Varicella zoster virus vaccine as well as preparation method and application thereof

Abstract

The present disclosure provides a vaccine against herpes zoster comprising or encoding a varicella zoster virus (VZV) antigen, capable of eliciting an effective neutralizing antibody response against the varicella zoster virus (VZV).

Description

Technical Field

[0001] The present disclosure relates to the field of gene medicine technology, and in particular to a nucleic acid vaccine encoding varicella zoster virus antigen. Background Art

[0002] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] The complete varicella-zoster virus consists of a core, a capsid, an inner membrane, and an envelope. There is only one serotype of varicella-zoster virus. The genome has 71 genes, encoding 67 different proteins, including 7 glycoproteins, named gE, gB, gH, gI, gC, gK, and gL. In infected cells, glycoproteins gE, gB, and gH are extremely abundant, and these glycoproteins are also present in the cell membrane of the virus body. The virus manifests as symptoms of chickenpox during the initial infection. After the virus is suppressed, it lurks in the human body, causing recurrent infections when the human immune system is low, manifested as herpes zoster, so it is called varicella-zoster virus (VZV). The main targets of infection are newborns, adults, and immunocompromised patients, and postherpetic neuralgia is a common complication. Humans are its only natural host, and the skin is the main target organ of the virus.

[0004] In 2006, the U.S. Food and Drug Administration (FDA) approved the use of Merck's live attenuated zoster virus (oka strain) vaccine for people over 60 years old. In order to further improve the protective efficiency of the vaccine in the elderly and replace the possible safety issues of the live attenuated vaccine, GSK designed a subunit vaccine HZ / su based on the oka strain surface glycoprotein gE after Merck's Zostavax. Since the vaccine only contains gE as a protein component, it is safer and can be used in pregnant women and immunocompromised people.

[0005] The inventors have realized that the traditional vaccine for preventing varicella zoster virus infection and recurrence is mainly a live attenuated varicella vaccine (oka strain), which has a certain protective effect, but has the disadvantages of safety and difficulty in storage and transportation, and the immune effect is not ideal in the elderly, with a protection rate of only about 50%. At the same time, the traditional vaccine lacks a cellular immune response, which leads to a high recurrence rate of VZV. Summary of the invention

[0006] The present disclosure provides a varicella-zoster virus vaccine, which contains or encodes a varicella-zoster virus (VZV) vaccine antigen and can induce an effective neutralizing antibody response against the varicella-zoster virus (VZV).

[0007] Some aspects of the present disclosure provide a varicella-zoster virus vaccine, which comprises or encodes a varicella-zoster virus (VZV) antigen, and the varicella-zoster virus vaccine is selected from a nucleic acid vaccine, a polypeptide vaccine, or a viral vaccine.

[0008] In some embodiments, the antigen is selected from VZV glycoprotein gE, VZV glycoprotein gB, VZV glycoprotein gH, VZV glycoprotein gl, VZV glycoprotein gC, VZV glycoprotein gK, and VZV glycoprotein gL.

[0009] In some embodiments, the antigen is a VZV glycoprotein gE variant; the amino acid sequence of the VZV glycoprotein gE variant includes at least one of a Y569A substitution, a Y582A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution.

[0010] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution.

[0011] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or intervening amino acid deletion at amino acids 570-592, 594, 597, or 599-623.

[0012] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and / or includes at least one consecutive or intervening amino acid deletion of amino acids 570 to 592, 594, 597, 599 to 623.

[0013] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes Y569A substitution, S593A substitution, S595A substitution, T596A substitution and T598A substitution, and at least one consecutive or intervening amino acid deletion from positions 570 to 592, for example, it may be but is not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 consecutive or intervening amino acid deletions from positions 570 to 592.

[0014] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and includes a deletion of amino acid position 594.

[0015] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and includes a deletion of amino acid position 597.

[0016] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution and a T598A substitution, and at least one consecutive or intervening amino acid deletion including amino acids 599 to 623, for example, it can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 consecutive or intervening amino acid deletions including amino acids 599 to 623.

[0017] In some embodiments, the varicella-zoster virus vaccine encodes an antigen of the varicella-zoster virus and the varicella-zoster virus vaccine is a nucleic acid vaccine.

[0018] In some embodiments, the nucleic acid vaccine comprises a nucleic acid molecule; the nucleic acid molecule includes a DNA molecule and / or an RNA molecule.

[0019] In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule.

[0020] In some embodiments, the RNA molecule comprises mRNA or circular RNA.

[0021] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b):

[0022] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 8 to 17;

[0023] (b) An mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (a).

[0024] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 70%, and the CAI value of the mRNA molecule is 0.91 to 0.97.

[0025] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 60%, and the CAI value of the mRNA molecule is 0.91 to 0.95.

[0026] In some embodiments, the nucleic acid molecule has any one of SEQ ID No. 8-17, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID No. 8-17.

[0027] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b):

[0028] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 8 to 12;

[0029] (b) An mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (a).

[0030] In some embodiments, the nucleic acid molecule has a nucleotide sequence encoding VZV glycoprotein gE as shown in any one of SEQ ID No. 8-12, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO. 8-17.

[0031] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b):

[0032] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID No. 8;

[0033] (b) An mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (a).

[0034] In some embodiments, the nucleic acid molecule has any one of SEQ ID No. 8, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID No. 8.

[0035] In some embodiments, the mRNA consists of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail from the 5' end to the 3' end.

[0036] In some embodiments, the 5' end cap is selected from ARCA, mCAP, dmCAP, m7G(5"")ppp(5"")(2""OMeA)pG, tmCAP, m7(3""OMeG)(5"")ppp(5"")(2""OMeA)pG, m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG, dmCAP or m7G(5"")ppp(5"")(2""OMeG)pG.

[0037] In some embodiments, the 5' cap is m7Gppp(5')(2'-OMeA)pG.

[0038] In some embodiments, the length of the 5'UTR is preferably 10 to 200 nucleotides.

[0039] In some embodiments, the 5'UTR is 15 to 100 nucleotides in length.

[0040] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1-3.

[0041] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1.

[0042] In some embodiments, the 3'UTR sequence is as shown in Seq ID NO. 4-6.

[0043] In some embodiments, the 3'UTR sequence is as shown in Seq ID NO.4.

[0044] In some embodiments, one or more uridines in the mRNA are replaced with modified nucleosides;

[0045] In some embodiments, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0046] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0047] In some embodiments, the mRNA includes at least one of a nucleotide sequence encoding a cis-regulatory element and a nucleotide sequence encoding a multimerization element.

[0048] In some embodiments, the amino acid sequence of the cis-regulatory element is as shown in Seq ID NO.18; preferably, the nucleotide sequence encoding the cis-regulatory element is as shown in Seq ID NO.19.

[0049] In some embodiments, the amino acid sequence of the multimerization element is shown as Seq ID NO.20 to Seq ID NO.24.

[0050] In some embodiments, the amino acid sequence of the multimerization element is as shown in Seq ID NO.24.

[0051] In some embodiments, the nucleotide sequence encoding the multimerization element can also be shown as Seq ID NO.25-29.

[0052] In some embodiments, the mRNA molecule comprises a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0053] In some embodiments, the varicella-zoster virus vaccine comprises a delivery formulation; preferably, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0054] In some embodiments, it includes: mixing the mRNA and a delivery formulation to form a varicella-zoster virus vaccine; the delivery formulation includes lipid nanoparticles or cationic liposomes.

[0055] In some embodiments, the coding region nucleic acid sequence of the mRNA encodes varicella-zoster virus VZV glycoprotein gE, and the amino acid sequence of the varicella-zoster virus VZV glycoprotein gE includes Y569A substitution, S593A substitution, S595A substitution, T596A substitution and T598A substitution, and / or includes at least one consecutive or intervening amino acid deletion of amino acids at positions 570 to 592, 594, 597, and 599 to 623. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0057] Figure 1 A schematic diagram of the VZV glycoprotein gE is shown.

