mRNA vaccine against rabies viruses and preparation method therefor

Abstract

The present application belongs to the technical field of biological medicines, and particularly relates to a mRNA vaccine against rabies viruses and a preparation method therefor, the nucleotide sequence of said vaccine being shown as any one of SEQ ID NO. 1-3. The mRNA vaccine obtained by the present application can generate an neutralizing antibody in a high level, has broad-spectrum neutralizing activity on representative strains of seven pandemic populations of rabies viruses in China, effectively induces B cell immunity and T cell immunity, and provides good protection effects for mice challenged with viruses in pre-exposure and post-exposure immune tests. In addition, the vaccine can be preserved stably for a long period under a condition of -20℃, thereby satisfying the requirements of use in countries and regions of various economic conditions.

Description

An mRNA vaccine against rabies virus and its preparation method Technical Field

[0001] The present application belongs to the field of biomedicine technology, and specifically relates to a rabies virus mRNA vaccine and a preparation method thereof. Background Art

[0002] Rabies virus (RABV) belongs to the genus Lyssavirus in the family Rhabdoviridae and is a single-stranded RNA virus. It has a bullet-shaped appearance, a helical nucleocapsid, an envelope, and contains single-stranded negative-strand RNA. The rabies virus genome is approximately 12 kilobases (KB) in size and primarily encodes five proteins: a glycoprotein (G protein), a nucleoprotein, a phosphoprotein, a matrix protein, and a transcriptase protein. The rabies virus relies on the G protein on its membrane to bind to acetylcholine receptors on the surface of nerve cells and invade the cells, causing severe damage to the central nervous system of humans or animals, ultimately leading to the death of the host. Research indicates that the rabies virus can infect a variety of mammals. Approximately 95% of human infections are caused by dog ​​bites, but other hosts include coyotes, skunks, and bats. According to recent data, 60,000 people die annually from the disease, or approximately one death every 10 minutes.

[0003] The complete rabies virus genome can express multiple proteins, including glycoprotein (Glycoprotein, G protein), nucleocapsid protein (N protein), phosphoprotein (P protein), matrix protein (M protein), and transcriptase protein. However, only the G protein is considered to be a protein that can induce the body to produce neutralizing antibodies, and the immune effect caused by the G protein is equivalent to that of the whole virus. Moreover, the G protein has an important function in the physiological cycle of the rabies virus: the G protein can form a homotrimeric spike structure on the rabies virus shell. This hollow structure allows signal molecules and genetic material to pass through, which is an important condition for the virus to uncoat. Therefore, the G protein is generally selected as the preferred antigen protein for the vaccine.

[0004] Specifically, the G protein is 1575 base pairs (bp) long and contains an open reading frame (ORF) and 524 amino acid residues. The mature G protein contains 505 amino acid residues, and the remaining 19 amino acid residues constitute the G protein signal peptide, which is removed by proteases during the maturation process. The G protein is presented on the surface of viral particles as a trimer of spikes. Each particle has approximately 1600 to 1900 spikes measuring 10 nm. Structurally, the G protein can be divided into an extracellular domain (amino acid residues 1 to 439), a transmembrane domain (amino acid residues 440 to 461), and an intracellular domain (amino acid residues 462 to 505). These domains play important roles in receptor recognition, membrane fusion, and stabilizing the spatial conformation of the G protein. The extracellular region contains three important antigenic determinants: the G1 region, the G2 region, and the G3 region. The G1 region contains both conformational and linear epitopes; the G2 region contains two discontinuous linear conformational epitopes; and the G3 region contains a conformational epitope that contains both B and T cell recognition sites. G proteins can induce specific immune responses in host cells and stimulate the proliferation of cytotoxic T lymphocytes (CTLs) and helper T cells.

[0005] Because the rabies virus has a strong affinity for nervous tissue, the mortality rate is almost 100% once infection occurs, and there is currently no effective treatment. Furthermore, my country has the second highest rabies incidence rate, after India. Due to the increasing number of pet dogs and a lack of awareness of rabies prevention, rabies continues to pose a serious threat to the lives, health, and safety of the Chinese people. In summary, vaccination remains the most effective method for preventing rabies, and the development of a more effective and less expensive rabies vaccine continues to have significant social and economic value.

[0006] Currently, the types of rabies vaccines approved for human use in my country include Vero cell-purified vaccines, human diploid cell vaccines, and hamster kidney primary cell-purified vaccines. The main advantages of these vaccines are: relatively mature technology, low incidence of adverse reactions, and good immune efficacy. However, their disadvantages are also very obvious, mainly including:

[0007] 1. There are safety issues with attenuated vaccines, and incomplete attenuation poses a risk of infection.

[0008] 2. The traditional vaccine preparation process is complex and requires contact with live viruses during the production process, which poses certain safety risks.

[0009] 3. The traditional vaccine production process involves a cell culture step, which requires high quality control and process amplification technology, and there is a risk of causing product quality accidents.

[0010] 4. The effective antigen amount produced by traditional vaccines is low. In order to ensure the effectiveness, it is often necessary to increase the virus titer, which increases the production cost.

[0011] In contrast, mRNA vaccines have unique advantages:

[0012] 1. mRNA vaccines are a non-infectious, non-integrating platform that does not directly inject intact viral genetic material or viral particles into the body. There is no potential risk of confirmed infection or insertional mutations. They are rapidly metabolized and degraded by normal cells, therefore ensuring safety.

[0013] 2. mRNA vaccines can effectively activate T cell immunity and B cell immunity, and are more effective than inactivated vaccines;

[0014] 3. The product design and production process of mRNA vaccines are relatively simple, and the difficulty and cost of large-scale production are relatively low;

[0015] Currently, some rabies mRNA vaccines have entered the clinical research stage. For example, CureVac AG has developed an mRNA vaccine CV7201 for rabies virus, but the clinical trials of this product have been suspended. CureVac AG's other product CV7202 (NCT03713086) has also not progressed smoothly. As can be seen, there are currently very few mRNA vaccine products for rabies virus vaccines.

[0016] In summary, the rabies vaccines currently available on the market have a cumbersome immunization procedure, require many injections, and have poor patient compliance. This creates an unmet medical need for a safe and effective rabies vaccine that is more affordable, faster to prepare, and more affordable than currently available vaccines. Furthermore, existing mRNA vaccines targeting rabies virus are extremely scarce, and there is an urgent need for more effective mRNA vaccines with fewer side effects.

[0017] In order to overcome the defects of the existing technology, the inventors of this patent, after years of research, selected the rabies virus-encoded protein G as the antigen, and developed an mRNA vaccine by optimizing the design of the mRNA sequence. This mRNA vaccine can effectively prevent rabies virus infection in animals, and can simultaneously induce humoral immunity and cellular immunity. High concentrations of neutralizing antibodies still exist in the body for up to one year, and it has long-term immune protection against rabies virus, achieving unexpected technical effects.

[0018] Summary of the Invention

[0019] In a first aspect, the present application provides an immune composition (e.g., an mRNA vaccine) comprising RNA encoding a highly immunogenic antigen capable of eliciting an effective neutralizing antibody response against rabies virus antigens (e.g., glycoprotein G).

[0020] In some embodiments, the mRNA comprises a coding region encoding at least one antigenic peptide or protein derived from rabies virus glycoprotein G (RAV-G or RABV-G), nucleoprotein N (RAV-N), phosphorylated protein P (RAV-P), matrix protein M (RAV-M) or RNA polymerase L (RAV-L) or a fragment or variant thereof.

[0021] In some embodiments, the mRNA comprises a coding region that encodes all or part of SEQ ID NO. 4 or an amino acid sequence that is 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 4.

[0022] In a second aspect, the present application provides a polynucleotide (e.g., mRNA) encoding at least one antigenic peptide or protein derived from rabies virus or an immunogenic fragment or immunogenic variant thereof, wherein the nucleotide comprises at least one heterologous untranslated region (UTR) having a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-3.

[0023] In some embodiments, the polynucleotides are suitable for use in the production of vaccines.

[0024] In some embodiments, the at least one antigenic peptide or protein comprises or consists of at least one protein derived from a structural protein, an accessory protein, or a replication protein, or a fragment or immunogenic variant of any of the foregoing.

[0025] In some embodiments, the antigenic peptide or protein is selected from at least one antigenic peptide or protein of rabies virus glycoprotein G (RAV-G or RABV-G), nucleoprotein N (RAV-N), phosphorylated protein P (RAV-P), matrix protein M (RAV-M) or RNA polymerase L (RAV-L) or fragments or variants thereof.

[0026] In some embodiments, the polynucleotide of the present application may comprise or consist of a nucleotide sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NO. 1, 2, or 3.

[0027] In some embodiments, at least one coding region of the polynucleotide of the present application encodes one or more mutations, and the coding region includes a nucleotide sequence optimized for G / C content.

[0028] In some embodiments, the heterologous untranslated regions (UTRs) include at least one heterologous 3'UTR and / or 5'UTR.

[0029] In some embodiments, the polynucleotide comprises at least the following structure:

[0030] (a) 5'-cap structure;

[0031] (b) a 3' polyadenylation sequence, preferably 50-200 A, more preferably 80-200 A;

[0032] (c) a 5'UTR of 10-200 nucleotides, preferably 15-150 nucleotides in length, preferably alpha-1-globin 5'UTR, KOZAK sequences;

[0033] (d) 3'UTR, preferably comprising human gp130, DH143, hHBB and hHBA1 3'UTR sequences;

[0034] (e) The optimized mRNA sequence has a higher GC base content than the natural sequence (wild type).

