WO2024017250A1

WO2024017250A1 - Mrna vaccine for novel coronavirus variants and use thereof

Info

Publication number: WO2024017250A1
Application number: PCT/CN2023/107926
Authority: WO
Inventors: 李林鲜; 杜爽
Original assignee: 深圳深信生物科技有限公司
Priority date: 2022-07-19
Filing date: 2023-07-18
Publication date: 2024-01-25
Also published as: CN117414418A

Abstract

The present invention relates to an mRNA vaccine for novel coronavirus variants and a use thereof. The mRNA vaccine can protect against infection by SARS-CoV-2 virus and Delta virus strain, can cause a strong and continuous novel coronavirus antibody titer, and has small toxic side effects and high safety.

Description

A kind of mRNA vaccine of new coronavirus mutant strain and its application

Technical field

The invention belongs to the field of biopharmaceuticals, and specifically relates to a novel coronavirus mutant strain mRNA vaccine and its application.

Background technique

The new coronavirus (SARS-COV-2) belongs to the Coronaviridae family and can cause serious infectious diseases. It is highly contagious and has a high fatality rate. It has also mutated and evolved into many different mutant strains, especially such as Delta, Mutated strains such as Omicron are more transmissible and more dangerous. According to existing data, the new coronavirus (SARS-COV-2) is likely to become a long-term, annual epidemic virus, and an effective vaccine is the best way to deal with coronavirus infection.

Compared with traditional vaccines such as recombinant protein subunit vaccines, inactivated or DNA vaccines, mRNA vaccines have a higher effective protection rate and higher safety, and can be quickly updated and iterated to deal with the emerging mutant strains. Faster development and large-scale production of vaccines against new viruses can more effectively respond to outbreaks of new viruses and control emergencies of infectious diseases.

Although mRNA vaccines have good specificity and in vivo expression, using mRNA as the active ingredient of the vaccine has the disadvantages of short circulation time, easy degradation, and difficulty in entering target cells. Although cationic lipids or protonable lipids are used as delivery systems Other methods can realize the functions of nucleic acid drugs, but there are still problems such as high cytotoxicity and low transfection efficiency.

Therefore, it is particularly important to develop a safe and effective new mRNA vaccine against new coronavirus mutant strains.

Contents of the invention

In order to achieve the above purpose, the present invention provides an mRNA vaccine with significant immune effect against new coronavirus mutant strains, especially against Delta and Omicron mutant strains. The vaccine has good biological safety and can efficiently express antigen proteins in the body. More effectively induce the body's neutralizing antibody response to the new coronavirus mutant strain to achieve the purpose of prevention or treatment.

In a first aspect, the present invention provides a novel mRNA vaccine containing S protein-encoding mRNA and a delivery vector.

In some embodiments, the mRNA encodes the new coronavirus S protein, and the S protein includes the amino acid sequence shown in SEQ ID NO: 3. Preferably, the amino acid sequence of the S protein is as shown in SEQ ID NO: 3. .

Preferably, the mRNA includes the nucleic acid sequence shown in SEQ ID NO: 2; more preferably, the nucleic acid sequence of the mRNA is shown in SEQ ID NO: 2.

In some embodiments, some or all of the cytosine and/or uracil in the mRNA are chemically modified to improve the stability of the mRNA in vivo.

In some embodiments, the chemical modifications include the following:

One or more uracil nucleosides in the mRNA, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 uracil nucleosides or at least 50%, at least 60%, at least 70 %, at least 80%, at least 90% or 100% of the uridine nucleosides are replaced by at least one nucleoside selected from the following: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2 -Thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudine, 2-thio-dihydrouridine, 2-thio-pseudouridine Glycoside, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudo Uridine or at least one of 5-methoxyuridine and 2′-O-methyluridine, preferably pseudouridine or N1-methylpseudouridine or N1-ethylpseudouridine, and further preferably N1 -methylpseudouridine; and/or

One or more cytosine nucleosides in the mRNA, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cytosine nucleosides or at least 50%, at least 60%, at least 70 %, at least 80%, at least 90% or 100% of the cytosine nucleosides are replaced by 5-methylcytosine nucleosides.

In some embodiments, all or part of the uridine nucleosides in the mRNA are replaced by pseudouridines, preferably N1-methylpseudouridines; preferably, in the nucleic acid sequence shown in SEQ ID NO: 2 All or part of the uridine nucleosides are replaced by pseudouridine, preferably N1-methylpseudouridine.

In some embodiments, all uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 2 are replaced by N1-methylpseudouridine.

In certain embodiments, the mRNA further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR, and polyA.

Preferably, the 5'-UTR includes an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4 or consists of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4; the 3'-UTR includes SEQ The RNA sequence corresponding to the nucleic acid sequence shown in ID NO: 5 may consist of the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 5; and/or the polyA includes the RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 6 The RNA sequence may consist of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 6.