[0058] Figure 2The expression of the mRNA encoding VZV glycoprotein gE in Example 4 in cells and on the membrane surface is shown.

[0059] Figure 3 A schematic diagram of the mouse experiment evaluation process is shown.

[0060] Figure 4 The TNF-α+CD4+ and TNF-α+CD8+ T cell response levels in mice induced by the varicella-zoster virus vaccine of Example 6 are shown.

[0061] Figure 5 The varicella-zoster virus vaccine of Example 6 induces the secretion of IFN-γ and IL-2 by mouse spleen lymphocytes.

[0062] Figure 6 The varicella-zoster virus vaccine of Example 6 induces IL-10 secretion by mouse spleen lymphocytes.

[0063] Figure 7 The varicella-zoster virus vaccine of Example 6 induces humoral immune responses in mice.

[0064] Figure 8 The intracellular expression of mRNA encoding VZV glycoprotein gE of Example 7 is shown.

[0065] Fig. 9 The membrane surface expression of mRNA encoding VZV glycoprotein gE in Example 7 is shown.

[0066] Fig.10 The TNF-α+CD4+T cell response level of mice induced by the varicella-zoster virus vaccine of Example 8 is shown.

[0067] Fig.11 The varicella-zoster virus vaccine of Example 8 induces IFN-γ secretion in mouse spleen lymphocytes.

[0068] Fig.12 The varicella-zoster virus vaccine of Example 8 induces IL-10 secretion by mouse spleen lymphocytes.

[0069] Fig.13 The humoral immune response of the varicella-zoster virus vaccine of Example 8 in mice is shown.

[0070] Fig.14 The membrane surface expression of mRNA encoding VZV glycoprotein gE in Example 9 is shown.

[0071] Fig.15The TNF-α+CD4+ and TNF-α+CD8+ T cell response levels in mice induced by the varicella-zoster virus vaccine of Example 10 are shown.

[0072] Fig.16 The varicella-zoster virus vaccine of Example 10 induces the secretion of IFN-γ and IL-2 by mouse spleen lymphocytes.

[0073] Fig.17 The varicella-zoster virus vaccine of Example 10 induces the secretion of IL-4 and IL-10 by mouse spleen lymphocytes.

[0074] Fig.18 The humoral immune response of the varicella-zoster virus vaccine of Example 10 in mice is shown. DETAILED DESCRIPTION

[0075] The varicella or herpes zoster vaccines currently available or about to be available on the market are all developed based on the genome of the VZV-oka strain, which was isolated and attenuated by the Institute of Microbial Diseases of Osaka University, Japan in 1992. Because of its mild clinical reaction and good immunogenicity, the VZV oka strain is currently the only attenuated strain approved by the WHO for vaccine production. The strain used in the present disclosure is the VZV oka strain.

[0076] VZV glycoprotein gE protein is composed of 623 amino acids encoded by ORF68 gene, and exists on the surface of virus particles, as well as on the surface and cytoplasm of infected cells. The protein molecule can be divided into the extracellular region (1-543), the transmembrane region (544-568) and the intracellular region (569-623). In the convalescent serum of patients with herpes zoster, VZV antibodies are mainly directed against three viral glycoproteins, gE, gB and gH, among which gE is the main one. Figure 1 Schematic diagram of VZV glycoprotein gE. VZV glycoprotein gE includes a signal peptide sequence consisting of 21 amino acids ("SP" in the figure, positions 1 to 21), a TM sequence consisting of 22 amino acids ("TM" in the figure, positions 544 to 568), and an AC sequence consisting of 55 amino acids ("AC" in the figure, positions 569 to 623).

[0077] The VZV glycoprotein gE variant described in the present disclosure is a VZV glycoprotein gE that has been artificially mutated and modified, and the mutation and modification (including but not limited to amino acid residue substitution, amino acid residue insertion and / or addition, amino acid residue deletion and covalent modification of the amino acid sequence, etc.) is obtained by mutation and modification of the VZV glycoprotein gE wild type to obtain an amino acid sequence that conforms to VZV glycoprotein gE.

[0078] In some embodiments, the present disclosure provides a varicella-zoster virus vaccine, which comprises or encodes a varicella-zoster virus (VZV) vaccine antigen and can induce an effective neutralizing antibody response against varicella-zoster virus (VZV).

[0079] Some aspects of the present disclosure provide a varicella-zoster virus vaccine, which comprises or encodes a varicella-zoster virus (VZV) antigen, and the varicella-zoster virus vaccine is selected from a nucleic acid vaccine, a polypeptide vaccine, or a viral vaccine.

[0080] In some embodiments, the antigen is selected from VZV glycoprotein gE, VZV glycoprotein gB, VZV glycoprotein gH, VZV glycoprotein gl, VZV glycoprotein gC, VZV glycoprotein gK, and VZV glycoprotein gL.

[0081] In some embodiments, the antigen is VZV glycoprotein gE.

[0082] In some embodiments, the antigen is a VZV glycoprotein gE variant.

[0083] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of a Y569A substitution, a Y582A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution.

[0084] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution.

[0085] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or intervening amino acid deletion at amino acids 570-592, 594, 597, or 599-623.

[0086] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and / or includes at least one consecutive or intervening amino acid deletion of amino acids 570 to 592, 594, 597, 599 to 623.

[0087] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes Y569A substitution, S593A substitution, S595A substitution, T596A substitution and T598A substitution, and at least one consecutive or intervening amino acid deletion from positions 570 to 592, for example, it may be but is not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 consecutive or intervening amino acid deletions from positions 570 to 592.

[0088] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and includes a deletion of amino acid position 594.

[0089] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and includes a deletion of amino acid position 597.

[0090] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution and a T598A substitution, and at least one consecutive or intervening amino acid deletion including amino acids 599 to 623, for example, it can be, but is not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 consecutive or intervening amino acid deletions including amino acids 599 to 623.

[0091] The positions of the amino acid sequences described in the present disclosure, such as Y569A substitution, S593A substitution, S595A substitution, T596A substitution, T598A substitution, "amino acids 570 to 592" and the like are located based on the full-length amino acid sequence of the VZV glycoprotein gE wild type (as shown in Seq ID NO.30). For example, the Y569A substitution is based on the VZV glycoprotein gE wild type, and the Y amino acid residue at position 569 of the full-length amino acid of the VZV glycoprotein gE wild type is replaced with an A amino acid residue. For another example, "amino acids 570 to 592" are amino acids 570 to 592 of the VZV glycoprotein gE wild type (as shown in Seq ID NO.31).

[0092] The "continuous or intermittent amino acid deletions" described in the present disclosure refer to "continuous amino acid deletions" or "intermittent amino acid deletions". For example, n consecutive amino acid deletions are amino acids at positions (m), (m+1), ... (m+n-1), where m, m+1, ... m+n-1 are consecutive natural numbers. For another example, n intermittent amino acid deletions are amino acids at positions (m1), (m2), ... (mn), where m1, m2, ... m n are n increasing natural numbers, and m1, m2, ...m n At least 2 adjacent natural numbers are discontinuous and differ by at least 2.

[0093] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is as shown in Seq ID NO.7, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO.7, for example, it can be but is not limited to an amino acid sequence that is at least 80%, 85%, 90%, 95% or 98% identical to SeqID NO.7.

[0094] In some embodiments, the varicella-zoster virus vaccine is a nucleic acid vaccine; the nucleic acid vaccine comprises a nucleic acid molecule; the nucleic acid molecule comprises a DNA molecule and / or an RNA molecule.

[0095] In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule.

[0096] In some embodiments, the RNA molecule comprises mRNA or circular RNA.

[0097] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b):

[0098] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 8 to 17;

[0099] (b) An mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (a).

[0100] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 70%, and the CAI value of the mRNA molecule is 0.91 to 0.97.

