MRNA vaccines encoding varicella-zoster virus glycoprotein e

Abstract

The present application provides an RNA encoding a varicella-zoster (VZV) glycoprotein E (gE) or a variant thereof, the RNA comprising a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the nucleotide sequence represented by any one of SEQ ID NO: 11-18. The eight mRNAs provided by the invention can translate in cells, the expression level of the generated VZV gE protein is high, and the mRNAs can induce a mouse to generate a high-titer gE specific IgG antibody in a manner of intramuscular injection into the body of the mouse through a preparation formed by encapsulating the lipidosome nanoparticles.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological vaccines, and in particular relates to an mRNA vaccine encoding varicella-zoster virus glycoprotein E and an application thereof. Background Art

[0002] Vaccines are the most effective public health intervention to prevent infectious diseases. The World Health Organization estimates that vaccinations prevent two to three million deaths each year. Although traditional vaccine types (such as inactivated vaccines, attenuated vaccines, and subunit vaccines) have played a huge role in public health, humans are still facing the threat of new infectious diseases and their variants. Developing vaccines for new infectious diseases, innovating vaccine production methods, improving vaccine effectiveness, and shortening vaccine production cycles are urgent needs to promote progress in public health.

[0003] Varicella-zoster virus (VZV) is a DNA virus belonging to the alpha-herpes subfamily of the Herpesviridae family. VZV virus particles are spherical with a diameter between 180-200nm. Its outer shell is an envelope composed of a lipid bilayer with glycoproteins with multiple functions dispersed on the surface. The stroma separates the envelope and the nucleocapsid, which is composed of 162 hexamers and pentamers arranged in an icosahedral form and contains double-stranded DNA genetic material. The virus is neurotropic and can lurk in the human body for a long time. Once it recurs, the patient will feel burning pain and electric shock-like pain, which brings great inconvenience to the patient's life.

[0004] At present, in the field of herpes zoster, researchers have explored different vaccine technology routes, such as recombinant protein vaccines, inactivated vaccines, and vector vaccines. The production of recombinant protein vaccines requires large-scale in vitro cell culture and the separation and purification of antigenic proteins from the culture. The process requirements for protein expression and purification are high. In addition, in order to achieve the ideal immune effect, the addition of immune adjuvants is necessary. For example, the AS01B adjuvant required by GlaxoSmithKline's herpes zoster vaccine "Shingrix" is mainly composed of QS21 extracted from the tropical Chilean soap tree. Due to the limited source of raw materials, it is not sustainable, which is not conducive to the large-scale production and application of vaccines; inactivated vaccines have high process requirements and may have potential safety issues. mRNA vaccines have high immunogenicity and safety, and are easy to mass produce. They are widely used in various disease fields such as infectious diseases, tumors and cardiovascular diseases. Therefore, they have received widespread attention from academia and industry.

[0005] The gE protein is the glycoprotein with the highest content produced during the VZV virus infection period. It not only mediates the replication and spread of the virus but also contains neutralizing antibody and CD4+ T cell-related epitopes, which can induce VZV-specific humoral immunity and cellular immunity. The incidence rate of VZV is relatively high globally. The current main strains of the VZV virus can be divided into several types such as J, E1, E2, M1, and M2. Summary of the Invention

[0006] The present application provides an RNA mainly encoding varicella-zoster virus glycoprotein gE, and a vaccine comprising the RNA. The mRNA or vaccine of the present application has one or more of the following characteristics: (1) higher protein expression level, (2) capable of stimulating a stronger immune response, (3) generating more cytokines, (4) having reduced immunogenicity, (5) generating a higher level of neutralizing antibody, and / or (6) the immune response generated has protective efficacy against different strains of varicella-zoster.

[0007] On the one hand, the present application provides an RNA encoding varicella-zoster virus (VZV) glycoprotein E (gE) or a variant thereof, wherein the RNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the nucleotide sequence shown in any one of SEQ ID NOs: 11-18.

[0008] In certain embodiments, the RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 11-18.

[0009] In certain embodiments, the RNA is mRNA.

[0010] In certain embodiments, the nucleotide sequence of the RNA is codon-optimized.

[0011] In the present application, the amino acid sequence of the varicella-zoster virus (VZV) glycoprotein E (gE) is as shown in SEQ ID NO: 10.

[0012] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 18.

[0013] In certain specific embodiments, the variant further has the immediate early protein IE62 at the C-terminus, and the variant is linked to the antigen IE62 through a linker.

[0014] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 17.

[0015] In some embodiments, the variant is a truncated polypeptide with the extracellular domain fragment truncated. For example, the variant comprises positions 1-593 of the amino acid sequence shown in SEQ ID NO: 10.

[0016] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 11.

[0017] In some embodiments, the variant is a truncated polypeptide with the extracellular domain fragment truncated. For example, the variant comprises positions 1-573 of the amino acid sequence shown in SEQ ID NO: 10.

[0018] In certain specific embodiments, the variant further has a Y569A mutation.

[0019] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 12.

[0020] In certain specific embodiments, the variant further has a ubiquitin tag at the C-terminus, and the variant is linked to the tag through a linker.

[0021] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 15.

[0022] In certain specific embodiments, the variant further has a LAMP1 tag at the C-terminus, and the variant is linked to the LAMP1 tag through a linker.

[0023] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 16.

[0024] In some embodiments, the variant is a truncated polypeptide with the transmembrane domain and extracellular domain truncated. For example, the variant comprises positions 1-546 of the amino acid sequence shown in SEQ ID NO: 10.

[0025] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 13.

[0026] In some embodiments, the variant is a truncated polypeptide with the intracellular domain fragment, transmembrane domain and extracellular domain truncated. For example, the variant comprises positions 1-162 of the amino acid sequence shown in SEQ ID NO: 10.

[0027] In certain specific embodiments, the RNA comprises the nucleotide sequence shown in SEQ ID NO: 14.

[0028] In certain embodiments, the RNA further comprises one or more structures selected from the group consisting of: a 5' Cap structure, a 5' untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly(A) tail.

[0029] In certain embodiments, the poly(A) tail of the RNA comprises more than 30 adenosines.

[0030] In certain embodiments, in the RNA, from the 5' end to the 3' end, the structure of the poly(A) tail is: 30 adenosines, a linker sequence, and 70 adenosines. For example, the length of the poly(A) tail can be 100 - 200 nucleotides, such as about 120 nucleotides. Such as about 110 nucleotides.

[0031] In certain embodiments, the length of the linker sequence can be 5 - 20 nucleotides. In certain embodiments, the linker sequence is GCAUAUGACU.

[0032] In certain embodiments, the poly(A) tail of the RNA comprises the nucleotide sequence shown in SEQ ID NO:9. In certain embodiments, the poly(A) tail has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the nucleotide sequence shown in SEQ ID NO:9.

[0033] In certain embodiments, the RNA comprises at least one modified nucleotide.