Preferably, the mRNA includes the nucleic acid sequence shown in SEQ ID NO:7; preferably, the nucleic acid sequence of the mRNA is shown in SEQ ID NO:7. SEQ ID NO: 7 sequence itself already contains a cap structure: cap G ¹ G ² =m ⁷ G ⁺ -5′-ppp-5′-Gm ^2′ -3′-p-[m ⁷ =7-CH ₃ ; m ^2′ =2′-O-CH ₃ ;-ppp-=-PO ₂ HO-PO ₂ HO-PO ₂ H)-; -p-=-PO ₂ H-]. It should be noted that according to WIPO standard ST.26 for nucleotide or amino acid sequence listings, t (thymine) in the RNA sequence (such as SEQ ID NO: 2, SEQ ID NO: 7) in the sequence listing is actually u ( uracil).

In some embodiments, all or part of the uridine nucleosides in the mRNA are replaced by pseudouridines, preferably N1-methylpseudouridines; preferably, in the nucleic acid sequence shown in SEQ ID NO: 7 All or part of the uridine nucleosides are replaced by pseudouridine, preferably N1-methylpseudouridine.

In some embodiments, all uridine nucleosides in the nucleic acid sequence shown in SEQ ID NO: 7 are replaced by N1-methylpseudouridine.

In some embodiments, the S protein comprises the amino acid sequence shown in SEQ ID NO: 3. Preferably, the amino acid sequence of the S protein is shown in SEQ ID NO: 3.

In some embodiments, the delivery vehicle includes one of cationic liposomes, cationic proteins, cationic polymers or cationic lipid nanoparticles, preferably cationic lipid nanoparticles.

Preferably, the cationic lipid nanoparticles include one or more of protonatable cationic lipids, auxiliary lipids, structural lipids and PEG-lipids.

Preferably, the protonatable cationic lipid is an amino lipid compound, selected from compound (I), SM102, ALC-0315, Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, c12-200 and DlinDMA One or more, preferably compound (I), wherein the structure of compound (I) is as follows:

The auxiliary lipids are phospholipids, which are usually semi-synthetic, can also be derived from natural sources, or can be chemically modified, including but not limited to DSPC, DOPE, DOPC, DOPS, DSPG, DPPG, DPPC, DGTS, lysophospholipid, etc., preferably DSPC.

Preferably, the structural lipid is one or more selected from the group consisting of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, cholesterol, ergosterol, β-sitosterol and oxidized cholesterol derivatives. species, preferably cholesterol (e.g. CHO-HP).

Preferably, the PEG-lipid is selected from DMG-PEG and DSPE-PEG, preferably DMG-PEG; preferably, the average molecular weight of PEG in the DMG-PEG is about 2000 to about 5000 Daltons, and the DMG-PEG is preferably DMG-PEG2000 (PEG2000-DMG).

Preferably, the lipid nanoparticles contain one or more of protonatable cationic lipids, auxiliary lipids, structural lipids and PEG-lipids.

In certain embodiments, the lipid nanoparticles comprise 25-75% protonable cationic lipids, auxiliary lipids, structural lipids, and PEG-lipids on a molar percent basis. The cationic lipid is preferably 45%-55%, more preferably 49.5%.

In certain embodiments, the lipid nanoparticles comprise 5-20% auxiliary lipids on a molar percentage basis based on the total amount of protonatable cationic lipids, auxiliary lipids, structural lipids, and PEG-lipids. The quality is preferably 8%-12%, and more preferably 10%.

In certain embodiments, the lipid nanoparticles comprise 0-50% structural lipids on a molar percent basis based on the total amount of protonatable cationic lipids, helper lipids, structural lipids, and PEG-lipids. The quality is preferably 35%-45%, and more preferably 39%.

In certain embodiments, the lipid nanoparticles comprise 0.5-5% PEG on a molar percent basis based on the total amount of protonatable cationic lipids, helper lipids, structural lipids, and lipid conjugates. - Lipid, preferably 1.0%-3.0%, more preferably 1.5%.

In certain embodiments, the mass ratio of protonatable cationic lipids and mRNA in the mRNA vaccine is (5-30):1, for example, it can be but is not limited to 5:1, 10:1, 15:1 , 20:1, 25:1 or 30:1, preferably (8-12):1, more preferably 10:1.

In certain embodiments, the mRNA vaccine may also contain a buffer. In certain embodiments, the buffer may include phosphate buffer, Tris buffer, preferably phosphate buffer. In certain embodiments, the buffer concentration may be 5 mmol/L-30 mmol/L, preferably 10 mmol/L.

Preferably, in certain embodiments, the mRNA vaccine may also contain a cryoprotectant. In certain embodiments, the cryoprotectant can be selected from sucrose, trehalose, preferably sucrose. In certain embodiments, the concentration of cryoprotectant may range from 5 mg/mL to 100 mg/mL, preferably 80 mg/mL.

In a second aspect, the present invention provides a method for preparing the above-mentioned mRNA vaccine. For example, the method may include the following steps:

(1) Prepare an aqueous phase containing nucleic acid;

(2) Preparing an organic phase (e.g., ethanol phase) containing one or more of protonatable cationic lipids, auxiliary lipids, structural lipids, and PEG-lipids;

(3) Encapsulation: Mix an appropriate amount of the aqueous phase and the organic phase for encapsulation;

(4) Replacement: Replace the solution in the two-phase mixture with another buffer containing a cryoprotectant; and

(5) Sterilize and filter to obtain the mRNA vaccine.