[0035] In some embodiments, the 3'UTR can also be selected from the 3'UTR of PSMB3, ALB7, α-globin, CASP1, COX6B1, GNAS, NDUFA1, DH143, gp130, hHBB, hHBA1, CYBA (cytochrome b-245 alpha chain), rabbit β-globin, hepatitis B virus (HBV), VEEV (Venezuelan equine encephalitis virus) virus, rps9 (Ribosomal Protein S9), FIG4 (FIG4 Phosphoinositide 5-Phosphatase), human albumin hHBB (human hemoglobin subunit beta), HBA1 (human Hemoglobin Subunit Alpha 1), or a homolog, fragment or variant from any of these genes.

[0036] In some embodiments, the 5'UTR is selected from the group consisting of African Xenopus or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, and tobacco etch virus, alpha-1-globin, HSD17B4, RPL32, ASAH1, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B, and UBQLN2 5'UTR, or a homolog, fragment, or variant from any of these genes.

[0037] In some embodiments, the KOZAK sequence is shown as SEQ ID NO.5.

[0038] In some embodiments, the alpha-1-globin 5'-UTR sequence is the nucleotide sequence shown in SEQ ID NO.6.

[0039] In some embodiments, the 3'UTR sequence comprises the nucleotide sequence shown in SEQ ID NO: 8 and / or 9.

[0040] In some embodiments, the 3'-poly(A) sequence is preferably 60-120 A, more preferably 80-110 A, and even more preferably 100 A, such as SEQ ID NO.7.

[0041] In some embodiments, the mRNA further comprises a 5' guanosine cap selected from: m7Gppp(2′OMeA)pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7Gpppm6ApG, m7G3’OmepppApA, m7G3’OmepppApC, m7G3’OmepppApU, m7G3’OmepppApG, m7G3’OmepppCpA, m7G3’OmepppCpC, m7G3’OmepppCpG, m7G3’OmepppCpU, m7G3’OmepppUpA, m7G3’OmepppUpC, m7G3’OmepppUpG, m7G3’OmepppUpU, m7G3’OmePppA2’OmepG, m7G3’OmepppA2’OmepC, m7G3’OmepppA2’OmepU, m7G3’OmepppA2’OmepA, m7G3’OmepppC2’OmepA, m7G3’OmepppC2’OmepU, m7G3’OmepppC2’OmepG, m7G3’OmepppC2’OmepC, m7G3’OmepppG2’OmepA, m7G3’OmepppG2’OmepU, m7G3’OmepppG2’OmepG, m7G3’OmepppG2’OmepC, m7G3’OmepppU2’OmepA, m7G3’OmepppU2’OmepU, m7G3’OmepppU2’OmepG, m7G3’OmepppU2’OmepC.

[0042] In some embodiments, the mRNA comprises chemically modified bases or analogs, including 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), and pseudouridine; preferably, 1-methyl pseudouridine is used for chemical modification; preferably, 1-methyl pseudouridine is used to replace uracil by 70%, 80%, 85%, 90%, 95%, 99% or 100%, and most preferably, 1-methyl pseudouridine is used to completely replace uracil, that is, natural uracil is not used, and 1-methyl pseudouridine is used 100%.

[0043] In some embodiments, the aforementioned polynucleotide is DNA or RNA.

[0044] In some embodiments, the aforementioned polynucleotide encodes RNA.

[0045] In some embodiments, the coding RNA is mRNA, self-replicating RNA, circular RNA, or replicon RNA, preferably mRNA.

[0046] In some embodiments, the mRNA comprises at least one 3'-poly(A) sequence comprising 30 to 200 adenosine nucleotides and the 3' terminal nucleotide is adenosine.

[0047] In some embodiments, the mRNA preferably comprises a 5′-cap structure, and the 5′-cap structure is preferably m 7 G, cap0, cap1, cap2, modified cap0 or modified cap1 structure, preferably cap1 structure, most preferably m7G(5′)ppp(5′)(2′OMeA)pG.

[0048] In a third aspect, the present application provides a composition, preferably an immunogenic composition comprising at least one nucleotide of the first aspect. Suitably, the composition may comprise at least one nucleotide, such as at least one encoding RNA, the nucleic acid being complexed with one or more lipids, encapsulated in one or more lipids, or associated with one or more lipids to form lipid nanoparticles.

[0049] In some embodiments, the present application relates to a rabies virus nucleic acid vaccine, characterized in that the vaccine carrier is a lipid nanoparticle (LNP), comprising an ionizable cationic lipid, a structural lipid, a helper lipid and a surfactant. In some embodiments, the molar content of the ionizable cationic lipid, the structural lipid, the helper lipid and the surfactant is 100% in total, calculated by mole percentage (mol%);

[0050] In some embodiments, the lipid nanoparticles comprise 20-60 mol% ionizable cationic lipid, 25-55 mol% structural lipid, 5-25 mol% helper lipid, and 0.5-15 mol% surfactant.

[0051] In some embodiments, the cationic lipid is selected from SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA (also known as MC3), DODMA, DLin-KC2-DMA, DlinDMA, preferably SM-102; SM-102 (Heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy) hexyl)amino)octanoate) has the following structure:

[0052] In some embodiments, the structured lipid comprises cholesterol, and cholesterol derivatives, preferably cholesterol.

[0053] In some embodiments, the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC.

[0054] In some embodiments, the surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

[0055] In some embodiments, the lipid nanoparticles comprise 20-50 mol% ionizable cationic lipids. For example, the lipid nanoparticles may comprise 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mol% ionizable cationic lipids.

[0056] In other embodiments, the lipid nanoparticles comprise 50-60 mol% ionizable cationic lipids. For example, the lipid nanoparticles may comprise 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 mol% ionizable cationic lipids.

[0057] In some embodiments, the lipid nanoparticle comprises 5-25 mol% DSPC; for example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 4, or 15 mol% DSPC.

[0058] In some embodiments, the lipid nanoparticle comprises 25-55 mol% cholesterol.For example, the lipid nanoparticle may comprise 30, 31, 32, 33, 34, 35, 36, 37, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.

[0059] In some embodiments, the lipid nanoparticles comprise 0.5-15 mol% DMG-PEG. For example, the lipid nanoparticles may comprise 0.5, 1, 1.5, or 2 mol% DMG-PEG.

[0060] In some embodiments, the lipid nanoparticles comprise 50 mol% ionizable cationic lipid, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0061] In some embodiments, the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% PEG2000-DMG.

[0062] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 2:1 to about 30:1.

[0063] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 6:1.

[0064] In some embodiments, the lipid nanoparticles of the present application comprise an N:P ratio of about 3:1.

[0065] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 10:1 to about 100:1.

[0066] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 20:1.

[0067] In some embodiments, the lipid nanoparticles of the present application comprise a wt / wt ratio of the ionizable cationic lipid component to the RNA of about 10: 1. In some embodiments, the lipid nanoparticles of the present application have an average diameter of from about 50 nm to about 150 nm.

[0068] In some embodiments, the lipid nanoparticles of the present application have an average diameter of about 70 nm to about 120 nm, preferably 100-120 nm, and most preferably 100 nm.

[0069] In some embodiments, the rabies virus nucleic acid vaccine of the present application further comprises: a buffer component and a cryoprotectant.

[0070] In some embodiments, the buffer can be selected from: Examples of buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium gluconate, calcium gluconate, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium levulinate, valeric acid, dibasic calcium phosphate, phosphoric acid, tricalcium phosphate, dibasic calcium phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, tromethamine, dibasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate, magnesium hydroxide, aluminum hydroxide, alginic acid, citric acid, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, lauryl magnesium sulfate, sodium lauryl sulfate, and combinations thereof.

[0071] In some embodiments, the cryoprotectant can be selected from substances such as sugars / polyols, polymers, surfactants, amino acids and salts, wherein the sugar can be selected from lactose, sucrose, trehalose, galactose and the like.

[0072] In some embodiments, the amount of the cryoprotectant is 1 to 50% w / w, such as from 2 to 50% w / w, or from 4 to 45% w / w, or from 6 to 12% w / w, or preferably from 6 to 10% w / w, or most preferably from 7 to 9% w / w.

[0073] In some embodiments, the pharmaceutical composition of the present application comprises the aforementioned lipid nanoparticle composition and an external phase buffer.

[0074] In some embodiments, the aqueous buffer comprises: tromethamine, sodium acetate, sucrose, pH=7-8.

[0075] In some embodiments, the content of tromethamine is selected from 10-30mmol / L, preferably 15-25mmol / L, preferably 15mmol / L, 15.5mmol / L, 16mmol / L, 16.5mmol / L, 17mmol / L, 17.5mmol / L, 18mmol / L, 18.5mmol / L, 19mmol / L, 19.5mmol / L, 20mmol / L, 20.5mmol / L, 21mmol / L, 21.5mmol / L, 22mmol / L, 22.5mmol / L, 23mmol / L, 23.5mmol / L, 24mmol / L, 24.5mmol / L, 25mmol / L, and most preferably 20mmol / L.