[0034] In certain embodiments, the RNA comprises a modification at one or more positions selected from the group consisting of: a 5' Cap structure, a 5' untranslated sequence, an open reading frame, a 3' untranslated sequence, and a poly(A) tail. For example, the 5' untranslated sequence may comprise the 5' untranslated sequence of a gene selected from the group consisting of: the β - globin gene, the heat shock protein 70 gene, the axonemal dynein heavy chain 2 gene, the hydroxysteroid (17 - β) dehydrogenase gene, and / or a KOZAK sequence, or its homolog, fragment, or variant. For example, the 3' untranslated sequence may comprise the 3' untranslated sequence of a gene selected from the group consisting of: the albumin gene, the α - globin gene, the β - globin gene, the tyrosine hydroxylase gene, the heat shock protein 70 gene, the lipoxygenase gene, and the collagen α gene, or its homolog, fragment, or variant.

[0035] In certain embodiments, the chemical structural formulas of the first and second structural units in the 5' Cap structure are as follows:

[0036]

[0037] In certain embodiments, the modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyluridine.

[0038] In certain preferred embodiments, the modified nucleotide of the RNA comprises one or more nucleotides selected from the group consisting of: N1-methylpseudouridine triphosphate (N1-Methylpseudo-UTP), pseudouridine triphosphate (pseudo-UTP), 5-methoxyuridine triphosphate (5-Methoxy-UDP), and 5-methylcytidine triphosphate (5-Methyl-CTP).

[0039] In certain preferred embodiments, all of the U bases of the structural units in the sequence of the RNA are replaced by m1Ψ (1-methyl-3'-pseudouridylyl).

[0040] In certain embodiments, the RNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence shown in any one of SEQ ID NO:1-8.

[0041] In certain embodiments, the RNA comprises the nucleotide sequence shown in any one of SEQ ID NO:1-8.

[0042] The RNA of the present application can be formulated in nanoparticles or other delivery carriers, for example, to avoid their degradation when delivered to a subject. In the present application, the mRNA can be encapsulated within the nanoparticles. In a specific embodiment, the nanoparticles comprise lipids. The lipid nanoparticles can include, but are not limited to, liposomes and micelles. In the present application, the lipid nanoparticles can include cationic and / or ionizable lipids, anionic lipids, neutral lipids, amphiphilic lipids, polyethylene glycolated lipids, and / or structural lipids, or a combination of the above. In certain embodiments, the lipid nanoparticles contain one or more RNAs described in the present application, such as mRNA, and for example, mRNA encoding varicella-zoster virus (VZV) glycoprotein E (gE).

[0043] There is no particular limitation on the specific delivery method, and those commonly used in the art can be adopted. For example, reference can be made to the methods disclosed in the published patent US20160376224A1 or WO2015199952A1.

[0044] The delivery carrier in the composition described in the present application can be a nano-lipid particle. The nano-lipid particle may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) cationic and / or ionizable lipids. "Cationic lipid" generally refers to a lipid that carries any number of net positive charges at a certain pH (e.g., physiological pH). The cationic lipid may include, but is not limited to, SM102, 3-(dodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(dodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-tricosyl-15,18,21,24-tetraazaoctaporphyrin (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, Octyl-CLinDMA, Octyl-CLinDMA(2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA and DMRIE.

[0045] In certain embodiments, the molar ratio of the cationic lipid in the lipid nanoparticle is about 40 - 70%, for example, about 40 - 65%, about 40 - 60%, about 45 - 55% or about 48 - 53%.

[0046] In the present application, the nano-lipid particle may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) non-cationic lipids. The non-cationic lipid may include anionic lipids. The anionic lipids applicable to the lipid nanoparticles of the present application may include phosphatidylglycerol, cardiolipin, diacyl phosphatidylserine, diacyl phosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaroyl phosphatidylethanol phosphate, and other neutral lipids linked with anionic groups.

[0047] The non-cationic lipid may include neutral lipids. Neutral lipids applicable to the lipid nanoparticles of the present application may include phospholipids, such as distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. Additionally, lipids having a mixture of saturated and unsaturated fatty acid chains may be used. For example, the neutral lipids described in the present application may be selected from DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine.

[0048] In certain embodiments, the molar ratio of the phospholipid in the lipid nanoparticles is about 5-20%.

[0049] In the present application, the nano-lipid particles may comprise lipid conjugates, such as polyethylene glycol (PEG)-modified lipids and derived lipids. PEG-modified lipids may include, but are not limited to, polyethylene glycol chains with a length of up to 5 kDa covalently linked to lipids having an alkyl chain with a length of C6-C20. The addition of these components can prevent lipid aggregation, increase the circulation duration, facilitate the delivery of the lipid-nucleic acid composition to target cells, or rapidly release the nucleic acid. For example, the polyethylene glycol (PEG)-modified lipid molecule may be PEG-ceramide having a shorter acyl chain (e.g., C14 or C18). In certain embodiments, the molar ratio of the polyethylene glycol (PEG)-modified lipid molecule in the lipid nanoparticles is about 0.5-2%, for example, about 1-2%, about 1.2-1.8%, or about 1.4-1.6%. In certain embodiments, the polyethylene glycol (PEG)-modified lipid molecule may be PEG2000-DMG.

[0050] In the present application, the nano-lipid particles may further comprise cholesterol. In certain embodiments, the molar ratio of the cholesterol in the lipid nanoparticles is about 30-50%, for example, about 35-45%, or about 38-42%.

[0051] In the present application, the nano-lipid particles may include cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules. In certain embodiments, the molar ratio of the cationic lipid, cholesterol, phospholipid, and polyethylene glycol-modified lipid molecule may be 45-55:35-45:5-15:0.5-2.

[0052] On the other hand, the present application provides a composition, which may contain the mRNA described in the present application and may also contain a delivery vector.

[0053] In certain embodiments, the delivery vector includes liposomes.

[0054] In certain embodiments, the delivery vector includes lipid nanoparticles (LNPs).

[0055] In certain embodiments, the lipid nanoparticles contain PEG-modified lipids, non-cationic lipids, cholesterol, cationic lipids, or any combination thereof.

[0056] In certain embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and / or the sterol is cholesterol.

[0057] On the other hand, the present application provides a lipid nanoparticle preparation that coats the RNA.

[0058] In certain embodiments, the liposome nanoparticle preparation contains PEG-modified lipids, non-cationic lipids, cholesterol, cationic lipids, or any combination thereof.

[0059] In certain embodiments, the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and / or the sterol is cholesterol.

[0060] On the other hand, the present application provides a vaccine that includes the RNA, the composition, and / or the lipid nanoparticle preparation.

[0061] In certain embodiments, the vaccine is multivalent. For example, the vaccine contains RNA encoding two antigens, VGV gE and IE62.

[0062] On the other hand, the present application provides a method for preparing an RNA vaccine, the method comprising: a. dissolving an ionized lipid, a polyethylene glycolated lipid, cholesterol and derivatives, and a phospholipid in an ethanol solution; b. mixing the lipid ethanol solution with an RNA aqueous solution through a microfluidic mixer to obtain lipid nanoparticles (LNPs); c. separating and purifying the LNPs obtained in step b to obtain the RNA vaccine, wherein the RNA is as defined in the present application.

