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 may 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 body's immunity 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, or VZV glycoprotein gL.

[0009] In some embodiments, the antigen is a VZV glycoprotein gE variant.

[0010] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0011] (a) at least one consecutive or intervening amino acid deletion from amino acids 569 to 623;

[0012] (b) at least one of Y569A substitution, Y582A substitution, S593A substitution, S595A substitution, T596A substitution, and T598A substitution;

[0013] (c) The C-terminus is connected to a multimerization element, and the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.24.

[0014] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0015] (a) at least one of the following: a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 623, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 579, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 574 to 623, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 588 to 623, and a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 579;

[0016] (b) at least one of Y569A substitution, Y582A substitution, S593A substitution, S595A substitution, T596A substitution, and T598A substitution;

[0017] (c) The C-terminus is connected to a multimerization element, and the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.24.

[0018] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution and at least one consecutive or intervening amino acid deletion from amino acids 588 to 623.

[0019] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution and a deletion of amino acids 588 to 623.

[0020] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a multimerization element connected to the C-terminus of the sequence, a Y569A substitution, and at least one consecutive or intervening amino acid deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24.

[0021] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.24.

[0022] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution and the amino acid deletions at positions 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24.

[0023] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution and the deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.24.

[0024] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a Y582A substitution, and at least one consecutive or intervening amino acid deletion from amino acids 588 to 623.

[0025] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a Y582A substitution, and a deletion of amino acids 588 to 623.

[0026] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, Y569A substitution, Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24.

[0027] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, Y569A substitution, Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.24.

[0028] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, Y569A substitution, Y582A substitution and amino acid deletions at positions 588 to 623, and the amino acid sequence of the multimerization element is shown in SeqID NO.20 to Seq ID NO.24.

[0029] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, Y569A substitution, Y582A substitution and amino acid deletions at positions 588 to 623, and the amino acid sequence of the multimerization element is shown in SeqID NO.24.

[0030] 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.

[0031] In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule.

[0032] In some embodiments, the RNA molecule comprises mRNA or circular RNA.

[0033] In some embodiments, the mRNA molecule comprises an open reading frame, and the nucleotide sequence of the open reading frame comprises any one of (a) to (b):

[0034] (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 or SEQ ID Nos. 35 to 44;

[0035] (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).

[0036] In some embodiments, the mRNA molecule comprises an open reading frame, and the nucleotide sequence of the open reading frame (ORF) comprises any one of (a) to (b):

[0037] (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 or SEQ ID Nos. 35 to 39;

[0038] (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).

[0039] In some embodiments, the mRNA molecule comprises an open reading frame, and the nucleotide sequence of the open reading frame (ORF) comprises any one of (a) to (b):

[0040] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in either SEQ ID No. 8 or SEQ ID No. 35;

[0041] (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).

[0042] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 70%, and the CAI value of the mRNA molecule is 0.78 to 0.97.

[0043] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 60%, and the CAI value of the mRNA molecule is 0.78 to 0.93.

[0044] In some embodiments, the nucleic acid molecule has any one of SEQ ID No. 8-17 or SEQ ID No. 35-44, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO. 8-17 or SEQ ID No. 35-44.

[0045] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No. 8-12 or SEQ ID No. 35-39, or an mRNA molecule encoding VZV glycoprotein gE having a nucleotide sequence at least 80% identical to Seq ID NO. 8-17 or SEQ ID No. 35-39.

[0046] In some embodiments, the nucleic acid molecule has any one of SEQ ID No. 8 or SEQ ID No. 34, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID No. 8 or SEQ ID No. 34.

[0047] 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. The 5' 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.

[0048] In some embodiments, the 5' cap is m7Gppp(5')(2'-OMeA)pG.

[0049] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1-3; in some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1.

[0050] In some embodiments, the 3'UTR sequence is as shown in Seq ID NO.4-6; in some embodiments, the 3'UTR sequence is as shown in Seq ID NO.4.

[0051] In some embodiments, one or more uridines in the mRNA are replaced with modified nucleosides; in some embodiments, the modified nucleosides are pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U); in some embodiments, the modified nucleosides are N1-methyl-pseudouridine (m1ψ).

[0052] In some embodiments, the mRNA includes a nucleotide sequence encoding a cis-regulatory element; in some embodiments, the nucleotide sequence encoding the cis-regulatory element is shown in Seq ID NO.19.

[0053] In some embodiments, the nucleotide sequence encoding the multimerization element can also be shown as Seq ID NO.25-29.

[0054] In some embodiments, the varicella-zoster virus vaccine comprises a delivery formulation.

[0055] In some embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] In some embodiments, the protonatable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA.

[0060] In some embodiments, the structured lipid comprises cholesterol and / or a cholesterol derivative.

[0061] In some embodiments, the helper lipid includes at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0062] In some embodiments, the surfactant includes at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0063] In some embodiments, the liposome nanoparticles include 20% to 50% cationic lipids by mole percentage.

[0064] In some embodiments, the liposome nanoparticles include 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-DMG by mole percentage.

[0065] In some embodiments, the present disclosure provides a method for preparing a varicella-zoster virus vaccine as described in any of the aforementioned embodiments, comprising: mixing the mRNA and a delivery formulation to form a varicella-zoster virus vaccine; the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0066] 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.