In a third aspect, the present invention provides the use of the above-mentioned mRNA vaccine in preventing or treating diseases caused by the new coronavirus. Preferably, the new coronavirus includes Alpha (such as the B.1.1.7 variant and its descendant lineage), Beta (such as the B.1.351 variant and its descendant lineage), Gamma (such as the P.1 variant and its descendant lineage) lineage), Delta (such as B.1.617.2 and AY lineage), and Omicron, etc.

In a fourth aspect, the present invention provides a method for preventing or treating novel coronavirus infection in an administration subject, or controlling, preventing, or treating infectious diseases caused by the novel coronavirus, which includes administering to the administration subject a preventive or therapeutic agent. An effective amount of the above-mentioned mRNA vaccine.

In some embodiments, the subject of administration is a human or non-human mammal. In some embodiments, the subject of administration is an adult, elderly person, child, or toddler. In some embodiments, the subject is at risk for or susceptible to coronavirus infection. In some embodiments, the subject has been diagnosed positive for coronavirus infection or is asymptomatic.

In some embodiments, the use of the above-mentioned mRNA vaccine in the preparation of products for preventing or treating novel coronavirus infection (such as preventive vaccines or therapeutic vaccines) is provided.

In some embodiments, the mRNA vaccine is administered intravenously, intramuscularly, or intradermally.

It has been confirmed by in vivo animal experimental studies that the mRNA vaccine can stimulate animals to produce novel coronavirus S1 antibodies (IgG), novel coronavirus Delta strain (pseudovirus) and novel coronavirus Omicron strain (pseudovirus) neutralizing antibodies in vivo. At the same time, it can fight against infection by SARS-CoV-2 virus and Delta strain virus, effectively reduce the replication of the virus in the body, protect tissues from virus intrusion, and can induce strong and sustained new coronavirus antibody titers, effectively protecting tissues from virus invasion; at the same time, the particle size distribution (PDI), particle size, and morphology of the prepared LNP are more in line with delivery requirements. The vaccine has small side effects, high safety, and good clinical application prospects.

Description of drawings

Figure 1 is a plasmid spectrum of plasmid H in the present invention.

Figure 2 is a spectrum of the engineering plasmid in the present invention.

Figure 3 is a transmission electron microscope image of the mRNA vaccine prepared in Example 1.

Figure 4 shows the protective effect of the mRNA vaccine prepared in Example 4 in the ACE2-IRES-luc transgenic (hACE2) mouse SARS-CoV-2 virus infection model.

Figure 5 shows the protective effect of the mRNA vaccine prepared in Example 4 in the K18-hACE2 KI transgenic mouse 2019-nCov Delta variant (B.1.617.2) virus infection model.

Detailed ways

The technical solution of the present invention will be described clearly and completely below with reference to the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as familiar to one of ordinary skill in the art. In addition, any methods or materials similar or equivalent to those described can also be used in the present invention.

The structures of the compounds used in the biological experiments of the present invention: SM102, ALC-0315, and compound (I) are as follows:

Among them, SM102 and ALC-0315 are commercially available or can be prepared according to known techniques in the art; compound (I) can be prepared according to the following steps:

step one

9-Heptadecyl-8-bromooctanoate reacts with (1S, 3R)-3-aminocyclohexanol in ethanol at 50°C for 15 hours. After the reaction is completed, the solvent is evaporated, EA is added, and washed with water. The organic phase was concentrated, and the concentrate was purified by column chromatography to obtain 9-heptadecyl-8-(((1R,3S)-3-hydroxycyclohexyl)amino)octanoate.

NMR data:

¹ H NMR (600MHz, CDCl ₃ ): δ4.91-4.81 (m, 1H), 3.86-3.82 (m, 1H), 2.84-2.81 (m, 1H), 2.69-2.54 (qt, J=11.2, 7.3 Hz, 2H), 2.29-2.26 (t, J=7.5Hz, 2H), 1.93-1.86 (m, 1H), 1.77-1.74 (d, J=12.0Hz, 1H), 1.72-1.57 (m, 6H) , 1.54-1.45 (m, 6H), 1.37-1.30 (m, 8H), 1.30-1.22 (m, 24H), 0.88 (t, J=7.0Hz, 6H).

LCMS: 496.7[M+H].

Step 2

The above-mentioned 9-heptadecyl-8-(((1R,3S)-3-hydroxycyclohexyl)amino)octanoate is mixed with 8-octanoate in a mixed solvent of acetonitrile and cyclopentyl methyl ether under the action of potassium carbonate and potassium iodide. - Nonyl bromooctanoate was reacted at 90°C for 24 hours. After the reaction was completed, the crude product was filtered and concentrated. The crude product was finally purified by column chromatography to obtain compound (I).

NMR data:

¹ H NMR (600MHz, CDCl ₃ ): δ4.93-4.84 (m, 1H), 4.09 (t, J=6.8Hz, 2H), 3.68-3.61 (m, 1H), 2.62-2.54 (m, 1H) , 2.53-2.41 (m, 4H), 2.31-2.26 (q, J=7.4Hz, 4H), 1.97 (m, 1H), 1.91-1.78 (m, 2H), 1.71-1.61 (m, 7H), 1.54 (d, J=6.0Hz, 4H), 1.43-1.37(m, 4H), 1.39-1.23(m, 52H), 0.91(m, 9H).