[0063] On the other hand, the present application provides a pharmaceutical composition comprising the RNA, the composition, the liposome nanoparticle formulation, the vaccine, and / or a pharmaceutically acceptable carrier thereof.

[0064] On the other hand, the present application provides a method for treating and / or preventing a disease or disorder associated with novel coronavirus infection, the method comprising administering to a patient in need thereof the RNA, the composition, the liposome nanoparticle formulation, the vaccine, and / or the pharmaceutical composition.

[0065] In some embodiments, the pharmaceutical composition of the present application can be administered to a subject by any method known to those skilled in the art, such as parenterally, orally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, vaginally, or intratumorally.

[0066] In the present application, the RNA, the carrier, the cell, the composition, the liposome nanoparticle formulation, the pharmaceutical composition, and / or the vaccine can be administered singly, multiply, or continuously, and all have safety. After the first administration, readministering the composition and / or the pharmaceutical composition can shorten the onset time. In the present application, the RNA, the carrier, the cell, the composition, the liposome nanoparticle formulation, the pharmaceutical composition, and / or the vaccine can be co-administered with other active substances or therapeutic / preventive components. In the present application, the RNA, the carrier, the cell, the composition, the liposome nanoparticle formulation, the pharmaceutical composition, and / or the vaccine can be administered before or after other active substances or therapeutic / preventive components.

[0067] On the other hand, the present application provides the use of the RNA, the composition, the liposome nanoparticle formulation, the vaccine, and / or the pharmaceutical composition in the preparation of a medicament for treating and / or preventing a disease or disorder associated with varicella-zoster virus infection.

[0068] In certain embodiments, the varicella-zoster virus-related disease or disorder includes a painful rash.

[0069] On the other hand, the present application provides a carrier comprising the RNA.

[0070] On the other hand, the present application provides a cell comprising the RNA and / or the carrier. In the present application, the cell can be a prokaryotic cell, such as, for example, Escherichia coli. In the present application, the cell can be a eukaryotic cell such as a yeast cell, an insect cell, a plant cell, and an animal cell. In the present application, the cell can be a mammalian cell, such as a mouse cell, a human cell, etc.

[0071] On the other hand, the present application provides a kit, which includes the RNA, the composition, the liposome nanoparticle preparation, the vaccine, and / or the pharmaceutical composition.

[0072] On the other hand, the present application provides a method for generating antibodies against varicella-zoster virus, which includes administering the RNA, the composition, the liposome nanoparticle preparation, the vaccine, and / or the pharmaceutical composition.

[0073] In certain embodiments, the method is for non-therapeutic purposes.

[0074] In certain embodiments, the method is an in vitro or ex vivo method.

[0075] On the other hand, the present application provides the use of the RNA, the composition, the liposome nanoparticle preparation, the vaccine, and / or the pharmaceutical composition in the preparation of a reagent for generating antibodies against varicella-zoster virus.

[0076] On the other hand, the present application provides a method for activating immunity, which includes administering the RNA, the composition, the liposome nanoparticle preparation, the vaccine, and / or the pharmaceutical composition.

[0077] In certain embodiments, the method is for non-therapeutic purposes.

[0078] In certain embodiments, the method is an in vitro or ex vivo method.

[0079] On the other hand, the present application provides the use of the RNA, the composition, the liposome nanoparticle preparation, the vaccine, and / or the pharmaceutical composition in the preparation of a reagent for activating immunity.

[0080] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0081] 1. The eight mRNAs provided in the embodiments of the present invention can be translated intracellularly, and the produced VZV gE protein has a high expression level. The preparation formed by encapsulating the mRNA with liposome nanoparticles can be injected into mice by intramuscular injection, which can induce mice to produce high-titer gE-specific IgG antibodies. Compared with other mRNAs or unoptimized mRNAs, the mRNAs of the present application can stimulate stronger cellular immunity and humoral immunity.

[0082] 2. The eight mRNAs provided in the embodiments of the present invention are CVG199, CVG200, CVG201, CVG202, CVG203, CVG204, CVG205, and CVG206 respectively. The open reading frames of the eight mRNAs have been codon-optimized. The full-length sequence of CVG199 is as described in SEQ ID NO:1, the full-length sequence of CVG200 is as described in SEQ ID NO:2, the full-length sequence of CVG201 is as shown in SEQ ID NO:3, the full-length sequence of CVG202 is as shown in SEQ ID NO:4, the full-length sequence of CVG203 is as shown in SEQ ID NO:5, the full-length sequence of CVG204 is as shown in SEQ ID NO:6, the full-length sequence of CVG205 is as shown in SEQ ID NO:7, and the full-length sequence of CVG206 is as shown in SEQ ID NO:8. Among them, CVG206 contains an open reading frame encoding the full-length VZV gE protein. CVG199, CVG200, CVG201, and CVG202 are mRNA sequences of different truncated forms of CVG206. CVG199 truncated the sequence encoding amino acids 594 - 623 in the CVG206 CDS, CVG200 truncated the sequence encoding amino acids 574 - 623 in the CVG206 CDS and replaced the amino acid "tyrosine (Y)" at position 569 with "alanine (A)", CVG201 truncated the sequence encoding amino acids 547 - 623 in the CVG206 CDS, and CVG202 truncated the sequence encoding amino acids 163 - 623 in the CVG206 CDS. The remaining sequence is the antigenic epitope of gE with high immunogenicity. CVG203 is based on CVG200 and has a linker and a ubiquitin tag added at the 3' end. CVG204 is based on CVG200 and has a linker and a lysosome-associated membrane protein 1 (LAMP1) tag added at the 3' end. CVG205 is the full-length sequence of gE plus a linker and an antigenic sequence encoding IE62. CVG206 encodes the full-length protein of VZV gE. The eight mRNAs all contain a 5' Cap structure, a 5' untranslated sequence (5'UTR), a 3' untranslated sequence (3'UTR), and a poly(A) tail. All U bases in the structural units of the eight mRNA sequences are replaced by m1Ψ (1-methyl-3'-pseudouridylyl). Description of the Drawings

[0083] Figure 1 Flow cytometry expression results of CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 in HEK 293T cells in Example 2 of the present invention.

[0084] Figure 2 Particle size (a), Z-potential (b), and polydispersity index of particle size (c) of mRNA-LNP encapsulating CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 mRNA after freeze-thawing at -80°C in Example 3 of the present invention.

[0085] Figure 3 Encapsulation efficiency of mRNA-LNP encapsulating CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 mRNA after freeze-thawing at -80°C in Example 3 of the present invention.

[0086] Figure 4 Integrity test results of CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 in Example 3 of the present invention.

[0087] Figure 5 Immunogenicity evaluation experimental design of CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 in C57BL / 6 naive mouse model.

[0088] Figure 6 (a) shows gE-specific antibodies induced by CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 in C57BL / 6 naive mouse model (D14); (b) shows gE-specific antibodies induced by CVG 199, CVG 200, CVG 201, CVG 204, and CVG 206 in C57BL / 6 naive mouse model (D21).