[0101] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 60%, and the CAI value of the mRNA molecule is 0.91 to 0.95.

[0102] The GC base pair percentage content described in the present disclosure is the percentage content of the number of GC bases in the open reading frame of the mRNA to the total number of bases in the open reading frame. For example, the open reading frame of RNA [M] contains 120 bases, of which the number of GC bases is 60, then the GC content of the RNA [M] is 50%.

[0103] The CAI value (Codon Adaptivity Index) described in the present disclosure refers to the degree of consistency between the usage frequency of synonymous codons in the coding region and the optimal codon, and can be used to evaluate the expression level of the exogenous gene in the host. The value ranges from 0 to 1: the higher the CAI value, the higher the expression level of the exogenous gene in the host.

[0104] In some embodiments, the nucleic acid molecule has any one of SEQ ID No. 8 to 17, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO. 8 to 17, for example, it can be but is not limited to an mRNA molecule comprising a nucleotide sequence encoding VZV glycoprotein gE that is at least 80%, 85%, 90%, 95% or 98% identical to Seq ID NO. 8 to 17.

[0105] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b):

[0106] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 8 to 12;

[0107] (b) An mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (a).

[0108] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 70%, and the CAI value of the mRNA molecule is 0.91 to 0.97.

[0109] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 60%, and the CAI value of the mRNA molecule is 0.91 to 0.95.

[0110] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No. 8 to 12, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO. 8 to 17, for example, it can be but is not limited to an mRNA molecule comprising a nucleotide sequence encoding VZV glycoprotein gE that is at least 80%, 85%, 90%, 95% or 98% identical to Seq ID NO. 8 to 17.

[0111] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b):

[0112] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID No. 8;

[0113] (b) An mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (a).

[0114] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 70%, and the CAI value of the mRNA molecule is 0.91 to 0.97.

[0115] In some embodiments, the GC base pair percentage content of the mRNA molecule is 55% to 60%, and the CAI value of the mRNA molecule is 0.91 to 0.95.

[0116] In some embodiments, the nucleic acid molecule has any one of SEQ ID No.8, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO.8, for example, it can be but is not limited to an mRNA molecule comprising a nucleotide sequence encoding VZV glycoprotein gE that is at least 80%, 85%, 90%, 95% or 98% identical to Seq ID NO.8.

[0117] In some embodiments, nucleic acid molecules can optimize mRNA sequences by sequence optimization means to improve properties related to expression efficacy after in vivo administration: for example, improving mRNA stability, increasing translation efficacy in target tissues, reducing the number of truncated proteins expressed, improving the folding of expressed proteins or preventing their misfolding, reducing the toxicity of expression products, reducing cell death caused by expression products, increasing and / or reducing protein aggregation, and obtaining mRNA with improved properties. The purpose of sequence optimization also includes: optimizing the formulation and delivery characteristics of nucleic acid-based therapeutic agents while maintaining structural and functional integrity; overcoming expression thresholds; increasing expression rates; half-life and / or protein concentrations; optimizing protein localization; and avoiding adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization methods include: (1) codon optimization based on codon frequencies in specific organs and / or host organisms to ensure proper folding and appropriate expression; (2) adjustment of G / C content to increase mRNA stability or reduce secondary structure; (3) minimization of tandemly repeated codons or base runs that may impair gene construction or expression; (4) customization of transcription and translation control regions; and (5) reduction or elimination of problematic secondary structures within polynucleotides.

[0118] The "sequence identity" between two nucleotide sequences indicates the percentage of identical nucleotides between the sequences. The "sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0119] The term "% identity" or similar terms refers to the percentage of identical nucleotides or amino acids between the sequences to be compared under optimal alignment. This percentage is purely statistical, and the differences between the two sequences may (but are not necessarily) randomly distributed over the entire length of the sequences to be compared. The comparison of two sequences is usually performed by comparing the sequences with respect to fragments or "comparison windows" after optimal alignment to identify local regions of corresponding sequences.

[0120] In some embodiments, based on the mRNA open reading frame sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding circular RNA open reading frame sequence, and according to the contents of the open literature such as CN202180048567.4, a circular RNA complete sequence capable of encoding the same amino acid sequence is prepared. In some embodiments, based on the mRNA sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding DNA sequence (e.g., uracil is converted to thymine). Similarly, based on the DNA sequence provided, a person of ordinary skill in the art will obtain the corresponding RNA sequence (e.g., thymine is converted to uracil). In some embodiments, based on the RNA or DNA sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.

[0121] The open reading frame (ORF) described in the present disclosure is a continuous stretch of DNA or RNA that begins with a start codon (such as ATG or AUG) and ends with a stop codon (such as TAA, TAG or TGA, or UAA, UAG or UGA).

[0122] In some embodiments, the mRNA described in the present disclosure consists of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail in sequence from the 5' end to the 3' end.

[0123] In some embodiments, the 5' end cap is selected from ARCA, mCAP, dmCAP, m7G(5"")ppp(5"")(2""OMeA)pG, tmCAP, m7(3""OMeG)(5"")ppp(5"")(2""OMeA)pG, m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG, dmCAP or m7G(5"")ppp(5"")(2""OMeG)pG;

[0124] In some embodiments, the 5' cap is m7Gppp(5')(2'-OMeA)pG.

[0125] In some embodiments, the length of the 5'UTR is preferably 10 to 200 nucleotides.

[0126] In some embodiments, the 5'UTR is 15 to 100 nucleotides in length.

[0127] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1-3.

[0128] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1.

[0129] In some embodiments, the 3'UTR sequence is as shown in Seq ID NO. 4-6.

[0130] In some embodiments, the 3'UTR sequence is as shown in Seq ID NO.4.

[0131] In some embodiments, one or more uridines in the mRNA are replaced with modified nucleosides.

[0132] In some embodiments, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0133] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0134] In some embodiments, the mRNA includes at least one of a nucleotide sequence encoding a cis-regulatory element and a nucleotide sequence encoding a multimerization element.

[0135] In some embodiments, the amino acid sequence of the cis-regulatory element is as shown in Seq ID NO.18.

[0136] In some embodiments, the nucleotide sequence encoding the cis-regulatory element is as shown in Seq ID NO.19.

[0137] The "cis-regulatory element" described in the present disclosure refers to a region in a nucleic acid molecule that can promote the translation of a nucleic acid coding sequence (e.g., via cap-dependent or cap-independent translation) into a protein or polypeptide. Cis-regulatory elements are generally located in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhance the translation level of a coding sequence located upstream or downstream. For example, a cis-regulatory element in the 5'-UTR of a nucleic acid molecule can be located between the promoter and the start codon of the nucleic acid molecule. The cis-regulatory element sequence disclosed before the application date of the present disclosure can be introduced into the present disclosure.

[0138] In some embodiments, the amino acid sequence of the multimerization element is shown as Seq ID NO.20 to Seq ID NO.24.

[0139] In some embodiments, the amino acid sequence of the multimerization element is as shown in Seq ID NO.24.

[0140] In some embodiments, the nucleotide sequence encoding the multimerization element can also be shown as Seq ID NO.25-29.

[0141] The 5' untranslated region (UTR) described in the present disclosure refers to a sequence located immediately upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome) of an mRNA that does not encode a polypeptide. When an RNA transcript is produced, the 5'UTR may include a promoter sequence. Such promoter sequences are known in the art. The RNA sequence of the 5'UTR is shown in one of Seq ID NO.1, Seq ID NO.2, or Seq ID NO.3, or a 5'UTR sequence disclosed before the filing date of the present disclosure may also be introduced into the present disclosure.

[0142] The 3' untranslated region (UTR) described in the present disclosure refers to a sequence located downstream of the stop codon of an mRNA that does not encode a polypeptide. The RNA sequence of the 3'UTR is shown in one of Seq ID NO.4, Seq ID NO.5 or Seq ID NO.6, or a 3'UTR sequence disclosed before the filing date of the present disclosure may be introduced into the present disclosure.