[0067] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2-4, and in some embodiments, 1:3.

[0068] In some embodiments, the buffer comprises citrate buffer or sodium acetate, and in some embodiments, citrate buffer.

[0069] In some embodiments, the pH of the buffer is 3-7, and in some embodiments, 4.

[0070] In some embodiments, the concentration of mRNA in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, and in some embodiments is 0.1 mg / mL.

[0071] In some embodiments, the present disclosure provides an isolated mRNA encoding a VZV glycoprotein gE variant, wherein the amino acid sequence of the VZV glycoprotein gE variant comprises at least one consecutive or intervening amino acid deletion of amino acids 588-623.

[0072] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant encoded by the isolated mRNA includes a Y569A substitution and a Y582A substitution, and / or includes at least one consecutive or intervening amino acid deletion of amino acids 588-623.

[0073] 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):

[0074] (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 or SEQ ID Nos. 35 to 44;

[0075] (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).

[0076] 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.

[0077] In some embodiments, the present disclosure provides a method of administering the shingles vaccine and at least one of the isolated mRNAs described above to a subject to induce an immune response against VZV in the subject.

[0078] In some embodiments, the immune response to VZV includes production of cytokines in lymphocytes.

[0079] In some embodiments, the lymphocytes are CD4+ T cells and / or CD8+ T cells.

[0080] In some embodiments, the cytokine is one or more of IFN-γ, IL-2, IL-4, IL-10, TNF-α.

[0081] In some embodiments, the immune response to VZV includes increased amounts of cytokines produced in lymphocytes.

[0082] 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.

[0083] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] 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.

[0085] Figure 1 A schematic diagram of the VZV glycoprotein gE is shown.

[0086] Figure 2 The expression of the mRNA encoding VZV glycoprotein gE in Example 4 in cells and on the membrane surface is shown.

[0087] Figure 3 A schematic diagram of the mouse experiment evaluation process is shown.

[0088] 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.

[0089] Figure 5 The varicella-zoster virus vaccine of Example 6 induces the secretion of IFN-γ and IL-2 by mouse spleen lymphocytes.

[0090] Figure 6 The varicella-zoster virus vaccine of Example 6 induces the secretion of IL-4 and IL-10 by mouse spleen lymphocytes.

[0091] Figure 7 The varicella-zoster virus vaccine of Example 6 induces humoral immune responses in mice.

[0092] Figure 8 The intracellular expression of mRNA encoding VZV glycoprotein gE of Example 7 is shown.

[0093] Fig. 9 The membrane surface expression of mRNA encoding VZV glycoprotein gE in Example 7 is shown.

[0094] Fig.10 The TNF-α+CD8+T cell response level of mice induced by the varicella-zoster virus vaccine of Example 8 is shown.

[0095] Fig.11 The varicella-zoster virus vaccine of Example 8 induces IFN-γ secretion in mouse spleen lymphocytes.

[0096] Fig.12The varicella-zoster virus vaccine of Example 8 induces IL-10 secretion by mouse spleen lymphocytes.

[0097] Fig.13 The varicella-zoster virus vaccine of Example 8 induces IL-4 secretion by mouse spleen lymphocytes.

[0098] Fig.14 The humoral immune response of the varicella-zoster virus vaccine of Example 8 in mice is shown.

[0099] Fig.15 The intracellular expression of mRNA encoding VZV glycoprotein gE in Example 9 is shown.

[0100] Fig.16 The membrane surface expression of mRNA encoding VZV glycoprotein gE in Example 9 is shown.

[0101] Fig.17 The TNF-α+CD4+T cell response level of mice induced by the varicella-zoster virus vaccine of Example 10 is shown.

[0102] Fig.18 The varicella-zoster virus vaccine of Example 10 induces IL-2 secretion by mouse spleen lymphocytes.

[0103] Fig.19 The varicella-zoster virus vaccine of Example 10 induces IL-10 secretion by mouse spleen lymphocytes.

[0104] Fig. 20 The humoral immune response of the varicella-zoster virus vaccine of Example 10 in mice is shown. DETAILED DESCRIPTION

[0105] 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.

[0106] 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), an AC sequence consisting of 55 amino acids ("AC" in the figure, positions 569 to 623), etc. VZV gE is a typical type I membrane glycoprotein that moves from the endoplasmic reticulum to the Golgi apparatus for post-translational modification and then transfers to the plasma membrane on the cell surface. Then, through endocytosis, gE is transported back to the TGN (trans-Golgi apparatus) for assembly and encapsulation of virion formation.

[0107] 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, etc.) are obtained by mutation and modification of the wild-type VZV glycoprotein gE to obtain an amino acid sequence that conforms to VZV glycoprotein gE. The mutation sites of the VZV glycoprotein gE variant described in the present disclosure are mainly concentrated at the AC end (the domain responsible for endocytosis), and the endocytosis of VZV glycoprotein gE is prevented by mutation, so that VZV glycoprotein gE is more displayed on the membrane surface. For example, VZV glycoprotein gE is substituted at the 569th amino acid, and amino acid Y is replaced with other amino acids, which can destroy the phosphorylation of the 569th amino acid site, prevent the 568-AYRV-571 motif from folding correctly, thereby preventing VZV glycoprotein gE from targeting the reverse Golgi tubular reticular structure (TGN) and destroying endocytosis. For another example, substitution of amino acid Y at position 582 of VZV glycoprotein gE with other amino acids can disrupt phosphorylation at position 582 and thus endocytosis.