LCMS: 765.2[M+H].

Example 1 Construction and preparation of novel coronavirus S protein coding sequence

The mutation site of the new coronavirus was analyzed and the nucleic acid sequence encoding the S protein of the new coronavirus was designed as shown in SEQ ID NO: 1. Its encoded amino acid sequence is shown in SEQ ID NO: 3, and its mRNA sequence is shown in SEQ ID NO: 2, in which all uridine nucleosides are replaced by N1-methylpseudouridine. The nucleic acid sequence (SEQ ID NO: 1) was provided and synthesized by Nanjing GenScript Biotechnology Co., Ltd., and GenScript QC released it.

The characterization data is: Genscript QC release results show that the sequence is correct.

Example 2 Construction of engineering plasmid

Through PCR, homologous arm sequences were introduced at both ends of the new coronavirus S protein coding sequence shown in SEQ ID NO: 1, and then the PCR product was subjected to BamHI and KpnI double enzyme digestion with plasmid H (Figure 1). The macromolecular fragments recovered from the gel were subjected to homologous recombination, transformed into competent cells with DH5a, single clones were selected, and sequenced for verification, and finally the correct engineering plasmid was obtained. This engineering plasmid uses the 5’UTR shown in the sequence SEQ ID NO: 4; the 3’UTR shown in the sequence SEQ ID NO: 5; and the polyA shown in the sequence SEQ ID NO: 6.

Data characterization: Starting from plasmid H, perform sequencing verification and confirmation. The results showed that the nucleotide sequence was completely inserted into the correct position and was completely consistent with the theoretical sequence. The plasmid spectrum of the engineering plasmid is shown in Figure 2.

Example 3 Preparation of novel coronavirus vaccine mRNA

1. Extract the engineering plasmid template prepared in Example 2 to ensure that the plasmid supercoiling rate reaches more than 90%.

2. Plasmid linearization

3. Linearized plasmid purification: recovery using Takara recovery kit

4. Phenol-chloroform extraction (removal of RNase, proteins, etc.)

5. In vitro transcription of mRNA (refer to Novozant IVT Reaction Kit)

(1) Cleaning of the experimental area: First, use ultraviolet rays to sterilize the biological safety cabinet for 0.5 hours, then wipe the safety cabinet clean with alcohol cotton balls and spray RNase inhibitor. After 5 minutes, wipe the inhibitor clean with an alcohol cotton ball and start the RNA experiment.

(2) System configuration: Before preparing the system, take out the reagents of each component, vortex and mix on a oscillator, centrifuge, and place in an ice box for later use.

Note: During the system configuration process, the entire process is performed on ice, and the plasmid template and IVT system are prepared separately.

(3) Preparation of IVT system: Add all components except plasmid template to below the liquid level of the system, finally add T7 enzyme, vortex and mix, in which uracil (U) is replaced with methylpseudouracil (N1-Me- Pseudo UTP).

(4) Take the required amount of plasmid into a new PCR tube, incubate it with the IVT system for 5 minutes, finally mix the two systems together, vortex to mix, centrifuge and incubate at 37°C for 2 hours.

(5) Prepare the system into a 0.2 mL thin-walled tube with a flat cover, mix well and place it in a PCR machine for reaction. The reaction conditions are 37°C for 2 hours. Note: The reaction system can also be synchronously scaled up according to the required production volume.

(6) Use DNase to remove the linearized plasmid template, add (20 μL system) 1 μL/(100 μL system) 5 μL DNase to the reaction system, and incubate at 37°C for 15 minutes.

6. Purification method: refer to the Thermo MEGAclear Kit, and use onedrop or Qubit to determine the concentration (diluted 10 measured after multiple times).

7. Identification: Mix 5 μl of diluted purified sample with NorthemMax Formaldehyde Load Dye and incubate at 75°C for 10 minutes, then store on ice. Then use 1% agarose gel to perform gel electrophoresis to confirm that the size of the mRNA is correct and the band is not diffuse, and the next step of capping reaction can be carried out.

8. Capping reaction

mRNACap type 1 capping reaction:

The structure and reaction principle of Cap1 type hat are as follows:

pppN1(p)Nx-OH(3′)→ppN1(pN)x-OH(3′)+Pi

ppN1(pN)x-OH(3′)+GTP→G(5′)ppp(5′)N1(pN)x-OH(3′)+PPi

G(5′)ppp(5′)N1(pN)x-OH(3′)+AdoMet→m7G(5′)ppp(5′)N1(pN)x-OH(3′)+AdoHyc

m7GpppN1(pN)x-OH(3′)+AdoMet→m7Gppp[m2’-O]N1(pN)x-OH(3′)+AdoHyc

5‘-Cap1 type cap structure:

cap G ¹ G ² =m ⁷ G ⁺ -5′-ppp-5′-Gm ^2′ -3′-p-[m ⁷ =7-CH ₃ ; m ^2′ =2′-O-CH ₃ ;- ppp-=-PO ₂ HO-PO ₂ HO-PO ₂ H)-; -p-=-PO ₂ H-], 37°C 5min or 65°C 5min (CELL SCRIPT).