[0089] Figure 7 One week after the second immunization in Example 4 of the present invention Intracellular cytokine staining data of mouse spleen lymphocytes stimulated by peptide libraries. The data was statistically analyzed for the results of CVG199, CVG200, CVG201, CVG204, and CVG206 using the Kruskal-Wallis non-parametric analysis method (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Among them, (a) is the IFNγ content in CD4+ cells, and the IFNγ content in different experimental groups is roughly equivalent; (b) is the IL2 content in CD4+ cells, and the IL2 content in CVG199, CVG200, and CVG206 is equivalent and higher than that in CVG201, CVG204, and the DPBS group; (c) is the IFNγ content in CD8+ cells; (d) is the IL2 content in CD8+ cells.

[0090] Figure 8 In Example 4 of the present invention, one week after the second immunization, IFNγ (a) and IL2 (b) enzyme-linked immunospot data of mouse spleen lymphocytes stimulated by peptide libraries. The data was statistically analyzed for the results of CVG199, CVG200, CVG201, CVG204, and CVG206 using the Kruskal-Wallis non-parametric analysis method (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0091] Figure 9 Schematic diagram of the body weight change of mice one day after the primary immunization with different doses of CVG206 in Example 5 of the present invention.

[0092] Figure 10 In Example 4 of the present invention, three weeks after the primary immunization and one week after the second immunization, The gE-specific IgG antibody titer in the serum of mice; from left to right on the abscissa are CVG206 0.5 μg / mouse, CVG206 2.5 μg / mouse, CVG206 5 μg / mouse, CVG206 10 μg / mouse, CVG206 20 μg / mouse, live attenuated vaccine from Shanghai Institute of Biological Products 1000 PFU / mouse, "Shinanlishi" from GlaxoSmithKline (GSK) 5 μg / mouse, empty LNP, and DPBS.

[0093] Figure 11This is the data of intracellular cytokine staining of mouse spleen lymphocytes stimulated by a peptide library one week after the second immunization with different doses of mRNA in Example 4 of the present invention. The Kruskal-Wallis non-parametric analysis method was used to perform statistical analysis on the results of different dose experimental groups of CVG206 and "Xinan Lishi" (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Among them, (a) is the IFN content in CD4+ cells. As the dose increases, the IFNγ content in CD4+ cells shows a positive correlation with the administered dose. The IFN content in the 20 μg dose group is significantly higher than that in the Xinan Lishi group; (b) is the IL2 content in CD4+ cells. Among them, the IL2 content in the 0.5 μg dose group is significantly lower than that in the Xinan Lishi group, and the IL2 content in the remaining dose groups is comparable to that in the Xinan Lishi group; (c) is the IFN content in CD8+ cells. The IFN content in different dose groups is comparable to that in the Xinan Lishi group; (d) is the IL2 content in CD8+ cells. The IFN content in different dose groups is comparable to that in the Xinan Lishi group.

[0094] Figure 12 This is the enzyme-linked immunospot data of mouse spleen lymphocytes stimulated by a peptide library one week after the second immunization with different doses of mRNA in Example 4 of the present invention. The Kruskal-Wallis non-parametric analysis method was used to perform statistical analysis on the results of different experimental groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Among them, in the 20 μg dose group, more cells in the mouse spleen secrete IL2 and IFNγ.

[0095] Figure 13 This is the quantitative data of cytokines in the cell supernatant of mouse spleen lymphocytes stimulated by a peptide library one week after the second immunization with different doses of mRNA in Example 4 of the present invention. The Kruskal-Wallis non-parametric analysis method was used to perform statistical analysis on the results of different experimental groups (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). Among them, the contents of IL2, IFNγ and TNFα in the cell supernatant of mouse spleen cells after stimulation by the peptide library show a positive correlation with the immunization dose.

[0096] Figure 14 This is the experimental design for the immunogenicity evaluation of CVG206 in the C57BL / 6 priming mouse model.

[0097] Figure 15It is the gE-specific IgG antibody titer in the serum of priming mice four weeks (D28) after the first immunization, two weeks (D42) and four weeks (D58) after the second immunization in Example 8 of the present invention; the horizontal axis corresponds to CVG206 0.5 μg / mouse, CVG206 2.5 μg / mouse, CVG206 5 μg / mouse, CVG206 10 μg / mouse, CVG206 20 μg / mouse, Shanghai Institute of Biological Products attenuated live vaccine 1000 PFU / mouse, GlaxoSmithKline (GSK)'s "Shingrix" 5 μg / mouse, empty LNP and DPBS from left to right.

[0098] Figure 16 The data of intracellular factor staining of mouse spleen lymphocytes after stimulation with peptide library four weeks after two immunizations with different doses of mRNA in Example 8 of the present invention. The data were statistically analyzed by Kruskal-Wallis nonparametric analysis method for the results of different dose experimental groups of CVG206 and "Xin An Li Shi" (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0099] Among them, (a) is the IL2 content in CD4+ cells. As the dose increases, the IL2 content in CD4+ cells shows a positive correlation with the dose of administration, among which the IL2 content in the 0.5ug dose group is significantly lower than that in the Xinanlisi group; (b) the IFNγ content in CD4+ cells. Among them, the IFNγ content in the 0.5ug dose group is significantly lower than that in the Xinanlisi group, and the IFNγ content in the other dose groups is equivalent to that in the Xinanlisi group; (c) the IL2 content in CD8+ cells. Among them, the IL2 content in the 0.5ug and 2.5ug dose groups is significantly lower than that in the Xinanlisi group; (d) the IFNγ content in CD8+ cells. The IFNγ content in different dose groups is equivalent to that in the Xinanlisi group. DETAILED DESCRIPTION

[0100] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0101] In the present application, the full English name of the term "varicella zoster virus" is varicella zoster virus (VZV). VZV belongs to the herpesvirus subfamily, i.e. human herpesvirus type 3, and is a double-stranded DNA virus. It can lurk and recur in the human body, causing symptoms related to herpes zoster.

[0102] In the present application, the term "gE protein", also known as "glycoprotein E", generally refers to the membrane protein on the surface of varicella-zoster virus.

[0103] In the present application, the term "variant" refers to a molecule whose amino acid sequence differs from the native or reference sequence. The amino acid sequence variant may have substitutions, deletions and / or insertions at certain positions within the amino acid sequence as compared to the native or reference sequence. The variant may be any truncated polypeptide, the variant may have at least one mutation, and the variant may have another sequence at the C-terminus.

[0104] In the present application, "RNA" generally includes mRNA, and also includes analogs of RNA, and their hybrids. In the present application, the term "mRNA" generally refers to an RNA transcript that has been processed to remove introns and is capable of being translated into a polypeptide.