[0108] 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).

[0109] 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.

[0110] 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.

[0111] In some embodiments, the antigen is VZV glycoprotein gE.

[0112] In some embodiments, the antigen is a VZV glycoprotein gE variant.

[0113] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0114] (a) at least one consecutive or intervening amino acid deletion from amino acids 569 to 623;

[0115] (b) at least one of Y569A substitution, Y582A substitution, S593A substitution, S595A substitution, T596A substitution, and T598A substitution;

[0116] (c) The C-terminus is connected to a multimerization element, and the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.24.

[0117] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one of (a) to (c):

[0118] (a) at least one of the following: a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 623, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 579, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 574 to 623, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 588 to 623, and a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 579;

[0119] (b) at least one of Y569A substitution, Y582A substitution, S593A substitution, S595A substitution, T596A substitution, and T598A substitution;

[0120] (c) The C-terminus is connected to a multimerization element, and the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.24.

[0121] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0122] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a deletion of amino acids 588 to 623.

[0123] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution.

[0124] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y582A substitution.

[0125] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution and a Y582A substitution.

[0126] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0127] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution and a deletion of amino acids 588 to 623 (VE4).

[0128] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0129] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y582A substitution and a deletion of amino acids 588 to 623.

[0130] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0131] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a Y582A substitution, and a deletion of amino acids 588 to 623.

[0132] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a multimerization element connected to the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24) and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0133] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a multimerization element connected to the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24) and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0134] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a multimerization element connected to the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24) and amino acids 588 to 623 are deleted.

[0135] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant includes a multimerization element connected to the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24) and amino acids 588 to 623 are deleted.

[0136] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24), a Y569A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0137] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24), a Y569A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0138] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24), Y569A substitution and amino acid deletions at positions 588 to 623.

[0139] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24), Y569A substitution and amino acid deletions at positions 588 to 623.

[0140] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the multimerization element amino acid sequence is shown in Seq ID NO.20 to Seq ID NO.24), a Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0141] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24), a Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0142] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24), Y582A substitution and amino acid deletions at positions 588 to 623.

[0143] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24), a Y582A substitution, and a deletion of amino acids 588 to 623.

[0144] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24), Y569A substitution, Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0145] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24), Y569A substitution, Y582A substitution and at least one consecutive or intervening amino acid deletion of amino acids 588 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 consecutive or intervening amino acid deletions of amino acids 588 to 623.

[0146] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24), Y569A substitution, Y582A substitution and amino acid deletions at positions 588 to 623.

[0147] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence (the amino acid sequence of the multimerization element is shown in Seq ID NO.24), Y569A substitution, Y582A substitution and amino acid deletions at positions 588 to 623.

[0148] 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).

[0149] 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, ... mn are n increasing natural numbers, and m1, m2, ...m n At least 2 adjacent natural numbers are discontinuous and differ by at least 2.

[0150] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant is as shown in Seq ID NO.7 or Seq ID NO.34, or comprises an amino acid sequence that is at least 80% identical to Seq ID NO.7 or Seq ID NO.34, 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 Seq ID NO.7 or Seq ID NO.34.

[0151] 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.

[0152] In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule.

[0153] In some embodiments, the RNA molecule comprises mRNA or circular RNA.

[0154] 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):

[0155] (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 or SEQ ID Nos. 35 to 44;

[0156] (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).

[0157] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 70%, and the CAI value of the mRNA molecule is 0.78 to 0.97.

[0158] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 60%, and the CAI value of the mRNA molecule is 0.78 to 0.95.

[0159] 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%.

[0160] 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.

[0161] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No.8-17 or SEQ ID No.35-44, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO.8-17 or SEQ ID No.35-44, 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.8-17 or SEQID No.35-44.

[0162] 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):

[0163] (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 or SEQ ID Nos. 35 to 39;

[0164] (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).

[0165] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 70%, and the CAI value of the mRNA molecule is 0.78 to 0.97.

[0166] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 60%, and the CAI value of the mRNA molecule is 0.78 to 0.95.

[0167] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No.8-12 or SEQ ID No.35-39, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO.8-17 or SEQ ID No.35-39, 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.8-17 or SEQID No.35-39.

[0168] 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):

[0169] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in either SEQ ID No. 8 or SEQ ID No. 35;

[0170] (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).

[0171] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 70%, and the CAI value of the mRNA molecule is 0.78 to 0.97.

[0172] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 60%, and the CAI value of the mRNA molecule is 0.78 to 0.95.

[0173] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No.8 or SEQ ID No.34, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to Seq ID NO.8 or SEQ ID No.34, 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 or SEQ ID No.34.

[0174] 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):

[0175] (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;

[0176] (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).

[0177] 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 0.90 to 0.97.

[0178] In some embodiments, the GC base pair percentage content of the mRNA molecule is 56% to 58%, and the CAI value of the mRNA molecule is 0.92 to 0.93.

[0179] 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.

[0180] 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):

[0181] (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;

[0182] (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).

[0183] 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 0.90 to 0.97.