[0143] The poly (A) tail described in the present disclosure is a sequence located downstream of the 3'UTR of mRNA containing multiple consecutive adenosine monophosphates. The poly (A) tail may contain 10 to 300 adenosine monophosphates. In cells and / or in vivo, the poly (A) tail is used to protect mRNA from enzymatic degradation and to help transcription termination, and / or mRNA export and translation from the nucleus.

[0144] In some embodiments, the mRNA molecule comprises a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0145] In some embodiments, the mRNA described in the present disclosure is a self-replicating mRNA, which carries a sequence capable of expressing RNA polymerase (RNA-dependent RNA polymerase, RdRP). Specifically, the sequence design content of self-replicating mRNA in Chinese patent CN202110424124.2 is introduced into the present disclosure.

[0146] In some embodiments, the varicella-zoster virus vaccine comprises a delivery formulation.

[0147] In some embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0148] In some embodiments, the components of the lipid nanoparticles include at least one of a protonatable cationic lipid, a structural lipid, a helper lipid, and a surfactant.

[0149] In some embodiments, the components of the lipid nanoparticles include, by weight: 40 to 60 parts of protonable cationic lipids, 20 to 40 parts of structural lipids, 10 to 30 parts of auxiliary lipids, and 0.5 to 5 parts of surfactants.

[0150] In some embodiments, the components of the lipid nanoparticles include, by weight: 40 to 60 parts of protonable cationic lipids, 10 to 40 parts of structural lipids, 10 to 30 parts of auxiliary lipids, and 0.5 to 5 parts of surfactants.

[0151] In some embodiments, the protonatable cationic lipid is selected from at least one of: Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA.

[0152] In some embodiments, the structured lipid comprises cholesterol and / or a cholesterol derivative.

[0153] In some embodiments, the helper lipid includes at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0154] In some embodiments, the surfactant includes at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0155] In some embodiments, the liposome nanoparticles include, by mole percentage, 20% to 50% of cationic lipids, such as but not limited to 20%, 25%, 30%, 35%, 40%, 45% or 50%; 10% to 50% of DSCP, such as but not limited to 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%; 5% to 20% of cholesterol, such as but not limited to 5%, 10%, 15% or 20%; and 1% to 5% of PEG-DMG, such as but not limited to 1%, 1.5%, 2%, 3%, 4% or 5%.

[0156] In some embodiments, the liposome nanoparticles include 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-DMG by mole percentage.

[0157] In some embodiments, the present disclosure provides a method for preparing a varicella-zoster virus vaccine as described in any of the aforementioned embodiments, characterized in that it comprises: mixing the mRNA and a delivery preparation to form a varicella-zoster virus vaccine; the delivery preparation comprises lipid nanoparticles or cationic liposomes.

[0158] In some embodiments, in the method for preparing the varicella-zoster virus vaccine, mRNA is dissolved in a buffer to obtain an aqueous phase, each lipid component of the liposome nanoparticle is measured and dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed and then the organic phase is removed to obtain the varicella-zoster virus vaccine.

[0159] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2 to 4, preferably 1:3. In some embodiments, the buffer comprises a citrate buffer or sodium acetate, preferably a citrate buffer. In some embodiments, the pH of the buffer is 3 to 7, preferably 4. In some embodiments, the concentration of mRNA in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL.

[0160] In some embodiments, the organic solvent is selected from C1 to C4 low-carbon alcohols, preferably anhydrous ethanol. In some embodiments, the concentration of the lipid component in the organic phase is 5 mg / mL to 7 mg / mL, preferably 6 mg / mL. In some embodiments, microfluidics is used to mix the aqueous phase and the organic phase, and the organic solvent is filtered by tangential flow. Preferably, the flow rate of the microfluidics is> 3 ml / min, and more preferably 12 ml / min.

[0161] In some embodiments, the step of concentration is further included after mixing, wherein the final concentration of the mRNA is 50 μg / mL to 200 μg / mL, preferably 100 μg / mL.

[0162] In some embodiments, the diameter of the lipid nanoparticle is less than about 200 nm. In some embodiments, the diameter of the lipid nanoparticle is less than about 150 nm. In some embodiments, the diameter of the lipid nanoparticle is less than 100 nm. In some embodiments, the diameter of the lipid nanoparticle is about 55 nm to about 90 nm.

[0163] In some embodiments, the varicella-zoster virus vaccine is a polypeptide vaccine. The polypeptide vaccine is an immune composition prepared by chemical synthesis technology or genetic engineering technology according to the amino acid sequence of a known or predicted segment of an antigenic epitope in the varicella-zoster virus antigen gene. In some embodiments, the polypeptide vaccine described in the present disclosure is an immune composition prepared by chemical synthesis technology according to the amino acid sequence of the varicella-zoster virus VZV glycoprotein gE, combined with artificial mutation and modification. In some embodiments, the polypeptide vaccine described in the present disclosure is obtained by fermentation according to the amino acid sequence of the varicella-zoster virus VZV glycoprotein gE, combined with artificial mutation and modification, and then by genetic engineering technology (such as constructing genetically engineered bacteria by means of genetic engineering technology, etc.).

[0164] In some embodiments, the varicella zoster virus vaccine is a viral vaccine. In some embodiments, the viral vaccine described in the present disclosure loads the varicella zoster virus VZV glycoprotein gE gene into the human body through harmless microorganisms to induce the body's immune system to make an immune response. Important viruses used in induced cellular immunity tests include variants of bovine disease virus, polio virus, etc.

[0165] In some embodiments, the present disclosure provides an isolated mRNA, the coding region nucleic acid sequence of which encodes varicella-zoster virus VZV glycoprotein gE, and the amino acid sequence of varicella-zoster virus VZV glycoprotein gE includes at least one of Y569A substitution, Y582A substitution, S593A substitution, S595A substitution, T596A substitution, and T598A substitution.

[0166] In some embodiments, the present disclosure provides an isolated mRNA, wherein the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution.

[0167] In some embodiments, the present disclosure provides an isolated mRNA, wherein the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or intervening amino acid deletion of amino acids 570-592, 594, 597, 599-623.

[0168] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and / or includes at least one consecutive or intervening amino acid deletion of amino acids 570 to 592, 594, 597, 599 to 623.

[0169] In some embodiments, the isolated mRNA comprises an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) comprises any one of (a) to (b):

[0170] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 8 to 17;

[0171] (b) An mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or several nucleotides in the nucleotide sequence defined in (a).

[0172] In some embodiments, the GC base pair percentage content of the isolated mRNA is 55% to 70%, and the CAI value of the mRNA molecule is 0.91 to 0.97.

[0173] In some embodiments, the present disclosure provides an isolated DNA, characterized in that the DNA is obtained by reverse transcription of the mRNA or mRNA molecule nucleic acid isolated as described in any of the above items, or the DNA is a sequence or mRNA molecule that can transcribe the mRNA isolated as described in any of the above items.

[0174] In some embodiments, the isolated DNA is isolated linear DNA or isolated circular DNA.

[0175] In some embodiments, the present disclosure provides a biological material, comprising any one of an expression cassette, a vector, an engineered bacterium or a cell line, wherein the biological material contains or expresses the mRNA isolated as described in any of the foregoing or the DNA isolated as described in any of the foregoing.

[0176] In some embodiments, the present disclosure provides a method comprising administering to a subject an effective amount of at least one of the shingles vaccine described in any of the foregoing, the isolated mRNA described in any of the foregoing, the isolated DNA described in any of the foregoing, or the biological material described in any of the foregoing, so as to induce a neutralizing antibody response against varicella-zoster virus in the subject.

[0177] In some embodiments, the method comprises administering to the subject at least once an effective amount of at least one of the shingles vaccine described in any of the preceding claims, the isolated mRNA described in any of the preceding claims, the isolated DNA described in any of the preceding claims, or the biological material described in any of the preceding claims.

