has dropped significantly. However, we still face challenges such as low vaccine penetration in economically underdeveloped areas and high number of vaccine immunizations with limited effectiveness.

[0003] The glycoprotein (G protein) of rabies virus is the most important antigen on the surface of the virus, and plays a key role in the virus infecting host cells and inducing the body's immune response. The widely used inactivated rabies vaccine requires repeated injections to induce protective neutralizing antibodies. Although it can prevent the occurrence of rabies to a certain extent, its immunogenicity needs to be further improved, and it needs repeated injections and may cause a variety of adverse reactions.

[0004] mRNA vaccines are safe and highly immunogenic, and do not require additional adjuvants to enhance immunogenicity. Therefore, developing rabies virus mRNA vaccines with better performance is of great clinical significance. Summary of the invention

[0005] The purpose of the present invention is to provide a rabies virus mRNA vaccine and a preparation method thereof, so as to more effectively stimulate an immune response in the body and more efficiently resist the attack of the virus to achieve a protective effect.

[0006] The present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a rabies virus mRNA vaccine is provided, comprising mRNA expressing rabies virus G protein, wherein the nucleotide sequence of the mRNA transcribing the rabies virus G protein is shown in SEQ ID NO.1.

[0008] Furthermore, the mRNA expressing rabies virus G protein further comprises the following elements: at least one or more of a 5'-cap structure, a 5'-UTR, a promoter element, a 3'-polyadenylic acid sequence and a 3'-UTR.

[0009] Furthermore, the nucleotide sequence encoding the rabies virus G protein also includes:

[0010] A sequence in which one or more bases are substituted, deleted, added and / or replaced based on the above nucleotide sequence; or a sequence with a homology of more than 90% with the above nucleotide sequence.

[0011] Furthermore, the vaccine also includes: a vaccine carrier.

[0012] Furthermore, the vaccine carrier includes one of cationic liposomes, cationic proteins, cationic polymers or cationic lipid nanoparticles.

[0013] Furthermore, the mass ratio of the cationic liposome to the mRNA expressing the rabies virus G protein is 10-30:1, preferably 15-25:1, and more preferably 20:1.

[0014] Furthermore, the cationic lipid nanoparticles, in molar percentage, include 20-50% ionizable cationic lipids, 20-50% structural lipids, 5-20% auxiliary lipid phospholipids and 1-5% surfactant, wherein the molar content of ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants totals 100%.

[0015] Preferably, the ionizable cationic lipids include SIM7, SIM40, SIM74, SIM102, preferably SIM102; the preparation method of the ionizable cationic lipids can be found in patent CN202410262430.4.

[0016] The cationic lipid is selected from the following compounds I-IV:

[0017]

[0018]

[0019] Preferably, the structural lipid comprises cholesterol, cholesterol ester, steroid hormone, steroid vitamin or bile acid, preferably cholesterol;

[0020] Preferably, the helper lipid phospholipid comprises DSPC, DOPE, DOPC or DOPS, preferably DSPC.

[0021] Preferably, the surfactant comprises PEG-DMG or PEG-DSPE, preferably PEG-DMG.

[0022] In a second aspect of the present invention, a method for preparing the mRNA vaccine is provided, the method comprising:

[0023] The components of the vaccine carrier are dissolved in an organic solution to obtain an organic phase; specifically, the cationic lipid, the structural lipid, the auxiliary lipid and the surfactant are dissolved in the organic solution according to the formula ratio to obtain an organic phase.

[0024] The mRNA expressing rabies virus G protein was dissolved in PBS to obtain an aqueous phase;

[0025] The organic phase and the aqueous phase are mixed, and the solvent in the mixed solution is replaced with a buffer solution to obtain a rabies virus mRNA vaccine.

[0026] Preferably, the organic solvent comprises anhydrous ethanol, tetrahydrofuran, acetone or DMSO, preferably anhydrous ethanol;

[0027] Preferably, the total concentration of ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants in the organic phase is 5-7 mg / ml;

[0028] Preferably, the concentration of optimized RVG mRNA in the aqueous phase is 0.08-0.12 mg / ml;

[0029] Preferably, the volume ratio of the organic phase to the aqueous phase is 1:2-4;

[0030] Preferably, the mixing is performed using a microfluidic device with a flow rate ranging from 6 to 24 ml / min, preferably 10.5 ml / min;

[0031] Preferably, the solvent in the mixed solution is replaced with a buffer solution, the mixed solution is diluted 40-100 times with the buffer solution, and then concentrated.