[0105] In the present application, the term "modification", when used with respect to a nucleic acid (such as RNA), generally means that the nucleic acid has different nucleotide molecules, a different nucleotide sequence, is composed of different bonds and / or incorporates non-natural moieties in its structure as compared to the corresponding wild-type. For example, the modification may include modification of nucleotides, for example, the nucleotide may contain a modified base, sugar or phosphate group. For example, the modification may include different nucleotide sequences but encoding a polypeptide or protein of the same amino acid sequence, or a polypeptide or protein of the same function. The modification may be a chemical modification and / or a biological modification. "Chemical modification" may include the introduction of modifications different from those found in wild-type or naturally occurring nucleic acids, for example, covalent modifications, for example, the introduction of modified nucleotides (such as nucleotide analogs, or the introduction of side groups not naturally found in these nucleic acid molecules). The term "modified nucleotide" generally refers to a unit in a nucleic acid polymer that contains a modified base, sugar or phosphate group, or incorporates a non-natural moiety in its structure.

[0106] According to the present invention, naturally occurring or modified nucleosides and / or nucleotides, or optimized nucleotides can be used to prepare modified nucleic acids, such as modified mRNA. For example, modified mRNA may include one or more natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); modified nucleosides (e.g., 2'-aminoadenosine, 2'-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynyl-cytidine, C5-propynyl-uridine, 2'-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2'-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine, (e.g., N1-methyl-pseudouridine), 2'-thiouridine, and 2'-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages), or any combination thereof. These modified nucleotides can be natural nucleotides or artificially optimized or modified nucleotides.

[0107] An RNA molecule (e.g., mRNA) can include at least 0.1% modified nucleotides. The fraction of modified nucleotides can be calculated as: the number of modified nucleotides / the total number of nucleotides × 100%. In some embodiments, the RNA molecule includes from about 0.1% to about 100% modified nucleotides. In some embodiments, the RNA molecule includes about 0.1% modified nucleotides, about 0.2% modified nucleotides, about 0.5% modified nucleotides, about 1% modified nucleotides, about 2% modified nucleotides, about 5% modified nucleotides, about 10% modified nucleotides, about 20% modified nucleotides, about 50% modified nucleotides, or about 100% modified nucleotides.

[0108] In the present application, when the term "codon optimization" is used for nucleic acids, it generally refers to a nucleic acid encoding a polypeptide that has been improved for expression in a cell, such as a mammalian cell or a bacterial cell, by replacing one, at least one, or more than one codon in the parental polypeptide-encoding nucleic acid with a codon encoding the same amino acid residue that has a different relative usage frequency in the cell.

[0109] In the present application, the term "delivery vehicle" generally refers to a transfer vehicle capable of delivering a reagent (e.g., mRNA) to a target cell. The delivery vehicle can deliver a reagent (e.g., mRNA) to a specific cell subclass. For example, the delivery vehicle can be targeted to certain types of cells by virtue of the inherent characteristics of the delivery vehicle or by a moiety conjugated to the vehicle, a moiety contained therein (or a moiety bound to the vehicle such that the moiety and the delivery vehicle remain together and the moiety is sufficient to target the delivery vehicle). The delivery vehicle can also increase the in vivo half-life and / or bioavailability of the reagent to be delivered (e.g., mRNA). The delivery vehicle can include viral vectors, virus-like particles, polycationic carriers, peptide carriers, liposomes, and / or hybrid carriers. For example, if the target cell is a hepatocyte, the properties of the delivery vehicle (e.g., size, charge, and / or pH) can effectively deliver the delivery vehicle and / or the molecule encapsulated therein (e.g., mRNA) to the target cell, reduce immune clearance, and / or promote residence in the target cell.

[0110] In the present application, the term "pharmaceutical composition" generally refers to a preparation in a form that permits the biological activity of the active ingredient (e.g., the vaccine, composition, pharmaceutical composition, carrier, cell, or RNA of the present application) to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is to be administered. These preparations can be sterile.

[0111] In the present application, the term "vaccine" generally refers to a reagent or composition containing an active component that effectively induces a therapeutic level of immunity against a specific pathogen or disease in a subject.

[0112] In the present application, the term "multivalent" vaccine refers to an immunogenic composition / vaccine containing two or more different antigens. The vaccines provided herein can include RNA or multiple RNAs encoding two or more antigens of the same or different species. In some embodiments, two or more different RNAs encoding antigens (e.g., mRNA) 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 formulated in a single lipid nanoparticle). The lipid nanoparticles can then be combined and administered in the form of a single vaccine composition (e.g., containing multiple RNAs encoding multiple antigens) or can be administered separately.

[0113] In the present application, the term "lipid nanoparticle" generally refers to a particle comprising a plurality (i.e., more than one) lipid molecules physically bound to each other by intermolecular forces (e.g., covalent or non-covalent). Lipid nanoparticles can be, for example, microspheres (including monolayer and multilayer vesicles, such as liposomes), the dispersed phase in an emulsion, micelles, or the internal phase in a suspension. Lipid nanoparticles can comprise one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0114] In the present application, the term "liposome" generally refers to a vesicle having an internal space isolated from the external medium by a membrane of one or more bilayers. For example, the membrane of the bilayer can be formed by amphiphilic molecules, such as synthetic or natural-source lipids comprising spatially segregated hydrophilic and hydrophobic domains; alternatively, the membrane of the bilayer can be formed by amphiphilic polymers and surfactants.

[0115] In the present application, the term "pharmaceutically acceptable carrier" generally refers to any adjuvant, excipient, or other pharmaceutical carrier such as a solvent, dispersion medium, coating, isotonic agent, and absorption delaying agent that is compatible with the active ingredient being administered.

[0116] In the present application, the term "carrier" is intended to include any solvent, dispersion medium, coating, diluent, buffer, isotonic agent, solution, suspension, colloid, inert body, etc., or combinations thereof, that are pharmaceutically acceptable for administration to the relevant animal or, if applicable, acceptable for therapeutic or diagnostic purposes.

[0117] In the present application, the term "effective amount" refers to an amount capable of treating or ameliorating a disease or condition or capable of producing a desired therapeutic effect.

[0118] As used herein, the terms "homology" or "identity" refer to the degree of complementarity between two or more polynucleotides. When a first nucleic acid sequence has the exact same primary sequence as a second nucleic acid sequence, the term "identity" can be used in place of the term "homology". Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods can be used to assess whether a given sequence has identity or homology with another selected sequence.

[0119] In the context of two or more nucleic acid sequences, the term "identical" or "percent identity" refers to the percentage of nucleotides that are the same or have a specific percentage of identity when two or more sequences or subsequences are compared and aligned for maximum correspondence using one of the sequence comparison algorithms described below (or other algorithms available to one of ordinary skill in the art) or by visual inspection measurement.

[0120] In the present context, the meaning of "SEQ ID NO:1-18" is specifically shown in the following table:

[0121]

[0122]

[0123]

[0124] As used herein, the term "kit" can be used to describe a variant of a portable self - contained housing that includes at least one set of the reagents, components, or pharmaceutically formulated compositions of the present invention. Optionally, such a kit can include one or more sets of instructions regarding, for example, the use of the encapsulated composition in laboratory or clinical applications.

[0125] As used herein, the terms "prevent" or "treat" refer to the administration, either alone or in a pharmaceutical composition, of a compound prior to the onset of the clinical symptoms of a disease state to prevent any symptoms, aspects, or features of the disease state. Such prevention and inhibition need not be absolute to be considered medically useful.