[0178] In some embodiments, the time interval between the first administration and the second administration in the at least two administrations is no less than 14 days.

[0179] In some embodiments, the effective amount disclosed herein is as low as 40 μg, 30 μg, 25 μg, 20 μg, 15 μg, 10 μg, 5 μg, 3 μg or 1 μg of any of the aforementioned shingles vaccines, any of the aforementioned isolated mRNA or any of the aforementioned isolated DNA, or any of the aforementioned biological materials.

[0180] In some embodiments, the present disclosure provides a method of administering to a subject at least one of the herpes zoster vaccine, the isolated mRNA described in any of the above, the isolated DNA described in any of the above, or the biological material described in any of the above, to induce an immune response against VZV in the subject, wherein the immune response in the subject is equivalent to the immune response in the subject vaccinated with a traditional vaccine against VZV at a dose level 2 to 100 times higher than that of the RNA vaccine.

[0181] In some embodiments, the immune response to VZV includes production of cytokines in lymphocytes.

[0182] In some embodiments, the lymphocytes are CD4+ T cells and / or CD8+ T cells.

[0183] In some embodiments, the cytokine is one or more of IFN-γ, IL-2, IL-4, IL-10, TNF-α.

[0184] In some embodiments, the immune response to VZV includes increased amounts of cytokines produced in lymphocytes.

[0185] In some embodiments, the immune response against VZV includes the production of antibodies that specifically bind to VZV glycoprotein gE encoded by the nucleic acid.

[0186] In some embodiments, the antibody is a neutralizing antibody against VZV or cells infected by VZV. In some embodiments, the serum titer of the antibody is increased in the subject.

[0187] Example 1

[0188] A lipid nanoparticle comprising RNA encoding a VZV glycoprotein gE variant, wherein the lipid nanoparticle comprises Dlin-MC3-DMA 50%, DSPC 10%, cholesterol 38.5% and PEG-DMG 1.5% by mole percentage.

[0189] The preparation method is as follows:

[0190] (a) RNA was dissolved in a pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0191] (b) Dlin-MC3-DMA, DSCP, cholesterol and PEG-DMG were dissolved in anhydrous ethanol according to the formula amount, and the concentration of the lipid component in the organic phase was adjusted to 6 mg / mL to obtain an organic phase.

[0192] (c) The aqueous phase of step (a) and the organic phase of step (b) were mixed at a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 ml / min. The mixed solution was immediately diluted 100 times with a pH 7.4 PBS solution, and the ethanol component in the solution was removed by tangential flow filtration (TFF). The solution was then concentrated to an mRNA concentration of 55 ug / ml to obtain lipid nanoparticles containing RNA encoding the VZV glycoprotein gE variant.

[0193] Example 2

[0194] Using luciferase as a reporter gene, the formulas of different vaccine carriers (as shown in Table 1 below, "MC3" refers to Dlin-MC3-DMA, "+": luciferase expression in mice was detected by the small animal in vivo fluorescence imaging system after administration) were studied to determine the efficiency of delivering mRNA encoding the luciferase gene in mice, and the physicochemical indicators of different composite preparations (see Example 1 for the preparation method) were detected. The results are shown in Table 1. Through research, it was found that increasing the mass ratio of lipids to mRNA is beneficial to increasing the encapsulation rate of mRNA in lipid nanoparticles, thereby making it more stable. In addition, moderately increasing the content of polyethylene glycol (PEG) in the formula is beneficial to improving the expression efficiency of mRNA in vivo. Therefore, considering factors such as mRNA encapsulation rate and mRNA in vivo delivery efficiency, formulas 3 and 4 were selected for subsequent mRNA vaccine research.

[0195] Table 1

[0196]

[0197] Example 3

[0198] The ability of cationic lipid nanoparticles of different formulations to encapsulate mRNA encoding luciferase and the particle size data of the formed nanoparticles are shown in Table 2. Several formulations can compress luciferase mRNA into nanoparticles with a particle size of less than 100 nm and a net neutral surface potential, and can encapsulate at least 50% of the mRNA, so they can all have a certain in vivo delivery effect. "MC3" refers to Dlin-MC3-DMA.

[0199] Table 2

[0200]

[0201] Example 4

[0202] The mRNA encoding the VZV glycoprotein gE variant was transfected into the cells, and the expression of the VZV glycoprotein gE variant in the cells was detected.

[0203] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.8, Seq ID NO.32 and Seq ID NO.33, and are denoted as VE2, VE1 and VE3, respectively. In addition to the open reading frame, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced with a 5' pseudouracil.

[0204] The amino acid sequence of VZV glycoprotein gE encoded by mRNA (VE1) with an open reading frame sequence as shown in Seq ID NO.32 is shown in Seq ID NO.30, which is the VZV wild-type glycoprotein gE.

[0205] The open reading frame sequence of the mRNA (VE3) shown in Seq ID NO. 33 encodes a VZV glycoprotein gE variant with a deletion of amino acid positions 574 to 623 and a Y569A substitution.

[0206] The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE1) shown in Seq ID NO.8 is shown in Seq ID NO.7, which is based on the VZV wild-type glycoprotein gE and has Y569A, S593A, S595A, T596A and T598A substitutions.

[0207] The method for detecting the in vitro expression of antigen VZV glycoprotein gE variant by immunoblotting experiment is as follows: HEK293 cells are spread on multi-well plates, 4 μg of mRNA and transfection reagent Lipofectamine RNAiMAX (purchased from Thermo Fisher Scientific) are mixed and then transfected into the cells. After 24 hours, the cells are collected and treated with lysis buffer. 20 μg of total protein are loaded on SDS-PAGE gel, and immunoblotting is performed with anti-varicella-varicella zoster virus glycoprotein E antibody and internal reference antibody (β-actin or GAPDH) to bind to the target protein, followed by incubation with secondary antibody, addition of chemiluminescent solution, and visualization of the target protein using a chemiluminescent instrument. Cells not transfected with mRNA are used as negative controls (see results). Figure 2 ).

[0208] The method for detecting antigen expression on the surface of HEK293 cells by flow cytometry is as follows: HEK293 cells are plated into multi-well plates, 2 μg of mRNA and transfection reagent Lipofectamine RNAiMAX (purchased from Thermo Fisher Scientific) are mixed and transfected into the cells. The specific transfection operation is carried out according to the instructions of the transfection reagent product. HEK293 cells after transfection for 24 hours are immunolabeled with anti-varicella-varicella zoster virus glycoprotein E antibody and fluorescent secondary antibody, and then the fluorescence signal is detected by flow cytometry (results are shown in Figure 2 ).

[0209] In the immunoblotting experiment, Ne is the cell without mRNA transfection, negative control; M is the marker. From the immunoblotting experiment, we can see that VE2, VE1 and VE3 were successfully transfected and expressed VZV glycoprotein gE in HEK293 cells; however, compared with VE1 and VE2, the expression level of VE3 was lower.

[0210] From the antigen detection map by flow cytometry, it can be seen that VE2, VE1 and VE3 all express VZV glycoprotein gE on the surface of HEK293 cell membrane, among which the expression level of VE2 on the cell membrane is slightly higher than that of VE1 and VE3.

[0211] Example 5

[0212] 7-week-old BALB / c mice were selected, and each mouse was intramuscularly injected with 10 μg of lipid nanoparticles encoding RNA of VZV glycoprotein gE variant, and the injection volume was 75 μL / mouse. The negative control was an equal volume of PBS injection (labeled as PBS). The positive control was varicella-varicella zoster virus glycoprotein E (labeled as gE) encapsulated in Freund's adjuvant, and the protein amount was 5 μg. The above various vaccine preparations were administered twice on day 0 and day 14, and the specific IgG antibody titer in the mouse serum was detected 14 days after the last administration; the neutralizing antibody titer; and the varicella-varicella zoster virus-specific CD4+ and CD8+ T lymphocyte signals.