[0184] In some embodiments, the GC base pair percentage content of the mRNA molecule is 56% to 58%, and the CAI value of the mRNA molecule is 0.92 to 0.93.

[0185] 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.

[0186] 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):

[0187] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID No. 8;

[0188] (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).

[0189] 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 0.90 to 0.97.

[0190] In some embodiments, the GC base pair percentage content of the mRNA molecule is 56% to 58%, and the CAI value of the mRNA molecule is 0.92 to 0.93.

[0191] 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):

[0192] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID Nos. 35 to 44;

[0193] (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).

[0194] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 55%, and the CAI value of the mRNA molecule is 0.78 to 0.85.

[0195] In some embodiments, the GC base pair percentage content of the mRNA molecule is 52% to 53%, and the CAI value of the mRNA molecule is 0.80 to 0.82.

[0196] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No.35 to 44, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to SEQ ID No.35 to 44, 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.35 to 44.

[0197] 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):

[0198] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID No. SEQ ID No. 35 to 39;

[0199] (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).

[0200] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 55%, and the CAI value of the mRNA molecule is 0.78 to 0.85.

[0201] In some embodiments, the GC base pair percentage content of the mRNA molecule is 52% to 53%, and the CAI value of the mRNA molecule is 0.80 to 0.82.

[0202] In some embodiments, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No.35 to 39, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to SEQ ID No.35 to 39, 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.35 to 39.

[0203] 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):

[0204] (a) an mRNA molecule having a nucleotide sequence encoding a VZV glycoprotein gE variant as shown in any one of SEQ ID No. 35;

[0205] (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).

[0206] In some embodiments, the GC base pair percentage content of the mRNA molecule is 50% to 55%, and the CAI value of the mRNA molecule is 0.78 to 0.85.

[0207] In some embodiments, the GC base pair percentage content of the mRNA molecule is 52% to 53%, and the CAI value of the mRNA molecule is 0.80 to 0.82.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] 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.

[0212] 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).

[0213] 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.

[0214] 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;

[0215] In some embodiments, the 5' cap is m7Gppp(5')(2'-OMeA)pG.

[0216] In some embodiments, the length of the 5'UTR is preferably 10 to 200 nucleotides.

[0217] In some embodiments, the 5'UTR is 15 to 100 nucleotides in length.

[0218] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1-3.

[0219] In some embodiments, the 5'UTR nucleotide sequence is as shown in Seq ID NO.1.

[0220] In some embodiments, the 3'UTR sequence is as shown in Seq ID NO. 4-6.

[0221] In some embodiments, the 3'UTR sequence is as shown in Seq ID NO.4.

[0222] In some embodiments, one or more uridines in the mRNA are replaced with modified nucleosides.

[0223] In some embodiments, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U).

[0224] In some embodiments, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0225] In some embodiments, the mRNA includes a nucleotide sequence encoding a cis-regulatory element.

[0226] In some embodiments, the nucleotide sequence encoding the cis-regulatory element is as shown in Seq ID NO.19.

[0227] 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.

[0228] In some embodiments, the nucleotide sequence encoding the multimerization element can also be shown as Seq ID NO.25-29.

[0229] 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.

[0230] 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.

[0231] 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.

[0232] In some embodiments, the mRNA molecule comprises a self-replicating mRNA molecule or a non-self-replicating mRNA molecule.

[0233] 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.

[0234] In some embodiments, the varicella-zoster virus vaccine comprises a delivery formulation.

[0235] In some embodiments, the delivery formulation comprises lipid nanoparticles or cationic liposomes.

[0236] 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.

[0237] 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.

[0238] 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.

[0239] In some embodiments, the protonatable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA.

[0240] In some embodiments, the structured lipid comprises cholesterol and / or a cholesterol derivative.

[0241] In some embodiments, the helper lipid includes at least one of DSPC, DOPE, DOPC, DOPG, and DOPS.

[0242] In some embodiments, the surfactant includes at least one of PEG-DMG, PEG-DSPE, and TPGS.

[0243] 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%.

[0244] In some embodiments, the liposome nanoparticles include 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-DMG by mole percentage.

[0245] 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.

[0246] 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.

[0247] 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.

[0248] 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.

[0249] 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.

[0250] 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.

[0251] 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.).

[0252] 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.

[0253] 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.

[0254] 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 and a Y582A substitution.

[0255] 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 588-623.

[0256] In some embodiments, the amino acid sequence of the VZV glycoprotein gE variant encoded by the isolated mRNA includes a Y569A substitution and a Y582A substitution, and / or includes at least one consecutive or intervening amino acid deletion of amino acids 588-623.

[0257] 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):

[0258] (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 or SEQ ID Nos. 35 to 44;

[0259] (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).

[0260] 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.

[0261] 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.

[0262] In some embodiments, the isolated DNA is isolated linear DNA or isolated circular DNA.

[0263] 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.

[0264] 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.

[0265] 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.

[0266] 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.

[0267] 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.

[0268] 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.

[0269] In some embodiments, the immune response to VZV includes production of cytokines in lymphocytes.

[0270] In some embodiments, the lymphocytes are CD4+ T cells and / or CD8+ T cells.

[0271] In some embodiments, the cytokine is one or more of IFN-γ, IL-2, IL-4, IL-10, TNF-α.