9. Purification method: The same as the purification method of in vitro transcription products.

Characterization data: Determine concentration using onedrop or Qubit.

Experimental results: The final mRNA product encoding the S protein of the new coronavirus was obtained. Its sequence is the sequence obtained by replacing all uracil (U) nucleosides in SEQ ID NO: 7 with N1-methylpseudouridine.

Example 4 Preparation of mRNA vaccine

Preparation of the aqueous phase: Weigh 190 mg of the mRNA prepared in Example 3 and 89.06 mL of acetic acid-sodium acetate buffer (0.2 mol/L, pH=5), add enzyme-free water to make the volume to 712.5 mL, and prepare the mRNA The aqueous phase has a concentration of approximately 0.267 mg/mL.

Preparation of the alcohol phase: Weigh 1.9078g compound (I), 0.3985g DSPC lipid, 0.7575g CHO-HP lipid, and 0.1972g M-DMG-2000, according to the molar ratio of 49.5:10:39: The ratio of compound (I) to mRNA is 10:1, and the mass ratio of compound (I) and mRNA is 10:1. Dissolve it with absolute ethanol and adjust the volume to 237.5mL to prepare compound (I), DSPC lipid, CHO-HP lipid and M- The total concentration of DMG-2000 is 13.73 mg/mL in the alcohol phase.

Encapsulation: Encapsulate with a volume ratio of water phase:alcohol phase of 3:1 and an encapsulation flow rate of 20 mL/min. Discard the initial encapsulation liquid and collect it for later use.

Tangential flow filtration: The encapsulated medicinal liquid is replaced by ultrafiltration according to the parameters of TMP of 0.2bar and inlet flow rate of 300mL/min. The dialysate used for replacement contains 8 mg/mL sodium chloride, 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium dihydrogen phosphate, 1.15 mg/mL sodium hydrogen phosphate dihydrate and 80 mg/mL sucrose.

Sterilizing filtration and filling: Use a 0.2μm PES filter for sterilizing filtration, fill into sterilized vials, and seal. An mRNA vaccine with an mRNA concentration of 0.2 mg/mL was prepared.

The test results are shown in Table 1:

Table 1 mRNA vaccine test results

The transmission electron microscope image is shown in Figure 3.

The results showed that the particles were in good shape, with designed spherical shape, uniform particle size, good dispersion, and the encapsulation rate met the requirements.

Example 5 Preparation of blank liposome (LNP) vaccine

No mRNA was added in the preparation step of the water phase. The other procedures were the same as in Example 4 to prepare a blank liposome (LNP) vaccine that did not contain mRNA.

Example 6 Toxicology Test

The mRNA vaccine prepared in Example 4 and Example 5 and the blank liposome (LNP) vaccine were used to conduct toxicological tests, in which the blank liposome (LNP) vaccine was used as a control to conduct the following tests:

Test 1: Immunogenicity of mRNA vaccine in Balb/c mice

36 Balb/c mice were randomly divided into 3 groups, half male and half female, respectively as blank liposome (LNP) control group, low-dose group, and high-dose group. Each group had 12 animals and were administered intramuscular injection. once. The doses given to the low-dose and high-dose groups were 5 and 10 μg/animal respectively. On days 14/21/28/35/42 after injection, blood was collected and serum was separated to detect novel coronavirus S1 antibodies (IgG) and novel coronavirus Delta strain (pseudovirus) neutralizing antibodies.

The test results are shown in Table 2 and Table 3:

Table 2 Effect of mRNA vaccine on serum novel coronavirus S 1 antibody (IgG) titers in mice

Note: “H1 group and L1 group” were given the mRNA vaccine prepared in Example 4; “CV1 group” was given blank LNP.

Table 3 Effect of mRNA vaccine on neutralizing antibody titers of new coronavirus Delta strain (pseudovirus) in mouse serum

As can be seen from Table 2 and Table 3, after each dose of injection, on the 21st day, the antibody positivity rate was 100%, and the new coronavirus S1 antibody (IgG) and the new coronavirus Delta strain (pseudovirus) neutralized Antibodies showed a strong immune response after the 21st day after immunization and maintained a high level for 42 days. This shows that the mRNA vaccine of the present invention can induce strong immune titers. Published preclinical studies show that after immunizing animals with new coronavirus mRNA vaccines such as BNT162b2 and mRNA-1273, the new coronavirus S1 antibody (IgG) titer or neutralizing antibody titer usually reaches a plateau on the 28th and 14th day. , and then the corresponding titers decreased to varying degrees, and this product also had a strong immune effect on the 42nd day.

Test 2: Immunogenicity of mRNA vaccine in rhesus monkeys

18 rhesus monkeys were randomly divided into 3 groups, half male and half female. They were divided into low-dose group, medium-dose group and high-dose group of mRNA vaccine. There were 6 animals in each group, which were administered by intramuscular injection. The doses given to the low, medium and high dose groups of the mRNA vaccine were 10, 30 and 90 μg/animal, once every 2 weeks for 4 consecutive weeks (3 times in total), with a 4-week recovery period. During the trial, the neutralizing antibody titers of the new coronavirus Delta and Omicron strains (pseudoviruses) in the animal serum were detected.