[0126] This application also provides one or more of the following embodiments:

[0127] 1. An RNA encoding varicella - zoster virus (VZV) glycoprotein E (gE) or a variant thereof, wherein the RNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 11 - 18.

[0128] 2. The RNA according to embodiment 1, wherein the RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 11 - 18.

[0129] 3. The RNA according to embodiment 1 or 2, which is mRNA.

[0130] 4. The RNA according to any one of embodiments 1 - 3, wherein the nucleotide sequence is codon - optimized.

[0131] 5. The RNA according to any one of embodiments 1 - 4, which comprises one or more structures selected from the group consisting of: 5' Cap structure, 5' untranslated sequence, open reading frame, 3' untranslated sequence, and poly(A) tail.

[0132] 6. The RNA according to embodiment 5, wherein the poly(A) tail comprises more than 30 adenylate residues.

[0133] 7. The RNA according to embodiment 5 or 6, wherein, from the 5'-end to the 3'-end, the structure of the poly(A) tail is: 32 adenylate, a linker sequence, and 74 adenylate.

[0134] 8. The RNA according to any one of embodiments 5-7, wherein the poly(A) tail comprises the nucleotide sequence shown in SEQ ID NO:9.

[0135] 9. The RNA according to any one of embodiments 1-8, which comprises at least one modified nucleotide.

[0136] 10. The RNA according to embodiment 9, which comprises a modification at one or more positions selected from the group consisting of: 5' Cap structure, 5' untranslated sequence, open reading frame, 3' untranslated sequence, and poly(A) tail.

[0137] 11. The RNA according to embodiment 9 or 10, wherein the modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0138] 12. The RNA according to any one of embodiments 1-11, which comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence shown in any one of SEQ ID NO:1-8.

[0139] 13. The RNA according to any one of embodiments 1-12, which comprises the nucleotide sequence shown in any one of SEQ ID NO:1-8.

[0140] 14. A composition, which comprises the RNA according to any one of embodiments 1-13, and a delivery vector.

[0141] 15. The composition according to embodiment 14, wherein the delivery vector comprises liposomes.

[0142] 16. The composition according to embodiment 14, wherein the delivery vector comprises lipid nanoparticles (LNP).

[0143] 17. The composition according to embodiment 16, wherein the lipid nanoparticles comprise a PEGylated lipid, a non-cationic lipid, a sterol, a cationic lipid, or any combination thereof.

[0144] 18. The composition according to embodiment 17, wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and / or the sterol is cholesterol.

[0145] 19. A liposomal nanoparticle formulation encapsulating the RNA according to any one of embodiments 1-13.

[0146] 20. The liposomal nanoparticle formulation according to embodiment 19, which comprises a PEGylated lipid, a non-cationic lipid, a sterol, a cationic lipid, or any combination thereof.

[0147] 21. The liposomal nanoparticle formulation according to embodiment 20, wherein the cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and / or the sterol is cholesterol.

[0148] 22. A vaccine comprising the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, and / or the lipid nanoparticle formulation according to any one of embodiments 19-21.

[0149] 23. The vaccine according to embodiment 22, which is multivalent.

[0150] 24. A pharmaceutical composition comprising the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23, and / or a pharmaceutically acceptable carrier thereof.

[0151] 25. A method for treating and / or preventing a disease or disorder associated with varicella-zoster virus infection, the method comprising administering to a patient in need thereof the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23, and / or the pharmaceutical composition according to embodiment 24.

[0152] Use of the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23 and / or the pharmaceutical composition according to embodiment 24 in the preparation of a medicament for treating and / or preventing a disease or disorder associated with varicella-zoster infection.

[0153] 27. A vector comprising the RNA according to any one of embodiments 1-13.

[0154] 28. A cell comprising the RNA according to any one of embodiments 1-13 and / or the vector according to embodiment 27.

[0155] 29. A kit comprising the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23 and / or the pharmaceutical composition according to embodiment 24.

[0156] 30. A method of generating against varicella-zoster virus, comprising the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23 and / or the pharmaceutical composition according to embodiment 24.

[0157] 31. Use of the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, and / or the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23 and / or the pharmaceutical composition according to embodiment 24 in the preparation of a reagent for generating an antibody against varicella-zoster virus.

[0158] 32. A method of activating immunity, comprising the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23 and / or the pharmaceutical composition according to embodiment 24.

[0159] 33. Use of the RNA according to any one of embodiments 1-13, the composition according to any one of embodiments 14-18, the lipid nanoparticle formulation according to any one of embodiments 19-21, the vaccine according to embodiment 22 or 23 and / or the pharmaceutical composition according to embodiment 24 in the preparation of a reagent for activating immunity.

[0160] 34. The method as described in embodiment 30 or 32, which is an in vitro or ex vivo method.

[0161] Examples

[0162] The technical solutions of the present invention will be described in detail below in conjunction with examples. The reagents and biological materials used below are all commercial products unless otherwise specified.

[0163] The specific experimental procedures of the present invention are as follows: 1. Obtain the gene sequence of varicella-zoster virus; 2. Obtain the protein sequence of varicella-zoster virus; 3. Design the DNA template sequence encoding the antigen protein; 4. Transcribe mRNA in vitro; 5. Encapsulate mRNA with liposome nanoparticles; 6. Immunize mice with the liposome nanoparticle preparation encapsulating mRNA; 7. Detect the antigen-specific IgG antibodies in the mouse serum; 8. Detect the intracellular factor contents of IL2 and IFNγ in CD4+ T cells and the intracellular factor contents of IL2 and IFNγ in CD8+ T cells in the lymphocytes of the mouse spleen after stimulation with the antigen peptide library; 9. Detect the number of lymphocytes secreting IL2 and IFNγ in the mouse spleen after stimulation with the antigen peptide library; 10. Detect the contents of IL2 and IFNγ secreted by the lymphocytes in the mouse spleen after stimulation with the peptide library.

[0164] Example 1 Preparation of mRNA

[0165] After the RNA described in the present invention is sequence-optimized, using the experimental methods well-known to those skilled in the art, the DNA template for in vitro transcription of RNA is cloned into the modified pUC57 vector, transformed into Escherichia coli competent cells, and the positive clone strains are preserved. The strains are cultured on a large scale, the DNA template plasmid is extracted and purified, the plasmid is linearized by enzymatic digestion, and RNA in vitro transcription (IVT) is carried out using the linearized plasmid as a template, and the RNA product is purified. The specific steps are as follows:

[0166] The commissioning company synthesized eight kinds of encoded gene sequences with the glycoprotein gE of varicella-zoster virus VZV as the main body. The gene sequences were codon-optimized (host: human), and the sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. The proteins translated from SEQ ID NO:1, 3, 4, 5, 6, and 7 are the same as some fragments of SEQ ID NO:8. Preferably, the eight genes are synthesized together with the 5'UTR DNA sequence, 3'UTR DNA sequence, and poly(A) tail. The synthetic gene products containing 5'UTR, open reading frame, 3'UTR, and poly(A) tail (the sequences are shown in SEQ ID NO:1-8 respectively) are cloned into the pUC57 vector. After amplifying the vector, it is digested with a restriction endonuclease to obtain a linearized vector. The purified linearized vector is used as a template for in vitro transcription of RNA, and T7 RNA polymerase, CTP, GTP, ATP, m1Ψ (1-methyl-3'-pseudouridylyl)-modified UTP, a Cap analog, and other necessary components well-known to those skilled in the art are added, and incubated at 37 °C for 1-5 hours. After the reaction is completed, it is digested with DNase to remove DNA, and further purified to obtain the mRNA product. The concentration and integrity of the mRNA are detected by methods well-known to those skilled in the art. Eight kinds of mRNAs are prepared and named CVG199, CVG200, CVG201, CVG202, CVG203, CVG204, CVG205, and CVG206, and the nucleic acid sequences are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 respectively.