[0272] In some embodiments, the immune response to VZV includes increased amounts of cytokines produced in lymphocytes.

[0273] 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.

[0274] 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.

[0275] Example 1

[0276] A lipid nanoparticle comprising RNA encoding a VZV glycoprotein gE variant, wherein the lipid nanoparticle comprises, by mole percentage, 50% of Dlin-MC3-DMA, 10% of DSPC, 38.5% of cholesterol and 1.5% of PEG-DMG.

[0277] The preparation method is as follows:

[0278] (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.

[0279] (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.

[0280] (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.

[0281] Example 2

[0282] Using luciferase as a reporter gene, the efficiency of delivering mRNA encoding the luciferase gene in mice with different vaccine vector formulations (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) was studied by in vivo fluorescence imaging technology, and the physicochemical indicators of different composite preparations (preparation methods refer to Example 1) were detected.

[0283] Table 1

[0284]

[0285] 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.

[0286] Example 3

[0287] 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.

[0288] Table 2

[0289]

[0290] Example 4

[0291] 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.

[0292] The open reading frame sequences of the mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.35, Seq ID NO.32, Seq ID NO.33 and Seq ID NO.8, and are denoted as VE4, VE1, VE3 and VE5, 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.

[0293] 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.

[0294] 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.

[0295] The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE4) shown in Seq ID NO.35 is shown in Seq ID NO.34, which is based on the VZV wild-type glycoprotein gE, with amino acid positions 588 to 623 deleted and Y569A substituted.

[0296] The amino acid sequence of the VZV glycoprotein gE variant encoded by the mRNA (VE4) with the open reading frame sequence shown in Seq ID NO.8 is shown in Seq ID NO.7, which is based on the VZV wild-type glycoprotein gE, with amino acid positions 588 to 623 deleted, and Y569A and Y582G substitutions occurring.

[0297] 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 ).

[0298] 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 ).

[0299] 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 VE4, VE1, VE3 and VE5 were successfully transfected and expressed VZV glycoprotein gE in HEK293 cells; compared with VE1 and VE3, the expression levels of VE4 and VE5 were low.

[0300] From the antigen detection map by flow cytometry, it can be seen that VE4, VE1, VE3 and VE5 all express VZV glycoprotein gE on the surface of HEK293 cell membrane, among which the expression levels of VE4 and VE5 on the cell membrane are slightly higher than those of VE1 and VE3 on the cell membrane.

[0301] Example 5

[0302] 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.

[0303] 1. Multi-function CD + 4 and CD + 8T lymphocyte response level detection

[0304] 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.

[0305] 2. Detection of extracellular cytokines IL-10, IL-2, IFN-γ and IL-4

[0306] Mouse spleen lymphocytes (4×10 6 cells / 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.

[0307] 3. Detection of specific IgG antibody titer in mouse serum

[0308] 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 IgG Fc (HRP) anti-mouse IgG 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 read 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.

[0309] 4. Varicella-zoster virus neutralization activity test

[0310] 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.

[0311] Example 6

[0312] The sequences of the open reading frames of mRNA encoding the VZV glycoprotein gE variant are shown in Seq ID NO.35, Seq ID NO.31, Seq ID NO.33 and Seq ID NO.8. The four 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 VE4, VE1, VE3 and VE5. 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.

[0313] The vaccine immunogenicity was evaluated for lipid nanoparticles containing RNA encoding VZV glycoprotein gE variants (ie, varicella-zoster virus vaccines VE4, VE1, VE3 and VE5) prepared according to the method described in Example 5.

[0314] 1. Detection of TNF-α+CD+4 and CD+8 T lymphocyte response levels

[0315] Results Figure 4The results showed that varicella-zoster virus vaccines VE4, VE1, VE3, VE5 and gE protein could induce TNF-α+CD+4 and CD8+T cell responses in mice.

[0316] 2. Detection of extracellular cytokines IL-10, IL-2, IFN-γ and IL-4

[0317] 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 vaccine VE4, VE1, VE3, VE5 and gE protein can induce mice to produce higher levels of IFN-γ and IL-2. Combined with the results of the multifunctional CD+4 and CD+8 T lymphocyte response level detection, the varicella-zoster virus vaccine VE4, VE1, VE3, VE5 and gE protein can induce mice to produce higher levels of Th1 cell response.

[0318] The results of extracellular cytokines IL-4 and IL-10 are shown in Figure 6 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 VE4, VE1 and VE5 could induce mice to produce higher levels of IL-10 (P<0.01). The levels of IL-4 induced by varicella zoster virus vaccines VE4, VE1 and VE5 were slightly higher than those induced by gE protein and varicella zoster virus vaccine VE3.

[0319] 3. Detection of specific IgG antibody titer in mouse serum

[0320] 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 7 As shown, when the varicella zoster virus vaccine VE4, VE1, VE3 and VE5 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 VE4, VE1, VE3 and VE5 was significantly different from that of the gE protein (P<0.01).

[0321] 4. Varicella-varicella zoster virus neutralization activity test

[0322] 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 titers of virus neutralizing antibodies against VE4, VE5 and VE1 of varicella zoster virus vaccine were 899, 791 and 789 respectively, and the geometric mean titer of virus neutralizing antibodies against VE3 of varicella zoster virus vaccine was 416. The geometric mean titers of virus neutralizing antibodies against VE4 and VE5 of varicella zoster virus vaccine were higher than those against VE3 and gE protein of varicella zoster virus vaccine.