[0032] The present invention has the following advantages and beneficial effects:

[0033] 1. In the rabies virus mRNA vaccine of the present invention, the G protein (RVG) coding region of the rabies virus CTN-1 strain is optimized, and the sequence optimization includes: adjusting the codon preference for expression in the human body, adjusting the usage frequency of common codons, setting the GC content, optimizing the secondary structure, etc. The nucleotide sequence corresponding to the optimized RVG mRNA sequence is shown in SEQ ID NO. 1. The nucleotide sequence makes the transcribed mRNA structure more stable in the cell and the target protein translation efficiency in mammals and humans is higher.

[0034] 2. The rabies virus mRNA vaccine provided by the present invention comprises a vaccine vector and an optimized sequence of RVG mRNA. The vaccine vector LNP uses a preferred ionizable cationic lipid and conventionally used cholesterol, DSPC and PEG-DMG, which can stimulate immune responses faster and more effectively in vivo, resist virus attacks more efficiently and achieve protective effects, and is superior to existing rabies vaccine technologies in terms of effectiveness.

[0035] 3. The present invention provides a method for preparing the above-mentioned rabies virus nucleic acid vaccine, wherein the vaccine vector and the sequence-optimized RVG mRNA are mixed to obtain a rabies virus mRNA vaccine. The method is simple and easy to operate, reduces the process difficulty of the vaccine, and has good vaccine protection efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0037] Figure 1 The expression results of RVG mRNA transfected HEK293 cells in Example 1;

[0038] Figure 2 This is a result diagram of the neutralizing antibody titer after mice were immunized with vaccines encapsulated with different ionizable cationic lipids in Example 3;

[0039] Figure 3 This is a graph showing the weight changes of mice after they were immunized with vaccines encapsulated with different ionizable cationic lipids in Example 3.

[0040] Figure 4 This is the antiviral protection efficacy of mice after vaccination in Example 4. DETAILED DESCRIPTION

[0041] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0042] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0043] The present application will be described in detail below with reference to embodiments and experimental data.

[0044] Example 1, sequence optimization of RVG mRNA

[0045] The sequence optimization scheme of RVG mRNA in this embodiment is as follows: based on the G protein amino acid of CTN-1 strain (GenBank: ACR39382.1, nucleic acid sequence GenBank: JN234418.1), based on the codon preference when expressed in the human body, the frequency of use of common codons, based on the optimization of GC content, secondary structure, MFE (minimum free energy) and CAI (codon adaptation index), etc. The nucleotide sequence corresponding to the sequence of the optimized RVG mRNA is shown in SEQ ID NO: 1, and the amino acid sequence of the antigen protein finally translated from the sequence is consistent with the G protein amino acid of the CTN-1 strain.

[0046] The above DNA sequence was synthesized by a bioengineering company, plasmid was constructed, cultured, bacteria were collected, alkaline lysed, linearized and purified, and mRNA was obtained by in vitro transcription under optimized reverse transcription conditions. The mRNA obtained from the unoptimized sequence and the optimized sequence were named RVG-WT and RVG-1, respectively. The two mRNAs were transfected into HEK293 cells. First, HEK293 cells were plated (density of 3×10 5 Cells / ml), after 24 hours, when the cell confluence is about 80%, the cells are transfected. The transfection system includes 2 μg mRNA and transfection reagent Lipofectamine MessagerMAX in one well of a 6-well plate. TM (ThermoFisher Scientific), and the specific transfection operation was carried out according to the instructions of the transfection reagent product. HEK293 cells were immunolabeled with rabies virus G protein antibody (Anti-Rabies glycoprotein, clone D1-25, purchased from Merck) 24 hours after transfection, and then the G protein expression was detected by flow cytometry. Untransfected cells were used as negative controls, and the experiment was repeated three times.

[0047] The results are as follows Figure 1 As shown, from Figure 1 The results showed that the number of positive cells transfected in each group was above 80% (0.82±0.035 vs 0.83±0.051), with no significant difference (ns, p=0.7944), indicating that there was no significant difference in transfection efficiency among the groups. The mean fluorescence intensity (MFI) of RVG-1 transfected cells was significantly higher than that of RVG-WT transfected cells (11288±863.9 vs 39723±3286, n=3, ***p<0.0001), indicating that the optimized mRNA sequence encoding G protein significantly increased the expression level of G protein in mammalian cells.

[0048] Example 2. Preparation of rabies virus mRNA vaccine

[0049] RVG-1 mRNA was dissolved in a citrate buffer at pH 4 to adjust the concentration to 0.1 mg / ml to obtain an aqueous phase.