[0076] In some embodiments, the content of sodium acetate is selected from 0-20mmol / L, preferably 5-11mmol / L, preferably 5mmol / L, 5.5mmol / L, 6mmol / L, 6.5mmol / L, 7mmol / L, 7.5mmol / L, 8mmol / L, 8.5mmol / L, 9mmol / L, 9.5mmol / L, 10mmol / L, 10.5mmol / L, 10.6mmol / L, 10.7mmol / L, 10.8mmol / L, 10.9mmol / L, 11mmol / L, 11.5mmol / L, 12mmol / L, 12.5mmol / L, 13mmol / L, and most preferably 10.7mmol / L.

[0077] In some embodiments, the sucrose content (w / w) is selected from: 5-15%, preferably 7.5-10%, more preferably 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 9%, 9.5%, 10%, and most preferably 8.7%.

[0078] In some embodiments, the pharmaceutical composition of the present application includes an mRNA having a sequence as shown in SEQ ID NO.1, a lipid nanoparticle composition, 20 mmol / L tromethamine, 10.7 mmol / L sodium acetate, 8.7% sucrose, pH 7.0-8.0, and the lipid nanoparticles contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG2000-DMG, N:P=6.

[0079] In a fourth aspect, the present application provides a rabies virus nucleic acid vaccine comprising a nucleic acid sequence encoding a rabies virus protein or a fragment thereof;

[0080] In some embodiments, the rabies virus proteins include but are not limited to: glycoprotein (G protein), nucleocapsid protein (N protein), phosphoprotein (P protein), matrix protein (M protein), and transcriptase protein.

[0081] In some embodiments, the nucleic acid vaccine comprises a sequence as shown in SEQ ID NO.1-3 or a nucleotide sequence that is at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to SEQ ID NO.1-3.

[0082] In some embodiments, the nucleic acid vaccine comprises a nucleotide sequence encoding SEQ ID NO. 4 or having at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity thereto.

[0083] In some embodiments, the nucleic acid includes but is not limited to DNA, RNA, preferably mRNA.

[0084] In some embodiments, the vaccine is administered by intravenous injection, intramuscular injection or subcutaneous injection, preferably intramuscular injection.

[0085] In some embodiments, the dosage form of the vaccine can be selected from lyophilized powder injection, liquid injection, and inhalation preparation.

[0086] In a fifth aspect, the present application relates to a method for preparing a nucleic acid vaccine, which comprises mixing a vaccine vector and mRNA to obtain a nucleic acid vaccine.

[0087] In some embodiments, the vaccine carrier is a cationic lipid nanoparticle, and the preparation method specifically comprises the following steps:

[0088] (1) dissolving a protonable cationic lipid, a structural lipid, an auxiliary lipid, and a surfactant in an organic solution according to a formula ratio to obtain an organic phase;

[0089] (2) dissolving the optimized mRNA in citrate buffer or sodium acetate solution to obtain an aqueous phase;

[0090] (3) The organic phase of step (1) and the aqueous phase of step (2) are mixed to form a mixed solution to obtain a rabies virus vaccine.

[0091] In some embodiments, the organic solution comprises anhydrous ethanol.

[0092] In some embodiments, the total concentration of the protonatable cationic lipid, structural lipid, helper lipid, and surfactant in the organic phase is 10-15 mg / ml.

[0093] In some embodiments, the concentration of the mRNA is 0.01-1 mg / ml, preferably 0.1-0.2 mg / ml.

[0094] In some embodiments, the volume ratio of the organic phase to the aqueous phase is 1:2-4.

[0095] In some embodiments, the mixing is performed using a microfluidic device with a flow rate controlled to be ≥12 ml / min.

[0096] In a sixth aspect, the present application also relates to the use of the mRNA or the composition in preparing a vaccine.

[0097] In some embodiments, the vaccines include combination vaccines and multivalent vaccines.

[0098] In some embodiments, the vaccine is a rabies virus mRNA vaccine.

[0099] In a seventh aspect, the present invention also relates to a method for pre-exposure or post-exposure immunization, comprising administering to a subject the composition of claims 7-16, which can effectively induce an immune response against rabies virus antigens in the subject.

[0100] In an eighth aspect, the present invention also relates to the nucleic acid molecule corresponding to the mRNA (DNA sequence encoding the mRNA).

[0101] In some embodiments, the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO.10.

[0102] In one embodiment, the present invention also relates to biological materials of said nucleic acid molecules.

[0103] In one embodiment, the biological material includes: an expression cassette, a recombinant vector, a recombinant plasmid, a transgenic cell line, etc. containing the nucleic acid molecule.

[0104] In one embodiment, the recombinant vector is preferably: pVAX.1 vector, pcDNA3.1 vector.

[0105] In the first to eighth aspects, the rabies virus strains include but are not limited to: Pasteur strain (PAS), Flury strain, SAD strain, aG strain, CTN strain, SC16 (CSC1016D), GD1 (GDZAQ45), NM3 (CNM1103C), QH2 (CQH1202D), LY (CNX8601), YN3 (CYN1009D), XZ17 (CXZ1704H) and their derivatives; preferably CTN strain, most preferably CTN-1 strain.

[0106] Compared with the existing technology, the anti-rabies virus mRNA vaccine of the present invention has the following characteristics:

[0107] 1. Taking into account the prevalence of local rabies virus, the CTN-1 strain was selected as the starting strain for the development of mRNA vaccine;

[0108] 2. In order to achieve the best expression effect and stability of the mRNA sequence in vivo, we optimized the nucleotide coding sequence of the G protein of the CTN-1 strain, including: adjusting the codon preference for expression in the human body, adjusting the frequency of use of commonly used codons, increasing the GC content of the sequence, etc., and screening the optimized nucleotide sequence to finally determine the optimal sequence. The nucleotide sequence obtained in this application makes the transcribed mRNA structure more stable, and the translation efficiency of the target protein in mammals and humans is higher. It can effectively produce neutralizing antibodies, and induce the body to produce humoral and cellular immunity, and produce memory B cells and T cells; using a smaller dose can induce animals to produce a protective effect against multiple strains of rabies virus and its mutants.

[0109] 3. The present invention uses LNP to deliver mRNA. The mRNA has good stability in LNP. After storage at a relatively high temperature of 5±3°C for 12 weeks, all test items are maintained at a relatively high level. Similarly, at -20°C, all test indicators are also very good, and the vaccine efficacy does not decrease significantly after storage, which can meet the storage requirements for large-scale use of mRNA vaccines.

[0110] definition

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as understood by those of ordinary skill in the art. For definitions and terminology in this field, professionals are specifically referred to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0112] Although the numerical ranges and parameter approximations shown in the broad scope of this application, the numerical values ​​shown in the specific examples are recorded as accurately as possible. However, any numerical value is necessarily contained in a certain error, which is caused by the standard deviation present in their respective measurements. In addition, all ranges disclosed herein should be understood to cover any and all sub-ranges contained therein. For example, a range of "1 to 10" should be considered to include any and all sub-ranges between a minimum of 1 and a maximum of 10 (including endpoints); that is, all sub-ranges starting with a minimum of 1 or greater, such as 1 to 6.1, and sub-ranges ending with a maximum of 10 or less, such as 5.5 to 10. In addition, any reference referred to as "incorporated herein" should be understood to be incorporated in its entirety.

[0113] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, many different polynucleotides can encode the same polypeptide. It will also be understood that the skilled artisan can use conventional techniques to make nucleotide substitutions that do not affect the polypeptide sequence encoded by the nucleic acid molecule to reflect the codon usage of any particular host organism in which the polypeptide is expressed. Therefore, unless otherwise indicated, "polynucleotides encoding the protein or immunogenic fragment of the present application" include all polynucleotide sequences that are degenerate to each other and encode the same amino acid sequence.

[0114] antigen

[0115] Antigen used herein is a protein that can induce an immune response (for example, causing the immune system to produce antibodies to the antigen). In this article, unless otherwise indicated, the use of the term "antigen" includes immunogenic proteins and immunogenic fragments (immunogenic fragments that induce or can induce an immune response to at least one rabies virus). It should be understood that the term "protein" includes peptides and the term "antigen" includes antigenic fragments. Other molecules may also be antigenic, such as bacterial polysaccharides or a combination of protein and polysaccharide structures, and viral vaccine antigens as described herein include viral proteins, viral protein fragments, and proteins designed and / or mutated from rabies virus.

[0116] Furthermore, the term "antigen," as used herein, will be recognized and understood by those of ordinary skill in the art to mean a substance that can be recognized by the immune system, preferably the adaptive immune system, and that is capable of triggering an antigen-specific immune response, for example, by the formation of antibodies and / or antigen-specific T cells as part of an adaptive immune response. An antigen can be or include a peptide or protein that can be presented to T cells by MHC. This also includes fragments, variants, and derivatives derived from, for example, a peptide or protein.

[0117] Antigenic peptide or protein: The term "antigenic peptide or protein" or "immunogenic peptide or protein" will be recognized and understood by those of ordinary skill in the art; it refers to a peptide or protein derived from a (antigenic or immunogenic) protein that stimulates the body's adaptive immune system to provide an adaptive immune response. Thus, an antigenic / immunogenic peptide or protein encompasses the protein from which it is derived.

[0118] As will be appreciated and understood by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of rabies virus antigens of interest. For example, any protein fragment of rabies virus or a mutant thereof, provided that the fragment is immunogenic and confers a protective immune response against rabies virus; in addition to variants that are identical to a reference protein but truncated, in some embodiments, the antigen comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations, and the length of the antigen / antigenic polypeptide may range from about 4, 6, or 8 amino acids to the full-length protein.