Table 4 Effect of mRNA vaccine on novel coronavirus Delta and Omicron (pseudovirus) neutralizing antibody titers

The results showed that the geometric mean neutralizing antibody titers of the new coronavirus Delta (pseudovirus) and Omicron (pseudovirus) in the animals in the mRNA vaccine group (10μg/animal, 30μg/animal, and 90μg/animal) increased with the increase in the number of immunizations. Basically dose dependent. The antibodies induced by the mRNA vaccine have neutralizing activity against two different mutant strains of Delta and Omicron pseudoviruses, and have preventive potential.

Test 3: Safety of intramuscular injection of mRNA vaccine in cynomolgus monkeys

60 cynomolgus monkeys were randomly divided into 6 groups, half male and half female, which were respectively the negative control (sodium chloride injection) group; the blank LNP low and high dose group and the mRNA vaccine low and high dose group. The blank LNP low-dose and high-dose groups, each group has 5 animals/sex, and the low- and high-dose groups were given doses of 150 and 600 μL/animal respectively. The mRNA vaccine low- and high-dose groups, each group has 5 animals/sex, with low and high doses. The doses given to the dosage groups were 30 and 120 μg/animal respectively, administered by intramuscular injection, once every 2 weeks, for 4 consecutive weeks (a total of 3 administrations). During the test, clinical observations, body weight, body temperature, hematological indicators, etc. were conducted on the animals. At the end of the test, gross anatomy observation and histopathological examination were conducted.

The results showed that during the trial, there were no obvious clinical observations or abnormalities in weight, body temperature and other indicators in each administration group. Hematological index testing, gross anatomy observation and histopathological examination found that except for some recoverable mild inflammation at the injection site in some animals, there were no other obvious changes in each group. It shows that when the mRNA vaccine of the present invention is administered to cynomolgus monkeys multiple times, except for the reversible inflammatory lesion changes at the injection site, there are no other safety risks. Comparing the existing public information on the new coronavirus mRNA vaccine, in repeated dosing studies, there is usually an increase in the body temperature of the animals; more common blood routine and lymphocyte abnormalities (neutrophils, monocytes, eosinophils) etc.); weight, histopathological changes of spleen, liver and other organs, as well as erythema, edema at the injection site, etc., and the toxicity changes displayed by the mRNA vaccine of the present invention are far lower than those of the existing mRNA vaccines, and have better safety sex.

Table 5 Safety test of intramuscular injection in cynomolgus monkeys

Test 4: Protective effect of mRNA vaccine in ACE2-IRES-luc transgenic (hACE2) mouse SARS-CoV-2 virus infection model

Experiments were conducted using the mRNA vaccine prepared in Example 4. 20 human ACE2-IRES-luc transgenic (hACE2) female mice were randomly divided into 4 groups, half male and half female, respectively: PBS control group, mRNA vaccine low-dose group, medium-dose group, and high-dose group, with 5 mice in each group. Animals, administered via intramuscular injection. The doses given to the low, medium and high dose groups of the mRNA vaccine were 1, 5 and 10 μg/animal, and were administered twice on the 35th and 14th days before challenge. Intranasal infection with SARS-CoV-2 virus was performed 14 days after the last dose, and mouse lung tissue was taken 3 days after infection to evaluate the virus copy number in the mouse lung tissue.

The test results are shown in Figure 4. It can be seen from Figure 4 that 3 days after infection, the virus copy number in the mouse lung tissue was significantly reduced, with a significant statistical difference (P<0.01), indicating that the mRNA vaccine of the present application can protect SARS-CoV-2 virus infection. The results are shown in Figure 5.

Test 5: Protective effect of mRNA vaccine in K18-hACE2KI transgenic mouse 2019-nCov Delta variant (B.1.617.2) virus infection model

Experiments were conducted using the mRNA vaccine prepared in Example 4. 50 K18-hACE2 KI transgenic female mice were randomly divided into 4 groups, half male and half female, respectively: blank control group, empty LNP control group, mRNA vaccine low-dose group, medium-dose group, high-dose group, blank control group, The empty LNP control group has 7 animals per group, and the IN002 low, medium and high dose groups have 12 animals per group, and the drugs are administered by intramuscular injection. The doses given to the low, medium and high dose groups of the mRNA vaccine were 1, 5 and 10 μg/animal, and they were immunized twice with an interval of 21 days. 14 days after the last dose, 2019-nCov Delta variant (B.1.617.2) virus was used for intranasal infection. On the 7th day after infection, all mice in the blank control group, empty LNP control group, and half of each dose group of the mRNA vaccine were treated. Mice. On the 14th day after infection, mouse tissues were collected from the remaining mice in each dose group of the mRNA vaccine to evaluate the virus copy number.

The test results are shown in Figure 5. It can be seen from Figure 5 that the viral load in the tissue of mice in each dose group completely disappeared 14 days after infection. It shows that the mRNA vaccine of this application can protect against infection by the new coronavirus Delta variant virus.

Example 7 Mass ratio screening of protonatable cationic lipid compound (I) and mRNA

Preparation of the aqueous phase: Pipette 1mg of mRNA, 0.469mL sodium acetate buffer (0.2mol/L, pH=5), dilute to 3.75mL with enzyme-free water, mix well and set aside.