[0323] Based on the results of specific IgG antibody titers, neutralizing antibody titers, and Th1 and Th2 cell response levels in the above serum, the overall performance of varicella-zoster virus vaccines VE4 and VE5 is better.

[0324] Example 7

[0325] 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.

[0326] 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 VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, VE5e 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.

[0327] The CAI values ​​of the open reading frame sequences Seq ID NOs. 8 to 17 of the mRNA encoding the VZV glycoprotein gE variant were 0.82, 0.80, 0.82, 0.80, 0.80, 0.92, 0.92, 0.92, 0.93 and 0.93, and the GC contents were 52.83%, 51.85%, 52.98%, 52.7%, 52.47%, 57.71%, 57.48%, 57.54%, 57.82% and 57.94%.

[0328] According to the method described in Example 4, 4 μg of mRNA stock solution was transfected to detect the in vitro expression of mRNA VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, VE5e and VE3 encoding VZV glycoprotein gE variants; according to the method described in Example 4, 4 μg of mRNA stock solution was transfected to detect the cell surface expression of mRNA VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, VE5e and VE3 encoding VZV glycoprotein gE variants.

[0329] In vitro expression of mRNA encoding VZV glycoprotein gE variant Figure 8 From the imprint, the expression levels of mRNA VE5, VE52, VE53, VE54 and VE55 were slightly higher than those of mRNA VE5a, VE5b, VE5c, VE5d and VE5e.

[0330] 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 mRNAVE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d and VE5e were all higher than those of mRNAVE3; the cell surface expression levels of mRNAVE5, VE52, VE53, VE54 and VE55 were higher than those of mRNA VE5a, VE5b, VE5c, VE5d and VE5e; among them, the cell surface expression level of mRNAVE5 was the highest.

[0331] Example 8

[0332] 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 VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, and VE5e. 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.

[0333] The vaccine immunogenicity was evaluated for lipid nanoparticles containing RNA encoding VZV glycoprotein gE variants (i.e., varicella-zoster virus vaccines VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, and VE5e) prepared according to the method described in Example 5.

[0334] 1.CD+8T lymphocyte response level detection

[0335] Results Fig.10 The results showed that varicella-zoster virus vaccines VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, VE5e and VE3 and gE protein could induce CD8+T cell responses in mice.

[0336] 2. Detection of extracellular cytokines IL-10, IFN-γ and IL-4

[0337] 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 VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, VE5e, VE3 and gE protein can induce mice to produce high levels of IFN-γ. Combined with the results of CD+8T lymphocyte response level detection, the varicella-zoster virus vaccine VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, VE5e, VE3 and gE protein can induce mice to produce high levels of Th1 cell response.

[0338] The results of extracellular cytokines IL-10 and IL-4 are shown in Fig.12 and Fig.13 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 vaccine VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, and VE5e could induce mice to produce higher levels of IL-10 (P<0.01). The level of IL-4 induced by varicella zoster virus vaccine VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, and VE5e was slightly higher than that induced by gE protein and varicella zoster virus vaccine VE3.

[0339] 3. Varicella-varicella zoster virus neutralization activity test

[0340] 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.14 As shown, the geometric mean titer of virus neutralizing antibodies against gE protein is approximately 8.8, and the geometric mean titers of virus neutralizing antibodies against varicella-zoster virus vaccines VE5, VE52, VE53, VE54, VE55, VE5a, VE5b, VE5c, VE5d, and VE5e are all over 600.

[0341] 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 vaccine VE5, VE52, VE53, VE54 and VE55 have better overall performance than VE5a, VE5b, VE5c, VE5d and VE5e; varicella-zoster virus vaccine VE5 has the best overall performance.

[0342] Example 9

[0343] 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.

[0344] The open reading frame sequences of mRNA encoding VZV glycoprotein gE variants are shown in Seq ID NO.35-44 and Seq ID NO.33, and are denoted as VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40 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.35-44 is shown in Seq ID NO.34.

[0345] The CAI values ​​of the open reading frame sequences Seq ID NOs. 35 to 44 encoding the mRNA encoding the VZV glycoprotein gE variant were 0.93, 0.93, 0.92, 0.92, 0.93, 0.80, 0.78, 0.80, 0.80 and 0.80, and the GC contents were 57.82%, 57.82%, 57.60%, 57.54%, 57.77%, 52.24%, 52.24%, 52.47%, 51.85% and 51.90%.

[0346] According to the method described in Example 4, 0.25 μg of mRNA stock solution was transfected to detect the in vitro expression of mRNA VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40 and VE3 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 VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40 and VE3 encoding VZV glycoprotein gE variants.

[0347] In vitro expression of mRNA encoding VZV glycoprotein gE variant Fig.15 As shown. From the imprint, mRNA VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, and VE40 can be expressed in vitro.

[0348] The cell surface expression of mRNA encoding the VZV glycoprotein gE variant is shown in Fig.16 As shown. The cell surface expression levels of mRNAVE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49 and VE40 were all higher than those of mRNAVE3; the cell surface expression levels of mRNAE4, VE42, VE43, VE44, VE45 were higher than those of mRNAVE46, VE47, VE48, VE49 and VE40; among them, the cell surface expression level of mRNAVE4 was the highest.