[0119] Epitope

[0120] Epitope: The term "epitope" (also referred to in the art as "antigenic determinant") as used herein will be recognized and understood by those of ordinary skill in the art to mean T cell epitopes and B cell epitopes. A T cell epitope is a portion of an antigenic peptide or protein and may include fragments preferably having a length of about 6 to about 20 or more amino acids, for example. A fragment processed and presented by an MHC class I molecule preferably has a length of about 8 to about 10 amino acids, for example: 8, 9 or 10 (or 11 or 12 amino acids) or a fragment processed and presented by an MHC class II molecule preferably has a length of about 13 to about 20 or more amino acids. These fragments are typically recognized by T cells in the form of a complex consisting of a peptide fragment and an MHC molecule, i.e., these fragments are not typically recognized in their native form. A B cell epitope is typically a fragment located on the outer surface of a (native) protein or peptide antigen, preferably having 5 to 15 amino acids, more preferably 5 to 12 amino acids, even more preferably 6 to 9 amino acids, which can be recognized by an antibody, i.e., recognized in its native form. Such epitopes of proteins or peptides may also be selected from any variants of such proteins or peptides mentioned herein. In the present context, an epitope may be a conformational or discontinuous epitope, consisting of fragments of a protein or peptide as defined herein that are discontinuous in the amino acid sequence of the protein or peptide as defined herein but are aggregated together in a three-dimensional structure or a continuous or linear epitope consisting of a single polypeptide chain.

[0121] Nucleic Acids

[0122] The term "nucleic acid" or "nucleic acid molecule" will be recognized and understood by those of ordinary skill in the art. As used herein, the term "nucleic acid" or "nucleic acid molecule" preferably refers to a DNA (molecule) or RNA (molecule). It is preferably used synonymously with the term polynucleotide. Preferably, a nucleic acid or nucleic acid molecule is a polymer comprising or consisting of nucleotide monomers, which are covalently linked to each other via phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid molecule" also includes modified nucleic acid molecules, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules as defined herein.

[0123] Specifically, the compositions disclosed herein comprise (at least one) RNA having an open reading frame (ORF) encoding a rabies virus antigen. In some embodiments, the RNA is messenger RNA (mRNA).

[0124] In some embodiments, the nucleic acid comprises at least one heterologous untranslated region (UTR). The term "untranslated region" or "UTR" or "UTR element" will be recognized and understood by those of ordinary skill in the art to mean a portion of a nucleic acid molecule, typically located at the 5' or 3' end of a coding sequence. The 5' end is referred to as a 5'UTR, and the 3' end is referred to as a 3'UTR. Generally speaking, UTRs are not translated into proteins; UTRs can be part of a nucleic acid, such as DNA or RNA. UTRs can comprise elements for controlling gene expression, also referred to as regulatory elements. Such regulatory elements can be ribosome binding sites, miRNA binding sites, etc.; RNA (e.g., mRNA) can further comprise a 5'UTR, a 3'UTR, a 3'-poly(A) and / or a 5' cap analog.

[0125] In some embodiments, the 5'UTR is a heterologous UTR, i.e., a UTR found in nature that is associated with a different ORF; in another embodiment, the 5'UTR is a synthetic UTR; the 5'UTR is a region of the mRNA that is located upstream (5') of the start codon (the first codon of the mRNA transcript translated by the ribosome). The 5'UTR does not encode a protein. The natural 5'UTR has characteristics that play a role in translation initiation, such as the Kozak sequence, which has a consensus CCR(A / G)CCAUGG; exemplary 5'UTRs also include African clawed frog or human α-globin or β-globin, human cytochrome b-245a polypeptide, hydroxysteroid (17b) dehydrogenase, tobacco etch virus, alpha-1-globin 5'UTR, and the like.

[0126] In some embodiments, the 3'UTR can be heterologous or synthetic; for example, globin UTRs, including African clawed frog β-globin UTRs and human β-globin UTRs; other 3'UTRs can also be CYBA (cytochrome b-245 alpha chain), rabbit β-globin, hepatitis B virus (HBV), α-globin 3'UTR, and VEEV (Venezuelan equine encephalitis virus) virus 3'UTR sequences. In some embodiments, rps9 (Ribosomal Protein S9) 3'UTR, FIG4 (FIG4 Phosphoinositide 5-Phosphatase), gp130, DH143, and human albumin hHBB (human hemoglobin subunit beta), HBA1 (human Hemoglobin Subunit Alpha 1) 3'UTRs can also be used.

[0127] In some embodiments, the 3'-poly (A) tail is also called a poly (A) tail; the poly (A) tail is located downstream of the 3' UTR, for example, the mRNA region immediately downstream (i.e., 3'), which contains multiple consecutive adenosine monophosphates. The poly (A) tail may contain 10 to 300 adenosine monophosphates, and may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some preferred embodiments, the poly (A) tail contains 50 to 250 adenosine monophosphates, more preferably 50-100 adenosine monophosphates; most preferably 100 adenosine monophosphates; in relevant biological environments (e.g., in cells, in vivo), the function of the 3'-poly (A) tail is to protect the mRNA from enzymatic degradation, for example in the cytoplasm, and to facilitate transcription termination and / or export of the mRNA from the nucleus and translation.

[0128] In some embodiments, the RNA (e.g., mRNA) further comprises a 5' guanosine cap; the 5' guanosine cap is a eukaryotic mRNA transcript, and the 5' cap is composed of an inverted 7-methylguanosine, connected to the rest of the eukaryotic mRNA via a 5'-5' triphosphate bridge, namely cap 0 (cap 0), which primarily serves as a quality control for correct mRNA processing and helps stabilize the eukaryotic mRNA; based on cap 0, the first nucleotide is methylated with 2'-OH, referred to as cap 1 (cap 1); in addition to cap 0 and cap 1, the second nucleotide can also be further methylated, referred to as cap 2; generally speaking, the 5'-cap can be synthesized by different synthetic routes of 5'-capped mRNA based on enzymatic, chemical, or chemoenzymatic methods;

[0129] In some embodiments, during in vitro transcription, a cap analog is directly added to the in vitro transcription (IVT) system, and the 5' cap analog includes but is not limited to: m7Gppp(2'OMeA)pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7GpppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7Gpppm6ApG, m7G3'OmepppApA, m7G3'OmepppApC, m7G3'Ome pppApU, m7G3'OmepppApG, m7G3'OmepppCpA, m7G3'OmepppCpC, m7G3'OmepppCpG, m7G3'OmepppCpU, m7G3'OmepppUpA, m7G3'OmepppUpC, m7G3'Omep ppUpG, m7G3'OmepppUpU, m7G3'OmePppA2'OmepG, m7G3'OmepppA2'OmepC, m7G3'OmepppA2'OmepU, m7G3'OmepppA2'OmepA, m7G3'OmepppC2'OmepA, m7G3'OmepppC2'OmepU, m7G3'OmepppC2'OmepG, m7G3'OmepppC2'OmepC, m7G3'OmepppG2'OmepA, m7G3'OmepppG2'OmepU, m7G3'Omepp pG2'OmepG, m7G3'OmepppG2'OmepC, m7G3'OmepppU2'OmepA, m7G3'OmepppU2'OmepU, m7G3'OmepppU2'OmepG, m7G3'OmepppU2'OmepC, etc.

[0130] In some embodiments, the capped analogs may also be other structures, such as tetramers, pentamers, hexamers, heptamers, octamers, nonamers, or decamers, etc. The specific sequence thereof may be determined according to the conditions of the template.

[0131] It should also be understood that the rabies virus vaccine disclosed in the present application may include any 5' untranslated region (UTR) and / or any 3' untranslated region (UTR). Nucleic acids comprise polymers (nucleotide monomers) of nucleotides. Therefore, nucleic acids are also referred to as polynucleotides. Nucleic acids can be or can include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNAs), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0132] Messenger RNA (mRNA) is any RNA that encodes (at least one) protein (a naturally occurring, non-naturally occurring, or modified amino acid polymer) and can be translated in vitro, in vivo, in situ, or ex vivo to produce the encoded protein. The skilled artisan will understand that, unless otherwise indicated, the nucleic acid sequences listed in this application may reference "T" in the representative DNA sequence, but when the sequence represents RNA (e.g., mRNA), the "T" will be replaced with a "U." Therefore, any DNA disclosed and identified herein by a particular sequence identification number also discloses the corresponding RNA (e.g., mRNA) sequence complementary to the DNA, wherein each "T" of the DNA sequence is replaced by a "U."

[0133] open reading frame

[0134] An open reading frame (ORF) is a continuous stretch of DNA or RNA that begins with a start codon (e.g., methionine (ATG or AUG)) and ends with a stop codon (e.g., TAA, TAG, or TGA, or UAA, UAG, or UGA). Generally speaking, an ORF typically encodes a protein. It will be understood that the sequences disclosed herein may also contain additional elements, such as 5' and 3' UTRs, but unlike ORFs, these elements are not necessarily present in the RNA polynucleotides disclosed in this patent.

[0135] In some embodiments, the composition comprises RNA (e.g., mRNA) comprising the nucleotide sequence of any one of SEQ ID NOs. 1-6 having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identity.