[0213] 1. Multi-function CD + 4 and CD + 8T lymphocyte response level detection

[0214] Splenic lymphocytes prepared from isolated mouse spleens were added to cell culture plate wells, stimulated by adding varicella-varicella zoster virus glycoprotein E peptide library, and stimulated by adding BrefeldinA (purchased from Biolegend) after 48 hours of culture. The cell suspension was collected and added with a blocker (purchased from BD Pharmingen) to block the Fc receptor, followed by incubation with each specific monoclonal fluorescent antibody (CD3, CD4 and CD8). After washing twice, the cells were permeabilized. Then, intracellular staining was performed using fluorescent antibodies to IFN-γ and TNF-α (purchased from Biolegend), and the fluorescent signal was detected by flow cytometry after washing.

[0215] 2. Detection of extracellular cytokines IL-10, IL-2, IFN-γ and IL-4

[0216] Mouse spleen lymphocytes (4×10 6cells / well), collect the cell culture supernatant. After diluting the cell supernatant, add 100 μL / well to the pre-coated plate, incubate at room temperature for 90 minutes, wash with the cleaning solution, add the detection antibody diluted by the diluent, incubate at room temperature, wash with the cleaning solution, add the enzyme conjugate Streptavidin-HRP diluted by the diluent, incubate at room temperature for 30 minutes, wash with the cleaning solution, add 100 μL TMB, react in the dark for 15 minutes, terminate the reaction and read the plate for detection.

[0217] 3. Detection of specific IgG antibody titer in mouse serum

[0218] The ELISA method was used to detect the titer of VZV-specific total IgG antibody in mouse serum. VZVgE protein was added to a 96-well plate, placed at 4°C overnight, blocked with 2% BSA solution at room temperature, and mouse serum samples diluted in multiples were added and placed at room temperature for 2 hours, then washed with PBST. Goat anti-mouse IgGFc (HRP) anti-mouseIgG antibody was added, incubated at room temperature, washed, and incubated with TMB. After terminating the reaction with 2M sulfuric acid aqueous solution, the reading was taken at a wavelength of 450nm / 630nm. Twice the arithmetic mean of the detection signal of the negative serum sample was taken as the Cutoff value.

[0219] 4. Varicella-varicella zoster virus neutralization activity test

[0220] After inactivation, the serum samples were diluted in multiples, incubated with varicella-varicella zoster virus Oka strain (purchased from ATCC) for 1 hour, and then added to a 96-well plate containing MRC-5 monolayer cells for 2 hours, and then the medium was replaced and cultured in each well. The supernatant was discarded, and the cells were fixed and the fluorescently labeled detection antibody was added. The plate was read using a CTL instrument, and the antibody titer with an inhibition rate of 50% was calculated.

[0221] Example 6

[0222] The sequences of the open reading frames of mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.8, Seq ID NO.31 and Seq ID NO.33. The three mRNAs were prepared into lipid nanoparticles containing RNA encoding the VZV glycoprotein gE variant according to the method described in Example 1, that is, varicella zoster virus vaccines, which were respectively recorded as VE2, VE1 and VE3. In addition to the open reading frame, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced with a 5' pseudouracil.

[0223] The vaccine immunogenicity was evaluated for lipid nanoparticles containing RNA encoding VZV glycoprotein gE variants (ie, varicella-zoster virus vaccines VE2, VE1, and VE3) prepared according to the method described in Example 5.

[0224] 1. Detection of multifunctional CD+4 and CD+8 T lymphocyte response levels

[0225] Results Figure 4 The results showed that varicella-zoster virus vaccines VE2, VE1, VE3 and gE protein could induce CD+4 and CD8+ T cell responses in mice, among which VE2 induced stronger TNF-α+CD+4 and TNF-α+CD+8 signals.

[0226] 2. Detection of extracellular cytokines IL-10, IL-2 and IFN-γ

[0227] The results of extracellular cytokines IFN-γ and IL-2 are shown in Figure 5 . IFN-γ and IL-2 are important antiviral cytokines that reflect the level of Th1 cell response. The results showed that the varicella-zoster virus vaccines VE2, VE1, VE3 and gE protein could induce mice to produce higher levels of IFN-γ and IL-2, among which VE2 had a more obvious effect. Combined with the results of the multifunctional T lymphocyte response level test, the varicella-zoster virus vaccines VE2, VE1 and VE3 could induce mice to produce higher levels of Th1 cell response.

[0228] The results of extracellular cytokine IL-10 detection are shown in Figure 6 IL-10 is an immunomodulatory cytokine that reflects the level of Th2 cell response. The results showed that compared with gE protein, varicella zoster virus vaccines VE2, VE1 and VE3 could induce mice to produce higher levels of IL-10 (P<0.01).

[0229] 3. Detection of specific IgG antibody titer in mouse serum

[0230] 14 days after the second immunization, ELISA was used to detect VZV glycoprotein gE-specific IgG antibodies in mouse serum. The serum was diluted from 100 times to 1 million times, and the OD450 was measured by indirect ELISA. The antigen used in this example is an antigen that can specifically recognize the amino acid sequence of the wild type of VZV glycoprotein gE. Figure 7As shown, when the varicella zoster virus vaccine VE2, VE1 and VE3 were diluted to 1 million times, the sample reached the final dilution titer; when the gE protein was diluted to 10,000 times, the sample reached the final dilution titer. The results showed that the IgG antibody titer of the varicella zoster virus vaccine VE2, VE1 and VE3 was significantly different from that of the gE protein (P<0.01).

[0231] 4. Varicella-varicella zoster virus neutralization activity test

[0232] 14 days after the second immunization, the VZV live virus tropism reduction experiment was used to detect the VZV neutralizing antibody titer in the mouse serum. Figure 7 As shown, the geometric mean titer of virus neutralizing antibodies against gE protein was about 8.8, the geometric mean titer of virus neutralizing antibodies against VE2 and VE1 of varicella zoster virus vaccine was higher than 1300, and the geometric mean titer of virus neutralizing antibodies against VE3 of varicella zoster virus vaccine was about 1090. The geometric mean titer of virus neutralizing antibodies against VE2 and VE1 of varicella zoster virus vaccine was higher than that against VE3 and gE protein of varicella zoster virus vaccine (P<0.01).

[0233] Based on the results of specific IgG antibody titers, neutralizing antibody titers, and Th1 and Th2 cell response levels in the above serum, the varicella-zoster virus vaccine VE2 has the best overall performance.

[0234] Example 7

[0235] The mRNA encoding the VZV glycoprotein gE variant was transfected into the cells, and the expression of the VZV glycoprotein gE variant in the cells was detected.

[0236] The open reading frame sequences of mRNA encoding VZV glycoprotein gE variants are shown in Seq ID NO.8-17 and Seq ID NO.33, and are denoted as VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28, VE29 and VE3. In addition to the open reading frame, the mRNA encoding VZV glycoprotein gE variants also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO.1), a 3' UTR (as shown in Seq ID NO.4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced with a 5' pseudouracil. The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE1) having the open reading frame sequence shown in Seq ID NO.8-17 is shown in Seq ID NO.7.

[0237] The open reading frame sequences of mRNA encoding VZV glycoprotein gE variants Seq ID NO.8-17 have CAI values ​​of 0.93, 0.92, 0.92, 0.93, 0.92, 0.81, 0.81, 0.80, 0.79 and 0.81, and GC contents: 57.43%, 57.75%, 57.64%, 57.91%, 57.59%, 51.31%, 53.24%, 52.22%, 52.11% and 53.50%

[0238] According to the method described in Example 4, 1 μg of mRNA stock solution was transfected to detect the in vitro expression of mRNA VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28, and VE29 encoding VZV glycoprotein gE variants; according to the method described in Example 4, 0.25 μg of mRNA stock solution was transfected to detect the cell surface expression of mRNA VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28, and VE29 encoding VZV glycoprotein gE variants.