Preparation of the alcohol phase: Prepare the alcohol phase according to the molar ratio of compound (I): DSPC: cholesterol: M-DMG-2000: 50: 10: 38.5: 1.5. Dissolve each lipid in 1.25 mL of absolute ethanol. , mix well and set aside; the mass ratio prescriptions of different mRNAs and compound (I) are shown in Table 6.

Encapsulation: Use microfluidic equipment to inject the two phases into the microfluidic chip. According to the volume ratio of water phase:alcohol phase of 3:1, the encapsulation flow rate is 12mL/min, encapsulate, discard part of the front-end volume, and collect Encapsulated mRNA-LNP solution.

Dialysis: Put the encapsulated mRNA-LNP solution into a dialysis bag and place it in a dialysis bag containing 8 mg/mL sodium chloride, 0.2 mg/mL potassium chloride, 0.2 mg/mL potassium dihydrogen phosphate, 1.15 mg /mL disodium hydrogen phosphate dihydrate and 80 mg/mL sucrose were replaced with the dialysate to remove residual ethanol and other components. Dialyze with magnetic stirring for 2 hours at room temperature in the dark (replace the dialysate every hour). .

Sterile filtration: Filter the dialyzed mRNA-LNP solution using a disposable sterile syringe filter to obtain vaccines with different mass ratios of mRNA and compound (I).

Table 6 Prescriptions with different mass ratios of mRNA and compound (I)

M-DMG2000 is PEG2000-DMG

Table 7 Vaccine test results of different mass ratio formulations of mRNA and compound (I):

It can be seen from Table 7 that in each mass ratio of compound (I) to mRNA, the particle size and PDI are both good, and the encapsulation efficiency meets the requirements.

Example 8 Activity comparison of protonatable cationic lipids

Test sample: the mRNA vaccine prepared in Example 4;

Preparation of control samples:

Compound (I) was replaced with ALC-0315, and other preparation methods were the same as those of the test sample. An mRNA vaccine with lipid ALC-0315 and other components that were the same as the test sample was prepared.

The test sample and control sample were administered to 5 mice respectively to detect their activity. The data are shown in Table 8:

Table 8 Activity data (lgG binding titer) of test samples and control samples

As can be seen from Table 8, both the test sample and the control sample showed good activity in mice, proving that the composition can effectively deliver and express mRNA into the body, while the average binding titer of the control sample was lower than Test samples indicate that the use of Compound (I) as a protonatable cationic lipid for the in vivo delivery of mRNA vaccines has better advantages.

As proven by in vivo animal experimental studies in the above examples, the mRNA vaccine can stimulate animals to produce novel coronavirus S1 antibodies (IgG), novel coronavirus Delta strain (pseudovirus) and novel coronavirus Omicron strain (pseudovirus) in vivo. virus) neutralizing antibodies, which showed a strong immune response after the 21st day after immunization and remained at a high level for 42 days. At the same time, the mRNA vaccine can fight against infection by SARS-CoV-2 virus and Delta strain virus, and can induce strong and sustained new coronavirus antibody titers, with fewer side effects and higher safety.

Claims

An mRNA vaccine, which includes mRNA expressing the new coronavirus S protein and a delivery vector, wherein the S protein includes the amino acid sequence shown in SEQ ID NO: 3; preferably, the mRNA includes the amino acid sequence shown in SEQ ID NO: 2 The nucleic acid sequence; more preferably, the nucleic acid sequence of the mRNA is as shown in SEQ ID NO: 2.
The mRNA vaccine according to claim 1, wherein one or more uracil nucleosides in the mRNA, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 uracil nucleosides Or at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the uridine nucleosides are replaced by at least one nucleoside selected from: pseudouridine, N1-methylpseudouridine Glycoside, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-de Aza-pseudouridine, 2-thio T-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydropseudine Hydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine Glycoside, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine or at least one of 5-methoxyuridine and 2'-O-methyluridine, preferably pseudouridine Uridine or N1-methylpseudouridine or N1-ethylpseudouridine, further preferably N1-methylpseudouridine; and/or,

One or more cytosine nucleosides in the mRNA, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 cytosine nucleosides or at least 50%, at least 60%, at least 70 %, at least 80%, at least 90% or 100% of the cytosine nucleosides are replaced by 5-methylcytosine nucleosides.
The mRNA vaccine according to claim 1 or 2, wherein all or part of the uridine nucleosides in the mRNA are replaced by pseudouridine, preferably N1-methylpseudouridine; Preferably, the SEQ ID NO: 2 All or part of the uracil nucleosides in the nucleic acid sequence shown are replaced by pseudouridine, preferably N1-methylpseudouridine; more preferably, all uracil nucleosides in the nucleic acid sequence shown in SEQ ID NO: 2 The nucleoside is replaced by N1-methylpseudouridine.
The mRNA vaccine according to any one of claims 1-3, wherein the mRNA further comprises at least one of a 5'-cap structure, a 5'-UTR, a 3'-UTR and polyA.
The mRNA vaccine according to claim 4, wherein the 5'-UTR includes an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4 or consists of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 4; and / or

wherein the 3'-UTR includes an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 5 or consists of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 5; and/or