[0136] In some embodiments, the open reading frame is preferably at least partially codon optimized. Codon optimization is based on such discovery: translation efficiency can be determined by the different frequencies of transfer RNA (tRNA) occurring in the cell. Therefore, if there is so-called "rare codon" of increasing degree in the coding region of the nucleic acid of the application defined herein, the translation of the corresponding modified nucleotide sequence is less efficient than when there is a codon encoding relatively "common" tRNA. Those skilled in the art can carry out codon optimization for sequence to be translated with the characteristics of its in vitro expression system.

[0137] Chemically modified or unmodified nucleotides

[0138] In some embodiments, RNA (e.g., mRNA) is not chemically modified, but rather comprises standard ribonucleotides consisting of adenosine, guanosine, cytidine, and uridine. In some embodiments, the nucleotides and nucleosides disclosed herein comprise standard nucleoside residues, such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, the nucleotides and nucleosides disclosed herein include standard deoxyribonucleosides, such as those present in DNA (e.g., dA, dG, dC, or dT);

[0139] In some embodiments, the compositions disclosed herein include RNA with an open reading frame encoding a rabies virus antigen, wherein the nucleic acid includes standard (unmodified) or modified nucleotides and / or nucleosides known in the art. In some embodiments, the nucleotides and nucleosides disclosed herein include modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally occurring modified nucleotides and nucleosides or non-naturally occurring modified nucleotides and nucleosides. Such modifications can include modifications of the sugar, backbone or core base moieties of nucleotides and / or nucleosides known in the art.

[0140] In some embodiments, the modified nucleic acid bases in a nucleic acid (e.g., an RNA nucleic acid, e.g., an mRNA nucleic acid) include 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methoxy-uridine, 5-methyl-cytidine, and pseudouridine.

[0141] In vitro transcription system (IVT)

[0142] In vitro transcription is the process of using DNA as a template in an in vitro cell-free system containing components such as RNA polymerase and NTP to mimic the in vivo transcription process to generate mRNA. Generally speaking, the capped RNA synthesized in the in vitro transcription reaction can be used for subsequent experiments such as microinjection, in vitro translation, and transfection. The in vitro transcription system usually includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. NTPs can be synthesized by oneself or selected from a supplier. NTPs can be natural or non-natural NTPs. Optional polymerases include, but are not limited to, phage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, and / or polymerase mutants thereof, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. Some embodiments exclude the use of DNA enzymes. In some embodiments, the RNA contains a 5′ guanosine cap.

[0143] In addition to synthesis by in vitro transcription systems, chemical synthesis methods can also be used, including solid-phase chemical synthesis and liquid-phase chemical synthesis; with respect to solid-phase chemical synthesis, the nucleic acids disclosed in this application can be prepared in whole or in part using solid-phase technology; solid-phase chemical synthesis of nucleic acids is an automated method in which molecules are fixed on a solid support and synthesized stepwise in a reactant solution. Solid-phase synthesis can be used for site-specific introduction of chemical modifications in nucleic acid sequences; with respect to liquid-phase chemical synthesis, the nucleic acids disclosed in this application can be synthesized in liquid phase by sequentially adding monomer constructs. In addition, the above-mentioned synthesis methods can also be used in combination, because the synthesis methods discussed above each have their own advantages and limitations, and attempts can be made to combine these methods to overcome the above-mentioned limitations. Combinations of these methods are within the scope disclosed in this application.

[0144] Antigenic variants

[0145] In some embodiments, the compositions disclosed herein comprise RNA encoding rabies virus antigen variants. Antigen variants or other polypeptide variants refer to molecules whose amino acid sequences are different from wild-type, natural or reference sequences. Compared to natural or reference sequences, antigen / polypeptide variants may have substitutions, deletions and / or insertions at certain positions within the amino acid sequence. Typically, the variant has at least 50% identity with the wild-type, natural or reference sequence. In some embodiments, the variant has at least 80% or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the wild-type, natural or reference sequence.

[0146] Variant antigens / polypeptides encoded by the nucleic acids disclosed herein can include amino acid changes that confer any of a variety of desired properties, for example, enhancing their immunogenicity, enhancing their expression and / or improving their stability or PK / PD properties. Conventional mutagenesis techniques can generally be used to prepare variant antigens / polypeptides, and analysis is performed as appropriate to determine whether they have the desired properties. Determination of expression levels and immunogenicity is well known in the art, and exemplary such determinations are described in the Examples section. Similarly, art-recognized techniques can be used to measure the PK / PD properties of protein variants, for example, by determining the expression of the antigen in the inoculated subject over time and / or by observing the persistence of the induced immune response. The stability of the protein encoded by the variant nucleic acid can be measured by measuring thermal stability or stability during urea denaturation, or can be measured using computer prediction. Methods for such experiments and computer determinations are known in the art.

[0147] Identity

[0148] The term "identity" refers to the relationship between the sequences of two or more polypeptides (e.g., antigens) or polynucleotides (nucleic acids) determined by comparing sequences. Identity also refers to the degree of sequence relatedness between or among sequences determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences, where gap alignments (if any) are solved by a specific mathematical model or computer program (e.g., an "algorithm"). The identities of the related antigens or nucleic acids can be easily calculated by known methods. "Percent (%) identity" for polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in the amino acid sequence or the nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum percentage identity. Methods and computer programs for comparison are well known in the art. It is understood that identity is determined by the calculation of percent identity, but its value may vary due to the gaps and penalties introduced in the calculation. Typically, variants of a particular polynucleotide or polypeptide (e.g., an antigen) have 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a particular reference polynucleotide or polypeptide as determined by the sequence alignment programs and parameters described herein and known to those of skill in the art.

[0149] Lipid nanoparticles (LNPs)

[0150] RNA disclosed herein (for example, mRNA) is formulated in lipid nanoparticles (LNPs). Lipid nanoparticles generally include ionizable cationic lipids, helper lipids, cholesterol and PEG lipid components and nucleic acid of interest. Lipid nanoparticles disclosed herein can use components commonly known in the art, and compositions and methods can be generated.

[0151] Multivalent vaccines

[0152] Compositions provided herein can comprise RNA or multiple RNAs encoding two or more antigens of the same or different types. In some embodiments, compositionss comprise RNA or multiple RNAs encoding two or more rabies virus antigens. In some embodiments, RNA can encode 1,2,3,4,5,6,7,8,9,10,11,12 or more rabies virus antigens.

[0153] Two or more different RNAs (e.g., mRNAs) encoding antigens can be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNAs encoding antigens can be formulated in separate lipid nanoparticles (each RNA is formulated in a single lipid nanoparticle). The lipid nanoparticles can then be combined and administered as a single vaccine composition (e.g., comprising multiple RNAs encoding multiple antigens), or can be administered separately.

[0154] Combined vaccines

[0155] Compositions provided herein can include RNA or multiple RNAs encoding two or more antigens of the same or different virus strains. Combination vaccines are also provided herein, which include RNAs encoding one or more rabies viruses and one or more different organisms' antigens. Therefore, vaccine disclosed herein can be a combined vaccine targeting one or more antigens of the same strain / species, or one or more antigens of different strains / species, such as inducing an antigen that may be exposed to the immunity of an organism or individual found in the same geographic area where rabies virus infection is dangerously high when being exposed to rabies virus.

[0156] pharmaceutical preparations

[0157] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits, and reagents for preventing or treating, for example, rabies virus in humans and other mammals. ... that can be used as therapeutic or prophylactic agents. They can be used in medicines for preventing and / or treating rabies virus infection.

[0158] Pharmaceutical composition

[0159] The term "pharmaceutical composition" refers to the combination of an active agent and an inert or active carrier such that the composition is particularly suitable for in vivo or in vitro diagnostic or therapeutic use. A "pharmaceutically acceptable carrier" does not cause undesirable physiological effects upon administration to or after administration to a subject. The carrier in a pharmaceutical composition must be "acceptable" in the sense that it is compatible with the active ingredient and capable of stabilizing it. One or more solubilizing agents may be used as pharmaceutical carriers for delivering the active agent. Examples of pharmaceutically acceptable carriers include, but are not limited to, biocompatible carriers, adjuvants, additives, and diluents to obtain a composition that can be used as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Other suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for them, are described in Remington's Pharmaceutical Sciences.

[0160] Sequence information

[0161] The relevant sequences involved in this application are as follows: Note: In SEQ ID NO. 1-3, U can be 100% replaced by 1-methylpseudouracil. BRIEF DESCRIPTION OF THE DRAWINGS

[0162] Figure 1: Bioanalyzer analysis of the purity of G protein-encoding mRNA.

[0163] Figure 2: Experimental results of LNP formulation optimization.

[0164] Figure 3: Transmission electron microscopy shows the morphology of the G protein mRNA and lipid nanoparticle (LNP) complex.

[0165] Figure 4: Immunoblotting experiments verified the expression of G protein in host cells.

[0166] Figure 5: FACS flow cytometry detected the expression of target antigen protein in cells.

[0167] Figure 6: Neutralizing antibody detection experiment in mice after single immunization.

[0168] Figure 7: Neutralizing antibody detection experiment in mice after immunization with different immunization schedules.

[0169] Figure 8: Results of the neutralizing antibody experiment in cynomolgus monkeys.

[0170] Figure 9: Rabies mRNA vaccine hepatotoxicity results.

[0171] Figure 10: Results of long-acting neutralizing antibody concentration of rabies mRNA vaccine.