[0239] In vitro expression of mRNA encoding VZV glycoprotein gE variant Figure 8 From the imprint, the expression levels of mRNA VE2, VE21, VE22, VE23 and VE24 were slightly higher than those of mRNA VE25, VE26, VE27, VE28 and VE29.

[0240] The cell surface expression of mRNA encoding the VZV glycoprotein gE variant is shown in Fig. 9 As shown. The cell surface expression levels of mRNAVE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28 and VE29 were all higher than those of mRNAVE3; the cell surface expression levels of mRNAVE2, VE21, VE22, VE23 and VE24 were higher than those of mRNAVE25, VE26, VE27, VE28 and VE29; among them, the cell surface expression level of mRNAVE2 was the highest.

[0241] Example 8

[0242] The sequences of the open reading frames of mRNA encoding the VZV glycoprotein gE variants are shown in Seq ID NOs. 8 to 17. The 11 mRNAs were prepared into lipid nanoparticles containing RNA encoding the VZV glycoprotein gE variants according to the method described in Example 1, that is, varicella zoster virus vaccines, which were respectively recorded as VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28 and VE29. In addition to the open reading frame, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp (5')), a 5' UTR (as shown in Seq ID NO. 1), a 3' UTR (as shown in Seq ID NO. 4) and a 3' poly A tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced with a 5' pseudouracil.

[0243] The vaccine immunogenicity was evaluated for lipid nanoparticles containing RNA encoding VZV glycoprotein gE variants (i.e., varicella-zoster virus vaccines VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28 and VE29) prepared according to the method described in Example 5.

[0244] 1.CD+4T lymphocyte response level detection

[0245] Results Fig.10 The results showed that varicella-zoster virus vaccines VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28, VE29 and VE3 and gE protein could induce TNF-α+CD+4T cell response in mice.

[0246] 2. Detection of extracellular cytokines IL-10 and IFN-γ

[0247] The results of extracellular cytokine IFN-γ detection are shown in Fig.11 . IFN-γ is an important antiviral cytokine that reflects the level of Th1 cell response. The results showed that the varicella-zoster virus vaccine VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28, VE29, VE3 and gE protein can induce mice to produce high levels of IFN-γ. Combined with the results of CD+4T lymphocyte response level detection, the varicella-zoster virus vaccine VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28, VE29, VE3 and gE protein can induce mice to produce high levels of Th1 cell response.

[0248] The results of extracellular cytokine IL-10 detection are shown in Fig.12 IL-10 is an immunoregulatory cytokine that reflects the level of Th2 cell response. The results showed that compared with gE protein, varicella-zoster virus vaccines VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28 and VE29 could induce mice to produce higher levels of IL-10.

[0249] 3. Varicella-varicella zoster virus neutralization activity test

[0250] 14 days after the second immunization, the VZV live virus tropism reduction experiment was used to detect the VZV neutralizing antibody titer in the mouse serum. Fig.13 As shown, the geometric mean titer of virus neutralizing antibodies against gE protein is about 8.8, and the geometric mean titers of virus neutralizing antibodies against varicella-zoster virus vaccines VE2, VE21, VE22, VE23, VE24, VE25, VE26, VE27, VE28 and VE29 are all high.

[0251] Based on the results of specific IgG antibody titers, neutralizing antibody titers, and Th1 and Th2 cell response levels in the above serum, varicella-zoster virus vaccines VE2, VE21, VE22, VE23 and VE24 have better overall performance than VE25, VE26, VE27, VE28 and VE29; varicella-zoster virus vaccine VE2 has the best overall performance.

[0252] Example 9

[0253] The mRNA encoding the VZV glycoprotein gE variant was transfected into the cells, and the expression of the VZV glycoprotein gE variant in the cells was detected.

[0254] The amino acid sequence of the VZV glycoprotein gE variant is sequentially connected as Seq ID NO.7+Seq ID NO.18 (i.e., the C-terminus of sequence Seq ID NO.7 is connected to the N-terminus of sequence Seq ID NO.18), wherein the amino acid sequence shown in sequence Seq ID NO.18 encodes the cis-regulatory element Exin21, and the mRNA open reading frame sequence corresponding to the variant is sequentially connected as SeqID NO.8+Seq ID NO.19 (i.e., the 3' end of sequence Seq ID NO.8 is connected to the 5' end of sequence Seq ID NO.18), and is recorded as VE2E.

[0255] The amino acid sequence of the VZV glycoprotein gE variant is sequentially connected as Seq ID NO.7+Seq ID NO.24 (i.e., the C-terminus of sequence Seq ID NO.7 is connected to the N-terminus of sequence Seq ID NO.24), wherein the amino acid sequence shown in sequence Seq ID NO.24 encodes the multimerization element GCN4, and the mRNA open reading frame sequence corresponding to the variant is sequentially connected as Seq ID NO.8+Seq ID NO.29 (i.e., the 3' end of sequence Seq ID NO.8 is connected to the 5' end of sequence Seq ID NO.29), denoted as VE2F.

[0256] The amino acid sequence of the VZV glycoprotein gE variant is Seq ID NO.7 sequence with a deletion of amino acid at position 594, and the mRNA open reading frame sequence corresponding to the variant is Seq ID NO.8 sequence with a deletion of positions 1782 to 1784, denoted as VE2G.

[0257] In addition to the open reading frame, the mRNA encoding the VZV glycoprotein gE variant also includes a 5' cap (m7Gppp(5')), a 5'UTR (as shown in Seq ID NO.1), a 3'UTR (as shown in Seq ID NO.4) and a 3'polyA tail of 100 adenine nucleotides; and the uracil of the mRNA is replaced by a 5'pseudouracil.

[0258] According to the method described in Example 4, 0.5 μg of mRNA stock solution was transfected to detect the expression of mRNA VE2E, VE2F and VE2G encoding VZV glycoprotein gE variant on the cell surface.

[0259] The cell surface expression of mRNA encoding the VZV glycoprotein gE variant is shown in Fig.14 As shown in Figure 3, mRNA VE2, VE2E, VE2F and VE2G are all highly expressed on the cell surface.

[0260] Example 10

[0261] The sequences of the mRNA open reading frame encoding the VZV glycoprotein gE variant are shown in Seq ID NOs. 8 to 17. The four mRNAs VE2, VE2E, VE2F and VE2G described in Example 9 were prepared into lipid nanoparticles containing RNA encoding the VZV glycoprotein gE variant according to the method described in Example 1, that is, varicella-zoster virus vaccines, which were respectively recorded as VE2, VE2E, VE2F and VE2G.

[0262] The vaccine immunogenicity was evaluated for lipid nanoparticles containing RNA encoding the VZV glycoprotein gE variant (ie, varicella-zoster virus vaccines VE2, VE2E, VE2F and VE2G) prepared according to the method described in Example 5.

[0263] 1. Detection of CD+4 and CD+8 T lymphocyte response levels

[0264] Results Fig.15 The results showed that varicella-zoster virus vaccines VE2, VE2E, VE2F and VE2G could induce TNF-α+CD+4 and TNF-α+CD8+T cell responses in mice.

[0265] 2. Detection of extracellular cytokines IL-10, IL-2, IFN-γ and IL-4

[0266] The results of extracellular cytokines IFN-γ and IL-2 are shown in Fig.16 . IFN-γ and IL-2 are important antiviral cytokines that reflect the level of Th1 cell response. The results showed that the varicella-zoster virus vaccine VE2, VE2E, VE2F, VE2G and gE proteins can induce mice to produce high levels of IFN-γ and IL-2. Combined with the results of CD+4 and CD+8 T lymphocyte response level detection, the varicella-zoster virus vaccine VE2, VE2E, VE2F, VE2G and gE proteins can induce mice to produce high levels of Th1 cell response.