[0050] Cationic lipid (any one of SIM07, SIM40, SIM74, SIM102), cholesterol, DSPC and PEG-DMG were dissolved in anhydrous ethanol according to the formula ratio (molar ratio 50:10:38.5:1.5), and the total lipid concentration was adjusted to 6 mg / ml to obtain an organic phase. Cationic lipid SM-102 was used as a positive control.

[0051] The organic phase and the aqueous phase were mixed in a volume ratio of 1:3 using a microfluidic device. The flow rate during mixing using the microfluidic device was 10.5 ml / min.

[0052] The mixed solution was immediately diluted 40-100 times with a pH 7.4 PBS solution, and tangential flow filtration (TFF) was used to remove the ethanol component in the solution and concentrate it to an mRNA concentration of about 200 μg / ml to obtain lipid nanoparticles encapsulating mRNA, which is the rabies virus nucleic acid vaccine. The vaccines using cationic lipids SM-102, SIM07, SIM40, SIM74, and SIM102 were named RVG-SM-102, RVG-SIM07, RVG-SIM40, RVG-SIM74, and RVG-SIM102, respectively.

[0053] Example 3, Neutralizing Antibody Titer Determination

[0054] The RVG vaccines (RVG-SM-102, RVG-SIM07, RVG-SIM40, RVG-SIM74, RVG-SIM102) in Example 2 were selected for mouse immunization experiments, with 6 BALB / C mice in each group, and each mouse was injected at a dosage of 10 μg, with an injection volume of 50 μl / mouse. The positive control was RVG-SM-102, a vaccine containing SM-102 as a cationic lipid, and the negative control was an equal volume of PBS injection.

[0055] The rapid fluorescent focus inhibition test (RFFIT) was used to detect the rabies virus-specific neutralizing antibody titer in serum samples. The specific experimental operation was as follows:

[0056] 1) Pre-dilute the complement-inactivated serum to be tested 5-fold with DMEM medium, and then perform a series of 3-fold dilutions, for a total of 8 dilutions. Dilute the standard to 1 IU / mL and then perform a 3-fold gradient dilution with DMEM. Take 100 μL / well of each dilution and add it to a 96-well plate.

[0057] 2) Add CVS-11 virus suspension that can cause about 80% cell infection, 100 μL / well. 37°C, 5% CO 2 Incubate for 1 h under the same conditions.

[0058] 3) After the incubation, add 50 μL of 1×10 6 cells / mL golden hamster kidney cell (BSR) suspension, mix well. 37℃, 5% CO 2 Incubate for 24 h.

[0059] 4) After 24 h, discard the supernatant and add cold 4% PFA, 50 μL / well, and fix at 4°C for 30 min.

[0060] 5) Discard the fixative and air dry. Add 1:800 diluted FITC-Rabies virus nucleoprotein antibody (Merck, Cat. No. 5500), protect from light, and incubate at 37°C for 1 hour.

[0061] 6) Wash the 96-well plate three times with PBS to remove unbound antibodies and non-specifically bound antibodies. Observe the percentage of fluorescent foci in each well under a fluorescence microscope.

[0062] 7) Calculate the neutralizing antibody titer of serum samples by the Reed-Muench method.

[0063] After rabies vaccination, the speed of neutralizing antibody production is the key to preventing rabies. Rapid production of neutralizing antibodies in the early stage is conducive to clearing the virus in the early stage of infection. In order to investigate the early antibody response speed and neutralizing antibody level after immunization, the above vaccines were administered once, and the RFFIT method was used to detect the neutralizing antibody level in the mouse serum 3, 5, 7, and 14 days after a single immunization.

[0064] from Figure 2 The results show that the lipid nanoparticle preparations all induced the production of neutralizing antibodies after a single injection. Among them, compared with the other three RVG vaccines (RVG-SM01, RVG-SM40, RVG-SM74) and the vaccine with SM-102 as the cationic lipid component (RVG-SM-102), RVG-SIM102 had the best effect, producing neutralizing antibodies on the 5th day, and was significantly better than the other groups on the 7th and 14th days, indicating that the combination of cationic lipid SIM102 and the optimized G protein expression mRNA sequence of the present invention produced an unexpected synergistic effect. Figure 3 These are the results of body weight monitoring after mice were immunized with RVG-SIM102. Importantly, compared with the negative control PBS and the positive control RVG-SM-102, RVG-SIM102 immunization had no significant effect on the body weight of mice, indicating that the vaccine is relatively safe.