[0172] Figure 11: Experimental results of neutralizing activity of rabies mRNA vaccine against mainstream strains in China. DETAILED DESCRIPTION

[0173] The embodiments of the present application will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present application and should not be considered as limiting the scope of the present application. In the examples, if no specific conditions are specified, the conditions according to conventional conditions or manufacturer recommendations are used. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0174] Example 1: Antigen protein sequence optimization and in vitro transcription (IVT)

[0175] 1.1 Antigen protein sequence optimization

[0176] Based on the natural coding region sequence of rabies virus G protein, this application designed the SY mRNA sequence. In addition to the coding region, the SY mRNA sequence features also include the alpha-1-globin 5'UTR, gp130 and DH143 3'UTRs, and 100 polyA residues. The SY-1, SY-2, and SY-3 mRNA sequences were all optimized in the G protein coding region. Compared with the original rabies virus G protein sequence, the GC content of the optimized sequences was increased, while the UTR region remained consistent.

[0177] The specific experimental steps are as follows: The present invention designed multiple optimized sequences and cloned them into the vector pVAX.1 (GenScript Biotech Co., Ltd.). Using the linearized vector (customized by GenScript Biotech Co., Ltd.) as a template, mRNA was synthesized in vitro and transfected into COS7 cells. At set time points, G protein expression levels in cells were measured by flow cytometry to evaluate the effect of codon optimization on mRNA expression levels and stability. The expression level results are as follows:

[0178] Table 1 G protein expression data of different codon optimization strategies Note: Relative expression means: COS7 cells transfected with SY-01, SY-02, or SY-03 mRNA were used as the experimental group, and cells not transfected with mRNA were used as the control group. The expression level of G protein was detected in vitro according to the method in Example 3, and the mean fluorescence intensity (MFI) of the experimental and control groups was recorded. The relative expression was calculated according to the following formula:

[0179] Based on the above experimental results, SY-01 (SEQ ID NO. 1) with the highest relative expression level was selected as the preferred mRNA sequence for subsequent vaccine research and development and the sequence used in the following examples.

[0180] 1.2 In vitro transcription (IVT) of G protein antigen

[0181] Here are the steps:

[0182] 1. According to the instructions of the IVT kit (Novozymes kit catalog number: DD4201-P-01), the IVT reaction system was prepared by mixing 10× Transcription Buffer, ATP, GTP, CTP, PseudoUTP (1-methylpseudouracil, Zhaowei Technology, catalog number R5-064), 5' cap analog (m7G(5′)ppp(5′)(2′OMeA)pG, Zhaowei Technology, catalog number ON-134), water for injection, plasmid template (linearized plasmid with T7 promoter, the template is the DNA sequence corresponding to SEQ ID NO.1, i.e., SEQ ID NO: 10), and Enzyme Mix.

[0183] 2. The mixed reaction system was placed at 37°C for 40 minutes.

[0184] 3. Add the corresponding proportion of DNase I to terminate the reaction.

[0185] 4. Add an equal volume of a certain concentration (7.5M) LiCl solution and precipitate twice. Then wash twice with 75% ethanol solution, dry at room temperature in a clean bench, and dissolve with an appropriate amount of RNase-free H2O.

[0186] The purity of the obtained mRNA was analyzed using a Bioanalyzer. The experimental results showed (see Figure 1) that after the G protein mRNA was synthesized in vitro, it was separated and purified to obtain high-purity mRNA.

[0187] Example 2: Optimal mRNA-LNP formulation and preparation method

[0188] 2.1 Formulation screening experiment

[0189] The formulation was optimized by comprehensively considering the following four aspects: lipid ratio, buffer system, N / P (nitrogen-to-phosphorus ratio), and whether to freeze-dry. Specific formulation options are shown in Tables 2.1 and 2.2.

[0190] Normal mice were immunized with 5 μg of mRNA (encapsulated in LNPs of different formulations 1-9) once, and neutralizing antibodies were measured in mouse sera on days 7 and 14. Based on the neutralizing antibody titers, formulation 9 was ultimately determined to be the preferred formulation (see Figure 2).

[0191] Table 2.1 LNP formula ingredients list

[0192] Table 2.2 Lipid ratio list

[0193] 2.2 Preparation of mRNA-LNP Preparations

[0194] Taking the above-mentioned formula 9 as an example, the preparation steps of the G protein antigen mRNA-LNP preparation are as follows:

[0195] 1. Accurately weigh a certain amount of SM-102, 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG2000 lipids at a molar mass ratio of 50% and dissolve them in an appropriate amount of anhydrous ethanol to prepare a lipid working solution (final concentration of the lipid working solution is 12 mg / mL).

[0196] 2. Prepare 130 mM sodium chloride in citric acid buffer (10 mM, pH 4.0), Tris-NaOAc buffer (20 mM, 10.7 mM, pH 7.5), and 60% sucrose in Tris-NaOAc buffer (20 mM, 10.7 mM, pH 7.5).

[0197] 3. Take an appropriate amount of mRNA stock solution (sequence shown in SEQ ID NO. 1) and dilute it with the sodium chloride-citrate buffer solution prepared above to adjust the final concentration of the mRNA working solution to 0.18 mg / mL;

[0198] 4. Using a microfluidic instrument and supporting chip, the lipid working solution and the mRNA working solution were mixed in a volume ratio of 1:3 to prepare an mRNA-loaded LNP solution;

[0199] 5. Dilute the prepared LNP solution with 9 times the volume of Tris-NaOAc buffer solution, and concentrate and purify it using TFF (tangential flow ultrafiltration) to remove the ethanol solution in the system;

[0200] 6. Use UV spectrometry to detect the mRNA content in the LNP solution and add an appropriate amount of Tris-NaOAc buffer solution (20mM, 10.7mM, pH 7.5) containing 60% sucrose to adjust the final mRNA concentration in the finished LNP solution to 100μg / mL and the sucrose content in the external aqueous phase system to 8.7%.

[0201] The experimental results show (see Figure 3): G protein mRNA and lipid working solution are mixed by microfluidics to form LNP complexes with uniform particle size, mostly around 100 nm, which meets the basic requirements for the next step of the experiment.

[0202] Example 3: G protein expression experiment in vitro

[0203] 3.1 Immunoblotting experiment:

[0204] G protein mRNA was transfected into COS-7 cells (Note: African green monkey kidney fibroblasts, purchased from Nanjing Kebai Biotechnology Co., Ltd.). 24 hours after transfection, cells were harvested and analyzed by immunoblotting. Cells not transfected with G protein mRNA served as a negative control (Blank). The experimental results (see Figure 4) demonstrated that the G protein-encoding mRNA obtained in this application can be effectively and stably expressed in COS-7 cells.

[0205] 3.2 Flow cytometry detection:

[0206] After transfecting 293T cells or COS-7 cells with G protein mRNA, FACS flow cytometry was used to detect the expression of the target antigen protein in the cells. Cells not transfected with G protein mRNA served as a negative control (Blank). The specific method is as follows (using COS-7 cells as an example):

[0207] 1. To detect the expression of rabies virus G protein mRNA in human cells, we digested COS-7 cells cultured for more than 24 hours and transferred them into 6-well plates at a cell density of 250,000 per well.

[0208] 2. Incubate the six-well plate at 37°C for 24 hours and observe the cells under a microscope. mRNA transfection can be performed when the cell confluence reaches 80% or above.

[0209] 3. Transfect the corresponding mRNA into COS-7 cells using the Lipofectamine 2000 kit (4 μg of mRNA per well). Refer to the kit instructions for specific procedures. Continue to culture at 37°C for 24 hours.

[0210] 4. After 24 hours of sustained mRNA expression, when expression levels approach peak, remove the cell supernatant, wash once with PBS, digest with 0.05% trypsin for 1 minute, neutralize with complete culture medium, and harvest the cells. Centrifuge the harvested cells at 350g for 5 minutes, and discard the supernatant. Resuspend the cells in 2 ml of PBS, harvest the cells, centrifuge at 350g for 5 minutes, and discard the supernatant. Finally, resuspend the cells in 100 μl of PBS, and control the cell count to between 200,000 and 1,000,000.

[0211] 5. Use 150μl Cyto-Fast TM Fix the collected cells with Fix / Perm Buffer and incubate on ice for 20 minutes.

[0212] 6. Add 1 ml of 1×Cyto-Fast TM Perm Wash solution, 350g, centrifuge for 5 minutes. Discard the supernatant and use 1ml 1×Cyto-Fast TM Resuspend in Perm Wash solution, rinse once more, and centrifuge to discard the supernatant.

[0213] 7. Use 100 μl 1× Cyto-Fast TM Dilute the primary antibody to 1:100 in Perm Wash solution, add it to the cells, shake to mix, and incubate on ice for 30 minutes.

[0214] 8. Add 1 ml of 1× Cyto-Fast to the cells after incubation with the primary antibody. TM Perm Wash solution, 350g, centrifuge for 5 min, discard the supernatant and use 1 ml 1× Cyto-Fast TM Resuspend in Perm Wash solution, rinse once more, and centrifuge to discard the supernatant.

[0215] 9. Use 250 μl 1× Cyto-Fast TM Dilute the secondary antibody to 1:1000 in Perm Wash solution, add it to the cells, shake to mix, and incubate on ice for 30 minutes.

[0216] 10. Add 1 ml of 1× Cyto-Fast to the cells after incubation with the secondary antibody. TM Perm Wash solution, 350g, centrifuge for 5 min, discard the supernatant and use 1 ml 1× Cyto-Fast TM Resuspend in Perm Wash solution, rinse once more, and centrifuge to discard the supernatant.