[0167] Example 2 Detection of mRNA Cell Translatability

[0168] Prepare the mRNA according to the steps described in Example 1. After encapsulating with lipid nanoparticles (LNP) and transfecting into HEK293T cells, the gE expression level after mRNA transfection into cells is detected 24 hours later, as Figure 1 shown Figure 1 (a) shows the gE expression of the CVG199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, and CVG 205 mRNAs in HEK 293T cells. Figure 1(b) shows the gE flow expression results of the mRNAs of CVG199, CVG 200, CVG 201, CVG 204, and CVG 206 in HEK293T cells. Figure 1 (a) and 1(b) together illustrate that CVG199, CVG200, CVG204, and CVG206 have relatively high expression levels on HEK293T cells.

[0169] Example 3 Coating of CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG204, CVG205, and CVG206 mRNAs with Liposome Nanoparticles

[0170] The mRNA obtained in Example 1 was encapsulated: The cationic lipid (MC3), DSPC, cholesterol, and PEG-lipid were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. Lipid nanoparticles (LNPs) were prepared at a total lipid to mRNA weight ratio of approximately 10:1 to 30:1. Briefly, the mRNA of Example 1 and the four-component lipid mixture were made into mRNA-LNP using a microfluidic nanoprecipitation process, in which an aqueous mRNA solution at acidic pH was rapidly mixed with an ethanol solution of the lipid. Then, ethanol in the crude product was removed by tangential flow ultrafiltration (TFF), and then buffer exchange was performed using PBS solution (1x, pH 7.4). Next, the mRNA in the neutralized product was diluted to 0.5 mg / mL using a sucrose solution. Finally, the product was sterile filtered through a 0.22 μm Sartopore PES membrane, and then aliquots were stored at room temperature or frozen at -80 °C (1.0 mL filled).

[0171] Characterization of mRNA-LNP: The particle size, polydispersity (PDI), and Zeta potential of mRNA-LNP were measured by dynamic light scattering (Malvern Nano ZS Zetasizer). The diameter was characterized by Z average. The encapsulation efficiency (E.E.) of mRNA in LNP was defined as the mass ratio of the encapsulated mRNA to the total mRNA in the final mRNA-LNP product. Specific intercalating fluorescent dyes (Quant-iT TM RNA Reagent) were used to quantitatively detect nucleic acids such as mRNA. The mRNA concentration was calculated according to the calibration curve generated using an mRNA standard. The relative proportion of the encapsulated mRNA in mRNA-LNP was determined by the ratio of the fluorescence signals in the absence and presence of a surfactant for the dispersed LNP. The signal in the absence of the surfactant indicates the level of free mRNA, while the signal in the presence of the surfactant serves as a measure of the total mRNA in the sample.

[0172] The particle sizes of mRNA-LNPs encapsulating CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 mRNA after freeze-thawing at -80 °C are as Figure 2 (a) shown, the Z-potential is as Figure 2 (b) shown, and the homogeneity is as Figure 2 (c) shown. The encapsulation efficiency is as Figure 3 . The particle sizes of all encapsulated LNPs are between 70 - 100 nm, the Zeta potential is between -10 mV and 0 mV, and the PDI is between 0.15 - 0.20. The encapsulation rate of mRNA is greater than 90%, as Figure 4 shown. The integrity of the prepared mRNA is greater than 85% except for CVG205 (78.8%).

[0173] Example 4 Animal Immunization

[0174] After filtering the liposome nanoparticle preparations encapsulating CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 mRNA in Example 3, they were intramuscularly injected into C57BL / 6 mice (the experimental protocol is as Figure 5 shown). The control group was injected with DPBS, and the injection dose of the liposome nanoparticle preparation was 5 μg / mouse. Fourteen days after the first immunization, mouse blood was collected to detect the titer of IgG antibodies specific to VZV gE in the serum. The results are as Figure 6 a shown. On the 14th day after the primary immunization, the mice were reinjected with the filtered liposome nanoparticle preparation of Example 3. This was the secondary immunization. One week after the secondary immunization, mouse blood was collected to detect the titer of gE-specific antibodies in the serum. The results are as Figure 6 b shown. The experimental results show that the liposome nanoparticles prepared from CVG 199, CVG 200, CVG 201, CVG 202, CVG 203, CVG 204, CVG 205, and CVG 206 mRNA and liposome components provided by the present invention, when injected into mice, induce the production of high levels of specific IgG antibodies against the membrane protein gE of varicella-zoster virus.

[0175] The CVG199 mRNA, CVG200 mRNA, CVG201 mRNA, CVG204, and CVG206 mRNA with nucleic acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:8 have similar immune effects. After the eight mRNAs are encapsulated by liposome nanoparticles and the resulting preparations are injected into mice, different titers of gE-specific IgG antibodies can be induced in the mice. Compared with other mRNAs or non-codon-optimized mRNAs, the eight mRNAs of the present application can stimulate a stronger immune response and produce higher levels of gE-specific IgG antibodies. In addition, the CVG199 mRNA, CVG200 mRNA, CVG201 mRNA, CVG204 mRNA, and CVG206 mRNA with nucleic acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:6, and SEQ ID NO:8 can elicit cellular immunity in mice. For example, after stimulation with a gE peptide library, the IL2 content in CD4+ cells of CVG206 is higher than that of CVG199, CVG200, CVG201, and CVG204( Figure 7 ). In addition, in mice immunized with CVG199, CVG200, and CVG206 mRNA, there are more lymphocytes in the spleen that can secrete IL2 and IFNγ( Figure 8 ).

[0176] Example 5 Weight changes caused by CVG206 in a mouse model

[0177] In this example, the weight changes of mice before and after immunization with different doses of CVG206 were detected( Figure 9 ).

[0178] Example 6 Humoral immunity in a mouse model induced by CVG206

[0179] In this example, the gE-specific IgG antibodies in the sera of mice were detected three weeks after the first immunization (D21) and two weeks after the second immunization (D35). The level of this IgG antibody showed a dose-dependent correlation( Figure 10 ).