The polyA includes an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 6 or consists of an RNA sequence corresponding to the nucleic acid sequence shown in SEQ ID NO: 6.
The mRNA vaccine according to any one of claims 1-5, wherein the mRNA comprises the nucleic acid sequence shown in SEQ ID NO: 7; Preferably, the nucleic acid sequence of the mRNA is shown in SEQ ID NO: 7.
The mRNA vaccine according to claim 6, wherein all or part of the uridine nucleosides in the mRNA are replaced by pseudouridine, preferably N1-methylpseudouridine; Preferably, the SEQ ID NO: 7 All or part of the uridine nucleosides in the nucleic acid sequence are replaced by pseudouridine, preferably N1-methylpseudouridine; more preferably, all the uracil nucleosides in the nucleic acid sequence shown in SEQ ID NO: 7 Replaced by N1-methylpseudouridine.
The mRNA vaccine according to any one of claims 1-7, wherein the S protein includes the amino acid sequence shown in SEQ ID NO: 3. Preferably, the amino acid sequence of the S protein is as shown in SEQ ID NO: 3. Show.
The mRNA vaccine according to any one of claims 1 to 8, wherein the delivery carrier includes one of cationic liposomes, cationic proteins, cationic polymers or cationic lipid nanoparticles, preferably cationic lipid nanoparticles. Particles.
The mRNA vaccine according to claim 9, wherein the cationic lipid nanoparticles comprise one or more of protonatable cationic lipids, auxiliary lipids, structural lipids and PEG-lipids.
The mRNA vaccine according to claim 10, wherein the protonatable cationic lipid is selected from the group consisting of compound (I), SM102, ALC-0315, Dlin-MC3-DMA, Dlin-KC2-DMA, DODMA, c12-200 and One or more of DlinDMA is preferably compound (I), wherein the structure of compound (I) is as follows:
The mRNA vaccine according to claim 10 or 11, wherein the auxiliary lipid is selected from one or more of DSPC, DOPE, DOPC, DOPS, DSPG, DPPG, DPPC, DGTS and lysophospholipids, preferably selected from One or more of DSPC, DOPE, DOPC and DOPS, more preferably DSPC.
The mRNA vaccine according to any one of claims 10-12, wherein the structural lipid is selected from the group consisting of cholesterol, cholesterol esters, sterol hormones, sterol vitamins, bile acids, cholesterol, ergosterol, β - one or more of sitosterol and oxidized cholesterol derivatives, preferably cholesterol (CHO-HP).
The mRNA vaccine according to any one of claims 10-13, wherein the PEG-lipid is selected from DMG-PEG and DSPE-PEG, preferably DMG-PEG; preferably, the PEG in the DMG-PEG The average molecular weight is about 2000 to about 5000 Daltons; the DMG-PEG is preferably M-DMG-2000.
The mRNA vaccine according to any one of claims 10 to 14, wherein based on the total amount of protonatable cationic lipids, auxiliary lipids, structural lipids and PEG-lipids, in molar percentage,

The lipid nanoparticles comprise 25-75% protonatable cationic lipids, preferably 45%-55%, more preferably 49.5%;

The lipid nanoparticles contain 5-20% auxiliary lipid, preferably 8%-12%, more preferably 10%;

The lipid nanoparticles comprise 0-50% structural lipid, preferably 35%-45%, more preferably 39%; and/or

The lipid nanoparticles comprise 0.5-5% PEG-lipid, preferably 1.0%-3.0%, more preferably 1.5%.
The mRNA vaccine according to any one of claims 10-15, wherein the mass ratio of protonatable cationic lipids and mRNA in the mRNA vaccine is (5-30):1, preferably 5:1, 10: 1, 15:1, 20:1, 25:1 or 30:1, preferably (8-12):1, more preferably 10:1.
The mRNA vaccine according to any one of claims 1-16, wherein the mRNA vaccine also contains a buffer, preferably, the buffer includes phosphate buffer or Tris buffer, preferably phosphate buffer; preferably Preferably, the buffer concentration is 5mmol/L-30mmol/L, preferably 10mmol/L.
The mRNA vaccine according to any one of claims 1-17, wherein the mRNA vaccine also contains a cryoprotectant; preferably, the cryoprotectant is selected from sucrose or trehalose, preferably sucrose; more preferably, The concentration of the cryoprotectant is 5 mg/mL-100 mg/mL, preferably 80 mg/mL.
The mRNA vaccine according to any one of claims 1 to 18, wherein the administration method of the mRNA vaccine includes intravenous injection, intramuscular injection or intradermal injection.
Use of the mRNA vaccine according to any one of claims 1-19 in the preparation of products for preventing or treating novel coronavirus infection.
The method for preparing the mRNA vaccine according to any one of claims 1 to 19 is characterized in that it includes the following steps:

(1) Prepare an aqueous phase containing mRNA;

(2) Formulating an organic phase containing one or more of protonatable cationic lipids, auxiliary lipids, structural lipids, and PEG-lipids;

(3) Mix and encapsulate appropriate amounts of the aqueous phase and the organic phase;

(4) Replace the solution in the two-phase mixture with another buffer containing a cryoprotectant; and

(5) Sterilize and filter to obtain the mRNA vaccine.