[0217] 11. Dilute the 4% paraformaldehyde fixative to 1% paraformaldehyde using PBS. Resuspend the cells in 200 μl of 1% paraformaldehyde fixative and analyze the expression of rabies glycoprotein in the cells using a flow cytometer.

[0218] 12. Flow cytometry: Set cells without mRNA transfection as a negative control. Analyze each sample sequentially, and read the FITC fluorescence intensity signal using a histogram. Record the mean fluorescence intensity (MFI) of the cells, reading 10,000 signals for each sample.

[0219] The experimental results show (see FIG5 ) that the mRNA encoding the G protein obtained by the present invention can be effectively and stably expressed in COS-7 cells and 293T cells.

[0220] Example 4: Mouse neutralizing antibody detection experiment

[0221] Healthy, well-grown 6-8-week-old BALB / c mice (purchased from Shanghai Southern Model Organisms Co., Ltd.) were selected and randomly divided into 10 groups after weight measurement, with half male and half female. Intramuscular injection of the mRNA prepared in Example 2.2 for LNP delivery was used. Dosages were 2.5 μg, 5 μg, and 10 μg, respectively, on day 0.

[0222] Mice were immunized with different doses of G protein mRNA (2.5μg, 5μg, and 10μg), and neutralizing antibodies were measured in their sera 14 days later. The results (see Figure 6) showed that even in the low-dose group, high concentrations of neutralizing antibodies were detected in the sera of mice. These concentrations were well above the WHO standard of 0.5 IU / mL.

[0223] Example 5: Neutralizing antibody detection experiment in mice after immunization with different immunization schedules

[0224] Healthy, well-grown 6-8 week old BALB / c mice (purchased from Shanghai Southern Model Organisms Co., Ltd.) were selected and randomly divided into groups after weight measurement, with 10 mice per group, half male and half female. Intramuscular injection was used for administration (mRNA delivered by LNP prepared in Example 2.2). The administration time was day 0, day 0 and day 3, day 0 and day 7; the dosages were 0.125 μg, 0.5 μg, 1 μg, 2 μg (injection on day 0), 0.0625 μg, 0.25 μg, 1 μg (injection on day 0 and day 3), and 1 μg (injection on day 0 and day 7). Normal mice were immunized with different doses (0.125μg, 0.5μg, 1μg, 2μg and 0.0625μg, 0.25μg, 1μg) and different immunization methods (0D injection, 0 / 3D injection, 0 / 7D injection), and the neutralizing antibodies in the mouse serum were detected after 5 and 14 days.

[0225] The experimental results show (see Figure 7): Under the condition of a single injection of 1 μg mRNA, neutralizing antibodies higher than the WHO standard (0.5 IU / mL) can be detected in mice after 5 days. Under the condition of two injections of 0.0625 μg mRNA on days 0 and 3, neutralizing antibodies higher than the WHO standard (0.5 IU / mL) can also be detected in mice after 14 days. This shows that at an extremely low injection dose (0.0625 μg), the mRNA vaccine of this application can also produce high levels of neutralizing antibodies.

[0226] Example 6: NIH titer detection experiment

[0227] According to the standard NIH potency test process specified by the WHO, the NIH potency of the mRNA vaccine finished preparation (vaccine to be tested) prepared based on Example 2.2, the Yangshen mRNA vaccine (prepared with reference to Example 1 of patent CN110714015A - Preparation method of rabies virus nucleic acid vaccine) and the reference vaccine standard (the standard vaccine of the China National Institute for Food and Drug Control, which is an inactivated vaccine with a nominal potency of 11.4 IU / ml) were respectively measured. The specific operation process is as follows:

[0228] 1. Take a vial of freeze-dried reference vaccine, add 1.0 ml of sterile water for injection, and mix until completely dissolved before use; the labeled potency after reconstitution is 11.4 IU / ml; dilute the reconstituted reference vaccine 5-fold in PBS (pH 7.4), and take samples of 4 dilutions, namely 1:25, 1:125, 1:625, and 1:3125, and store them in an ice bath until used;

[0229] 2. Take the vaccine to be tested and the Yangshen vaccine, perform a 5-fold serial dilution of the samples, take the finished vaccine, and 5 dilution samples of 1:5, 1:25, 1:125 and 1:625, and store them in an ice bath for later use;

[0230] 3. For the first immunization, administer the reference vaccine and the test vaccine at various dilutions, starting from the highest dilution and continuing with the lowest dilution. Intraperitoneally inject 0.5 ml of each dilution into 16 female mice, 3-4 weeks old and weighing 11-16 g. One week after the first immunization, administer the reference vaccine and the test vaccine at various dilutions, using the method and dosage described above, to the mice for a second immunization.

[0231] 4. Fourteen days after the first immunization, all immunized mice were inoculated intracerebrally with 50 LD50 / 0.03 ml of rabies virus (CVS-24 strain) at 0.03 ml per mouse. All mice were observed for 14 consecutive days after challenge. Mice that died within the first four days (including the fourth day) were counted as nonspecific deaths. Starting from the fifth day, the number of mice that died from rabies in each group was recorded daily. Mice that developed typical rabies symptoms such as paralysis or convulsions on the 14th day were also counted as dead. The number of mice that died and survived between days 5 and 14 was counted.

[0232] 5. Calculate the ED50 of the test vaccine and the reference vaccine using the Reed-Muench method, and calculate the relative potency (RP, also known as the NIH potency) of the test vaccine using the following formula.

[0233] The specific results are as follows:

[0234] Table 3 NIH titer test results

[0235] The above results show that the potency of the rabies mRNA vaccine obtained in this application was measured to be 25.49 IU / mL, which is much higher than the Yangshen mRNA vaccine (9.71 IU / mL) and the reference vaccine (11.4 IU / mL). In addition, at the same dose, the mortality rate of mice immunized with the rabies mRNA vaccine of this application was much lower than that of the Yangshen vaccine.

[0236] Example 7: LNP formulation stability test

[0237] The finished rabies mRNA vaccine formulation (prepared by the method of Example 2.2) was stored at -20°C and 5°C, and the vaccine stability was evaluated using different physical and chemical indicators. After six months of storage under both conditions, the physical and chemical properties of the finished mRNA vaccine formulation did not change significantly, the RNA concentration did not change significantly, and the encapsulation efficiency remained above 75%. These results indicate that the vaccine formulation has long-term stability. See Table 4 for details:

[0238] Table 4 LNP vaccine stability test results

[0239] Example 8: Cynomolgus monkey neutralizing antibody experiment

[0240] Normal cynomolgus macaques (3 monkeys per dose group) were immunized twice on days 0 and 7 with different doses (10 μg and 30 μg) of the mRNA vaccine LNP formulation (prepared by the method in Example 2.2), and neutralizing antibodies were measured in the macaque sera on day 14. The results showed (see Figure 8) that compared with the marketed Rabipur, the rabies mRNA vaccine of the present invention produced higher titers of neutralizing antibodies with fewer immunizations.

[0241] Example 9: Post-exposure immunity experiment

[0242] Mice were immunized twice with 3 μg of the LNP formulation of the mRNA vaccine (prepared by the method of Example 2.2) on days 0 and 3 after challenge, and the survival rate of each group of mice was measured at days 7, 14, and 21. The protective efficacy of the mRNA vaccine of this application was significantly higher than that of the standard Yangshen mRNA vaccine (obtained by referring to the preparation method of rabies virus nucleic acid vaccine in Example 1 of patent CN110714015A). Specific results are shown in Table 5.1:

[0243] Table 5.1 Results of post-exposure immune tests

[0244] In addition, the present invention also focuses on the mRNA vaccine and inactivated vaccine of the present application Related experiments were conducted: female BALB / c mice were selected, 10 in each group, and the mice were immunized after exposure. On the day after the challenge (D0), the mRNA vaccine and Afterwards, the survival of the animals was observed until D28. The specific results are shown in Table 5.2.

[0245] Table 5.2 Post-exposure immune survival results Note: 1. The specification of the Rabipur inactivated vaccine for human use is 300ul, and the mouse injection dose is 1 / 10 and 1 / 30 of the human dose; 2. The tentative specification of the mRNA vaccine for human use in this application is 30ug, and the mouse injection dose is 1 / 10 and 1 / 30 of the human dose.

[0246] It can be seen that the survival rate of mice injected with the mRNA vaccine of the present application was significantly higher than that of the Rabipur group, proving that the immune effect of the vaccine of the present application is significantly better than that of Rabipur.

[0247] Example 10: Rabies mRNA vaccine liver toxicity experiment

[0248] Cynomolgus macaques were immunized three times with low-dose (30 μg) and high-dose (90 μg) LNP formulations of the mRNA vaccine (prepared by the method of Example 2.2) on days 0, 7, and 14. On day 17, liver alanine aminotransferase and aspartate aminotransferase activities were measured in each group of cynomolgus macaques. The results (see Figure 9) showed that the rabies mRNA vaccine prepared by the present invention showed no significant liver toxicity in either the low-dose or high-dose groups, demonstrating excellent safety.