[0349] Example 10

[0350] The sequences of the open reading frames of the mRNA encoding the VZV glycoprotein gE variants are shown in Seq ID NOs. 35 to 44 and Seq ID NO. 33. 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 VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40 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.

[0351] The vaccine immunogenicity was evaluated for lipid nanoparticles containing RNA encoding VZV glycoprotein gE variants (i.e., varicella-zoster virus vaccines VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40 and VE3) prepared according to the method described in Example 5.

[0352] 1. Detection of TNF-α+CD+4T lymphocyte response level

[0353] Results Fig.17 The results showed that varicella-zoster virus vaccines VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40 and VE3 and gE protein could induce TNF-α+CD+4T cell response in mice.

[0354] 2. Detection of extracellular cytokines IL-10, IL-2 and IL-4

[0355] The results of extracellular cytokine IL-2 detection are shown in Fig.18 . IL-2 is an important antiviral cytokine that reflects the level of Th1 cell response. The results showed that the varicella-zoster virus vaccine VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40, VE3 and gE protein can induce mice to produce high levels of IL-2. Combined with the results of CD+4 lymphocyte response level detection, the varicella-zoster virus vaccine VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49, VE40, VE3 and gE protein can induce mice to produce high levels of Th1 cell response.

[0356] The results of extracellular cytokine IL-10 detection are shown in Fig.19 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 VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49 and VE40V could induce mice to produce higher levels of IL-10.

[0357] 3. Varicella-varicella zoster virus neutralization activity test

[0358] 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. 20As shown, the geometric mean titer of virus neutralizing antibodies against gE protein is about 8.8, and the geometric mean titer of virus neutralizing antibodies against varicella-zoster virus vaccines VE4, VE42, VE43, VE44, VE45, VE46, VE47, VE48, VE49 and VE40 are all over 600.

[0359] 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 vaccine VE4, VE42, VE43, VE44, and VE45 have better overall performance than VE46, VE47, VE48, VE49, and VE40; varicella-zoster virus vaccine VE4 has the best overall performance.

[0360] 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 comprises or encodes a varicella-zoster virus (VZV) vaccine antigen, wherein the antigen is selected from VZV glycoprotein gE, VZV glycoprotein gB, VZV glycoprotein gH, VZV glycoprotein gI, VZV glycoprotein gC, VZV glycoprotein gK or VZV glycoprotein gL; Preferably, the varicella-zoster virus vaccine 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.

2. The varicella-zoster virus vaccine according to claim 1, characterized in that The antigen is a VZV glycoprotein gE variant; Preferably, the amino acid sequence of the VZV glycoprotein gE variant comprises at least one of (a) to (c): (a) at least one consecutive or intervening amino acid deletion from amino acids 569 to 623; (b) at least one of Y569A substitution, Y582A substitution, S593A substitution, S595A substitution, T596A substitution, and T598A substitution; (c) a C-terminal connected to a multimerization element, wherein the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.24; Preferably, the amino acid sequence of the VZV glycoprotein gE variant comprises at least one of (a) to (c): (a) at least one of the following: a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 623, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 579, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 574 to 623, a deletion of at least one consecutive or intermittent amino acid at amino acid positions 588 to 623, and a deletion of at least one consecutive or intermittent amino acid at amino acid positions 569 to 579; (b) at least one of Y569A substitution, Y582A substitution, S593A substitution, S595A substitution, T596A substitution, and T598A substitution; (c) The C-terminus is connected to a multimerization element, and the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.

24.

3. The varicella-zoster virus vaccine according to claim 2, characterized in that The amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution and at least one consecutive or intervening amino acid deletion from amino acids 588 to 623; Preferably, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution and a deletion of amino acids 588 to 623; Preferably, the amino acid sequence of the VZV glycoprotein gE variant comprises a multimerization element connected to the C-terminus of the sequence, a Y569A substitution, and at least one consecutive or intermittent amino acid deletion of amino acids 588 to 623, and the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.24; Preferably, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution and at least one consecutive or intermittent amino acid deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.24; Preferably, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution and the deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24; Preferably, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution and the deletion of amino acids at positions 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.

24.

4. The varicella-zoster virus vaccine according to claim 2, characterized in that The amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a Y582A substitution, and at least one consecutive or intervening amino acid deletion of amino acids 588 to 623; Preferably, the amino acid sequence of the VZV glycoprotein gE variant includes a Y569A substitution, a Y582A substitution, and a deletion of amino acids 588 to 623; Preferably, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution, the Y582A substitution and at least one consecutive or intermittent amino acid deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.20 to Seq ID NO.24; Preferably, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, the Y569A substitution, the Y582A substitution and at least one consecutive or intermittent amino acid deletion of amino acids 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.24; Preferably, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, Y569A substitution, Y582A substitution and amino acid deletions at positions 588 to 623, and the amino acid sequences of the multimerization element are shown in Seq ID NO.20 to Seq ID NO.24; Preferably, the amino acid sequence of the VZV glycoprotein gE variant is connected to the multimerization element at the C-terminus of the sequence, Y569A substitution, Y582A substitution and amino acid deletions at positions 588 to 623, and the amino acid sequence of the multimerization element is shown in Seq ID NO.