[0267] The results of extracellular cytokines IL-10 and IL-4 are shown in Fig.17 IL-4 and IL-10 are immunoregulatory cytokines that reflect the level of Th2 cell response. The results showed that compared with gE protein, varicella zoster virus vaccines VE2, VE2E, VE2F and VE2G could induce mice to produce higher levels of IL-10 (P<0.01). The levels of IL-4 induced by varicella zoster virus vaccines VE2, VE2E, VE2F and VE2G were slightly higher than those induced by gE protein and varicella zoster virus vaccine VE3.

[0268] 3. Varicella-varicella zoster virus neutralization activity test

[0269] 14 days after the second immunization, the VZV live virus tropism reduction experiment was used to detect the VZV neutralizing antibody titer in the mouse serum. Fig.18 As shown, varicella-zoster virus vaccines VE2, VE2E, VE2F and VE2G all achieved good geometric mean titers of virus neutralizing antibodies.

[0270] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them. Although the present disclosure has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. A varicella-zoster virus vaccine, characterized in that: The varicella zoster virus vaccine contains or encodes an antigen of the varicella zoster virus, and the varicella zoster virus vaccine is selected from a nucleic acid vaccine, a polypeptide vaccine or a virus vaccine; The antigen is selected from one or more of VZV glycoprotein gE, VZV glycoprotein gB, VZV glycoprotein gH, VZV glycoprotein gI, VZV glycoprotein gC, VZV glycoprotein gK or VZV glycoprotein gL.

2. The varicella-zoster virus vaccine according to claim 1, characterized in that The antigen is a VZV glycoprotein gE variant; the amino acid sequence of the VZV glycoprotein gE variant includes at least one of a Y569A substitution, a Y582A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution; Preferably, the amino acid sequence of the VZV glycoprotein gE variant comprises a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution and a T598A substitution.

3. The varicella-zoster virus vaccine according to claim 1, characterized in that The amino acid sequence of the VZV glycoprotein gE variant includes at least one continuous or intermittent amino acid deletion at amino acids 570 to 592, 594, 597, and 599 to 623.

4. The varicella-zoster virus vaccine according to claim 2 or 4, characterized in that The amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and / or includes at least one consecutive or intervening amino acid deletion of amino acids at positions 570 to 592, 594, 597, and 599 to 623; Preferably, the amino acid sequence of the VZV glycoprotein gE variant includes Y569A substitution, S593A substitution, S595A substitution, T596A substitution and T598A substitution, and at least one consecutive or intervening amino acid deletion from positions 570 to 592, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 consecutive or intervening amino acid deletions from positions 570 to 592; Preferably, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and includes a deletion of amino acid 594; Preferably, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a S593A substitution, a S595A substitution, a T596A substitution, and a T598A substitution, and includes a deletion of amino acid 597; Preferably, the amino acid sequence of the VZV glycoprotein gE variant includes Y569A substitution, S593A substitution, S595A substitution, T596A substitution and T598A substitution, and at least one consecutive or intervening amino acid deletion including amino acids 599 to 623, for example, may be but is not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 consecutive or intervening amino acid deletions including amino acids 599 to 623.

5. The varicella-zoster virus vaccine according to claim 1, characterized in that The varicella zoster virus vaccine encodes the antigen of varicella zoster virus, and the varicella zoster virus vaccine is a nucleic acid vaccine; Preferably, the nucleic acid vaccine comprises a nucleic acid molecule; the nucleic acid molecule comprises a DNA molecule and / or an RNA molecule; Preferably, the DNA molecules include chain DNA molecules and / or circular DNA molecules; Preferably, the RNA molecule comprises mRNA or circular RNA.

6. The varicella-zoster virus vaccine according to claim 5, characterized in that The mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) includes any one of (a) to (b): (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 8 to 17; (b) an mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or more nucleotides in the nucleotide sequence defined in (a); Preferably, the GC base pair percentage content of the mRNA molecule is 55% to 70%, and the CAI value of the mRNA molecule is 0.91 to 0.97; Preferably, the GC base pair percentage content of the mRNA molecule is 55% to 60%, and the CAI value of the mRNA molecule is 0.91 to 0.95; Preferably, the nucleic acid molecule has any one of SEQ ID No. 8 to 17, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID No. 8 to 17; Preferably, the mRNA molecule comprises an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) comprises any one of (a) to (b): (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 8 to 12; (b) an mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or more nucleotides in the nucleotide sequence defined in (a); Preferably, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No. 8 to 12, or an mRNA molecule encoding VZV glycoprotein gE having a nucleotide sequence at least 80% identical to Seq ID No. 8 to 17; Preferably, the mRNA molecule comprises an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) comprises any one of (a) to (b): (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID No. 8; (b) an mRNA molecule derived from (a) which has the function of encoding a VZV glycoprotein gE variant and has the same GC base pair percentage content and / or CAI value as the original nucleotide sequence, after substitution, deletion or addition of one or more nucleotides in the nucleotide sequence defined in (a); Preferably, the nucleic acid molecule has any one of SEQ ID No. 8, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO.

8.

7. The varicella-zoster virus vaccine according to claim 5, characterized in that The mRNA is composed of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail in sequence from the 5' end to the 3' end; Preferably, the 5' end cap is selected from ARCA, mCAP, dmCAP, m7G(5"")ppp(5"")(2""OMeA)pG, tmCAP, m7(3""OMeG)(5"")ppp(5"")(2""OMeA)pG, m7(3""OMeG)(5"")ppp(5"")(2""OMeG)pG, dmCAP or m7G(5"")ppp(5"")(2""OMeG)pG; preferably, the 5' end cap is m7Gppp(5')(2'-OMeA)pG. Preferably, the length of the 5'UTR is preferably 10 to 200 nucleotides; preferably, the length of the 5'UTR is 15 to 100 nucleotides; preferably, the nucleotide sequence of the 5'UTR is as shown in Seq ID NO.1 to 3; preferably, the nucleotide sequence of the 5'UTR is as shown in Seq ID NO.1; Preferably, the 3'UTR sequence is as shown in Seq ID NO.4 to 6; Preferably, the 3'UTR sequence is as shown in Seq ID NO.4; Preferably, one or more uridines in the mRNA are replaced with modified nucleosides; preferably, the modified nucleosides are pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U); preferably, the modified nucleosides are N1-methyl-pseudouridine (m1ψ). Preferably, the mRNA comprises at least one of a nucleotide sequence encoding a cis-regulatory element and a nucleotide sequence encoding a multimerization element; preferably, the nucleotide sequence encoding the cis-regulatory element is as shown in Seq ID NO.19; Preferably, the amino acid sequence of the multimerization element is as shown in Seq ID NO.20 to Seq ID NO.24; preferably, the amino acid sequence of the multimerization element is as shown in Seq ID NO.24; preferably, the nucleotide sequence encoding the multimerization element may also be as shown in Seq ID NO.25 to 29; Preferably, the mRNA molecule comprises a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

8. The varicella-zoster virus vaccine according to claim 1, characterized in that The varicella-zoster virus vaccine comprises a delivery preparation; preferably, the delivery preparation comprises lipid nanoparticles or cationic liposomes.

9. The method for preparing the varicella zoster virus vaccine according to claims 1 to 8, characterized in that: It includes: mixing the mRNA and a delivery formulation to form a varicella zoster virus vaccine; The delivery formulation includes lipid nanoparticles or cationic liposomes.

10. An isolated mRNA, characterized in that The coding region nucleic acid sequence of the mRNA encodes VZV glycoprotein gE, and the amino acid sequence of the VZV glycoprotein gE includes Y569A substitution, S593A substitution, S595A substitution, T596A substitution and T598A substitution, and / or includes at least one continuous or intervening amino acid deletion of amino acids at positions 570 to 592, 594, 597, and 599 to 623.

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