[0249] Example 11: Long-term effect of rabies mRNA vaccine

[0250] Select 6-8 week old Balb / C male and female mice, 5 each, 10 mice / group, and administer by intramuscular injection. 10 μg was injected on day 0 and day 21, respectively. Afterwards, mouse serum was taken on days 0, 14, 28, 35, 90, 180, 270, and 360, and the neutralizing antibody concentration in the mouse serum was detected. The results showed (see Figure 10, the horizontal axis is days): After injection of 10 μg of mRNA vaccine LNP preparation (prepared by the method of Example 2.2) on days 0 and 21, high concentrations of neutralizing antibodies can be produced in mice for a long time (at least one year), and are far higher than the WHO specified standard of 0.5 IU / mL, which has a long-term protective effect against rabies virus.

[0251] Example 12: Neutralization activity test of rabies mRNA vaccine against mainstream strains in China

[0252] The mRNA vaccine LNP formulation was administered intramuscularly to crab-eating macaques. The first immunization of the animals was recorded as D0, and the animals were immunized twice with an interval of 7 days (D0 and D7). The monkey serum was collected 14 days after the second immunization (D21), and the serum was tested for in vitro neutralizing activity against representative strains of seven Chinese populations, including SC16, GD1, NM3, QH2, LY, YN3, and XZ17 (Figure 11). The results of molecular epidemiological studies have shown that the rabies viruses prevalent in China can be divided into the above seven populations (SC16, GD1, NM3, QH2, LY, YN3, and XZ17). The experimental results showed that the neutralizing antibody positive conversion rate (GMT>0.5IU / mL) of all animals for each strain was 100%, indicating that the mRNA vaccine of this application has broad-spectrum neutralizing activity against rabies virus.

[0253] The above experimental results show that the mRNA vaccine obtained by the present invention can produce high levels of neutralizing antibodies and effectively induce B cell immunity and T cell immunity. Its comprehensive immune effect is far superior to that of the Yangshen reference product. In addition, the vaccine has moderate storage conditions and can be stably stored for a long time at -20°C. It is relatively convenient to transport and does not require high equipment. It is more suitable for economically underdeveloped or underdeveloped areas that are in the most urgent need of vaccination. In addition, the rabies mRNA vaccine obtained by the present invention has a broad spectrum of neutralizing activity against representative strains of the seven populations of rabies virus prevalent in China, and provides good protection for challenged mice in pre-exposure and post-exposure immunization experiments.

Claims

1. An mRNA encoding a rabies virus antigen comprising amino acids having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identity with the amino acid sequence of SEQ ID NO.

4. 2 . The mRNA of claim 1 , wherein the mRNA comprises a 5′UTR, a 3′UTR, and a 3′-poly(A).

3. The mRNA of claim 2, wherein the 3'-poly(A) comprises the nucleotide sequence of SEQ ID NO.

7. 4 . The mRNA according to claim 2 , wherein the 5′UTR comprises the nucleotide sequence of SEQ ID NO. 6 and / or the 3′UTR comprises the nucleotide sequence of SEQ ID NO. 8 and / or SEQ ID NO.

9.

5. The mRNA of any one of claims 2-4, comprising a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% identical to the nucleotide sequence of any one of SEQ ID NOs. 1-3.

6. The mRNA according to any one of claims 1 to 5, further comprising a 5' guanosine cap selected from the group consisting of: m7Gppp(2'OMeA)pG, m7GpppApA, m7GpppApC, m7GpppApG, m7GpppApU, m7GpppCpA, m7GpppCpC, m7GpppCpG, m7GpppCpU, m7GpppGpA, m7GpppGpC, m7GpppGpG, m7GpppGpU, m7G pppUpA, m7GpppUpC, m7GpppUpG, m7GpppUpU, m7Gpppm6ApG, m7G3'OmepppApA, m7G3'OmepppApC, m7G3'OmepppA pU, m7G3'OmepppApG, m7G3'OmepppCpA, m7G3'OmepppCpC, m7G3'OmepppCpG, m7G3'OmepppCpU, m7G3'OmepppUpA , m7G3'OmepppUpC, m7G3'OmepppUpG, m7G3'OmepppUpU, m7G3'OmePppA2'OmepG, m7G3'OmepppA2'OmepC, m7G3' OmeppA2'OmepU, m7G3'OmepppA2'OmepA, m7G3'OmepppC2'OmepA, m7G3'OmepppC2'OmepU, m7G3'OmepppC2'Ome pG, m7G3'OmepppC2'OmepC, m7G3'OmepppG2'OmepA, m7G3'OmepppG2'OmepU, m7G3'OmepppG2'OmepG, m7G3'Ome pppG2'OmepC, m7G3'OmepppU2'OmepA, m7G3'OmepppU2'OmepU, m7G3'OmepppU2'OmepG, m7G3'OmepppU2'OmepC.

7. The mRNA according to any one of claims 1 to 6, wherein the rabies virus antigen encoded by the mRNA is G protein.

8. A composition comprising lipid nanoparticles and mRNA, wherein the mRNA is the mRNA according to any one of claims 1 to 7.

9. The composition of any one of claim 8, comprising an ionizable cationic lipid, a structural lipid, a helper lipid and a surfactant.

10. The composition of any one of claims 8-9, wherein the lipid nanoparticles comprise, in terms of molar percentage (mol%), 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 5-25 mol% auxiliary lipids and 0.5-15 mol% surfactants.

11. The composition according to any one of claims 8 to 10, wherein the structured lipid is selected from cholesterol and cholesterol derivatives, preferably cholesterol.

12. The composition of any one of claims 8 to 11, wherein the cationic lipid is selected from the group consisting of SM-102, ALC-0315, ALC-0519, Dlin-MC3-DMA, DODMA, DLin-KC2-DMA, DlinDMA, preferably SM-102.

13. The composition of any one of claims 8 to 12, wherein the helper lipid is selected from DSPC, DOPE, DOPC, DOPG or DOPS, preferably DSPC.

14. The composition according to any one of claims 8 to 13, wherein the surfactant is selected from PEG2000-DMG, PEG-DSPE, DTDA-PEG2000, TPGS, preferably PEG2000-DMG.

15. The composition of any one of claims 8 to 14, wherein the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG2000-DMG in terms of molar percentage (mol%).

16. The mRNA of claims 1-7 or the composition of claims 8-15, wherein the mRNA comprises a chemically modified base or analogue selected from 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, 5-methoxy cytidine, 1-methyl pseudouridine (N1-Methyl-Pseudo-UTP), pseudouridine; preferably 1-methyl pseudouridine.

17. Use of the mRNA according to any one of claims 1 to 7 or the composition according to any one of claims 8 to 16 in the preparation of a rabies virus nucleic acid vaccine.

18. The use according to claim 17, wherein the vaccine comprises a combined vaccine and a multivalent vaccine.

19. The use according to any one of claims 17-18, wherein the vaccine is an anti-rabies virus mRNA vaccine.

20. A method for pre-exposure or post-exposure immunization, comprising administering to a subject the composition of claims 7-16, which is effective in inducing an immune response against rabies virus antigens in the subject.

21. A pharmaceutical composition comprising an mRNA having a sequence as shown in SEQ ID NO.1, a lipid nanoparticle composition, 20 mmol / L tromethamine, 10.7 mmol / L sodium acetate, 8.7% sucrose, pH 7.0-8.0, wherein the lipid nanoparticles contain 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol and 1.5 mol% PEG2000-DMG, N:P=6.

22. A method for preparing a nucleic acid vaccine, comprising the steps of: (1) dissolving a protonable cationic lipid, a structural lipid, an auxiliary lipid and a surfactant in an organic solution in a certain proportion to obtain an organic phase; (2) dissolving the mRNA having a sequence as shown in SEQ ID NO.1 in a citrate buffer or a sodium acetate solution to obtain an aqueous phase; (3) The organic phase of step (1) and the aqueous phase of step (2) are mixed to generate a mixed solution to obtain a rabies virus vaccine.

23. The preparation method according to claim 22, wherein the organic solution comprises anhydrous ethanol.

24. The preparation method according to any one of claims 22 to 23, wherein the total concentration of the protonable cationic lipids, structural lipids, auxiliary lipids and surfactants in the organic phase is 10-15 mg / ml.

25. The preparation method according to any one of claims 22 to 24, wherein the concentration of the mRNA is 0.01-1 mg / ml, preferably 0.1-0.2 mg / ml.

26. The preparation method according to any one of claims 22 to 25, wherein the volume ratio of the organic phase to the aqueous phase is 1:2-4.

27. The preparation method according to any one of claims 22 to 26, wherein the mixing is performed using a microfluidic device and the flow rate is controlled to be ≥12 ml / min.

28. The nucleic acid molecule as shown in SEQ ID NO.

10.

29. A biological material expressing the nucleic acid molecule of claim 28; preferably, the biological material comprises: An expression cassette, recombinant vector, recombinant plasmid or transgenic cell line containing the nucleic acid molecule; the recombinant vector is preferably: pVAX.1 vector, pcDNA3.1 vector.

30. The mRNA of any one of claims 1-7, the composition of any one of claims 8-16, the use of claims 17-19, the method of claim 20, wherein the strains of rabies virus include but are not limited to: Pasteur strain (PAS), Flury strain, SAD strain, aG strain, CTN strain, SC16 (CSC1016D), GD1 (GDZAQ45), NM3 (CNM1103C), QH2 (CQH1202D), LY (CNX8601), YN3 (CYN1009D), XZ17 (CXZ1704H) and their derivatives; preferably CTN strain, most preferably CTN-1 strain.

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