[0180] Example 7 Cellular immunity in a mouse model induced by CVG206

[0181] In this example, the T cell immune responses of mice immunized with different doses of CVG206 were detected. Intracellular staining of cytokines induced by restimulation with the full-length VZV gE peptide library was used to evaluate the gE protein-specific cellular immune function. Spleen cells were collected for analysis one week after the second vaccination. The results showed that CVG206 could induce antigen-specific responses in CD8 and CD4 T cells, as evidenced by the increased intracellular factor IFNγ in CD4+T and CD8+ cells after treatment with the gE peptide library respectively ( Figure 11 Figure 12 ). The results indicated that the cellular immunity was activated in mice immunized with different doses of CVG206.

[0182] Example 8 Cellular and Humoral Immunity in the Priming Mouse Model Induced by CVG206

[0183] In this example, the T cell immune responses of mice immunized with different doses of CVG206 in the priming mouse model were detected (the experimental design is as Figure 14 shown). After the first immunization for four weeks (D28), two weeks (D42) and four weeks (D58) after the second immunization, the results of the gE-specific IgG antibody titers in the sera of the priming mice showed a dose correlation, and the higher the immunization dose, the higher the IgG antibody level ( Figure 15 ). The spleens of the mice were collected for detection four weeks after the second immunization, and the intracellular factor IL2+ and IFNγ contents of CD4+ T cells showed a certain dose dependence on the immunization dose. ( Figure 16 )

[0184] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the content of the embodiments. For those skilled in the art, various changes and modifications can be made within the scope of the concept of the technical solution of the present invention, and any changes and modifications made are within the protection scope of the present invention.

[0185] The sequences used in the present invention are shown in Table 1.

[0186] Table 1

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

Claims

1. An RNA encoding varicella-zoster virus (VZV) glycoprotein E (gE) or a variant thereof, characterized in that, The RNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 11-18.

2. The RNA according to claim 1, characterized in that, The RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 11-18.

3. The RNA according to claim 1 or 2, characterized in that, The RNA is mRNA.

4. The RNA according to any one of claims 1-3, characterized in that, The nucleotide sequence of the RNA is codon-optimized.

5. The RNA according to any one of claims 1-4, characterized in that, The RNA further comprises one or more structures selected from the group consisting of: 5' Cap structure, 5' untranslated sequence, open reading frame, 3' untranslated sequence, and poly(A) tail.

6. The RNA according to claim 5, characterized in that, The poly(A) tail comprises more than 30 adenylates.

7. The RNA according to claim 5 or 6, characterized in that, The structure of the poly(A) tail from the 5’ end to the 3’ end is: 32 adenylates, a linker sequence, and 74 adenylates.

8. The RNA according to any one of claims 5-7, characterized in that, The poly(A) tail comprises the nucleotide sequence shown in SEQ ID NO:

9.

9. The RNA according to any one of claims 1-8, characterized in that, The RNA comprises at least one modified nucleotide.

10. The RNA according to claim 9, characterized in that, The modified nucleotide is at one or more positions selected from the group consisting of: 5' Cap structure, 5' untranslated sequence, open reading frame, 3' untranslated sequence, and poly(A) tail.

11. The RNA according to claim 9 or 10, characterized in that, The modification is selected from the group consisting of: pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxypseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyluridine.

12. The RNA according to any one of claims 1-11, characterized in that, The RNA comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 1-8.

13. The RNA according to any one of claims 1-12, characterized in that, The RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-8.

14. A composition, characterized in that, The composition comprises the RNA according to any one of claims 1-13 and a delivery vector.

15. The composition according to claim 14, characterized in that, The delivery vector comprises liposomes.

16. The composition according to claim 14, characterized in that, The delivery vector comprises lipid nanoparticles (LNPs).

17. The composition according to claim 16, characterized in that, The lipid nanoparticles comprise PEGylated lipids, non-cationic lipids, sterols, cationic lipids, or any combination thereof.

18. The composition according to claim 17, characterized in that, The cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and / or the sterol is cholesterol.

19. A liposomal nanoparticle formulation, characterized in that, The lipid nanoparticle formulation coats the RNA according to any one of claims 1-13.

20. The liposomal nanoparticle formulation according to claim 19, characterized in that, The lipid nanoparticle formulation comprises PEGylated lipids, non-cationic lipids, sterols, cationic lipids, or any combination thereof.

21. The liposome nanoparticle preparation according to claim 20, characterized in that The cationic lipid is an ionizable cationic lipid, the non-cationic lipid is a neutral lipid, and / or the sterol is cholesterol.

22. A vaccine, characterized in that The vaccine comprises the RNA according to any one of claims 1-13, the composition according to any one of claims 14-18, and / or the liposomal nanoparticle formulation according to any one of claims 19-21.

23. The vaccine according to claim 22, characterized in that The vaccine is multivalent.

24. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the RNA according to any one of claims 1-13, the composition according to any one of claims 14-18, the liposomal nanoparticle formulation according to any one of claims 19-21, the vaccine according to claim 22 or 23, and / or a pharmaceutically acceptable carrier thereof.

25. Use of the RNA according to any one of claims 1-13, the composition according to any one of claims 14-18, the liposome nanoparticle preparation according to any one of claims 19-21, the vaccine according to claim 22 or 23, and / or the pharmaceutical composition according to claim 24 in the preparation of a drug for treating and / or preventing diseases or disorders related to varicella-zoster virus infection.

26. A vector, characterized in that The carrier contains the RNA according to any one of claims 1-13.

27. A cell, characterized in that The cell contains the RNA according to any one of claims 1-13 and / or the carrier according to claim 26.

28. A kit, characterized in that The kit comprises the RNA according to any one of claims 1-13, the composition according to any one of claims 14-18, the liposomal nanoparticle formulation according to any one of claims 19-21, the vaccine according to claim 22 or 23, and / or the pharmaceutical composition according to claim 24.

29. A method for generating antibodies against varicella-zoster virus, which comprises administering the RNA according to any one of claims 1-13, the composition according to any one of claims 14-18, the liposome nanoparticle preparation according to any one of claims 19-21, the vaccine according to claim 22 or 23, and / or the pharmaceutical composition according to claim 24.

30. The method according to claim 29, characterized in that The method is an in vitro or ex vivo method.

31. Use of the RNA according to any one of claims 1-13, the composition according to any one of claims 14-18, the liposome nanoparticle preparation according to any one of claims 19-21, the vaccine according to claim 22 or 23, and / or the pharmaceutical composition according to claim 24 in the preparation of a reagent for generating antibodies against varicella-zoster virus.

32. A method for activating immunity, which comprises administering the RNA according to any one of claims 1-13, the composition according to any one of claims 14-18, the liposome nanoparticle preparation according to any one of claims 19-21, the vaccine according to claim 22 or 23, and / or the pharmaceutical composition according to claim 24.

33. The method according to claim 32, characterized in that The method is an in vitro or ex vivo method.

34. Use of an RNA according to any one of claims 1-13, a composition according to any one of claims 14-18, a liposomal nanoparticle formulation according to any one of claims 19-21, a vaccine according to claim 22 or 23 and / or a pharmaceutical composition according to claim 24 in the preparation of a reagent for activating immunity.

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