24.

5. The varicella-zoster virus vaccine according to claim 1, characterized in that The varicella zoster virus vaccine is a nucleic acid vaccine; the nucleic acid vaccine comprises nucleic acid molecules; the nucleic acid molecules include DNA molecules and / or RNA molecules; Preferably, the DNA molecules include chain DNA molecules and / or circular DNA molecules; Preferably, the RNA molecule comprises mRNA or circular RNA; Preferably, the mRNA molecule comprises an open reading frame, and the nucleotide sequence of the open reading frame 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 17 or SEQ ID Nos. 35 to 44; (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 mRNA molecule comprises an open reading frame, 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 or SEQ ID Nos. 35 to 39; (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 mRNA molecule comprises an open reading frame, 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 either SEQ ID No. 8 or SEQ ID No. 35; (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); The GC base pair percentage content of the mRNA molecule is 50% to 70%, and the CAI value of the mRNA molecule is 0.78 to 0.97; Preferably, the GC base pair percentage content of the mRNA molecule is 50% to 60%, and the CAI value of the mRNA molecule is 0.78 to 0.93; Preferably, the nucleic acid molecule has any one of SEQ ID No. 8 to 17 or SEQ ID No. 35 to 44, 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 or SEQ ID No. 35 to 44; Preferably, the nucleic acid molecule has a nucleotide sequence as shown in any one of SEQ ID No. 8 to 12 or SEQ ID No. 35 to 39, or an mRNA molecule encoding VZV glycoprotein gE having a nucleotide sequence at least 80% identical to Seq ID No. 8 to 17 or SEQ ID No. 35 to 39; Preferably, the nucleic acid molecule has any one of SEQ ID No.8 or SEQ ID No.34, or an mRNA molecule having a nucleotide sequence encoding VZV glycoprotein gE that is at least 80% identical to SeqID NO.8 or SEQ ID No.

34.

6. 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; 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 5'UTR nucleotide sequence is as shown in Seq ID NO.1 to 3; Preferably, the 5'UTR nucleotide sequence 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 a nucleotide sequence encoding a cis-regulatory element; preferably, the nucleotide sequence encoding the cis-regulatory element is as shown in Seq ID NO.19; Preferably, the nucleotide sequence encoding the multimerization element can also be shown as Seq ID NO.25-29.

7. The varicella-zoster virus vaccine according to claim 5, characterized in that The varicella zoster virus vaccine comprises a delivery formulation; Preferably, the delivery formulation comprises lipid nanoparticles or cationic liposomes; Preferably, the components of the lipid nanoparticles include: at least one of a protonable cationic lipid, a structural lipid, an auxiliary lipid and a surfactant; Preferably, 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; Preferably, 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; Preferably, the protonable cationic lipid is selected from at least one of Dlin-MC3-DMA, DODMA, C12-200 and DlinDMA; Preferably, the structured lipid comprises cholesterol and / or cholesterol derivatives; Preferably, the helper lipid comprises at least one of DSPC, DOPE, DOPC, DOPG and DOPS; Preferably, the surfactant comprises at least one of PEG-DMG, PEG-DSPE and TPGS; Preferably, the liposome nanoparticles comprise 20% to 50% of cationic lipids in terms of molar percentage; Preferably, the liposome nanoparticles comprise 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol and 1.5% PEG-DMG in molar percentage.

8. The method for preparing the varicella zoster virus vaccine according to claims 1 to 7, 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; Preferably, 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; Preferably, the volume ratio of the aqueous phase to the organic phase is 1:2 to 4, preferably 1:3; Preferably, the buffer comprises citrate buffer or sodium acetate, preferably citrate buffer; Preferably, the pH of the buffer is 3 to 7, preferably 4; Preferably, the concentration of mRNA in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL.

9. An isolated mRNA, characterized in that The isolated mRNA encodes a VZV glycoprotein gE variant, the amino acid sequence of the VZV glycoprotein gE variant comprising at least one consecutive or intervening amino acid deletion from amino acids 588 to 623; Preferably, the amino acid sequence of the VZV glycoprotein gE variant encoded by the isolated mRNA comprises a Y569A substitution and a Y582A substitution, and / or comprises at least one consecutive or intervening amino acid deletion of amino acids 588 to 623; Preferably, 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): (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 or SEQ ID Nos. 35 to 44; (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); The GC base pair percentage content of the separated mRNA is 55% to 70%, and the CAI value of the mRNA molecule is 0.91 to 0.

97.

10. A method of administering to a subject at least one of the herpes zoster vaccine according to claims 1 to 7 and the isolated mRNA according to claim 9 to induce an immune response against VZV in the subject; Preferably, the immune response against VZV includes the production of cytokines in lymphocytes; Preferably, the lymphocytes are CD4+T cells and / or CD8+T cells; Preferably, the cytokine is one or more of IFN-γ, IL-2, IL-4, IL-10, and TNF-α; Preferably, the immune response against VZV includes an increase in the amount of cytokines produced in lymphocytes; Preferably, the immune response against VZV comprises the production of antibodies that specifically bind to VZV glycoprotein gE encoded by the nucleic acid; Preferably, the antibody is a neutralizing antibody against VZV or cells infected by VZV; preferably, the serum titer of the antibody is increased in the subject.

Images