[0065] Example 4: Animal protection experiment

[0066] The rabies virus mRNA vaccine prepared in Example 2 was used, with an immunization dose of 10 μg / mouse and a dosing volume of 50 μl / mouse, to examine the protection of mice against virus attack after vaccination. The inactivated whole virus commercial vaccine (high dose, i.e., 0.5 times the human dose) and the vaccine RVG-SM-102 with SM-102 as the cationic lipid component were used as positive controls, and the negative control was an equal volume of sterile PBS. To further determine whether the mRNA vaccine can effectively protect mice against RABV virus attack, immunization was performed according to the scheme of Example 3, using 50LD 50 The CVS-11 strain of rabies virus was injected into the brain cavity on the 14th day. After the injection, the number of deaths of mice in the experimental group was recorded and observed for 4 consecutive weeks.

[0067] The survival of mice Figure 4 As shown, all mice in the PBS group died 31 days after the virus attack (i.e., 45 days after vaccination); the positive control, i.e., a high-dose inactivated rabies vaccine, can protect 100% of the mice, with 0 mice dying; some mice died in the RVG-SIM07, RVG-SIM40, and RVG-SIM74 groups, and the protection rate did not reach 100%, only the RVG-SIM102 group had 0 mice die, achieving 100% protection, and its protection effect was also better than that of the RVG-SM-102 group.

[0068] Based on the above test results, RVG-SIM102 showed 100% protection for mice after challenge, and the protective efficacy was the same as that of relatively high doses of inactivated vaccines, and was better than that of RVG-SIM07, RVG-SIM40, RVG-SIM74 and RVG-SM-102. What is important is that there was no significant difference in the weight gain of mice in the RVG-SIM102 group after immunization compared with the negative control group, and it had better safety, so RVG-SIM102 was preferred as the ideal rabies mRNA candidate vaccine.

[0069] In summary, the rabies virus nucleic acid vaccine provided by the present invention includes a vaccine vector and an optimized sequence of RVG mRNA. The vaccine vector LNP uses a preferred ionizable cationic lipid and conventionally used cholesterol, DSPC and PEG-DMG, which stimulates immune response faster and more effectively in vivo, thereby more efficiently resisting the attack of the virus to achieve a protective effect, and is superior to existing rabies vaccine technology in terms of effectiveness.

[0070] Finally, it should be noted that the terms "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0071] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0072] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A rabies virus mRNA vaccine, characterized in that: It includes mRNA expressing rabies virus G protein, and the nucleotide sequence of the mRNA transcribing rabies virus G protein is shown in SEQ ID NO.

1.

2. A rabies virus mRNA vaccine according to claim 1, characterized in that: The mRNA expressing rabies virus G protein further comprises the following elements: at least one or more of a 5'-cap structure, a 5'-UTR, a promoter element, a 3'-polyadenylic acid sequence and a 3'-UTR.

3. A rabies virus mRNA vaccine according to claim 1, characterized in that: The nucleotide sequence encoding the rabies virus G protein also includes: A sequence in which one or more bases are substituted, deleted, added and / or replaced based on the nucleotide sequence shown in SEQ ID NO: 1; or a sequence with a homology of more than 90% to the above nucleotide sequence.

4. The mRNA vaccine according to claim 1, characterized in that The vaccine also includes: a vaccine carrier.

5. The mRNA vaccine according to claim 4, characterized in that The vaccine carrier includes one of cationic liposomes, cationic proteins, cationic polymers or cationic lipid nanoparticles.

6. The mRNA vaccine according to claim 4, characterized in that The mass ratio of the cationic liposome to the mRNA expressing the rabies virus G protein is 10-30:1, and the cationic liposome is selected from the following compounds I-IV:

7. The mRNA vaccine according to claim 5, characterized in that The cationic lipid nanoparticles, measured by molar percentage, include 20-50% ionizable cationic lipids, 20-50% structural lipids, 5-20% auxiliary lipid phospholipids and 1-5% surfactant, wherein the molar content of ionizable cationic lipids, structural lipids, auxiliary lipids and surfactants totals 100%.

8. A method for preparing the mRNA vaccine according to any one of claims 1 to 7, characterized in that: The method comprises: Dissolving the components of the vaccine carrier in an organic solution to obtain an organic phase; The mRNA expressing rabies virus G protein was dissolved in PBS to obtain an aqueous phase; The organic phase and the aqueous phase are mixed, and the solvent in the mixed solution is replaced with a buffer solution to obtain a rabies virus mRNA vaccine.

9. The preparation method according to claim 8, characterized in that: The concentration of mRNA expressing rabies virus G protein in the water phase is 0.08-0.12 mg / ml.

10. The preparation method according to claim 8, characterized in that: The volume ratio of the organic phase to the aqueous phase is 1:2-4.

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